The diversity observed among crop wild relatives (CWRs) and their ability to flourish in unfavorable and harsh environments have drawn the attention of plant scientists and breeders for many decades. However, it is also recognized that the benefit gained from using CWRs in breeding is a potential rose between thorns of detrimental genetic variation that is linked to the trait of interest. Despite the increased interest in CWRs, little attention was given so far to the statistical, analytical, and technical considerations that should guide the sampling design, the germplasm characterization, and later its implementation in breeding. Here, we review the entire process of sampling and identifying beneficial genetic variation in CWRs and the challenge of using it in breeding. The ability to detect beneficial genetic variation in CWRs is strongly affected by the sampling design which should be adjusted to the spatial and temporal variation of the target species, the trait of interest, and the analytical approach used. Moreover, linkage disequilibrium is a key factor that constrains the resolution of searching for beneficial alleles along the genome, and later, the ability to deplete linked deleterious genetic variation as a consequence of genetic drag. We also discuss how technological advances in genomics, phenomics, biotechnology, and data science can improve the ability to identify beneficial genetic variation in CWRs and to exploit it in strive for higher-yielding and sustainable crops. More is expected from the world’s food systems than at any previous time in human history . The demHumans have domesticated hundreds of plant species over the millennia, transforming wild forms into a domesticate by fixing traits of importance in the agricultural system . Over tiSubsequently, one of the major limitations of using CWR in breeding is hybridization barriers between undomesticated germplasm and the crop which increases along with evolutionary divergence . UsuallyWild germplasm that has been collected and conserved over the last century is already available in genebanks . Howeverwww.gbif.org and www.genesys-pgr.org). Moreover, performing species distribution model analysis based on observations of existing populations can provide indications also for potential unobserved occurrence of populations across the studied area  has been the main instrument to regulate and ensure national sovereignty over biological resources including propagation material of CWRs in contrast to the previous situation of free access and sharing . The CBDDespite the wide phenotypic diversity observed among crop varieties, the majority of genetic diversity found in the wild did not pass the genetic bottleneck of domestication or was eroded later in strive for higher yields during the improvement phase. CWRs hold many benefits for breeding especially through the reintroduction of lost genetic diversity and new adaptive alleles that can increase crop production . This geThe ultimate goal of collecting and exploring CWR is to identify genetic variants that contribute to adaptation in natural environments and can also be beneficial in agricultural systems. Thus, the motivation in this context is mainly to identify genes and alleles with a potential benefit for crop breeding. Despite the contextual difference from ecological and evolutionary perspectives, the considerations, methodologies, and principles are akin. Generally, two types of approaches could be used to identify genes of interest in CWR: phenotype-independent approaches, such as genome scans  and genoSampling designs impact all approaches attempting to identify beneficial genetic variation and therefore should be given considerable attention and planning . The samA key consideration in the sampling strategy is the number of genotypes that should be sampled to facilitate the identification of adaptive genetic variation. This is a probabilistic problem; more sampling will increase the chances of detecting beneficial genetic variation albeit with additional cost. Formerly, genotyping was the main limiting factor for sample size; however, advent in genomic sequencing technologies has made ultra-high throughput genotyping accessible; hence, genotyping a large number of individuals is now affordable . The nexEnvironmental heterogeneity along space requires that sampling of individuals will properly represent the ecological gradient across the distribution range including the extremes. Occasionally, substantial adaptive genetic variation can be obtained from a single sampling location where micro-climatic conditions result in a range of environmental stressors within the same population. Moreover, theory predicts that adaptive genetic variation could also be obtained across different time points because non-random dispersion will lead to some level of environmental heterogeneity . Thus, sClearly, the sampling strategy and the subsequent analytical approach should be planned with much attention to the ecological and genetic characteristics of the target species. While abundant species can benefit from sampling across a spectrum of environmental gradients, species that are characterized by a constrained distribution may require an adjusted strategy of temporal repeated sampling or fine-resolution sampling of microenvironments to allow exploring the breadth of genetic variation.Implementation of beneficial genetic variation obtained from CWR in breeding often requires identifying the causative mutation or tightly linked polymorphism to the trait of interest in the wild germplasm. Analytical approaches to identify the genetic variation of interest can be divided between approaches where phenotypic data are not mandatory (phenotype-independent) and approaches that rely on high-quality phenotypic data (phenotype-dependent). A short list of tools and packages is provided as an example in a priori genome screening approaches. Genome scan methods are conducted by calculating population genetics statistics using a sliding window frame in a target population  that hinders adaptation to increasing environmental stress. Therefore, enhancing adaptation in elite varieties by introgression of new genetic variation from wild relatives is a promising venture. Introgression of beneficial genetic variation from CWR is not a new concept, and there have been many successful attempts to enhance adaptation mainly through increasing biotic and abiotic stress tolerance in various crop species . Some ofIn eukaryotes, the recombination rate varies significantly across species, populations, and individuals. The recombination rate also varies across the chromosome which appears as regions of elevated recombination (hot-spots) or suppressed recombination (cold-spots) along the genome. Likewise, recombination landscape varies between crop species and their wild relatives, thus recombination hot-spots and cold-spots do not necessarily occur at overlapping positions in both species . IntrogrSecond, introgression from a congener species through hybridization and backcrossing could be efficiently cleaved in recombination hot-spots and consequently purged by selection. Hence, the deleterious effect of genetic drag can be depleted efficiently while the beneficial allele is fixed . NeverthAs the ultimate goal is to be able to introgress the traits into a breeding material, sampling ecotypes that are very diverged from the cultivated gene pool may pose challenges to successful introgression of the anticipated beneficial genetic variation. Thus, identifying the progenitor wild population of the cultivated gene pool or at least the genetically closest can be critical for an efficient and successful introgression. Due to local environmental variation, beneficial alleles could potentially also be found in a genetically closer population to the cultivated gene pool . The higAnother consideration for the implementation of CWR in crop improvement is the breeding strategy of the target species. In many crops, breeders are crossing distinct inbred parents to produce a hybrid variety with increased vigor and yield. In these cases, the interaction between the introgressed wild allele and the cultivated allele at the adaptive gene should also be considered. Overdominance heterotic interactions are difficult to predict in advance and, in some cases, dominance of the cultivated allele may mask the effect of the wild allele. Nevertheless, at least some of the heterotic effects observed in hybrid crops are caused by genetic complementation from the wild parent , thus alDomestication is a long endeavor, which includes the fixation of many quantitative traits. Therefore, to develop a fully domesticated crop from a wild species, a long breeding process is required before all domestication syndrome traits are fixed and acceptable yields and biomass are obtained. Thus, it is not surprising that only a few plant species went through a complete process of domestication to meet modern agriculture standards . Among tde novo domestication . Other levels of genetic variation, including gene inversions, deletions, insertions, and duplications, are emerging as key factors in evolutionary biology and the generation of phenotypic variation within breeding material . Thus, himeframe .post hoc analysis especially in complex traits (Data collecting technologies are emerging as interesting means to monitor phenotypes in large-scale field trials. Moreover, the integration of continuous data recording instruments and remote sensing devices is now allowing researchers and breeders to monitor and perform detailed phenotyping of CWR in their natural habitats . In the x traits . Other ax traits . Rapid tx traits .Genomic and phenomic data could now be generated in large quantities and reasonable budget, thus the genotype-phenotype gap is quickly shrinking thanks to technology . This haCrop wild relatives have long been recognized as a highly valuable resource of genetic variation that could be exploited in breeding. Many examples of successful implementation of wild genetic variation in breeding exist yet much more could be exploited to improve the nutritional value of crops, increase their resilience to biotic and abiotic stress, and enhance their economic yield. Targeting beneficial genetic variation in nature is challenging and requires a careful sampling design that considers the ecological and evolutionary properties of the target species. Advent in high-throughput genotyping technology coupled with ongoing developments in computation power and machine-learning algorithms is allowing to identify beneficial genetic variation in CWR at the finest resolution. Thus, the implementation of wild genetic variation in breeding is expected to increase in the near future thanks to the ability to narrow the introgressed region and reduce the effect of genetic drag. Genome editing technology is quickly emerging as a promising approach to introduce beneficial genetic variation and avoid some of the complications associated with crossing. Although this technology is still not fully functional in many crop species, new protocols are emerging, thus CWRs are becoming more relevant for breeding than ever before.