Aedes aegypti is the most important arboviral disease vector worldwide. In Africa, it exists as two morphologically distinct forms, often referred to as subspecies, Aaa and Aaf. There is a dearth of information on the distribution and genetic diversity of these two forms in Sudan and other African Sahelian region countries. This study aimed to explore the distribution and genetic diversity of Aedes aegypti subspecies using morphology and Cytochrome oxidase-1 mitochondrial marker in a large Sahelian zone in Sudan. An extensive cross-sectional survey of Aedes aegypti in Sudan was performed. Samples collected from eight locations were morphologically identified, subjected to DNA extraction, amplification, sequencing, and analyses. We classified four populations as Aaa and the other four as Aaf. Out of 140 sequence samples, forty-six distinct haplotypes were characterized. The haplotype and nucleotide diversity of the collected samples were 0.377–0.947 and 0.002–0.01, respectively. Isolation by distance was significantly evident . The SAMOVA test indicated that all Aaf populations are structured in one group, while the Aaa clustered into two groups. AMOVA showed 53.53% genetic differences within populations and 39.22% among groups. Phylogenetic relationships indicated two clusters in which the two subspecies were structured. Thus, the haplotype network consisted of three clusters. Aedes aegypti (Ae. aegypti), which is commonly known as the yellow fever mosquito, is recognized for the transmission of the most significant arboviral diseases, including dengue, chikungunya, and zika viruses Aaf subspAedes aegypti populations was different in different sites. The lowest variation was observed in the Aaa population in the coastal city, Port Sudan. Similar results were reported by Elnour [Aedes aegypti populations in Port Sudan. This lower genetic variation in Port Sudan may be due to the fact that the Red Sea provides consistent high humidity throughout the year, thus reducing the impact of dryness on the vector population. In contrast, other sites in the country had a higher impact of natural selection, as predicted by Tajima D and Fu’s Fs statistics. It was evident that the level of genetic variation of y Elnour , who repAedes aegypti are found in Sudan, with clear variation in their distribution. Whereas the Aaa subspecies appears to be more abundant in the eastern part of the country, the Aaf subspecies is the main form of the vector found in western Sudan. There was a clear distinction in the genetic structure of the two subspecies. Further work is urgently required to elucidate the role of each subspecies in the transmission of dengue fever, chikungunya, yellow fever, and other arboviruses in Sudan and other countries of the African Sahelian region. Furthermore, work is needed to understand the ecological determinants of the distribution of the two subspecies and develop suitable control programs for the viruses they transmit. In conclusion, our results show that both subspecies of Mosquitoes were collected from eight study sites in Sudan, where dengue and other arboviral disease outbreaks were reported. As shown in Seven of the study sites  have a tropical continental climate, characterized by a long (9 month) dry season between October and June and a short rainy season between July and September. The eighth site (Port Sudan), located on the coast of the Red Sea, has a hot desert climate with high levels of humidity and a short rainy season during the cooler months of November to February . Apart from Barakat, all study sites have a consistent history of dengue fever, which results in more frequent outbreaks in Port Sudan, Tokar, and Kassala. Other major arboviral diseases in the study sites are yellow fever in Kadugli, Al Fashir, Nyala and Al Junaynah, and chikungunya, which was recently reported in all areas of the country. Aedes mosquitoes  were collected from their natural habitats indoors and outdoors in all the study sites throughout (January 2014–April 2017). Different breeding sites were surveyed. The samples were transferred to the insectary at the Sudan national public health laboratory for rearing at the optimum temperature (25 ± 2 °C) and relative humidity (80–90%) with a photoperiod of 12:12 (L:D) until the adults emerged. Samples from each collection were morphologically identified to their species using appropriate taxonomic keys [Aedes aegypti females and males were classified into their subspecies/forms, either Aaa or Aaf, according to the appropriate morphological key, depending on the scale pattern system [Aaa form, while adults that possessed white scales in the first abdominal tergite were classified as Aaf form. Female mosquitoes of Aaa and Aaf from different sites were preserved individually in labelled microfuge tubes with 70% isopropanol and then kept in a −20 °C freezer. Preserved samples were transferred to the Universiti Sains Malaysia (USM) where the DNA extraction was performed.Immature stages of mic keys . After tn system . The mosGenomic DNA was extracted from single female mosquitoes using Qiagen DNeasy Blood and Tissue Extraction Kit  following the manufacturer’s instructions with slight modifications in extending the incubation period to overnight at 65 °C. Extracted DNA was eluted in nuclease-free water and preserved in a −20 °C freezer until use. The DNA quantity and quality for all extracted samples were checked using Nanodrop Quawell UV spectrophotometer Q3000. Further confirmation of species identification was executed using internal transcribed spacer region 1 (ITS1) using forward primer ITS1A, 5 -CCT TTG TAC ACA CCG CCC GTC G-3, and reverse primer ITS1B, 5-ATG TGT CCT GCA GTT CAC A-3 described by  with mod2, 1 μL of 25 mM dNTP, 0.3 μM from each primer, 0.5 μL of Taq polymerase and 2 μL (>50 ng) from the sample DNA [Cytochrome oxidase 1 mitochondrial marker was used to detect polymorphisms among the different populations; a minimum of 10 mosquito samples from each site were tested . Partialmple DNA . The themple DNA . The cleaned PCR products were chosen, isolated, and sent to Apical sequencing services  for the Sanger DNA sequencing process. The sequencing services were done with the ABI 3130XL automatic sequencer . https://blast.ncbi.nlm.nih.gov/Blast.cgi). The nucleotides obtained were aligned by using the sequence alignment tool ClustalW in the MEGA. Sequences were translated to amino acids  to check for unexpected stop codons or frameshift mutations implemented in the same software. Sequences were generated and checked manually by using Molecular Evolutionary Genetics Analysis software MEGA v7.0  and compAedes aegypti were determined using DNAsp 5.10.1 [Unique haplotypes in the aligned matrix for both forms of p 5.10.1  and wereThe relationship between individuals of all population studies was illustrated using polar unrooted maximum-likelihood phylogenetic trees built using IQ-TREE 1.6.9  and shapπ)—were estimated within Aaa and Aaf populations using Arlequin v3.5 [Standard molecular diversity indices—the number of haplotypes per locality (h), the number of variable sites (S), haplotype diversity (Hd), and nucleotide diversity (uin v3.5 .ST) were estimated between the different populations and their significance.Genetic diversity and molecular variance of the populations assessed using Analyses of Molecular Variance (AMOVA) in Arlequin v3.5 . The paiST in the different haplotypes independently to obtain a linear regression relationship. The Mantel test was carried out to prove the significance of the association between genetic variations and geographical distance between study sites using Arlequin v3.5 [Geographical distances between the different study sites were approximated using a ruler tool in Google Earth TM . Genetic isolation by distance (IBD) was assessed using pairwise geographical distance and was matched to pairwise Fuin v3.5 .CT (diversity between groups) while the lowest FSC (diversity among populations within a group) were considered the most possible populations grouping. Since the FCT values in the study were found to have no significant difference, other methods were used following [CT (genetic differentiation among groups) was found to be relatively stable with very low differences; nevertheless, FSC  revealed a noticeable decrease from two to three clusters of groups); from this, we conclude that the best grouping (best homogeneity among groups) is three-group clustering.The genetic structure was estimated using Spatial Analyses of Molecular Variance (SAMOVA), SAMOVA v2.0 , to inteollowing . The FCTAfter that, SAMOVA structuring results were used to estimate the genetic molecular variance using Analyses of Molecular Variance (AMOVA) in Arlequin v3.5 .Tajima’s D and Fu’s F statistics were used to detect deviation from neutrality and population expansion using Arlequin v3.5 .


