Halichoerus grypus) breeding colonies in eastern Canada. Human analysts counted and classified individual seals in imagery manually. Concurrently, an automated classification and detection algorithm discriminated seals based upon temperature, size, and shape of thermal signatures. Automated counts were within 95–98% of human estimates; at Saddle Island, the model estimated 894 seals compared to analyst counts of 913, and at Hay Island estimated 2188 seals compared to analysts’ 2311. The algorithm improves upon shortcomings of computer vision by effectively recognizing seals in aggregations while keeping model setup time minimal. Our study illustrates how UAS, thermal imagery, and automated detection can be combined to efficiently collect population data critical to wildlife management.Estimating animal populations is critical for wildlife management. Aerial surveys are used for generating population estimates, but can be hampered by cost, logistical complexity, and human risk. Additionally, human counts of organisms in aerial imagery can be tedious and subjective. Automated approaches show promise, but can be constrained by long setup times and difficulty discriminating animals in aggregations. We combine unmanned aircraft systems (UAS), thermal imagery and computer vision to improve traditional wildlife survey methods. During spring 2015, we flew fixed-wing UAS equipped with thermal sensors, imaging two grey seal ( Estimating the abundance of animal populations is essential for both fundamental and applied wildlife ecology1Generating abundance information about a species usually relies on human survey effort to either census a population, or estimate its numbers through a statistically robust sampling program. In many cases, these efforts employ real-time visualAerial surveys are particularly useful for species that make use of predictable habitats for resting, mating, breeding, feeding or other social activities. This is especially true for marine species such as seabirds and pinnipeds that spend significant time at sea where they are cryptic and largely unavailable for visual or remotely-sensed detection. For these species, ground-based or aerial surveys at terrestrial/on ice aggregation sites provide for efficient abundance assessments.Traditional surveys of many animal colonies are conducted using human-occupied helicopters or fixed-wing aircraft, which is riskyThe use of UAS in terrestrial research applications is now wide-spread and transforming spatial ecological science18While UAS surveys collect detailed information rapidly, they do not overcome existing data analysis bottlenecks. Specifically, manual counting of animals in imagery is time consuming and inefficient22th and 20th centuries. Subsequent conservation actions have enabled the recovery of many populationsHalichoerus grypus) populations were once depleted through bounties and hunts27Like many terrestrial carnivores, coastal predators in North America were severely depleted during the 19This paper illustrates the combination of (1) efficient UAS surveys, (2) thermal imaging and (3) a simple to use GIS-based workflow, to enhance the detection and enumeration of a marine predator at predictable terrestrial breeding colonies. Specifically, the present study estimates the abundance of grey seals at two breeding colonies in eastern Canada through automated counting of thermal imagery, classifying each individual seal as either an adult (>1 year old) or pup/young of the year . These counts are then compared to traditional human-generated counts of seals in both thermal and RGB imagery.Surveys for adult and YOY grey seals were conducted during the breeding season  at Hay Island  and Saddle Island , two breeding colonies in the Gulf of St. Lawrence, Nova Scotia, Canada .All UAS Surveys were conducted with the senseFly eBee, a commercially available fixed wing UAS. These modular devices are light-weight foam airframes powered by a single brushless electric motor supplied by lithium polymer batteries. They have a wing-span of 96 cm, weigh 0.7 kg and are highly portable, fitting into a case capable of being carried as cabin baggage on airliners for transport.The single engine and propeller are rear-mounted to ensure the safety of both the UAS and the people operating it. The aerodynamic profile allows the UAS to cruise at speeds of 36–57 km/h (10–16 m/s) and resist winds of up to 45 km/h (12 m/s). The UAS was pre-programmed in eMotion 2 software package  to follow a 3-dimensional flight path guided by a precision GPS sensor, a high-resolution barometer, ground-sensing camera and wind-speed sensor. Failsafe logic within the autopilot was programmed to return the UAS to the landing zone if it experienced anomalies in sensor performance or extreme wind conditions. All flight data was telemetered to the operator over VHF frequencies in real-time. The instrument was launched by hand and recovered after either a linear approach/landing at a predetermined 5 m radius region.th of a second at 3 cm ground sampling resolution with a photo taken approximately every 3 seconds. Thermal imagery was captured at sub-second intervals and at 8 cm ground sampling resolution at each location.Seal surveys were conducted at each colony using both a 12 megapixel RGB camera (Canon S110) and a 640 × 512-pixel thermal infrared camera . This sensor is self-calibrating, with a marketed precision of 0.1 °C (senseFly LLC). Comparisons with ground-based temperature measurements indicate that it is accurate within 1 °C. RGB imagery was captured at a shutter speed of 1/2000Images collected by the UAV were processed using Pix4d software to create RGB orthomosaics and thermal spatial indices (°C) of both colonies. All orthomosaics and thermal indices were corrected for any inconsistencies due to animal movement between adjacent images, exported as GeoTIFF files and then then imported into the iTag software package. Human analysts manually counted the number of seals and classified each as either an adult or a YOY. Temperature index GeoTIFFs were also imported into ArcMap GIS software  for subsequent automated detection and counting of adult and YOY seals.The seal detection model was built in the ArcGIS model builder programming environment and was designed to scan thermal imagery, detect seals and count them. The tool used spectral thresholds and pixel cluster size sorting to detect grey seal adults and YOY, and integrated object recognition and high pass filtering (i.e. edge detection) to discriminate individuals within closely packed aggregations. The tool’s logical overview can be seen in Thermal and RGB mosaic spatial indices of Hay Island were first masked by excluding areas around the southern and eastern edge of the island where the ambient temperature of landscape features overlapped with the temperature of seals.All pixels in the Hay Island thermal imagery greater than or equal to 9 °C were selected. We chose this value after visually estimating a lower thermal boundary for seal identification that excluded warmer landscape features. Selected pixels were then converted into polygons Panel B,  and the Because seals touching one another in aggregations resulted in single polygons with irregular shapes, we built convex hulls around all polygons Panel B, . A ratioResulting polygons were then classified as individual YOYs, individual adults, YOY aggregations or adult aggregations Panel C, . ClassifAggregation polygons were additionally processed with a high pass filter. High pass filters are neighborhood functions that accentuate the differences between pixels and are effective at detecting edges  of the model on Saddle Island. The simplified version used only planer area and polygon shape to discriminate individual YOY and adult polygons  where ambient landscape temperatures were lower, allowing for better contrast between seals and the environment. Discrepancies between human and model counts were due to humans overlooking YOYs, the lower thermal detection boundary of the model, size/temperature similarities between large YOYs and small adults, and an inability to distinguish between YOY aggregations and pup/mother pairs.Much of the difference in counts between the two methods was due to the detection of individual YOYs. Some of the apparent false positives , or to correct for misclassification error within a larger statistical model. However, the model is not suitable for analysis of imagery containing multiple species and, in this study, would likely misclassify any homeotherm with a body size similar to a seal. As such, this approach is suitable for enumeration of single species colonies where the target species represents the vast majority of animals present. The model is also sensitive to large shifts in ground sampling resolution. Our analysis is performed on corrected, spatially rectified imagery and individual seal classification relies largely on the planar area of objects, which should remain relatively constant with resolution shift. However, aggregation detection relies on convex hull ratios, which may be more sensitive to large resolution changes.Automated image classification is often considered a means to save time and costs associated with human-driven analysisAutomated image classification models like the one in this study synergize well with UAS. Appropriate use of UAS can output more precise counts than traditional methodsHow to cite this article: Seymour, A. C. et al. Automated detection and enumeration of marine wildlife using unmanned aircraft systems (UAS) and thermal imagery. Sci. Rep.7, 45127; doi: 10.1038/srep45127 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pericallis (Asteraceae) exemplify the ADE. In this study we used morphometric, Amplified Length Polymorphisms, and bioclimatic data for herbaceous Pericallis lineages endemic to the Azores and the Canaries, to test two key hypotheses proposed to explain the ADE: i) that it is a taxonomic artefact or Linnean shortfall, ie. the under description of taxa in the Azores or the over-splitting of taxa in the Canaries and (ii) that it reflects the greater ecological homogeneity of the Azores, which results in limited opportunity for ecological diversification compared to the Canaries.Oceanic archipelagos typically harbour extensive radiations of flowering plants and a high proportion of endemics, many of which are restricted to a single island . The Azores represents an anomaly as overall levels of endemism are low; there are few SIEs and few documented cases of intra-archipelago radiations. The distinctiveness of the flora was first recognized by Darwin and has been referred to as the ‘Azores Diversity Enigma’ (ADE). Diversity patterns in the Macaronesian endemic genus In both the Azores and the Canaries, morphological patterns were generally consistent with current taxonomic classifications. However, the AFLP data showed no genetic differentiation between the two currently recognized Azorean subspecies that are ecologically differentiated. Instead, genetic diversity in the Azores was structured geographically across the archipelago. In contrast, in the Canaries genetic differentiation was mostly consistent with morphology and current taxonomic treatments. Both Azorean and Canarian lineages exhibited ecological differentiation between currently recognized taxa.Pericallis. Whilst variation in genetic data and morphological data in the Canaries were largely congruent, this was not the case in the Azores, where genetic patterns reflected inter-island geographical isolation, and morphology reflected intra-island bioclimatic variation. The combined effects of differences in (i) the extent of geographical isolation, (ii) population sizes and (iii) geographical occupancy of bioclimatic niche space, coupled with the morphological plasticity of Pericallis, may all have contributed to generating the contrasting patterns observed in the archipelagos.Neither a Linnean shortfall nor the perceived ecological homogeneity of the Azores fully explained the ADE-like pattern observed in The online version of this article (doi:10.1186/s12862-016-0766-1) contains supplementary material, which is available to authorized users. Aeonium alliance in the Canary Islands [sensu Dansereau [Watson’s paper on [the] Azores has surprised me much; do you not think it odd, the fewness of peculiar species…?” [A key question for biologists is: why do different geographic regions harbour contrasting levels of biodiversity? –4. Ocean Islands . Howeveransereau  that alsansereau . Furtheransereau –10. Thisansereau  and noteecies…?” . Carine ecies…?”  