However, having been developed in different national contexts, these projects have had distinct scientific and social trajectories: in Mexico, the genomic mestizo is mobilized mainly in relation to health, while in Brazil the key arena is that of race. We show the importance of the nation as a frame for mobilizing genetic data in public policy debates, and demonstrate how race comes in and out of focus in different Latin American national contexts of genomic research, while never completely disappearing.This article explores the relationship between genetic research, nationalism and the construction of collective social identities in Latin America. It makes a comparative analysis of two research projects – the ‘Genoma Mexicano’ and the ‘Homo Brasilis’ – both of which sought to establish national and genetic profiles. Both have reproduced and strengthened the idea of their respective nations of focus, incorporating biological elements into debates on social identities. Also, both have placed the unifying figure of the mestizo/ Jiménez-Sánchez presented the gift as ‘the book of life … for Mexicans, by Mexicans, of Mexicans’. In his speech, president Calderón described the ‘map of the Mexican genome’ as a vital instrument for understanding, preventing and treating serious diseases that affect the Mexican population. The symbolism and language of the presentation conjured issues of national identity: the president was enacting metaphorical ownership of biological clues to the identity of ‘reignty’ .Ciência Hoje. Pena and colleagues emphasized the mixed genetic ancestry of self-identified white Brazilians, as evidence of the mixed nature the Brazilian population in general. They presented genetic knowledge as a potential antidote to racism, which had become seen as one of the main social problems of the country:It might be naïve on our part, but if the many white Brazilians who have Amerindian and African mitochondrial DNA became aware of this, they would better value the exuberant genetic diversity of our population, and, who knows, they might construct a more just and harmonious society in the twenty-first century. : 25In April 2000, amid the celebrations of the 500th anniversary of the Portuguese arrival in Brazil, geneticist Later, mestiço (mixed person) at the centre of the nation. However, while both research endeavours hinted at solutions to some of their countries’ most crucial problems, such as health, sovereignty and national integration, each privileged local issues: concerns about health took centre stage in Mexico, while dilemmas about how to confront racism and racial inequalities were paramount in Brazil.Mexico and Brazil were, in the first decade of the 21st century, well situated, economically and scientifically, to profit from the biotechnological possibilities enabled by the race for the human genome. In both countries, the context for national genomic projects included access to high-tech sequencing and bioinformatics, and well-trained scientists who were already participating in cutting-edge research. In both places, projects analysed similar scenarios, using the conceptual and technical tools of population genetics and the guiding frames of questions about genetic admixture. These projects addressed the past, present and future of the nation, placing the figure of the mestizo/There were other fundamental differences between the two endeavours. What has generally been referred to as ‘the Genoma Mexicano project’ (henceforth GM) was a large-scale, coordinated, centralized and government-led endeavour, which sought to address nation-wide biomedical priorities, which were linked to rocketing rates of obesity and diabetes. The GM mobilized an extant stable social mestizo identity and redrew it in population genomic idiom, preserving its basic ready-made outline; race was implicit in the figure of the mestizo, but racism was not a central concern . In cont1Both GM and HB reproduce the nation by generating national genetic profiles, deploy biology in discourses on national identity and locate (or relocate) the figure of the mestizo at the heart of such identity. But, given their different national socio-political contexts, GM and HB have distinct trajectories, which have established different kinds of relationships between genetics and social identity. This article analyses the convergences and differences of GM and HB.This analysis addresses the complex ways in which genetic research becomes articulated with socially imagined identities and nations. Since the 1990s, the development of new molecular techniques has enabled direct biological connections between contemporary populations and (a proportion of) their ancestors, as well as the production of genetic profiles that indicate degrees of mixture and the relative contribution of ancestral populations . Both geneticists and non-scientists have at times used genetic knowledge as an element in their interpretations of social groups, their histories and their futures e.g. . In thisAcademic debates concerned with the relationship between genetics and identity – particularly those in the United States, but also those in Europe – have often focused on race and ethnicity. Some social scientists have emphasized the role of genetics in reproducing racial categories, albeit in altered form . Others 2 The sections of this article cover: (1) the historical background of relationships between biology and nation in both Mexico and Brazil, (2) the context of genetic research for each country, (3) the characteristics of the GM and HB projects, and (4) the convergences and divergences between the two projects, in terms of how they construct the nation and relate to public policy.While arguing for the specificity of Latin America, this article qualifies the homogeneity of race and identity often projected onto the region. Although the GM and HB projects are similar in many respects, their trajectories and the public projections of their results have been quite different. Therefore, it is necessary to guard against overly broad regional generalizations, and avoid juxtaposing Latin America as a block with the US context.In Brazil and Mexico, there is a long tradition of thinking about the nation through its biological constitution . In the The period from 1910 to 1930 saw the emergence of nationalist projects in Brazil and Mexico, with some resistance to theories that condemned racial mixture. Physical anthropologists, along with other intellectuals, re-interpreted mixture in positive terms, and discourses that emphasized common features and harmony became central to debates on national identity. In these discussions, mixture was conceived in terms of history and culture, as well as in terms of biology. Academics and intellectuals who engaged in nation-building efforts reflected on the national character, by systematically describing the physical and psychological peculiarities of the Mexican and Brazilian people – their forms of life, gestures, temperament and history. From the 1930s, the hybrid figure of the mestizo (in Mexico) and the mestiço (in Brazil) became central to the construction of national identity.indigenismo, was facilitated by biological crossing and educational acculturation. Physical anthropology, which emerged as the key scientific discipline for forging national identity  ‘meta-race’ was discussed, somewhat later, by Gilberto Freyre  (During the post-Revolutionary period (1940–2000) in Mexico, a ‘mestizo ideology’  emerged mixture) .More recently, there has been growing critical inquiry into the biological and cultural aspects of the mestizo, much of which treats overemphasis on the mestizo as an obstacle to the appreciation of diversity in Mexico . Multicu3 These ‘racial mixture studies’ aimed to establish the relative contributions of ‘white/Caucasian’, ‘black/Negroid’ and ‘Indian’ populations to the gene pool of Brazilian populations. Key authors – such as Francisco Salzano, Pedro Saldanha and Newton Freire-Maia – highlighted the importance of the genetic diversity of the Brazilian population for understanding of admixture and inter-ethnic relations on a global scale . In contrast to Brazil, where research like Pena’s was decentralized and carried out by individuals and small teams, in Mexico in the early 2000s, a group of politically influential physicians set out to organize the dispersed population genetics endeavours, to define the strategy that Mexico would follow in order to take advantage of the newly generated tools and knowledge. This led to the creation of the National Institute for Genomic Medicine (INMEGEN) in July 2004. INMEGEN’s medically oriented Mexican Genome Diversity Project (MGDP) was, like biomedical projects of the previous century, accompanied by anthropo-historical questions. The GM, which encompassed this project and its resulting ‘Map of the Mexican Genome’, aspired to become both a biomedical research tool for the genetic basis of diseases specific to the Mexican population, and a molecular portrait of the Mexican mestizo  and African (28%) ancestry. Since then, working with different sets of collaborators from various universities in Brazil, Pena has produced a number of further studies on the genetic ancestry of the Brazilian population.research , Pena haresearch . At otheresearch , 2011; wresearch ; or whenresearch .In 2002, Pena published an edited book on the Brazilian population, which featured contributions by anthropologists, linguists and historians, in addition to several chapters on genetics. He justified the term HB, in the title, as follows: ‘[in Brazil] a process of genetic mixture was initiated that is unique in the entire history of Humanity, generating the contemporary Brazilian, which we decided to call, a little irreverently, Homo Brasilis’ : v. Althpreto, pardo and branco . Results revealed that while variation in genetic ancestry between individuals within each category was considerable, such variation between categories taken as a whole was relatively small. The authors concluded that, in Brazil, there is only a weak correlation between people’s physical appearance  and their genetic ancestry , as well as the lower than expected proportions of African ancestry (40%–50%) among those categorized as preto , 2011. FIn 1999, Gerardo Jiménez-Sánchez , supported by his senior colleague Guillermo Soberón Acevedo, whose career included terms as Mexican Secretary of Health (1982–1988) and rector of Mexico’s National University (UNAM), began to propose a national research programme on medical genomics. From the start, and in contrast to comparable projects in Brazil, the initiative was linked to mainstream state institutions. Lobbying efforts for