We present direct evidence of early grape domestication in southern Italy via a multidisciplinary study of pip assemblage from one site, shedding new light on the spread of viticulture in the western Mediterranean during the Bronze Age. This consist of 55 waterlogged pips from Grotta di Pertosa, a Middle Bronze Age settlement in the south of the Italian peninsula. Direct radiocarbon dating of pips was carried out, confirming the chronological consistency of the samples with their archaeological contexts (ca. 1450–1200 BCE). The extraordinary state of conservation of the sample allowed to perform geometric morphometric (GMM) and paleogenetic analyses (aDNA) at the same time. The combination of the two methods has irrefutably shown the presence of domestic grapevines, together with wild ones, in Southern Italy during the Middle/Late Bronze Age. The results converge towards an oriental origin of the domestic grapes, most likely arriving from the Aegean area through the Mycenaeans. A parent/offspring kinship was also recognised between a domestic/wild hybrid individual and a domestic clonal group. This data point out a little known aspect of the diffusion of the first viticulture in Italy, and therefore in the western Mediterranean, which involved the hybridization between imported domestic varieties with, likely local, wild vines. Vitis vinifera L.) domestication, several studies have consistently shown that this process originated in Western Asia and Caucasus11, and it resulted into the differentiation of phenotypically distinct domestic (Vitis vinifera subsp. vinifera) and wild (Vitis vinifera subsp. sylvestris) subspecies. A recent study clarified that two separate domestication events took place concurrently around the advent of agriculture (~ 10.500 to 12.500 BP) in the two areas12. There is uncertainty however regarding what happened outside the primary area of domestication, especially concerning the role played by local wild populations present in those areas where viticulture developed over time. Numerous genetic studies have highlighted a complex relationship between the two subspecies, variously interpreted as the result of introgressive hybridisation following the introduction of domestic varieties from the eastern Mediterranean or secondary domestication of local wild vines14. In addition, the timing of grapevine domestication and of its early spread in the Mediterranean is poorly known. Observing the current geographical distribution of wild vines17, it is worth exploring the relationship between the two subspecies in the areas where viticulture spread.In order to reconstruct the history of grape vines  analysis of ventral and lateral outlines of the pips using the Elliptic Fourier Transform method allow the identification of modern cultivars and a powerful discrimination of wild and domesticated grape pips29. Paleogenetic analysis has also proved to be a powerful tool, even if strictly dependent on the taphonomic processes and the state of conservation. Up to now the best results, for both paleogenetics and morphometrics, come from the extraction and analysis from waterlogged material. This type of remains is not very common but some studies have been done, mainly regarding advanced steps in the history of viticulture in Mediterranean France31 but also other areas32, including Italy33.Archaeobotany can potentially play a fundamental role in understanding the phenomenon as it allows us to have a direct look at the plant remains within past anthropic contexts that testify the process. Nevertheless, the spread and domestication of grapevine are not easy to read, mainly because recognition of the characteristics that enable a For its geographic position, the Italian peninsula—especially its southern part—presumably played a major role in the spread of viticulture in the Mediterranean, and from here to western and central Europe.34. The use of non-comparable methods is not the only problem with the research on the diffusion of viticulture in this important geographical region. The lack of large grape pip assemblages from Southern Italy constitutes the main limit on our ability to read the phenomenon in detail and assess its implications for the morphology of grape pips  BCE  was takenThe linear discriminant analysis (LDA) performed on a balanced collection of modern wild and domestic grape pips allowed to classify a majority of the archaeological pips in the wild morphotype. The domestic morphotype is nevertheless clearly identified. With a probability threshold of p ≥ 0.75, 38 grape pips were assigned to the wild group (69.1%), 9 (16.4%) to the domestic morphotype and 8 (14.5%) remained unclassified Fig. . It must29 and in the Aegean area44. Although these results are poorly representative due to the limited number of pips of the domesticated morphotype, we