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila suzukii Matsumura, which is widely spread in the main soft-skinned fruits production areas in China, presents a threat to importing countries. In order to develop a phytosanitary cold treatment measure for preventing the movement of this drosophila fly, cold tolerance of six immature life stages of D. suzukii was compared followed by time-mortality and large-scale confirmatory tests on the most tolerant stage in grape fruit. Egg was defined as the most cold-tolerant stage by comparing the mortality of all the immature stages  treated at 0 and 2°C. The minimal lethal time (LT) for 99.9968% mortality (95% confidence level [CL]) estimated by the probit model was 10.47 d at 0°C and 11.92 d at 2°C, respectively. Hence, 11 d (at 0°C) and 12 d (at 2°C) were chosen as the target time to conduct the confirmatory tests. No survivors were found among the estimated 50,385 and 57,366 treated eggs, which resulted in the efficacy of 99.9941 and 99.9948% mortality (95% CL) at 0 and 2°C, respectively. Our study suggests a technical basis for cold disinfestation on D. suzukii in cage-infested Chinese ‘Red Globe’ (Vitis vinifera L.) grape, which could provide flexible phytosanitary treatment for control of D. suzukii in the international trade of grape. The spotted wing drosophila,  Drosophila suzukii Matsumura, is a worldwide pest that originated from Asia and that has infested more than 60 plant species of berries and stone fruits, with economic losses of up to 100% reported in more than 30 countries in Asia, the Americas, and Europe , Comité de Sanidad Vegetal (COSAVE), the Eurasian Economic Union (EAEU), Jordan, Kazakhstan, Turkey, Morocco, and Mexico  are suitable hosts for D. suzukii and can be heavily infested in laboratory conditions  of D. suzukii in ‘Red Globe’ grapes to ensure quarantine security  with an artificial diet  modified from the larvae diet of papaya fruit fly, Bactrocera papayae (The  papayae . All staD. suzukii egg laying for 24 h. Every three to four infested grapes (40 ± 5 g for each) were put into one glass jar (0.42 liter) with filter paper at the bottom and covered with gauze to prevent contamination. Each jar was as one replication in the cold treatment and each treatment had three replications (three jars).Table grapes (cultivar of ‘Red Globe’) were bought from the local market near the Chinese Academy of Inspection and Quarantine, Beijing, China. Pesticides were not applied directly on the fruit and there were no pesticide residues. To prevent microbial contaminations, grapes with similar size, hardness, and color were washed with distilled water, dried at room temperature, and stored in the cold chamber at 0°C. For each time of egg collection, the grapes were taken out and warmed up to room temperature, and a longitudinal cut was made with a sterilized scalpel , and theD. suzukii eggs. When larvae began to pupate, wheat bran was put under the grapes to prevent the larvae and pupae from drowning. Stages were checked every 12 h in the first 2 d and then at an interval of 24 h. At each time point, grapes were removed from three jars and dissected to count the number of each stages under a microscope.The infested grapes in jars were placed in the rearing room for the development of the D. suzukii were treated at 0°C (0.027 ± 0.168) for 12 h and 2°C (2.034 ± 0.181) for 24 h via three replicates for each stage. Fruit pulp temperatures were recorded every 90 s by a temperature recorder  with six probes . All the probes were calibrated at the beginning and the end of each trial with a certified mercury glass thermometer. The starting time was recorded as all probes reached the target temperature. Twelve or 24 h later, the infested grapes were transferred to the rearing room for 4 d and the mortality of treated and controlled insects was checked under a microscope. These treatments were repeated three times each.To determine the most tolerant stage against cold, grapes infested with different immature stages based on above rearing experiment of The criteria for mortality assessment in this research study were as follows: eggs: blacking or unhatched; larvae: no movement when prodded with a blunt probe; pupae: blacking, half-eclosion, deformation of eyes and wings.The grapes containing 1-d-old eggs, which were defined as the most tolerant stage after the cold tolerance test, were kept into the refrigerator chamber to be cold treated at 0 or 2°C. The core temperatures of the infesting grapes were monitored with six probes. Treatment was deemed to start after all the fruits had reached the target temperature. The grapes were transferred to the rearing room every day till 10-d treatment at 2°C and on the day of 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 for 0°C treatment. The mortality of all treatments and controls was checked 4 d later by using the methods mentioned above. The temperature monitored in the treatment was 0.0 ± 0.2 and 2.0 ± 0.2°C (mean ± SE) for the treatment at 0 and 2°C, respectively. These treatments were repeated three times each.For each trial of the confirmation test, grapes were put into the adult cages which contained about 2,000 adults from one batch of the colony and kept for 24 h. Then, the infested grapes were put into the cold refrigerate chamber for cold treatment at 0°C for 11 d and 2°C for 12 d according to the result of the efficacy test. The mortality was checked by using the method mentioned above. These treatments were repeated three times at each temperature.99) and 99.9968% mortality (LT99.9968) at the 95% confidence level  by SPSS  . Means wce level .Pu) of the large-scale confirmatory tests was calculated by the equation (1):The mortality proportion , and n is the number of treated D. suzukii with zero survivors  and 2°C for 24 h   . The eggEach growth state observation of immature stage was done in triplication and each replication of eggs and first instar included more than 100 test individuals. For second and third instars and early pupae, each replication included about 70 individuals, while late pupae contained more than 50 individuals.D. suzukii during the time–response test increased significantly with the increase of time when treated at 0°C  and 2°C  for 8 d. The minimum days to 100% mortality were 6 d at 0°C and 7 d at 2°C, respectively  and the logit model without log-transformed data (2.34–2.73) indicated that the estimations matched the data. Both the LT99 and the LT99.9968 estimated by probit analysis (with log-transformation) were about 4 d longer than those estimated by the logit model (without log-transformation). The heterogeneity data in these two models were similar. Therefore, 7- and 11-d cold treatment at 0°C, and 9- and 12-d cold treatment at 2°C were all selected as the target lethal time for conducting the following confirmatory large-scale test , respectively. For the treatment at 2°C for 9 d, eight living larvae were found in the treated grapes containing more than 24,500 eggs in the first trial, suggesting that the logit model was not appropriate. However, there were no survivors from a total of 57,366 eggs and 50,385 eggs treated at 0°C for 11 d and 2°C for 12 d, respectively  of ectively , indicatD. suzukii was firstly observed in ‘Red Globe’ grapes to select the appropriate stages used in the cold tolerance test , whose mixed stages were treated in host fruit , Bactrocera tryoni (Froggatt), B. dorsalis (Hendel), and B. zonata (Saunders)  . In addiBesides the disinfestation of the infesting insects in the commodity, there should be no significant deleterious effects on the quality of the fruit. In general, the ‘Red Globe’ grape could be stored at an optimum temperature of −1 to 0°C to keep the quality for more than 1 month . SeveralD. suzukii in ‘Red Globe’ grape were examined. The results show that among six developmental stages  of D. suzukii in grapes, egg was the most cold-tolerant stage. The time–response test and large-scale confirmatory test suggest that a minimum of 11-d cold treatment at 0°C and 12-d at 2°C were recommended for disinfesting D. suzukii in grapes with efficacy of 99.9941 and 99.9948%, in order to provide postharvest pest control and quarantine security for international trade. All results suggest that cold treatment could be used as a methyl bromide alternative for quarantine control of D. suzukii in ‘Red Globe’ grape.In this research, the effects of phytosanitary cold treatment of


Rucervus eldii) is one of the most endangered cervids of Southeast Asia. Geographically, it has three distinct subspecies; Sangai or Manipur's brow-antlered deer (R. e. eldii), Siamese brow-antlered deer (R. e. siamensis) and Thamin or Myanmar’s brow antlered deer (R. e. thamin). We examined the genetic diversity of wild and captive populations of R. e. eldii and compared its relationship with other subspecies using mtDNA control region gene. During the analysis, only one haplotype was detected in 30 samples of R. e. eldii. No genetic variation was observed among the R. e. eldii populations. The reduced genetic diversity indicates that the population has passed through the bottleneck effect that might have resulted in the inbreeding depression.The Eld’s deer or brow-antlered deer ( Rucervus eldii) is the most localized cervid species found in India occurring as a single isolated population in a 40 km2 area of the Keibul Lamjao National Park (KLNP), Manipur . The tissue samples were stored at −20 °C and fresh faecal samples were carefully collected and stored in 70% ethanol at room temperature. DNA was extracted from all the samples using the phenol/chloroform method  using primer Cerv.tPro and CervCRH  positions  and hog deer (Axis porcinus) that is closely related to the chital (Axis axis), and were grouped together as monophyly (Angom et al. R. e. eldii from Manipur showed a closest relationship with R. e. thamin than the R. e. siamensis. During the analysis, only one haplotype was detected in wild and captive populations of R. e. eldii. The lack of genetic diversity within the R. e. eldii indicates that the population is under inbreeding depression.In the present study, no nucleotide diversity was found in wild and captive populations of sus tree . The phyClick here for additional data file.


