Sus scrofa meridionalis) population inhabiting the island of Sardinia , and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow  within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar  population inhabiting the island of Sardinia . A sharp genetic structure into different subpopulations was observed, after removing the confounding effect of hybridization. Isolation by resistance due to main roads and land cover explained the observed genetic discontinuities.Genetic structure and landscape genetics were investigated in the wild boar ( The impact of anthropogenic barriers and habitat fragmentation on gene flow was investigated in different wild ungulates , based on both morphological and genetic evidence, as it is characterized by a relevant genetic differentiation, due to its long‐lasting isolation, reported by Scandura et al. . Sardinian wild boar, a dwarf form of the European wild boar, is believed to have originated during the Neolithic following a human introduction from the mainland , Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR) were tested using a landscape genetic approach by comparing alternative landscape resistances, as suggested by Balkenhol et al.  and Fran22.12). Human population density is relatively low for Europe (around 68 inhabitants/km2) and people mainly live in major cities and along the coasts, while small villages and large uninhabited areas characterize the interior. Climate is Mediterranean‐temperate at low elevations and along the coast, more continental inland and at higher elevations. Temperature is mild and relatively constant throughout the year . Annual precipitations range from less than 400 mm in the dry south to 1500 mm in the eastern mountains. Such climatic conditions are suitable for wild boar all over the island.The island of Sardinia is the second largest in the Mediterranean Sea , and, based on habitat suitability analyses, higher densities were expected to occur in the central and northern part of the island .A total of 393 wild boar samples were obtained from all over Sardinia by local hunters during the period 2001–2019. Tissue or hair samples were collected from hunted animals and stored, respectively, in absolute ethanol or frozen until analysis. Sampling locations . Each PCR was performed in a 10 μl reaction volume, containing 3 μl of DNA solution, 0.5 U of Taq DNA polymerase (Euroclone), 1× PCR buffer (Euroclone), 2.5 mM MgCl2, 100 μM of each dNTP, and 2 pmol of each primer. Forward primer of each pair was labeled with an ABI fluorescent dye . The amplification profile was set up with an initial step of denaturation at 95°C for 3 min, followed by 35 cycles of 92°C for 45 s, annealing T ranging between 62–52°C for 45 s, and 72°C for 30 s, with a further final extension step of 72°C for 10 min. Amplicons were sized using capillary electrophoresis in an ABI PRISM 3100 or 3730XL Avant automatic sequencer (Applied Biosystems) by the BMR‐Genomics sequencing service . Appropriate calibrations were made to standardize microsatellite scoring results when the automatic sequencer was changed. Peak Scanner software v. 1.0 (Applied Biosystems) was used to analyze electrophoretic data.DNA was extracted using GenElute kit  for tissue samples and Instagene Matrix  for hair samples, and then stored at −20°C. All samples were genotyped with a panel of 16 microsatellites: S090, SW122, SW2532, S355, SW1492, SW461, IGF1, SW951, SW2021, SW2496, S026, S215, SW72, SW857, S155, and SW24 (details at 2.3OH) and expected (EH) heterozygosity, mean number of alleles per locus (A), and FIS were computed in GENETIX v. 4.05  and from domestic pigs in Sardinia was already reported by Scandura et al. . As distn = 568) was analyzed by Bayesian cluster analysis in STRUCTURE v. 2.3.4  ranging from 1 to 10, with settings: admixture model, no population information, correlated allele frequencies, 500,000 burn‐in and 500,000 iterations of data collection. Again, the optimal K‐value was chosen according to the ΔK statistics in Structure Harvester  as belonging to the corresponding subpopulation. Genotypes were plotted in a two‐dimensional space based on their genetic proximity. Pairwise Rousset's ar genetic distance  was also performed using Adegenet package in R v, 4.0.2 Jombart,  to detecRousset, , shown tRousset, , was comRousset, .2.4www.hpc.cineca.it/). This approach uses stochastic search algorithms that solve optimization problems by simulating natural selection processes , Isolation‐By‐Barrier (IBB), and Isolation‐By‐Resistance (IBR). To assess the relevance of each driver, the resistance optimization process described by Peterman et al.  was implScrucca,  to find n = 270) using Circuitscape 5.0 implemented in Julia . To test the IBR hypothesis, we optimized a categorical land‐cover grid surface. Land‐cover data were obtained from a digital map of Sardinia .To test the IBB hypothesis, we optimized a binary grid surface with a 500 × 500 m resolution where cells crossed by main roads had a value equal to 1 while all other cells had a value equal to 0. Main roads were identified as those with an Average Daily Traffic (ADT) higher than one standard deviation of the mean ADT from all the sampling stations in Sardinia , at all loci observed heterozygosity (Ho) was lower than expected heterozygosity (eH), thus revealing an excess of homozygotes that was confirmed by FIS values. Not surprisingly, GENEPOP analysis detected a significant deviation from Hardy‐Weinberg equilibrium due to heterozygote deficiency in the overall population , except for locus S026 (p = .031) when performing HW test for each locus. Several pairs of loci resulted in linkage disequilibrium .The total number of alleles detected in the Sardinian wild boar sample was 154, ranging from 6 to 16 per locus and an average of 9.63 ± 3.18  per locus. Missing alleles represented 2.17% of the dataset. MICRO‐CHECKER did not find any scoring error in the dataset or evidence of allele dropout. Properties of the 16 microsatellite loci used in this study and the variability observed at each locus are shown in Table From the original dataset of 393 Sardinian wild boar samples analyzed with 16 microsatellite loci, 75 individuals showed a high relatedness  to other individuals in the dataset and were likely to represent full‐siblings or parent/offspring. Therefore, they were removed from the dataset to obtain a cleaned pool of 318 unrelated individuals to perform the following analyses.3.2K = 5 (or higher) the Bayesian analysis in STRUCTURE sharply distinguished the main source populations in the overall sample of 568 individuals . However, in order to identify individuals with a clear signature of genetic introgression in Sardinia, we selected K = 4 as it showed a higher support than K = 5 , as the most likely, but local maxima were detected also at K = 5 and K = 8  and wild boars from the rest of the island (north and east), with the main discontinuity between the two clusters apparently represented by the SS131. At K = 5, five subpopulations were clearly identified  detected a partition in two clusters  were assigned to one of the five subpopulations with q > 0.70, and specifically 23 individuals were assigned to NW, 37 to CW, 39 to SW, 57 to NCE, and 54 to SE. No individual was assigned to a population different from that expected on the basis of its sampling site. Such pattern of population differentiation seemed to identify the presence of genetic discontinuity in coincidence with the SS131 and the Campidano plain. The consistency of results obtained by different analytical approaches points to a sharp structuring in the island with a limited ongoing gene flow between subpopulations.On the basis of their The PCA plot Figure  confirme3.4Comparing the best models obtained through the optimization process revealed that the IBR, accounting for land cover and the presence of main roads, was by far the best‐supported hypothesis Table . The IBBThe resistance surface associated with the best‐supported model under the IBR hypothesis revealed minimum resistance to wild boar movement in coniferous and broadleaved forests, and water bodies  and confirmed expectations (Table p ≤ .001) and were significantly lower in pairs including locations classified in the same cluster than in pairs including locations classified in different clusters .As discussed by Reiner et al. , detectiCervus elaphus, Frantz et al., Lynx rufus, Serieys et al., According to our data, the Sardinian wild boar population should not be managed as a single panmictic unit, rather subpopulations should be treated as separate management units. The lack of gene flow across barriers  should be taken into account in the definition of spatial units for disease prevention. Results may also have implications for the management of other wild species in Sardinia. Given that urban areas, main roads and the most intensively cultivated areas apparently play a role as barriers to gene flow in the wild boar population, they could also represent a cause of fragmentation for other mammals (including endemic and endangered species), promoting isolation and genetic drift. However, the effect on other species should be tested by targeted studies, as landscape features might have various impacts on different species ; Data curation ; Methodology ; Software (lead); Writing – original draft (lead). Olivia Dondina: Conceptualization (supporting); Methodology ; Software (lead); Writing – original draft . Valerio Orioli: Conceptualization (supporting); Methodology ; Software ; Writing – original draft . Daniela Biosa: Data curation ; Investigation ; Writing – original draft . Antonio Canu: Data curation ; Investigation ; Methodology ; Writing – review & editing . Giulia Fabbri: Methodology (supporting); Software ; Writing – review & editing . Laura Iacolina: Data curation ; Investigation ; Writing – review & editing . Antonio Cossu: Data curation (supporting); Investigation (supporting); Writing – review & editing . Luciano Bani: Conceptualization (supporting); Methodology (supporting); Supervision ; Writing – review & editing . Marco Apollonio: Conceptualization (supporting); Funding acquisition (lead); Supervision ; Writing – review & editing . Massimo Scandura: Conceptualization (lead); Data curation ; Funding acquisition (lead); Methodology (lead); Project administration (lead); Writing – original draft ; Writing – review & editing .Appendix S1‐S2Click here for additional data file.