decided to include them in our study anyway as we have the possibility to compare them to aDNA results.Despite their small number, the 9 pips identified as domesticated by this first LDA with p-values ≥ 0.75 were submitted to a second LDA performed at cultivar level  and the expected patterns in cytosine deamination and DNA fragmentation SI. Fig. . Damage-45 projects two archaeological samples among wild V. vinifera sylvestris (pips P51 and P55), one near the boundary of wild and domesticated accessions (P52), and six within the PCA-space of domesticated grapevines  of modern grapevine diversitynes Fig. A. PCA wanes Fig. B, revealUsing the called genotypes, KING found no kinship relationships between the archaeological samples and modern accessions, however it detected kinship within Grotta di Pertosa specimens: one parent–offspring relationship between P50 and P52 and a clonal relationship between P54 and P57. Kinship among all archaeological samples was further explored in a genotype likelihood framework, with ngsRelate identifying the three clusters observed in PCA to be genetic clones: P50 identical to P53, P56 and P59, P51 identical to P55, and P54 identical to P57  has close relationships with many sylvestris accessions, with the highest in vines from Slovakia and Germany. The third clonal group is mostly closely related to admixed grapes, with the closest being Greek variety ‘Muscat à petits grains blancs’. P52, the only specimen not a part of a clonal group, has genetic affinity to many wild and domesticated grapes, with the closest being ‘Riesling bleu’, but with nearly the same genetic distance to a wild vine from Algeria. While these genetic links show affinity toward the local region, the specimens have even closer genetic links to other archaeological samples. In particular, the first clonal group and P52 show close relationships to Roman seeds from Terrasses de Montfau from southern France and the third clonal group is related to early Roman seeds from Mas de Vignoles XIV near Nîmes31  has the closest genetic affinity with domesticated vines, with the closest being ‘Landroter’, a variety identified as admixed by Laucou et alSI Table ..47, the deeper history of the cultivated vines was explored in PCA by projecting cultivars and archaeological samples onto sylvestris diversity , which, given the characteristics of their sediments, conserve organic remains even in the absence of combustion. In contrast, in the south and the islands, mainly charred grape pips are discovered, their preservation probably being the result of fortuitous and unusual conditions. However, an increase in grapevine interest has also been recorded by pollen analyses conducted on cores taken from sites in both Apulia, in the Alimini Piccolo lake82, it may be wondered whether viticulture in Italy was in some way triggered or accelerated by allochthonous inputs from the eastern Mediterranean, at its very dawn. The answer is not obvious. In order to pursue this aim it is worthwhile to establish certain key points regarding Mycenaean visits to the Italian coasts during the Bronze Age . After this period the Mycenaean palatial system collapsed, although relations between the eastern Mediterranean and Southern Italy continued, as attested by the abundant discoveries of LH III B-C materials in Sub-Apennine contexts (Late Bronze Age). The period saw the rise of new cultural manifestations such as the imitative ceramics known as Italo-Mycenaean productstimopheevii wheat) show a certain degree of integration in a network of contacts on the regional and transregional scale89. Furthermore sporadic Aegean ceramic fragments have been discovered in underground funerary contexts very close to Grotta di Pertosa99.Grotta di Pertosa is located in an area not directly exposed to Mycenaean traffic, but several archaeological indicators  and their hybridisation with local wild grapes. This previously unknown data for the western Mediterranean, supported both by GMM and ancient DNA results, seems to be correlated to the first contacts with Mycenaean people, who have probably already experimented with this model and spread it where there is suitable local wild material.The results of the aDNA analyses showed no evidence of independent domestication of local wild grapes. Nonetheless, a parent–offspring relationship emerged between a domestic/wild hybrid individual (P52) and a domesticated clonal group . Besides GMM showed that vines producing morphologically wild pips were surely exploited. This highlight the contribution of wild grapes in the early viticulture of this area. We cannot state whether the crossing was voluntary or accidental and we do not know the extent of the phenomenon. However, the presence of clonal groups and hybrids within the sample, as well as the coexistence of wild and domesticated