Phaseolus vulgaris L.) has resulted in the development of local populations/cultivars in restricted Italian rural areas. Many common bean landraces, still cultivated in small mountain areas from Sicily, have become outdated and endangered due to the commercial varieties spreading. These accessions are poorly known but often represent a genetic heritage to be preserved and enhanced. The ex situ conservation of fifty-seven Sicilian common bean landraces was carried out at the “Living Plants Germplasm Bank” at Ucria , founded by the Nebrodi Regional Park, together with the “Sicilian Plant Germplasm Repository” of University of Palermo (SPGR/PA). To assess the germplasm genetic diversity, nineteen morphological traits and eight Simple Sequence Repeats (SSRs) were used. Genetic distances among landraces were calculated to construct a clustering tree by using unweighted pair group method arithmetic (UPGMA). Seed germplasm diversity of Sicilian common bean varied from 80.7% to 93.3%, based on six seed descriptors and six leaf, flower, and pod descriptors, respectively, while cluster genetic analysis depicted a clear separation among all the 57 landraces. Principal coordinates (PCoA) and STRUCTURE analyses showed a prevalent rate of admixture between Mesoamerican and Andean gene pools in Sicilian common bean collection, confirming its heterogeneity. The observed high level of diversity evidenced the needs to adopt accurate criterion to plan a definitive ex situ germplasm collection to share agrobiodiversity with local farmers and to avoid any further loss of genetic resources in rural and protected areas.The historical cultivation of common bean ( Phaseolus vulgaris L.) is a major grain legume consumed worldwide as important source of proteins, minerals (mostly zinc and iron) and vitamins for human diets [Common bean  which together with Madonie Mountains form the Sicilian portion of the Apennine mountain chain characterized by rural small-scale traditional communities, typical of the Mediterranean basin. Established on 1993, the “Regional Park of Nebrodi” is engaged in wild biodiversity and agrobiodiversity preservation highly interconnected with local peasant agriculture. The Sicilian Plant Germplasm Repository at the University of Palermo (SPGR/PA), together with the “Living Plant Germplasm Bank” (LPGB) of Ucria (ME) [Hortus Botanicus Panormitanus seed bank from 1993 [The “Regional Park of Nebrodi” is one of the largest protect area in Sicily (860 kmria (ME)  currentlrom 1993 , collectrom 1993 .The aim of this study was firstly the assessment of genetic diversity of common bean germplasm collection at SPGR/PA, mainly representative of Nebrodi Mountains area. Further, the data analysis was used to define a core collection throughout morphological and genetic characterization.A total of eighteen morphologic traits, twelve qualitative and six quantitative, were used to evaluate 57 common bean landraces from the germplasm collections at SPGR/PA  collecteThe seed, pod, flower, and leaf descriptors are reported in Three landraces  showed a different color of strips each, while one (‘Nanu Carrazzu’) harbored different seed coat color background . To checSeed morphology on the whole collection  describeDescriptive analysis of quantitative traits among all landraces are reported in p < 0.001) for all variables (−2) not significant different by the mean value. The higher value for 100-seed weight was recorded by ‘Facigghuini’ (80.3 g) and the lowest by ‘Nanu Niuru’ (24.9 g), the latter was not significantly different from ‘Rampicante Nerella’ (25.1 g) and ’Carrabbaru Niuru’ (31.2 g). The post-hoc Tukey’s test on dry bean yield identified significant differences between landraces and the standard cultivar Borlotto. In particular, ’Ucchittu di Zappa’ (5.2 kg m−2) and ’Fasolu di Padre Bernardinu’ (4.56 kg m−2) showed a significantly higher yield than cv. Borlotto (3.79 kg m−2), while ’Nanu di Castania’ shows the lowest value of dry bean yield (0.43 kg m−2) despite a 100-seed weight (53.0g) close to mean value. Fourteen landraces showed a dry bean yield values not significantly different compared to cv. Borlotto (3.79 kg m−2) and 43% of landraces show a value above the mean (2.32 kg m−2)  and the results revealed a significant effect  .Principal component analysis (PCA) displayed two principal components that contributed for 58.4% to overall variability among the Sicilian common bean landraces . A heatmEight SSR markers , chosen among the most informative and useful for common bean genetic analyses, allowed us to record a total of 75 alleles and the number of alleles  per locus ranges from 5 (BM159) to 13 (PVBR25) as reported in −8, meaning that the probability of getting the same genotype in two accessions is almost null. The percentage of homozygosity (Hom) at each locus, as expected for an autogamous species, ranged from 56% (PVBR163) to 98% (BM210 and BM160).The lowest polymorphic information content (PIC) was 0.463 (PV-ag001) and the highest 0.853 (BM160) and the detected value of power discrimination (PD) at all the loci (0.8656) indicated a high SSR efficiency . BM160 wRare and private alleles within each accession were also evaluated . The gerSamples belonging to the Sicilian common bean germplasm collection were grouped in six main clusters by UPGMA analysis based on Bruvo’s distance . In clusPCoA and STRUCTURE analysis are in agreement, grouping the germplasm in four groups/pools . PCoA sep > 0.05) correlation.The Mantel test between genetic and morphological distance matrices showed a low negative (r: −0.019), albeit not significant  from 58 countries . MoreoveGermplasm diversity of Sicilian common bean collection increased from 80.7% to 93.3%, adding six leaf, flower, and pod traits to seed descriptors. The high level of morphological variability across the 57 accessions is comparable to that observed in others Italian collection ,54,55. IThe heatmap based on Euclidean distance is mainly defined by two morphological descriptors (seed coat color and seed coat color pattern) and the different geographic collection site. Landraces collected in Nebrodi Mountains were included in all five morphological clusters as well as in all six clusters obtained by UPGMA analysis.2) compared to those related to other Regional Italian collections . The efficiency of each SSRs in genetic fingerprinting was rather high for all the loci (mean 0.8656) and BM160 appeared the most discriminant locus. The PIC value of locus BM160 showed similar values to those detected in previous Italian germplasm studies [The genetic analysis of 57 Sicilian landraces showed a high degree of homozygosity, as expected by a self-pollinating species , but not studies ,49,58, b studies ,59. The  studies ,48,49. F studies ,47,48,49 studies ,60,61,62 studies .The presence of 36 rare alleles, 21 of which private, detected in about 58% of landraces underlined the high differentiation of our germplasm collection. High priority in SPGR/PA seed bank will be reserved to the landraces containing rare alleles, and even more private alleles, whose presence is an important feature for germplasm collection because proven to be informative for genetic conservation .PCoA and STRUCTURE analysis indicated a large rate of admixture 61%) confirming the differentiated origin of the Sicilian common bean collection here reported. The germplasm spread from Andean and Mesoamerican gene pools in Europe aided the gene flow between them, by increasing the genetic diversity found in thousand landraces grown in small farms . Our fin% confirmNot significant correlation (r = 0.01) was found between morphological and genetic distances by using the Mantel test. This could be due to the limited number of morphological traits and/or SSR loci analyzed.Analysis of variance (ANOVA) indicated the high yield variability of the Sicilian common bean landraces when cultivated under the same pedoclimatic condition. Among the collection, 25% of the landraces showed a comparable yield performance to the standard modern cultivar ’Borlotto’ widely cultivated in Sicily. Two landraces, ’Ucchittu di Zappa’ and ’Fasolu di Padre Bernardinu’, showed a higher level of dry bean production.The high genetic diversity detected in our Sicilian common bean germplasm highlight its potential economic importance for finding adaptive traits to stressful environments and low inputs condition frequently present in the marginal areas ,70,71, wMorphological and molecular techniques were able to detect high level of phenotypic and genetic diversity in the common bean collection from Sicily at SPGR/PA; these data set allowed us to detect some redundancies useful to define an ex situ core collection. Community-based conservation should be shared with local farmers, whose could directly benefit from this research to curb biodiversity loss and to maintain the genetic variation as evolution of the landrace itself in response to environmental changes in rural and protected areas. More interestingly, this study well reported the key role of the regional parks in conserving local agrobiodiversity for supporting a model to sustain the economy of local communities.Phaseolus vulgaris L.), consisting of fifty-one indeterminate climbing and six determinates, were collected from local growers in different geographical areas of Sicily  in 2014. In particular, fifty-four landraces were representative of Province of Messina including Regional Park of Nebrodi Mountains, two were collected from single farms located in Madonie Regional Park  and one from a farm of Province of Agrigento , which is useful for future reference.To renew plant material in the SPGR/PA and to increase seed availability for request of local farmers, each accession was sown and grown for following growing seasons at the Experimental Station of “Living Plants Germplasm Bank” of Ucria  every year. The germplasm collection is conserved at SPGR/PA, while the voucher specimens are housed at Harvested seeds from each accession were divided in two lots: One assigned for long term storage in SPGR/PA, after dehydration, at low humidity and low temperature storage at −20 °C , the othPhaseolus vulgaris L. descriptor list [Morphological traits assessment was carried out on five randomly plants for each landrace, according to International Board for Plant Genetic Resources (IBPGR) tor list  and to Btor list  , according to manufacture procedures. DNA quality and quantity were analyzed using a Thermo Scientific™ NanoDrop 2000c spectrophotometer.Eight SSR loci  were selQuantitative variables were analyzed using one-way ANOVA and mean comparison with the Tuckey-b test by using R package multcomp. Quantitative and qualitative variables were normalized during the analysis to balance the influence of each set of variables.https://cran.r-project.org/package=factoextra). A cluster analysis, based on Euclidean distances, for qualitative morpho-phenotypic traits, was performed on all the investigated accessions and UPGMA (unweighted pair group method with arithmetic mean) tree was developed using the function heatmap.2 in R package gplots .Principal component analysis (PCA) was performed to define the most determinant quantitative variables able to discriminate among accessions by using the R packages FactoMiner  and factGenetic diversity per locus was evaluated through several parameters, such as number of alleles per locus (Na), number of rare and private alleles , observeGenetic relationships among samples were investigated through UPGMA cluster analysis. Phylogenetic tree was developed by using R package poppr , performTo study the relationships between genetic and Euclidean distance of samples, the Mantel test  was also