the creation of INMEGEN began under the aegis of the Fundación Mexicana para la Salud (FUNSALUD), a politically powerful institution that links private and public interests around health-related issues, then headed by Soberón. Initial meetings brought together further potential funders: the Mexican Ministry of Health, the National Autonomous University (UNAM) and the Mexican Council of Science and Technology .The first results of the Human Genome Project in 2000 indicated to Jiménez-Sánchez and Soberón that Mexico could not afford to be outside the genomic revolution, and needed to create a genomic research institute. A 2001 Feasibility Study (IFS) helped potential investors to foster informal support for the development of INMEGEN, and became the platform for circulating the promises of genomic medicine to strategic audiences, including members of Congress.Soon after, the Consortium for the Creation of the Institute for Genomic Medicine was created to promote the project, with the backing of eminent physicians such as Juan Ramón de la Fuente  and Julio Frenk , and corporate figures such as pharmaceutical tycoon Antonio López de Silanes. Due to his experience and networks, Jiménez-Sánchez was chosen to lead an initiative that INMEGEN lobbyists deliberately compared with the Human Genome Project. From July 2004, when INMEGEN was created, to November 2010, when he stepped down as director, Jiménez-Sánchez was the face of Mexican genomic medicine. The task was to bring Mexican medical genomic research to a global level of excellence.Under Jiménez-Sánchez, the MGDP aimed to be the catalyst of this change, by attempting to describe the genetic heterogeneity and the specific genetic characteristics of the Mexican population, and exploring haplotype–disease associations . Once thAlthough the project set out to measure diversity, the mestizo, as an object recognizable by both political and scientific communities, remained a dominant figure: ‘The initial results of the MGDP published in 2009 show that although some regional genetic differences exist between Mexican subpopulations, these are similar enough to be considered as a single group’. Scientists further concluded that, despite considering Mexicans a relatively homogeneous national population , there were some ‘genetic differences between mestizos of some regions of Mexico’, mainly due to ‘differences in ancestral contributions by European and Amerindian groups’ : 1186–7.During all the phases of the MGDP, INMEGEN devoted resources to public engagement activities: conferences, media coverage, museum exhibits, publications (including comic books and public science articles). Three discursive elements were central to these efforts: (a) the idea that the Mexican population has distinctive genetic peculiarities; (b) the idea that such peculiarities derive from the events of admixture starting 500 years ago; and (c) the idea that the distinctive genetic make-up of Mexicans can be represented as a ‘Map of the Mexican Genome’. Few critical reactions were triggered by these campaigns. INMEGEN succeeded in mobilizing a nationalist rhetoric that was already in place in order to bolster the concept of a ‘GM’. The genetic portrayal of Mexicans as bodies assembled from portions of European, Amerindian and (to a lesser extent) African ancestry was not news. INMEGEN’s research translated the notion of national mestizo identity, a concept already underwritten by older genetic studies, such as those by Lisker , into thAs simultaneously scientific and socio-political projects, the GM and HB each established more pronounced exchanges between biological and social repertoires than is usually the case in genetic research. Both projects focussed on nations and their mixtures, placing figurative genomic mirrors in front of national populations, and calling on members to recognize their ‘real’ characters. In this way, both the GM and HB framed relationships between genetic ancestry and social identity in ways that essentialized conceptions of the nation and the mestizo.Pena’s research relies on constructs such as Brazil and its population, HB and the Molecular Portrait of Brazil, and the nation as a discrete unit of sampling and analysis. In his publications, Pena has systematically used his genetic perspective to reject alternative categories, particularly those based on race, and, more recently, region. Notably, singular. Pena has attempted international dialogue, using the Brazilian population in order to think about universal issues, such as race relations, and to explore the lessons that Brazil has to teach to the world , ‘[t]he perception of mixture, which profoundly permeates Brazilians, in a way reflects realities proven by genetic studies’  designed to recruit mestizo blood donors in three regions of the country: north, south and centre , non-academic books  and some influential social scientists have also drawn on Pena’s studies to argue against race-focused, as opposed to class-focused policies . Mexican scientists have been aligned firmly with the state’s approach to framing health priorities, especially the idea that obesity and diabetes are major problems in need of genetic solutions. Political campaigning by interested geneticists was a crucial aspect of the initial take-off of INMEGEN. Scientists and lobbyists justified investment of public money in INMEGEN, emphasizing the potential of genomic research to improve public health outcomes and reduce costs: genomic research promised to enhance genomic sovereignty, by tailoring future health provision to the Mexican citizens’ individual genetic profiles and reducing the nation’s dependence on foreign genomic research and pharmaceutical companies. In early 2009, INMEGEN scientists were conveniently on hand to offer a powerful scientific response to an unexpected outbreak of swine flu: on the brink of publication, INMEGEN’s Mexican Genome paper demonstrated that the national health system anticipated health hazards, such as the flu, and had cutting-edge genetics and local scientific talent to develop adequate responses. A newspaper report commented, ‘Only a story as strong as the decoding of the Mexican Genome could compete in Geneva, Switzerland, with the attention that the World Health Organization (WHO) had given to the health alert for AH1N1 flu’ cited by : 241.In Mexico, genetics is now intertwined with notions of crisis and the health of the nation. In the first decade of the 2000s, much of the country’s political discourse emphasized rising rates of obesity and diabetes as signals of a national medical crisis. The GM project was presented as a no-nonsense, radical, high-tech solution, one that could cut straight to genetic causes, located deep in the mestizo body: science and technology could help cure the ailing medical body of the nation see , and INMindio/mestizo division that has long been central to the Mexican social imaginary. Meanwhile, the Mexican figure of the mestizo acts as a screen, hiding the racial underpinnings of typological categorizations in Mexico. The centrality of the mestizo to the GM has linked genetics and implicit racial concepts, rekindling the concept of race in the idiom of genetic ancestry. Researchers commonly use bio-geographical populations – Africans, Amerindians, Europeans – to represent genetic ancestries , which avoids the kinds of divisive issues of race and multiculturalism that characterize genomic research in Brazil.While the opacity of race dissociates Mexican mestizo bodies from racial markers  associated with the past, the persistence of old ideas about mestiçagem – and the mestizo. In the early and middle parts of the 20th century in both Brazil and Mexico, the mestizo became central to self-conscious processes of state-supported ideological nation building. The two countries both have long-standing motivations to define themselves in ways that resist cultural dominance by the United States, and genomic research in both countries has thus attempted to develop genomic resources, techniques and knowledge that produce distinctive national profiles. Moreover, Mexico and Brazil both participate in a global scientific enterprise that primarily recognizes them as countries that can offer examples of admixed populations  has struggled to command a public space.There were also important differences between Brazil and Mexico in their respective political contexts for post-1990 multiculturalist reforms. A pioneer of constitutional and legal reform, Brazil has explicitly moved towards multiculturalism through its 1988 constitution, officially acknowledged racism as a significant problem in the 1990s, and rapidly adopted race-based affirmative action programmes. Mexico has been slower and less radical in these areas. Mexican measures to recognize indigenous communities – while offering departures from mid-century assimilationist These differences have led to distinct Brazilian and Mexican mobilizations of the genomic mestizo. In Mexico, the state, geneticists and the medical community agree that improvements to health policy are the main goal of genomic research, despite disagreements over strategies and the roles of INMEGEN. And while multiculturalism and indigenous rights are certainly at issue in Mexico – witness the Zapatista movement – race is not a major controversy in genomic research. INMEGEN has had little to say openly about multiculturalism or race. As we have illustrated, the GM project implicitly reproduced the old divide between indigenous and mestizo identities, repackaging it in genetic idiom. This divide continues to guide approaches to national identity in post-multicultural reform Mexico, framing the genetic mestizo as paradigmatic. The presence of race is still significant, but implicit.In contrast, in Brazil, race has been the main public arena of genomic research. Even debates about genomics and health – for example, in relation to provisions for sickle cell anaemia – have focused to some extent on the question of whether public health policies should differentiate by race . In suchOur comparison of Mexico and Brazil shows the complex ways genetic data get entangled in debates about public policy, national identities and destinies. In many ways, GM and HB have been driven by common concerns in the field of global genetic medicine and population genetics; they have also been shaped by long-standing interests in mixture as a defining feature of the cultures and biological profiles of Latin American nations. Yet, the specific features of national histories, demographics and politics, as well as the ways states have decided to support genomic research, have resulted in different genomic mestizos in each country.