grapes, leads us to suppose that this first stage of viticulture in Middle to the Late Bronze Age Southern Italy included both asexual and sexual propagation of the vines, as well as the introduction of non-native varieties and the use of local wild vines. Selective pressure had to be rather mild and this could explain the proportions between domesticated and wild morphotypes in the Grotta di Pertosa assemblage. Indeed, GMM analyses shows that the percentage of domestic morphotypes reaches 16.5%, while the wild ones are the 69.1%.100, according to which, viticulture in its initial stage, requiring maintenance of the plants involving low selective pressure and occasional recourse to sexual reproduction, does not necessarily result in drastic changes in the shape of the seeds. This is seen only gradually, in a non-linear manner, as the selective pressure exerted on the plants by human beings increases. It is manifested on the archaeobotanical level by mixed assemblages composed of variable percentages of fully domestic types, wild types and weakly selected types, as in the case presented here, according to a trend that persists even in subsequent historical periods in other areas21. Our study adds a piece to this reconstruction and suggests that, in some cases, the less clear morphotypes could represent wild-domestic hybrids, rather than weakly selected types.Such a consideration is in line with what has emerged from recent studiesThis work has allowed us to establish some fixed points on the diffusion of viticulture in the western Mediterranean. The presence of domestic vines in the south of the Italian peninsula between the Middle and Late Bronze Age, already presumed from other archaeobotanical indicators, has been confirmed beyond any doubt. In this historical phase the indigenous Italic communities experience a growing economic and social complexity and contacts with Mycenaean peoples constitute an important factor of cultural contamination. Within this framework, the introduction of domestic grape varieties from the Aegean area, as well as the growing interest in the cultivation of vines, could represent a reflection of the adhesion by local élites to a system of uses and values spread by Mycenaean sailors throughout the Mediterranean. Local societies, however, do not passively absorb the new models, but make them their own and reinterpret them. It is suggestive to note how this mechanism, observed for some aspects of material culture, is in a certain way also valid for viticulture practices: the domestic grapes introduced from the East, in fact, hybridize with the local wild ones, harvested and exploited in this region for thousands of years, originating something new.A total of 55 grape pips from Grotta di Pertosa, dated to the latter period of the Middle Bronze Age to the beginning of the Late Bronze Age (from 1440 to 1200 BCE), were analysed in this study by geometric morphometrics. Although the total number of specimens discovered in the site was greater, only the pips that were perfectly preserved could be taken into account. All the grape pips analysed were waterlogged and were from an inhabited context, precisely from a single stratigraphic unit, formed by the collapsed floor of a pile-dwelling. From these, a group of ten seeds, showing a certain degree of shape diversity, were selected for paleogenetic analysis. All plant experiments were carried out with relevant institutional, national, and international guidelines and legislation.95. The 55 selected grape pips were photographed in dorsal and lateral views using a stereomicroscope (Olympus SZ-ET) connected to a digital camera (Olympus DP12). Subsequently, these images were semi-automatically converted into black shapes on white backgrounds by means of photo editing software. All the morphometric analyses were then performed using the Momocs package101 in R environment (R Development Core Team 2021). The x and y coordinates of 360 equidistant points were sampled along the outline of each profile. The outlines were normalized by centring and scaling according to their centroid size and the first point was positioned above the centroid. The computation of EFT allows to decompose the seed outlines into a harmonic serie of trigonometric functions described by numerical coefficients that can be used as shape descriptors in statistical analyses. Only the 6 first harmonics were considered, as a good compromise between the need to precisely describe the shape and to minimise the errors24. Each harmonic is associated with 4 coefficients, and thus each outline is described by 24 coefficients, bringing the total number of coefficients used to describe each individual seed to 48.Geometric morphometrics were based on seed outline analysis using the Elliptic Fourier Transform (EFT) method, which is by now widely known in the