coined tHypotheses to explain the ADE have included the proposal that the Azorean islands, or the lineages inhabiting them, are too young for extensive radiations to have occurred - with ca. 62 % of the land area being less than 1 million years old –15, thatPericallis  is endemic to Macaronesia. With sixteen species and a distribution spanning the Azores, Canaries and Madeira, it exemplifies the ADE, since diversity in this genus is unevenly distributed across the region: 14 taxa occur in the Canaries, 11 of which are SIEs, two SIEs occur in the Madeira archipelago [The genus hipelago  and one P. cruenta, P. papyracea, P. murrayi, P. steetzii and P. echinata) all exhibit broad and overlapping altitudinal and habitat ranges  on Santa Maria, São Miguel, Pico, Faial and São Jorge; P. malvifolia subsp. caldeirae is restricted to higher altitudes (>500 m) and is found on São Miguel, Faial, Terceira and Pico [Jones et al.  identifiand Pico ; see FigPericallis need to be re-assessed.Phylogenetic analyses of chloroplast and nuclear ribosomal ITS data provided only limited resolution within these clades but they did not support current taxonomic treatments in the Azorean clade . The twoPericallis lineages endemic to each archipelago. Using morphology, Amplified Fragment Length Polymorphisms (AFLPs) and bioclimate data we test two hypotheses to explain the ADE  thamalvifolia from the central islands and ssp. caldeirae was evident  from all other taxa (>10 florets), subsequent dimensions provided no useful information regarding the differences amongst the populations investigated. Non-parametric permutational multivariate analyses of variance (perMANOVA) of morphological data in the Azores revealed a significant difference between taxa and islands (P = 0.001). R2 values were 0.19 and 0.35 for the analyses of variation between species and between islands, respectively . R2 values were 0.65 and 0.43 for analyses between species and between islands respectively  were analysed using Principal Component Analysis (PCA). A number of clusters were separated along the first and second axis  São Miguel subsp. malvifolia (iii) central sub-archipelago subsp. malvifolia, (iv) São Miguel subsp. caldeirae and (v) central sub-archipelago subsp. caldeirae . PC1 explained a much higher percentage of the variation than PC2 . The most significant ecological variables that contributed to PC1 were all related to precipitation ; those contributing to PC2 were precipitation of the driest quarter and the driest month and isothermality  was used to assign individuals to genetic clusters [K-means clustering analyses of the Azorean AFLP data suggested that the best K value was 3 ; this reveals a pattern that broadly corresponded to the currently accepted taxa but with more sharing of genetic material between taxa than in the Azorean dataset  revealed a significant positive correlation between geographic distance and genetic distance but no significant correlation between geographical and morphological distance Table . There wIn the Canaries, full dbRDA revealed significant positive correlations between both geographic distance and genetic distance, and geographical distance and morphological distance Table . Full dbPericallis lineages that are endemic to each archipelago. We specifically tested two hypotheses  thePericallis, contrary to the suggestion of Schaefer et al. [Pericallis in the Canaries (65 % of the variation) with species circumscriptions in the Canaries also broadly supported by FAMD analysis of morphological variation. However, P. steetzii and P. murrayi were not differentiated in the FAMD analysis, which suggests some level of over-splitting . Analyscaldeirae individuals, with the exception of one individual from São Miguel were differentiated from subsp. malvifolia. Populations of subsp. malvifolia from Santa Maria, São Miguel and the central group were also differentiated in the PCA of climatic data. Whilst morphology showed no correlation with geographical distance, it was correlated with climate PC2, and strongly so according to the partial dbRDA when geographical distance was taken into account .Pericallis diversity patterns in the two archipelagos concerns the relationship between morphological and molecular (AFLP) data. We observe isolation by distance (IBD) for AFLP data in both archipelagos  that likely facilitates long distance dispersal to islands [Pericallis occur is ~60 km. In the Azores, the distances between Santa Maria and São Miguel (~80 km) and between São Miguel and the central group (~120 km) are both greater, and this is likely to promote greater genetic differentiation by geographic isolation . Thus, morphologically differentiated clusters in the Canarian lineage tend to be both geographically isolated and climatically differentiated .Pericallis are uncorrelated.In the Azores, morphology showed no correlation with geographical distance but was correlated with climate when the possible noise caused by geographical distance was taken into account in the partial dbRDA Table . In contcaldeirae ‘morphotype’ on separate islands in the central group and São Miguel may reflect underlying phenotypic plasticity, i.e. the property of a genotype to express distinct phenotypes in different environments [The independent origins of the high altitude subsp. ronments . The rolronments  and referonments . The maironments –54. Receronments . The potEuphorbia stygiana subsp. stygiana shows geographical structuring of molecular data yet morphological differences to support this have not been identified [Ammi lineage [Juniperus [Pericallis populations in both archipelagos, the number of samples with both morphological and genetic data was limited  and Canarian  lineages were sampled across the distribution ranges of each taxon, as recommended by Caujapé-Castells et al. [P. cruenta and P. echinata are known to hybridize [Individuals from both the Azorean  model may not account for the complex topological variation and micro-climatic conditions and climate predictions in regions with poor station density and varied topography [http://www.diva-gis.org/; Additional file .Whilst bioclimatic datasets from the WorldClim  and MyTaq™  were used during the selective amplification phase .To assess the reproducibility and reliability of AFLP fragments, we replicated 5–7 % of the individuals  at all stages from DNA isolation to AFLP production, according to the recommendations of Bonin et al.  and HollPericallis are challenging due to the various assumptions of statistical analyses linked with the difficulty in characterising the allelic variation within each individual, and the differing inheritance patterns between loci [Population genetic analyses of polyploids such as een loci . FurtherK-means clustering algorithm  using the “find.clusters” function in the R package adegenet, based on the Bayesian Information Criterion  was used to assign individuals to genetic clusters , 24. Thimination . ConsideFor each archipelago, we used hierarchical analyses of molecular variance (AMOVA) , 26 to iTo investigate IBD for each archipelago, we regressed geographic distances against morphological and genetic distances by carrying out full dbRDA using the Vegan package in R  and calcThe first two PCs (PC1 and PC2) from the PCA analyses of bioclimatic variables were used as a measure of bioclimatic conditions to avoid autocorrelation between individual bioclimatic variables, and a full dbRDA was used to test for any correlation between bioclimate and morphological variation, and genetic variation as above. We used partial dbRDA to test the influence of geographical distance on the relationship between (i) genetic diversity and bioclimate and (ii) morphology and bioclimate.


Oxynoemacheilus argyrogramma collected from Sepidbarg, Gamasiab and Ghaleji rivers, in the west of Iran. Analyses using three microsatellite loci indicated that the average number of alleles in the population was 12, which was well above the reported values for freshwater fishes. The expected (He) and observed (Ho) heterozygosity means were 0.865 and 0.576, respectively. Almost all loci showed deviation from the Hardy-Weinberg equilibrium (HWE). The results demonstrated that Oxynoemacheilus argyrogramma had desirable genetic diversity in the investigated regions.This study was carried out to investigate the genetic diversity and population structure of 90 specimens of  Oxynoemacheilus argyrogramma species  belongs to the Nemacheilidae family stone loaches of the West of Iran. Like most river loaches of the Nemacheilidae family, these fish are small benthic species that live in swift-flowing water, mainly on gravel or stony substrata [Confirmed freshwater fishes of Iran comprise of 202 species in 104 genera, 28 families, 17 orders and 3 classes. The dominant order is Cypriniformes with 120 species comprising 59.4% of the fauna, followed by Perciformes , Cyprinodontiformes  and Clupeiformes . The most diverse family is the Cyprinidae with 93 confirmed species (46.0%) followed by Gobiidae with 22 species (10.9%), Nemacheilidae with 22 species (10.9%), Clupeidae with 9 species (4.5%), Cyprinodontidae with 8 species (3.9%) and Salmonidae with 7 species (3.5%) [Oxynoemacheilusargyrogramma populations from three different river systems was examined using SSR markers. Three polymorphic microsatellite markers were developed to assess the genetic breeding of Oxynoemacheilus argyrogramma. Genetic diversity enabls environmental adaptation can assure survival chances of one species or population and is considered essential for the long-term survival of species . MicrosaSamplingand DNA extraction: Analyses were carried out based on 90 collected specimens from Sepidbarg , Gamasiab, Kermanshah Province  and Ghaleji, Kurdistan Province  rivers  rivers . CollectSSR marker amplification: Three primer pairs for SSR markers, Bbar5, Bbar8 and Bbar9 [2, 0.2 μL forward and reverse primers, 0.2mM deoxyribonucleotide triphosphates (dNTP) and 0.5U Taq polymerase. Amplification conditions were as follows: a pre-denaturation for 3 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at the selected higher annealing temperature, 30 s at 72°C, and 5 min at 72°C. Amplification products were separated by electrophoresis through 8% denaturing polyacrylamide gels. Detection of allele sizes obtained by the silver staining method was determined by comparing them to known DNA sequencing ladders.nd Bbar9  were useScoring and statistical analyses: Sizes of individual alleles were determined in relation to a 50 bp DNA size standard using the GenePro Analysis software. GenAlex software package, version 6.5 [o) and expected (He) heterozygosities and also to test for significant deviations from the Hardy-Weinberg equilibrium. Observed and expected genotype frequencies were then compared for each locus. The genetic distance between population pairs was estimated from the Nei standard genetic distance and the genetic similarity index [ST values.sion 6.5 , was usety index . GeneticOxynoemacheilus argyrogramma populations, the number of effective alleles ranged from 4.84 to 14.23, and the average for each population was 8.3 for Sepidbarg (Se), 8.07 for Gamasiab (Ga) and 8.07 for Ghaleji (Gh). The observed alleles ranged from 6 to17, the average for each population being 13.66 (Se), 11.66 (Ga) and 10.66 (Gh). The expected heterozygosity range was from 0.793 to 0.930 and the average for each population was 0.8783 (Se), 0.874 (Ga) and 0.843 (Gh). The average observed heterozygosity of each population was 0.636 (Se), 0.667 (Ga) and 0.424 (Gh). The Bbar8 locus had the highest number of alleles (17) and the Bbar9 had the lowest  were polymorphic in all populations of this study. The number of observed and effective alleles, observed heterozygosity, expected heterozygosity and fixation index are shown in e lowest . ST values and genetic distances, calculated based on the reduced set of three microsatellite loci, are given in FST) metric for the Sepidbarg and Gamasiab populations was 0.017, while for Sepidbarg-Ghaleji and Gamasiab-Ghaleji, it was found to be 0.045  were detected . Pair-wibe 0.045 . The estbe 0.045 . Analysibe 0.045 .Species adapt to various environments based on biological variation, which is one of the important factors for evaluating species' resources. A precondition to maintaining the highest level of genetic variation is making persistent use of a species' resources . HeterozOxynoemacheilusargyrogramma populations . The results of the study were comparable in variability to those reported by DeWoody and Avise [Paraschistura bampurensis  which hThe Hardy-Weinberg disequilibrium is common in many fishes, but deviations to the equilibrium generally prevail over heterozygote deficits , 18, 19 ST)/4 FST [According to the expressions of Nm=(1−FT)/4 FST , the Nm ST index, the genetic diversity between the populations was 4% and the mean of the FST index was about 0.039, which represent the low differentiation between the three populations. According to Wright (1987) [Violations of the Hardy-Weinberg assumptions can cause deviations from expectation. Reduction in size of a population is considered to be one of the few factors that might be responsible for such deviations. Small population size causes a random change in genotypic frequencies, particularly if the population is very small due to genetic drift. Analysis of molecular variance (AMOVA) is a suitable criterion to assess population structure and to determine the differentiation and genetic similarity between populations . Accordit (1987) , an Fst Oxynoemacheilusargyrogramma in these rivers was probably caused by past migrations. To characterize and distinguish Oxynoemacheilusargyrogramma populations, microsatellite loci should be preferred because of their generally higher variability and better performance, particularly if populations are within geographical regions. Our study indicated that the three populations had moderate levels of polymorphism and genetic variation. This information should be taken into account for future genetic conservations and stock improvement plans.Clustering order reflects relationships between populations. Using an UPGMA dendrogram, two separated communities were identified in these rivers. The genetic structure of