Climate change is a great threat to global biodiversity and has resulted in serious ecological consequences. Although the potential effects of climate change on genetic diversity have recently received much research attention, little research has focused on the impacts of climate change on genetic connectivity and the relationship between climate stability and genetic divergence. Here, we combined population connectivity with genetic data to predict the impacts of future climate change on genetic connectivity. Coupled with climatic variables and genetic data, we used POPS software to create spatially explicit simulations and predict the dynamics in genetic clusters in response to climate changes. A generalized additive model was employed to test the correlation between climatic stability and genetic diversification. Our findings indicated that a reduction in species distribution due to severe climate change would lead to a substantial loss of genetic connectivity. More severe future climatic scenarios would likely cause greater loss of variability or more distinct homogenization in genetic variation of species. Relatively low interpolated genetic distances are generally associated with areas of greater losses in climatic suitability from the present to the future. The displacement of climatic genetic clusters will challenge species adaptation to future climate change because of the loss of fundamental evolutionary potential. The persistence capacity of plant species may be weakened in the face of future climate change. Modern reliance on fossil fuels has caused unprecedented climate change, ushering in extreme temperatures globally and abnormal precipitation patterns in many regions  is endemic , 2011. Tf forest . It was f forest . What isf forest . The accf forest , and is f forest . AlthougCornus kousa subsp. chinensis as our model species. It is broadly distributed across subtropical and warm-temperate deciduous forests of mainland China. The species grows on environmental heterogeneities, such as variation in elevation, precipitation, temperature, moisture, and soil-nutrient composition, occurring from the subtropical zone to the temperate zone across its natural distributional range in central and eastern China  were downloaded from the WorldClim database. Different general circulation models (GCMs) vary considerably in future projections (1. The Chinese vegetation (VEG) dataset was obtained from the Environmental and Ecological Science Data Center for West China National Natural Science Foundation of China (see text footnote 1). Land cover data were obtained from the Moderate Resolution Imaging Spectroradiometer (MODIS)2. To construct species distribution models (SDMs), we gathered a total of 492 occurrence records of C. kousa subsp. chinensis covering the entire range known for the species. These occurrences were mainly collected from the IUCN Red List Data3, Chinese Virtual Herbarium (CVH)4, and previous literature. To ensure optimum SDM performance, the occurrence data must be spatially independent; therefore, the elimination of spatially auto-correlated occurrence points was crucial for model evaluation. Spatially independent occurrences can avoid the prediction over-fitting problem and improve model performance  for the environmental variables (Projected climate data layers for the current (∼1950–2000) and the 2080 period (average for 2061–2080) based on 19 bioclimatic variables at a resolution of 2.5 arc-min   via ArcGtability .C. kousa subsp. chinensis throughout its distribution across central and eastern China. Whole genomic DNA was extracted from the dried leaf tissue Plant DNA-Easy kit . For the DNA survey, two intergenic spacer regions (IGSs) (rpl14-rpl36 and trnL-trnF) of chloroplast DNA (cpDNA) were sequenced for all C. kousa subsp. chinensis samples (n = 186). The sequences were edited, assembled, and aligned in GENEIOUS version 4.8.4  was analyzed using GENEPOP version 4.0.7 (Ne); expected heterozygosity (He) under Hardy–Weinberg equilibrium following Ne, we removed populations with sample sizes less than 5 (T), gene diversity (h), and the number of haplotypes (Nh) (To forecast the impacts of climate change on genetic clusters of on 4.0.7 . GenAlExon 4.0.7  was empls than 5 . For seqpes (Nh)  for eachpes (Nh) .C. kousa subsp. chinensis in the future, we recalculated the genetic parameters of diversity mentioned above using LPT for both future climate change scenarios (RCP45 and RCP85). For nuclear microsatellites, the simulations were executed for each locus and overall loci with the software GenAlex 6.502  of habitat suitability will persist and generate the gene pool of individuals for the next generation. To predict the genetic diversity of ex 6.502 , whereasex 6.502 .Fst values [Fst/(1−Fst)]. Landscape resistance values were exacted from a friction layer from inverted SDMs. Then, the matrices were generated with GenAlEx v 6.5  method, which computed the least dispersal costs between two localities. In this approach, we inverted the SDMs produced by Biomod2 to obtain a friction layer  for the current and future (2080) periods. That is, areas of high suitability had a low dispersal cost through the landscape, whereas regions with low or no probability of occurrence were converted to areas of high dispersal costs. Subsequently, we used the friction layer to generate a cost distance layer for each locality. Finally, based on the cost distance layer, we created pairwise populations and shared a cpDNA haplotype network by adding the LCPs between two populations. The models were generated using SDM toolbox version 1.2 in ArcMap 10.2 .We used the POPS software  to execu2 . To avoid extrapolating beyond the original sample locations, the raster was clipped to the spatial extent of sample locations. High values represent areas of high genetic differentiation between sampled locations, and low values mean relatively low genetic divergence between collection locations. A generalized additive model (GAM) was used in R 3.6.1 to test the correlation between climatic stability and genetic diversification. Interpolated genetic distance was used as the response variable, and change between the present and the future was used as the predictor variable. These values were calculated by extracting the value of the difference between the current and present climatic suitability at each point at which an interpolated genetic distance was calculated.We used the Genetic Landscapes Toolbox  to genertrnL-trnF and rpl14-rpl36) was surveyed across the 186 individuals of C. kousa subsp. chinensis was 1,566 bp in length, including four indels and 31 substitutions. Together, all the polymorphisms identified seven chloroplast haplotypes  (T) was 1.08 × 10–3 and that haplotype diversity (Hd) was 0.447. Microsatellite data were obtained across the 215 individuals of C. kousa subsp. chinensis genotyped at eight microsatellite loci , 2.98 (range: 1.6–4.9), and 1.05 (range: 0.45–1.56), respectively   across ts, H1–7) . At the ite loci . All eigectively . There wC. kousa subsp. Chinensis was highly related to annual mean temperature and precipitation in the driest month  had no genetic connectivity with the central and eastern populations from the present to the year 2080 . AlthougK = 7 with a DIC value of 9,676  can be found in the article/BG: conceptualization, supervision, funding acquisition, writing the review and editing, sample collection, and data curation. JG: writing – original draft. WC: field investigation, leaf sample collection, and data curation. XG: formal analysis, software, and writing the review and editing. GG: revising the manuscript. BG and GG: ensuring that the descriptions are accurate and agreed upon by all other authors. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PTGS2 and PLA2G4A genes on chr10 were related to reproduction efficiency. This is the first study mapping genetic variation in autochthonous turkey populations. Breeds were genetically different among them, with the Roma breed proving to be a mixture of the other breeds. The ROH_islands identified harboured genes peculiar to the selection that occurred in heritage breeds. Finally, this study releases previously undisclosed information on existing genetic variation in the turkey species.Italian autochthonous turkey breeds are an important reservoir of genetic biodiversity that should be maintained with an in vivo approach. The aim of this study, part of the TuBAvI national project on biodiversity, was to use run of homozygosity (ROH), together with others statistical approaches , to investigate the genomic diversity in several heritage turkey breeds. We performed a genome-wide characterization of ROH-rich regions in seven autochthonous turkey breeds, i.e., Brianzolo (Brzl), Bronzato Comune Italiano (BrCI), Bronzato dei Colli Euganei (CoEu), Parma e Piacenza (PrPc), Nero d’Italia (NeIt), Ermellinato di Rovigo (ErRo) and Romagnolo (Roma). ROHs were detected based on a 650K SNP genotyping. ROH_islands were identified as homozygous ROH regions shared by at least 75% of birds (within breed). Annotation of genes was performed with DAVID. The admixture analyses revealed that six breeds are unique populations while the Roma breed consists in an admixture of founder populations. Effective population size estimated on genomic data shows a numeric contraction. ROH_islands harbour genes that may be interesting for target selection in commercial populations also. Among them the  According to historical evidence, turkey domestication originated in Mexico and Central America. After the Spanish conquerors brought the turkeys to Europe, the novelty and the appreciated meat characteristics permitted a rapid spread across Europe, starting from the 16th century. Since their diffusion in Europe, turkey populations were then bred divergently in the next centuries [The Italian autochthonous turkey populations show a wide phenotypic variation among breeds, spanning plumage colour, body size and weight . HeritagSince these turkey breeds are reared in rural and family farms, the size of the populations is at present very limited. To date, many actions have already been taken to preserve the turkey’s biodiversity. The Italian Ministry of Agriculture (MIPAAF) established the Registro Anagrafico delle Razze Avicole Autoctone  . Furthermore, the European Agricultural Fund for Rural Development funded the TuBAvI project “Tutela della Biodiversità nelle razze AVicole Italiane”. The aim of this project was to safeguard, preserve and improve the Italian poultry genetic resources, which are represented by all the autochthonous breeds. This project has been granted a follow-up, the TuBAvI-2, broadening and consolidating the knowledge on Italian breed populations gained with TuBAvI.Costs to obtain SNP high-throughput genotypes have decreased in recent years, favouring the production of genomic data and the application of genomic analyses in livestock populations. This occurred either where genomic selection  has been adopted or in small and endangered populations where in situ genetic conservation plans are desired. Heritage turkey breeds were recently studied and, even if still limited, some indication about their genomic variability and diversity is nowadays available ,6,7,8,9.ROHs are continuous genomic regions defined by adjacent markers in a homozygous state that are identical by descent as a result of processes such as inbreeding, population size reduction and natural selection . ROH’s fIn this study, we analysed the genetic variability among seven Italian turkey breeds registered in the RAA  and we performed a genome-wide characterization of ROH-rich regions in order to identify the genome tracts under recent and ancient inbreeding as determined by ROH.® TurkeyHD Genotyping Array of seven Italian turkey breeds  were available from Strillacci et al.  and from Strillacci et al.  [In this study, 181 birds’ genotypes from the AxiomFiltering was applied to retain for the analyses SNPs with genome position on autosome (first 30 chrs) and having a call rate >99%.A total of 346,155 SNP markers passed the quality control, and their location on the genome was in concordance with the Turkey_5.0 genome assembly—GCA_000146605.1.The number of monomorphic SNPs, the expected and observed heterozygosity (Exp Het and Obs Het), the expected and observed number of homozygous SNPs (Exp Hom and Exp Hom) and minor allele frequency for each breed were calculated.A principal components analysis (PCA) was performed based on allele genotypes using the SNP & Variation Suite (SVS) v8.9 . The graphical visualization of PCA was obtained by the ggplot2 R package .ST) was estimated using the dedicated module implemented in SVS. The FST was estimated for all possible pairs of breed combinations.The pairwise fixation index  was constructed—using the distance matrix as input file—by Phylip software implemented in the online WebPHYLIP tools . A NJ trThe ADMIXTURE analysis: The most probable number of ancestral populations was identified in conjunction with the lowest cross-validation error (CV), setting the analysis with optimal number of clusters  from 2 to 8. PLINK 1.9 software  was usedEffective population size (Ne) for each breed was predicted using the SNeP_111 software  based onThe genetic diversity within and among breeds was determined using the following approaches:ROHs were computed using the consecutive run method implemented in SVS 8.9 software. We restricted ROH segments to longer than 1 Mb to avoid identification of ROH resulting from LD. In addition, a minimum of 150 homozygous SNPs was required to be in each run, and no heterozygote or missing SNPs were allowed in the ROH, and the maximum gap between SNPs was set to 1000 Kb.The mean number, the average length of ROH, the average sum of ROH segments per breed, as well as the ROHs distribution into four classes of length  were estimated per breed.The genomic regions most commonly associated with ROH (ROH_islands) were defined within breed as clusters of runs found in more than 75% of samples using a specific option implemented in SVS software during the ROH detection: samples within the same cluster had identical ROH .The completed list of turkey genes annotated according to the most recent reference genome was downloaded from NCBI online Database (NCBI Annotation Release: 103). Genes with an official gene name and “LOC” ID associated both with a protein coding and official gene name were catalogued within the identified ROH_islands using the “intersectBed” command of BEDTools software . The DatAdditionally, considering that to date no quantitative trait loci (QTLs) database is available for turkeys, the one accessible for the chicken species, i.e., the Chicken QTLdb—based on Galgal 6.0 genome assembly, was used to identify—using the “Search by associated gene” option—QTLs overlapping the genes found in the ROH_islands.HOM—the difference between the observed and expected numbers of homozygous genotypes within the population calculated using the routine implemented within SVS;FROH—the ratio calculated between the total length (sum) of all ROH of an individual and the total length of the autosomal genome covered by the SNP marker dataset, 902,020,024 bp in this study.FTwo genomic inbreeding coefficients for all the birds were estimated based on:The proportion of monomorphic SNPs was variable according to breed, spanning from 21% (NeIt) to 77% in ErRo .The observed (Obs Het) and expected (Exp Het) heterozygosity values were in concordance with the monomorphic SNPs and with minor allele frequency (MAF) values ranging from 0.06 (ErRo) to 0.20 (PrPc).The principal component analysis (PCA) results are shown in the graphs of ST values) .Comparable results were found by NJ tree analysis C and by  values) D, confirThe ADMIXTURE analysis revealed that at K = 6 , six breeds seem to be mostly unique populations, with an ancestral genetic composition defined by a proportion of 90% in CoEu and equal (PrPc) or larger than 98% . The Roma breed appeared to have a composite ancestral genetic contribution from CoEu (39%), PrPc (30%), NeIt (7%), Brzl (21%) and BrCI (3%) E.ROHs were identified in all birds of each breed for a total of 20,858 homozygous regions . The larThe graphical representations of ROH statistics by breeds are shown in In order to explore how the mating plans in each breed have affected their genetic structure, the ROH_island regions were considered and analysed. Using the SNP incidences, the ROH_islands were defined as a genomic homozygous region with (i) the same boundaries (and as such the same chromosome length) and (ii) shared in at least 75% of samples of a given breed .As shown in A different proportion of proper (identified only in one breed) and common ROH_islands (shared by at least two breeds) were observed among the populations a,b. Two p-value <0.05) is reported in When we considered all birds as a unique population, an ROH_island on chr10 was identified  in at lePTGS2 (n.2) and PLA2G4A (n.16) genes, the only ones annotated in this genomic region. Among the 200 SNPs defining this ROH_island in all 136 samples , 18 SNPs were mapped in the intronic position of the Although no ROH_islands for the PrPc breed were found at the threshold considered , two homozygous regions were shared by a large proportion of individuals, 60%, corresponding to 15 birds. These two regions mapped on chr 1  and on chr 21   and harbHOM and FROH have been calculated and are reported in HOM mean values were calculated for CoEu, NeIt, and PrPc while positive close to 0 ones were found for the remaining breeds. CoEu birds showed the largest variability in FHOM values.Two genomic inbreeding coefficients FROH values were calculated based on ROH proportion over the genome length, they reflect the ROH distribution in each sample, its average length across the classes and the total genome length covered by ROH  obtained for each breed are reported in As the Fd by ROH . Differel breeds . Linear Autochthonous turkey breeds have undergone to a population size contraction after the diffusion of meat type hybrids, more efficient in growth performance and carcass yield. Nowadays, heritage turkey breeds represent a unique genetic biodiversity reservoir. Their in situ conservation is particularly important as the autochthonous breeds own unique characteristics, including adaptability to extensive farming in harsh environments. Their characterization, as envisaged in the TuBAvI project, allows the identification of the phenotypic and the genetic characteristics related to their intrinsic value, especially if compared to selected hybrids that, even though very efficient in meat production performances, may exhibit suboptimal performance in other traits, e.g., reproduction efficiency ,25.ST values, the ErRo is the one more differentiated from all the other ones: the FST values were 0.47 and 0.63 when ErRo was compared with PrPc and CoEu, respectively. A possible explanation is that the feather colour of this breed, white with black streaks, clearly different from plumage colour of other breeds, has been chosen as a selection criterion by the breeders and farmers causing, as such, a divergent selection with respect to the other breeds. The pairs Roma–PrPc (FST = 0.18) and Roma–CoEu were less differentiated (FST = 0.21). The relative closeness of Roma and PrPc could be related to the geographic origin of these breeds, the northern area of Italy below the Po river in the Emilia-Romagna region . The Roma breed standard is not as stringent as the one of the other breeds, in fact it does not include a specific trait such as the feather colour as in the ErRo. This may have facilitated the cross of birds of the breeds geographically closer to the Romagna region, i.e., Veneto (CoEu), Emilia (PrPc) and Lombardia (Brzl), but not the ErRo due to its colour. It is interesting to note that the proportion of ancestors found in the Roma breed using the ADMIXTURE analysis was inversely proportional to the geographical distance of Veneto, Emilia and Lombardia from Romagna. With the exclusion of the Roma breed, the ADMIXTURE analysis revealed for all the other ones the uniqueness of the genetic background for each of the Italian breeds with a proportion of each ancestor >90%.This study showed that the genetic composition of each of the seven autochthonous turkey breeds differs from others. Based on the PCA and the NJ tree analyses, birds of each breed clustered together revealing a consistent genetic composition. The ErRo and BrCI are the two more genetically distant breeds from the other ones and, according to the FIn the last decades, the in situ maintenance, i.e., the reproduction of Italian autochthonous turkeys, was mostly carried on by private breeders. They had two main selection goals: (i) to maintain the morphological characteristics of each breed; (ii) to improve birds’ performance in a semi-extensive farming system.The meat of these breeds is mainly used for traditional cuisine recipes for private consumption; the backyard farming as a consequence is not centred on growth performance but on meat quality  and resilience of birds to pastoral extensive farming.Generally, the reproductive scheme used by private breeders is avoiding as much as possible increases in inbreeding, adopting as strategy of toms’ exchange between farms. Although the selection in autochthonous breeds is not as focused and intense as the one in other livestock species in pure breeding, it determined consequences in the genome makeup of the turkey breeds, including the formation of ROH_islands.The population size of each breed is particularly small because its contraction occurred in the last 4 decades: assuming a generation interval of 1 year , the graph in It is interesting to note that the Ne of the ErRo was the smallest among all, with a fairly constant value in the last 30 generations. This is a further evidence supporting the closeness of the nucleus of the ErRo population in the last decades. The Roma breed, in accordance with the ADMIXTURE results, exhibited the largest Ne possibly because of the inclusion of birds from different breeds.KITLG gene, associated with melanin patterning [The population size of these breed is very small as reported by the FAO DAD-IS database and by Castillo et al. ranging from 9 (PrPc) to 445 (BrCI) birds across the breeds and all considered at critical risk of extinction ,27. Due tterning  and invotterning . AdditioThe Roma breed is the one with the smallest proportion of homozygous SNPs in ROH. Additionally, there are no ROHs longer than 8 Mb. This result agrees with the ADMIXTURE one, indicating a possible introgression in the breed from various ones as hereinbefore supposed.A similar high level of homozygosity has been identified in Italian local chicken breeds , where lAs shown in PRGS2 and PLA2G4A genes , as shown in 4A genes .PTGS2 and PLA2G4A genes were found by several authors to be related to reproductive physiology in avian and other livestock species [To assess the genetic variability existing in this species, it is important to characterize the heritage breeds’ specific genetic biodiversity, especially in comparison to the highly selected heavy turkey lines. Comparing the evidence of the ROH_Islands in this region to existing data on ROH in commercial hybrids, it is interesting to note that the  species ,34,35,36 species . The int species . Interes species ,9 reachi species  and, to TRAF2) [NOXA1) [GRIN1) [The second region mapped on chr19 in four breeds harboured genes connected to heat-stress-related functions (TRAF2)  and invo [NOXA1) , meat qu [NOXA1) , feeding [GRIN1) .MMRN2 [ADIRF [Opn4 [To the best of our knowledge, only three studies ,11,38 peMMRN2 ,44), fat2 [ADIRF ), and ciRF [Opn4 ).HOM mean values range from −0.118 to 0.141 while FROH mean values vary from 0.126 to 0.401. The variability of inbreeding values within breeds is also very different across them: considering FROH, the SD of the mean values ranges from 0.035 to 0.153 showing that the effect of selection and the management of reproductive practices breeders are influencing in a variable manner the genetic makeup of these populations.The possibility of calculating genomic inbreeding values is useful in the absence of pedigree information, a very common condition in avian species. This is particularly important in local and endangered populations such as autochthonous Italian turkey breeds. In our study, populations showed genomic inbreeding with a large variability of mean values as shown in ROH are the Roma and the PrPc, reflecting the reproductive structure and management of the two populations. The Roma breed is an admixture of different turkey breeds as hereinbefore commented. The PrPc is, together with the Roma, the breed with largest Ne as reported in the graph of The two breeds with lower value of FHOM and FROH in all breeds, with R values ranging from moderate to strong (>0.90 in BrCI and Roma). These results, attributable to a higher proportion (from 62% to 77%) of short ROHs in all breeds, are consistent with those reported in the literature on livestock [A positive correlation was found between Fivestock ,47,48,49Information on the genetic variability of turkey breeds is very limited and, to the best of our knowledge, this is the first study on autochthonous turkey populations. The wide diffusion of commercial, fast-growing, heavy hybrids in the last 40 years has impacted the farming of autochthonous populations, affecting in the same manner the existing genetic variability in the turkey species. The wide spread of commercial hybrids has in fact reduced the farming of heritage breeds, causing a loss of genetic variation and biodiversity. Local breeds are well adapted and selected over centuries to deal with environmental harsh conditions of semi-extensive farming system. The knowledge of their genomic variability is indeed an important step forward with respect to the state of the art, today mainly based on selected heavy turkeys. This study is in fact part of the nation-funded project TuBAvI that aims to investigate the existing biodiversity in local avian populations using genomic data and to provide insights for their in situ conservation. This study releases previously unavailable information on the genomic variation existing in autochthonous turkey populations, showing regions under selection. The results of this project represent as such a unique resource to compare the genetic variability of autochthonous turkey breeds with the one of selected commercial turkeys. The use of ROHs to exploit genomic variation in autochthonous populations revealed genomic regions under selection (ROH_islands) that harbour genes potentially interesting also for a targeted selection in commercial lines. The results here obtained are, as such, a unique resource in mapping genomic variation in turkey species.