We utilized 4,267 25- to 35-year-old European larch trees growing in 21 reforestation installations across four distinct climatic regions in Austria. With the aid of marker-based pedigree reconstruction, we applied multi-trait, multi-site quantitative genetic analyses that enabled the identification of broadly adapted and productive individuals. Height and wood density, proxies to fitness and productivity, yielded in situ heritability estimates of 0.23 ± 0.07 and 0.30 ± 0.07, values similar to those from traditional “structured” pedigrees methods. In addition, individual trees selected with this approach are expected to yield genetic response of 1.1 and 0.7 standard deviations for fitness and productivity attributes, respectively, and be broadly adapted to a range of climatic conditions. Genetic evaluation across broad climatic gradients permitted the delineation of suitable reforestation areas under current and future climates. This simple and resource-efficient management of gene resources is applicable to most tree species.Sustainable and efficient forestry in a rapidly changing climate is a daunting task. The sessile nature of trees makes adaptation to climate change challenging; thereby, ecological services and economic potential are under risk. Current long-term and costly gene resources management practices have been primarily directed at a few economically important species and are confined to defined ecological boundaries. Here, we present a novel  The composition, function, and service of terrestrial ecosystems are increasingly threatened by the steady global warming trend . Plants'Forest tree gene resource management, with concurrent selective breeding and gene conservation, are long-term endeavors involving hundreds of parents and thousands of offspring tested at multiple locations, requiring substantial resources, elaborate logistics, and sustained organizational commitment, and more importantly, are predominantly encapsulated within specific ecological boundaries known as breeding zones . These eL. decidua has the finest wood characteristics among temperate European conifers, it has been widely planted throughout the continent in artificial plantations, thus facilitating translocations of genetic materials for more than three centuries  through the hilly landscape of the alpine foreland up to mountains of 900 m in the northern calcareous Alps. Present climate conditions are represented by four climatic zones: 1) pannonical continental climate with hot summers and frequent droughts in low elevations of the East and Northeast, 2) temperate Atlantic climate with warm temperatures and frequent precipitations in the western Alpine foreland, 3) temperate climate with continental influence at low elevations of the Eastern Alps, and 4) harsh mountain conditions at higher elevation with lower temperatures and low winter temperatures. Global warming in the Alpine region has already resulted in a significant 2)∘C temperature increase since 1880, about twice as high as the global average .Utilizing the European larch breeding program in Austria, we investigated a feasible alternative that would efficiently address the global climate change issues and overcome the major limitations of assisted gene flow, and deliver substantial production and social benefits to the human society. We analyzed 25- to 35-year-old larch progenies originating from open pollination in a common parental source (a seed orchard) and growing in 21 reforestation installations . Utilizing the “Breeding-without-Breeding” methodology with phenotypic preselection , we recoThe seed orchard [Nat. Reg. No. Lä P3 (4.2/sm-tm)] for which we aimed to conduct accelerated gene-resource management is located at an altitude of 520 m a.s.l., and its seed material is considered to be the most valuable larch seeds for the mountainous areas of the northern alpine foreland. The orchard was established in 1954 over 3.15 ha, with 1,666 vegetative propagules of 53 phenotypically selected parent trees. Since its establishment, the main objective of the Austrian larch seed orchard program was to secure seed supply with minimal genetic testing; thus, controlled pollinations and progeny tests were not conducted.Next, we identified 21 reforestation installations (sites) within comparable tree ages (25 to 37 years), sufficient size (at least 200 remaining trees in more or less regular planting designs), low level of environmental variation within the site, and composition in which larch is the single or dominant tree species. These 21 reforestation installations all originated from mixed seedlots harvested from the seed orchard, are located at altitudes between 250 and 800 m, and span through a geographical space of about 170 km W-E and 110 km N-S.Climate data for the 21 reforestation installations with putative seed deployment areas under the present and future climate conditions were obtained from the locally downscaled high-resolution WorldClim models ; WorldClPhenotyping was conducted on the 21 reforestation sites after excluding individuals with damage and/or bad form. In total, 4,267 trees were measured for height [m] and scored for wood density using the pilodyn penetration method . IndividTissue samples for DNA extraction were collected using a 15-mm hole-punch to obtain cambium cell layers. Samples were immediately dried and stored in silica gel. DNA extraction followed a modified CTAB protocol , using aWe performed pedigree reconstruction, yielding 1,024 offspring in 491 full-sib families, representing the largest known forest tree pedigree assembly. The likelihood-based method Cervus  was usedPedigree-based genetic analyses were used and variance components, heritabilities, genetic correlations, and individual tree breeding values were estimated/predicted using the bivariate animal model , combiniy is the vector of observations for the two traits; X is the incidence matrix for the fixed effect β (trait means); Z is the corresponding incidence matrix related to random additive genetic effects  interaction where The covariance matrix for the random additive genetic effects was modelled using the heterogeneous covariance structure asA is the average numerator relationship matrix, aσ1a2 is the additive covariance between traits 1 and 2, and ⊗ is the Kronecker product operator. A corresponding structure was used for the dominance effects with A by D, i.e., the dominance genetic relationship matrix. The covariance matrix for the random site effects (genotype by environment interaction) was modelled using a heterogeneous general correlation matrix  suitable for such a complex correlation structure aswhere I is the identity matrix.where The random residual error effect was modelled using an unstructured covariance matrix structure aseσ1e2 is the residual covariance between the two traits. Random effects were assumed to be independent. The above genetic evaluation was conducted in ASReml software  estimates of 0.25 (SE = 0.065) and 0.30 (SE = 0.072) for height and wood density, respectively, corresponding to reported estimates from Larix species “structured” testing trials  interaction was observed across the 21 studied sites, leading us to conclude that individuals' additive genetic values are indicative of their general performance across the range of studied sites and that the studied population consists of generalists that performed well under wide site and climatic conditions and further demonstrating that the selected individuals would form appropriate seed production population for planting over a wide climate regime range including potential future conditions Figure 1Following the genetic evaluation, we selected 25 unrelated offspring individuals with which to establish the new seed production population with improved climate change adaptability and productivity; thus, the phenotyping and genotyping effort were sufficient in capturing the target effective population size. Selected individuals yielded genetic responses of 1.1 and 0.7 standard deviations for fitness and productivity attributes, respectively. Scions collected from selected offspring were grafted onto rootstocks, and the second-generation seed orchard was established (the advanced generation seed production population).in situ forest gene resource management scheme to identify productive and climatically adapted individuals originating from an Austrian European larch seed production population (seed orchard), utilizing traditional reforestation installations planted widely across the landscape and spanning three decades. Our thesis is based on the expectation that thriving individuals within these installations have been spatially and temporary challenged and thus have effectively dealt with the negative impacts of climate change. In conventional selective breeding programs, a structured pedigree is produced from controlled pollinations following specific mating designs  and extended the testing timeframe, thus speeding the selection of adapted individuals and matching them with the most appropriate planting location. The approach is simple and cost-efficient, enabling improvement and conservation of commercial and non-commercial species under rapidly changing environmental conditions.https://github.com/mlstiburek/European-larch.The datasets analyzed for this study can be found in ML, SS, YE-K, and TG conceived the project and designed the study. ML estimated sample sizes. SS, HK, and TG carried out the genotyping. PŠ coordinated the fieldwork. JK conducted the pedigree reconstruction. GH, JS, and ML ran statistical analyses. ML, SS, TG, YE-K, JK, and JS contributed to writing the manuscript.This research was funded by OP RDE grant Extemit-K, No. CZ.02.1.01/0.0/0.0/15003/0000433 (ML), the Austrian Research Promotion Agency (FFG) and the Cooperation Platform Forst Holz Papier (FHP) and LIECO nurseries and the Austrian Federal Forests (ÖBf) , the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant and the Johnson's Family Forest Biotechnology Endowment (YE-K), Camcore, Department of Forestry and Environmental Resources, NC State University (GH).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.


Lanping black-boned sheep was first discovered in the 1950s in Lanping county of China and characterized by black pigmentation on skin and internal organs. Due to the novel and unique trait, the genetic background of Lanping black-boned sheep is of great interest. Here, we genotyped genome-wide SNPs (single nucleotide polymorphisms) of Lanping black-boned sheep and Lanping normal sheep using Illumina OvineSNP50 BeadChip to investigate the genetic diversity and genetic origin of Lanping black-boned sheep. We also downloaded a subset SNP dataset of two Tibet-lineage sheep breeds and four other sheep breeds from the International Sheep Genomics Consortium (ISGC) as a reference for interpreting. Lanping black-boned sheep had a lower genetic diversity level when compared to seven other sheep breeds. Principal component analysis (PCA) showed that Lanping black-boned sheep and Lanping normal sheep were clustered into the Asian group, but there was no clear separation between the two breeds. Structure analysis demonstrated a high ancestry coefficient in Lanping black-boned sheep and Lanping normal sheep. However, the two populations were separated into two distinct branches in a neighbor-joining (NJ) tree. We further evaluated the genetic divergence using population  Attributable to their tender and tasty meat quality and no special smell of mutton, LPBB stands out among Lanping local sheep breeds and has been designated as a novel genetic resource by the Chinese Ministry of Agriculture .LPBB has the same morphology with LPN such as coat color and horns and tail shape;there are slight phenotypic differences between the two breeds seen by carefulvisible inspection: LPBB has dark teeth and gums and visible mucosa. However, there are great differences between the two breeds: the skin,muscle, and inner organs – including kidney, heart, lung, and trachea of LPBB –were dark colored compared to the red coloration in LPN . The husbandry system of LPBB is similar to semi-feral sheep breeds of Tibet sheep, which graze freely with more natural selection and less human intervention such as selective breeding and nutrition complements . Due to the phenotypic similarity with LPN and less human intervention, it is supposed by scientists that the formation of LPBB was the result of advantageous genetic mutations from LPN in the process of adaptation to the local harsh environments, which include high altitude (approximately 3000 m), strong radiation, steep terrain, and low temperatures during winter . Meanwhile, some scholars believe that gene flow is present in Lanping local sheep and Tibet sheep because Lanping is located on the Tea Horse Road leading to Tibet in Yunnan and is adjacent to Diqing Tibetan Autonomous Prefecture . Furthermore, previous studies reported that Yunnan-Kweichow Plateau sheep has closer relation with Qinghai-Tibetan Plateau sheep compared to northern Chinese sheep . Thus, investigating the genetic relation of LPBB with these sheep breeds may provide informative knowledge on genetic origin of LPBB.Genetic diversity is an important population genetics parameter that helpsto explain the process of evolution  and is a major concern,considering the necessity of