HT = 0.7080 and the genetic differentiation value was GST = 0.1730. STRUCTURE software allocated 90.1% of the accessions in five subpopulations, one including all modern cultivars, and the four containing landrace related to their geographic origin: eastern Mediterranean, eastern Balkans and Turkey, western Balkans and Egypt, and western Mediterranean. Mean yield of subpopulations ranged from 2.6 t ha-1 for the western Balkan and Egyptian landraces to 4.0 t ha-1 for modern cultivars, with the remaining three subpopulations showing similar values of 3.1 t ha-1. Modern cultivars had the highest number of grains m-2 and harvest index, and the shortest cycle length. The diversity was lowest in modern cultivars (HT = 0.4835) and highest in landraces from the western Balkans and Egypt (HT = 0.6979). Genetic diversity and AMOVA indicated that variability between subpopulations was much lower (17%) than variability within them (83%), though all subpopulations had similar biomass values in all growth stages. A dendrogram based on simple sequence repeat data matched with the clusters obtained by STRUCTURE, improving this classification for some accessions that have a large admixture. landraces included in the subpopulation from the eastern Balkans and Turkey were separated into two branches in the dendrogram drawn with phenotypic data, suggesting a different origin for the landraces collected in Serbia and Macedonia. The current study shows a reliable relationship between genetic and phenotypic population structures, and the connection of both with the geographic origin of the landraces.A collection of 172 durum wheat landraces from 21 Mediterranean countries and 20 modern cultivars were phenotyped in 6 environments for 14 traits including phenology, biomass, yield and yield components. The genetic structure of the collection was ascertained with 44 simple sequence repeat markers that identified 448 alleles, 226 of them with a frequency lower than 5%, and 10 alleles per locus on average. In the modern cultivars all the alleles were fixed in 59% of the markers. Total genetic diversity was  Triticum turgidum L. var. durum) is a traditional Mediterranean crop. It originated in the Fertile Crescent  and spread over the northern side of the Mediterranean, reaching the Iberian Peninsula in about 7000 BP . Although Mediterranean landraces have been shown to be particularly valuable due to their huge genetic diversity and the presence of accessions with high resilience to abiotic stresses, resistance to pests and diseases and high grain quality [Genetic diversity in wheat was increasingly narrowed down during the second part of the 20 quality , the larHe values between 0.55 and 0.68) [He values between 0.54 and 0.63 [Exploiting the variability of wheat landraces requires previous knowledge of their genetic diversity. In this study, we used 44 SSR markers to quantify the genetic diversity existing in a set of 172 durum wheat landraces from the Mediterranean Basin and 20 modern cultivars. The number of alleles identified in this study, and the value estimated for the genetic diversity of the collection (0.71), were higher than the values reported by previous studies involving durum wheat collections (nd 0.68) –47, and and 0.63 . The higGST) is directly proportional to the amount of variation among populations [DST) obtained in the current study, GST was also low, showing that only 17% of the variability was due to differences between subpopulations, while the remainder was a consequence of the genotypic variability within each subpopulation. The results of the analysis of molecular variance were in agreement with the low value of GST, as only 13% of the variation in the durum wheat accessions was due to variation between subpopulations. These results indicate that, though landraces of different geographic origin were polymorphic enough to trace a consistent geographical pattern, the genetic variability within the set of genotypes of a common origin was much wider. The presence of a large number of unique putative alleles in this collection, which have already been identified for glutenin subunits [The coefficient of gene differentiation  jointly with modern cultivars (subpopulation 2) in the phenotypic tree may be due to its extensive use by Italian breeders as a hallmark founder for the development of drought-tolerant cultivars .The clustering of accessions in dendrograms based on SSR and phenotypic data were essentially coincident. However, in cases of accessions with large admixture, the dendrograms concurred in locating them in branches corresponding to different subpopulations than those assigned by STRUCTURE. This was the case of the Greek LR ‘Rapsani’ and the Egyptian LR ‘31’. Interestingly, 5 of the 19 accessions not assigned to any subpopulations by STRUCTURE were located in dendrograms in branches close to accessions of similar geographic origin. This was the case of the landraces ‘Marques’ and ‘Lobeiro de grao escuro’ from Portugal, ‘Hamira’ from Tunisia, and ‘Haj Mouline’ from Morocco, which were all located jointly with western Mediterranean accessions in both dendrograms.In addition, some of admixed cultivars from STRUCTURE that originated in Israel, Jordan, Lebanon and Syria were placed close to the cluster defined by DARWin with molecular data containing accessions from the eastern Balkans and Turkey (subpopulation 3).Accessions included in subpopulation3  by STRUCTURE were separated into two different branches in the dendrogram drawn with phenotypic data. One of them contained the accessions from Cyprus and Turkey ‘Vroulos’, ‘IG-82549’, ‘BGE-018192’, ‘BGE-018353’, ‘BGE-019262’ and ‘BGE-019264’, and the other clustered all Serbian and Macedonian landraces included in subpopulation 3. This result not only supports the hypothesis that subpopulation 3 contains accessions from the Volga region, but identifies them on the basis of their agronomic performance.A previous study  using a The current study aimed to understand the genetic, phenotypic and geographic structures of a collection of Mediterranean durum wheat cultivars and the relationships between them. The results demonstrated the usefulness of the methodologies used to determine the structure of Mediterranean durums. Landraces and modern cultivars were split into two different groups using either molecular or phenotypic data. Based on data from 44 SSR markers, STRUCTURE software assigned 90% of the accessions to five subpopulations, with the four ones having landraces showing a geographical structure. Unexpectedly, the genetic diversity was greater within subpopulations than between them, which denotes the large variability existing in landraces of a common geographic origin. The study identified a large number of alleles (448), about half of which had a very low frequency and were therefore rare alleles, and an average number of 10 alleles per locus. Gene diversity increased from the eastern to the western Mediterranean Basin, in agreement with the dispersal pattern of wheat from its domestication area.The un-rooted neighbour-joining dendrogram based on SSR data coincided in essence with the clusters obtained by STRUCTURE, but complemented the information provided by it when accessions showed large admixture. The strong coincidence between dendrograms based on molecular and phenotypic data indicates i) the suitability of the phenotypic traits used in the current study for differentiating groups of accessions with similar field performance, and ii) the robust relationship between the phenotypic expression of traits and the genetic background underlying them. Among the subpopulation that included durums from the eastern Balkans and Turkey, the assessment of phenotypic traits based on yield, yield components and crop phenology was also useful for separating those from Macedonia and Serbia from those from Turkey, Cyprus and Greece, which very likely had a different origin.This is the first study using durum wheat Mediterranean landraces and modern cultivars that shows a reliable relationship between genetic and phenotypic population structures, and the connection of both with the geographic origin of landraces. The results of the current study demonstrate that, when appropriate markers in number and distribution are used and phenotyping is adequately conducted, high similarities may be found between genetic distances and the adaptive response of durum wheat.S1 TableDurum wheats included in the study. Entries from 001 to 172 correspond to landraces and those from 173 to 192 correspond to modern cultivars.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableGenetic distance based on  between (DOCX)Click here for additional data file.


