Cymodocea nodosa, in the eastern Mediterranean Sea, to identify the historical dynamics, demography, and connectivity of the species in the area. Eighteen microsatellite loci were used in conjunction with coalescent methods to investigate the genetic structure and demographic history of C. nodosa meadows. Approximate Bayesian computation (ABC) modeling was used to examine the pattern of divergence over time in the context of environmental change over the course of the Quaternary period. ABC analysis revealed an initial split of the C. nodosa populations between the north‐western, northern, and north‐eastern Aegean Sea during the Pleistocene epoch, followed by a more recent divergence of the north‐western population and the central‐western part of the Aegean Sea. According to the results, the most parsimonious historical scenario is that of a pervasive genetic signature of the effects of the drop in sea level during the Pleistocene epoch. This scenario supports the isolation of the north‐western, north, and north‐eastern area, and the subsequent recolonization after post‐glaciation sea level rise that may explain the north‐western differentiation as well present‐day detected dispersion of C. nodosa.We investigated the population dynamics of a highly clonal marine angiosperm,  Cymodocea nodosa, in the eastern Mediterranean Sea, to identify historical dynamics, demography, and connectivity of the species in the area. The most parsimonious historical scenario is that of a pervasive genetic signature of the effects of the drop of Aegean Sea level during the Pleistocene epoch. This scenario supports the isolation of the north‐western, north, and north‐eastern area, and the subsequent recolonization after post‐glaciation Aegean Sea level rise that may explain the north‐western differentiation as well present day detected dispersion of C. nodosa.We investigated the population dynamics of a highly clonal marine angiosperm,  However, studying past environmental conditions such as the glaciation cycles during the Quaternary period can provide us with hints of the current population structure of many marine organisms , the western basin was affected mainly by the lower export production fluxes, suggesting that different processes have resulted in contrasting conditions in each basin .The population structure of seagrasses has also supported this hypothesis of a strong influence of the Quaternary period. Arnaud‐Haond et al.  indicateo et al.  have shoC. nodosa within the eastern Mediterranean waters are closely related .Following our recent studies of transcriptomic responses  were collected haphazardly, at least 10 m apart from each other. Shoots were dissected and individually stored in dry silica gel until further analysis. In total 335 SUs were analyzed.2.2Shoots tissue samples were extracted following a standard phenol/chloroform extraction protocol  and observed heterozygosity (HOBS), along with the departure from Hardy–Weinberg through FIS calculation using the formulations described by Weir and Cockerham  were estimated using the θ estimator of Weir and Cockerham , nearly all of the 335 genotyped sampling units (SUs) were unique MLG. Only 3 MLLs were detected across the whole data set Table . Analyse3.2OBS = 0.599) and were comparatively lower than overall expected heterozygosity values (mean HEXP = 0.663). Values of HOBS, HEXP, FIS, number of alleles, and allelic richness are shown in Table A moderately high level of polymorphism was detected in all tested loci, ranging from a few loci 1–10 loci) for the majority of populations, to 16 alleles at the Cn4‐27 locus  and Vrasidas (north Aegean) to 0.2937 between Cyprus and Lemnos (north‐eastern Aegean). Most of the values were highly significant after correction for multiple tests  as shown in Table F3.3Factorial correspondence analysis showed the geographical clustering of the 12 sampled meadows Figure . At K = 3.4The estimation of the contemporary and historical migration rates between all groups was performed using BayesAss and Migrate software, respectively Table . Histori3.5Among the 16 different demographic scenarios examined, the most likely scenario emphasized by DIYABC analysis as seen in Figure 4Cymodocea nodosa meadows of the eastern Mediterranean Sea exhibit a relatively high genetic differentiation. A key finding here is the existence of relatively strong barriers to dispersal between north‐eastern and other Aegean meadows, the former appearing genetically slightly closer to Cyprus meadows than to their Aegean counterparts, both through clustering and FST analysis. Such a recorded pattern is not in line with geographic distances or proximity, but is logical since a re‐colonization pattern of the north‐eastern Aegean Sea from Cyprus, gradually through the coasts of Asia Minor, has also been hypothesized for other marine plants  may have also been re‐colonized later on by Lemnos strain though Samothraki Island anti‐cyclone drift that also limited species westward migration. Also, perhaps the deep waters (basins) of the Chalkidiki peninsula and the Sporades islands  could have served as a reservoir for preserving the species during the last glacial period , and then fits the LGM and Holocene conditions for the split between north‐western and central‐western. Moreover, historical and contemporary migration analyses showed a directionality from north to north‐western and north‐western to central‐western, respectively, in agreement with the most likely scenario supported by our coalescent scenario.Factorial component analysis clearly formed three distinct groups in the Aegean Sea and ABC analysis revealed an initial split of the Zostera marina ; Methodology ; Software ; Writing – original draft . Georgios A. Gkafas: Data curation ; Investigation ; Methodology ; Software ; Validation ; Writing – original draft . Vasillis Papathanasiou: Data curation ; Investigation ; Software . Sotiris Orfanidis: Data curation ; Formal analysis ; Investigation ; Methodology ; Validation ; Writing – review & editing . Frithjof C. Küpper: Data curation ; Formal analysis ; Investigation ; Validation ; Writing – review & editing . Sophie Arnaud‐Haond: Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Writing – review & editing . Athanasios Exadactylos: Conceptualization ; Data curation ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Software ; Supervision ; Validation ; Writing – review & editing .Appendix S1Click here for additional data file.


To identify the scope and nature of agricultural biodiversity actions within the climate adaptation plans of a sample of large world cities.I evaluated data from the 2021 Cities Climate Adaptation Actions database curated by the Carbon Disclosure Project. Cities with a population over 1 million and reporting at least one adaptation action were included. I identified actions involving agriculture and biodiversity using a framework consisting of five agrobiodiversity categories: urban and peri-urban land use and water management, and urban food supply chains, food availability and food environments. I also identified reported health co-benefits and health sector involvement.Of 141 cities reviewed, 61 cities reported actions on agricultural biodiversity, mostly supporting land use or water management. Key health outcomes addressed were illnesses linked to air pollution and excessive heat and vector-borne diseases, corresponding with cities’ major health concerns. Greenhouse gas mitigation was also addressed by many cities. Fewer cities reported actions in food categories or concern for noncommunicable diseases or poor nutrition. Nearly two thirds of cities (40/61) reported health co-benefits or health-sector involvement for at least one intervention. A higher proportion of the 43 cities in low- and middle-income countries reported agrobiodiversity actions and health co-benefits than the 18 cities in high-income countries.Cities are key partners in achieving sustainable global agriculture that promotes health and supports climate and biodiversity goals. Cities can enhance this role through climate adaptation plans with strong health engagement, a focus on nature-based solutions and greater emphasis on food and nutrition. Cities are home to over half the world’s population, and are responsible for about 70% of greenhouse gas emissions.,,One way in which cities are addressing agricultural biodiversity is through climate adaptation planning. These adaptation goals often overlap with the sustainable development goals (SDGs), particularly in low- and middle-income country cities, most notably those targeting ending poverty (SDG 1); good health and well-being (SDG 3); clean water and sanitation (SDG 6); sustainable cities and communities (SDG 11); and climate action (SDG 13).Another way in which cities are responding is by sharing experiences via knowledge networks. The Milan Urban Food Policy Pact aims to develop sustainable urban food systems,However, few systematic assessments of adaptation actions to address agricultural biodiversity and its relationship with health have been published. A review of national adaptation plans in 50 lower-income countries found that few plans incorporated health in a comprehensive way.For this descriptive study I evaluated data from the 2021 Cities Climate Adaptation Actions database curated by the Carbon Disclosure Project. This organization hosts a suite of databases forming the unified reporting framework for a consortium of city climate change networks, including: Local Governments for Sustainability, the Global Covenant of Mayors, C40 Cities and others.I aimed to focus on a city sample with a sizeable population vulnerable to climate hazards, biodiversity loss and significant greenhouse gas emissions. I therefore chose large cities as the focus for this study, selecting cities reporting to the 2021 database if the population exceeded 1 million and if a description of at least one adaptation action was provided. For cities meeting these criteria, I downloaded each city’s descriptions of their climate adaptation actions, their implementation progress, and the climate hazards their city faces from the Carbon Disclosure Project Cities, States and Regions Open Data Portal on 12 September 2022. I also extracted cities’ responses to survey questions regarding the sectors that the adaptation action applies to  and co-benefit areas . I collated these data on an Excel spreadsheet .Lancet strategic goals and provide a structure for considering city activities and health impacts. As adapted for my study, the framework consisted of five categories: (i) urban and peri-urban land use; (ii) urban and peri-urban water management; (iii) urban food supply chains; (iv) urban food availability; and (v) urban food environments.The structure for the analysis was an urban agricultural biodiversity framework which I adapted from another study.To conduct the analysis, I reviewed all descriptions of climate adaptation actions reported by the selected cities (using translations for non-English language descriptions) and identified actions involving agriculture , and ecosystems or biodiversity. For each action, I determined which category it fitted in the urban agrobiodiversity framework. In some cases, reported actions contained more than one different intervention or policy and I therefore separated these and eliminated any duplicate actions. I subsequently re-checked the analysis for any errors or misclassifications. I then identified and aggregated, and subsequently re-checked, the reported health and safety co-benefits; health and safety sector involvement; and implementation status for each city. I also extracted and analysed the means of implementing the actions and associated climate hazards. Finally, as context, I extracted data on climate-related health issues and vulnerable groups reported by a subset of these large cities to the 2020 Carbon Disclosure Project Cities Adaptation Actions database. The main study results are shown here as number and share of cities reporting at least one adaptation action that came