Washingtonia and Brahea palms had low levels of genetic diversity and were highly structured, with the majority of species and major geographic regions being characterized by distinct haplotypes. We also found strong support for currently recognized species in Washingtonia, but our results were less clear cut for Brahea due to haplotype sharing. Furthermore, patterns of population structure were broadly consistent with historical vicariant events such as the inundation of the Isthmus of La Paz, the formation of the Sea of Cortez, and the more recent colonization and isolation of Guadalupe Island's palms. Our findings contribute toward a growing appreciation of the complexity of plant responses to past geological changes and also provide valuable baseline genetic data on relict American palm species.The complex geological and ecological processes that have generated high levels of biodiversity and endemism in the Baja California Peninsula have been the subject of intensive study. However, relatively little is known about phylogeography of the iconic endemic palm species of this region. We therefore analyzed a total of 2,294 bp of chloroplast and 738 bp of nuclear sequence data in 169 samples of five native palm species from Baja California, Sonora and Guadalupe Island. We found that  Current species distributions arise from the interplay of various evolutionary, ecological and geological forces as well as from stochastic effects acting on species and populations. Multiple factors have been implicated in shaping species distributions including contemporary climate conditions  are one of the most distinctive of all plant families and are emblematic of the tropics  are currently present on the Baja Peninsula  and one (B. edulis) to Guadalupe Island. The remaining Brahea species are absent from the Baja California Peninsula but can be found on the Mexican mainland, with some species (B. nitida and B. dulcis) extending their range into Central America. Brahea is the least studied genus of American palms and the relationships between and within its species are not clearly described  to Sierra La Laguna at the southernmost tip of the peninsula  Do sympatrically occurring Washingtonia and Brahea palms exhibit similar patterns of genetic structure along the Baja Peninsula? (ii) Is the restricted distribution of desert palms reflected in low genetic diversity and high divergence between major geographic regions? (iii) Are patterns of geographically structured genetic variation concordant with major historical vicariant events that shaped the region such as the formation of the Gulf of California, the inundation of the Isthmus of La Paz and the formation of Guadalupe Island?Here we used 22.1Washingtonia and Brahea) identified on the basis of their morphology in the field. Specimens were collected from virtually all accessible oases throughout the Baja California Peninsula as well as from Guadalupe Island and two sites in Sonora, Mexico , of which eight samples corresponded to W. filifera and 83 to W. robusta. For Brahea, we collected 85 samples from nine populations (sierras) and tenth popilation from Guadalupe Island, of which 17 samples belonged to B. edulis, 29 to B. armata and 39 to B. brandegeei. As some authors . As we are unaware of any population‐level genetic studies of 2.3http://popart.otago.ac.nz, June 2016). We also used Gengis 2.1.1  with a total of 10,000 permutations. Molecular diversity statistics including the number of variable sites, the number of haplotypes, nucleotide and haplotype diversities, and the number of shared haplotypes were then computed using DnaSP 5.1 , Cabo Region , Sierra del Mechudo (SM), Sierra la Giganta (SG), Sierra San Pedro (SSP), combined sites of Sierra Libertad and Sierra San Francisco (SLI + SSF), Cataviña (Cat) and Sierra Juárez (SJ). For Brahea, we specified ten geographical regions. Nine of these corresponded to different sierras on the Baja Peninsula: Cabo Region (SL), Sierra del Mechudo (SM), Sierra San Francisco (SSF), Sierra San Pedro (SSP), Sierra Libertad (SLI), Sierra Asamblea (SA), Cataviña (Cat), Sierra San Pedro Martir (SSPM) and Sierra Juárez (SJ); while the final geographical region corresponded to Guadalupe Island (GI). For further details including sampling coordinates, see Tables To assess levels of genetic divergence between putative taxonomic species and  Kimura, . The for Kimura,  while thWashingtonia and Brahea using ARLEQUIN. We used AMOVA to estimate relative support for three alternative scenarios . Second, for the Washingtonia palms, we constructed a scenario based on the isolation of the mainland and peninsular populations after the formation of Sea of Cortez into three major regions: the Mexican mainland, Baja Peninsula and samples of W. filifera. Third, for the Brahea palms, we partitioned all of the samples into three groups: Guadalupe Island, Cabo region and rest of the Baja Peninsula. Fourth, for both Washingtonia and Brahea palms, we tested the hypothesis that relict populations persisted within each of the sierras, which should be reflected in each sierra harboring a genetically distinctive population. Eight groups were specified for Washingtonia and ten for Brahea, each corresponding to a different sierra  to partition the genetic variation within s Figure . Each scK) within the data. K was set from 1 to 10 and five replicates were performed for each value of K. An admixture analysis was then carried out with 50 reference individuals and 1,000 iterations. Finally, we spatially clustered the individuals using Voronoi tessellation and geo‐referenced sample location data. All of the spatial analyses were conducted separately for Washingtonia and Brahea.Finally, we used a Bayesian approach to estimate the number of genetic clusters present within the data as implemented in BAPS 6  using the maximum entropy method implemented in Maxent 3.4.0 . Each model included seven bioclimatic variables that we considered might have the strongest effect on the distribution of these species . By restricting this analysis to a subset of the most promising variables, we avoided potential overfitting of the Maxent model  and the following three historical time periods: mid Holocene , the last glacial maximum  and the last interglacial . All of the bioclimatic data were downloaded from WorldClim (www.worldclim.org) as a set of raster layers with a spatial resolution of 30 s (ca. 1 km) or in the case of the LGM data, 2.5 min (ca. 5 km)   to 1  , we performed a niche identity test using ENMtools 1.4.4 . In total, we obtained 89 nuclear and 81 plastid sequences for Washingtonia palms and 80 nuclear and 80 plastid sequences from Brahea palms, including a single sample of B. elegans from Mexican mainland  and two (nuclear) fixed nucleotides and carried no shared haplotypes , a group from the Baja California Peninsula and a group of mainland populations.Parsimony networks were used to visualize relationships among the samples based on the concatenated chloroplast and nuclear sequences  and the peninsular and mainland W. robusta populations. In contrast, none of the comparisons among the sierras of the Baja Peninsula were significant  and plastid (94%) data   Table .3.4K = 4, Figure Washingtonia, corresponding to the taxonomically defined W. filifera and W. robusta. A further two clusters were found within Brahea, corresponding to B. brandegeei samples from the SL population plus the single sample of B. elegans from the mainland, and B. edulis plus all of the B. brandegeei samples north of the Cabo region and all of the B. armata individuals. Based on the nuclear data, only three clusters were identified  and W. robusta throughout the rest of the Baja Peninsula. Based on the nuclear data, an additional cluster was recovered corresponding to samples from the mainland Washingtonia populations .During the mid Holocene  the predicted geographical distribution of n Figure b. Furthe Figures c. AccordM Figure d. The esr Figure , regardl4Washingtonia and Brahea were only partially confirmed. In accordance with expectations, disjunct and isolated populations of Baja palms contained low haplotype diversity. Additionally, ecological barriers like sea channels appear to be responsible for high levels of isolation between groups on either side of the barrier. In contrast to expectation however, genetic structure and diversity distribution of Washingtonia and Brahea along the Peninsula were not the same. Detailed ecological niche modeling analysis provided an explanation for this trend, suggesting slightly different re‐colonization routes after the LGM and sensitivity to different ecological variables. Our work demonstrates the importance and advantages of a multispecies approach, as some relevant conclusions about each species can only be reached using a comparison among different, yet similar and related species.We have generated baseline molecular data on species boundaries and the population structure of native palms of Baja California and Guadalupe Island, which provide new insights into the histories of these unique species. Our initial prediction regarding the levels of genetic diversity and phylogeography of 4.1Washingtonia and Brahea palms in the study area. The distribution of Washingtonia inferred from our sequence data is broadly in line with previously published morphological information  of the Peninsula. Our study also included B. edulis samples from Guadalupe Island, allowing a comparison to be made with the peninsular Brahea populations. We found that B. edulis was strongly differentiated from the peninsular populations at the nuclear sequence data, being fixed for a unique haplotype, but that at the chloroplast sequence data B. edulis could not be distinguished from the northern peninsula populations corresponding to B. armata.In contrast, our sequence data revealed mixed support for previous morphological species definitions within h et al.  recentlyBrahea. First, introgression of chloroplast DNA cannot be discounted, but this seems unlikely given the degree of geographic isolation of Guadalupe Island and its geological origin , whereas in Brahea palms, the mid peninsular sierras (SSF and SLI) had the highest nuclear sequence diversity  and northern (SSPM) refugia during the LGM. This hypothesis would also help to explain why it has been hard to established precise species boundaries between B. brandegeei from the southern and B. armata from the northern peninsula using morphological characteristics (Felger & Joyal, The pattern of sequence diversity found in M Figure . AnotherB. edulis individuals respectively. This pattern is in contrast to results found for other tree species of the Island (e.g., Pinus radiata, Karhu, Vogl, Moran, Bell, and Savolainen (Cupressus guadalupensis, Rosas‐Escobar et al. (B. edulis is not surprising. For example, although the total area of Guadalupe Island is about 240.00 km2, B. edulis occupies only 0.8 km2 at the foggy northern extreme of the Island (Garcillán et al., B. edulis is the low diversity of Brahea populations from the Baja Peninsula. For example, only two chloroplast haplotypes were detected in over 60 peninsular Brahea individuals. Additionally, the long generation time of Brahea (Bullock & Heath, B. edulis (Stuessy et al., Moving to the Guadalupe Island palms, we found comparably much lower levels of chloroplast and nuclear diversity. Only one chloroplast and one nuclear haplotype was recovered for 17 and 14 volainen  and Cuprr et al. . Althougr et al. . When al4.3W. robusta at both nuclear and chloroplast sequences. Our results from the chloroplast data for Brahea were similar, but as only one individual from the Mexican mainland was analyzed, we could not draw any firm conclusions. Nonetheless, although we lacked samples from the Mexican mainland for Brahea, we were able to include an endemic Brahea palm species from Guadalupe Island. This volcanic island has never been in contact with the Baja Peninsula and as a consequence almost 16% of its native plant species are endemics (Batiza, Brahea from its peninsular sister species is consistent with a strong effect of dispersal barriers like sea channels on the population structure of Brahea palms (Karhu et al., During the last six million years, the Baja California Peninsula has undergone many geological changes that have profoundly affected the distribution and genetic structure of plant and animals species (Dolby et al.,  Batiza, . Deep nuWashingtonia's nuclear and plastid sequence data did not provide any support for either of these breaks, with clustering results indicating the presence of a single genetically homogeneous population along the peninsula. By contrast, for Brahea, the chloroplast data strongly supported the presence of two genetically differentiated clades along the peninsula (Figures Historically, the Baja has also undergone a series of submersions and uplifts (Lindell et al.,  Figures b, providNone declared. Click here for additional data file.