Lactic acid fermentation is one of the oldest methods used worldwide to preserve cereals and vegetables. Europe and Asia have long and huge traditions in the manufacturing of lactic acid bacteria (LAB)-fermented foods. They have different cultures, religions and ethnicities with the available resources that strongly influence their food habits. Many differences and similarities exist with respect to raw substrates, products and microbes involved in the manufacture of fermented products. Many of them are produced on industrial scale with starter cultures, while others rely on spontaneous fermentation, produced homemade or in traditional events. In Europe, common LAB-fermented products made from cereals include traditional breads, leavened sweet doughs, and low and non-alcoholic cereal-based beverages, whereas among vegetable ones prevail sauerkraut, cucumber pickles and olives. In Asia, the prevailing LAB-fermented cereals include acid-leavened steamed breads or pancakes from rice and wheat, whereas LAB-fermented vegetables are more multifarious, such as kimchi, sinki, khalpi, dakguadong, jiang-gua, soidon and sauerkraut. Here, an overview of the main Euro-Asiatic LAB-fermented cereals and vegetables was proposed, underlining the relevance of fermentation as a tool for improving cereals and vegetables, and highlighting some differences and similarities among the Euro-Asiatic products. The study culminated in “omics”-based and future-oriented studies of the fermented products. A huge percentage of these functional products are usually produced by lactic acid bacteria (LAB) via fermentation.Globally, the choice and consumption of healthier and safe food products has steadily increased in the past few decades, emphasizing the diet–human health connectivity. In this sense, functional products are excellent food options, as they aim to improve life quality by preventing nutrition-related diseases . A huge Fermentation is a cheap food biotechnological process used worldwide, a tool that is helpful in the production of several functional foods and beverages. The pyramidal base for the production of traditional fermented foods is in the household, in which an enormous amount of microbial and raw matter inoculants are used in a haphazard manner . The lowEurope and Asia have long and huge traditions in the manufacturing of several LAB-fermented foods and, in the last years, thanks to scientific progress and “omic” studies, the knowledge, safety and regulation of many fermented products have increased.Trade between European and Asian countries accounts for about half of the global trade, and though intra-regional trade is four times higher than cross-regional trade, the Euro-Asian regions trade more between them than between any other regions of the world . As signIn general, natural fermentations are performed by lactic acid bacteria and yeasts. These microorganisms often act in synergy. Yeasts play an important role in the production of numerous fermented beverages and leavened breads thanks to their ability to produce high levels of ethanol, carbon dioxide and highly desirable aromatic compounds, while lactic acid bacteria are particularly important in the fermentation of cereals, vegetables, meat and dairy products for their acidifying and proteolytic activity, and for their ability to produce aromatic compounds.In this review, our interest has focused only on the study of lactic acid fermentation, which, although having a secondary role for some fermented products, is however decisive in defining the final quality of the products.This review considers cereals and vegetable products obtained via lactic acid bacteria fermentation in Europe and Asia. The review includes the relevance of fermentation as a tool for improving cereals and vegetables, the concept of lactic acid bacteria fermentation and the Euro-Asian LAB-fermented cereals and vegetables list, highlighting differences and similarities among products. It further describes an outlook on prospective studies and considerations.Cereals are the main consumed staple-food in most parts of the world. Their derived products can also be consumed alongside popular vegetables to make exotic delicacies. Being rich sources of carbohydrates and mineral nutrients, cereals and vegetables proffer numerous sensory, nutritional and physiological potentials when fermented. Natural or spontaneous fermentation was used by our ancestors for the preparation and preservation of foods like cereals and vegetables, without having any knowledge of microbes involved in the process .In Europe, Asia or in other parts of world, people still practice the old traditional method of preparation of breads or loaves without using any commercial strains of baker’s yeast . FurtherA general classification of food fermentation includes different categories: (a) fermentations, producing textured vegetable protein as meat substitutes in legume/cereal mixtures; (b) high salt/savory meat flavored/amino acid/peptide sauce and paste fermentations; (c) lactic acid, alcoholic, acetic acid/vinegar, alkaline fermentations; (d) leavened breads/sour dough breads; (e) flat unleavened breads .LAB fermentation enables the preservation of numerous cereals and vegetables, as well as the transformation of the raw material into a new product with unique sensorial properties, new flavors, aromas, better textures and enhanced nutritional value. During fermentation, raw substrates become colonized by autochthonous microorganisms that use their enzymes to digest the food components into acceptable final products to the prospective consumers ,11.Cereals, for example, lack the essential amino acids and some other vital compounds for proper nutrition, thus fermentation helps in releasing these compounds by decreasing the levels of carbohydrates and non-digestible polysaccharides and oligosaccharides in cereals, and increasing the essential amino acids’ production and bioavailability ,13. PolyThe use of a desirable starting culture for the making of fermented foods may prevent contamination, and helps retain consistent food quality . TherefoFermentation is a way to exploit the activities of microorganisms so as to preserve and transform raw materials into high-quality products. Lactic acid fermentation is the oldest and most popular way to improve the functionality, nutritional value, taste, appearance, shelf-life and safety of cereal foods, and reduce the energy required for cooking .Examples of lactic acid-fermented products, i.e., products primarily fermented by lactic acid bacteria, include yoghurt, cheese, sourdough, olives in the western world, sauerkraut , thai pickled vegetables (fermented cabbage from Thailand), sausages, Chinese steamed bun, kimchi (fermented and spiced Napa cabbage from Korea), fermented soybean and rice .Responsible for this kind of fermentation are the lactic acid bacteria, non-spore forming, acid-tolerant bacteria classified as anaerobic, facultative anaerobic bacteria, aerobic or microaerophilic, catalase-negative, and Gram-positive with coccoid or rod-shaped cells, which may form chains, pairs or tetrads .Firmicutes, class Bacilli, order Lactobacillales, and refers to five families: Lactobacillaceae (which now includes the family Leuconostocaceae), Streptococcaceae, Enterococcaceae, Carnobacteriaceae and Aerococcaceae [The new bacterial taxonomy classifies the lactic acid bacteria in the phylum occaceae .Lactic acid bacteria are among the most familiar bacterial groups to humans, since they are widespread in nature, occurring in numerous nutrient-rich habitats such as plant, soil, animals and humans. They can as well be found fermenting already consumed food products in the gut . The isoMany genera/species of microorganisms have been reported in relation to various fermented foods and beverages across the world.Lactobacillus (old classification), Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella.The lactic acid bacteria associated with foods include species of the genera Lactobacillus into 25 genera, including the emended genus Lactobacillus, which includes host-adapted organisms that have been referred to as the L. delbrueckii group, Paralactobacillus and 23 novel genera: Acetilactobacillus, Agrilactobacillus, Amylolactobacillus, Apilactobacillus, Bombilactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, Lactiplantibacillus, Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacilus, Paucilactobacillus, Schleiferilactobacillus and Secundilactobacillus [Lactobacillus to avoid any confusion in the reader. However, the links for the appropriate name conversions are as follows: http://lactobacillus.ualberta.ca; http://lactobacillus.uantwerpen.be; http://lactotax.embl.de/wuyts/lactotax/).Actually, scientists have recently reclassified the genus bacillus . , or to a mixture of lactic acid, carbon dioxide and acetic acid and/or ethanol (heterofermentation). Other compounds, such as diacetyl, acetaldehyde and hydrogen peroxide, are also produced. These compounds contribute to the flavor and texture of fermented foods, and may also contribute to the inhibition of undesirable microbes . In addiThe richness and diversity of microbial species, with their relative abundances and interactions, determine the establishment of distinctive and unique characteristics of each fermented product.Despite common features, however, many differences exist with respect to substrates and products, and the types of microbes involved in the manufacture of fermented foods and beverages produced globally.In European countries, the fermentation of cereals is used mainly for the obtainment of bakery products  and for different beverages, including the ales and beers of northern Europe, the lagers of central and eastern Europe and numerous low or non-alcoholic beverages that contain a maximum of 0.5% volume of ethyl alcohol .The most common and uncommon European products made from grains and obtained mainly by lactic acid fermentation are observed in broa, a home-baked sourdough bread, still following ancient protocols [In Europe, countless different types of “Breads”, including traditional typical breads ,30,31,32rotocols ,35,36. Irotocols ,38,39. Mrotocols ,41,42. Trotocols .Sourdough is obtained by the fermentation of autochthonous microorganisms (bacteria and yeast) that are naturally present in the environment, or in raw materials or ingredients, conferring each sourdough a unique characteristic. Sourdoughs are characterized by more than 50 different species of lactic acid bacteria, but it is very likely that a non-identifiable and perhaps new sourdough LAB species exists .In Europe, among fermented cereal products other than bakery products, different low and non-alcoholic cereal-based beverages have been developed. The development of cereal-based fermented beverages is in response to the growing prevalence in consumer vegetarianism, lactose intolerance, cholesterol content