conservation in local breeds . The ability of a population to respond adaptively to environmentalchanges depends on its level of genetic diversity, and a species withoutenough genetic diversity is thought to be unable to cope with changingenvironments or evolving competitors and parasites . Lanping black-boned sheep was found initially with about 200 individuals and the population number increased slowly to 2000 from 2001 to 2005 . Due to the small population and isolatedhabitat, LPBB was expected to have low genetic diversity and increasedincidence of inbreeding, which was not good for this unique and rare sheepbreed. Thus, investigating genetic diversity of LPBB will provide a guide forthe strategies on this genetic resource conservation.TYR , MC1R , TYRP1, TYRP2 , and EDN3 . However, the genetic background of LPBB has not yet been reported. In this study, we aimed to investigate the genetic diversity and genetic origin of Lanping black-boned sheep by genotyping LPBB and LPN using Illumina OvineSNP50 BeadChip. We also downloaded a subset of publicly available SNP (single-nucleotide polymorphism) BeadChip data  from the Ovine HapMap project of International Sheep Genomics Consortium (ISGC)  to use this established resource as a reference for interpreting genetic diversity and phylogenetic relationship within Lanping local breeds and for developing assumption about their relationship with worldwide breeds . The results obtained in this study will be informative for future research and conservation of this unique breed.To date, genes related to the mechanism of black pigmentation traits on LPBBhave been studied such as 22.1We collected 101 LPBB  and 106  LPN sheep blood samples from Lanping County, Yunnan Province, China. Each individual was more than 1 year old and also carefully confirmed to match the breed standard. We then selected 15 LPBB  and 15 LPN  individuals randomly from the above collected blood samples to genotype; one LPN blood sample was lost for some reason. Genomic DNA was extracted from 500 All the experimental procedures were conducted under the InternationalAnimal Care and Use Committee of the Yunnan Agricultural University. Thecare and use of animals fully complied with local animal welfare laws,guidelines, and policies.2.2http://www.sheephapmap.org (last access: 24 June 2020). Then, we merged and generated a 182-individual dataset (Table 1) with 49 034 overlapping SNPs for subsequent analysis. SNPs that cannot pass any of the following criteria were excluded from the analysis using PLINK 1.9 : (1) with minor allele frequency (MAF) In total, 29 genomic DNA samples were genotyped using Illumina OvineSNP50 BeadChip. The second dataset used in this study, including two Tibetan-lineage breeds (Tibetan and Changthangi sheep), another two Asian breeds (2.3Pn) gives the fraction of total SNP that displayed both alleles within each population . Pn was calculated as the proportion of SNPs with average minor allele frequency (MAF) greater than 1 % within each breed . MAF is the frequency estimate of the least common allele per breed and estimated using the “freq” flag. Expected heterozygosity (He) and observed heterozygosity (Ho) were estimated using the “hardy” flag, and inbreeding coefficient . Theproportion of polymorphic SNP  , was used to investigate the genetic structure. Principal component analysis (PCA) was performed with GCTA software  . Population structure was evaluated using Structure software (version 2.3.4) . The neighbor-joining (NJ) tree was constructed using MEGA software (version 7.0) on the basis of all SNPs . The pairwise 33.1Pnpresent in LPBB population was 0.8390, which was slightly lower than LPNpopulation but greatly lower than the other six populations; this reflectedthat LPBB population had lower loci displaying polymorphism compared to theremaining populations (Table 1). The distribution of MAF per breed is givenin Fig. 1 and shows that the LPBB population has the lowest level of highlyvariable SNPs , followed by LPN population (0.2870); compared to pure breeds, the cross breed of CME had the highest level of He (0.3551). The inbreeding coefficients in all populations were detected with weak to moderate levels, ranging from 0.0267 to 0.2835 and the level in the LPBB population was moderate (0.1847).Inbred individuals were not observed in the 182 studied sheep according tothe IBS score  based on the prunedgenotype data of 36 711 SNPs among all individuals to examine the geneticrelationship between LPBB and its geographic neighbors and worldwide sheepbreeds. The largest PC  separated eight breedsinto three groups consistent with their genetic origin which were Asianbreeds  and five individuals of CHA were outside of their clusters. The phylogenetic tree shows that LPBB and LPN breeds are separated into two clear clusters and positioned in one clade with TIB and CHA breed. Furthermore, LPBB and LPN breeds had the shortest branch length compared to the TIB breed, which revealed that LPBB and LPN had a relatively close relationship with TIB. The fact that they had common ancestry or gene flow between them was confirmed by the structure analysis.Due to the morphology and genetic similarity of LPBB and LPN breeds, it wasquestionable that LPBB and LPN were distinct enough to be considered adifferent breed. To investigate their genetic difference, we usedpopulations To better understand the population variation, we performed linkagedisequilibrium (LD) decay analysis, which can be informative for populationdemography and assessing the number of markers required to associate geneticvariation with traits. Estimates of LD based on the 4Pn, He, In this study, we evaluated for the first time the genetic diversity andpopulation structure of LPBB with seven other populations. The mean values ofthe three metrics (He) of 0.2861 and polymorphic SNPs (Pn) of 0.8390 (Table 1) when compared to other breeds. The lower level of genetic diversity of LPBB population was also detected in the distribution of MAF and confirmed by the lower rate of LD decay in LPBB. CME, a crossbreed of Australian Merino He of 0.3551 and Pn of 0.9740; this was consistent with the report that the crossbreed has a higher genetic diversity level compared to the pure breed . Furthermore, Australian Merinos were reported as the most diverse sheep population . The low genetic diversity in the LPBB population can be explained by their geographically separated habitat and small population size . Lanping black-boned sheep have distributes in three towns of Yuping Mountain of Lanping. There was dozens of Lanping black-boned sheep individuals when first discovered in the 1950s, and until 2000 their number increased slowly to several hundred. In addition, due to the high elevation and steep road of Yuping Mountain, there is a geographically isolated area, and no sheep genes were introduced. The higher LD level and slow decay of LPBB population also demonstrated that the population size was relatively small. Genetic diversity is correlated with fitness : populations with low level of genetic diversity are expected to be less able to adapt to environmental changes  or evolving competitors and parasites ; thus, it is suggested to conserve this unique breed.Analysis of genetic diversity revealed that the LPBB population had the lowestlevel of diversity with an estimated genetic diversity  and carpet wool . To date, there are more than 42 native sheep breeds established in China, and they are classified into three major lineages by their geographic distribution known as northern China, Qinghai-Tibetan Plateau, and Yunnan-Kweichow Plateau sheep ; Yunnan-Kweichow Plateau sheep had closer affinitywith Qinghai-Tibet sheep compared with northern China sheep and could beexplained by their thin-tail origin . Lanping is located onthe Tea Horse Road leading to Tibet in Yunnan and is adjacent to DiqingTibetan Autonomous Prefecture. This study provided the genetic evidence thatLPBB sheep had close affinity with Tibet sheep. For a more accurate understanding of the origin of LPBB sheep breeds, the relation with otherYunnan-Kweichow Plateau breeds should be studied.5In this study, we estimated for the first time the genetic diversity and genetic origin of Lanping black-boned sheep by using genome-wide SNP data. We observed that the LPBB population had the lowest genetic diversity compared to its geographical neighbors and four other world breeds. This finding improved our understanding of the genetic diversity in LPBB sheep breed and suggested that strategies should be implemented to maintain or increase genetic diversity in this breed. The PCA and structure analysis revealed that LPBB and LPN populations had great genetic similarity, whilst the NJ tree and genetic differentiation showed that there are differences between LPBB and LPN populations, and LPBB can be considered a specific separate breed. In addition, this study demonstrated that LPBB and LPN populations had common ancestry with the TIB population, which provided insight into the genetic origin of LPBB sheep and will be useful for the novel breed formation research.


Olive is considered one of the oldest and the most important cultivated fruit trees in Albania. In the present study, the genetic diversity and structure of Albanian olive germplasm is represented by a set of 194 olive genotypes collected in-situ in their natural ecosystems and in the ex-situ collection. The study was conducted using 26 microsatellite markers (14 genomic SSR and 12 Expressed Sequence Tag microsatellites). The identity analysis revealed 183 unique genotypes. Genetic distance-based and model-based Bayesian analyses were used to investigate the genetic diversity, relatedness, and the partitioning of the genetic variability among the Albanian olive germplasm. The genetic distance-based analysis grouped olives into 12 clusters, with an average similarity of 50.9%. Albanian native olives clustered in one main group separated from introduced foreign cultivars, which was also supported by Principal Coordinate Analysis (PCoA) and model-based methods. A core collection of 57 genotypes representing all allelic richness found in Albanian germplasm was developed for the first time. Herein, we report the first extended genetic characterization and structure of olive germplasm in Albania. The findings suggest that Albanian olive germplasm is a unique gene pool and provides an interesting genetic basis for breeding programs. Olea europaea L.) is one of the most important and oldest cultivated plants in the Mediterranean Basin and it is an emblematic tree crop, even in Albania. Olive has been grown in Albania since ancient times due to the favorable climatic and ecological conditions. The cultivation of olive in Albania is thought to have begun approximately at the same time as in neighboring regions. The presence of olives in Albania has been historically demonstrated by records of olive oil trade by 300–150 BC, finds at archeological sites of olive mills dating back to the 4th Century BC [Olive  were collected in situ and in the ex-situ collection in Albania, encompassing the representatives of local olive cultivars (130), cultivars of foreign origin (45) that have been introduced , and oleasters (19), which are used mainly as rootstocks and are pollen donors for many cultivated olive cultivars. A group of 74 olive trees represent very old plants, with an estimated age of 500–3000 years, maintained in situ in their natural ecosystem at olive growing regions throughout Albania. The ancient olive genotypes were previously morphologically characterized and selected to construct an Albanian in situ olive germplasm collection under the auspices of the Gene Bank of Albania.A set of 194 samples of olive accessions  method . DNA conThe SSR analysis was performed following the approach of Schuelke , by appenull) were calculated with Cervus 3.0 software [For each locus, particular attention was paid to genotyping errors, such as potential allele dropout, which can lead to a decrease in sample heterozygosity and to stuttering patterns, which can hide the true allele peak. The resulting low-frequency alleles occurring ≤ 5 times were double checked on the original pherogram and any genotyping errors were corrected accordingly. The number of alleles per locus (No), the observed and expected heterozygosity (Ho and He), Hardy-Weinberg equilibrium (HW), polymorphic information content (PIC), and frequency of null alleles  was carried out to determine the relative partitioning of the total