Enantia jethys complex that has been studied for over a century; many debates exist regarding the species composition of this complex. Currently, three species that live sympatrically in the Gulf slope of Mexico  are recognized in this complex . Where these species live in sympatry, some cases of interspecific mating have been observed, suggesting hybridization events. Considering this, we employed a multilocus approach  to study hybridization in sympatric populations from Veracruz, Mexico. Genetic diversity parameters were determined for all molecular markers, and species identification was assessed by different methods such as analyses of molecular variance (AMOVA), clustering, principal coordinate analysis (PCoA), gene flow, and PhiPT parameters. ISSR molecular markers were used for a more profound study of hybridization process. Although species of the Enantia jethys complex have a low dispersal capacity, we observed high genetic diversity, probably reflecting a high density of individuals locally. ISSR markers provided evidence of a contemporary hybridization process, detecting a high number of hybrids (from 17% to 53%) with significant differences in genetic diversity. Furthermore, a directional pattern of hybridization was observed from E. albania to other species. Phylogenetic study through DNA sequencing confirmed the existence of three clades corresponding to the three species previously recognized by morphological and molecular studies. This study underlines the importance of assessing hybridization in evolutionary studies, by tracing the lineage separation process that leads to the origin of new species. Our research demonstrates that hybridization processes have a high occurrence in natural populations.Hybridization events are frequently demonstrated in natural butterfly populations. One interesting butterfly complex species is the  Pygaera pigra and P. curtula were chromosomally incompatible and short-lived. Current studies have shown that hybridization is not a rare process in nature and is evidence of the evolutionary speciation process, wherein population splitting lead to the subsequent divergence of entire lineages . C. CEnanties (COI)  and studes (COI) –74, likeousquets  suggesteE. albania to E. jethys and E. mazai. Some field observations  combined with molecular information support this hypothesis. First, in the field, interspecific mating events were observed between E. albania and E. jethys, and between E. mazai and E. jethys, but no genetic profiles corresponding to a combination of E. jethys and E. mazai were identified among the 117 hybrid individuals detected in this study. Second, very few hybrids were observed in the E. albania group; and third, all hybrid individuals from the two other morphospecies contained a genetic component of E. albania. These results could suggest some reproductive barriers among species of the complex and differences in the viability of hybrids, with the existence of a prezygotic barrier  that present premating reproductive isolation [Our molecular results suggest a directional pattern for hybridization from lization ) betweenterflies . Many stterflies  for revipulatory , or betwsolation .Enantia jethys complex, considering that closely related species are more likely to hybridize [Enantia jethys complex are subject to the same environmental pressures as they share the same oviposition and larval host plants [Many cases of interspecific hybridization have been reported for Lepidoptera .ybridize , and thaybridize . Furthert plants , which mEnantia jethys complex did not show intermediate morphology, however E. mazai males have a wide variation of morphotypes  than non-hybrids, probably a reflection of sample size. Second, hybrid individuals of E. jethys presented a low but significant decrease in genetic diversity value (h), which could reflect a reduction in hybrid fitness. Finally, hybrid individuals of E. mazai showed an important and significant increase in genetic diversity value (h). This is not surprising because non-hybrid E. mazai individuals presented the lowest value of genetic diversity. Therefore, when they cross with individuals with higher genetic diversity, hybrids presented higher genetic diversity values (h), which could result in novel adaptations. Nevertheless, additional ecological, behavioral, chromosomal, and genomic studies will be necessary to test our hypothesis.A study on Lepidoptera showed that hybrid individuals may present intermediate morphology . Hybrid photypes . The hybEnantia jethys complex using morphological data supported the existence of three distinct species: E. albania, E. jethys, and E. mazai, subsequently subdivided in subspecies E. mazai mazai and E. m. diazi [COI barcode) [E. albania, E. jethys, and E. mazai, without any evidence for subspecies or deep intraspecific differentiation. However, these authors discovered a sister clade of E. mazai composed of specimens that were morphologically indistinguishable from E. albania, suggesting the existence of a potential cryptic species originating in a specific geographic area . Using a multilocus approach , we confirmed the existence of three clades in the Enantia jethys complex in agreement with the results reported by Jasso-Matínez and colleagues [Wg marker   validatelleagues . The difS1 FigWg, B) RpS5, and C) COI. Black circles are the mutational steps.A) (TIF)Click here for additional data file.S2 FigCOI, RpS5, and Wg for the Enantia jethys complex. E. albania (red), E. jethys (green), and E. mazai (blue).(TIF)Click here for additional data file.S3 FigE. albania (red), E. jethys (green), and E. mazai (blue).(TIF)Click here for additional data file.S4 FigE. albania (red), E. jethys (green), and E. mazai (blue).Obtained after removing hybrid individuals from the dataset. (TIF)Click here for additional data file.S5 FigE. albania; B, C, and D) Common phenotypes; E) Phenotype very similar to E. jethys males.Pictures are organized according to a gradient of brown wing pigmentation, from lower to higher extent of brown spots. A) Phenotype very similar to a male of (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TableCOI) and nuclear (RpS5 and Wg) DNA sequences.Evaluated by analysis of molecular variance using mitochondrial ((DOC)Click here for additional data file.S3 Table(XLS)Click here for additional data file.


Extensive genetic variation underpinning phenotypic traits exists in natural plant populations, and this is intimately connected to the process of evolutionary adaptation. Importantly, such genetic variation also exists in the close relatives and progenitor species of many of our staple crops, which has great potential significance in agriculture. Therefore, the study of plant natural genetic variation can provide new insights into biological mechanisms, in addition to providing a vital source of genetic diversity in crop breeding.Arabidopsis thaliana (Arabidopsis)  . Sequencing over 1000 individuals collected from different geographic locations would allow a fuller characterization of the genetic variation within the species, providing a powerful resource for further investigations.As more whole-genome seque nces were completed for various wild-collected accessions of Arabidopsis it became clear that the concept of the single species A. thaliana accessions used in the 1001 genomes project.The image shows an educational installation within the Cruickshank Botanic Gardens (University of Aberdeen) focused on genomics called ‘a living-plant double helix’ created by Liam Salt from the diverse set of over 1000 different These and similar projects in other plant species, including many crop species, are being greatly strengthened by new DNA sequencing technologies that allow unprecedented depths of sequencing and longer reads. This is particularly valuable when repetitive elements of the genome, such as transposons and centromeres, are of interest . The comIn order for natural genetic variation to be utilized in agriculture, backgrounds of interest are typically crossed to domesticated strains, via either intra- or inter-specific hybridization. However, the phenotypic expression of traits can be complex, and the inheritance of characteristics of interest may not be easy to predict. Hence, the interaction observed between genotypes at different loci that determine phenotype has emerged as an area of great interest. FLC by vernalization, which involves the Polycomb repression system and long non-coding RNAs (Quantitative trait loci (QTL) analysis is a powerful approach to understand complex multigenic traits in plants and animals. Indeed, QTL mapping has been a successful approach for dissecting the molecular basis of flowering-time control in Arabidopsis, as reviewed by ing RNAs .Flowering is a key trait that can determine the extent of plant inter- and intra-specific hybridization . Sequenccis- and trans-acting effects of variation, and QTL-mapping approaches have been successful in identifying genes controlling recombination, including the conserved E3 ligase gene HEI10 (Once hybrids form, natural variation can be further recombined via the meiotic cell division . Meiosisne HEI10 . ImportaNatural genetic and epigenetic variation in plants is extensive, and remains to be fully described and harnessed in crop species. Nevertheless, new sequencing technologies provide opportunities to explore plant variation at greater depth and accuracy. This is particularly important in species with large, repetitive genomes, or in chromosomal regions typified by repeated sequences. For example, centromeres and ribosomal RNA clusters remain poorly understood due to our inability to completely sequence these regions. As technology advances it will be possible to obtain near-complete descriptions of genetic variation in natural populations. This will provide the opportunity to examine patterns of variation in relation to environmental adaptation at unprecedented resolution in space and time. As our climate continues to change, understanding global patterns of natural genetic variation and its relationship to plant adaptation will thus be of critical importance.