under one of the framework categories. Also shown are number and share of cities with at least one action with health sector involvement or health co-benefits. To examine differences across cities, I stratified the results by World Bank country income category,A total of 141 large world cities reporting 973 adaptation actions met the population and data inclusion criteria of the study. Of these, 94 (67%) of cities were located in low- and middle-income countries  and 47 (33%) in high-income countries (mainly in Europe and North America). The climate hazards most frequently being addressed were extreme heat , followed by flooding , extreme precipitation  and drought . A subset of 70 cities reported climate-related health issues; the sample was similarly distributed across low-, middle- and high-income countries as the main sample. These cities most frequently expressed current concern for air pollution-related illness , vector-borne disease  and heat-related illness . Concern for noncommunicable diseases  and food- and waterborne diseases  were also reported, while nutrition outcomes and mental illness were among the least reported health concerns . Among 72 cities reporting climate-vulnerable populations, elderly people and children were most frequently reported , followed by people with low incomes  and those living in poor-quality housing .A total of 61 of the 141 cities (43%) reported 142 adaptive actions that fitted within the urban agrobiodiversity and health framework. Every world region was represented, although Brazil, Canada, Colombia, Italy, Malaysia, Mexico, Peru, Philippines, Türkiye, the United Kingdom of Great Britain and Northern Ireland, and the United States of America each had two or more cities represented (more details in the online repository).Of 142 agrobiodiversity actions, 118 actions (83%) were under implementation, operational or being monitored, while 23 actions (17%) were in the earlier stages (scoping or pre-feasibility studies of actions). The largest single means of implementation was through infrastructure actions , while other important types of action were awareness raising, policy and regulation, stakeholder engagement and capacity-building.Among the 61 cities with agrobiodiversity actions, urban and peri-urban land use was the category most frequently reported , followed by urban and peri-urban water management , the peri-urban erosion reduction programme maps flood-related erosion risk, identifies afforestation priorities and supports farmers to improve terracing, pasturing and wildfire risk identification. Among other actions are a forest management programme in Amman (Jordan) and financing for private sector investment in urban and peri-urban tree planting in Manchester (United Kingdom of Great Britain and Northern Ireland).The restoration programme for mangrove forests in Seberang Perai  supports the fishing industry and enhances carbon retention. Other actions are programmes for water reuse, water-efficient irrigation and drought-resistant crops in Casablanca (Morocco) and the beach recovery plan in Barcelona (Spain) that addresses conservation and the effects of warming seawater on fish and marine species.In Toronto (Canada), an examination of the city’s food supply chain used an assessment of the risks from climate-related extreme weather to develop peri-urban farming alternatives. Other initiatives include an agricultural drought early warning programme in Izmir (Türkiye) and a training programme for agriculture companies and farmers on the business implications of climate resilience in Ekurhuleni (South Africa).Urban agriculture and community garden programmes in Curitiba (Brazil) make use of abandoned spaces for organic farming, renewable energy, reuse of materials and greenhouse gas absorption. Paris (France) funds organic farming on city reservoirs via its water company, while Lima (Peru) reports a campaign on awareness of healthy food and Cali (Colombia) has launched a formal food independence policy.Milan  has an urban greening programme that provides for biodiversity, greenhouse gas retention, water conservation, heat management and nutrition benefits. Kaohsiung  reported a pilot study of a roof garden, while Quezon City (Philippines) provides food for humanitarian shelters in the context of climate-related extreme flooding, and has enhanced food safety monitoring for periods of extreme heat.Source: The examples were extracted from the 2021 Cities Climate Adaptation Actions database curated by the Carbon Disclosure ProjectA higher share of the cities in low- and middle-income countries  reported any urban agrobiodiversity action compared with cities in high-income countries . The agrobiodiversity actions were also more likely to have health sector involvement or health co-benefits in cities in low- and middle-income countries  than cities in high-income countries  was rarely reported by cities as a health issue of concern. Given the relative newness of city-based food or agriculture and biodiversity policies, these mixed results are not surprising.,,Just under half of cities reported health sector involvement in agrobiodiversity actions (higher for food choices and food environment) and over half reported health co-benefits (higher for peri-urban land and water management). Health therefore played a greater role in cities’ agrobiodiversity activity than might be expected based on previous research.The impact of support from past and current international programmes and collaborative initiatives was evident from this analysis. Some of the strongest urban agrobiodiversity performers were low- and middle-income country cities which have benefited from FAO and other related programmes. Examples are Lima in Peru and Quezon City and Davao in the Philippines, which have been Resilient Cities target countries,This study provides a systematic comparison of urban agrobiodiversity actions based on the most comprehensive available international database of cities’ climate adaptation actions. However, there are several limitations. The Carbon Disclosure Project database reports actions being implemented by a sample of participating network member cities and is not a representative sample of large world cities. While the database’s validation procedures reduce the risk of error in self-reported responses, other shortcomings exist, including different interpretations of the meaning of action across cities. For this reason, my study relied on the share of cities reporting any action. Lack of survey questions regarding agrobiodiversity may also have hindered responses . Over time, further refinements to the database may be helpful in addressing this issue. In addition, some sources suggest that urban agrobiodiversity activity, particularly food-related action, is more extensive than reflected here. For example, more comprehensive food and nutrition programmes are reported for Barcelona, which has hosted the Milan Urban Food Policy Pact network.,The findings suggest that there are opportunities for promoting policies in support of healthier, more sustainable food and biodiversity programmes through city climate adaptation plans. One opportunity is building on health sector engagement in city adaptation plans to further enhance the interconnections between agriculture and biodiversity. Research suggests that when the public health sector is involved, climate adaptation outcomes may be better targeted towards those in greatest need.,A second opportunity is to integrate agrobiodiversity goals more fully into current and future nature-based solutions. Existing efforts may already be contributing to the goals of urban agrobiodiversity, even if the descriptions of cities’ adaptation actions do not show this. Current approaches may be readily modifiable, such as by incorporating urban agriculture;The findings presented here underline that cities are key partners in building a more sustainable global agriculture system that addresses the twin challenges of climate change and biodiversity loss. The results also suggest that city climate action plans with a strong emphasis on health present a promising opportunity to strengthen policy and practice focus on agrobiodiversity towards more sustainable human and planetary health.


Despite the widespread uptake of Lewontin's conclusion that racial classification is of ‘virtually no genetic or taxonomic significance’, the biomedical research community continues to grapple with whether and how best to account for race in its work. Nowhere is this struggle more apparent than in the latest attempts to translate genetic associations with complex disease risk to clinical use in the form of polygenic risk scores, or PRS. In this perspective piece, we trace current challenges surrounding the appropriate development and clinical application of PRS in diverse patient cohorts to ongoing difficulties deciding which facets of population structure matter, and for what reasons, to human health. Despite numerous analytical innovations, there are reasons that emerge from Lewontin's work to remain sceptical that accounting for population structure in the context of polygenic risk estimation will allow us to more effectively identify and intervene on the significant health disparities which plague marginalized populations around the world.This article is part of the theme issue ‘Celebrating 50 years since Lewontin's apportionment of human diversity’. Nowhere is the latter struggle more apparent than in the latest attempts to translate genetic associations with complex disease risk to clinical use: the polygenic risk score, or polygenic risk scores (PRS). The hope is that PRS can be used to triage high-risk patients for intervention of preventable health problems. But such methods must account for human population structure . While PRS attempt to deal with population structure by using principal components of genetic diversity in tests of association with disease, risk estimates are usually applied to patients stratified by self-identified race and/or ethnicity, with a range of consequences arising from the elision of genetic ancestry and social identity. In this paper, we offer some reflections on our ongoing inability to reconcile these complexities of human population structure and discuss the potential implications for clinical use of genetic information in the context of PRS.. 2‘The Apportionment of Human Diversity’ was situated at a particular moment in the history of population genetic investigation and in the face of historical and philosophical recognition of the role that anthropology and biomedicine played in legitimizing claims of racial distinction. Far from being a value-neutral description of population difference, research on the creation of the modern ‘race’ concept has highlighted repeatedly the ways in which it was, from the start, used as a way to justify (for example) colonialism, chattel slavery and genocide as well as other, similarly exclusionary and frankly monstrous, practices e.g. ,9). This. This9])within ‘conventional racial’ groups . Only 6% of human genetic diversity appeared to distinguish the seven human races he defined. From this estimate, Lewontin concluded that ‘our perception of relatively large differences between human races and subgroups, as compared to the variation within these groups, is indeed a biased perception and that, based on randomly chosen genetic differences, human races and populations are remarkably similar to each other’ [Human racial classification is of no social value and is positively destructive of social and human relations. Since such racial classification is now seen to be of virtually no genetic or taxonomic significance either, no justification can be offered for its continuance , p. 397.This context explains the move between Lewontin's technical (population genetic) observations and his sociopolitical conclusion. Lewontin noted that of the (roughly) 15% of human genetic diversity that is not attributable to differences between individuals within populations, the larger part of that 