Malus sylvestris) from European forests and 339 apple cultivars . We made use of genetic, environmental, and ecological data . We provide the first evidence that both human activities, through apple production, and human disturbance, through modifications of apple flower visitor diversity, have had a significant impact on crop-to-wild interspecific introgression rates. Our analysis also revealed the impact of previous natural climate change on historical gene flow in the nonintrogressed wild apple M. sylvestris, by identifying five distinct genetic groups in Europe and a north–south gradient of genetic diversity. These findings identify human activities and climate as key drivers of gene flow in a wild temperate fruit tree and provide a practical basis for conservation, agroforestry, and breeding programs for apples in Europe.Gene flow is an essential component of population adaptation and species evolution. Understanding of the natural and anthropogenic factors affecting gene flow is also critical for the development of appropriate management, breeding, and conservation programs. Here, we explored the natural and anthropogenic factors impacting crop-to-wild and within wild gene flow in apples in Europe using an unprecedented dense sampling of 1889 wild apple ( Over the last century, researchers in evolutionary and applied biology have paid much attention to gene flow, which is considered to play a key role in species persistence, evolution, and diversification. Many studies have investigated the consequences of intra- and interspecies gene flow for evolution  is the one of the most important fruit tree crop in terms of worldwide production (http://faostat.fao.org/). It was introduced into Europe 1500 years ago and has been subject to the introgression of genes from the local wild European apple, Malus sylvestris, to the extent that M. domestica is now more closely related to M. sylvestris than to its primary progenitor in the Tian Shan, M. sieversii , with a focus in France , unique for a temperate fruit tree. We also sampled a large number of M. domestica varieties (339 varieties), and combined genetic (microsatellites), ecological, and environmental data, to investigate the natural and anthropogenic drivers of gene flow in apples. Using genetic data across Europe, we estimated the degree of crop-to-wild introgressions into the European wild apple, across its geographic distribution and investigated the impact of climate on population structure and diversity, through estimates of historical gene flow within the nonintrogressed wild apple gene pool. Furthermore, by estimating recent crop-to-wild introgressions in France, and using environmental and ecological data, we investigated the effects of apple production on these crop-to-wild introgressions. Finally, we determined whether human activities had indirectly impacted rates of gene flow through their effects on the flower visitor community.We report here an unprecedented dense sampling of in situ and ex situ conservation management for this emblematic fruit tree species in Europe, for agroforestry programs aiming at integrating wild apples in agrosystems and for future breeding programs, the European wild apple being a major contributor to the cultivated apple genome.Altogether, we highlighted the impact of previous natural climate change on historical gene flow across Europe in the wild apple and identify human activities as key drivers of gene flow in a wild temperate fruit tree. Our landscape genetics study provides useful information for M. sylvestris from Europe previously studied , mostly in France , but also in Denmark , Italy  and Belgium . In total, 1889 M. sylvestris trees from 66 sites were analyzed for this study. In addition to the 299 M. domestica previously analyzed  and in French apple orchards (N = 16). In total, we analyzed 339 M. domestica trees in this study. The dataset for this study thus corresponds to 2228 individuals .In addition to the 599 samples of DNA was extracted with the NucleoSpin® plant II DNA extraction kit . Multiplex microsatellite PCR amplifications were performed with a multiplex PCR kit (Qiagen Inc.) as previously described  simulations to infer the proportion of ancestry of genotypes from K distinct clusters. The underlying algorithms attempt to minimize deviations from Hardy–Weinberg within clusters and linkage disequilibrium among loci. A lack of consideration of intraspecies genetic structure in STRUCTURE analyses can bias the interpretation of introgression rates  and M. domestica (N = 339). Based on 10 repeated runs of MCMC sampling comprising 500 000 iterations after a burn-in of 50 000 steps, we determined the amount of additional information explained by increasing K, using the ΔK statistic , we assumed that the total membership coefficient of M. sylvestris individuals in the M. sylvestris gene pool was the sum of their membership coefficients in each M. sylvestris cluster. All M. sylvestris individuals with a cumulative membership coefficient for the M. sylvestris gene pool >0.9 were considered to be pure M. sylvestris trees. Conversely, all M. sylvestris individuals with a cumulative membership coefficient >0.1 in the M. domestica gene pool were considered to be admixed with M. domestica. Malus sylvestris individuals with a cumulative membership coefficient <0.1 in the M. sylvestris gene pool were considered to be misidentified individuals actually belonging to M. domestica. The proportion admixture of the M. domestica gene pool with the M. sylvestris gene pool is hereafter denoted PDOM→SYL and referred as ‘introgression rate’. In the absence of genomewide genotyping data, we probably failed to identify advanced generation backcrosses. However, our approach aimed to identify early generations of admixture and introgression, because our focus here was the effect of recent human disturbance on gene flow.We used the individual-based Bayesian clustering methods implemented in STRUCTURE 2.3.3  and used the individual-based spatially explicit Bayesian clustering method implemented in TESS 2.3.1  to 0.6. These parameters (ρ and trend) affect the weight given to spatial distance when clustering genotypes. Ten independent analyses were carried out for each number of clusters K (2 ≤ Kmax ≤ 9), using 500 000 MCMC iterations after a burn-in of 50 000 steps. We determined the amount of additional information explained by increasing K, using the rate of change of the deviation index criterion (DIC) with increasing K.To investigate the population structure and admixture of K value.For both TESS and STRUCTURE analyses, outputs were processed with CLUMPP v1.1.2  and each population .For the analysis of within-species genetic variation, we excluded hybrids identified as displaying introgression from AR) and private allelic richness (AP) were calculated with ADZE  and N = 100 , respectively, corresponding to the minimal number of observations across sites and populations. Heterozygosities (expected and observed), Weir and Cockerham F-statistics and deviations from Hardy–Weinberg equilibrium were calculated with GENEPOP 4.2 (Raymond and Rousset AR), for 43 sites  and genetic diversity (HO) for 51 sites across space, with the kriging and surface functions (field R package).Allelic richness (Fij) were regressed against the natural logarithm, ln(dij), to obtain the regression slope, b. The spatial positions of individuals were permuted 9999 times to test for the existence of a spatial genetic structure. We compared the extent of spatial genetic structure between sites over the same spatial scale, by calculating bLd, the regression slope of Fij against ln(dij) for dij = 10 km . We then calculated the Sp statistic, defined as Sp = −b(Ld10)/(1−FN), where FN is the mean Fij between neighboring individuals, which was approximated by F(d) for the first distance interval ., PDOM→SYL). We first investigated the effect of the density of cultivated apple trees (based on apple production in tons/ha/year for the French administrative region concerned) on PDOM→SYL in wild apple. We then included cultivated apple density as a fixed factor in the models 1 and 2 described below, as the source of the crop-to-wild gene flow. Because the number and area of apple orchards were highly correlated , we investigated the effects of the number and area of apple orchards on PDOM→SYL independently in two distinct models , controlling for cultivated apple tree density. With this approach, we aimed to determine the relative contributions of orchard number and orchard area to crop-to-wild gene flow in apples. For these analyses, we used the dense sample obtained in France , and we ran a generalized linear model (GLM) with a quasi-Poisson distribution, as the data appeared to be overdispersed. Apple production (tonnes/year) and the number of and area covered by apple orchards were extracted, for each French administrative region, from the AGRESTE (http://agreste.agriculture.gouv.fr/).We expected the density of cultivated apple trees to be the principal determinant of recent crop-to-wild introgression rate detected by STRUCTURE . We investigated whether the frequency of apple taxa visiting flowers depended on the intensity of cultivation and management. We ran a GLM with a quasi-Poisson distribution, as the data appeared to be overdispersed. In total, 306 observations corresponding to different intensities of cultivation were recorded .http://edit.csic.es/Soil-Vegetation-LandCover.html). We defined two classes of cultivation and management intensity, distinguishing between areas with higher and lower intensities of cultivation and management, corresponding to the 1–15 and 17–18 classes of the GLC2000 dataset, respectively. These two classes were defined qualitatively on the basis of the standard classification in the worldwide GLC database as ‘natural’ or ‘cultivated and managed’. Individual geographical coordinates were assigned to these two classes with the extract function (raster R package).The intensity of cultivation and management was obtained from the GLC2000 dataset  was obtained from a nationwide monitoring exercise based on observations by citizens. Using the database generated by this exercise, entomology experts used the photographs provided to check the preliminary classifications obtained with a simple online key by the citizens participating in the project . The use of K values >6 uncovered no further structure within M. sylvestris, indicating that the M. sylvestris × M. domestica hybrids detected at K = 6 were not artifacts due to genetic structure within the wild apple gene pool. We therefore used cumulative membership coefficient in the five M. sylvestris clusters in subsequent analyses to identify crop-to-wild hybrid genotypes.Failing to take the population structure of the wild species into account can lead to spurious signals of introgression from crop species , 436 genotypes  showed signs of introgression from M. domestica  and 77 genotypes were found to correspond to misidentified M. domestica trees . The mean introgression rates and numbers of hybrids are presented in Fig.For the total dataset for individuals identified tris N = 889, 436 M. domestica cultivars , as previously demonstrated  were removed. The final dataset included 1376 individuals (62 sites). For each site, the summary statistics for genetic diversity are shown in Table S4. On average, gene diversity was high across sites and across markers [HE = 0.79 ± 0.07 (min–max: 0.57–0.89)] and FIS values were low [FIS = 0.10 ± 0.06 (0–0.21)], although there was a highly significant species-wide heterozygote deficit compared to Hardy–Weinberg expectations (P < 0.001). Mean FST across loci were small [FST = 0.025 ± 0.04 (0–0.56)], but significant for 1349/1831 pairs of sites (P < 0.05). Overall, these results indicate that spatial genetic structure is weak in M. sylvestris.For analyzing the population structure of AR . A