genetic variation among and within different groups of olive genotypes by using GenAlEx 6.5 ,39. The 6 MCMC (Monte Carlo Markov Chain) replicates, assuming an admixture model and correlated allele frequencies.The model-based Bayesian method in STRUCTURE version 2.3.4 software  was applFinally, we assembled a core collection from the large database of individual molecular profiles of our sample set, using the M strategy ,46, applThe genetic variation among 183 unique olive genotypes identified by identity analysis was estimated using 26 SSR markers. The list of unique genotypes is presented in the The unique Albanian genotypes (183) were compared with 80 olive genotypes from 11 olive growing countries (deposited in the World Olive Database) at ten microsatellite loci and with 19 olive cultivars from Slovenian collection at 26 loci. The comparative analysis did not find cases of synonymy among compared microsatellite profiles.The parameters of diversity calculated for 183 unique olive genotypes  are presented in the The clustering analysis highlighted 12 groups of olive genotypes with mean similarity of 0.509. Each of the main groups subdivides into several small clusters consisting of related genotypes. In many cases, the high level of genetic similarity between genotypes of the same subcluster (>85%) was observed. The genetic relationships among 183 olive genotypes were visualized by a dendrogram, shown in The majority of genotypes used as table olives and dual use were dispersed in clusters I, II, and IV (subcluster II). Most genotypes used for oil production were dispersed in clusters IV (subcluster I), V, VI, and XII. Only native genotypes, especially those from the central region, showed fairly good clustering according to their fruit end-use. Only five oleasters out of 18 in cluster VI were subclustered distinctly. Most of the oleasters (13 out of 18) clustered together with Albanian cultivated forms, especially with foreign cultivars, in the different groups , but fivPrincipal coordinate analysis (PCoA), available in GenAlEx v 6.501 software ,39, was The PCoA explained a total variation of 23.21% . The firp < 0.001, indicating low genetic differentiation among groups. The p-values were calculated for a random permutation test of 9999 permutations.PCoA in pairwise comparison of olive groups  revealedBased on a pairwise population matrix of Nei’s genetic distances , the higp < 0.05). Nei’s genetic distance between the group of genotypes of the Ionic region and that of the Adriatic region was 9.4%.Analysis of principal coordinates based on assignment of two geographical groups according to their main area of distribution/origin (Ionic vs. Adriatic region) explained a total of 26.30% of genetic variation. The first two principal coordinates accounted for 10.57 and 9.03 of variation, respectively . The slip < 0.05). The admixture observed in the principal coordinate analysis suggests that the abundant diversity existing in olives cannot be differentiated based on the end-use of the product.The links among these three groups of olive genotypes based on their product end-use was evaluated by PCoA . The oliPCoA indicated that, despite slight overlapping, the olive genotypes showed grouping according to their origin and their area of distribution. The differentiation of genotypes according to their fruit end-use obtained in UPGMA analysis was not supported in the PCoA analysis.A model-based Bayesian approach was performed to infer the genetic structure of individuals under K = 2 clusters, which, as estimated by Evanno’s ΔKs statistics , resulteHierarchical levels of population structure could hardly be recognized. Both groups, A and B, are of mixed usage origin  and no well-assigned population designations could therefore be recognized. We observed only weak differentiation, as in group A (red color), containing 81 olive genotypes, there is 9 oleasters, the majority of foreign imported olive cultivars (39), and some native cultivars (33), while group B (green color) is composed of 53 native cultivars and lacks the oleaster genotypes and foreign imported cultivars. This kind of differentiation was also observed in PCoA and AMOVA, where only 7% of variation was observed among native cultivars, oleasters, and foreign cultivars, indicating a mixed genetic structure of the olive genotypes.The overall assignment of the samples to each of two clusters were 58.9% and 41.1% for cluster A and B, respectively. Average distances (expected heterozygosity) among individuals within a cluster were 0.708 and 0.559 for clusters A and B, respectively. The low-differentiation structure (K = 2) detected within our analyzed set of samples may be due to complex relationships among the olive cultivars.The dataset of microsatellite profiles of 183 unique olive cultivars for 26 microsatellite loci was used to construct an olive core collection, using the M strategy ,46, implThe entire allelic richness of the actual ex situ core collection (117 accessions) may be represented by a minimum number of 45 accessions (38%), while the diversity of the in situ collection (66 accessions) could be represented by 32 accessions (48%).However, for better management of the two olive collections in Albania and the preservation of the germplasm in a thorough and efficient way, the collections were unified to sort out a core collection of all Albanian olive germplasm. CoreFinder analysis showed that 203 alleles identified by characterization of 183 unique genotypes with 26 microsatellite loci could be represented by a core collection of 57 accessions (31%) of all genotypes .Herein, this study examined genetic diversity and structure of Albanian olive germplasm, consisting of native and introduced foreign cultivated olives and of oleasters. In total, 26 microsatellites including 12 EST-SSRs were employed in this study. The analyses revealed in total 203 alleles, with an average 7.8 alleles per locus, revealing a high level of variability within a sample set. The obtained average number of alleles per locus is in the same range as that reported for: 489 Italian olive varieties (7.6) , 84 TuniThe observed heterozygosity resulted higher than the expected one at the majority of loci (19), while we only observed lower observed heterozygosity on 7 loci compared to the expected heterozygosity. This observed heterozygosity deficiency may be related to the presence of null alleles, whose frequency values were positive at six of these loci . HoweverThe PIC value indicates the level of polymorphism information provided, as well as the usefulness of the microsatellite primers for genotyping, gene mapping, molecular breeding, and germplasm evaluation. In this sense, the most suitable loci for genetic characterization of the analyzed set of olive genotypes were DCA03, DCA09, DCA18, SNB03, and SNB11, which showed PIC values equal to or higher than 80% .The comparative analysis of 183 unique genotypes with the allelic profiles of 99 olive genotypes belonging to 12 olive growing countries did not find cases of synonymy, supporting the hypothesis of an autochthonous origin of Albanian cultivars ,8,66 andO. europaea subsp. europaea: the cultivated form var. sativa and wild form var. sylvestris, also called ‘oleaster’. Both varieties are diploid species (2n = 46) [Botanists have reported two varieties of 2n = 46) ,68. True2n = 46) . Presume2n = 46) . Several2n = 46) ,12,13 by2n = 46) , but low2n = 46) , the mea2n = 46) ,13,63,71Based on the results of genetic relatedness, our sample set showed an average similarity of 0.509, which was higher than the average similarity found in olive cultivars from the Slovenian olive collection (0.26)  and withThe area of origin can be identified because of the climatic and pedological conditions under which populations experienced a secondary structuration of variability. Distinguishing among varieties belonging to different gene pools would enable selection of the most adapted cultivars for the new breeding policies for each geographically defined area . The oliWeak clustering in relation to the fruit end-use was observed in the overall sample set, in line with the findings of Abdelhamid et al. . Only naThe model-based method, PCoA, and analysis of molecular variance showed a wide range of variability within our olive groups. The geographical grouping according to olive origin and area of distribution was confirmed, though not in a clear-cut way. There was weak differentiation of olive genotypes based on fruit end-use. No hierarchical differentiation based on olive end-use products was revealed by PCoA, either . This leThe clear separation of native cultivars from foreign cultivars and the presence of oleasters suggest that the most native olive cultivars originated from native oleasters. However, some of the present Albanian olive cultivars were admixed with foreign ones, suggesting that they might be derived from the crossing of native and introduced foreign cultivars A. The diThe exchange of olive plant material that may have occurred between Italy and Albania is also supported by the high similarity observed between some native olive cultivars or oleasters and Italian cultivars grown in Albania, as well as with the cultivars ‘Frantoio’, ‘Leccino’, ‘Carolea’, ‘Ascolana Tenera’, and those from the olive database. The Italian cultivars showed similarity with some native genotypes collected in situ at the central and northern parts of Albania, suggesting that the trade developed in ancient times between these two countries through the port city of Durrësi in Albania, or the well-known trading activities of northern Albania with Venice, indicate how the material exchange took place. Further studies employing a larger number of oleasters, including those of neighboring countries, will elucidate the origin of olive origin in Albania as well as the direction of olive exchange among countries.A model-based Bayesian approach used to infer the genetic structure of samples resulted in K = 2 clusters as the best fit to the model for olive genetic structuring. The set of genotypes that were assigned to different gene pools showed no clear differentiation based on olive end-use, and while based on their origin, a weak differentiation of Albanian native cultivars was observed, this weak differentiation was also observed in PCoA and AMOVA, where only 7% of variation was observed among native cultivars, oleasters, and foreign cultivars, indicating a mixed genetic structure of the olive genotypes. The low-differentiation structure (K = 2) detected within our analyzed set of samples may be due to complex relationships among the olive cultivars or due to the inclusion of oleasters in the cluster. A weak differentiation of cultivars (51) and wild olives (107) was also reported by Belaj et al. , where wOur population was not structurally based on the end-use of the fruit (oil or table), in contrast to Do Val et al. , who repThe conservation of cultivated plants by the establishment of a core collection is essential for the optimal management and use of their genetic resources . Two worThis study is the most representative analysis of olive germplasm in Albania to date, reporting the existing levels of genetic diversity and structure at a broad national scale. In addition, the present study is the first report on the molecular characterization of Albanian germplasm and an assessment of genetic relationships of oleasters, native cultivars, and imported foreign cultivars. The geographical grouping according to olive origin and area of distribution was confirmed, although not in a clear-cut way. There was weak differentiation of olive genotypes based on their fruit end-use. The analysis of genetic relatedness between native and foreign olive genotypes gives an insight into the origin of Albanian olive cultivars. The native olive genotypes showed tight clustering, suggesting their autochthonous origin. The molecular data provides for the first time a broad inventor of olive genetic resources, as essential information to construct the reference molecular database of Albanian olive germplasm. Herein, we assembled the core collection to represent the entire diversity of the analyzed set of olive genotypes as a valuable source for the development of an improved breeding strategy and for safeguarding the wealth of genetic diversity. The findings will provide a useful resource as well as guidance for better germplasm utilization in genetic improvement and serve as a database for identification and traceability purposes.