This year marks the 48th anniversary of Francis Crick’s seminal work on the origin of the genetic code, in which he first proposed the “frozen accident” hypothesis to describe evolutionary selection against changes to the genetic code that cause devastating global proteome modification. However, numerous efforts have demonstrated the viability of both natural and artificial genetic code variations. Recent advances in genetic engineering allow the creation of synthetic organisms that incorporate noncanonical, or even unnatural, amino acids into the proteome. Currently, successful genetic code engineering is mainly achieved by creating orthogonal aminoacyl-tRNA/synthetase pairs to repurpose stop and rare codons or to induce quadruplet codons. In this review, we summarize the current progress in genetic code engineering and discuss the challenges, current understanding, and future perspectives regarding genetic code modification. In 1968, Francis Crick first proposed the frozen accident theory of the genetic code , pyrrolyGenetic code engineering refers to the modification, or the directed evolution of cellular machineries, in order to incorporate NCAAs into the proteome of an organism. In general, NCAAs can be artificially incorporated in a site-specific or proteome-wide manner. In the former, scientists have attempted to artificially engineer organisms for compatibility with various NCAAs by employing orthogonal tRNA/aminoacyl-tRNA synthetase pairs ,30,31,32In this review, we first give a brief introduction of current studies on both site-specific and proteome-wide incorporation of NCAAs. Next, we will focus on the challenges of engineering organisms to use modified genetic codes and their implications, such as inhibitory effects caused by NCAAs. Finally, we will discuss current trends in this research area.E. coli BL21 rarely use the amber stop codon (UAG) (only 275 of 4160 stop codons in BL21 are amber codons), which minimizes disturbances to existing protein termination signals, this codon has been preferably selected for NCAA encoding pyrrolyl  was fed to a tryptophan (Trp)-auxotrophic E. coli capable of synthesizing Tpa  to generate mutants that could propagate on l-β-alanine  Tpa)   .Regardless of approach, the incorporation of NCAAs in the proteome may negatively affect the growth of an organism. The inherent toxicities of many unnatural amino acids could suppress propagation of the wild-type strain and select mutants that respond favorably to the NCAA, ultimately causing rejection of the expanded genetic code ,35. In tThe growth inhibitory effects caused by NCAAs, which have been demonstrated in different species including bacteria ,66,67, yBacillus subtilis str. QB928) via sequential mutagenesis in an early attempt to modify the genetic code. The resultant HR23 strain could propagate indefinitely on 4-fluoro-tryptophan (4FTrp) but became inviable on canonical Trp [Trp [The growth inhibitory potentials of NCAAs create negative selective pressure, while the organism adapts to the modified genetic code. One effective strategy for overcoming this evolutionary barrier comprises an increase in the mutation rate via mutagenesis with the expectation of generating beneficial mutations that would favor the NCAA. Wong and colleagues isolated mutants from a Trp auxotroph  that was absent from all other intermediate strains that could still propagate on Trp, suggesting a potential role for this mutation in switching membership of the UGG codon from Trp to 4-FTrp [E. coli, a rpoB mutation was found to confer rifampicin resistance via amber suppression at Gln513 [RNA polymerase might also play a key role in controlling the genetic code. The above-mentioned o 4-FTrp . In an it Gln513 , and thet Gln513 . In thatt Gln513 . These sB. subtilis HR23 mutant (unpublished data). Here, a gene ontology analysis of the gene expression profiles of these strains demonstrated enrichment of genes related to reactive oxygen species responses and branched-chain amino acid biosynthetic processes among upregulated genes, and enrichment of genes related to siderophore biosynthetic processes among downregulated genes (unpublished data). Unsurprisingly, stress response genes were modulated in response to the new genetic code, and the downregulation of siderophore biosynthetic process related genes was consistent with a previous observation of the reduced growth rate of HR23 cells grown on 4-FTrp [In addition to mutations, gene and protein expression profiles might also reveal key factors needed to fine-tune the use of modified genetic codes. Technologies such as RNA-seq and mass spectrometry can be used to investigate the cellular responses of organisms in high resolution. RNA-seq was used to compare the cellular responses between mutant (grown on 4-FTrp) and wild-type strains of the above-mentioned n 4-FTrp  because n 4-FTrp . This unMethanosarcina acetivorans is a methanogenic archaea strain that uses the alternative genetic codon UAG to encode pyrrolysine (Pyl) [Pyl, thus blocking the incorporation of Pyl in the proteome. A comparison of the proteomes of mutant and wild-type M. acetivorans strains revealed that most upregulated peptides were related to methanogenesis, protein synthesis, and the stress response [ne (Pyl) . O’Donogresponse , suggestVery few transcriptomic and proteomic studies of organisms with modified genetic codes have been conducted, and we have only glimpsed the potential factors involved in adaptation to modified genetic codes. Additional genes that contribute to this adaptation might remain to be discovered. In the future, studies of gene and protein expression in organisms with modified genetic codes will be necessary.Environmental factors, such as the growth medium and selection method, are important when optimizing the use of a modified genetic code. The amino acid source is the first and most obvious factor, as an organism can either take up NCAAs directly from the environment or synthesize them using environmentally available molecules. If the source of NCAAs is from the environment, mutations in amino acid transporters are often needed to facilitate NCAA uptake ,77.E. coli strains, the site-specific incorporation of two tyrosine analogs in β-lactamase was selected, and enzymatic function was found to depend on the presence of these analogs [E. coli mutants that could utilize NCAAs enjoyed a selective advantage under growth mediums containing certain classes of antibiotics [Acetohalobium arabaticum [A. arabaticum used the standard genetic code when grown on pyruvate, but gained the ability to use an expanded genetic code that included Pyl in the presence of the alternative carbon source trimethylamine [Positive selection pressure is also needed to maintain stability of the modified genetic code. In a previous study, incorporation of the methionine analog azidohomoalanine (Aha) into the coat protein of a human adenovirus and the subsequent addition of a folate group to Aha facilitated adenoviral infection in mouse hosts . In othe analogs . As descibiotics . In one abaticum . A. arabhylamine .Current efforts in genetic code engineering have reshaped our ideas regarding genetic code evolution and have paved the way for expanding the genetic alphabet. Based on these studies, we have outlined the key steps by which an organism accommodates a modified genetic code . During From the viewpoint of synthetic biology and xenobiology, genetic code engineering increases the repertoire of building blocks available for protein engineering, thus enabling the development of novel proteins that would be impossible with canonical amino acids ,93. XenoHigh-throughput genome editing technologies, such as MAGE  and the


Non-alcoholic fatty liver disease (NAFLD) is an epidemic metabolic condition driven by an underlying lipid homeostasis disorder. The lipid droplet (LD), the main organelle involved in neutral lipid storage and hydrolysis, is a potential target for NAFLD therapeutic treatment. In this review, we summarize recent progress elucidating the connections between LD-associated proteins and NAFLD found by genome-wide association studies (GWAS), genomic and proteomic studies. Finally, we discuss a possible mechanism by which the protein 17β-hydroxysteroid dehydrogenase 13 (17β-HSD13) may promote the development of NAFLD. Non-alcoholic fatty liver disease (NAFLD) is characterized by a pathological accumulation of triacylglycerol (TAG) in hepatocytes (i.e. hepatic steatosis) without excessive alcohol consumption  in eukaryotic organisms) are mainly stored in an organelle termed the lipid droplet (LD) Murphy, . The LD Cidea and Fsp27 (Cidec) mRNA levels are significantly increased in fatty liver dystrophic mice  are the major LD proteins in mammalian cells , genomic and proteomic research. As the mechanisms are still unknown, we also propose a hypothesis based on recent studies.PNPLA3 (rs738409), GCKR (rs780094), NCAN (rs2228603), LYPLAL1 (rs12137855), PPP1R3B (rs4240624), CPN1-ERLIN1-CHUK gene cluster, as well as others. These gene polymorphisms may influence NAFLD via hepatic carbohydrate and lipid metabolism, protein modification or signaling pathways  associated with diseases. Under the HapMap project, haplotype maps were established using tag SNPs. Disease associations with novel genes can be established by GWAS using a non-candidate driven method  is a LD-associated protein that is also distributed on other membranes  is possibly the most important. A comparative, quantitative proteomic study in humans detected a dramatic elevation of 17β-HSD13 in patients with NAFLD, compared with healthy controls , the main marker of hepatocellular injury (possibly representing fatty liver disease)  near HSD17B13 (encoding 17β-HSD13) and MAPK10  is strongly associated with the concentration of plasma alanine transaminase (ALT) (+/NAD(P)H binding domain (TGxxxGxG) and an enzymatic activation site (YxxxK) at the N-terminus. The 17β-HSDs are regarded as potential therapeutic targets for diseases such as breast cancer, endometriosis, osteoporosis, prostate cancer and even Alzheimer’s disease. All 17β-HSDs can modify the keto and hydroxy groups of steroids at the position C17 in vitro. However, the real physiological function of 17β-HSD13 in vivo is still unknown   with 17β-HSD11 , a kind of proandrogen, is associated with advanced NAFLD (Charlton et al., A significant fraction of estrogens and androgens are synthesized locally inside of target peripheral tissue cells, a process described as intracrine regulation. Almost all sex steroids in postmenopausal women and half of androgens in adult men are synthesized in peripheral intracrine tissues (Labrie et al., Circulating DHEA and DHEA-S originating from the adrenal gland are the major substrates for intracrine sex hormone synthesis and the conversion involves enzymes belonging to 3β-HSD, 17β-HSD, 5α-reductase and aromatase families Labrie, . MembersWe propose a hypothesis to explain how increased 17β-HSD13 promotes lipid synthesis and expands LDs in hepatocytes (Fig. The mechanisms involved may be more complicated. 17β-HSD enzymes interconvert estrogens in both directions. Therefore, 17β-HSD13 might convert E2 to E1, thereby reducing the E2 concentration in the cellular microenvironment, resulting in a decrease in metabolic protection afforded by E2 and activating lipid anabolic genes. Besides steroid metabolism, some subtypes of 17β-HSDs also play roles in fatty acid elongation and β-oxidation, synthesis of prostaglandin and retinol metabolism. These other activities of 17β-HSD13 may influence fatty liver development via distinct metabolic pathways. In addition, 17β-HSD13 is also present as a major protein on hepatic LDs. Thus it may also act as a structural protein, protecting lipids from hydrolysis by preventing lipases from gaining access to the interior lipids. The mechanisms regulating 17β-HSD13 and its involvement in the progression of NAFLD still need to be elucidated.PNPLA3-I148M variant. PNPLA3-I148M seems to be a loss-of-function mutation, but its pathogenic effect appears to be mediated through a gain in function resulting in a suppression of lipase activity. The 17β-HSD13 enzyme was identified by a comparative proteomic study of patients with normal and simple hepatic steatosis liver. Increased 17β-HSD13 on LDs greatly elevates lipid content in both hepatocytes and mouse liver. Since 17β-HSD family proteins catalyze the key step in sex hormone synthesis, we speculate that 17β-HSD13 may promote lipid synthesis by producing hormones which regulate lipogenic gene expression. However, the detailed underlying mechanisms by which LDs and LD-associated proteins induce NAFLD remain to be elucidated.The LD is the central organelle regulating lipid homeostasis and disorders of LD-associated proteins induce metabolic diseases like NAFLD. GWAS studies reveal that SNPs linked with PNPLA3 are strongly associated with NAFLD, especially the