significant positive correlation between longitude and AR was also found . The map of interpolated HO ; no correlation between latitude and HO was also found . This latter map highlights the main admixture zones among M. sylvestris populations as regions of high diversity.The map of interpolated AR Fig. showed td HO Fig. showed aM. sylvestris : two of these clusters spanned Western (green) and Central Europe (yellow); one spread over Eastern Europe and the Balkan Peninsula (red); one covered Northern Europe (blue); the last one was located in Italy (purple). DIC values decreased monotonically from Kmax = 2 to Kmax = 9, indicating that increases in the number of clusters continually improved the fit of the model to the data, with a sharper decrease in DIC was observed at Kmax = 8  indicating that Kmax = 8 was the best-fit model. However, at Kmax = 8, only five clusters still appeared , as in three of the eight clusters, the membership coefficients of all individuals were close to 0. We therefore concluded that the biologically most relevant number of clusters was five, as observed in STRUCTURE analyses; we nevertheless used the TESS membership coefficient inferred at Kmax = 8 to define the five populations used in subsequent analyses, as it had the highest DIC value and therefore the best fit to the data. Individuals were assigned to populations for which their membership coefficients exceeded 0.55. The five populations are hereafter referred to as the Western , Central , Southern , Eastern , and Northern  populations. In total, 88 genotypes could not be assigned to any population; N = 1288 individuals were thus retained for population-specific analyses.Spatially explicit clustering analyses carried out with TESS confirmed the existence of five well-defined clusters within HE and HO,AR and AP) differed significantly between all populations (Wilcoxon signed rank (WSR) test, P < 0.001) other than the Western and Central populations , and the Eastern and Southern populations . Overall, the Eastern and Southern populations displayed significantly higher levels of genetic variability, in terms of allelic richness (AR and AP), than the Northern, Western, and Central populations. Higher heterozygosities (HE and HO) were considered to be footprints of recent zones of admixture between different genetic clusters .For each population, summary statistics for genetic diversity are shown in TableN = 321 admixed individuals) and then for the Northern and Western populations (N = 41) (TableN = 31), Northern (N = 21), and Central (N = 21) populations. The Southern population displayed admixture mostly with the Eastern (N = 31) population and the Western population (N = 23). The admixture detected is indicative of gene flow between these populations. Genetic differentiation (FST) was greatest between the Central/Western/Northern and Southern/Eastern populations .Proportions of admixture differed between the five populations Fig. and 2. SWithin each of the five populations, genetic differentiation and geographical distance were significantly correlated, consistent with an IBD model Table. The Sp M. domestica gene pool into the M. sylvestris gene pool (PDOM→SYL) was found to be dependent on the interaction between cultivated apple density and the number of apple orchards .Apple species were visited by 86 taxa from six orders of insects Fig., after cM. sylvestris, the extent of which was influenced by the intensity of apple production, and (ii) a decrease in the diversity of apple flower visitors with increasing intensity of cultivation and management practices, suggesting an indirect impact of land-use on pollen flow.Previous studies investigating crop-to-wild gene flow in fruit trees have provided estimates of introgression rates, but did not identify the factors influencing these rates , in France (between the green and yellow clusters), and in the Carpathian Mountains (between the red and purple clusters). These results suggest that previous contractions of the distributional range of wild apple during the last glacial maximum in southern Europe were followed by a recolonization of Northern Europe via two main routes: a western front  and an eastern front . In particular, the additional Italian cluster detected appears to have acted as a bridge for the recolonization of the western and eastern parts of Europe after glaciation. Barriers to dispersal during recolonization and bottlenecks may also have affected the existing population structure. For instance, populations from northern Germany, Denmark, and Norway have a low allelic richness, and a bottleneck might account for these populations belonging to a distinct gene pool.Past climate changes and spatial barriers to dispersal across Europe can explain the population structure of wild apples revealed here. Over the last 2.6 million years, trees species have experienced dynamic alternations between the contraction and expansion of their ranges in Europe, in response to climate changes Hewitt . Our invSorbus domestica , suggesting relatively high dispersal capacities and/or effective population sizes (Vekemans and Hardy Cocos nucifera), in which introgression from domesticated populations led to the disappearance of all genetically pure wild individuals (Ellstrand The consequences of crop-to-wild gene flow for the wild apple remain unclear, particularly as concerns the possibility of genetic swamping (Lenormand in situ conservation could also be combined with the ex situ conservation of genetic resources through the creation of gene banks for a core collection of nonintrogressed M. sylvestris populations from diverse European regions.The implementation of conservation programs is challenging, in terms of the application of the recommended practices in the field. Landscape genetics can provide useful information for conservation management, in the form of fundamental knowledge for the identification of high-priority populations for the conservation of genetic variation. Our landscape genetic studies of within-species gene flow and crop-to-wild gene flow in apples in Europe should facilitate the allocation of priority zones for the conservation of the European wild apple, across its natural distribution. We detected five main genetic groups, within which it would be optimal to conserve populations. Southern Europe, housing the main glacial refugia with the highest levels of genetic diversity, should also be considered a priority area for conservation programs, together with France and the Carpathian Mountains, the main suture zones, with their original allelic combinations. The identification of highly introgressed European wild apple populations and of the anthropogenic drivers of crop-to-wild introgressions will also help to dynamically manage these priority conservation zones. This Our landscape genetic investigations would also help to identify the optimal provenance for seeds or germplasm for the reintroduction of wild apples into agroforestry programs (Krauss et al. In conclusion, the relatively recent introduction of the cultivated apple into Europe, only about 1500 years ago, has particularly facilitated the detection of wild-to-crop and crop-to-wild gene flow and the identification of its determinants. The cultivated-wild apple complex in Europe thus appears to be a good model in which to address both fundamental and applied issues relating to the impact of human landscape changes on gene flow.AC, PG, and TG conceived and designed the experiments; AC, PG, and TG obtained funding; AC and AF performed the experiments; AC and TG supervised the Master's thesis of AF; AC, AF, and UG analyzed the data. KV and JR provided samples. The manuscript was written by AC with critical input from UG and TG.


Pediococcus pentosaceus and Lactobacillus graminis were the most abundant LAB on grapes, while Lactobacillus plantarum dominated in fermenting musts from both regions. No particular structure of Lactobacillus plantarum populations according to the region of origin was observed, and strain distribution seems random. LAB species diversity in winery tanks differed significantly from that in vineyard samples, consisting principally of Oenococcus oeni. Different strains were analysed as per their enological characteristics and the ability to produce biogenic amines (BAs). Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4 than vineyard-associated isolates. The frequency of BA-producing strains was relatively low but not negligible, considering that certain winery-associated Lactobacillus hilgardii strains were able to produce BAs. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in wine.Vineyard- and winery-associated lactic acid bacteria (LAB) from two major PDO regions in Greece, Peza and Nemea, were surveyed. LAB were isolated from grapes, fermenting musts, and winery tanks performing spontaneous malolactic fermentations (MLF). Higher population density and species richness were detected in Nemea than in Peza vineyards and on grapes than in fermenting musts.  In winemaking, a secondary fermentation known as malolactic fermentation (MLF) often takes place following the cease of yeast activity. During MLF, L-malate is converted into L-lactate by the lactic acid bacteria (LAB) of wine. This bioconversion is a desirable process in red winemaking and also in the production of certain white wines of high acidity, due to the organoleptic advantages that LAB activity confers. These include a decline in the total acidity and an increase of soft mouth feel, flavour, and microbiological stability of the wine . HoweverCurrently, there is a growing concern regarding the limits of BAs in wines because of their potential health implications . Althoug Oenococcus oeni, the principal malolactic bacterium, have been launched in the market over the last decades. Nevertheless, wineries often face difficulties when conducting MLF by current commercial starters, as the induction of the process is not always successful [ O. oeni, since native strains can deal with microbial incompatibilities and are better acclimatized to the local wine and practices [Selected strains ofccessful . Still sccessful . In thesractices , 12. In  O. oeni is also being considered [To this end, the wine industry seeks for novel MLF starters bearing positive technological and flavouring attributes . The usensidered . Grape rVitis vinifera) varieties “Vilana” (white), “Mandilaria” (red), and “Kotsifali” (red) were collected from 16 vineyards (1VP–16VP) within the Peza PDO region in Crete. Grapes of the “Agiorgitiko” cultivar (red variety) were collected from 11 vineyards (1VN–11VN) in the Nemea PDO region, Peloponnese. Samples consisting of healthy grape bunches were collected from at least 3 distant sampling points (sites) within each vineyard, placed into sterile plastic bags and transferred at 4°C to the laboratory. Grapes were crushed with a stomacher and let to ferment spontaneously in sterile bottles. Fermentation progress was daily followed by weight determinations. LAB were isolated from grapes or fermenting grape juice at the middle stage (MF) when about 50% of sugars were consumed, the final stage (EF) when sugars were depleted, and after the end of alcoholic fermentation. LAB were also isolated from wine samples collected from 9 tanks (T1–T9) of a winery in Nemea during spontaneous MLF. No spontaneous MLF was conducted in Peza winery. For bacteria enumeration, appropriate dilutions were spread onto MRS agar medium (pH 5.5) supplemented with 100 mg/L cycloheximide and incubated in anaerobic jars at 28°C for 3–8 days. Colonies were randomly selected from plates and examined microscopically. Bacterial colonies were further examined for Gram stain and catalase reaction. Isolates were maintained in liquid cultures in MRS broth with 30% glycerol at −80°C until further analysis.Grape samples belonging to the Greek grapevine . BLAST searches of sequences were performed at