and economic reasons that are associated with dairy products. In fact, cereal-based beverages fermented with LAB could be a perfect alternative for people who are allergic to milk proteins . Such prSaccharomyces cerevisiae) and LAB such as Lactobacillus casei and Leuconostoc mesenteroides [“Kvass” is a cereal-based beverage traditionally made from fermented barley and rye malt, rye flour and stale rye bread that is mainly produced in eastern countries such as Russia and Poland, and Baltic countries, especially Lithjuania and Latvia . It is ateroides ,59. As iteroides .Another traditional ancient fermented cereal, produced in different East European, countries is “Boza”, a cereal beverage that is obtained from the fermentation of whole grain or flour of different cereals , but boza of the best quality and taste is made of millet flour. It is very popular in Turkey, Albania, Bulgaria, Macedonia, Montenegro, Bosnia and Herzegovina, and parts of Romania and Serbia, as well as in South Russia. It is a viscous low-alcoholic drink with a pleasant sweet taste and slightly acid flavor, varying in color from creamy-white and beige to light brownish .Due to boza’s pleasant flavor and taste and high nutritional value, it has become a very popular drink consumed daily by people of all ages. The compositional variations of boza samples result from the utilization of different types and amounts of cereals as raw materials, and uncontrolled fermentation conditions .L. plantarum, L. brevis, L. fermentum, Leuc. mesenteroides, L. casei, L. acidophilus, etc), which provide the acid character of boza, and yeasts responsible for alcohol fermentation [The microbiota responsible for the fermentation of Boza seem to be particularly heterogeneous, including both homo- or hetero-fermentative LAB , liver and bile diseases, and even cancer treatment.Romanian borş was investigated for the first time regarding its microbiological and physicochemical characteristics by Grosu-tudor , who shoOriganum vulgare and Ledum palustre) are added during the fermentation process [There are many other cereal-fermented products typical of the traditions of the East European countries, still little studied. Historically, several low-alcoholic drinks were made in Estonia such as “Taar” and “Kali”. “Taar” is made of rye and barley, rarely also oats. Grains are milled into flour and mixed with boiled water. The beverage has to turn sour for 2–3 days, and the resulting drink is “taar”. To prolong the preservation of taar and to add specific taste, aromatic species  preparations from fermented cereals. Different fermented dishes and drinks made of oat were widely consumed as regular meals in Estonia until the end of 19th century, and sporadically until the middle of 20th century, but nowadays their popular use is limited to special groups trying to restore national food heritage. For example, oat seeds were macerated in warm water, left to ferment for a few days, and then the milky water was squeezed out and the resulting sour liquor, called “kaera kiesa” or “kaerapiim”, was drunk on the side of the meal .A bit of a different beverage, called “kile”, was made of oat flour mixed with water; it was let to stay in warmth for a night. This filtered sour beverage was consumed instead of sour milk on the side of the meal. If the filtrate was boiled, it became a kind of gruel, which was also called kile, but also “kiisel” or “kisla”, and eaten hot with butter or fat, or later as a cold jelly. Similar gruels  were prepared from rye or from rye and potatoes .In Europe, a total of 21 different vegetables are fermented, in addition to an unspecified number of variably composed vegetable blends and fermented vegetable juices . Most veMost fermented vegetables typical of European countries such as pickles, capers, cucumber, sea fennel, turnip shreds and hardaliye are still fermented on a small scale, either in the home as side dishes or as ingredients. Instead, “table olives” and “sauerkrauts” are the major fermented vegetables produced in western countries, are of significant commercial importance and will be treated in depth in this paragraph (Table.1).Olea europaea subsp. europaea var. europaea trees, and their production has expanded to many countries around the world [“Table olives” are a traditional food of the Mediterranean countries , with many centuries of history. They are prepared with fruits obtained from cultivated he world ,61,89.After harvest, olives are inedible due to bitterness, and have to be processed to remove the fruit bitterness by hydrolysis of the phenolic compounds, especially oleuropein.Globally, there are three main methods of obtaining table olives: Spanish- or Sevillian-style green olives in brine; Californian-style black ripe olives and Greek-style natural black olives in brine. However, there are many other traditional table olive elaboration recipes, which are less known in the international market  and consAll methods rely on the autochthonous or indigenous microbiota for fermentation, with LAB and yeasts as the primary organisms. Lactic acid bacteria are involved in the fermentation of Spanish-style treated olives, while yeasts are responsible for the fermentation of Greek and Californian processed black olives, with LAB representing a small proportion of the total microflora .Lactic acid bacteria, which convert fermentable sugars to lactic acid and other organic acids depending on their metabolic pathways, are the most important group of bacteria in olives . LAB ferTogether with LAB, yeasts play a substantial role in fermented olive production, contributing to the determination of their aroma and taste due to the production of desirable metabolites and volatile compounds, while at the same time they enhance the growth of LAB and degrade phenolic compounds ,94.Different studies highlighted that the use of selected starter cultures, and more particularly the use of autochthonous LAB strains isolated from a spontaneous fermentation of the respective olive cultivar, could lead to a more controllable fermentation resulting in a product of high and constant quality, which is safer for the consumers and has a longer lifetime, and this process also influences the volatile organic compounds (VOC) profiles of olives ,96.Like table olives, “Sauerkraut”, primarily from cabbage, is one of the most well-known varieties of fermented vegetables associated with central and eastern European cultures, though it can be found in western European cuisine as well. For this product, the crucial importance of LAB fermentation has also been confirmed.In fact, though the microbial composition of sauerkraut can vary during the initial stages of fermentation, appropriate fermentation conditions, such as temperature and relative ingredient concentration, ensure that LAB are the dominant microorganisms in the final fermented product. Zabat et al.  highlighLAB guarantee the success of cabbage fermentation by the production of organic acids, bacteriocins, vitamins and flavor compounds responsible for many of the characteristic sensory qualities of the final fermented products, including extended shelf life. Moreover, numerous LAB isolated from sauerkraut were found to be probiotic or putative probiotic cultures ,97.Several other varieties of vegetables , which are mainly cultivated in southern Italy or, more generally, in the Mediterranean area, may increase their safety, nutritional, sensory and shelf-life properties through lactic acid fermentation under standardized industrial conditions .Among these minor fermented products occurring in the European countries are “caper”, ”sea fennel”, ”turnip shreds” and ”cucumber”, regarding which few scientific studies have yet been carried out, focusing on their microbial communities and population dynamics during lactic acid fermentation.Capparis species (mainly Capparis spinosa L.), a Mediterranean shrub cultivated for its buds and fruits.For example, “Caper” berries are the fruits of L. plantarum is the main species, which is isolated from the brine of capers [The main producers of fermented caper berries are Mediterranean countries, especially Greece, Italy, Turkey, Morocco and Spain, and the final products are exported mainly to central European countries, the United States and the United Kingdom as a delicatessen product . The ferf capers .Crithmum maritimum L.), is a salt-tolerant plant, widely grown in maritime rocks and more rarely sandy beaches in Mediterranean countries and Atlantic coasts. This plant has several usage areas such as culinary, medicine and cosmetics because of the content of the nutrients and phytochemicals. In the Mediterranean region, their leaves are consumed fresh or fermented as salad or with yoghurt [“Sea fennel”, known as maritime rock  and in the northeast Italian region (known as Brovada).Brassica rapa). It is similar to sauerkraut, but in some aspects the technology of production is different. Turnips are cleaned and put in vats, alternating them with layers of grape skins. Before covering the vat, a mix of water and salt or water only is added. The fermentation is a spontaneous process caused by the microorganisms present on the various components (turnip and grape skins) and goes on for about 30 days. After that, the turnips are extracted from the vats, peeled, cut into pieces and wrapped up in plastic bags ready for selling. The product is generally eaten after cooking, but sometimes even raw [Brovada is obtained by natural fermentation of turnips  and L. vaccinostercus [Another non-alcoholic fermented beverage produced in a traditional way in Thrace, the European part of Turkey, is “Hardaliye”. Hardaliye is a lactic acid-fermented traditional beverage produced from grape juice and crushed grapes, with the addition of different concentrations of whole/ground or heat-treated mustard seeds and sour cherry leaves . The preostercus .In Asia, especially in China, Japan, Korea and other Far East regions, where pastoral agricultural practices and animal husbandry are more limited, fermented foods that evolved were based more often on rice and grains, soybeans, vegetables and fish as the primary substrates .Lactic acid fermentation of cereals is a long-established processing method and is used in Asia for the production of foods in various forms, such as (semi)-solid cooked dough, gruels, porridge and liquid beverages.Although differences exist between countries, processing the cereals may involve soaking, heating, mashing, filtering and backslopping. In general, cereals are soaked in water for a couple of days to soften the grains for easier crushing and wet-milling into slurry, from which hulls, bran particles and germs can be removed by sieving procedures. During the slurring or doughing stage, which lasts for 1 to 3 days, mixed fermentations including lactic acid fermentation take place .In Asia, the main fermented cereals are millet, maize, barley, oats, rye, wheat, rice and sorghum, easily fermented by naturally occurring microorganisms. In particular, rice and soybean are the commonest foods in Asia, and are popularly fermented. In Rice noodles, called “Nombonchock” in Cambodia or “Khanom jeen” in Thailand, are made by lactic acid bacteria fermentation, which gives them a low fermented odor, flexibility and smoothness ,104,105.For these rice noodles, broken rice grains, usually stored/fermented over a six-month period, are naturally fermented for a few days, wet milled, and then left to precipitate as rice flour, which is then placed in a cotton bag under an applied weight for excess water removal. The fermented rice flour is then pre-cooked, kneaded and extruded into noodles in boiling water, and is immediately cooled and dried .Fermentation in nombonchock/khanom jeen is achieved by lactic acid bacteria acting on the lipid, protein and ash contents to enhance the stringy mouth feel of the noodles, and a number of volatile compounds such as 3-methylbutanoic acid and diacetyl, 2-methylpropanoic acid, are synthesized by the metabolic activities of these microorganisms, which impart a characteristic flavor to nombonchock/khanom jeen .