Gadopsis marmoratus) within five streams separated by weirs or dams constructed 45–120 years ago. We found evidence of small-scale (<13 km) genetic structure within reaches unimpeded by barriers, as expected for a fish with low mobility. Genetic diversity was lower above barriers in small streams only, regardless of barrier age. In particular, one isolated population showed evidence of a recent bottleneck and inbreeding. Differentiation above and below the barrier (FST = 0.13) was greatest in this stream, but in other streams did not differ from background levels. Spatially explicit simulations suggest that short-term barrier effects would not be detected with our data set unless effective population sizes were very small (<100). Our study highlights that, in structured populations, the ability to detect short-term genetic effects from barriers is reduced and requires more genetic markers compared to panmictic populations. We also demonstrate the importance of accounting for natural population genetic structure in fragmentation studies.Habitat loss and fragmentation often result in small, isolated populations vulnerable to environmental disturbance and loss of genetic diversity. Low genetic diversity can increase extinction risk of small populations by elevating inbreeding and inbreeding depression, and reducing adaptive potential. Due to their linear nature and extensive use by humans, freshwater ecosystems are especially vulnerable to habitat loss and fragmentation. Although the effects of fragmentation on genetic structure have been extensively studied in migratory fishes, they are less understood in low-mobility species. We estimated impacts of instream barriers on genetic structure and diversity of the low-mobility river blackfish ( Widespread loss of habitat and associated fragmentation of wildlife populations is a major threat to global biodiversity  and lateral (notably onto adjacent floodplains during high flows) movements as a part of their life history. Along with other threats, such as overexploitation, water pollution, flow modification, and invasion by exotic species, these aspects of freshwater ecosystems have contributed to a proportionally higher number of threatened freshwater species per area compared to terrestrial and marine ecosystems  after accounting for natural genetic structure. The upper Yarra River system near Melbourne, Victoria, Australia presents an excellent opportunity to design an assessment of the impacts of barriers of known age on the genetic structure of a low-mobility freshwater fish. In response to Melbourne’s human population growth, a network of stream flow diversion weirs of known age has progressively increased in extent in the upper Yarra River system, with staged installation of water supply weirs and dams since 1893.In this study, we focus on the impacts of artificial barriers on the genetic structure and diversity of a non-migratory freshwater fish species, the southern river blackfish, G. marmoratus generations) and catchment sizes were 14–337 km2. To assess our ability to distinguish barrier effects from background genetic structure, we first estimated the background levels of differentiation across sample sites within connected parts of the catchment. We then simulated the effects of genetic drift on genetic diversity and differentiation (FST) across a barrier for different densities of populations with background genetic structure similar to those estimated in our observed data set. We also used simulations to investigate whether barrier effects on genetic differentiation were masked by unidirectional downstream gene flow  stronger differentiation between populations above and below barriers than would be expected under natural processes, and (2) reduced genetic diversity and inbreeding within populations above barriers compared to populations below barriers. More specifically, we predicted that loss of genetic diversity in populations above each barrier and genetic differentiation across the barrier would depend on (1) the age of the barrier, (2) the size of the above-barrier population (based on catchment area) and (3) the disturbance history  that could lead to bottlenecks compounding genetic drift due to fragmentation. To achieve our aims, we analysed microsatellite DNA marker data for populations above and below water supply barriers in five streams, where barrier ages were 45–120 years old  and the upper Yarra River sub-catchment (including Armstrong and McMahons creeks)  and surface area 777 ha; Watts River 165 km2, Maroondah Reservoir–22,179 ML, 199 ha) and creeks with small catchment areas and diversion weirs   Fig. . Land us2) Table , and wil2) Table .Fig. 1YaGadopsis marmoratus sensu lato has a range across south-eastern Australia, being widespread in Victoria and present in parts of south east Queensland, eastern New South Wales and south east South Australia , increasing with fish size  from 28 sites across five streams  was collected and preserved in 100% ethanol prior to being stored in the laboratory at −20 °C. Fish were released after sampling. In this study, total length of G. marmoratus was 38–455 mm  and weight 1–830 g   were removed from genetic analyses.The total length and the total weight of each captured individual were measured and a small piece of caudal fin  were monomorphic in all samples of this study (Supplementary Table Ho and He) and Weir and Cockerham’s . Using the rarefaction procedure implemented in FSTAT 2.9.3.2  for ≥6 individuals at single sample sizes, and ≥24 individuals in the pooled samples.Total DNA was extracted from kerham’s  estimate Goudet, , for eacK) within the complete data set, ten replicate runs of 2 × 106 Markov chain Monte Carlo (MCMC) iterations, after an initial burn-in period of 5 × 105 iterations, were performed for values of K ranging from 1 to 10 (the maximum set to the number of pooled samples). Results were summarised using the standard pipeline on the CLUMPAK web server  was explored using the estimated logarithm of likelihood (LnP(D)) and the Evanno et al. (K method that finds the point of greatest change in the distribution of LnP(D) with STRUCTURE HARVESTER Version 0.6.92  against river distance between sample sites . Spatial autocorrelation was investigated with SPAGEDI 1.5  above versus below each barrier, and (2) between the below-barrier samples in each sub-catchment (Watts River or Upper Yarra). The significance of FST values was determined using 45,000 permutations. We also explored Dest values, which more accurately account for differences in allelic diversity than does FST  was included as a fixed factor. Because progressive loss of genetic diversity through drift in small, isolated populations above barriers was expected to depend on: i) population size above the barrier and ii) the number of generations since isolation, we included a fixed factor  separating the two rivers with large catchments (Yarra and Watts rivers) from the three creeks with small catchments  as a proxy for population size, and the age of the barrier (weir_age) as a proxy for the number of generations since isolation of above-barrier populations. We also included the pairwise interactions weir_side:catchment_type and weir_side:weir_age. Locus identity was included as a random intercept. Models were validated a posteriori by checking plots of residuals. Significance of fixed effects was assessed with analysis-of-deviance tables (function Anova in the R package car). Post-hoc comparisons of mean allelic richness between populations above and below barriers  was performed using the “lsmeans” function implemented in the lsmeans package  of a population in question and He outbred is heterozygosity of an outbred population  were estimated using the linkage disequilibrium method (LDNe)  ‘population’ but reflect the effective size in the sampled area : 3, 10 and 30 individuals per node  to cover the range of population sizes in each study stream. According to the formula Ne = Nt / (1-FST) . Maximal dispersal distance was set to 40 nodes, the emigration rate (e) and geometric distribution parameter (g) were set to 0.5/0.95, 0.25/0.9 and 0.15/0.85 for De of 3, 10 and 30 individuals per node, respectively. To evaluate the impact of asymmetric migration across the barrier, we ran an IBD + asymmetric migration scenario with the same set of parameters, while allowing downstream gene flow at a rate of 0.2. To compare simulation results with a more typical scenario where there is no genetic structure along each stream prior to the installation of barriers, we also ran a Panmixia scenario, for which the maximum dispersal distance was set to 160 nodes (the size of the network), the emigration rate (e) and the geometric distribution parameter (g) were set to 1/1. For each dispersal scenario and effective density, 50 data sets were simulated assuming no barrier and assuming a complete barrier introduced at various number of generations prior to sampling , chosen to incorporate the range of barrier ages observed in our study area (45–120 years or ~9–24 generations).To evaluate our power to detect an effect of a barrier through time for various ) Wright , where NDe and age of the barrier), we calculated allelic richness in pooled sampled nodes above  and below the barrier , and IBD regression slopes for the simulations without a barrier using the GENEPOP 4.1 and adegenet package in R 3.1.3. Weir and Cockerham’s pairwise FST was calculated using genpop for each locus and globally among (i) pooled sampled nodes 130, 141, and 151 (above the barrier at node 126) and pooled sampled nodes 100, 110 and 120 (below the barrier at node 126), and (ii) among pooled sampled nodes 100, 110 and 120 and 70, 80 and 90 . To determine whether pairwise FST values between pooled nodes above and below the barrier were significantly higher than pairwise FST values between pooled nodes below the barrier, we used a paired Wilcoxon test and each locus as replicate unit. We also conducted similar Wilcoxon tests using the observed data, where pairwise FST values between pooled sites above and below each barrier were compared to pairwise FST values between pooled sites below barriers, but connected via the Yarra River mainstem, within each sub-catchment. Finally, to evaluate our power to detect an effect of a barrier with more markers, we ran the same simulations with 20 microsatellite loci and the same characteristics as above.For 50 data sets simulated with each combination of parameters  was highest for K = 3  with distance expressed in metres, Mantel test P-value = 0.069).Spatial genetic autocorrelation analysis between pairs of individuals below barriers in the Yarra River sub-catchment showed significant and positive kinship values for the first distance class only Fig. , suggestFST values between