15% (approximately 9%) was accounted for by differences between populations The taxonomic division of the human species into races places a completely disproportionate emphasis on a very small fraction of the total of human diversity. That scientists as well as nonscientists nevertheless continue to emphasize these genetically minor differences and find new ‘scientific’ justifications for doing so is an indication of the power of socioeconomically based ideology over the supposed objectivity of knowledge , p. 156.The point here seemed to be that the very small amount of human genetic diversity for which ‘racial’ classifications accounted was not significant enough to warrant either our usual racial ascriptions or treating racial categories as especially meaningful objects of biological study. In a subsequent publication, Lewontin made this point more explicitly:The technical result, and the sociopolitical implication that Lewontin drew from it, became part of the mainstream position that ‘race’ is not a legitimate biological category. So for example, authors such as Biondi & Rickards  argue thOn the other hand, others have resisted  the conclusion that biology cannot support the conception of race demanded by folk racial categories and usage and that race is therefore of ‘virtually no taxonomic significance’. In an article with the subtitle ‘Lewontin's Fallacy’ , EdwardsIrrespective of one's beliefs about the role that genes may or may not play in population health disparities (discussed further in the next section), questions about the scale and biomedical relevance of population genetic differences have continued to matter in genetic investigation. This is on account of the move away from linkage studies in families to investigations of genome-wide association in many thousands of cases and controls, where statistical confounding due to population stratification becomes a potential concern. Of course, the traditional epidemiological approach of matching cases and controls with respect to self-identified race and/or ethnicity (on an assumption of shared genetic background such that confounding can be minimized) has given way to approaches that instead consider genetic ancestry directly. Nevertheless, even with direct incorporation of principal components of genetic variation into association models, it is still the norm to conduct genome-wide association studies (GWASs) in ancestry subgroups and so to estimate effect sizes of gene–disease associations with respect to specific ancestral genetic backgrounds. The ensuing associations, when combined across thousands of loci in the form of a PRS, are tidy but not obviously translatable to patients who self-identify with (or are ascribed to) racial and/or ethnic categories rather than genetic ancestries.. 3A major rationale for continuing to study human populations subdivided with regard to racial classification is the observation that many common complex traits and diseases differ in their prevalence between racial and/or ethnic groups, particularly in the United States ,17. The APOL1 association with end-stage kidney disease risk [The difficulty with the pursuit of such research is twofold. First, despite a great deal of effort, relatively few plausible racially stratified gene variants with discernible effects on common complex disease risk have thus far been identified. The most prominent examples in this category include the 8q24 association with prostate cancer risk  and the ase risk ,24. Secoase risk –28. Not ase risk ,30.There is really no debate about the relative contributions of genetic and social determinants in this regard, just a stubborn refusal on the part of many in the human genetics community to give up on the potential of genetic discovery to contribute remedies to the problem. Having (for the most part) given up on the hope of identifying loci with reasonable effect sizes as contributing to health outcome differences, geneticists wishing to address health disparities have now turned instead to PRS.. 4APOE may be the best example of this, although its discovery also involved linkage analyses [The diseases of highest public health significance, so-called ‘common complex diseases’ such as coronary artery disease, type 2 diabetes mellitus and asthma, are multifactorial in aetiology, arising from the joint influence of multiple genetic risk factors and environmental exposures. The extent of the genetic component of common disease risk varies; heritabilities, estimated from twin, adoption and studies of other relatives, of between 30% and 50% are commonly cited . Whereasanalyses ). GWAS, analyses ), nearlyanalyses ,37.One of the problems with finding risk variants using GWAS is that, to reduce the chances of spurious associations, very stringent statistical standards must be maintained. It is possible that loci that do not meet these high standards nevertheless have a measurable association with the trait in question. PRS attempt to address this problem by using many different markers, most of which do not meet standard genome-wide significance thresholds, to generate a composite measure of the overall association between those markers and the trait in question ,39. By lslightly more likely to at some point suffer from a complex disease such as heart disease is not necessarily useful for individual clinical  decision-making.Hence, while the identification of sets of loci linked to complex disease risk has helped increase confidence that fewer genetic contributions are missed, it has changed how the risk information can be translated for public health benefit. Whereas previously, the goal of most GWAS was to identify specific disease-linked genes and biological pathways with the hope of informing drug development, PRS are better suited to risk stratification not genetic discovery. PRS calculation for patients with and without the trait of interest permits estimation of the relative risk of disease for specific scores, and those at the upper limits of the population risk distribution  can be et al. [For phenotypes where effect sizes are higher, or other clinical risk assessments less informative, PRS could be a useful screening tool. Lello et al.  argue, fet al. , and manet al. –49.Aside from the variable predictive power, and hence clinical utility, of PRS, there are at least two major stumbling blocks to moving PRS into clinical practice. First, PRS, in summing across many hundreds or thousands of low significance individual estimates of variant–disease association, are especially susceptible to confounding by cryptic population structure. In other words, even if stratification bias is small per locus, it can accumulate across loci in a PRS. Researchers working on GWAS generally address the difficulty of population structure by adjusting associations for a finite number of principal components of genetic variation . But sucBox 1.The sib-pair method is one way to test for the problem of traits covarying with populations, and genetic differences between the populations therefore being statistically associated with the trait in question, despite not being causally involved in the development of the trait . To adapt an example from Coop , imagineBut now, if one tried to use the resulting PRS to predict tea-drinking behaviour within families, it would suddenly become non-predictive, as within a family, all children usually either grow up in a heavily tea-drinking household or not, and any differences between children are the result of idiosyncratic differences, and not associated with the alleles that differ in frequency between people from Liverpool and those from Paris. When the associations found are reduced radically under sib-pair control, it is usually a sign that something has gone awry .why this is the case, that it is the case makes the clinical application of PRS fraught.The other problem, to which we turn in the next section, is that PRS developed for one population tend to be much less predictive when applied to other populations  ,60. And . 5Most currently validated PRS have been developed using data from samples of broadly ‘European’ origin and/or ancestry , and becBox 2.et al. [Duncan et al.  list six(i) True differences due to drift.(ii) True differences due to selection.(iii) True differences in genetic effects due to environmental differences (gene–environment interactions).(iv) Bias due to uncorrected population stratification in discovery and/or training samples.(v) Bias due to our discovery/training population data and/or polygenic scoring methods. Specifically, linkage disequilibrium (LD) structure and variant frequency are captured imperfectly with current methods (including genotyping and imputation), and they vary across populations, and currently available data resources are unequally representative of diverse worldwide populations.(vi) Random error in the estimation of GWAS betas.Both (i) and (ii) involve the possibility that differences in the genetic makeup of two populations might result in a polygenic risk score (PRS) that is predictive in one population not being predictive in the other, because of the differences in allele frequencies between the two populations (see  for a deFor (iii), the issue is that an allele that, in one environment, has a positive effect on the trait , might, in a different environment, have a much weaker effect, have no effect, or even have a negative effect, on the same trait. Here, Lewontin's ‘Analysis of Variance and Analysis of Causes’  is the cFor (iv), and as per For (v), recall that GWAS do not identify actual genes, but rather markers which are presumed to be associated with some (number of) gene(s). If, in one population, variation in a marker tends to be correlated with that of a particular gene (i.e. the marker and gene are in LD), but in another population LD is lower, a PRS developed using the first population will not be as predictive in the second.Point (vi) simply notes the possibility of random errors; while this concern is considerable in GWAS, it is generally thought that markers found to be associated at the standard, very strong, statistical significance levels are unlikely to be spurious. The same cannot be said of the weaker associations used in PRS, many of which are in fact likely to be mis-estimated or entirely spurious.Note that more than one of these problems may occur in any particular GWAS and that these different issues can interact in potentially complex ways.More recently, Mathieson  has suggFor example, while the immediate source of the transferability problem is the markedly skewed (Euro-centric) underlying population distribution of global GWAS discovery efforts ,60,71, tImportantly, no matter which approach is applied, complexities of human population structure  will stymie even good faith efforts to translate polygenic risk information for use with individual patients. First, there is no way to determine, ahead of actually testing them, which population or ancestry genetic clusters are biomedically relevant, making any choice of ancestry categories or subpopulations for effect size  estimation effectively arbitrary; a feature of human genetic variation that is well recognized . FurtherAlong with the lack of an objective way to identify populations of interest, there is the equally significant problem of the disconnect between human population genetic structure and non-genetic population arrangements such as, for example, socially ascribed racial and/or ethnic identity . Often, unlike actual human population structure. While it is true that sophisticated clustering algorithms can tease out elements of genetic population affinities [In other words, if the population structure that mattered for disease risk could be readily identified, and if sociocultural location mirrored genetic population structure, then population structure, understood to reflect both sociocultural position and genomic affinities, could be accounted for in the development and clinical application of PRS. Put another way, if human population structure was straightforward, i.e. if there were in fact only a few major population divisions within our species, little gene exchange between populations, and general homogeneity (of both genetic and non-genetic risk factors) within each of those populations, the problem would be easily solved. In such a world, it would be simple to develop separate PRS for each population, and easy to assign people to the correct referent population for risk analysis using a PRS. But part of the lesson from the kind of study of human populations that Lewontin made famous is precisely that this picture is utterly finities –80, therBut if developing and validating separate PRS for each population of interest is a losing proposition, there are good reasons to be sceptical that developing a PRS using a worldwide population sample could solve this problem either. Recall that predictive accuracy in populations with internal structure can be high, even if many of the variants on which the prediction is based have no causal effect whatsoever. That is, at the population level, we can successfully predict that specific alleles are associated with the trait in question, but we are doing so only because they are associated with genetic population structure, and it is the environmental differences associated with the stratified populations that are doing the real work of increasing disease risks.. 