Cryptosporidium parvum is a zoonotic pathogen worldwide. Extensive genetic diversity and complex population structures exist in C. parvum in different geographical regions and hosts. Unlike the IIa subtype family, which is responsible for most zoonotic C. parvum infections in industrialized countries, IId is identified as the dominant subtype family in farm animals, rodents and humans in China. Thus far, the population genetic characteristics of IId subtypes in calves in China are not clear.C. parvum isolates from dairy and beef cattle in six provinces and regions in China were characterized using sequence analysis of eight genetic loci, including msc6-7, rpgr, msc6-5, dz-hrgp, chom3t, hsp70, mucin1 and gp60. They belonged to three IId subtypes in the gp60 gene, including IIdA20G1 (n = 17), IIdA19G1 (n = 24) and IIdA15G1 (n = 5). The data generated were analyzed for population genetic structures of C. parvum using DnaSP and LIAN and subpopulation structures using STRUCTURE, RAxML, Arlequin, GENALEX and Network.In the present study, 46 C. parvum IId population. When isolates of various geographical areas were treated as individual subpopulations, maximum likelihood inference of phylogeny, pairwise genetic distance analysis, substructure analysis, principal components analysis and network analysis all provided evidence for geographical segregation of subpopulations in Heilongjiang, Hebei and Xinjiang. In contrast, isolates from Guangdong, Shanghai and Jiangsu were genetically similar to each other.Seventeen multilocus genotypes were identified. The results of linkage disequilibrium analysis indicated the presence of an epidemic genetic structure in the C. parvum than gp60 sequence analysis. Despite an epidemic population structure, there is an apparent geographical segregation in C. parvum subpopulations within China. Data from the multilocus analysis have revealed a much higher genetic diversity of  Cryptosporidium spp. are apicomplexan pathogens that can cause debilitating gastrointestinal illness in animals and humans with the main clinical symptom as diarrhea [Cryptosporidium. Among the nearly 40 Cryptosporidium species identified, C. parvum is the most important species causing zoonotic cryptosporidiosis [gp60) locus [diarrhea . There iridiosis . It has 0) locus . Among t0) locus , 4.C. parvum, with pre-weaned calves being considered the most important reservoir for zoonotic C. parvum infection [C. parvum have been observed among geographical regions [C. parvum in bovine studies identified an exclusive occurrence of IId subtypes in calves in China, mostly IIdA15G1 and IIdA19G1 [C. parvum IIa subtypes, especially IIaA15G2R1 [Cattle are among the most common hosts of nfection . Differe regions . SubtypiIIdA19G1 . MoreoveIIdA19G1 , 9. In caA15G2R1 , 7.C. parvum in disease endemic areas and compensate for the relatively low resolution of the single gp60 locus because of the likely occurrence of genetic recombination among loci and the existence of genetic determinants of other phenotypic traits [C. parvum, leading to the discovery of high genetic diversity, significant geographical segregation and complex population structure [C. parvum have been identified, including panmictic (unrestricted gene flow and linkage equilibrium among loci), clonal (largely restricted gene flow and linkage disequilibrium among loci), and epidemic  [Population genetic studies based on highly polymorphic loci can shed light on the true genetic diversity of c traits , 10. Multructure , 12. ThuC. parvum had focused on the IIa subtype family. A mostly panmictic population structure for C. parvum IIa subtype family has been found in humans and calves in many industrialized nations [C. parvum IId subtype family in China, Egypt and Sweden, which mostly has a clonal population structure.Most previous studies of the population genetics of  nations –21. This nations . Several nations , 22. ThiC. parvum in cattle in China using multilocus sequence typing (MLST) of isolates.The aim of this study was to explore the population genetic characteristics of IId subtypes of C. parvum IId subtypes including IIdA20G1 (n = 17), IIdA19G1 (n = 24), IIdA15G1 (n = 5) from beef and dairy cattle in Xinjiang, Heilongjiang, Hebei, Shanghai, Jiangsu and Guangzhou, China, were selected for the population genetics analysis. They were from previous and ongoing studies of molecular epidemiology of cryptosporidiosis in cattle [gp60 subtype designations are shown in Table C. parvum subtypes examined in the study are the most common subtypes in China, responsible for over 90% C. parvum infections in cattle. They were diagnosed by DNA sequence analysis of the gp60 gene [Forty-six isolates of n cattle , 23, 24.gp60 with simple tandem repeats were used in the characterization of C. parvum isolates in the present study. In addition to gp60, they included msc6-7 (serine repeat antigen), rpgr (retinitis pigmentosa GTPase regulator), msc6-5 , dz-hrgp (hydroxyproline-rich glycoprotein), chom3t (T-rich gene fragment), hsp70 (70 kDa heat shock protein), mucin1 (mucin-like protein). Nested PCR was used in the analysis of these genetic loci as previously described [C. parvum IOWA isolate (IIaA15G2R1 subtype) was used as a positive control. Positive PCR products were sequenced on an ABI 3730 Genetic Analyzer . The sequences generated were assembled using ChromasPro v.2.1.8 (http://technelysium.com.au/ChromasPro.html) and aligned with reference sequences from each locus using the program Clustal X v.2.1 .Eight polymorphic loci including escribed . Each isHd), linkage disequilibrium (LD) and recombination events (Rms) using software DnaSP version 6.12.03 (http://www.ub.edu/dnasp/) with consideration of both sequence length polymorphism and nucleotide substitutions [C. parvum IId subtypes was assessed by measuring the association of standard correlation index  [The sequences from the eight loci were tandemly concatenated for each isolate. The multilocus genotypes (MLGs) with the same sequences were analyzed for gene diversity (itutions . The genl/query) .http://epa.h-its.org/raxml/submit_single_gene) was used in clustering nucleotide sequences of all isolates using the General Time Reversible (GTR) model [C. parvum IId subtype family were identified using STRUCTURE v.2.3.4 (http://web.stanford.edu/group/pritchardlab/structure.html) [Fst) was calculated using Arlequin v.3.5 (http://cmpg.unibe.ch/software/arlequin3/) in the evaluation of the genetic differentiation between MLGs of C. parvum. Principal coordinates analysis (PCoA) via covariance matrix with data standardization was performed on the generated matrices with the software GENALEX v.6.501 (http://biology-assets.anu.edu.au/GenAlEx) [www.fluxus-engineering.com/sharenet.htm) to estimate the genetic segregation and evolutionary trend of C. parvum [Maximum likelihood analysis implemented in the software RAxML v.8.0.0 (R) model . Subpopure.html) . SeveralGenAlEx) . A media. parvum .dz-hrgp, rpgr and mucin1 had relatively higher sequence polymorphism, with 5, 4 and 4 subtypes being identified, respectively. In contrast, the 44 isolates generated the same sequence at the normally polymorphic chom3t locus  within C. parvum IId population in China (Table Hd = 0.94) was greater than isolates from Guangdong (Hd = 0.78) or Jiangsu (Hd = 0.67) (Table Hd = 0.48). Among them, isolates from Hebei and Heilongjiang showed high genetic homogeneity (Hd = 0.00) within each population. In contrast, IIdA15G1 isolates from Xinjiang were highly heterogeneous (Hd = 1.00)  Table . This co0) Table .Table 2GVD and L measurements, an epidemic genetic structure was obtained in the overall population  . In contrast, the differentiation among IIdA19G1 isolates from Guangdong, Shanghai and Jiangsu was low. Compared with IIdA20G1 isolates from Heilongjiang, there was also reduced differentiation of between IIdA20G1 isolates from Hebei and IIdA19G1 isolates from Jiangsu and Shanghai (Table The results of ce Table . Within C. parvum IId subtypes from different geographical areas in China. Although they were identical at the gp60 locus, the IIdA19G1 isolates differed at most other genetic loci including dz-hrgp, msc6-5, msc6-7, mucin1 and rpgr. Similarly, IIdA20G1 isolates from Hebei and Heilongjiang differed from each other at the hsp70 locus, while IIdA15G1 isolates from Xinjiang differed from each other at the dz-hrgp, hsp70, msc6-5 and msc6-7 loci.The population genetic analysis of eight polymorphic loci has unravelled a high genetic diversity among isolates of LD analysis indicate the presence of an epidemic genetic structure of C. parvum IId subtypes in the present study. This could be attributed to the high prevalence of C. parvum in calves as the result of concentrated animal feeding operations and limited number of IId subtypes in China [C. parvum IId subtypes from China, Egypt and Sweden were shown to have a clonal population structure with limited genetic recombination [C. parvum IId subtypes could be in fact an epidemic population.Results of the in China . Indeed,in China , 32. Prebination . The disFst analyses. Previous reports indicated that most IIa isolates of C. parvum form country-specific populations. For example, an eBURST-based analysis revealed geographical differences among isolates from Uganda, Israel, Serbia, Turkey and New Zealand [C. parvum isolates from Italy, Ireland and Scotland [C. parvum between China and Sweden in a previous MLST study [C. parvum populations, but they were conducted over smaller geographical areas within a country [C. parvum in the more geographically isolated Xinjiang and Harbin. In contrast, isolates from Shanghai, Jiangsu and Guangdong had frequent genetic exchanges with no significant geographical barriers among them. These regions were chosen with the consideration of both geographical representation and the intensity of cattle production. The three C. parvum subtypes examined in the study are the most common ones in China, responsible for over 90% C. parvum infections in cattle. Additional population genetic analysis of more C. parvum isolates from other areas and other subtypes is needed to support the observations in this study.Significant geographical segregation was observed in the IIdA15G1 isolates from Xinjiang and the IIdA20G1 isolates from Heilongjiang based on phylogenetic, substructure, PCoA and  Zealand . SimilarScotland . The samST study . Other s country , 18. In C. parvum. This was not revealed by gp60-based subtyping, as all isolates belonged to IIdA19G1. Nevertheless, this is in agreement with previous population genetic studies of C. parvum in European countries [C. parvum in cattle may be attributed to frequent animal trade among farms, which is known to increase the heterogeneity of C. parvum and the complexity of infections [The presence of multiple MLGs on almost all farms in Guangdong, Shanghai and Jiangsu suggests the presence of a significant intra-farm genetic diversity in ountries , 25, 34.fections , 19, 21.gp60 subtypes of C. parvum in cattle in China, a much higher genetic diversity is evident in MLST characterization of isolates at both farm and region levels. Nevertheless, biological selection has led to the dominance of limited numbers of geographically segregated MLGs of C. parvum in calves in China, with an apparent epidemic population structure. Currently, the veterinary and public health significance of this biological selection of C. parvum subpopulations is not entirely clear. Efforts should be made to monitor the genetic evolution of this unique zoonotic pathogen in China.Despite the presence of only a limited number of Additional file 1: Table S1.Cryptosporidium parvum isolates used in this study and their genetic identity at eight loci.