all pooled sites were all significantly different from zero  were similar to values between the below-barrier samples pooled within each sub-catchment (FST = 0.023–0.035). The exception was McMahons Creek, where FST between above and below-barrier pooled samples was 0.129.Pairwise ro Table . PairwisP-value = 0.086), although allelic richness in above-barrier sample sites was significantly lower in smaller catchments  compared to larger catchments (Watts and Yarra rivers)—that is, there was a significant weir_side:catchment_type interaction; estimate = −0.62 SE 0.23, P-value = 0.007 , while in larger catchments, there was no significant difference . There was no effect of barrier_age or interaction with catchment_type on allelic richness. The variance estimate for the random intercept on loci was 1.69 (SD 1.3).Linear mixed modelling did not identify a significant effect of weir_side on allelic richness across all sample sites (Fe) calculated in pooled samples above barriers within each stream was positive in Donnelly’s Creek (Fe = 0.055) and McMahons Creek , while in McMahons Creek, Ne was lower above than below the barrier .BOTTLENECK test results provided evidence for a recent bottleneck in the pooled samples above McMahons Creek weir under the TPM and SMM mutation models, but no bottlenecks were found in any other population Table . ConfideFST values among pooled sampled nodes above and below node 126 (barrier location) and IBD regression slopes among sampled nodes consistent with observed values between sample sites below barriers in the Yarra River sub-catchment (Table −6 (SD 1.992 × 10−6) to 3.275 × 10−6 (SD 1.587 × 10−6) and the percentage of significant Mantel tests ranged from 66 to 96. This result compares to the simulations with eight loci where regression slopes ranged from 2.395 × 10−6 (SD 3.431 × 10−6) to 3.417 × 10−6 (SD 2.562 × 10−6) and the percentage of significant Mantel tests ranged from 28 to 76. Even without a barrier, allelic richness in pooled upstream nodes was slightly lower than in pooled downstream nodes  and for each effective density, simulated data sets without a barrier yielded pairwise nt Table . SimulatDe) would be required for genetic drift to increase FST values substantially  over the number of generations since the installation of barriers in the study area (up to ~25 generations) regardless of the dispersal scenario , while 100 generations are likely to be required in the IBD scenario to reach a similar threshold  and the detection of a bottleneck. For Donnellys Creek, however, where the barrier is the oldest (~25 generations) and Ne was estimated to be similar to McMahons Creek above the barrier, the low level of differentiation (FST = 0.029) cannot be attributed to downstream gene flow only.Comparing these simulations with observed pairwise We sought to address a lack of studies that assess the genetic impacts of artificial barriers on freshwater fish species with low mobility. On the basis of spatially explicit simulations and an extensive empirical data set, our study highlights the importance of accounting for background genetic structure in species of low mobility to avoid falsely attributing genetic impacts to barriers. For example, a simple test of genetic differentiation across a barrier may be insufficient to establish the effect of a barrier alone. In these cases, barrier effects can only be identified if there is a significant increase in genetic differentiation that exceeds background levels of genetic structure. This can be assessed with temporal sampling  and the ‘Watts River sub-catchment’ (Watts River and Donnellys Creek). Given that the two sub-catchments are currently hydrologically connected via the mainstem of the Yarra River, differentiation between these two sub-catchments is likely due to the low mobility of s Koehn, . Within Ar) within upper reaches compared to the lower reaches, and (ii) significant differentiation between upper and lower reaches within our study streams. Hence, in non-panmictic populations such as G. marmoratus in our study area, significant genetic differentiation (FST) across barriers cannot be solely attributed to barrier effects.Theory and simulation-based studies predict that the effect of genetic drift on genetic diversity and structure depends on the effective population size and the number of generations since population isolation , substantial loss of diversity above a barrier and development of genetic differentiation across barriers will only occur in IBD populations when effective densities are small . In addition, when asymmetrical gene flow was taken into account in IBD populations, effects of barriers were unlikely to become evident even in the smallest simulated effective density of 15 individuals per km for up to 50 generations. Results from the IBD simulations with and without asymmetrical gene flow were in clear contrast to those for panmictic populations. In the simulations for panmictic populations, substantial loss of diversity above a barrier and development of genetic differentiation across barriers can be expected within much smaller timeframes  and for larger population densities . On the basis of our estimates of effective population sizes and number of generations since the presence of barriers (~9–24), an effect of barriers on genetic structure and diversity was only expected in the two smallest study streams (Donnellys and McMahons creeks).High variance among simulated data sets reflects the limited ability of a small number of microsatellite markers with low diversity to detect genetic effects  and cutthroat trout (Oncorhynchus clarkii clarkii) populations that the largest patches above barriers had higher genetic diversity than adjacent below-barrier patches, and Gouskov et al.  were important for sustaining genetic diversity in fragmented rivers. It is likely that large reservoirs in our system (Maroondah reservoir for the Watts River and upper Yarra reservoir for the Yarra River) sustain large populations and provide refuge habitats for G. marmoratus during droughts, such as those experienced in 1982–83 and late 1996–mid 2010.We originally hypothesised that populations isolated upstream of barriers would have lower genetic diversity compared to downstream populations that are more interconnected over a much larger area of habitat. In particular, we hypothesised that genetic diversity in isolated populations upstream of barriers would vary depending on barrier age, the size of the upstream population and disturbance history. Using our observed data set, we found support for the effect of population size, but not the age of the barrier. The size of the populations (based on catchment area) was found to be important for retention of genetic diversity above barriers. Importantly, we cannot relate this result directly to barrier effects, because simulations showed that even in the absence of a barrier upstream, genetic diversity is still expected to be lower above barriers. In addition, contrary to expectations based on simulations, we found more genetic diversity above barriers in the two largest streams (upper Yarra and Watts rivers)–indicating likely large densities of individuals above barriers in these two rivers. This finding is consistent with those of Whiteley et al. , 2013, wv et al. , who obsGalaxiella pusilla) and Weeks et al. Oncorhynchus mykiss) populations following catchment disturbance from fires, where genetic diversity was lower in populations upstream of barriers due to culverts , so it is plausible that there has also been some upstream migration of individuals over the weir during extreme stream flows or from undocumented translocations into the upstream weir pool. Homogenisation of gene pools of Roanoke logperch (Percina rex) above and below older (c. 1920), but smaller artificial barriers (~10 m high), was thought to be associated with at least one-way gene flow across the barrier . This finding highlights the importance of maintaining population connectivity in small streams where genetic impacts of a barrier due to restricted gene flow following a disturbance event can develop within just a few generations. Immediate actions to improve connectivity and gene flow could include the installation of fishways on the smaller streams showing signs of genetic drift above barriers, as demonstrated for European chub . The Salmo trutta) and recreational fishing  and are currently unable to recover via gene flow from downstream populations. In addition, the need for assisted gene flow is increased for species with low mobility that may take longer to recover from genetic impacts than more mobile species  barriers to assess temporal changes in http://dx.doi.org/10.5061/dryad.hn050.Data available from the Dryad Digital Repository: Supplemetal material


Jatropha curcas L. (Jatropha), a shrub species of the family Euphorbiaceae, has been recognized as a promising biofuel plant for reducing greenhouse gas emissions. However, recent attempts at commercial cultivation in Africa and Asia have failed because of low productivity. It is important to elucidate genetic diversity and relationship in worldwide Jatropha genetic resources for breeding of better commercial cultivars. Here, genetic diversity was analyzed by using 246 accessions from Mesoamerica, Africa and Asia, based on 59 simple sequence repeat markers and eight retrotransposon-based insertion polymorphism markers. We found that central Chiapas of Mexico possesses the most diverse genetic resources, and the Chiapas Central Depression could be the center of origin. We identified three genetic groups in Mesoamerica, whose distribution revealed a distinct geographic cline. One of them consists mainly of accessions from central Chiapas. This suggests that it represents the original genetic group. We found two Veracruz accessions in another group, whose ancestors might be shipped from Port of Veracruz to the Old World, to be the source of all African and Asian Jatropha. Our results suggest the human selection that caused low productivity in Africa and Asia, and also breeding strategies to improve African and Asian Jatropha. Cultivars improved in the productivity will contribute to expand mass commercial cultivation of Jatropha in Africa and Asia to increase biofuel production, and finally will support in the battle against the climate change. Jatropha curcas L. (Jatropha) is a shrub that produces non-edible seed oil suitable for biodiesel fuel. The oil content ranges from 30 to 50% of its seed weight  markers and eight retrotransposon-based insertion polymorphism (RBIP) markers were applied to assess the genetic diversity of 246 Jatropha accessions from eight Mexican states , Guatemala and countries of Africa and Asia. We also evaluated the genetic variability of accessions from each of nine regions in Chiapas, to identify the center of origin. We further identified the voyage from Chiapas to the Old World by tracing the progenies in Mesoamerica sharing the same ancestors with nowadays African and Asian accessions. Human selection causing a low genetic basis in Africa and Asia is discussed. Finally, we propose strategies to improve African and Asian Jatropha, in order to increase the phenotypic performance including productivity, which will lead us to relieve from the climate change by mass production of the biofuel.Figure 3 for the locations of Mexican states and regions in the state of Chiapas). The Mexican accessions and seven Guatemalan accessions were obtained from the collection of the Instituto Nacional de Investigaciones Forestales, Agropecuarias . Seven Philippine accessions were obtained from the collection of UPLB originating in four provinces of Luzon and Mindanao islands. Twenty-one Vietnamese accessions were obtained from plants grown in Quang Tri Province. Sudanese and Egyptian accessions were obtained from the University