An enzyme-linked immunosorbent assay (ELISA) can be used for quantitative measurement of proteins, and improving the detection sensitivity to the ultrasensitive level would facilitate the diagnosis of various diseases. In the present review article, we first define the term ‘ultrasensitive’. We follow this with a survey and discussion of the current literature regarding modified ELISA methods with ultrasensitive detection and their application for diagnosis. Finally, we introduce our own newly devised system for ultrasensitive ELISA combined with thionicotinamide adenine dinucleotide cycling and its application for the diagnosis of infectious diseases and lifestyle-related diseases. The aim of the present article is to expand the application of ultrasensitive ELISAs in the medical and biological fields. The enzyme-linked immunosorbent assay (ELISA) is an easy-to-use method for detecting proteins  on the basis of an enzyme reaction . The potBeyond this limitation, however, the ELISA method has many advantages, including simplicity, practicality , rapidness, and reasonable cost . The remIn the present review article, we first define what we mean by ‘ultrasensitive’, which is often misinterpreted, leading to confusion. Next, we survey and discuss the current literature about ultrasensitive ELISA and its applicability for diagnosis. Lastly, we introduce our own newly devised system for an ultrasensitive ELISA coupled with thionicotinamide adenine dinucleotide (thio-NAD) cycling and its application to diagnosis for infectious diseases and lifestyle-related diseases.The strongest requirement for conventional ELISA is the ability for ultrasensitive detection ,27,28. TA second reason for the misinterpretation of ‘ultrasensitive’ originates from comparison of the LOD among various other methods for protein measurements. As described above, many techniques can be used to quantify target proteins, including ELISA, LC-MS, and lab-on-a-chip methods . Whereas−13 moles/L or smaller can be referred to as ‘ultrasensitive’ detection. If we use a 96-well microplate for ELISA, one assay volume is generally equal to 100 μL. Thus, 10−13 moles/L is equivalent to 10−17 moles/assay, which might be challenging to achieve. Our own ELISA method, however, can detect proteins at zeptomolar detection limits, i.e., 10−21 moles/assay . Wh. Wh33]. The digital ELISA was modified to count single molecules. This technique is the dropcast single molecule assay , in whic−11 g/mL, which could even be observed with the naked eye. Avian influenza virus A was ultrasensitively detected with an ELISA using AuNPs and magnetic beads . Th. Th40]. ic beads . Virusesic beads . In thisMycobacterium tuberculosis ESAT-6-like protein esxB, showing that the LOD of 0.01 μg/mL was detectable by the naked eye [2). The structural properties of polyamidoamine dendrimers contribute to the amplification of the signal in the immunoassay. Using this assay, human chorionic gonadotropin was detected at 0.03 IU/L [Techniques using AuNPs were also applied to detect many biomarkers. A plasmonic ELISA with AuNPs was used to detect aked eye . This te.03 IU/L . Measuri.03 IU/L . The com.03 IU/L . AccuratThe use of silver nanoparticles (AgNPs) also increases the detection sensitivity of ELISAs. For example, AgNPs are thought to be an ideal colorimetric indicator because of the high extinction coefficient and strong plasmon resonance absorption property, e.g., for achieving excellent detection performance in surface-enhanced Raman scattering detection. Using an AgNP technique, an LOD for alkaline phosphatase (ALP) was 0.003 U/L, and that of carbohydrate antigen 125, which is found on ovarian cancer cells, was 1.75 U/mL . A combi42 (Aβ42) in the plasma of patients with Alzheimer disease [42 levels in both patients with Alzheimer disease and normal subjects. The sensitivity of the chemifluorescence ELISA was enhanced using photooxidation-induced fluorescence amplification [42, it enhanced the detection sensitivity by a factor of more than 10. Electrochemiluminescence ELISA combined with an Au nanocluster technology was developed, and the LOD for tumor necrosis factor-α (TNF-α) was two orders lower than that using a conventional ELISA [Chemiluminescence and electrochemiluminescence are also used for ELISA. A sandwich ELISA using a chemiluminescence substrate was applied to measure amyloid-β disease . The LODfication . When thal ELISA .3+) is an attractive rare-earth metal due to its bright fluorescence when chelated by organic ligands. A fluoroimmunoassay using Eu3+ was developed for the quantitative analysis of saikosaporin a, which is a traditional Chinese medicine, and had an LOD of 0.006 μg/mL [1, which is a key factor causing liver cancer, using Eu3+-labeled IgG as a tracer [50 was 94.73 pg/mL and the LOD was 3.55 pg/mL.The trivalent europium ion  level [In 2014, Watabe and Ito attempted to realize an ultrasensitive ELISA by combining a sandwich ELISA and thio-NAD cycling to achieve ultrasensitive, quantitative detection of proteins . Accordis) level . A sandwThe major challenge for protein quantification is that proteins cannot be amplified like nucleic acids using a method such as PCR . We hypoThe most suitable quantification technique for a trace amount of protein is a sandwich ELISA . In the We identified another assay for detecting a trace amount of molecule that utilizes an enzyme cycling method to amplify the signal . One of a is the turnover ratio of ALP per minute, b is the cycling ratio of 3α-HSD per minute, and n is the measurement time in minutes.We combined these two assays, sandwich ELISA and thio-NAD cycling . This co−18 moles/assay. If the virus has approximately 25 spike proteins on its surface, this value corresponds to 10−20 moles of virus/assay. In other words, it could detect 104 copies of the virus RNA/assay, which is almost the same as that of PCR assays because the average virus RNA load used for PCR assays is 105 copies per oro- or naso-pharyngeal swab specimen [−17 moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable amount of virus obtained was 104 RNA copies/assay. The minimum detectable value was smaller than that of other antigen tests targeting SARS-CoV-2 nucleocapsid proteins, indicating the validity of this detection system for COVID-19 diagnosis. More recently, an ELISA with europium enhancement was proposed to detect the recombinant spike and nucleocapsid proteins from SARS-CoV-2 viruses [The ultrasensitive ELISA with thio-NAD cycling has been used for the diagnosis of infectious diseases and lifestyle-related diseases. Among infectious diseases, we now face threats from COVID-19. To detect SARS-CoV-2, which causes COVID-19, its two major proteins were recently detected using the ultrasensitive ELISA with thio-NAD cycling . One of specimen . To our specimen . The LOD viruses . Even thMycobacterium tuberculosis is heated, only live bacilli secrete a specific protein, MPT64 [−19 moles/assay, corresponding to 330 CFU/mL in the culture method, which was almost the same high detection sensitivity as the culture method. This ultrasensitive ELISA with thio-NAD cycling required only 5 h, resulting in a same-day diagnosis of tuberculosis and enabling drug efficacy monitoring.In addition to COVID-19, there are three other major infectious diseases of public health concern: tuberculosis, HIV/acquired immunodeficiency syndrome (AIDS), and malaria, which are serious threats to global health . With ren, MPT64 ,82. The −18 moles/assay , which meets the qualification of 2 IU/mL established by the French Health Authority for a CE-marked HIV antigen/antibody assay in 2010. We are now devoting ourselves to the development of a specific detection system for malaria using the ultrasensitive ELISA with thio-NAD cycling.For HIV detection, fourth-generation tests needed to detect both HIV-1/2 antibodies and the HIV-1 p24 antigen, which allowed the diagnosis to be made 1 to 3 weeks earlier than tests based on antibody alone. Furthermore, fifth-generation tests detected the same markers but could distinguish samples that were positive for HIV-1 p24 antigen and were specific for the HIV-1 and HIV-2 antibodies. Because of these technological advances, the window period for HIV detection has been reduced to approximately 18 days. To further decrease the window period, the ultrasensitive ELISA with thio-NAD cycling was applied for the detection of HIV-1 p24 . The LOD−19 moles/assay [−16 moles/assay [Insulin is a peptide hormone in the blood that serves as a signal of the fed state. Diabetes mellitus (DM) is a disease in which the control of insulin levels has failed. DM occurs in two forms—insulin-dependent, i.e., type 1 DM, and non-insulin dependent, i.e., type 2 DM. Patients with type 2 DM are often insulin-resistant and thus suffer from a relative insulin deficiency. In both cases, an ultrasensitive immunoassay approach for monitoring insulin is highly desirable and beneficial. The LOD of the ultrasensitive ELISA for a recombinant insulin was 10es/assay . Anotheres/assay . The WHO−19 moles/assay [Adiponectin is a hormone secreted from adipocytes that demonstrates antidiabetic and anti-obesity activities. An increase in blood adiponectin levels is considered acceptable for good health; however, the normal urinary adiponectin levels were unknown because the urinary adiponectin levels in healthy subjects could not be determined using conventional ELISAs. The ultrasensitive ELISA with thio-NAD cycling was thus applied to detect a recombinant antigen of adiponectin and had an LOD of 10es/assay . This ades/assay ,86. The Nucleic acids can be amplified with PCR, making it is easy to detect a small change in the amount of nucleic acids in cells and viruses. On the other hand, proteins, peptides, and many biomarkers cannot be similarly amplified. Therefore, detection techniques with high sensitivity are needed. While ELISA is a promising technique for this purpose, greater detection sensitivity is needed. Many variations of ELISA have been developed to achieve ultrasensitive detection, such as our ultrasensitive ELISA with thio-NAD cycling ,88. We h