6within populations (causes of incidence) were usually distinct from the causes of differences in disease prevalence between populations . The other take-home message from Rose was that the causes of between-population prevalence were usually where the public health action was. If one wants to make major changes in population-level health outcomes, i.e. to identify and intervene on the risk factors responsible for health inequities, attention to the causes of prevalence usually makes more sense than a focus on identifying and intervening on individuals' exposure to the causes of incidence. But, of course, using PRS to guide clinical practice is to focus on causes of incidence (a very particular set of causes: genetic predispositions to disease), and hence to not only disregard more salient risk factors but to misdirect public health intervention.There is a certain irony in the utility of PRS being wrecked on the shoals of population structure. Twenty years ago, Rose  publisheThere are at least two potential negative consequences of this misdirected attention. First, in labelling only those at the furthest limits of the incident population distribution ‘high risk’, PRS  may contribute to false reassurance among those whose social identities nevertheless place them at higher  risk due to the experience of systemic disadvantage and discrimination. This can promote both individual complacency and broader harms, such as those associated with delayed diagnosis and treatment. Second, it may inappropriately lay the blame for poorer health outcomes on incident  susceptibilities, suggesting that the uptake of an earlier colonoscopy  by those at the highest end of the genomic risk distribution will compensate for myriad unmeasured and unaddressed social determinants of health. The degree to which these negative consequences come to pass is still an open question and, as has been argued elsewhere ,82, empi. 7The problem here, as was the case in Lewontin's intervention 50 years ago, is that neither health risks nor sociopolitical relationships can be adjudicated by scientific descriptions of human genetic variation. On its face, this is confusing: if racial discrimination is justified by supposed evidence of racial biological distinction, then surely definitive proof that such biological distinction is a fallacy would effectively, and permanently, undermine discriminatory beliefs and practices. But this ignores the complex ways in which population genetic observations are taken up and used, or alternatively ignored , for str


Marsilea drummondii and Eleocharis acuta, we investigated the proportions of sexual and asexual reproduction and self-fertilisation by employing next-generation sequencing approaches, and we used this information to understand the population genetic structure of a large inland floodplain in western New South Wales (NSW), Australia. Asexual vegetative reproduction and self-fertilisation were more common in M. drummondii, but both species used sexual reproduction as the main mode of reproduction. This resulted in a highly differentiated genetic structure between wetlands and a similar genetic structure within wetlands. The similarity in genetic structure was influenced by the wetland in the two species, highlighting the influence of the floodplain landscape and hydrology on structuring population genetic structure. The high levels of genetic variation among wetlands and the low variation within wetlands suggests that dispersal and pollination occur within close proximity and that gene flow is restricted. This suggests a reliance on locally sourced (persistent) seed, rather than asexual  reproduction or recolonisation via dispersal, for the population maintenance of plants in dryland rivers. This highlights the importance of floodplain inundation to promote seed germination, establishment, and reproduction in dryland regions.Aquatic plants share a range of convergent reproductive strategies, such as the ability to reproduce both sexually and asexually through vegetative growth. In dryland river systems, floodplain inundation is infrequent and irregular, and wetlands consist of discrete and unstable habitat patches. In these systems, life history strategies such as long-distance dispersal, seed longevity, self-fertilisation, and reproduction from vegetative propagules are important strategies that allow plants to persist. Using two aquatic plants,  Phragmites australis [Decodon verticillatus [Cladium jamaicense [Callitriche [Aquatic plants are an extremely heterogeneous group of species sharing a range of convergent reproductive and other life history traits, which allow them to live and thrive in aquatic environments ,2. One oustralis , Decodoncillatus , and Clamaicense ). The chmaicense , and pollitriche ).Sexual reproduction increases genetic diversity, while asexual reproduction maintains the genetic uniformity of well-adapted gene complexes ,7. The hEichhornia crassipes (water hyacinth), sub-optimal seed production was related to inefficient pollinator service and seedlings were only recorded at sites with saturated soil and shallow water conditions. In the aquatic macrophyte Phragmites australis (common reed), seed production was found to be highly variable between sites and years, and was related to rainfall and temperature [Decodon verticillatus (swamp loosestrife), where asexual reproduction became more dominant toward the geographical range limits of the species. This was caused by reduced sexual performance related to genetic traits (such as reduced pollen tube growth and ovule penetration) and ecological factors (primarily changes in temperature) [Asexual reproduction provides an alternative reproductive strategy when sexual recruitment is challenging or limited. Barrett  observedperature . Intersperature) . For thierature) .Carex platyphylla (broad-leaf sedge) [In vegetative  reproduction, there are two levels of population structure that can provide meaningful data on plant demography: the ‘ramet’, which is the unit of clonal growth , and the ‘genet’, which is the colony of all the genetically indistinct ramets  . The orgf sedge) .Cladium jamaicense (swamp sawgrass), with the genetic structure reflecting patterns from colonisation maintained through long-lived clones [Sparganium erectum (branching bur-reed).A range of factors are likely to influence the population genetic structure in aquatic plants, such as hydrology  and the d clones . Similard clones  observedOne reason for the high genetic differentiation often observed among populations is that long-distance dispersal (>100 m) is rare in plants ,22. DespLaminaria rodriguezii (kelp) [Chara spp. [The genetic distinctiveness of individuals has long been a challenge in plant demography, especially for species that are clonal, where a plant census is unsuitable . Recent i (kelp)  and Charara spp.  to be de2 in the southeast of Australia and is an important region of Australia for agricultural production as well as supporting around 2.3 million people and a range of environmental assets [The Murray–Darling Basin covers an area of 1 million kml assets . Many ril assets . These sl assets ,31. Marsilea drummondii A. Braun (common nardoo) and Eleocharis acuta R.Br. (common spike-rush). On the floodplains of the Murray–Darling Basin, M. drummondii and E. acuta are often the dominant groundcover during and after flooding events, forming monospecific stands or clumps (Pers Obs. W Higgisson and F Dyer). Being common and widely distributed, M. drummondii and E. acuta play an important role in maintaining functioning river floodplain ecosystems within the Murray–Darling Basin. Using these two species, we investigated the extent and spatial arrangement of (1) asexual vegetative propagules, (2) self-fertilisation, and (3) plants in a parent–offspring relationship across a large inland floodplain in western New South Wales (NSW), Australia using next-generation sequencing approaches. Using this information, we also explored the population genetic structure of both species. Understanding the population genetic structure of these plants provides important information on mating patterns, resource allocation, and the role of hydrology in the maintenance and establishment of these and other plants on floodplains in dryland regions.Two aquatic/semi-aquatic plants that reproduce sexually and asexually via vegetative growth and are common on the floodplains of the Murray–Darling Basin are This study was carried out in three wetlands on the floodplain of the lower Lachlan River, NSW, Australia , where bMarsilea drummondiiMarsilea drummondii (Marsileaceae) is an aquatic/semi-aquatic perennial fern widely distributed across inland, mainland Australia [ustralia . It is aMarsileaceae is an aquatic and semi-aquatic heterosporous perennial rooted water fern that forms rhizomatous spreading clumps [Marsilea produces a modified seed-like spore-bearing leaf known as a sporocarp, which has a thick sclerenchymatous wall that protects the enclosed sori against damage, such as insect attacks and dryness [Marsilea drummondii sporocarps are 4–9 mm long, on stalks 8–35 mm long [g clumps . Ferns s dryness . When st mm long .Marsilea is highly dependent on the flooding regime and the wetting and drying cycles, with the entire reproductive biology dependent on water for the release of spores from the sporocarp, the dispersal of spores following fertilisation, and the sinking of young developing embryos [Marsilea (including in M. drummondii) occurs primarily on mud as a habitat dries [Marsilea species have been reported to germinate from herbarium material up to 100 years old [Marsilea drummondii has germinated in soil seed bank studies, indicating their ability to remain viable and contribute to the soil seed bank .Reproduction in  embryos . Sporocaat dries . Viable ears old , highligM. mucronata have been shown to pass intact through the digestive tract of waterbirds [M. drummondii range from 4 to 9 mm long) are predicted to limit wind dispersal [The sporocarp can be transported by adhesion, such as to the external surfaces of birds, and can be ingested . The spoterbirds . The sizispersal . Hydrochispersal . Once thispersal .Eleocharis acutaEleocharis acuta (Cyperaceae) is a rhizomatous perennial plant that grows to 90 cm high [Eleocharis acuta occurs along the eastern and southwestern mainland of Australia, and across Tasmania as well as New Zealand, New Guinea, and Norfolk Island [ cm high ,42. EleoEleocharis acuta seeds can float on water (50% of seeds floated for >30 days), and the species is likely to disperse via hydrochory; the seeds are dormant, although the mechanism involved is unknown, and they require extended contact with water to germinate (>40 