the NCBI/GenBank database.DNA was extracted as previously described . The 16S MseI  and fragi et al. . PCR proRepetitive element sequence-based PCR (rep-PCR) using the single primer (GTG)5 or the primer pair REP1R-Dt and REP2R-Dt , 19 and hdc), tyramine (tyrdc), and putrescine (odc and agdi), a multiplex PCR assay was applied as described elsewhere [ hdc and tyrdc genes were targeted with the primer pairs HDC3/HDC4 and TD2/TD5, respectively, while the primers ODC1/ODC2 and AGD1/AGD2 were used for the detection of agdi and odc genes, respectively. The 16S rRNA gene was concomitantly targeted with the universal primers BSF8/BSR1541 [For simultaneous detection of four genes involved in the production of major BAs in wine by LAB, that is, histamine  plates  supplemented with 2% of either tyrosine, histidine, or arginine. The formation of biogenic amines was indicated by a purple halo around the bacterial colony as a result of amino acid decarboxylation [6 cells/mL) on the surface of agar medium. Growth was evaluated after anaerobic incubation for up to 8 days at 28°C.Tests were performed on MRS agar (pH 4) containing 7% ethanol unless otherwise stated. Ethanol tolerance was determined at ethanol contents of 10, 12, or 14%. SOxylation . Isolate in situ in winery tanks during spontaneous MLF.Grapes and wine fermentations constitute complex microbial ecosystems consisting of highly dynamic yeast and bacteria communities. Despite the importance of LAB populations in shaping the wine quality, our current knowledge on the spatiotemporal distribution of LAB populations in grapes and musts during the alcoholic or malolactic fermentation is still limited. Here we analyzed the LAB culturable communities in two distant viticultural zones in Greece, Peza in Crete and Nemea in Peloponnese. Samples included grapes and the respective fermenting musts. Sampling was also conducted after the end of the alcoholic fermentation (AF) andLAB were detected at relatively low frequencies on grapes. About 28% of grape samples from the Nemea region harbored bacteria at populations ranging from 1.4 to 3.8 log CFU/mL. In grapes from Peza, the bacterial populations were below the detection limit. The low incidence of LAB populations on wine grapes, as detected here, is in accordance with previous studies that suggest limited LAB population density (<3 log CFU/g) in vineyards, due to their nutritional requirements , 27–31. O. oeni [ Lactobacillus spp. [Musts from grape samples were allowed to ferment spontaneously and at the middle stage of the AF (MF stage) bacteria could be recovered from 16% of the samples from either region. In the case of Nemea, population densities were relatively low (1.4–3.7 log CFU/mL), except for a single population that reached 8.7 log CFU/mL. Similarly, in Peza samples, populations at stage MF ranged from 0.9 to 3.3 log CFU/mL, except for one sample (ca. 7.2 log CFU/mL). At the end of the AF (EF stage), the number of Nemea samples with detectable populations decreased to 9%, while counts ranged from 1.3 to 7.0 log CFU/mL. As opposed, the respective percentage of Peza samples increased (24%), with populations ranging from 1.9 to 4.3 log CFU/mL. No bacterial populations were detected in samples from Nemea or Peza regions after the completion of AF. Present results show that, with a few exceptions, the bacterial growth is limited during the AF. Similarly low bacterial densities during the AF, ranging from 2 to 4 log CFU/mL, have been recorded previously . These p O. oeni , 32–36.  O. oeni , 36. Conlus spp. . As oppo Lactobacillus graminis, Lactobacillus hilgardii, Lactobacillus pentosus/plantarum, Lactococcus lactis, Oenococcus oeni, Pediococcus parvulus, P. pentosaceus, Staphylococcus epidermidis, and Weissella sp. According to the above analysis, isolates within group III showed 100% sequence similarity to both Lactobacillus pentosus JCM 1558T (D79211) and Lactobacillus plantarum NRRL B-14768T (AJ965482) followed by 99.8% to Lactobacillus paraplantarum DSM 10667T. Since 16S rDNA sequence is identical or highly similar among these species, a multiplex PCR assay with recA gene-based primers was applied for the identification of isolates within group III, as previously suggested [ Lactobacillus plantarum.16S-ARDRA grouped 626 isolates according to their banding profiles (profiles I to IX) . PhylogePediococcus pentosaceus and Lactobacillus graminis were the most abundant LAB species in grape samples from Nemea , followed by Weissella sp. and Lactococcus lactis at percentages lower than 7%. Typically, LAB species diversity associated with grape surfaces is rather limited mainly due to their nutritional requirements [ Lactobacillus , Pediococcus, and Leuconostoc [ Enterococcus and Weissella. Here we also detected Lactococcus lactis, a species that is quite scarce on grapes and a potentially novel Weissella species.irements . Speciesconostoc , 37, 38.conostoc  revealed Pediococcus pentosaceus showed a higher level of persistence compared to the other species encountered on grapes. All other grape-associated populations were undetectable except for Lactobacillus graminis, which replaced S. epidermidis in one case. Lactobacillus plantarum emerged for the first time in two out of five samples, in which initial LAB populations on grapes were below the detection limit. At the EF stage, LAB were detected in three samples and all isolates were identified as Lactobacillus plantarum. Although in Peza grape samples bacteria were below the detection limit, LAB populations then emerged during the AF. At stage MF, Lactobacillus plantarum was the only species detected in all samples. At the EF stage, all samples were exclusively dominated by Lactobacillus plantarum, except for one sample in which P. pentosaceus thrived.At the MF stage in Nemea samples, Lactobacillus plantarum is scarce on grapes [ Oenococcus oeni, the principal malolactic bacterium often isolated from wines, was not detected on grapes or fermenting musts, collaborating previous suggestions about the absence or low population of this species in Greek vineyards [Previous studies have also shown thatn grapes , 30, butn grapes . Oenococineyards . O. oeni that could be recovered from all tanks performing spontaneous MLF. In 75% of the samples, Pediococcus parvulus was also isolated, albeit at significant lower populations than O. oeni. In one case, Lactobacillus hilgardii was also isolated along with P. parvulus, again at much lower population density than O. oeni . The high occurrence of P. parvulus in the present samples needs further consideration since it is often associated with ropiness and oiliness of wine [ P. parvulus and Lactobacillus hilgardii were identified as the main spoilage, high histamine producing bacteria [The dominant population in winery-associated samples was of wine . Furtherbacteria ; therefoFor the discrimination of different LAB genotypes, various PCR-based fingerprinting methods were initially evaluated, including rep-PCR using the primer (GTG)5 or the primer set REP1R-Dt/REP2R-Dt and RAPD analysis with various primers. Among them, PCR using the primer RAPD2 (RAPD2-PCR) generated clear and reproducible banding patterns and also showed the highest discriminatory capacity in our tests (data not shown). Therefore, it was retained as the fingerprinting method of choice in the present genotyping analysis. The primer RAPD2 has been successfully applied previously in RAPD-PCR assays to differentiate strains within various LAB species , 42. Lactobacillus plantarum isolates, RAPD2-PCR generated a total of 45 polymorphic bands and 14 distinct banding patterns (hereafter referred to as genotypes) were identified  and the existence of genetic structure between populations of the two geographical zones of origin (groups of Peza and Nemea) was evaluated. Results from UPGMA cluster analysis showed that the spatial distribution of genotypes within a vineyard is rather random (data not shown). Measures of genetic identity (Nei's coefficient) showed that most vineyard populations shared a relatively high degree of genetic similarity (>0.7). The UPGMA tree of vineyard populations showed no clustering according to the zone of origin  in wine. Therefore, LAB should be evaluated for their ability to produce BAs, before being used as malolactic starters. By using appropriate culture media , we anal Lactobacillus hilgardii strain that produced tyramine but the corresponding gene (tyrdc) was not amplified. Thus the percentage of mismatching was rather low (1.5% of the strains), being slightly lower than the one detected by Coton et al. [Recently, a PCR method was developed for the simultaneous detection of four genes involved in the production of the above BAs . We appln et al.  (2.5%). n et al. . The reln et al. . Lactobacillus hilgardii strains isolated from one winery tank performing spontaneous MLF produced tyramine and/or putrescine. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in the final product, since spontaneous fermentation may allow the occurrence of BA-producing strains.All three Lactobacillus plantarum was the most abundant species in both regions and dominated the alcoholic fermentations. However, there was not any genetic structure in the Lactobacillus plantarum populations examined. As expected, O. oeni was quantitatively the principal LAB in the winery tanks during the MLF. Present results point to relatively high genotypic and phenotypic diversity within most LAB species identified, including O. oeni. Most importantly, various strains of the same species may coexist in the same tank during the MLF. Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4 than vineyard-associated isolates. Most LAB strains did not produce BAs in our tests. Further PCR analysis targeting BA-producing genes verified that the frequency of BA-producing LAB was low. However, a few LAB strains isolated from a winery tank conducting MLF did produce major BAs, strengthening the need for novel superior LAB starters to control the MLF.The present study shows that the LAB species richness and population densities on grapes may differ considerably between regions or vineyards. Yet,