Plasmodium falciparum (PfAMA-1) is a leading malaria vaccine candidate antigen. However, the genetic diversity of pfama-1 and associated antigenic variation in global P. falciparum field isolates are major hurdles to the design of an efficacious vaccine formulated with this antigen. Here, we analyzed the genetic structure and the natural selection of pfama-1 in the P. falciparum population of Vietnam. A total of 37 distinct haplotypes were found in 131 P. falciparum Vietnamese isolates. Most amino acid changes detected in Vietnamese pfama-1 were localized in the ectodomain, domains I, II, and III. Overall patterns of major amino acid changes in Vietnamese pfama-1 were similar to those of global pfama-1, but the frequencies of the amino acid changes slightly differed by country. Novel amino acid changes were also identified in Vietnamese pfama-1. Vietnamese pfama-1 revealed relatively lower genetic diversity than currently analyzed pfama-1 in other geographical regions, and suggested a distinct genetic differentiation pattern. Evidence for natural selection was detected in Vietnamese pfama-1, but it showed purifying selection unlike the global pfama-1 analyzed so far. Recombination events were also found in Vietnamese pfama-1. Major amino acid changes that were commonly identified in global pfama-1 were mainly localized to predicted B-cell epitopes, RBC-binding sites, and IUR regions. These results provide important information for understanding the genetic nature of the Vietnamese pfama-1 population, and have significant implications for the design of a vaccine based on PfAMA-1.Apical membrane antigen-1 of  Despite remarkable reduction in global mortality and morbidity of malaria in recent years, the disease is still a global public health concern. Approximately 229 million clinical cases of malaria, with an estimated 409,000 deaths, have been reported in 2019 [Plasmodium species is a membrane protein consisting of a signal sequence, a cysteine-rich ectodomain, a conserved cytoplasmic region, and a transmembrane region [Plasmodium parasites [P. falciparum or P. vivax [Plasmodium field isolates, and the resulting variants in different geographic areas, are major hurdles in the development of a global malaria vaccine based on this antigen. Therefore, it is important to monitor genetic variations in the vaccine candidate antigen in the global Plasmodium isolates, since accumulated or newly emerging mutations can change the structure of the antigen, making it difficult to design optimized malaria vaccines.Apical membrane antigen-1 (AMA-1) of the e region . The ecte region . This prarasites ,5,6,7,8.P. vivax ,10,11,12P. vivax ,14. HoweP. vivax ,16,17. DP. vivax ,25,26,27P. falciparum is a dominant species circulating in the Central Highlands [P. falciparum AMA-1 (pfama-1) in P. falciparum isolates from Vietnam to expand our knowledge on genetic variations in the global pfama-1 applicable to the development of a vaccine targeting PfAMA-1.Malaria was one of the most prominent infectious diseases affecting high mortality in Vietnam until the early 1990s, but the malaria burden in the country has been dramatically reduced over the past few decades . Howeverighlands ,31. Concighlands ,33,34,35Plasmodium falciparum in Dak Lak Province, Central Highlands, Vietnam, in 2019 [P. falciparum infections were further validated by polymerase chain reaction (PCR) targeting the 18S ribosomal RNA (rRNA) gene [Blood samples were collected from malaria patients who were infected with  in 2019   gene ,37. The pfama-1 was amplified from the genomic DNA by nested PCR using primer sets and thermal cycles, as described previously [Escherichia coli DH5α competent cells, and positive clones were selected by colony PCR with the nested PCR primers [pfama-1 [pfama-1 were deposited at GenBank (accession numbers MW938322–MW938452).Parasite genomic DNA was isolated from the blood spots using QIAamp DNA Blood Kit  according to the manufacturer’s protocol. Full-length eviously . To mini primers . Nucleot[pfama-1 . To ensupfama-1 were analyzed using EditSeq and SeqMan programs in the DNASTAR package . Nucleotide sequence polymorphism of Vietnamese pfama-1 sequences was analyzed. The numbers of segregating sites (S), average number of pair-wise nucleotide differences within a population (K), haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (π) were analyzed using DnaSP ver. 5.10.00 [pfama-1 based on a sliding window of 100 bp with a step size of 25 bp. Non-synonymous (dN) and synonymous (dS) substitutions were estimated and were compared with Z-test (p < 0.05 was considered significant) in the MEGA4 program [2 index using DnaSP ver. 5.10.00 [Nucleotide and deduced amino acid sequences of  5.10.00 . The π v program  using th program  with Juk program , and Fu  program  were ana program . The rec program . Linkage 5.10.00 .pfama-1 in global P. falciparum isolates was analyzed. The full-length pfama-1 sequences available in public databases were included: Ghana ; Myanmar ; Papua New Guinea ; Philippines ; Solomon Islands ; Tanzania ; Thailand ; and Vanuatu . Nucleotide sequence polymorphism and a neutrality test for each population were analyzed using the DnaSP ver. 5.10.00 [STF) to estimate pair-wise DNA sequence diversity between and within populations using DnaSP ver. 5.10.00 [pfama-1 haplotypes, the haplotype network of 648 pfama-1 sequences, including 131 from Vietnam and the 517 from Ghana, Myanmar, PNG, Philippines, Solomon Islands, Tanzania, Thailand, and Vanuatu, was constructed using the program PopART version 1.7  with the Median Joining algorithm [pfama-1 within P. falciparum isolates and host immune pressure, genetic diversity in predicted B-cell epitopes, intrinsically unstructured/disordered regions (IUR), and red blood cell (RBC) binding regions in global pfama-1 were analyzed [The genetic diversity of  5.10.00  and MEGA 5.10.00  programs 5.10.00 . To detelgorithm . To evalanalyzed ,44,45. Tanalyzed .pfama-1 from 135 P. falciparum samples from Vietnam resulted in successful amplification of pfama-1 in 131 samples. No amplicon was detected in four P. falciparum isolates. The size of the amplified products was approximately 1.9 kbp, and no size variation was detected between and among the amplified products. Nucleotide sequence analysis of the 131 Vietnamese pfama-1 sequences based on pfama-1 from 3D7 reference strain (GenBank accession number: U65407) revealed 116 single nucleotide polymorphisms (SNPs), including 52 synonymous and 64 non-synonymous SNPs. The non-synonymous SNPs induced amino acid substitutions at 52 positions in Vietnamese pfama-1 sequences, resulting in 37 distinct haplotypes of pfama-1 in the amino acid levels , domain II (12 positions), and domain III (6 positions). Most amino acid changes were di-morphic (48 positions), but tri-morphic amino acid changes at three positions , and a penta-morphic amino acid change at one position (E197D/G/Q/V) were also detected. One amino acid change (D584H) was commonly detected in all Vietnamese pfama-1 sequences  were novel and never reported previously in global pfama-1, despite their low frequencies, ranging from 1.5 to 3.8%. Haplotype 23 was the most prevalent haplotype, accounting for 56.5% (74/131), followed by haplotype 34 , haplotype 12 , and haplotype 20 . Other haplotypes were detected in only one or two sequences resulting in low frequencies, respectively.Nested PCR of d levels . These aequences . Of the pfama-1 shared similar, but not identical, patterns of amino acid polymorphism with global pfama-1. Amino acid changes at 91 positions were identified in global pfama-1. Most amino acid changes were detected in domains I (33 positions), II (19 positions), and III (16 positions), but also at 5′-terminal (5′-T) and 3′-terminal (3′-T) regions ; E52Q/K ; H200D/L/R ; F201L/S/V ; K206E ; Y207D ; E267Q/P ; I282K/N ; S283L ; D296H ; and K544N . Amino acid changes at 12 positions were detected with lower frequencies in Vietnamese pfama-1 compared with pfama-1 sequences from other countries: G172E ; Y175D ; E187K/N ; I225N ; K230E/Q ; D242Y/A ; K243N/E ; Q285E/P ; Q308E/K ; T404R ; M496I ; and R503N/H . Amino acid changes at 11 positions , which were commonly identified in other countries, were not detected in Vietnamese pfama-1. Meanwhile, 12 amino acid changes at 11 positions  were unique to Vietnamese pfama-1, and not detected in global pfama-1 [pfama-1 analyzed.Vietnamese  regions . AlthougK) of full-length pfama-1 of Vietnam isolates was 8.062 (K was identified at domain I (K = 4.198), whereas the lowest was detected at 5′-T (K = 0.485). Haplotype diversity (Hd) for 131 full-length Vietnamese pfama-1 was 0.837 ± 0.034. This value was higher at domain I (0.575 ± 0.052) than at 5′-T (0.358 ± 0.055), domains II (0.465 ± 0.053), domain III (0.455 ± 0.053), and 3′-T (0.432 ± 0.053). The nucleotide diversity (π) of each fragment was 0.0011 ± 0.0002 (5′-T), 0.0091 ± 0.0014 (domain I), 0.0031 ± 0.0004 (domain II), 0.0076 ± 0.0012 (domain III), and 0.0023 ± 0.0003 (3′-T), respectively  and domain II (0.0020), implying the impact of mild natural selection. Tajima’s D value for full-length Vietnamese pfama-1 was −2.0046 (p < 0.05)  . When Ta domains b.pfama-1 showed a substantially lower K value compared with the global pfama-1 including the Southeast Asian countries, Myanmar and Thailand , whereas Asian and Pacific pfama-1, except PNG, showed relatively lower R values (2) for global pfama-1 also declined with increasing distance across the gene, suggesting the role of intragenic recombination in the genetic diversity of global pfama-1 (The estimated minimum number of recombination events between adjacent polymorphic sites (Rm) for Vietnamese 1 was 20 . The preR values . The LD  pfama-1 .pfama-1, a haplotype network was constructed. A dense network with 245 individual haplotypes with complicated relationships was established using 648 global pfama-1 sequences  to 0.42009 (between Vietnam and Solomon Islands). Interestingly, the Vietnamese pfama-1 exhibited large genetic differentiation from other global pfama-1.To further analyze the genetic differentiation and gene flow in global analyzed . The FSTpfama-1, the genetic polymorphisms in the predicted B-cell epitopes, RBC-binding sites, and IUR regions of pfama-1 were analyzed. Most major amino acid changes were detected in the predicted B-cell epitopes, RBC-binding sites, or IUR regions of pfama-1 (pfama-1 compared with the 3D7 sequence (GenBank accession No.: U65407), 72 were found at the predicted B-cell epitopes, RBC-binding sites, or IUR regions. Among 51 amino acid changes detected commonly in global pfama-1, 42 were located in the predicted B-cell epitopes, RBC-binding sites, or IUR regions. Twenty-nine less common amino acid changes in global pfama-1 were also detected in the predicted B-cell epitopes, RBC-binding sites, or IUR regions. Eight of eleven predicted B-cell epitopes were polymorphic. Particularly, B-cell epitopes 3, 4, 5, 8, and 10 had major polymorphic amino acid residues with high levels of π  were unique to Vietnamese pfama-1, but not detected in global pfama-1. By contrast, 11 amino acid changes , which were commonly identified in global pfama-1 with varying frequencies, were not detected in Vietnamese pfama-1. Haplotype network analysis also revealed that Vietnamese pfama-1 formed distinct clusters, which were clearly distinguished from other global populations, including pfama-1 from the neighboring Southeast Asian countries of Myanmar and Thailand. These findings suggest that the Vietnamese pfama-1 exhibited distinct patterns of polymorphism and genetic differentiation compared with other global isolates. The STF value, a measure of population substructure based on an analysis of the overall genetic differentiation among populations [pfama-1 from other global populations. Due to the limitations of currently available full-length pfama-1 sequences from