days) [E. acuta seeds were recorded in waterbird faecal samples by Green et al. [Cyperaceae are wind-pollinated [E. acuta, the spikelet of E. laeviglumis from southern Brazil is dichogamous and protogynous, with its reproductive organs maturing at different times [40 days) . Intact n et al. , suggestllinated . While tnt times , which rM. drummondii and E. acuta were collected from Lake Nooran, Oxley Lagoon, and Lake Noonamah. Lake Nooran is an open temporary wetland a few hundred metres from the main channel of the Lachlan River, in a complex of wetlands where the Lachlan River terminates. Oxley Lagoon is in close proximity (<50 m) to the Lachlan River up-stream of Lake Nooran, while Lake Noonamah is on an ephemeral channel off the main stem of the Lachlan River . DArTseq™ represents a combination of a DArT complexity reduction method and next-generation sequencing platforms [M. drummondii and 150,801 SNPs for E. acuta.DNA extraction was performed using protocols developed by Diversity Array Technology Pty Ltd. . Tissue samples (25 mg) were incubated overnight at 56 °C with lysis buffer and proteinase K. Lysed tissue was washed using a washing buffer to remove impurities, such as proteins and polysaccharides, and stored in an elution buffer. Genotyping was undertaken on samples from each species with a genome-wide profiling approach using restriction enzymes to produce DNA fragments, which were pooled (for each species) and sequenced using next-generation platforms by Diversity Arrays Technology, Canberra, Australia , which removed or significantly reduced the occurrence of SNP errors in the datasets. We also filtered the data for average repeatability of alleles at a locus of 99.8% and a call rate threshold of 0.95; all secondary SNPs where then removed. This filtering left 1241 SNPs for M. drummondii and 6618 SNPs for E. acuta, which were used to determine the three levels of pairwise relatedness. The SNP data supplied by DarTseq and associated metadata were read and filtered using the software package dartR, Version 1.9.1 . As thisE. acuta and S2 for M. drummondii, showing the number of inconsistent loci and the total number of loci used in the analysis between each pair of samples in each of the three relatedness scenarios. We reported only the pairs with no (0) locus differences in the results, but highlighted that those pairs with a single or few locus differences may also be in the relatedness scenarios.We have provided the results from the relatedness analysis for each species as S1 for M. drummondii and 90 E. acuta genotypes for further analysis. These datasets were then re-filtered as described above to produce a dataset of 1242 SNPs for M. drummondii and 6623 SNPs for E. acuta.Following the relatedness analysis, a single representative of each genotype  from each patch was retained , leaving 84 Ho) and expected (He) heterozygosity were calculated for each species across all samples, within each wetland and within each patch using the function gl.report.heterozygosity from the package dartR [FIS) was calculated for each patch and for each species, using the function boot.ppfis in the package Hierfstat, version 0.5.7 [FIS = 1−(Ho/He), with 1000 bootstraps.The observed  was undertaken on all genotypes for each species in dartR, version 1.9.1 . The gension 6.5 .M. drummondii and 90 for E. acuta  and the third from Patch 2 . No other patches contained clones for either species.The 91 samples for each species collected from two patches in Lake Nooran and Oxley Lagoon and three patches in Lake Noonamah (13 samples per patch) yielded 83 genotypes for E. acuta . Seven M Patch 2 . In E. aM. drummondii, we only detected pairwise samples in a parent–offspring relationship via self-fertilisation in Lake Noonamah   .M. drummondii, a total of 422  pairs were in a parent–offspring relationship through outcrossing . In E. acuta, there were three pairs that had between two and five inconsistent loci (out of 6588 loci) in the clonality scenario, while the next most similar pair had 41 locus differences  across all genotypes of 0.105 compared with E. acuta, which had a mean oH of 0.165  for M. drummondii was greater than 0 at Lake Noonamah’s Patches 1 and 2, and slightly greater than 0 at Oxley Lagoon’s Patch 2, which indicated that inbreeding had occurred at these patches. In E. acuta, FIS was less than 0 in all patches, demonstrating excess heterozygosity as compared to expected in all patches, and the widespread occurrence of outcrossing . Most samples were clustered within the wetland patch from which these were collected  and highlighted a strong differentiation between samples collected from Lake Noonamah and those from Oxley Lagoon and Lake Nooran , which was significant in both species  between Lake Noonamah’s Patch 2 and Lake Noonamah’s Patch 3 to the highest difference between Lake Nooran’s Patch 1 and Lake Noonamah’s Patch 3 (FST = 0.40). For E. acuta, the FST ranged from 0.03 between Lake Noonamah’s Patches 1 and 2 to ~0.31 between all the Lake Noonamah patches and Lake Nooran’s Patches 1 and 2  of a single genet (clone) in this species. A genotype in Lake Noonamah was sampled from two patches within the same wetland, highlighting the potential ability  of asexual dispersal via vegetative propagules within a wetland. In E. acuta, only one pair of identical samples was detected within a patch at a distance of 50 cm, demonstrating that vegetative reproduction also occurs in this species; however, the size of an individual genet is no more than 50 cm and is likely to be most commonly smaller.Seven samples contributed to the same genotype in M. drummondii and unlikely in E. acuta, and may be considered lower than what would be expected in aquatic macrophytes [Phragmites australis was recorded predominately from generative  diaspores while Potamogeton natans reproduced exclusively by vegetative propagules [E. acuta and M. drummondii is through the production of rhizomes, which have a low dispersibility compared to other vegetative organs such as stolons, runners, and bulbils [Phragmites australis, can float for at least six months under experimental conditions once removed from the soil [E. acuta and M. drummondii, a combination of destructive removal (such as by birds) coinciding with flooding conditions may be required.Although the results show that asexual vegetative reproduction does occur, the dispersal of asexual propagules away from the parent plant is rare in rophytes ,17. Boedopagules . The veg bulbils . Howeverthe soil . For vegE. acuta and M. drummondii have persistent soil seed or spore banks [Sexual reproduction appears to be the dominant reproductive strategy in both species, and patches consisted of numerous genets. This suggests that patches are regularly invaded by additional genotypes of sexual origin and that genets are rarely large and dominant. Both re banks ,43 and tM. strigosa growing in the Mediterranean basin was found to rely on sexual rather than vegetative reproduction, with the authors suggesting that sporocarps were the only means for this species to survive the harsh winter conditions [M. drummondii and E. acuta on dryland river floodplains. The longevity of E. acuta seeds and M. drummondii sporocarps may help to reduce genetic drift and retain genetic variation in populations.The reliance on sexual reproduction rather than vegetative shown here may be related to the infrequent and variable flooding regime and the extended dry phases experienced on river floodplain systems in dryland regions such as the lower Lachlan River , comparenditions . The resM. drummondii produce both male and female sex organs, megaspores , and microspores , and as such, they are partially hermaphroditic and have the ability to self-fertilise [M. drummondii can and does reproduce via self-fertilisation, with nine samples primarily from the same site in parent–offspring relationships via self-fertilisation. Self-fertilisation has been demonstrated in M. strigosa in the Mediterranean [M. drummondii and potentially other species where reproduction occurs in water. Among the population variation in plant mating systems (self-fertilisation vs. exclusive outcrossing), within-species variation has been observed in other plants [As with self-compatible flowering plants, heterosporous ferns such as ertilise . As theserranean . Interesr plants .E. acuta, only one pair of samples was in a parent–offspring relationship through self-fertilisation, suggesting that while this species may reproduce via self-fertilisation, mechanisms to reduce self-fertilisation such as dichogamy, as observed in E. laeviglumis [Cyperaceae, such as self-incompatibility in Rhynchospora ciliata [In viglumis , exist.  ciliata .Marsilea species, Schneider and Pryer [M. drummondii has no apparent adaptation to reduce selfing, apart from the opportunistic mixing of spores. The greater the number of spores from different genotypes on the water surface, the more likely is cross-pollination.During a reproductive experiment of multiple nd Pryer  observedM. drummondii and E. acuta were both highest at Oxley Lagoon, intermediate at Lake Nooran, and lowest at Lake Noonamah. These similar patterns, despite different life histories, highlight how genetic diversity in the aquatic plants of dryland river floodplains are driven by numerous factors, including geographic barriers and the hydrological regime. Oxley Lagoon is situated in very close proximity to the Lachlan River (<50 m) and Lake Nooran is approx. 200 m away, while Lake Noonamah is >5 km from the main channel, suggesting that the distance from the river may have influenced the levels of genetic diversity. Genetic diversity was found to increase in downstream populations of Sparganium emersum (unbranched bur-reed), which was related to the effects of unidirectional downstream flow on diversity patterns [The measures of within-wetland genetic diversity found in patterns . SimilarE. acuta, but more frequent in M. drummondii. This high genetic differentiation further suggests that colonisation  occurs from the local propagule pool (deme/wetland), and that the mixing of colonising individuals from different demes is unlikely [The high levels of genetic differentiation and clustering within patches and wetlands suggests that pollination and dispersal most likely occur in proximity to the parent plants. Colonisation (founder) events between wetlands appear to be very rare in unlikely . The limunlikely , as wellE. acuta seeds floated for at least 30 days in an experiment by Higgisson and Dyer [E. acuta, here we show that the realised seed dispersal is most likely within close proximity to the parent plant and within a wetland. Although the hydrochorous dispersal of sporocarps is unknown, these start to open within a few hours of contact with water [M. drummondii individuals were scattered beyond the patch they were sampled from in the PCoA suggests that gene flow is occurring between wetlands; this situation was not observed for E. acuta. Fifty percent of and Dyer , indicatth water , possiblM. drummondii, the sporocarps of M. mucronata have been shown to pass intact through the digestive tract of waterbirds [E. acuta were recorded in faecal samples of the Grey Teal waterbird, none of these seeds germinated during experimental trials [E. acuta, compared with M. drummondii.The floodplain of the lower Lachlan River is known as a nesting and feeding habitat for a range of waterbirds, which have been recorded traveling along the Lachlan River and between its wetlands . In the terbirds , showingl trials . A lowerM. drummondii and E. acuta, sexual reproduction is the dominant reproductive strategy. Dispersal between wetlands (and even between patches) is infrequent in M. drummondii and extremely rare in E. acuta. This suggests a reliance on local seed sources and seed or spore bank longevity. Low dispersal means that recolonising wetland patches is challenging, especially under a drying climate across the study region where the timing, frequency, and volume of precipitation is likely to dramatically change. In river floodplain systems in dryland regions, such as the lower Lachlan River, the recolonisation of patches following a disturbance such as an extended drought may be unlikely. These results highlight the importance of protecting existing populations and maintaining diverse and functioning wetlands. There may also be a need to collect and store propagules for future restoration activities. Once lost from a wetland, these species may not have the ability to recolonise, especially under regulated flow conditions.While asexual vegetative reproduction does occur in