In this work we evaluated molecular and studbook data for captive populations of black-lion-tamarin (BLT), an endangered primate endemic to Brazil’s Atlantic Forest. Pedigree analyses were performed using BLT studbook information collected from 1973 to 2018. We analyzed the whole captive population since its foundation; the current captive population (CCP); and all extant BLTs in the Brazilian captive population (BCP), separately. Microsatellite analyses were implemented on the BCP individuals from the eighth generation (BCP-F8) only to avoid generation overlap. The expected heterozygosity for BCP-F8, using molecular, data was 0.45, and the initial expected heterozygosity was 0.69. Kinship parameters showed high genetic relationships in both pedigree and molecular analyses. The genealogy-based endogamy evidenced a high inbreeding coefficient, while the molecular analyses suggested a non-inbreeding signature. The Mate Suitability Index showed detrimental values for the majority of potential pairs in the CCP. Nevertheless, some individuals evidenced high individual heterozygosity and allele representation, demonstrating good potential to be used as breeders. Thus, we propose the use of molecular data as a complementary parameter to evaluate mating-pairs and to aid management decision-making.Breeding strategies based on molecular markers have been adopted by  Often based on pedigree analyses, ex-situ management plans aim to maintain demographically stable populations, retaining genetic diversity, limiting inbreeding, and avoiding adaptation to captivity6. However, this is not an easy task, and consequently captive groups tend to present lower levels of genetic diversity and higher inbreeding rates than expected7, challenging the success of these captive breeding programs. On the other hand, wild endangered species often present small and fragmented populations subjected to bottleneck effects and absence of gene flow, and low genetic diversity levels are commonly also observed in nature9. This is the case for the black-lion-tamarin (BLT), Leontopithecus chrysopygus , an endangered primate inhabiting exclusively the Atlantic Forest of São Paulo state in Southeast Brazil10.Captive breeding programs have been recognized as a powerful alternative for rescuing endangered species and for biological conservationL. chrysopygus in nature is small11, currently estimated at a total of a thousand individuals living in a few small forest fragments12. This species was assumed to be extinct about 65 years, when a small population was rediscovered in the Morro do Diabo State Park 13. At that time, a population census estimated that only about 200 animals existed in nature. In 1973, the first seven wild individuals of two contiguous subgroups of BLT were brought into captivity, at the Biological Bank of Tijuca in Rio de Janeiro 14. In 1985, because of the construction of the Rosana Hydroelectric dam, invading about 3,000 ha of the protected Morro do Diabo State Park, eight wild groups were rescued. Of these animals, one group of six individuals was brought to the Rio de Janeiro Primatology Center , and the seven other groups, totaling 31 BLTs, were kept in a vivarium, and transferred to a nearby forest fragment later16. However, due to the poor health condition of these transferred animals, only sixteen BLTs  survived and were relocated to the Zoological Park Foundation of São Paulo State , starting a new group in captivity in 198617.The population size of 18. The first captive group of BLT overseas emerged in 1990, when six individuals were transferred from CPRJ to the Jersey Wildlife Preservation Trust , currently known as Durrell Wildlife Conservation Trust (DWCT). The animals kept at Jersey Zoo successfully produced offspring, and some L. chrysopygus were transferred to other institutions in Europe, North America and Australia. However, the majority of these individuals died17, and nowadays there are only extant captive BLT overseas in Jersey8.In 1987, the International Committee for the Preservation and Management of BLTs was organized in order to contribute to the management of the captive groups of this species. From this initiative, the studbook for the black-lion-tamarin, describing genealogical records for the captive animals, was created in the same yearex-situ breeding programs, the management of L. chrysopygus in captivity has been implemented based only on pedigree analyses8, aiming to minimize population average kinship and preserve representative genetic diversity21. Although this strategy has been considered appropriate to avoid inbreeding22, even if a pedigree has been properly scored for a captive group since its foundation, founder relationships are generally unknown, and for management purposes it is commonly assumed that the founders are unrelated23. Moreover, captive breeding programs often recruit few founders, in general from a single population, representing a small proportion of the total genetic diversity of a species1.Similarly to most 28, although studies integrating both types of data are still scarce29.To compensate for the lack of knowledge about the initial genetic diversity and relationships between the founders, various institutions that manage endangered species have recently tried to combine molecular data with pedigree analysesL. chrysopygus. We analyzed molecular and pedigree data and estimated genetic diversity for F0. The most common pedigree-based index used to choose mates in breeding programs was compared with the individual heterozygosity obtained by microsatellite markers. Our findings suggest that although genealogical analysis has been beneficial, an integrated approach including molecular data might be useful for a better understanding of genetic diversity and the structure of the BLT population in captivity, and for proper metapopulation management.In the present work, we performed studbook and microsatellite analyses to assess population genetic structure and infer demographic and genetic diversity parameters in the captive groups of 30. Of these, 17 individuals were maintained at the Primatology Center of Rio de Janeiro, 16 at the Zoological Park Foundation of São Paulo, and four at the São Carlos Ecological Park . However, some of these animals were relocated among the zoos during the years 2015–2018, including five BLTs that were recently transferred from both CPRJ  and FPZSP  to Jersey Zoo. In addition, other BLTs were born and one wild individual from Patrania municipality was brought to captivity. Currently, there are 55 living animals in the captive population. Eight of them are in Jersey Zoo, one in Magdeburg Zoo (German) and 46 are in Brazil  , which included the descendants from the prior generations without the parents , mortality (Qx), survival (Lx), expected lambda-λ, instantaneous rate of change of the population (r), and reproductive peaks in captivity are shown in Supplementary Information  and father-son for the CCP (11.04 years) populations. The average generation interval for WCP and CCP was 5.44 and 7.57 years, respectively. The generation intervals calculated across all pathways are presented in Table  years anon Figs. –S6.Tablefa) calculated following Boichard et al.31 was 10 for WCP and eight for both CCP and BCP. The effective number of founders (fe) was equal to 10 for WCP, CCP and BCP (Table rGD) based on the founder genome equivalent (fge) was 87.2%. When we considered only BCP, rGD was equal to 83.9% (Table The effective number of ancestors (F) was higher in BCP and CCP than in WCP  and the value of Nec, which assumes random mating occurring in the near future, are shown in Table Nec/Ne for CCP was equal to 0.30.Mean kinship statistics showed slightly superior values in the CCP and BCP Table . The reaF-statistics (FST between groups) and mean coancestry (fij) within and between zoos, is shown in Table F-statistics evidenced that Jersey Zoo and CPRJ are the most genetically distant captive groups, whereas mean coancestry and FST values showed that PESC and CPRJ are the most related ones.The genetic structure, based on Wright’s 34, no genetic inconsistencies were found for these loci, after following the technical procedures employed for DNA amplification and genotyping (see Supplementary Information). The obtained electropherograms (EPGs) evidenced specific allele patterns with proper quality , which were born between 2005 and 2012. Currently, 10 of these are alive and consequently are included in the CCP as well  based on pedigree data showed a high value concordant with that observed using the pedigree inbreeding index (F = 0.19). Kinship based on molecular data (rm) confirmed a high degree of relatedness. The molecular inbreeding coefficient was negative (f = −0.58), as a consequence of an excess of heterozygosis, the observed mean (Ho = 0.73) being higher than the expected heterozygosity (He = 0.45).In total, we computed 31 alleles, ranging from two to three per locus, with an average of 2.06 alleles per locus, and average allelic richness equal to 2.07. The equivalent genetic diversity estimators, based on both pedigree and molecular data, are shown in Table MSI estimated by PMx for all extant BLTs from CCP, including individuals from Jersey, resulted in a total of 480 simulated potential couples, in which 80% were considered at least as slightly detrimental   Fig. . In cont35. In this sense, pedigree analyses can provide relevant information for the management of species in captivity28. However, the effectiveness of pedigree-based approaches depends on its completeness and depth, since deeper pedigrees usually generate more accurate and robust inferences35. According to the present study, 92% of ancestral relationships in the whole captive population of BLTs are well known. Taking into account such high pedigree depth value, the demographic and genetic inferences raised herein, based on the BLT studbook data, should be considered as reliable.Setting up an efficient captive breeding program requires a precise knowledge of the genetic diversity and genealogical data of the populations to be managed36, that in this case are the oldest BLTs. In addition, we verified low rates of reproduction of founder and non-founder wild animals, and also a high number of unrelated captive animals that never reproduced at all. Thus, despite the huge efforts to avoid matings between closer relatives, or in recent years to decrease accumulation of inbreeding as much as possible, the BLT captive population has been showing an increase in inbreeding over time and high kinship values.Overall, the captive BLT population is well established; nevertheless, its age structure is typical for a slowly growing population (λ > 1), showing an explicit decline from the year 2001, in both sexes. The whole captive group reached a maximum population size of 114  in 2000, but in the subsequent years it suffered a drastic and continuous decline, though new births have contributed to the growth of the current captive population in Brazil. We also observed an increase in the average generation interval over the years. These results are quite probably due to management efforts aimed at minimizing inbreeding by the reuse of less related animals as reproducers37. Unfortunately, some possible evidence of inbreeding depression, such as bone deformities, low copulation rates, low sperm motility, high rates of infertility and cleft lip that cause infant deaths during tooth changes, have already been observed in the Brazilian captive population of BLTs . Additionally, the European population has experienced a high incidence of gallbladder problems . In spite of this, the short-term goal of the BLT breeding program in captivity has been achieved by the high survival rates in the infant and juvenile age groups. However, the long-term goal for BLT management consists of maintaining genetic diversity levels and avoiding inbreeding depression38.High rates of inbreeding and kinship can promote genetic diversity loss and inbreeding depression in future generations, compromising fitness-related features such as viability, birth weight and fecundity23. In our study, we know the origin of the founders, which came from two neighboring groups of the same population that lived in the Morro do Diabo State Park and might be genetically related. Consequently, the kinship and inbreeding values calculated by pedigree analyses were high, compromising the viability38 of the captive population of BLTs in the long-term.Captive populations often have a very small number of founders, which are considered unrelated and consequently have inbreeding rates assumed as zero39. Moreover, molecular data have shown a higher observed heterozygosity than expected, which leads us to infer that zoos are managing the population appropriately. Nevertheless, the PMx analyses showed that the MSI values are at least detrimental40 for the majority of potential couples of the CCP. From the total of simulated mate pairs, 174 and 30 showed values of MSI that were slightly detrimental and detrimental, respectively. The remaining ones were considered very detrimental and very highly detrimental. On the other hand, only 20% pairs had MSI values considered as