diverse geographical origins, the implications of substantial differentiation of Vietnamese pfama-1 from global pfama-1 is currently unclear. Further analysis of genetic polymorphisms of pfama-1 in a larger number of global P. falciparum populations is necessary to appreciate the polymorphic nature and evolutionary linkage of global pfama-1.In the present study, we analyzed the genetic polymorphisms and natural selection of Vietnamese ountries ,48,49,50morphism . Some ampfama-1 differed depending on the origin of the isolates. The nucleotide diversity in Vietnam pfama-1 (π = 0.0043) was relatively lower than in isolates from different geographical areas. Lower malaria transmission in restricted areas of Vietnam compared with other endemic countries may contribute to the low-level genetic diversity in Vietnamese pfama-1. However, it is also necessary to consider the global pfama-1 sequences analyzed in this study were obtained from parasites collected at different time points in each country, which do not reflect the π value of the pfama-1 populations in the same period. In fact, the genetic structure of Plasmodium populations in endemic areas or countries changes dynamically over time due to various factors [pfama-1 in each country. Therefore, a systematic analysis of the global P. falciparum isolates collected in the same period, especially current, may be necessary to delineate the genetic variations and evolutionary trends of global pfama-1. Although the π value of global pfama-1 differed by country, synchronized patterns of π across pfama-1 were recognized in global pfama-1, including Vietnamese pfama-1. A sliding window plot suggested that the nucleotide diversity was unevenly distributed across pfama-1, and high levels of π were similarly observed in domains I and III of global pfama-1, supporting that the domains are the main regions contributing to the genetic heterogeneity of pfama-1.The π value of global  factors ,54,55,56pfama-1 genetic diversity [pfama-1 showed different patterns of natural selection compared with pfama-1 from other countries. In contrasts to global pfama-1, which showed positive values of Tajima’s D, the Vietnamese pfama-1 revealed a negative Tajima’s D value, implying purifying selection. The dN/dS ratios of global pfama-1 were relatively high in all isolates, but the value was much lower in Vietnamese pfama-1, suggesting the absence of strong positive selection. These findings suggested that Vietnamese pfama-1 underwent different natural selection compared with pfama-1 from other countries, probably due to selective sweep or a bottleneck effect. However, regardless of these differences, the sliding window plot revealed that domains I and III exhibited high values of π and Tajima’s D across global pfama-1, implying that these regions represent possible dominant targets for natural selection by host immune response. Indeed, most common amino acid changes identified in global pfama-1 were mainly scattered in domains I and III, corresponding to B-cell epitopes 3, 4, 5, 9, and 10. The C1-L cluster, which is located near the hydrophobic pocket of domain I in PfAMA-1 [P. falciparum infection or vaccine trials [pfama-1 suggest strong natural selection, which further contributes to host immune escape [pfama-1, suggesting that this region might be a vaccine candidate based on PfAMA-1 [pfama-1 [pfama-1 derived from other geographical isolates have been reported [pfama-1 revealed a lower level of genetic diversity than the other global pfama-1, a comparable level of recombination events and decline of the LD index R2 were also detected in Vietnamese pfama-1. These findings suggest that interallelic recombination is another force generating genetic diversity of the Vietnamese pfama-1, and consistent with findings from other geographical areas.Natural selection probably affected by the host–immune response and recombination between genetically distinct alleles during meiotic replication in the mosquito midgut have been understood as the two main mechanisms underlying iversity ,57,58. I PfAMA-1 , is knowe trials ,59,60,61e escape ,61,62. Me escape , was hig PfAMA-1 . Meiotic[pfama-1 ,50,57. Sreported . Althougpfama-1 were similar to those seen in global pfama-1. However, the Vietnamese pfama-1 revealed relatively lower genetic diversity than pfama-1 populations from other geographical regions, and showed a distinct genetic differentiation profile. Although evidence for natural selection and recombination, which may contribute to the generation and maintenance of genetic diversity of pfama-1, were also found in Vietnamese pfama-1, it revealed a distinct trend of natural selection compared with other global pfama-1. These results have significant implications for understanding the genetic nature of the Vietnamese P. falciparum population, warranting continuous monitoring of the genetic diversity of global pfama-1 to elucidate the polymorphic nature and evolutionary aspects of pfama-1, and design an effective vaccine targeting P. falciparum populations globally.Overall patterns of nucleotide diversity and major amino acid changes in Vietnamese


Lasiurus cinereus). Because it is not possible to monitor population sizes for these solitary and cryptic species using traditional techniques, we must rely on other methods, such as acoustic and genetic monitoring, to provide information regarding population status and trends. Using these approaches, we can build a weight of evidence to assess whether mortality associated with wind turbines is sustainable and determine if and when mitigation measures should be implemented to reduce wind turbine mortality. To this end, we recommend that acoustic data be collected using the North American Bat Monitoring Program (NABat) protocols, and that genetic diversity be monitored at repeated time intervals to document species trends. There are no short-term measures to resolve population-level questions for migratory tree-roosting bats. Nonetheless, we discuss opportunities for relatively short-term investments that will lead to long-term success in reducing uncertainty for these species.Although wind energy provides valuable environmental and economic benefits, it has unintended negative consequences for wildlife, particularly birds and bats that collide with the rotating blades. In North America, for example, there are increasing concerns that wind turbines threaten the persistence of populations of migratory tree-roosting bats, such as the hoary bat (Lasiurus cinereus) in North America. These species are dispersed within their range, making it impractical to estimate census populations size using traditional survey methods. Nonetheless, understanding population size and trends is essential for evaluating and mitigating risk from wind turbine mortality. Using various sampling techniques, including systematic acoustic sampling and genetic analyses, we argue that building a weight of evidence regarding bat population status and trends is possible to (1) assess the sustainability of mortality associated with wind turbines; (2) determine the level of mitigation required; and (3) evaluate the effectiveness of mitigation measures to ensure population viability for these species. Long-term, systematic data collection remains the most viable option for reducing uncertainty regarding population trends for migratory tree-roosting bats. We recommend collecting acoustic data using the statistically robust North American Bat Monitoring Program (NABat) protocols and that genetic diversity is monitored at repeated time intervals to show species trends. There are no short-term actions to resolve these population-level questions; however, we discuss opportunities for relatively short-term investments that will lead to long-term success in reducing uncertainty.Wind turbine-related mortality may pose a population-level threat for migratory tree-roosting bats, such as the hoary bat ( Understanding the structure and dynamics of a population can assist in evaluating the scope and severity of a stressor, and the efficacy of mitigation measures. For most species of bats, population data are limited or nonexistent, making it difficult to assess natural and human-induced stressors ,2,3. DesLasiurus cinereus), eastern red bats , and silver-haired bats (Lasionycteris noctivagans). Note: we elected to use the traditional taxonomy recognized by the taxonomic nomenclature authority and database (www.batnames.org (accessed on 11 November 2021)); [The global expansion of wind energy development represents a potential threat to certain bat populations. In North America, wind energy development primarily impacts migratory tree-roosting species, including hoary bats ( 2021)); ); see [9 2021));  for sugg 2021)); ,13. Give 2021)); ,15, the Placing wind turbine-related bat mortality into context has been challenging because estimating census population size (Nc) using traditional methods is impractical for migratory tree-roosting bats. These species roost individually, are dispersed across the landscape, and migrate over long distances ,17. In lThe lack of available demographic data for many bat species, including hoary bats, emphasizes the importance of collecting data to determine vital rates; however, these studies are labor intensive and may require ≥5 years of data from capturing, marking, and tracking individuals . AlthougThere are no practical, short-term measures to determine Nc for migratory tree-roosting bats, but additional data can be collected to reduce uncertainty and assist in determining population trends ,28. The In 2012, a workshop on bat population monitoring and modeling was convened to design statistically robust and logistically feasible methods for monitoring changes in bat populations . At the 2 cells and probability-based sampling [Pipistrellus pipistrellus), whereas it would take approximately 15 years to detect a 25% decline in this species.For acoustic sampling, NABat uses a systematic grid-based framework of 100 kmsampling ,34,35,36sampling  generatesampling . Becausesampling . Based osampling  reportedMyotis lucifugus). The inference of the study was limited as it was regionally focused and included only 5% of the continental United States; thus, conclusions about the population status of the two species at larger spatial scales could not be determined. Rodhouse et al. [Repeated annual surveys at priority grid cells permits the extension of the model to unsampled areas. As a result, changes in occupancy dynamics, or the probability of a site being unoccupied in one year given it was occupied the previous year, can be estimated . Rodhouse et al.  indicateFor NABat, acoustic surveys can be either mobile or stationary. For stationary surveys, as were used in Rodhouse et al. , each reMobile transects assume each bat pass represents a unique individual because the vehicle is presumably traveling faster than most bats are flying. Since most bats in North America fly between 9 and 32 km/h, mobile transects are unlikely to encounter the same