STAT3 gene account for autosomal dominant hyper-IgE syndrome (AD-HIES). Patients typically present high IgE serum levels, recurrent infections, and soft tissue abnormalities. While current therapies focus on alleviating the symptoms, hematopoietic stem cell transplantation (HSCT) has recently been proposed as a strategy to treat the immunological defect and stabilize the disease, especially in cases with severe lung infections. However, because of the potentially severe side effects associated with allogeneic HSCT, this has been considered only for a few patients. Autologous HSCT represents a safer alternative but it requires the removal of the dominant negative mutation in the patients’ cells prior to transplantation. Here, we developed allele-specific CRISPR-Cas9 nucleases to selectively disrupt five of the most common STAT3 dominant negative alleles. When tested ex vivo in patient-derived hematopoietic cells, allele-specific disruption frequencies varied in an allele-dependent fashion and reached up to 62% of alleles harboring the V637M mutation without detectable alterations in the healthy STAT3 allele. However, assessment of the gene expression profiles of the STAT3 downstream target genes revealed that, upon activation of those edited patient cells, mono-allelic STAT3 expression  is not able to sufficiently restore STAT3-dependent signaling in edited T cells cultured in vitro. Moreover, the stochastic mutagenesis induced by the repair of the nuclease-induced DNA break could further contribute to dominant negative effects. In summary, our results advocate for precise genome editing strategies rather than allele-specific gene disruption to correct the underlying mutations in AD-HIES.Dominant negative mutations in the  STAT3 gene account for a variety of human conditions including immune deficiencies and cancer [STAT3 have been associated with hyper-immunoglobulin E syndrome (HIES), a rare primary immune deficiency characterized by elevated serum IgE levels and recurrent skin and lung infections [STAT3 missense mutation associated with AD-HIES [STAT3 allele are phenotypically normal [STAT3 allele might be beneficial for STAT3-HIES patients. The latter could be achieved with designer nucleases, engineered proteins capable of introducing a DNA double strand break (DSB) in the DNA helix at desired genomic locations [Signal transducer and activator of transcription 3 (STAT3) is a key cytoplasmic transcription factors that regulates immune cell activation and differentiation through the transduction of signals initiated by a wide variety of cytokines and growth factors . Given id cancer . In partfections . Since t AD-HIES , more th AD-HIES . Since S AD-HIES . STAT3-H AD-HIES . Treatme AD-HIES , data on AD-HIES ,12. The  AD-HIES  have to y normal , strategocations . This evocations . Therefoocations ,18. ConsSTAT3 allele harboring the autosomal dominant mutation causing STAT3-HIES. We selected five common dominant-negative STAT3 mutations in an attempt to validate a strategy that would be applicable to the majority of STAT3-HIES patients. We successfully developed allele-specific nucleases capable of inactivating the selected mutated alleles through the deposition of indel mutations with frequencies approaching 62%, without any effect on the healthy allele. However, when tested functionally, the edited cells were indistinguishable from non-edited cells, suggesting that STAT3 expression driven from a single allele is not sufficient to restore proper STAT3 signaling. Here, we explored the high-specificity of the CRISPR-Cas9 system to selectively disrupt, in an allele-specific manner, the Streptococcus pyogenes Cas9 was kindly provided by J. Keith Joung (Addgene plasmid #43861). All of the single guide RNA (sgRNA) expression plasmids were cloned through an oligo-cloning procedure. In brief two complementary oligonucleotides . First, a multiple cloning site was introduced into the plasmid #890 digested with SalI and NheI by oligo-cloning of the primers #2413 (5′-ctagccgacctgcaggtaccggtggatccatgcatg) and #2414 (5′-tcgacatgcatggatccaccggtacctgcaggtcgg), as described above, resulting in plasmid #890-MCS. Subsequently, the open reading frame (ORF) of a destabilized green fluorescence protein (dsGFP), which included a self-cleaving T2A peptide, was released from the plasmid #1265 via BamHI and AgeI digestion and ligated using the same restriction sites into the #890-MCS, resulting in #890-MCS-T2A-dsGFP. A gBlock (Integrated DNA Technology) containing the sequence of the EF1α short promoter (EFS) and the STAT3 ORF including the five selected mutations . To generate the reporter plasmid #1665 (pCCL_EFS_STAT3wt_T2A_E2Crimson), the mutated STAT3 and the dsGFP sequences from #1664 were replaced via Gibson Assembly using two gBlocks (IDT) containing either the normal STAT3 ORF (using NheI and SfbI restriction enzymes) or the E2-Crimson ORF , respectively.The expression plasmid for the utations A was clo2. The cell lines described in this study were authenticated using a 16 DNA marker profile . HEK293T cells and derivate reporters were kept in Dulbecco’s modified Eagle medium  supplemented with 10% FCS , 1% penicillin/streptomycin , 1 mM sodium pyruvate (Merck), and grown in plates for adherent cells  kept at 37 °C in 5% CO2 for 4 h before activation with the T Cell Activation/Expansion Kit (Miltenyi) according to the manufacturer’s protocol. After 3 days, beads were removed and the cells were expanded at a concentration of 1.0 × 106 cells/mL or 0.5 × 106 cells/cm². When necessary, the cell number and viability were measured using the NucleoCounter NC-250 (ChemoMetec) and Solution 18 containing acridine orange and DAPI according to the manufacturer’s protocol. For genome editing experiments, 1.0 × 106 PBMCs were transfected four days after thawing . To this end, 20 pmol Cas9 protein (Integrated DNA Technology) and 100 pmol of the indicated sgRNAs  were precomplexed at 37 °C for 10 min before transfection using the P3 Primary Cell 4D-Nucleofector (Lonza) and the EO115 program. Afterward, cells were seeded in a 48-well plate and expanded until further analysis. For functional assays, IL2 was removed one day before stimulating 0.5–1.0 × 106 the PBMCs for 1 h with 50 ng/mL IL21 (PeproTech).Human peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte reduction system (LRS) chambers by density gradient centrifugation (Ficoll-Paque) and kindly provided by the Blood Donation Center of the Medical Center, University of Freiburg after routine plateletpheresis and anonymized informed donor consent. PBMCs from AD-HIES patients were purified from whole blood samples received from the FREEZE Biobank of the Medical Center–University of Freiburg after anonymizing the informed donor consent and approval of the local ethics committee. In this case, PBMCs were isolated and purified on a Biocoll gradient  and used for allele-specific knock out experiments. PBMCs were kept in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS (Thermo Fisher Scientific), 1% penicillin/streptomycin (Merck), and 100 U/mL IL-2 in plates for suspension cells (Sarstedt) at 37 °C in 5% COAllele-specific nucleases were preselected in an episomal reporter assay. One day before transfection, 100′000 HEK293T cells were seeded in a 24-well plate in 450 μL medium. The day of transfection, the cells were transfected with a DNA mixture containing 100 ng of each reporter plasmid, 600 ng of Cas9, and 200 ng of sgRNA expression plasmids, respectively. To normalize for transfection efficiency, the DNA mixture contained 50 ng of a BFP expression plasmid to reach a total amount of 1250 ng of transfected DNA. Two days after transfection, the cells were analyzed by flow cytometry on a BD FACSCanto II (Becton Dickinson). After isolating the single (FSC-A/FSC-H) and living cells (FSC-A/SSC-A), the mean fluorescence intensity (MFI) of dsGFP and E2-Crimson were recorded in the BFP-positive cell population. All samples were analyzed in technical triplicates and the assay included at least three biological replicates. For allele-specificity validation in the HEK-STAT3mut cells, 200,000 cells were nucleofected using the CM130 program using the SF Cell Line 4D-Nucleofector (Lonza). The nucleofection mix included the Cas9 protein and the selected sgRNA, preassembled to form active RNPs at 37 °C for 10 min in a 1:1 molar ratio with 30 pmol each. Nucleofected cells were seeded in a 24-well plate and expanded until further analysis.STAT3 mutation  following the manufacture’s procedure. PCR amplicons encompassing the nuclease target sites were amplified from 50 ng of genomic DNA using primers specific for each mutation  using Q5HPRT1, SOCS3, BATF, and IRF4 expression are listed in HPRT1 gene.The effects of the STAT3-HIES allele-specific inactivation on STAT3 signaling were assessed via analysis of the expression levels of STAT3-specific transcriptional target genes by droplet digital PCR. At the indicated time points, the total RNA was extracted utilizing the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol and the RNA concentration and quality were determined using the Nanodrop 2000 device (Thermo Fisher Scientific). A total of 150 ng of RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s recommendations. The expression levels of selected STAT3-specific transcriptional targets were measured using the QX200 Droplet Digital PCR System (Bio-Rad Laboratories) and the QX200 ddPCR EvaGreen Supermix. The primers used to detect STAT3 mutations that accounted for 47% of the total patients identified in our cohort [To establish a targeting strategy that could be applicable to the majority of STAT3-HIES patients, we selected five frequent r cohort . These ir cohort .STAT3 alleles, we exploited various ways to favor the targeting of the mutated STAT3 allele. Since mismatches in the ‘seed sequence’ of the CRISPR-Cas protospacer have a stronger impact on Cas9 binding [We used the CRISPR-Cas9 genome editing system to introduce a DSB in proximity to the selected mutations. Targeted indel formation, consequent to the repair of the Cas9-induced DSB via NHEJ, eventually led to allele-specific inactivation. Given the high sequence identity between the two  binding , we firsSTAT3 allele, thus favoring the occurrence of DSBs in the mutated allele. To facilitate the screening of CRISPR-Cas9 nucleases capable of allele-specific targeting, we established a dual fluorescent reporter system. A first expression plasmid encoded a fusion protein that included the red fluorescent E2-Crimson protein fused by a T2A self-cleaving peptide to the normal STAT3. A second plasmid coded for a destabilized green fluorescent protein (dsGFP) fused to an artificial STAT3 harboring the five selected mutations. We then assessed the activity of the designer nucleases by co-transfection of their respective expression plasmids with the reporter plasmids in a HEK293T cell line. A reduction in the mean fluorescent intensity (MFI) of either E2-Crimson or dsGFP signals compared to the controls is therefore indicative of the nuclease ability to cleave the respective plasmid. In particular, reduction in the green signal with the absence of red signal alterations identifies an allele-specific nuclease  to normalize for transfection efficiency  via lentiviral transduction, as previously described [PTBP2) and of STAT3 were measured in human-derived peripheral blood mononuclear cells (PBMCs) and the parental HEK293T cells. While PBMCs harbored two copies of each