of Khartoum and University of Sadat City, respectively. Other accessions were the same as those used in our previous study , seven from Africa, and 32 from Asia  according to the manufacturer’s instructions. The DNA samples were diluted to a final concentration of 0.35 ng/μl and stored at -20°C until use.2, 0.8 μl dNTPs (2.5 mM), 0.08 U Bio-taq (5 units/μL), and Milli Q water. Touch-down amplification was performed as follows: 3 min hold at 94°C, followed by three cycles at 94°C for 30 s, and 68°C for 30 s, with the annealing temperature reduced by 2°C until 64°C every three cycles. Continuing with three cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s, the annealing temperature was reduced by 2°C until 58°C every three cycles. A further 30 cycles of amplification was performed at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The PCR products were stored at -20°C until separation by polyacrylamide gel electrophoresis (PAGE). Amplified bands were stained with ethidium bromide. For markers that showed ambiguous bands, experiments were repeated until clear bands were observed by increasing annealing temperatures.Polymerase chain reaction (PCR) for SSR markers was performed in a 10 μl total volume solution containing 1.4 ng of DNA template, 0.16 μl of SSR primer mix (25 μM each), 1× PCR buffer, 0.03 μM MgCl2, 0.4 μl dNTPs (2.5 mM), and 0.08 U Bio-taq (5 units/μL), made to the volume with Milli Q water. PCR reaction was started with denaturation at 94°C for 2 min, followed by 35 cycles of 45 s at 94°C, 45 s at 55°C and 2 min at 72°C, continued with a final elongation step at 72°C for10 min. Amplified bands were stained with ethidium bromide. The PCR products were stored and analyzed like the SSR markers.For RBIP markers, PCR was performed in a 5 μl total volume solution containing 0.7 ng of DNA template, 0.08 μl of primer mix (50 μM each), 1× PCR buffer, 0.03 μM MgClFor all the SSR markers and RBIP markers, bands were individually scored . All data analyses were performed based on the genotype data matrix.HE) and observed heterozygosity (HO) show the portion of heterozygotes in populations. They are measures of the extent of genetic variation in a population. HE is heterozygosity under Hardy–Weinberg equilibrium (HWE) by random mating of individuals  is the value to estimate the level of inbreeding of the population. FIS was calculated from HE and HO by the equation: FIS = 1 -HO/HE. The larger FIS shows the larger extent of inbreeding in a population. HE and HO were calculated with GenALEx 6.5 , ΔK method was applied. K varied from 1 to 10, after 10 independent runs for each K, ΔK was calculated based on the rate of change of the log likelihood for K values between successive K values following Model-based clustering analysis was performed using Mesoamerican accessions or all accessions including African and Asian accessions with STRUCTURE 2.3.4 . The anaPrincipal coordinates analysis (PCoA) of all individuals was performed with GenALEx 6.5. The final results were shown as a three-dimensional plot  of all the accessions.A distance) between groups  were also calculated with POPULATION 1.2.32.A neighbor-joining (NJ) tree of African and Asian accessions and non-admixed Mesoamerican accessions with more than 80% of the group membership in Mesoamerican structure analysis was constructed on the basis of genetic distances  with POPPresence of each of the eight retrotransposons was judged by presence of the amplified band using a FLK primer and a LTR primer, and in the case of full-length members, also by absence of the band using FLK primers at both sides of the retrotransposon. According to the results, number of retrotransposons present in each accession was counted.To characterize the Mesoamerican ancestral genetic group of African and Asian accessions, we assumed that African and Asian Jatropha originated from different Mesoamerican genetic groups and then constructed scenarios. To decide which scenario is the best supported by data, scenarios were compared in DIYABC v2.1.0 , 2010.Number of inflorescences per plant, number of female flowers per plant, ratio of female to male flowers and seed yield (g) per plant of 100 representative accessions from seven Mexican states  were measured individually at the Rosario Izapa Experimental Farm, INIFAP . Chiapas accessions were further sub-classified into those from six regions . To compare the variation among accessions from central Chiapas , peripheral areas of Chiapas (Selva and Soconusco), and other Mexican states, the mean value and standard deviation (SD) of each area were calculated.To survey the worldwide genetic variation of Jatropha, 59 effective SSR markers that showed the intraspecific polymorphism were selected from more than 500 Jatropha SSRs. The mean percentage of the missing data of SSR markers was 0.79%. We also identified six full-length members with two LTRs showing 100% sequence identity with each other, as well as two solo LTRs displaying 100% sequence identity with the consensus sequence, from Jatropha copia-type retrotransposon families  to use aThe SSR markers yielded 221 polymorphic bands ranging from two to seven with an average of 3.75 alleles per marker in all accessions. Among them, 161 alleles were specific to Mesoamerica, whereas no allele was specific to Africa or Asia (Supplementary Table HE) or genetic variation was observed in African and Asian accessions  . These results showed that the highest genetic diversity of Jatropha exists in Chiapas.In Mesoamerica, the highest genetic variation was found in Chiapas, with a clear decreasing cline of HE was examined among the regions within Chiapas. Five regions in central Chiapas had significantly higher HE values than four regions in the peripheral areas . Slight differences between expected and observed heterozygosity (HE versus HO) were observed among the accessions from central Chiapas, whereas large differences between these statistics were detected among the accessions from the peripheral areas of Chiapas, other Mexican states, and Guatemala . These resulted in lower inbreeding coefficients (FIS) in central Chiapas than in most of the peripheral areas of Chiapas, other Mexican states, and Guatemala . The numbers and ratios of accessions assigned to each group in each Chiapas region, other Mexican states and Guatemala are presented in Figure 3. The distribution of the groups revealed a distinct geographic cline. Accessions from central Chiapas were mostly in Group A (orange). Accessions in Group B (purple) were mainly distributed in the peripheral areas of Chiapas and in neighboring states and countries, whereas accessions in Group C (blue) were mainly distributed in states distant from Chiapas . This geographical distribution of Jatropha genetic groups in Mesoamerica has not been reported before. The distribution of Group B is especially interesting, because it seems to surround the area of Group A.The genetic constitutions of all the Mesoamerican accessions were examined by structure analysis. The ΔK value with respect to K (number of groups in the population) showed a peak when K equaled to 3 , and African and Asian accessions had the closest genetic and phylogenetic relationships with Group B , a phylogenetic tree of African and Asian accessions and non-admixed Mesoamerican accessions was constructed. The tree showed three clades, and interestingly, all of Mesoamerican accessions in each of the three clades  belonged to each of the three Mesoamerican Groups , respectively. One  of the eight Mesoamerican accessions that genetically close to African and Asian groups . PCoA analysis also revealed a close relationship between these seven accessions and African and Asian ones  is the closest to the ancestral genotypes of African and Asian Jatropha.After eliminating admixed accessions which showed < 80% of the membership of each Mesoamerican Groups  that correlated with geographic location in Mesoamerica, which has not been reported previously. Our results suggest that Group A, which distributes in central Chiapas represents the original genetic group. Specific genotypes of Group A might have spread from the Chiapas Central Depression to bordering areas, and subsequently formed distinct genotypes that comprise Group B and then C, according to their geographical distribution. We showed that African and Asian accessions were derived from Group B, rather than Group A or C. Further ABC analysis would be useful to make this conclusion more convincing.In our study, we found some Group B accessions from Veracruz and Guatemala were genetically close to African and Asian accessions . It is also noteworthy that this Chiapas accession is in Group B, even though Group A represents the major genetic background there . The two Veracruz accessions also belong to Group B. The absence of genetically similar accessions between central Chiapas and the port of Veracruz excludes a gradual natural expansion by seeds and favors the human-mediated spread of Jatropha from central Chiapas to the Port of Veracruz. These also suggest that there were selections when people brought Jatropha from central Chiapas. One important question is as follows: who first selected the ancestral African and Asian Jatropha in central Chiapas? The Spanish conquistadors normally did not enter the Chiapas Central Depression, where no mineral resources were expected to occur   .As a limited number of genotypes would have been brought to the Old World by Portuguese galleons, crosses could have occurred only among small populations in the Old World plantings, yielding very low genetic variation. This breeding bottleneck represents a typical example of the founder effect and is probably the major cause of the failure of Jatropha breeding in African and Asian countries. In addition, accumulation of mildly deleterious genes exhibited in the homozygous state by inbreeding might show inbreeding depression. In fact, one of the Veracruz accessions (No. 127), which has been suggested to share a common ancestor with African and Asian accessions as discussed above, had low values for the four yield-related traits  comparing with those in each region and group in Mexico (Supplementary Table 1 progenies. Second, select elite F1 or F2 progenies by phenotypic performance. Finally, use vegetative propagation (Based on our findings that the genetic basis of African and Asian accessions is Group B in Mesoamerica and that Group A shows the highest genetic diversity in Mesoamerica, we propose the following strategies for genetic improvement in African and Asian Jatropha. First, make wide crosses between elite lines from each country in Africa and Asia, which should have their epigenetic adaptations to the climatic conditions, and accessions of Group A from central Chiapas, which are genetically distant from African and Asian Jatropha and have the largest genetic diversity. Not only hybrid vigor, but also some extent of phenotypic variations which are derived from the highest heterozygosity of Group A, would be expected in Fpagation  of selecHL wrote the manuscript; HL, ST, KH, and KF discussed the results, organized and revised the manuscript; MY supported and conducted some statistical analyses; HS and NW commented the manuscript and supported the experiment; HL, AA, and TS performed the genotyping experiment; AT, HTs, TA, and HTo suggested and helped the data collection and analyses. SS and HH designed SSR markers and performed part of the marker analysis; VQ and AZ collected Mexican accessions and provided the phenotype data of Mexican accessions; PS collected and provided Philippines accessions; AH provided one Egyptian accession; AMA provided two Sudanese accessions. KF organized the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer NO declared a shared affiliation, with no collaboration, with one of the authors MY to the handling Editor.