bat twice ,45,46,47Each survey type, stationary vs. mobile, has advantages and biases. Neece et al.  reportedAlthough genetic data cannot provide direct estimates of census population sizes, they are useful for monitoring population status and trends, informing geographic ranges and seasonal movements, and detecting cryptic speciation . These aforementioned aspects of bat biology all require additional investigation and are necessary to properly plan and implement conservation strategies. By combining recent advances in the tools and analytical approaches to monitor genetic variation in wildlife populations with the abundance of bat carcasses that are recovered at certain times of the year in distinct geographic locations across the United States , these data have the potential to be especially informative. In addition, the ability to monitor for changes in genetic diversity over time could provide unique insights that reflect actual fluctuations in census population size. Although it is not possible to isolate changes in genetic diversity due to a single stressor, the scope and severity of these cumulative changes can guide management and mitigation decisions that are necessary for the conservation of migratory tree-roosting bats.Genetic variation in a population is essential for adaptation to environmental change, and it is well established that loss of genetic diversity impedes the long-term survival of species . When laSalmo salar) from Maine revealed significant decreases in Ne and several measures of genetic diversity from 1963 to 2001 [Population genetic analyses also can detect population bottlenecks, estimate historical and current effective population sizes, and identify evolutionarily unique subpopulations . The eff to 2001 . These r to 2001 .Genetic diversity and population structure of migratory tree-roosting bats likely affect resilience to sustained mortality from wind turbines and the likelihood of population persistence. Species with smaller Ne, for example, have lower genetic diversity which may limit their evolutionary potential to respond to selection and avoid inbreeding in the future . AlthougSeveral tools are available to characterize and monitor genetic variation in populations, and recent review papers illustrate how these data can be applied to the conservation of natural populations  provides some guidance on the number of detectors and duration for these surveys . StandarA primary objective for standard preconstruction acoustic surveys was to determine whether preconstruction activity predicts postconstruction mortality. Because no reliable relationship has been established between these variables ,83, it mThere are instances where following the probabilistic sample is not feasible, such as conducting presence/absence surveys to fulfill obligations under Section 7 of the U.S. Endangered Species Act (16 U.S. Code § 1536). The Range-Wide Indiana Bat Survey Guidelines , for exaWhile stationary acoustic data are valuable in determining occupancy probability, mobile acoustic surveys serve as inputs to both occupancy and abundance models. In support of the objective to determine measures of abundance for migratory tree-roosting bats and to place wind turbine mortality in the context of population size and trends, it is recommended to adopt mobile acoustic surveys in high-priority cells. Although offshore transects are not currently part of the NABat protocol, these could be considered, and protocols developed if they are deemed feasible in the offshore environment. It may be possible to conduct boat surveys by adapting the methods used by Whitby et al. . There aData collected in association with wind energy development can increase the quantity and quality of data available for analyses, offering in return data-driven updates to the probability of occupancy, species range maps, indices of abundance, and improved understanding of relationships with habitat features, available resources, and various stressors. To help achieve these goals, standard preconstruction bat data should be contributed to the NABat database, and where possible, transitions to mobile acoustic surveys following the NABat protocol are recommended.There is value in comparative genetic studies across species, geographic regions, and temporal scales. Continued genetic evaluation of opportunistically collected samples, such as those in Cornman et al. , with a For contemporary samples, it will be important to locate sources of existing genetic material . It also will be necessary to identify partners willing to share tissue samples or carcasses from mortality searches at wind turbines during the next several years, with the goal of filling in data gaps in species or geographic coverage that may exist with the samples that are already available.Future research can expand the geographic scope and temporal scale of hoary bat samples. It may be possible to use historical DNA samples to estimate the genetic parameters 10, 20, or even 100 years ago, which can be matched by geographic location to contemporary samples collected from wind energy facilities. The results of these analyses could be used to help answer the following questions: (1) is there evidence of a decline predating the development of wind energy?; (2) do changes in genetic diversity or Ne coincide with the expansion of wind energy development during the past 20–25 years? and (3) how do estimates of Ne and genetic diversity compare among different geographic regions that may be experiencing different stressors, such as wind energy development and climate change impacts?Estimates obtained from population genetic studies will not provide direct estimates of Nc, but they can be used to monitor populations and population impacts over time and to make predictions about relative impacts to different species. Obtaining these estimates for hoary, eastern red, and silver-haired bats can reduce uncertainty regarding the species-specific impacts of wind energy development. It is crucial to invest resources to store and manage genetic samples from bats collected at wind energy facilities during the next decade. This collection should target two different scales that are reflected herein: (1) sample intensively at select geographic areas that have high levels of bat mortality for hoary, eastern red, and silver-haired bats; and (2) sample broadly across North America to capture the range-wide level of impacts for hoary, eastern red, and silver-haired bats. By dedicating collection efforts at these two scales, we will ensure sufficient sample sizes and that we have sampled the entire population for each species.Strigops habroptilus) resulted from a recent population bottleneck associated with European colonization. In another example, Cammen et al. [Halichoerus grypus atlantica) and harbor seals (Phoca vitulina vitulina). These types of studies may also be informative for migratory tree-roosting bats.Taking multiple samples over time allows for monitoring genetic changes associated with various stressors . This tyn et al.  used RADWe recommend sampling several wind energy facilities in each U.S. Fish and Wildlife Service region for continuous long-term monitoring  for the three species of migratory tree-roosting bats with a focus on collecting tissue samples for subsequent genetic analyses. This could be achieved using a low-level monitoring effort, such as searching only gravel pads and roads at selected sites. The collection, processing, and storage of tissue samples and carcasses will require cooperation and coordination during standard postconstruction mortality monitoring efforts. The anticipated cost of tissue collection and storage are not anticipated to be a barrier to participation, but additional funding sources will be necessary to archive the samples and conduct the genetic analyses.Lasiurus ega) and the northern yellow bat (Lasiurus intermedius) using DNA from carcasses collected at wind energy facilities in south Texas, U.S. Yellow bats are in the same genus as hoary bats and eastern red bats, and also collide with wind turbine blades. Wind energy is relatively new in the region and represents a new source of mortality for these species. Chipps et al. [In addition to the strategic monitoring efforts described, we recommend that researchers continue to collect genetic samples from additional bat species that experience wind turbine mortality, especially as wind energy development expands into new areas. For example, Chipps et al.  examineds et al.  detecteds et al. . In addis et al. . FurtherNycticeius humeralis) [Tadarida brasiliensis) [Lasiurus seminolus) [Finally, we recommend the use of readily available and low-cost molecular methods to determine sex ,89,90 anmeralis) , Brazililiensis) ,98, and minolus) . ClearlyWhile migratory tree-roosting bats are experiencing a relatively new source of mortality associated with wind energy development, they are simultaneously experiencing other persistent and emerging stressors, such as climate change ,100, glo


Mimosa catharinensis – a narrow endemic, critically endangered plant species – we identified 1,497 unlinked SNP markers derived from a reduced representation sequencing method . This set of molecular markers was employed to assess intrapopulation genetic parameters and the demographic history of one extremely small population of M. catharinensis (N=33) located in the Brazilian Atlantic Forest. Contrary to what is expected for narrow endemic and threatened species with small population sizes, we observed a moderate level of genetic diversity for M. catharinensis . Interestingly, M. catharinensis, which is a lianescent shrub with no indication of seed production for at least two decades, presented high levels of outcrossing  and an apparent absence of inbreeding . However, the reconstruction of demographic history of M. catharinensis indicated that the population should be suffered a recent bottleneck. Our population genomic study tackles a central issue in evolution and conservation biology and we expect that it will be useful to help safeguard the remaining genetic diversity reported for this unique genetic resource.Intraspecific genetic variation plays a fundamental role in maintaining the evolutionary potential of wild populations. Hence, the assessment of genetic diversity patterns becomes essential to guide biodiversity conservation policies, particularly for threatened species. To inform management strategies for conservation of  Differences in DNA sequences among individuals within a population represent its genetic diversity, a key component for the long-term survival of natural populations that is related to the maintenance of its evolutionary potential in the face of environmental change e.g., . As a maAlthough the amount of genetic diversity in a population is commonly linked to its size and range e.g., , a betteMimosa catharinensis Burkart (Fabaceae), a rare, critically endangered (2) in the Brazilian Atlantic Forest , and based on the assumptions of population theory