Hippocampus zosterae) is widely distributed throughout near-shore habitats of the Gulf of Mexico and is of commercial significance in Florida, where it is harvested for the aquarium and curio trades. Despite its regional importance, the genetic structure of dwarf seahorse populations remains largely unknown. As an aid to ongoing conservation efforts, we employed three commonly applied mtDNA markers  to investigate the genetic structuring of H. zosterae in Florida using samples collected throughout its range in the state. A total of 1450 bp provided sufficient resolution to delineate four populations of dwarf seahorses, as indicated by significant fixation indices. Despite an overall significant population structure, we observed evidence of interbreeding between individuals from geographically distant sites, supporting the hypothesis that rafting serves to maintain a degree of population connectivity. All individuals collected from Pensacola belong to a single distinct subpopulation, which is highly differentiated from the rest of Floridian dwarf seahorses sampled. Our findings highlight the utility of mtDNA markers in evaluating barriers to gene flow and identifying genetically distinct populations, which are vital to the development of comprehensive conservation strategies for exploited taxa.The dwarf seahorse ( Hippocampus zosterae) is a diminutive species endemic to North America, distributed from southern Texas throughout the northern coast of the Gulf of Mexico, including the entirety of the Florida Peninsula and the northern islands of the Bahamas [H. zosterae is one of three seahorse species found in the western North Atlantic, but it is the only New World seahorse demonstrating dwarfed morphology, reaching a maximum length of only 38.0 mm [The dwarf seahorse  as tools for an initial assessment of genetic structuring within an exploited species.Here we assess the population structure of one such species, This study was conducted in accordance with American Veterinary Medical Association (AVMA) guidelines and all steps were taken to avoid needless suffering. The following protocol for all sampling procedures employed in this study was reviewed and specifically approved by the Columbia University Institutional Animal Care and Use Committee (IACUC) Protocol number: AC-AAAE9612 (Y1 M00) and was administered by the authors. Tricaine methane sulfonate (MS-222) was used as an anesthetic agent at a range of 25-50mg/L as suggested by the ICM veterinary consult. All field collections for this study were conducted in locations that permit public fishing, and nonresident fishing licenses for saltwater shoreline fishing issued through the Florida Fish and Wildlife Conservation Commission were obtained prior to all sampling efforts. Field collections did not involve endangered or protected species and no additional specific permissions were required for sampling procedures or experimental manipulations. Seahorses collected from Big Pine Key and Tampa Bay were collected through a partnership with licensed marine ornamental collectors and were fin-clipped while in holding tanks. Individuals collected in this manner could not be returned to the wild and were donated as breeding stock to an aquaculture facility in Ft. Pierce, Florida (Seahorse Source). Seahorses from all other locations were collected by dip netting and released after fin clipping to the site of collection.H. zosterae individuals from eight locations around the Florida Peninsula during August 2012 and January 2013  [20 and 2.0μl PCR product [DNA was extracted from preserved fin clips using a DNeasy Blood and Tissue Kit (Qiagen) and amplified through PCR. Amplification reagents consisted of: 22.3μl deionized water, 0.28 uM forward primer, 0.28 uM reverse primer, 301.55 mM BSA, one Illustra PuReTaq Ready-To-Go PCR bead and 1.0μl template DNA (25-50ng). The same thermocycler profile specified by Woodall  was used product . AnalysiSequencher v. 5.0.1 . All contiguous sequences below 90% quality were re-sequenced; those still below 90% quality were then excluded from the analysis. Aligned sequences from all three mtDNA markers were used in a BLAST search conducted on Geneious R6.06 using sequences from GenBank for reference. All CO1 sequences were estimated above 99% confidence as H. zosterae. No reference sequences existed on GenBank for ND4 and DLoop gene regions. Contiguous sequences were trimmed so that all were the same length and exported by mtDNA gene region as FASTA concatenated files. All sequences were checked for stop codons to ensure that no mitochondrial pseudogenes (NUMTs) were present in the sample. Concatenated files from all gene regions  were combined, aligned and further trimmed in Textwrangler  to produce a single concatenated file with a 1450 bp consensus region. Sequences were deposited in Genbank .Forward and reverse DNA sequences were aligned and edited in DNA Sequence Polymorphism v. 5.10.1 (DNA SP), which assigned 104 haplotypes from the overall population of 127 individuals [FaBox v.1.41 [Arlequin 3.5 [ST and FST values between all sampled populations [FST assumes distances between alleles to be equal and responds to vicariance more quickly, while ΦST considers pairwise distances between alleles and increases more slowly after population subdivision, presenting a more historical view of population dynamics [Arlequin. We also calculated two neutrality statistics, Tajima’s D and Fu’s F, to differentiate our results from neutral variation [The diversity of mtDNA sequences was estimated at the haplotype level using ividuals . This hax v.1.41 . Differequin 3.5 ,27. To aulations . Though dynamics ,30. To aariation ,32.Network v.4.611 to produce a median joining network with an epsilon value of zero [DNA SP v. 5.10.1 [xy) between all sampled sites  and the Florida Keys locations . Mean nucleotide diversity (π) and mean number of pairwise differences among sequences (K) was highest in individuals collected from the west coast of Florida . Tajima’s D and Fu’s F were negative for all sampled sites, and both measures were significantly negative for individuals collected from sites A, B and G ( B and G .Arlequin 3.5 was significant for molecular distance (ΦST) and haplotype frequencies (FST) in the overall sample . Pairwise comparisons between sampling sites demonstrated significant genetic structuring among Floridian dwarf seahorses . Seahorses from site F also demonstrated high and significant pairwise fixation index values when compared to seahorses from the majority of other sites, and were retained as the fourth discrete putative population (Big Pine Key). However, the Big Pine Key group was less statistically supported than the other identified populations, as fixation index data showed seahorses from Big Pine Key to be significantly differentiated from the Pensacola and West Coast populations, but not significantly different from all of the sampling sites included in the Eastern Keys population  conducted in eahorses . Four pupulation . SeahorsNetwork v.4.611 shows that, with the exception of individuals collected from site I, haplotype groups were broadly distributed throughout the sample   . Pairwiszations) . Similardistance .Hippocampus zosterae populations in Florida. We present strong evidence for the presence of a genetically distinct dwarf seahorse population (Pensacola), delineate two recognizable populations (Eastern Keys and West Coast), and suggest the presence of a fourth (Big Pine Key). The consistently high and strongly significant values for the various estimates of genetic differentiation employed in this study support the demarcation of the Pensacola sample as markedly genetically differentiated from the rest of the seahorses collected in this study and the consideration of the population as a discrete management unit [Our findings indicate significant population structuring among ent unit . Our anaent unit ,4,21,38.H. zosterae, in which geographically proximate populations are more genetically similar than those that are farther apart [Our results support an isolation by distance model as a contributing driver of population structuring in Floridian er apart . These fer apart .H. zosterae for active migration, these findings implicate passive dispersal as a common mechanism underpinning genetic connectivity in the species. Passive dispersal is likely accomplished through rafting, which has been previously documented in other seahorse species [The network visualization and genetic distance map suggest that, with the possible exception of Pensacola, some gene flow occurs between sampled locations. As the distances between these locations far exceed the capacity of  species ,40, and  species  reported species , 13.Palumbi  implicatST from west to east along the Florida coast identified through pairwise comparisons of genetic distance between contiguous sampling sites may signal migration facilitated by the Loop Current, in which the effective distance is compressed for eastwardly migrating individuals. The influence of the current may also increase the effective distance for westward migration, which would constitute what Palumbi [FST and ΦST measured between the adjacent sampling sites D and F. The considerable distance between Pensacola and the typical northern extent of the Loop Current may prevent passively dispersing individuals from gaining access to the current, potentially contributing to the lack of genetic intermixing observed between Pensacola and all other populations [The pattern of decreasing Φ Palumbi  describeulations . Howeverulations .b fragments from two large Hippocampus species (H. kuda and H. trimaculatus) with pelagic juvenile phases and found substantially lower measures [H. zosterae as a rapidly reproducing, short-lived species. Dwarf seahorses produce at least three generations annually throughout their range, exhibiting a seasonal boom-bust cycle of population expansion that typically peaks in September, likely coinciding with a similar rise in genetic variation [The ratio of haplotypes to individuals identified in this analysis could be considered uncommonly high in general terms. Goswami et al.  conductemeasures ,47,48. Tariation ,28. AddiHippocampus species. Species that produce pelagic offspring display prolonged periods of planktonic suspension that increase access to ocean currents and maximize capacity for long-range dispersal [H. zosterae, lack the ability to broadcast offspring over long distances, potentially resulting in greater retention of regional haplotypes and elevated heterozygosity within the overall population.Differences in developmental strategy may also contribute to the variation in haplotype diversity observed among ispersal . This maAnolis carolinensis) populations throughout the southeastern United States, which assigned 128 haplotypes to a total of 191 individuals in an analysis of concatenated 1172bp sequences from multiple mtDNA markers. Similar to H. zosterae, green anoles are widely distributed organisms with relatively short lifespans and strong potential for rapid population growth [H. zosterae populations could be achieved by analyzing nuclear DNA, which typically yields much lower levels of haplotype diversity than observed in analyses of mitochondrial DNA [A similarly high level of haplotype diversity was observed by Tollis et al.  in theirn growth . Greaterrial DNA .Assessments of population structure that reveal significant genetic differentiation in the absence of obvious population breaks or subdivisions often result in the designation of new management units . The recH. zosterae in Florida is regulated in the same manner as other near-shore marine fishes and is subject to the restrictions of recreational and commercial fishing licenses issued by the Florida Fish and Wildlife Conservation Commission [H. zosterae is likely small and can be sustained by dwarf seahorse populations. Implementation of seasonal restrictions on large-scale catches of dwarf seahorses, such as those for the curio trade, could be a useful management tool for curtailing collection during the peak of annual population contraction, which occurs from December to February [The trade in dwarf seahorses for both the aquarium and curio trades has historically been concentrated in Florida ,52. Collmmission . The resFebruary ,52.H. zosterae will be useful in resolving the full population dynamics of the species.Further study is needed to determine if the Pensacola population is truly genetically isolated or is part of a larger population spanning the northern coast of the Gulf of Mexico, including Mississippi, Alabama and the rest of the Florida panhandle. Future research incorporating both morphological and nuclear data with samples collected across the range of S1 FigDNA SP v. 5.10.1, showing the number of fixed differences (FD) and shared mutations (SM) observed in pairwise comparisons.DNA Divergence between sampling sites calculated in (TIF)Click here for additional data file.S2 FigDNA SP v. 5.10.1, showing the number of Jukes-Cantor corrected variances of the average pairwise number (±SE) of nucleotide differences per site (Dxy) between all sampled sites.DNA Divergence between subpopulations implemented in (TIF)Click here for additional data file.S1 TableSequences and properties of primer pairs for sequenced mtDNA gene regions .(TIFF)Click here for additional data file.S2 TableST) are shown on the lower diagonal.Background data tests conducted in IBD Web Service; pairwise estimates of geographic distance (rounded to the nearest km) between sampling locations are shown on the upper diagonal; pairwise estimates of genetic distance (Φ(XLSX)Click here for additional data file.