“Amazake” is a Japanese fermented sweet food beverage prepared from rice.There are two types of amazake depending on the preparation method: a low-alcoholic drink made with lees left from sake (sakekasu amazake) and a non-alcoholic drink made with koji rice that can be enjoyed by everyone including small children .Aspergillus oryzae, a mold that is widespread in Japan. It is at the heart of many different Japanese ferments. It is used to make miso, sake, amazake, rice vinegar, soy sauce and mirin [Koji rice is cooked rice that has been inoculated with nd mirin . In eithL. sakei strains  were reported as crucial for the promotion of amazake’s nutritional, sensorial and functional properties [Amazake is a sake precursor and known for its health benefits, including weight loss and stress relief, positive skin effects, innate immunestimulating activity, antihypertensive effects, cholesterol lowering effects, and tyrosinase-inhibiting activity ,110,111.operties .L. plantarum, L. paracasei, L. casei and L. acidophilus species was found in “Com me” samples from provinces along the Mekong river delta (South Vietnam).Further, “Com me” is a sour rice fermented paste, a Vietnamese traditionally fermented food. It is a sour fermented paste made from overcooked wet rice fermented in clean and tightly closed containers for 7–10 days. Sometimes, the fermented product from a previous batch is added to the present batch to expedite the souring process (backslopping). During fermentation, rice changes from the whole grain state to a macerated product, and finally to a milky cream substance with a light sour aroma. After fermentation, the product is crushed and sieved. Com me is used as a souring agent during cooking and gives a meal a specific flavor and sour taste . The preAnother traditional Asiatic cereal-fermented food is “Tarhana”. It is a traditional Turkish food prepared by mixing wheat flour, yoghurt and some vegetables and spices, followed by fermentation, drying and grinding .Lactococcus lactis spp. lactis, Leuc. mesenteroides, L acidophilus, Enterococcus durans, Pediococcus spp., L. delbrueckii ssp. lactis and L. paracasei bacteria played important roles in the fermentation of Tarhana dough. Furthermore, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia membranaefaciens, Pichia mexicana, Pichia angusta, Debaryomyces hansenii, Candida sorboxylosa, Candida fluviatilis and Saccharomyces cerevisiae were identified during Tarhana fermentation.It is a good source of nutrition for children and the elderly, since it provides increased digestibility and nutritional value due to its high bioactive proteins and prolonged fermentation process ,115,116.There are several products similar to Tarhana in European countries. They are known as Thanu in Hungary, Trahanas in Greece and Talkuna in Finland.Lactobacillus, Micrococcus, Leuconostoc and Streptococcus genera are the commonest LAB used [Further, “Boza” is a traditional Turkish beverage made by the fermentation of millet, cooked maize, wheat or rice semolina/flour. Two different types of fermentation occur simultaneously during boza fermentation. In one, alcohol fermentation produces carbon dioxide bubbles and increases the volume. The second is lactic acid fermentation, which produces lactic acid and gives the acidic character to boza. In either of the two fermentative processes, the cereals are boiled for 1–2 h, cooled and strained. Sugar is then added before fermentation for up to 24 h. Apart from the yeasts present, LAB used .Leuc. mesenteroides and L. confusus as the main fermentative LAB species [Boza is a thick liquid, pale yellow in color with a characteristic acidic–alcoholic odor, and it is a favorite drink, particularly on cold winter nights. It is a healthy and nutritious beverage, and its lactic acid content has positive effects on digestion and intestinal flora ,118. A p species .There are also similar beverages to boza that are produced in East European countries, known as braga or brascha, as well as in the Balkan region, also known as boza.Weissella spp, Pediococcus pentosaceus and Enterococcus spp [Tape ketan (fermented glutinous rice) and tape singkong (fermented cassava) are traditional Indonesian foods produced by fermenting carbohydrate sources using ragi as the starter culture . Mold, yccus spp .Tape ketan is a sweet alcoholic drink similar to the sweet Japanese sake. The alcoholic content could be as high as 10% and largely depend on fermentation time. Its production involves boiling glutinous rice, cooling and inoculation with crushed ragi yeast cake or other desired LAB, and then fermented for 1–3 days.Selroti is a Nepali sweet rice bread, mostly made and eaten during the Hindu festivals of Dashain and Tihar that take place in Nepal and some parts of India. The preparation involves the addition of milk, water, cooking oil, sugar, ghee, butter, cardamom, cloves, bananas and other flavors to a semi-liquid rice flour dough, which are thoroughly mixed, allowed to ferment for between 4 and 24 h, and fried.Leuc. mesenteroides, E. faecium, Pc. pentosaceus and L. curvatus as the predominant LAB found in selroti, alongside other yeasts. They also reported that Leuc. mesenteroides is the most prevalent LAB in selroti batters.Yonzan and Tamang  reportedMoreover, in different Asiatic countries, wheat is widely used to produce sourdough-based products. In fact, dough for the various flat breads  made in parts of Asia, as well as dough for the production of steamed bread in East Asia, is fermented in a process resembling that used for sourdough bread in Europe . FurtherThe Chinese steamed bun also called baozi is a traditionally fermented wheat product originating from China, but it is consumed in many other countries in Asia. The bun has been a staple food for more than 2000 years , and is Asians generally are lovers of fermented food products, particularly Southeast Asians.Fermented vegetables such as Inziangsang, Kimchi, Pak-Gard-Dong, Khalpi, Dakguadong, Jiang-gua, Burong mustala, Gundruk, Ca muoi, Soidon, Suan-tsai, Sayur asin, Paocai, Sauerkraut, Nozawana-Zuke, Yan-jiang and Yan-tsai-shin are habitually consumed by Thai, Indian, Chinese, Vietnamese, Japanese, Korean and Taiwanese people ,129,130.Most traditional preparatory and fermentation processes are kept secret, and are passed on from generation to generation in tribes and castes in different provinces, while many of them are made at the home scale using backslopping . DiffereMost of the vegetable fermentations occur spontaneously and provide conditions that favor the growth of LAB and inhibit undesirable microorganisms . Lactic The fermented vegetables in Asia are numerous. In this review, just the most widely consumed will be described in depth (Table.1). In Korea, a popular lactic acid bacteria fermented vegetable called “Kimchi” is widely consumed. It is traditionally prepared by immersing cabbage in a pool of vast ingredients including fish sauce, spices and radishes. This is followed by fermentation with various strains of LAB and other microorganisms, and it is consumed raw globally ,133. Kim“Sinki”, a non-salted fermented radish taproot product, is traditionally consumed as a base for soup and as a pickle in some north-eastern states of India, in Nepal and a few places in Bhutan . Sinki iL. plantarum, L. brevis and L. fermentum to be the predominant LAB in the fermentation of sinki.If it is consumed as a pickle, drying is not required; rather, sinki is mixed with spices and bottled. Tamang and Sarkar  reportedFermented cucumbers are produced both in Europe and Asia. Fermented cucumbers are turned into a product called jioang-gua in Taiwan, khalpi in Nepal and India, paocai in China, or oiji in Korea, and are referred to as pickles in many parts of the United States, Europe  and Canada . Most cuB. siamensis B44v are also consumed in Thailand [L. casei, L. plantarum, L. brevis, L. pentosus, L. fermentum, L. paracasei, Weissella koreenis, W. confusa, W. cibaria, Leuc. kimchi, Leuc. mesenteroides, L. fallax, Enterococcus and Pediococcus species [Thai pickled vegetables (Phak-dong) fermented with Thailand . These t species . The lac species .L. brevis, L. namurensis, L. acetotolerans, L. sakei, L. curvatus, L. collinoides, L. fructivorans, L. casei, L. casei subsp. pseudoplantarum, L. delbrueckii, L. fermentum, L. plantarum, L. citreum, Pc. pentosaceus and Pc. damnosus [“Tsukemono” refers to Japanese pickled vegetables that are traditionally fermented with salt and seasonings for the purpose of shelf-life-extension, as well as taste and flavor enhancements. Preparation involves washing and direct pickling in nukadoko , with proper mixing to effect anaerobic fermentation balance. Microbial growth activation takes more than 7 days, and the microbes that are involved in the fermented vegetables are mainly lactic acid bacteria, including damnosus .In addition, “Miang” is a fermented Thai vegetable. The production involves steaming Thai tea leaves, which are then wrapped tightly and packed in a container. The leaves are pressed tightly under a heavy weight covered with banana leaves and fermented for 4–7 days, or even up to a year depending on the maturity of the tea leaves .Lactobacillus, Streptococcus and Leuconostoc, as well as several other members of the LAB group.The fermented product is similar to Myanmar’s lak hpak, and is famous in northern Thailand. It is usually consumed together with peanuts, ginger, garlic or roasted coconut sliced as a side dish . The maiGenerally safe, high-quality and acceptable ethnic and traditional fermented cereals and vegetables are important because they have eminent roles in the traditions, histories, and social and economic activities of Euro-Asian