gene, hypotriploid HEK293T cells had four copies of STAT3 and three copies of PTBP2, respectively  assay, as previously described [Having identified at least one allele-specific nuclease for four out of the five escribed . Upon clectively . We perfectively . We usedescribed . In lineSTAT3 alleles, their cognate target sites could only be found in patient-derived cells. Before approaching this source of limited cells, we sought to establish the most efficient conditions to achieve high gene disruption frequencies in human PBMCs. As a control, we used a CCR5-specific nuclease (CCR5#2) that was previously established in our laboratory [STAT3 allele-specific nucleases for disruption of the non-mutated STAT3 gene in cells derived from an healthy donor. We activated the PBMCs with beads coupled with anti-CD2, -CD3 or -CD28 antibodies to expand the lymphocyte fraction. The beads were removed from the medium one day prior to nucleofection of the cells with the indicated RNPs. The indel frequency was determined ten days later via the T7E1 assay .Since the selected nucleases were specific for the mutated boratory . In addiE1 assay A. The CCE1 assay B. Indel ectively B. Then, STAT3 alleles represented about 50% of the total NGS reads. As the patients were heterozygous for the STAT3 mutations, we therefore concluded that all of the tested nucleases were highly selective and able to discriminate between the healthy and mutant STAT3 alleles. As expected, the non-selective H1 nuclease reduced the count of non-edited normal alleles by about 20%, which is in line with the results achieved with the reporter systems.All of the selected allele-specific nucleases were effective in introducing indel mutations in their respective target sites, with frequencies ranging from 30% to 60% C. ImportSOCS3, BATF, BCL6, FOSL2, IKZF2, IRF4, RORA, and SMAD7 upon activation of STAT3 signaling [SOCS3, BATF, and IRF4 , we chose to use this gene to monitor for any effects of mutated STAT3 allele disruption in the AD-HIES patient-derived cells. We first validated reduced SOCS3 upregulation in patient-derived cells, which ranged between 2- and 4-fold compared to healthy donor cells . To val exon 21 . We achi exon 21 . This ge exon 21 . As expetivation . Since Sor cells A. AlthouMCs VM1, C, we didth IL-21 B.STAT3 might represent a valuable therapeutic opportunity. However, genome editing strategies to precisely correct a genetic mutation such as those resulting in STAT3-HIES rely on the homology-directed repair (HDR) mechanism. This DNA repair pathway is generally inefficient in mammalian cells and as a consequence, therapeutic genome editing has mostly relied on NHEJ-based gene disruption strategies [Transplantation of hematopoietic stem cells of allogeneic source has shown unquestioned success for the treatment of multiple human conditions of the blood and immune system including inherited disorders. However, the intrinsic risks of this procedure require careful evaluation of the patient background, and it is often challenging for the physician to decide whether HSCT is a reasonable solution unless it represents the patient’s last resort. The use of autologous stem cells for transplantation represents a valuable alternative as it would mitigate many of the risks associated with allogeneic HSCT such as graft-versus-host disease as a result of incomplete histocompatibility between the cell donor and the receiving patient. However, this approach can be pursued only after correction of the monogenetic defect underlying the disease. First, evidence that CRISPR-Cas9-based therapeutics can be used in humans  has openrategies ,30.STAT3 allele in patient-derived cells. We assumed that the remaining healthy allele would be sufficient to ameliorate the disease in patient cells, similarly to what has been observed in mouse [STAT3 alleles, we explored multiple strategies to destabilize CRISPR-Cas9 binding to the healthy allele. In particular, we used truncated gRNAs as well as the addition of mismatches and achieved allele-specificity for four out of five selected DN-STAT3 mutations. For one mutation ), we experienced low targeting efficiency for all of the designed nucleases. Since low activity was observed for both the episomal reporter and the genomic alleles while the nuclease was efficiently cleaving the target site in vitro, we reasoned that the epigenetic context was not the cause of the lack of activity. Possibly, the affinity of the CRISPR-Cas9 nuclease for its intended target site was too low for efficient cleavage in cells in the context of a complex genome, but sufficient for in vitro activity on a relatively short DNA fragment. Further tests are necessary to clarify the reason behind the failure in targeting the R382W (c.1144C > T) mutation. Such experiments could provide novel insights on how to select DNA sequences for efficient editing.Taking advantage of the high specificity of the CRIPSR-Cas9 system, we explored this concept in order to selectively disrupt the mutant in mouse . To estaDN-STAT3 alleles harbored indels. Interestingly, we achieved the most efficient allele-discrimination with CRISPR-Cas9 nucleases that were not destabilized. In contrast, CRISPR-Cas9 complexes with truncated gRNAs were effective in the reporter cell lines, but almost completely lost their editing capacity in primary patient cells (Importantly, the nucleases selected from the reporter assays retained their allele selectivity when applied to patient-derived cells. NGS results confirmed the absence of indel formation at the healthy allele for all selected allele-specific CRISPR-Cas9 nucleases, whereas 25% to 62% of the mutated nt cells C. These STAT3 alleles, and hence causing a state of STAT3 haploinsufficiency in the genetically corrected cells, we failed to achieve a measurable benefit in restoring STAT3 signaling, indicating that insufficiency of STAT3 is likewise pathogenic. The expression levels of SOCS3, a direct transcriptional target of STAT3 signaling, were indistinguishable from those measured in non-edited cells. The reasons behind this failure might be multifarious. On one hand, haploinsufficiency might result in the inability to rescue the defect in the presence of a single healthy STAT3 allele [STAT3 pathogenic variance known so far [STAT3 alleles are still not enough to secure normal STAT3 signaling. Such possibilities have not been highlighted in a recent study aimed at the inactivation of a mutated KRT5 allele, causing epidermolysis bullosa simplex [STAT3 allele deletion might be tolerated in mouse [Despite the high efficiency in disrupting the mutant 3 allele . On the n so far , we tend simplex . While tin mouse , this coIn general, the presented results have important consequences for the genome editing field. Our findings suggest that using designer nucleases to target protein-coding regions with the goal of inactivating a mutant allele with dominant-negative effects entails a non-trivial risk of failure. First, haploinsufficiency needs to be excluded as being disease-causing. Furthermore, and maybe even more importantly, when pursuing HDR-mediated precision editing to correct a disease-underlying mutation, the simultaneous generation of alleles harboring indel mutations in the target cell population might represent a risk that has to be carefully evaluated. In conclusion, our results demonstrate that CRISPR-Cas9 nucleases offer the opportunity of allele-specific editing with single base resolution. However, when targeting protein-coding regions, imprecise cellular DNA repair pathways may represent a concern. The use of editing strategies that avoid the insertion of DNA double strand breaks such as base editing  or epige


Magnoliasubsect.Talauma in Cuba has long been debated and exclusively based on traditional morphological study of a limited number of individuals. A more accurate description of leaf morphology variation using geometric morphometrics combined with genetic data could bring consistency to taxa delimitation in this group. Leaf samples for the morphological (243) and genetic (461) analyses were collected throughout the entire distribution range. The variability of each taxon was analyzed through multivariate and geometric morphometry, and 21 genetic markers (SSR). The observed leaf morphological variability was higher than previously described. Morphological and genetic classifications were highly congruent in two out of four taxa. Our data brought evidence that Magnoliaorbiculata can be considered a true species with very clear genetic and morphological limits. The main taxonomic issues concern the north-eastern Cuban populations of Magnoliasubsect.Talauma. The data supported the existence of two clear groups: corresponding mainly to M.minor-M.oblongifolia and T.ophiticola. However, these two groups cannot be considered fully delimited since genetic markers provided evidence of genetic admixture between them. Due to the likely absence of, at least strong, reproductive barriers between these three taxa, we propose therefore to consider them as a species complex.An accurate taxa delimitation, based on a full understanding of evolutionary processes involved in taxa differentiation, can be gained from a combination of ecological, morphological, and molecular approaches. The taxonomy of  SC) over time   , 2007. DSC) .The combination of several species concepts to broadly support species limits is known as integrative taxonomy . Dayrat Magnolia L. is a good model for applying an integrative taxonomic approach. It is the largest genus of the family Magnoliaceae Juss. It includes three subgenera, 13 sections, and an equal number of subsections  of Central and South America, and the Caribbean Islands  R.A. Howard, previously described by Svenhediniatruncata Moldenke. Two years later, Talaumaminor with extremely oblong leaves that they named T.minorvar.oblongifolia León. In the Flora of Cuba, Talauma: T.minor var. minor, T.minorvar.oblongifolia, T.orbiculata and T.truncata. These authors distinguished T.orbiculata and T.truncata based on the largely truncate leaves of the latter, which inhabits only areas around Pico Turquino  Borhidi and T.minorsubsp.orbiculata (Britton and P. Wilson) Borhidi. Afterward, T.ophiticola Bisse and recognized T.oblongifolia (León) Bisse. Years later, T.orbiculata, T.minor, T.oblongifolia and T.ophiticola. The delimitation of each taxon was mainly supported, as in previous works, by leaf morphological characters.In the Supplemental Material of the Flora of Cuba, Talaumaminor to Magnoliaminor (Urb.) Govaerts and considered all the other names of the Cuban Talauma species as synonyms of M.minor  Palmarola, Magnoliaminor, and Magnoliaoblongifolia (León) Palmarola (T.ophiticola synonym of M.oblongifolia due to the existence of one specimen (Bisse and Kohler HFC 5358 HAJB) that has leaves with the characteristics used by M.orbiculata is the only species that could be considered ecologically distinct from the others.Based on anatomical and morphological (vegetative and reproductive) traits described by  M.minor . The latalmarola . In the Magnoliasect.Talaumasubsect.Talauma and aims to (1) assess their phenotypic variability of leaf morphological traits across their full geographic range (2) based on morphological data, evaluate the three main classification systems  of these taxa proposed up to date: the two taxa CS of (3) infer the genetic structure of Magnoliasubsect.Talauma in Cuba; (4) integrate morphological and genetic data to review taxon delimitation in Magnoliasubsect.Talauma in Cuba.All abovementioned taxonomic revisions e.g.,  were exc aims to  assess tMagnoliasect.Talaumasubsect.Talauma throughout their entire distribution range in the mountains of Nipe-Sagua-Baracoa and Sierra Maestra in eastern Cuba . Hence, in total 243 individuals of Magnoliasubsect.Talauma in Cuba .For the genetic analyses, young leaf samples of a total of 461 individuals, belonging to 26 of 30 known localities, were stored in self-sealed bags with silica gel for DNA extraction. The resulting number of DNA samples represented 52% of the known individuals of Magnoliaminor and M.orbiculata, of M.minor, M.orbiculata, M.oblongifolia, and Talaumaophiticola, of M.minor, M.orbiculata, and M.oblongifolia, of Analyses based on morphological variables were aimed at comparing the relevance of each of the three CS previously proposed: the two taxa CS, ci) for each leaf. The eleven variables were recorded for each leaf. Subsequently, the twelve variables were averaged per individual for the 4–8 leaves available per individual. These averages of the twelve variables were used for all the subsequent statistical analyses.In the multivariate morphometry analysis, linear and angular measures of leaf characters were automatically taken from the digital photographs using the R v. 3.4.1  package EFDs)  , to obtaEFDs) . The harEFDs) . The lanEFDs) . All anaEFDs) . Next, tPCA). Differences among taxa were tested according to a one-way nonparametric MANOVA, using Euclidian distance, with 10 000 randomizations. Correction of p-values for multiple testing was done using the Bonferroni method. The multivariate comparisons were done independently for each dataset . All statistical analyses were conducted in R v. 3.4.1  was assessed by a MonteCarlo analysis in PopTools v. 3.23  with 10 v. 3.4.1  and PASTv. 