beneficial for breeding programs, whereas the BLTs recently transferred from Brazil to Jersey showed MSI values moderately or slightly beneficial when we simulated pairing with BLTs from Jersey Zoo.Fortunately, according to measures of probabilities of gene origin, our data showed a greater value of effective size when compared to the effective number of founders, ancestors and founder genome equivalent values. The relationship between the effective size and the population size (NeI/N) shows that the pedigree-based recommendations are being directed to the equalization of the families, and the sex ratio and the number of individuals throughout the generations, aspects considered very advantageousMSI is the most common parameter used to select mates in captive breeding programs, and considers differences in genetic diversity, kinship, inbreeding coefficient and unknown ancestry, all calculated only by pedigree data42. Considering the MSI values found here, we estimated the Internal Relatedness index as a complementary parameter to the MSI, in order to gain some insights based on molecular data as well.The 43. In our study, despite the fact that the transferred BLTs have shown moderately or slightly beneficial MSI values, they all showed negative IR values, indicating that these individuals have high heterozygosity, besides allele representativeness, and consequently are valuable as breeders44. In fact, these animals have already mated and successfully produced offspring in Jersey . Alternatively, if only very homozygous individuals are available for forming mate-pairs, genetic differentiation among the potential breeders and their allele representativeness45 must be considered in addition to MSI scores.The IR index is a method for estimating individual heterozygosity and considers that rare alleles count more than common alleles. Negative IR values indicate higher heterozygosity, whilst positive values are attributed to more homozygous individuals45. Previous molecular analysis, using the same set of microsatellites used here, showed private alleles in each captive group from Brazil and Europe, evidencing genetic structuring among them8. In addition, the pedigree analyses performed here pointed to greater genetic differentiation between the Jersey and CPRJ captive groups. It is noteworthy that the animals that successfully mated in Jersey are from CPRJ (487) and FPZSP (472), these latter being descended from the CPRJ group. In this case, the genetic diversity increment was beneficial to the metapopulation management of BLTs46.Management decisions must take into account the possibility of changes in genetic diversity by mating between genetically more divergent individuals49.Notwithstanding this, changes in the genetic diversity of source and recipient populations, by movement of individuals, are not always beneficial to both populations. Such groups need to be carefully managed to maintain the maximum of allele richness, to avoid inbreeding, but also potential outbreeding depression. Therefore, combining multiple genetic diversity measures, based on both molecular and studbook data, might produce a more robust data set40. However, our findings suggest that despite the remaining genetic diversity is about 65% in both pedigree and integrated analyses, the expected heterozygosity represented in the founder individuals, based on the integrative approach, would be about 0.69. Such results show a more coherent value of genetic diversity for F0, reinforcing the idea that genetic diversity inferences must be specific for each breeding program and cannot be extrapolated from hypothetical assumptions48.When the initial genetic diversity of the founding captive population is unknown, it is hypothetically considered equal to 1, as proposed by the PMx model commonly used for calculating pedigree parameters53. According to recommendations of the ISFG  for the area of non-human DNA typing34, they have also potential to be used, by the community of forensic scientist, for investigations involving poaching, smuggling and illegal trade of protected species55Molecular analyses are essential for populations with unknown genetic diversity and can be relevant to monitoring genetic diversity across generations in conservation actions56. Genetic management of threatened species has experienced an increase in the last few years58, and more recently has been improved by a combination of pedigree and molecular information59. For the BLT captive breeding program, we highlight that an integrative approach could be of benefit in terms of allele representativeness and also for considering a more plausible genetic diversity estimate for the founding population.DNA-based studies can still simulate, estimate and compare genetic diversity levels in breeding programsMSI scores. Microsatellite-based values of expected heterozygosity, individual heterozygosity, allele richness, private alleles, population structure, inbreeding and kinship could be monitored over generations, helping to evaluate gains and losses of genetic diversity more effectively, and identifying individuals potentially better suited for reproduction and for relocation in captivity47. Finally, we must take into account that an integrated in situ and ex situ approach is strongly indicated for the metapopulation management of BLTs and to help shield this species from its imminent risk of extinction, since in nature L. chrysopygus has a small population size and a very low genetic diversity level.Overall, to promote the long-term success of the BLT conservation program, we recommend including genetic diversity parameters based on molecular data, in addition to the pedigree analyses and https://www.asp.org/society/resolutions/EthicalTreatmentOfNonHumanPrimates.cfm).The present study was approved by the Ethics Committee on Animal Experimentation , under CEUA-UFSCAR number 9805200815; the Authorization System and Biodiversity Information of the Chico Mendes Institute for Biodiversity Conservation , under SISBIO-ICMBio numbers 50616-1; and the National System of Genetic Patrimony Management and Associated Traditional Knowledge , under SISGEN number A411359. The approved experimental protocols included the capture of live animals in captivity, and the anesthesia using direct inhalation equipment and blood collection procedures. The animals were handled by a veterinarian who released them safely after blood collection. These procedures followed all ethical and legal recommendations proposed by the institutional and licensing committee and the American Society of Primatologists for the Ethical Treatment of Non-Human Primates . We considered all BLTs kept in captivity from 1973 to 2018, including the founders, ancestors and their offspring, and we carried out analyses separately for three set of individuals: the whole captive population (WCP), including all living or dead captive BLTs; the current captive population (CCP), comprising all extant captive individuals maintained in Brazil and overseas until 2018; and the Brazilian captive population (BCP), including only living captive BLT adults in Brazil8.We analyzed all records of 60. First, we calculated the pedigree depth by considering the proportion of known ancestors per generation for each offspring, and then we added the interval of generations, defined as the mean age of parents when their progeny is selected to be parent, considering the relationships between mother-daughter, mother-son, father-daughter and father-son61. Finally, we estimated the equivalent complete generations based on the proportions of individuals with both known parents. This parameter is also known as the mean equivalent generation (ge) and it is calculated as the sum of all known ancestors n is the number of the 62. The complete pedigree was constructed using Pedigree Viewer version 6.5.2.063.For demographic inferences we implemented three different analyses for evaluating the consequences of the applied random mating system and its evolution over time by using the Endog 4.8 softwareMx) was calculated considering the individual fertility or reproductive potential information for each age class. Mortality (Qx) was estimated as the proportion of individuals entering an age class versus animals that died before reaching the age class x + 1. Survival (Lx) was determined as the proportion of individuals surviving from birth to the beginning of the age group x. The proportional change in population size from one year to the next, based on life table calculations (expected lambda-λ), and the instantaneous rate of change of the population, averaged for males and females (r), were also estimated. A lambda value greater than one indicates an increase in the population. A value of r greater than one also means that the population is increasing. All these estimators were calculated using PMx software40.Fertility (fe)64 and total effective number of ancestors (fa)31, using the Endog 4.8 software60, and founder genome equivalents (fge)64 using PMx40. The degree of remaining genetic diversity  was calculated based on the following expression: H0 = 1. The inbreeding coefficient (F) was estimated to illustrate the trend in mean inbreeding across years. Likewise, mean kinship (Mk) was also calculated as complementary information to that provided by the inbreeding coefficient (F). FST and mean coancestry (fij) were calculated following Caballero and Toro66, considering the genetic divergence between each pair of zoos which hold the species based on the pedigree data. These latter parameters were calculated using PMx software40.For the pedigree-based genetic inferences, we determined genetic diversity by calculating the total effective number of founders (Ne) was estimated based on two approaches (NeI and Nec) implemented in Endog version 4.860. First, Ne was calculated to estimate the founder population size and to detect the existence of bottlenecks and possible consequences of the mating strategy, via the individual increase in inbreeding (NeI), as proposed by De la Rosa et al.67. To calculate NeI, the coefficient of individual increases in inbreeding (∆Fi), determined according to Falconer and Mackay68 and modified by Gonzales-Recio et al.69 and Gutiérrez et al.70, was used. The modified method proposed by Gutiérrez et al.70 is considered the most appropriate to analyze permanently subdivided populations. Ne was also calculated using the increase in coancestry (Nec) proposed by Cervantes et al.71, which is suitable when mixing of populations becomes a usual practice. We also calculated the ratio of the effective population size (NeI) to the census size of living captive-born individuals (NeI/N). Mate Suitability Index (MSI) was determined for all potential pairs in the current captive population of BLTs using PMx40.The effective population size (vacutainers containing EDTA (3.6 mg). The animals were anesthetized by direct induction using inhalation equipment calibrated with isoflurane (2–5%) and oxygen (2 L/min), and were then released back into their respective enclosures. Blood samples were stored at −20 °C for subsequent DNA extraction. Genomic DNA was obtained following the phenol protocol72. The DNA integrity was confirmed using 1% agarose gels under constant voltage (100 V for 45 min) . Each sample genotyped as homozygous was confirmed by a minimum of three replications. We also performed multiple PCRs for random samples, in order to identify genetic inconsistencies, according to recommendations proposed by the ISFG  for the area of non-human DNA typing34. More details related to the technical procedures employed for DNA amplification and genotyping of the STR (Short Tandem Repeats) loci are available in Supplementary Information.Polymerase chain reactions (PCRs) for the microsatellite amplifications followed procedures proposed by Ayala-Burbano 76. Subsequently, lack of linkage disequilibrium (LD) between loci was verified in Genepop version 4.0.1077. We used the linkage disequilibrium method to assess the effective population size. Genetic diversity parameters were inferred by calculating the number of alleles (Na), effective number of alleles (Nae), expected (He) and observed (Ho) heterozygosity using GenAlEx version 6.478.Before the statistical analyses, we estimated the occurrence of null alleles, allelic dropout and stuttering for all scored alleles using Micro-Checker79, where The proportion of remaining genetic diversity represented in the eighth generation of the BCP was calculated as 80. The mean relatedness (rm) between individuals was estimated using Coancestry81. This software calculates seven different relatedness estimators, and after testing all of them, we choose the estimator based on Triade likelihood (TrioML), which showed the smallest variance among all the estimators tested81.Allelic richness (Ra) and inbreeding coefficient (f) were calculated using Fstat version 2.9.3.282 was calculated for the individuals recently transferred from Brazil to England, using GENHET83, in order to add a relevant molecular genetic diversity parameter to the MSI obtained from pedigree data.Individual heterozygosity, based on the internal relatedness index (IR)Supplementary information.