Beilschmiedia roxburghiana within each plot. We observed a significant negative correlation between tree diversity and the genetic diversity of B. roxburghiana within the communities. Using structural equation modeling, we further determined that the inter-plot environmental characteristics (soil pH and phosphorus availability) directly affected tree diversity and that the tree diversity within the community determined the genetic diversity of B. roxburghiana. Increased soil pH and phosphorus availability might promote the coexistence of more tree species within community and reduce genetic diversity of B. roxburghiana for the reduced average niche breadth; alternatively, competition could reduce effective population size and therefore genetic diversity of B. roxburghiana within community.A negative species-genetic diversity correlation (SGDC) could be predicted by the niche variation hypothesis, whereby an increase in species diversity within community reduces the genetic diversity of the co-occurring species because of the reduction in average niche breadth; alternatively, competition could reduce effective population size and therefore genetic diversity of the species within community. We tested these predictions within a 20 ha tropical forest dynamics plot (FDP) in the Xishuangbanna tropical seasonal rainforest. We established 15 plots within the FDP and investigated the soil properties, tree diversity, and genetic diversity of a common tree species  The relations between species diversity and genetic diversity have long been proposed and observed2345678910111213141357351741920et al. summarized the causal effects between species and genetic diversity within communities, but more studies are required to investigate the mechanisms of how one level of diversity affects the other level5822When the local characteristics influence the two levels of diversity in parallel, a positive correlation occursOver the last decade, the majority of research on SGDCs concentrated on how neutral processes or disturbances affect parallel species diversity and genetic diversity3171819202425262728Beilschmiedia roxburghiana (Lauraceae) using microsatellite DNA markers, and measured the environmental properties (soil nutrients and topography) of each plot. Our goal was to answer two fundamental questions: 1) Are the tree diversity of a community and the genetic diversity of a common tree species correlated? 2) What are the possible drivers of the SGDC pattern in these species-diverse communities?In this study, we established 15 plots (40 m × 40 m) within a 20 ha forest dynamics plot (FDP) in the Xishuangbanna tropical seasonal rainforest . We surv2 each (Appendix S1). The total tree abundance among the plots varied from 680 to 1186 with an average of 902 trees per plot. An average of 116 tree species were recorded in each plot and the rarefied tree species richness ranging from 90 to 124. The rarefied allelic richness (Ra) varied from 1.90 to 4.48 across the 15 plots . The soil properties within each plot were not correlated with the effective population size of B. roxburghiana (B. roxburghiana was negative (P = 0.063) within each plot , whereas the soil pH was correlated with phosphorus availability   , Fig. S3 < 0.01) .B. roxburghiana within the plots was identified with the structural equation modeling (SEM) analysis. A number of SEM models were tested in our study, and the best-fit model  was selected  or EP   and the genetic diversity of urghiana . An incr= 0.444) , whereas= 0.444) . Thus, tB. roxburghiana (B. roxburghiana could contain less genetic diversityP = 0.063) between the tree richness in the plot and the effective population size of B. roxburghiana within community, and also a significant positive correlation between effective population size of B. roxburghiana and its genetic diversity , were established within the 20 ha (400 m × 500 m) FDP in the Xishuangbanna tropical seasonal rainforest in south-western China B. roxburghiana within the plot.Fifteen plots, each 1600 mBeilschmiedia roxburghiana Nees is an evergreen, small- to medium-sized tree that grows in the tropical, evergreen, broad leaf forests in south-eastern Xizang Province and Yunnan Province, China, and in north-eastern Myanmar and India. This tree is typically a forest-dwelling species that generally occupies the second and third layers of the canopy and that can reach 20 m in height. Insects pollinate the hermaphroditic flowers, and gravity and vertebrates, such as birds and small mammals, disperse the seedsB. roxburghiana have become increasingly fragmented in recent years because of the deforestation that is a consequence of the development of rubber tree plantations. In addition to B. roxburghiana, the common tree species in the plots include Pittosporopsis kerrii (Icacinaceae), Parashorea chinensis (Dipterocarpaceae), Knema furfuracea (Myristicaceae) and Garcinia cowa (Clusiaceae).et al.et al.fi is the number of the focal tree species divided by the number of samples within the community; and the inverse Simpson index = S_TD, calculated as 1/RTR) was calculated using a rarefaction procedure implemented with R statistical software package (R Development Core Team 2013).The Xishuangbanna Station for Tropical Rain Forest Ecosystem Studies of the Chinese Academy of Sciences provided all primary data for this section of the FDP.For each of the 15 plots, the elevation, aspect, slope and convex were calculated using the procedures described in Liu B. roxburghiana in each of the 15 plots was measured using ten pairs of microsatellite DNA primers. We considered each plot a “population”, although the distribution of B. roxburghiana is more or less continuous in the FDP. An average of 11 samples (7–22) were genotyped. The protocols for the microsatellite genotyping and the primer sequences were described in Liu et al.a), the Shannon-Wiener index , and the inverse Simpson index  using a rarefaction procedure implemented with HP-rare1.0Ne) of B. roxburghiana within each plot using an updated version of the heterozygote-excess methodPcrit = 0.05 for excluding rare alleles. To clarify the degree of genetic differentiation among the populations, the FST was calculated, and the significance was obtained with 999 permutations using GenAlEx6.5The genetic diversity of Ne were consequently log-transformed to improve normality. In our study, four data sets were developed , and each data set contained several variables that were possibly correlated with one another. Therefore, we analysed these data sets with principal component analysis (PCA) based on their correlation matrices. The principal components score (PCS) was calculated for the topographical variables and the soil nutrients because two or three principal components had eigenvalues above 1 during the PCA analysis. The PCS was calculated as follows: PCS = (λ1/(λ1 + λ2+λ3)) × F1 + (λ2/(λ1 + λ2 + λ3)) × F2 + (λ3/(λ1 + λ2 + λ3)) × F3, where λ1, λ2, and λ3 are the eigenvalues of the three components and F1, F2, and F3 are the first three components; λ3 and F3 equal zero when only two components have eigenvalues above 1 during a PCA analysis. We first explored the pairwise correlations between topography, soil nutrients, tree diversity and genetic diversity of B. roxburghiana in the plots using simple Pearson correlation analysis, and the significance differences were corrected for multiple comparisons following the Bonferroni procedure. An analysis with SEM was used to further generate and explore the model and to infer the causal correlations among topography, soil nutrients, tree diversity and genetic diversity of B. roxburghiana. To begin, we constructed three conceptual SEM models of the expected multivariate relationships based on theoretically developed hypotheses of the interactions among the variables .