3.4.1 , and theBIC) allowing to choose the best value(s) of G, without any information about individual assignation to the different clusters. The default “mclust” setting was used to assess the 14 types of models which all differ in the covariance matrix landscape  extraction protocol (SSR) , M.mayae Vázquez and Pérez-Farrera (MA40), M.dealbata Zucc. (MA41) and M.cubensissubsp.acunae Imkhan. (MA42) (DNA was extracted from dried leaf tissue using a modified cetyltrimethylammonium bromide (protocol  with Magprotocol . DNA qua. (MA42) , using f. (MA42) . PCR con. (MA42)  with theST   . Then, 1rvester)  was usedrvester) . An indiia sensu .DAPC analysis  was executed in R v. 3.6.1  were retained. Secondly, a discriminant analysis was executed using the number of genetic clusters defined in the previous step. Parallel to the STRUCTURE analysis, a second DAPC analysis was done without M.oblongifolia  and genetic (STRUCTURE) clusters were compared with a Chi v. 2.14 . A heatmci) showed significant differences between the defined species whatever the CS tested (p < 0.0001). Magnoliaorbiculata and Talaumaophiticola displayed the highest and the lowest mean Bci, respectively. Most of the eleven other variables showed significant differences between taxa, whatever the CS , and between M.oblongifolia and T.ophiticola (p = 0.132) when the four taxa CS was considered. Likewise, the leaf area between M.minor and M.oblongifolia (p = 0.115) did not show differences when the three taxa CS was used. When following the two taxa CS  showed an intra-taxon bimodal pattern within M.minor.The results of the multivariate morphometry analysis are summarized in Figs p < 0.000. Magnolip < 0.000. MagnoliM.minor and M.oblongifolia (p = 0.316) and between M.orbiculata and T.ophiticola (p = 0.406), when referring to the four taxa CS  between taxa for each of the CS in the linear and angular measures dataset , and G = 6 for the Landmark dataset (with other ghost clusters)  Fig. . It was M.oblongifolia, and most individuals of T.ophiticola and M.minor, were assigned to the same cluster (cluster 1), while most individuals of M.orbiculata were assigned to a different cluster (cluster 4). Therefore, Elliptic Fourier descriptors were efficient to discriminate between M.orbiculata on the one hand and the 3 other taxa on the other hand. Finally, the analysis carried out on the matrix of landmarks showed a similar pattern to that obtained with the linear and angular variables for M.minor, T.ophiticola and M.orbiculata. The main difference between these two analyses (matrix of landmarks and linear and angular dataset) was that in the first one, M.oblongifolia was split into two clusters, one of which was shared with M.minor and the other one with T.ophiticola .Thus, despite a continuous variation of leaf morphology across taxa, a clear delimitation of Magnoliaminor and Talaumaophiticola, while the lowest diversity was found in Magnoliaorbiculata. The expected heterozygosity was similar in the four taxa . Magnoliaorbiculata contributed mainly to this result since it was highly differentiated from the three other taxa, while M.minor and M.oblongifolia were the less differentiated taxa (Table M.oblongifolia)  M.oblongifoliaand  (16) individuals, but also included some individuals (14) of T.ophiticola.  displayed genetic admixture . Based on these “admixed” individuals, the level of genetic admixture varied according to taxa. The mean value of probability to belong to their a priori cluster (defined from their taxonomic status) was 0.624 (±0.104) for M.orbiculata, 0.678 (±0.172) for M.minor (including M.oblongifolia), and only 0.477 (±0.262) for T.ophiticola.The species with the greatest genetic diversity were xa Table . The genla. Fig. . The thila. Fig.  was predMagnoliaorbiculata was strongly homogeneous pertaining to ancestry coefficient values with only four individuals displaying genome admixture with the “minor-oblongifolia” cluster  of the individuals of M.minor showed genome admixture with the “ophiticola” cluster. In M.oblongifolia, 51% of the individuals exhibited an ancestry coefficient over 0.9 to the “minor-oblongifolia” cluster, the rest showed high admixture levels. Moreover, it is noticeable that one individual of M.oblongifolia displayed a very high ancestry to M.orbiculata. The localities of Cupeyal del Norte (CN), Monte Fresco (MF), Piedra la Vela (PV), and Cayo Guam (CG) show the highest levels of misclassification of M.minor and M.oblongifolia into the “ophiticola” cluster.ter Fig. . This isT.ophiticola, 56.4% (97/172) of individuals could be assigned to the “ophiticola” genetic cluster while 8.14% (14/172) could be assigned to the “minor-oblongifolia” genetic cluster . Similar to M.minor and M.oblongifolia, many individuals of T.ophiticola (61/172) also displayed signals of genetic admixture, mainly with the “minor-oblongifolia” cluster, but also, for a few of them, with the “orbiculata” cluster. The localities of Subida a la Melba (SM), Mina Iberia (MI), and Sur de las Delicias del Duaba (SDD) showed the lowest levels of misclassification. Four individuals from Cupeyal del Norte (CN) were clustered with the group of M.orbiculata. Most individuals from La Melba (MEL), Pico Cristal (PC), and Monte Fresco (MF) showed an ancestry coefficient similar to the “minor-oblongifolia cluster”. The clustering analysis without individuals of M.oblongifolia also provided K = 3 as the best solution  K = 3 and K = 4. When K = 3, individual assignment displayed a pattern very similar to that found with the Bayesian clustering approach; with one cluster predominantly composed by M.minor and M.oblongifolia, the other cluster with T.ophiticola, and the third one with the individuals of M.orbiculata. In the three clusters, some level of misclassification was found. Many individuals “misclassified” in the DAPC analysis were the same that were “misclassified” based on the STRUCTURE analysis. The DAPC analysis confirmed the correspondence of M.orbiculata to a unique genetic cluster as expected because of its high genetic differentiation from the three other taxa . The concordance between the two classifications (genetic and morphology) was especially high for Magnoliaorbiculata and M.minor, and to a lesser extent for M.oblongifolia and Talaumaophiticola  were similar to those reported by M.orbiculata and M.oblongifolia. For M.minor and T.ophiticola, the average values of BCI are slightly lower and slightly higher, respectively, than those reported by Magnolia, where very little variation is observed in flower and fruit characters . Moreover, our data showed that very few M.orbiculata individuals displayed genetic admixture with M.minor. Similarly, a few M.oblongifolia and T.ophiticola individuals displayed genome admixture with M.orbiculata. The levels of genetic differentiation among species are influenced by the time of separation and the amount of gene exchange  and Nipe-Sagua-Baracoa (habitat of the other species) may have acted and still acts as a barrier to gene flow by strongly limiting pollination and seed dispersal. MagnoliacubensisUrb.subsp.cubensis (from the Sierra Maestra) and Magnoliacristalensis Bisse (from Nipe-Sagua-Baracoa), both from subsection Cubenses. Magnolia diversification in the Neotropics. Therefore, it seems more likely that the admixture signal between M.orbiculata and the other taxa could rather be explained by shared ancestral polymorphism with other Cuban talaumas due to the likely recent diversification of the subsection in Cuba, that is less than 5 mya according to M.orbiculata from the other taxa. However, the possibility of rare events of inter-taxa hybridization involving M.orbiculata as one parent cannot be totally ruled out, especially because individuals displaying admixed genome involving M.orbiculata have intermediate ancestry coefficients, which is compatible with a hypothetical first- or early-generation hybrid status. M.orbiculata is differentiated from that of the other taxa considered in this study. This may suggest that selection against first- or early-generation hybrids due to local adaptation could also contribute to preventing genetic exchanges between that taxon and the other taxa of Magnoliasubsect.Talauma in Cuba.Distinguishing between those two causes from observed patterns is challenging, although coalescence modeling can help e.g. . HoweverMagnoliasubsect.Talauma. M.minor with more oblong leaves, considered by them as Talaumaminorvar.oblongifolia, may belong to a different species. However, the authors did not assign the species rank to this group because of the absence of reproductive structures in the available specimens. On the other hand, Magnoliaminor . Our morphological and genetic data did not support those two proposals. Indeed, concerning M.oblongifolia . This could be explained by a recent diversification of the three taxa that led to numerous genetic loci with incomplete lineage sorting and to overlaps in the distribution of morphological traits. In trees, factors such as long generation time, and large effective population sizes, increase the opportunity of sharing ancestral polymorphisms through incomplete lineage sorting which makes species identification based on neutral markers even more problematic (Undoubtedly, our data confirmed that the main taxonomic issues concern the north-eastern Cuban populations of or sensu , 1951 inia sensu , the fol species  and the izophora . Rather,see Figs –7 data nsee Figs  of the pia sensu , 1988 inblematic .SSR markers, as well as the few cases of reciprocal “miss-assignment“, suggest gene flow between the taxa of northeastern Cuba has occurred recently and may still be occurring, producing recombinant and therefore intermediate genotypes and phenotypes. This hypothesis is reinforced by the observation that reciprocal genetic admixture between the two genetic clusters, corresponding mainly to M.minor and T.ophiticola, is more frequent in the localities where both taxa occur. According to The taxa from the north-eastern part of Cuba live in the same habitats and in similar ecological conditions , a situaMagnoliasubsect.Talauma showed a high intra-specific leaf morphological variability, which reinforces the value of leaf characteristics in taxonomic studies of Cuban magnolias. As it has been shown in other groups of plants, the integrative approach was efficient to build an accurate classification in Magnoliasubsect.Talauma. Indeed, according to this study, Magnoliaorbiculata appears to be an evolutionary lineage separated from other Cuban magnolias of the subsection, with very clear genetic, morphological delimitations, which is consistent with its ecological delimitation already shown (M.minor-M.oblongifolia on the one hand and T.ophiticola (sensu The Cuban taxa of dy shown . This tala sensu  on the o