cultures and peoples. The fermented food products may be significantly improved using commercial starter cultures such as LAB, except that current molecular and technological developments provide much better and faster prospects, with increased accuracy and sensitivity. The culture-independent methods are critical for investigating the metagenomic compositions of diverse and complex microbial communities within fermented food products. Available databases serve as match references to microbial DNA sequences, ascertaining individual microbes’ identity and potential . A combiMetagenomics and metatranscriptomics, although still underexploited, promise to uncover the functionality of complex microbial consortia directly in the food matrix, and understand microbial behavior in response to different process conditions . In metaMetatranscriptomics is currently being considered as an option for doing an in-depth investigation into gene expressions of fermented foods’ microbial communities. The results may be coupled with other ‘omics’-based approaches, including proteomics and metabolomics, for a much deeper insight into the interactions of the microorganisms in association with the organoleptic and physicochemical properties of fermented foods ., and the bacterial diversity of kefir drinks from Bosnia and Herzegovina was obtained through rRNA gene amplicon sequencing by Garofalo et al. [Using a combination of 16S rRNA sequencing with tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS PAGE), certain fermented foods from Cambodia were determined to contain bacteriocin-producing LAB , and theo et al. . Denaturo et al.  and bozao et al. . Pla-somo et al. ,139.Ribosomal DNA sequencing, and molecular typing by random amplified polymorphic DNA (RAPD) techniques, are other methods used in the determination of the LAB and other bacterial strains in fermented foods such as nham  or boza Similarly, restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal DNA (rDNA) is a potential tool for the overall screening of the LAB-fermented cereals and vegetables in Euro-Asia and globally, as it was successfully employed in the identification of LAB used as starter cultures for sensorial, aroma and product quality studies involving Thai fish sausage .Another version of RFLP, terminal restriction fragment length polymorphism (T-RFLP), in conjunction with 16S rRNA gene sequencing may be utilized with broad potential in the investigation and analysis of LAB-fermented cereals and vegetables, because it was useful in determining LAB starter cultures for gamma-Aminobutyric acid (GABA) accumulation in Myanmar fermented fish with boiled rice .Other LAB identification methods, such as mass spectrometry (ESI-TOF MS) and matrix-assisted laser desorption/ionizing time-of-flight mass spectrometry (MALDI-TOF MS), can play very important roles in identifying the diversity of native lactic acid bacteria in Euro-Asian fermented cereals and vegetables, as found in selected Vietnamese and Thai fermented foods ,142. MetIt should be considered that LAB starter cultures need constant enhancements, which can ensure better-defined and -characterized strains meant for cereals and vegetables fermentation, and in turn lead to the production of functional fermented food products with sensorial qualities .Further, due to some local or small-scale production of the fermented products, unhygienic processing conditions are not uncommonly associated with them, thereby attracting pathogens with diverse contaminating avenues, ranging from vessels used, to raw materials and acidophilic pathogens. These health risks require cogent investigations. Other than LAB, some pathogens may be present to release harmful toxins in the fermented cereals and vegetables, such as mycotoxins, which are more common in tropical Southeast Asia.Ashaolu  highlighThere is a global demand for functional foods, and a cheap and green biotechnological means of attaining this is fermentation. Foods, especially cereals and vegetables, fermented with lactic acid bacteria offer valuable health-promoting functions to consumers. Europe and Asia have long and huge traditions in the manufacture of several LAB-fermented foods, and LAB starter culture improvement can aid in increasing the hygiene, safety and quality of fermented products.The use of diverse microbial strains, especially LAB groups, in the production of fermented cereals and vegetables can provide socio-economic benefits and several other prospective merits. Other than culture-dependent LAB fermentation, bioinformatics coupled with DNA sequencing methods are providing clearer insights into the make-up of the LAB metatranscriptomics, in order to decipher the relationships between fermented cereals and vegetables and LAB, and their products’ sensory qualities. The small scale and local production of several Euro-Asian LAB-fermented foods imply that their characterization, safety and hygiene must be improved.


Tabebuia aurea. Using a multi-scale approach, we sampled five landscapes with two sites of savanna in each. To obtain neutral genetic variation, we genotyped 60 adult individuals from each site using 10 microsatellite loci. We measured seed size and mass. Seeds were grown in nursery in completely randomized experimental design and 17 traits were measured in seedlings to obtain the average, additive genetic variance (Va) and coefficient of variation (CVa%), which measures evolvability, for each trait. We found that habitat loss increased genetic diversity (He) and allelic richness (AR), and decreased genetic differentiation among populations (FST), most likely due to longer dispersal distance of pollen in landscapes with lower density of flowering individuals. Habitat amount positively influenced seed size. Seeds of T. aurea are wind-dispersed and larger seeds may be dispersed to short distance, increasing genetic differentiation and decreasing genetic diversity and allelic richness. Evolvability (CVa%) in root length decreased with habitat amount. Savanna trees have higher root than shoot growth rate in the initial stages, allowing seedlings to obtain water from water tables. Landscapes with lower habitat amount may be more stressful for plant species, due to the lower plant density, edge effects and the negative impacts of agroecosystems. In these landscapes, larger roots may provide higher ability to obtain water, increasing survival and avoiding dying back because of fire. Despite the very recent agriculture expansion in Central Brazil, landscape changes are affecting neutral and adaptive variation in T. aurea. Several populations have low additive genetic variation for some traits and thus, may have limited evolvability, which may jeopardize species long-term persistence. The effect of habitat loss on highly variable neutral loci may only be detected after a certain threshold of population size is attained, that could become dangerously small masking important losses of heterozygosity endangering species conservation.Changes in landscape structure can affect essential population ecological features, such as dispersal and recruitment, and thus genetic processes. Here, we analyze the effects of landscape metrics on adaptive quantitative traits variation, evolutionary potential, and on neutral genetic diversity in populations of the Neotropical savanna tree  Agricultural landscapes are now occupying most of Neotropical savannas. The Brazilian Cerrado biome is the largest Neotropical savanna and one of the world’s biodiversity hotspots because of its high level of endemism and threatening . Over thChanges in landscape structure, such as habitat amount, edge effect and heterogeneity, can affect essential ecological processes such as dispersal , seed prIn agricultural landscapes, anthropogenic matrix characteristics may be as important as habitat size and amount due to the potential restriction the agroecosystems may impose to species movement, dispersal and gene flow . Matrix Most studies addressing the impacts of landscape composition and configuration in plant genetic diversity focus on genetic variation at neutral loci e.g., . AdaptivTabebuia aurea (Bignoniaceae); for this we used a multi scale approach. This species is broadly distributed across seasonal and wet savannas of Brazil, Bolivia and Paraguay, but locally it is distributed in well-delimited patches, usually with high density. It is hermaphroditic with mixed-mating reproductive system . The sites were either structurally isolated in different savanna fragments . Mapping was performed using visual digitalization and manual classification at a scale of 1:5,000 m, and we made exhaustive field inspection to assure high map accuracy. Our final map comprised 11 different land cover classes: (i) water courses; (ii) savanna; (iii) riparian forest; (iv) seasonal forest; (v) wetland; (vi) pasture; (vii) agriculture (corn or soybean); (viii) rural building; (ix) urban area; (x) road and train rail, and (xi) Eucalyptus spp. plantation  FIS obtained from an analysis of variance of allele frequencies , we estimated genetic diversity . We used the molecular co-ancestry method (Ne (see below).We used microsatellite markers to estimate effective population size  and mass and to seedling growth (height and diameter growth), size  and mass (root and aboveground). Seed size and mass are strongly related to seedling successful establishment and also with other adaptive traits such as plant height and specific leaf area . Height To obtain quantitative genetic variation, we sampled seeds from randomly chosen trees in each site, obtaining a total of 1070 seeds . The numPlant aboveground height and stem diameter were measured 76, 116, 133, and 145 days after seed germination, and growth rate was obtained from the regression coefficient (β) of height  and diameter . We also registered the initial and final seedling height  and diameter . We count the total number of leaves (NL), and leaf length  and width  were obtained from the mean value among three leaves per seedling, measured up to 145 days after germination . After 1r > 0.5. We kept for seed traits SLD, STD and SM, and for seedlings, NL, LW, RL, RDM, and ASL.For statistical analyses, we first performed Pearson correlation analyses among quantitative variables for sites and landscapes , S5, to Va) and the additive genetic coefficient of variation (CVa%), for each selected trait in each site, as a surrogate of population evolutionary potential or evolvability (h2) of each trait in each site. These parameters were estimated using restricted maximum likelihood (REML) analysis implemented in the software SELEGEN–REML/BLUP . To estimate additive genetic variance and the other parameters (a%CV and h2) we used We estimated the variance  and pollinators’ habitat  to generate the variables: (i) % savanna + pasture + wetlands and (ii) savanna + pasture + seasonal forest + riparian forest + wetlands. We also avoided