and the findings of empirical studies about genetic diversity in rare and endemic plant species occurring in small populations, we expected to find low levels of genetic diversity for M. catharinensis. Further, we expected to find indications of population retraction given the historic threats that have occurred in its biome (presented in the below section). To this end, and to contribute to in situ and ex situ conservation strategies, we used a high-throughput sequencing approach  occurs in one of the most heavily impacted areas of the PAERVE . As no seedlings were found in its area of occurrence, M. catharinensis may be considered an ecologically sterile plant species . As successful in vitro pollen germination had been reported for M. catharinensis  vegetation and is the main cause of degradation of the coastal ecosystem of Santa Catarina Island  is deposited at the FLOR herbarium at the Federal University of Santa Catarina – UFSC.We collected leaf samples from all 33 identified adult individuals of unknown age that constitute the only remaining MseI, 5,000 units of HF-EcoRI, and 1x CutSmart buffer (New England Biolabs) in a total 20μl reaction volume for 3h at 37°C, ending with a 20min deactivation step at 65°C  and 1.40μl of T4 ligase buffer in 30μl ligation reaction that was stored at a room temperature (23°C) for 30min After, heat-killed the reaction at 65°C for 10min and then, slowly cooled until reach room temperature. Details of barcode sequences can be found in 2 0.5U iProof™ High-Fidelity DNA Polymerase (Bio-Rad) and 2X of iProof buffer. The PCR protocol  was performed in an Eppendorf System. The amplicons were cleaned with the Agencourt AMPure XP system and quantified using Qubit dsDNA assay Kit. We used an automated size-selection technology  at 2% agarose cartridge to select DNA fragments at a target range size of 375–475bp. The library was sequenced (100-bp single-end) on a lane of Illumina HiSeq 2,500 flow cell  at The Centre for Applied Genomics in Toronto, Canada.DNA was extracted from leaf samples of all collected individuals employing the NucleoSpin® kit (Machereney-Nagel GmbH & Co. KG) following manufacture’s guidelines. After extraction, the quality of each sample was verified using Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc.) and the concentration of double-strand DNA (dsDNA) was obtained by using Qubit dsDNA Assay Kit (Invitrogen). The genomic library was prepared using a double-digest restriction site-associated DNA sequencing  protocol  with mod at 65°C . Reactiode novo assembly. Initially, we used the process_radtags program in Stacks to examine individual barcodes, enzyme cutsite integrity and to demultiplex the data. For barcodes rescue we admitted at most two mismatches (−barcode_dist 1 2). We filtered raw reads using the phred score 33 and used the option −t to trim all of it at 85 base pairs . With the process_radtags output, we run the Ustacks program, that uses a maximum likelihood framework to aligns short-read sequences and create putative alleles (stacks). The maximum distance allowed between “stacks” was two nucleotides. The minimum depth of coverage required to create a “stack” and the maximum number of stacks at a single de novo locus was set as three. We enabled the deleveraging algorithm (−d) to solve merged tags, and a bounded-error model to identify SNPs. An alpha value of 0.05 and an upper bound of 0.1 were used. In the next step, we performed Cstacks to merge alleles of all samples and create a catalog of consensus loci. Three mismatches were allowed between loci in catalog build. Each individual sample loci were then compared against the catalog through the Sstacks program. We ran tsv2bam program to organize the single-end reads by locus, instead by sample, creating a BAM file that was used as input to the Gstacks program. Gstacks uses the single-reads to build contigs and merges them into loci. It also aligns reads to the loci using Ukkonen’s algorithm, identifies SNPs for each locus and each individual genotype and converts SNPs into phased haplotypes. Finally, we used POPULATIONS in Stacks  and Acacia ligulata (NCBI accession number MH933866), respectively. We used the BLASTn programData quality was checked using the program FastQC version: 0.11.8.n Stacks  to genern Stacks  implemenn Stacks . Type I n Stacks , and SNPuHE; HO), and the inbreeding coefficient  based on standardized identity disequilibrium for all data sets. Significance for the identity disequilibrium coefficient was obtained with 1,000 permutations, and a jackknife over loci approach was applied to calculate the standard error of outcrossing estimates.We used the kinship coefficient  implemenM. catharinensis  for the complete single nucleotide polymorphism (SNP) data set  using the equation 1-PI, where PI represents the probability that two individuals drawn at random from a population will have the same genotype at multiple loci . This anrinensis , a clona package , 2015 in package . The “bi package . Applyin package . We chos package . In addiM. catharinensis was inferred using a composite likelihood-based approach implemented in FastSimCoal v. 2.6 . Once the best model was selected, its estimated parameters were used to simulate 100 SFS data sets to built confidence intervals based on bootstrap distribution.The demographic history of l v. 2.6 . VCF fill v. 2.6 . Assuminl v. 2.6 . We testl v. 2.6 . We perfthaliana . The maxthaliana , 1974 esthaliana . We alsoA. dealbata (96.47% identity) and it was excluded from the data set. The final numbers of unlinked SNPs used in each data set are shown in The Illumina sequencing generated a total of 79,759,227 raw reads, of which less than 1% was discarded due to low quality. The average (±SE), minimum, and maximum retained reads were 2,384,161 , 1,470,849, and 4,011,918. The total number of genotyped RADtag loci was 274,113 with a mean coverage depth of 12.7 (±2.7 SD). After Bonferroni adjustment, significant deviations from Hardy–Weinberg equilibrium were observed for all data sets . No linkEuH and OH) and Wright’s Fixation Index (FIS) for data sets with different percentages of missing data are shown in EuH, OH, and FIS) obtained for the M. catharinensis population, no statistical differences were observed among the data sets  has high discriminatory power to identify M. catharinensis individuals. The defined threshold of 0.04 did not cluster MLGs into multilocus lineages . The model with the best fit for the complete (N=33) and the reduced (n=23) data sets was the bottleneck , with a continuous decline in population size after the bottleneck. However, the 95% CIs were wide and overlapping.The highest likelihoods are summarized in ttleneck . The infM. catharinensis. This data was used to assess intrapopulation genetic variation, as well as the demographic history for this critically endangered, narrow endemic species of the Atlantic Rainforest.Genomic approaches are valuable as they can provide new insights and a better understanding of the amount, distribution, and functional significance of genetic variation in natural populations . As predM. catharinensis has moderate intrapopulation genetic diversity, expressed here by HE, with an apparent absence of inbreeding. Although the use of only one metric to assess genetic diversity is not an optimal condition, our results bring to light several factors that can influence the observed patterns of genetic diversity and inbreeding in relictual populations of threatened species. Our results underscore the importance of assessing the genetic patterns of each species in order to critically evaluate their genetic variability and the microevolutionary forces by which they are shaped , traits such as self-incompatibility, long-term persistence, clonal reproduction, and consistent population size may explain the high levels of genetic diversity observed for the species  suggests that the level of intrapopulation genetic diversity has remained static for at least two decades, even though our demographic inferences indicated a recent decline in population size.In addition, several studies have indicated that life-history traits  strongly influence the amount and distribution of genetic variation in natural populations e.g., . For Aec species . Specifi species . MoreovePetunia secreta and Petunia exserta, both of which are narrow endemic species, have high genetic diversity indices; however, these indices are lower than those reported for its congener Petunia axillaris, which has a wider geographic distribution range , we are unable to make generalizations about the genetic diversity of narrow endemics. Indeed, M. catharinensis displays higher levels of genetic diversity than self-compatible  and low estimates of selfing (s=0.075/0.155). For M. catharinensis, the interpretation of negative FIS values coupled with high t values should be viewed with caution due to the distinct biology of the species . Such biological characteristics observed for M. catharinensis suggest that a significant selective pressure against homozygotes must have occurred in previous generations, especially when taking into account the excesses of heterozygosity observed in its small population. As M. catharinensis population is constituted only by adult trees, a further study investigating inbreeding depression seems not to be a feasible task as a consequence of the lack of different ontogenic stages in its population.The  outcrossing rate observed in requency . Such a requency . As withes e.g., , includi species . Studiescabrella , for insM. catharinensis  in the only known M. catharinensis population, coupled with strong evidence for a lack of sexual reproduction, suggests that the conservation perspectives for this critically endangered species are concerning. Although no inbreeding was detected, the species is facing imminent risk of extinction because of its reduced population size, making it more susceptible to stochastic events. As a matter of fact, ex situ conservation activities must be implemented in order to safeguard the remaining genetic diversity of M. catharinensis. One potential strategy suggested in studies on biodiversity conservation of threatened species consists of the preservation of tissue culture, an in vitro cultivation of isolated live tissue, enabling the propagation of species facing difficulties with natural reproduction , one of the main issues threatening the flora of PAERVE, should be prioritized. Furthermore, it is widely reported that adequate restoration of the contaminated areas of the park are urgently needed .SNP data sets for M. catharinensis are available for download from the Dryad Digital Repository  to AN (429266/2018-9) for funding the field and molecular work associated with this project. Additional funds were provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through a PQ-2 grant to AN (306182/2020-3) and the Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for the scholarship to TT.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.