correlation subtracting the values of each metric in additional scales  available in Fragstats , which iris spp. . We consris spp. , S7. Theris spp. .BMQ = ∑PiQi/∑Pi, where Pi is the percentage of each land cover class in each landscape at different spatial scales, and Qi is the quality score of each land cover class considering the requirements and resources that each land cover can provide to focal species. We scored each land cover based in the characteristics of T. aurea’s pollinators, since T. aurea is wind-dispersed and seedlings can eventually establish in wetlands and grasslands. For this, we considered our personal experience, the literature and the opinion of other experts in bees’ behavior. Our scores values ranged from the 0.2 to 1.0, where low values corresponded to areas more impermeable to species  metric , BMQ = ∑ species , S7.r > 0.90), we kept only metrics for 2 km spatial scale. We then assessed collinearity between landscape metrics within 2 km spatial scale  and compositional heterogeneity , we modeled genetic parameters using both landscape metrics and Ne  for both neutral genetic parameters and adaptive quantitative traits . Becauses and Ne .We evaluated the distribution of the residuals of all models and used the Gaussian distribution that fitted better to our data. To find the best predictive model at node level we considered both the significance and the Akaike Information Criteria (AIC). We estimated AIC corrected for small sample sizes (AICc) and the difference of each model and the best model (ΔAICci). We also estimated Akaike’s Weight of Evidence (wAICc) as the relative contribution of each model to explain the observed pattern, given a set of competing models . Models bblme . Additionally, the sampling sites in P5  was high in all populations ranging from 0.874 to 0.921 (AR) was also high, ranging from 14.2 to 18.5 (f) was significant for all populations, ranging from 0.121 to 0.231. FST across landscapes ranged from 0.008 to 0.240, GST’ ranged from 0.115 to 0.304, and Jost’ D ranged from 0.104 to 0.277  was not significantly different from FST (p = 0.852). Inbreeding coefficients within landscapes (FIS) was also high  in quantitative traits, but some had low variance in several traits  was low in most of the landscapes (PST) was also low for most pairwise comparisons, ranging from 0.0003 to 0.0835 .Habitat amount was the model that best explained the variation in neutral genetic diversity . Populations within landscapes with higher amount of habitat had higher genetic differentiation (STG’ and Jost’s D were not explained by our model (p > 0.10). Inbreeding coefficient (FIS) was also not significantly related to habitat amount.At link level, genetic differentiation based on ntiation . G’ST anp = 0.099; eN, wAICc = 0.74) than by landscape structure, but was marginally significant .Habitat amount explained variation in seed size (SLD) between sites within landscapes, although marginally significant . NL  and RL  coefficients of variation were better explained by habitat amount (p = 0.082) and RDM  coefficients of variation were explained by compositional heterogeneity, although marginally significant . Populations within landscapes with higher amount of habitat had higher quantitative genetic differentiation in RL (p > 0.10) differentiation at other seedling or seed adaptive traits.At link level, genetic differentiation at root length (RL) based on on in RL . Our modT. aurea from the Cerrado biome. However, unexpectedly, habitat loss increased genetic diversity and allelic richness and decreased genetic differentiation among populations. This result is most likely due to pollinator behavior and pollen dispersal patterns. Tabebuia aurea has a mixed mating system, with potential long-distance pollen dispersal of at least 2.6 km .Our findings show that contemporary changes in landscape affect genetic diversity and differentiation at neutral microsatellite loci among populations of t 2.6 km . Pollinat 2.6 km . Howeverion size . Therefoion size . Habitatdistance . In factT. aurea  within landscapes, but not related to landscape structure. In fact, T. aurea have high proportions of self-pollination and biparental inbreeding , time to germinate , plant height , and plant growth rate . Thus, although seedlings from large seeds may growth faster, they may be dispersed to shorter distances. In the same manner, sites with higher habitat amount may provide more resources for larger seeds . Seeds of as e.g.,  and dispas e.g., . In facter seeds  that mayQST) was also higher in landscapes with higher habitat amount. In fact, root mass and length are important adaptive traits in savanna trees, which have higher root growth rates than shoot growth rates in the initial stages, allowing seedlings to obtain water from water tables  and root length (RL) evolvability increased with compositional heterogeneity and RL decreased with habitat amount. In addition, RL quantitative differentiation . On one hand, the loss of habitat increases neutral genetic diversity and decreases genetic differentiation, most likely due to long distance pollen dispersal. The lower genetic differentiation between sites with lower habitat amount may also indicate that agroecosystem matrix is somehow permeable to pollinators. On the other hand, habitat amount decreases adaptive quantitative traits and increases evolvability. It is important to note that, as habitat fragmentation process (sensuT. aurea’s microsatellite loci (see C. brasiliense;We found that landscape changes are affecting genetic diversity and adaptive variation in the savanna tree species ss sensu is a recloci see , may onlloci see . As suggSeveral adaptive quantitative traits show evolutionary potential, meaning that populations still have enough variation to respond to selection, and thus to environmental changes. However, it is important to note that several populations have already low additive genetic variation for some traits at seedling stage. Evolutionary potential at short-term depends on population additive genetic variance  and thusThe data and additional supporting information may be found in the online version of this article as supporting information.RC and MR conceived and funded the work. FR and TA obtained the data. RC, JS, MR, and LC designed the experiment, field sampling, and statistical analyses. JS carried out the analyses. RC, JS, and MR wrote the original draft. All authors contributed to manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stat3-floxed/Ltf-iCre (Stat3-eKO), Stat3-floxed/Amhr2-Cre (Stat3-sKO), and Stat3-floxed/Pgr-Cre (Stat3-uKO) mice to delete Stat3 in uterine epithelium, uterine stroma, and whole uterine layers, respectively. We found that both epithelial and stromal STAT3 have critical roles in embryo attachment because all the Stat3-eKO and Stat3-sKO female mice were infertile due to implantation failure without any embryo attachment sites. Stat3-eKO uteri showed indented structure of uterine lumen, indicating the role of epithelial STAT3 in slit-like lumen formation in the peri-implantation uterus. Stat3-sKO uteri exhibited hyper-estrogenic responses and persistent cell proliferation of the epithelium in the peri-implantation uterus, suggesting the role of stromal STAT3 in uterine receptivity. In addition, Stat3-uKO female mice possessed not only the characteristic of persistent epithelial proliferation but also that of indented structure of uterine lumen. These findings indicate that epithelial STAT3 controls the formation of slit-like structure in uterine lumen and stromal STAT3 suppresses epithelial estrogenic responses and cell proliferation. Thus, epithelial and stromal STAT3 cooperatively controls uterine receptivity and embryo attachment through their different pathways.Although it has been reported that uterine signal transducer and activator of transcription 3 (STAT3) is essential for embryo implantation, the exact roles of uterine epithelial and stromal STAT3 on embryo implantation have not been elucidated. To address this issue, we generated  Although the molecular mechanism of uterine receptivity is not fully clarified, several molecules and pathways have been identified as key regulators of embryo implantation such as progesterone (P4) signaling, leukemia inhibitory factor (LIF), signal transducer and activator of transcription 3 (STAT3) signaling7.Implantation failure is a major problem of patients with infertility undergoing in vitro fertilization and embryo transfer (IVF-ET). Successful embryo implantation necessitates an intimate crosstalk between the receptive uterus and the implantation-competent blastocyst. These molecular and cellular interactions initiate when embryonic development to blastocyst stage is synchronized with the endometrium being receptive8. LIF is crucial for embryo implantation and activates uterine STAT3 pathway9. Depending on the ligands and cell types, STAT3 exerts a variety of functions: for example, cell proliferation/differentiation and amplification/suppression of inflammatory responses10. In the process of acute and chronic inflammation, pro-inflammatory IL-6 produced by immune cells and fibroblasts activates STAT3 and induces a series of genes responsible for cell proliferation and inflammation11. At the same time, STAT3 also mediates the signal from anti-inflammatory IL-10 to suppress inflammation12. Since systemic Stat3 null mice show embryonic lethality13, a conditional knockout mouse model of STAT3 was recently established to investigate the function of STAT3 in the uterus15. Pgr-Cre mice show the specific expression of Cre recombinase in the whole uterine layers16, and Pgr-Cre mice were crossed with Stat3-floxed mice17 to create Stat3-deleted mice at the whole uterine layers (Stat3-floxed/Pgr-Cre (Stat3-uKO)). Stat3-uKO female mice showed infertility due to implantation failure with enhanced estrogenic responses, presenting increased expression of 17β-estradiol (E2) -responsive genes Ltf and Muc1 and persistent epithelial proliferation in the peri-implantation uteri. These findings indicate the role of uterine STAT3 in uterine receptivity by modulating E2 signaling, a critical determinant of uterine receptivity. In addition, our group recently demonstrated the key role of uterine STAT3 in uterine regeneration using Stat3-uKO mice15, suggesting that STAT3 acts on the physiological uterine reconstruction processes such as menstruation and postpartum. Thus, Stat3-uKO mice have been useful for the investigation of the role of uterine STAT3.STAT3 is a major transcription factor which transduces signals from interleukin (IL)-6 family cytokines including IL-6, IL-11, LIF, oncostatin M (OSM) and other growth factors19. Uterine epithelium and stroma have independent and mutual functions to support uterine receptivity and embryo implantation20. Although the previous literature demonstrated the significance of uterine STAT3 in embryo implantation14, the role of epithelial and stromal STAT3 in the process of embryo implantation remains unclear. In this study, we generated Stat3-floxed/Ltf-iCre (Stat3-eKO) and Stat3-floxed/Amhr2-Cre (Stat3-sKO) mice to delete Stat3 in the uterine epithelium and stroma, respectively, and investigated the detailed functions of epithelial and stromal STAT3 during embryo implantation. Stat3-eKO and Stat3-sKO female mice were infertile due to implantation failure without any embryo attachment sites. Stat3-eKO mice compromised the formation of slit-like structure in uterine lumen which enables normal embryo attachment. In contrast, Stat3-sKO mice showed epithelial hyper-estrogenic responses and persistent proliferation which impair uterine receptivity. In addition, Stat3-uKO mice showed both compromised luminal structure and persistent epithelial proliferation. Taken together, epithelial and stromal STAT3 supports uterine receptivity and embryo attachment through the regulation of different pathways.Uterine epithelium and stroma are two major components of the endometrium, and their interactions are involved in the process of the receptivity acquisition21, which is expressed in the uterine epitheliumon days 1 and 4 of pregnancy22 and activates uterine STAT3 pathway to induce embryo implantation in mice9.We examined spatiotemporal activation of STAT3 in the uteri of wild-type (WT) mice on days 1, 2, 3 and 4 of pregnancy. The expression of total STAT3 was observed in both uterine epithelium and stroma from days 1 to 4 of pregnancy  to clarify the roles of epithelial and stromal STAT3 in embryo implantation.Although the significance of STAT3 in the whole uterus in mouse embryo implantation has already been demonstrated in the former study using Stat3-eKO mice. We performed immunohistochemistry for pSTAT3 and confirmed that the nuclear accumulation of activated STAT3 was clearly diminished in the epithelium of Stat3-eKO uteri  mice) were mated with WT fertile male mice. Although vaginal plugs were equally observed both in Stat3-eCtrl and Stat3-eKO mice, all Stat3-eKO mice did not delivered any pups  mice) were mated with WT fertile males. As is the case with Stat3-eKO females, all Stat3-sKO females failed to produce offspring although vaginal plugs were normally observed  and progesterone (P4). Former investigations have clarified that appropriate balance between E2 and P4 signaling is a crucial factor for uterine receptivity28. Therefore, we performed quantitative PCR and examined the expression of Muc1, C3, and Ltf as E2-responsive genes in the epithelium, Indian hedgehog (Ihh) and Hoxa10 as P4-responsive genes in the epithelium and stroma on day 4 of pregnancy. As a result, Stat3-eKO uteri did not show significant differences in the mRNA levels of neither E2-responsive genes nor P4-responsive genes compared to Stat3-eCtrl ones, except the significant suppression of Ltf in Stat3-eKO uteri. ” in embryo implantationStat3-eKO mice, we focused on the morphology of uterine lumen. Previous studies have revealed that the slit-like structure of uterine lumen before embryo attachment is critical for normal embryo attachment32. Histological sections on day 4 showed that the uterine lumen was complicated and indented in both Stat3-eKO and Stat3-uKO uteri but not in Stat3-sKO ones  looks faint14, implying that LIF-LIFR pathway is not responsible for the stromal STAT3 activation. This speculation may account for the current findings of different functions of epithelial and stromal STAT3.As shown in this study, STAT3 is activated in both epithelium and stroma on days 1 and 4 of pregnancy, LIF is expressed in the luminal and glandular epithelium on day 1 and expressed in the glandular epithelium on day 4, and 14, suggesting that dysfunction of uterine LIF-LIFR-STAT3 signaling pathway may affect the phenotypes of Stat3-eKO mice such as indented uterine lumen and poor formation of slit-like structure. The previous studies reported that the indented luminal structure is involved in defective implantation33. These findings indicate that LIF-LIFR-STAT3 signaling in the uterine epithelium may control appropriate formation of luminal structure for successful embryo implantation.LIFR is strongly expressed in the epithelium on day 4 of pregnancyStat3 using Stat3-uKO mice causes the reduction of stromal PGR expression in the peri-implantation uterus7. However, stromal PGR expression was not decreased in both Stat3-eKO and Stat3-sKO mice in our study. These findings make us speculate that both epithelial and stromal STAT3 play a role in stromal PGR expression, and that even if either epithelial or stromal STAT3 is inactivated, stromal PGR is sufficiently maintained. In another previous study of uterine epithelial Stat3-deleted mouse model, Stat3-floxed Wnt7a-Cre (SWd/d) mice show the increased expression of E-cadherin encoded by Cdh134. Our study demonstrated that both stromal proliferation and uterine Cdh1 expression are normal in Stat3-floxed Ltf-iCre (Stat3-eKO) mice, another mouse model of uterine epithelial deletion of Stat3. There are temporal differences in epithelial Cre recombinase induction between Ltf-iCre and Wnt7a-Cre mice. Wnt7a is expressed in the uterine epithelium from fetal and neonatal stages35, while Ltf is expressed from the postpubertal stage23. These findings indicate that epithelial STAT3 in the developmental stage may suppress E-cadherin expression.A previous study has revealed that entire uterine deletion of Stat3-floxed/Amhr2-Cre female mice were subfertile due to resorption, although embryo implantation was normal36. In the present study, Stat3-floxed/Amhr2-Cre mice showed implantation failure. The discrepancy of the phenotypes between the previous and present studies might be derived from different Stat3-floxed mouse lines in which loxP-sites are inserted into the different loci. Therefore, it is possible that different efficiency of Stat3 deletion might cause the inconsistent phenotypes between these studies.Also, a previous study has reported that Stat3-sKO uteri have the enhanced estrogenic responses in the epithelium, suggesting the stromal-epithelial interactions through stromal STAT3. There is accumulated evidence showing that stromal transcriptional factors such as Hand2 and Hif2a affect epithelial functions to support embryo implantation38. In this sense, STAT3 could be another stromal transcriptional factor regulating epithelial conditions during embryo implantation.The present study showed that 40. Our study showed that neither epithelial nor stromal Stat3 deficiency affects blastocyst growth until day 4 of pregnancy. Since epithelial and stromal STAT3 may affect the intrauterine blastocyst growth and the blastocyst competency to embryo attachment from day 4 midnight to day 5, further investigations are needed to elucidate this issue.The interactions between the uterus and embryos provide blastocyst growth and competency to embryo implantation15, it is also unclear whether regeneration activity of uterine STAT3 is associated with embryo implantation. Further investigations are needed to answer these questions and to clarify the association of STAT3 with recurrent implantation failure in humans.The present study revealed that both epithelial and stromal STAT3 are critical for uterine receptivity and embryo attachment in different manners. However, it remains elusive how epithelial STAT3 regulates the formation of slit-like structure in uterine lumen, and how stromal STAT3 controls estrogen responsiveness in the epithelium. Although uterine STAT3 is involved in the uterine regeneration processStat3-floxed mice 17, Ltf-iCre mice23, Amhr2-Cre mice24, and Pgr-Cre mice16 were used in this study. Ltf, Amhr2 and Pgr are expressed in the luminal epithelium, the stroma, and all layers of the endometrium, respectively. Therefore, Ltf-iCre, Amhr2-Cre and Pgr-Cre mice have the specific expression of Cre recombinase in the luminal epithelium, the stroma, and the endometrium, respectively. We generated Stat3-floxed/Ltf-iCre (Stat3-eKO), Stat3-floxed/Amhr2-Cre (Stat3-sKO), and Stat3-floxed/Pgr-Cre (Stat3-uKO) mice to deplete Stat3 specifically in the luminal epithelium, the stroma, and the endometrium, respectively. For the experiments of pregnancy, female mice were mated with fertile WT males. Day 1 of pregnancy was defined as the day when we recognized vaginal plug. These female mice were sacrificed on days 1, 2, 3, 4 and 5 for the evaluation of embryo implantation and sample collection. Embryo attachment sites were identified as blue bands under the intravenous injection of Chicago blue dye solution26. When no embryo attachment sites were detected, uterine horns were cut and flushed with normal saline to obtain embryos to confirm whether embryo development and transportation from the oviduct into the uterus were normal. All mice were housed in the University of Tokyo Animal Care Facility according to the institutional guidelines for the use of laboratory animals. All animal experiments were approved by the Institutional Animal Experiment Committee of the University of Tokyo Graduate School of Medicine .WT (C57BL/6 N), Immunostaining was performed in 10% formalin-fixed paraffin-embedded sections (6 µm), using antibodies to Ki67 , MUC1 , ERα , PGR , total STAT3  and phosphorylated STAT3 . As an isotype control, rabbit IgG  was used.41. The intensity of each cell was graded with a value of 0, 1+, 2+, or 3+  in a high-power field, and then, the percentage of cells at each staining intensity level was calculated. H-score was obtained by the following equation: H-score = 1 × (% cells 1 +)+ 2 × (% cells 2 +) + 3 × (% cells 3 +) Five high-powered fields per respective section were analyzed, and the average value was assigned as the H-score for each section from different mice.The immunoreactivity of pSTAT3 and MUC1 staining was evaluated by a semiquantitative H-scoreKi67-positive (Ki67 +) epithelial and stromal cell number on five selected high-power fields in the uterine sections obtained from more than three different mice in each group were manually counted. PDS was assessed by the percentage of Ki67 + luminal epithelial cells and the total number of Ki67 + stromal cells.43. The complementary DNA were synthesized from the extracted RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO), and, and qPCR was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific). A housekeeping gene Actb was used as an internal standard for normalizing the relative mRNA expression. Sequences of qPCR primers which were used to detect each gene are shown in Table Total RNA extraction was performed as described previouslyt test, Mann–Whitney U test, and Fisher’s exact probability test as appropriate. P values less than 0.05 were considered statistically significant.Statistical analyses were performed using two-tailed Student’s Supplementary Information.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.