literature and has been consolidated over the last decade102. Based on the 48 shape descriptors, the morphology of the 55 archaeological seeds was characterized by comparison to modern populations of grape pips using Linear Discriminant Analyses (LDA). A two-step approach was applied using two nested LDAs and the modern reference collection of wild grapevines and cultivated varieties of ISEM44. These wild grapes and modern cultivars are originating from various countries from Western Asia to Central and Western Europe and around the Mediterranean. A first LDA was performed to identify the wild or domesticated status of the archaeological pips. The archaeological pips were compared to a balanced modern collection of randomly selected pips of wild (N = 2430) and domesticated (N = 2430) grapes. The power of this method in discriminating between domestic and wild morphotypes has been demonstrated in previous studies29. Using this balanced reference collection and leave-one-out cross-validation 95.7% of the pips are correctly classified according to their wild or domesticated status44.All statistical analyses were performed with the MASS packageThe archaeological pips identified as domesticated  by this first LDA were then submitted to a second LDA designed to compare their morphology to modern varieties. This second LDA was based on a balanced collection composed of 280 traditional cultivars whose main agronomic traits and areas of origins are known, with ca 30 pips per cultivar. In this case, leave-one-out cross-validation allows the correct cultivar identification of 77.18% of the pips., et al.31, with details described in supplementary information and data. Ultrashort DNA was extracted from ten individual seeds103, converted to Illumina libraries using a single-stranded DNA preparation104, treated with uracil–DNA–glycosylase to repair DNA damage105, and enriched using an in-solution kit which targets 10,000 single nucleotide polymorphisms (SNPs)31. Sequencing data were processed in the PALEOMIX pipeline following recommendations for degraded DNA106. Genetic structure and relatedness were investigated in the context of a grapevine reference panel of 783 cultivars46, 112 wild accessions107, and 31 archaeological samples31, using smartpca45, KING108, and ngsRelate109.Ancient DNA was recovered and analyzed following methods developed by Ramos-MadrigalSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Table S5.


Amaranthaceae species make them promising for introduction into agriculture and markets, particularly in arid conditions. Molecular genetic polymorphism analysis is the most powerful tool for studying plant resources; therefore, the current study aimed to investigate the polymorphisms of allelic variations in the ARF and SOD gene families, as well as the genetic diversity of six Amaranthaceae species, using retrotransposon-based fingerprinting with the multi-locus EPIC-PCR profiling approach. Additionally, the iPBS PCR amplification was employed for genome profiling, revealing variations in genetic diversity among the studied Amaranthaceae samples. The observed genetic diversity in Amaranthaceae species contributes to their enhanced tolerance to adverse environmental conditions. The knowledge about the genetic diversity of genes crucial in plant development and stress resistance can be useful for the genetic improvement of cultivated Amaranthaceae species.The adaptive potential and biochemical properties of the  Amaranthaceae family, are relatively recent additions to contemporary agriculture, originating from the New World. Many members of the family are classified as pseudo-cereals . They have gained widespread attention in recent years due to their highly nutritious grain and green mass, particularly their valuable amino acid composition  [Amaranths, belonging to the stidine) ,2,3. Amastidine) ,5,6,7. Tstidine) . Moreovestidine) ,10,11. Pstidine) ,10,11,12Amaranthaceae plants exhibit remarkable resilience to adverse environmental conditions such as nutrient-poor soil, water scarcity, and severe defoliation [oliation . Unlike oliation ,14,15,16A. hybridus through multiple independent domestication events [Understanding plant responses to environmental stress necessitates comprehensive investigations into physiological, cellular, and transcriptome alterations that ensure plant survival ; howevern events ,20,21,22A. caudatus, A. deflexus, A. hypochondriacus, A. retroflexus, A. spinosis (epinard), and Ch. quinoa (cultivar “Vahdat”) selected for this study are undoubtedly related, originating in Central and South America but with varying degrees of domestication: from malicious weeds like A. deflexus [A. spinosis (epinard) [A. retroflexus [A. caudatus [A. hypochondriacus [Ch. quinoa [Species deflexus  and A. sepinard)  to foragroflexus , A. caudcaudatus , A. hypondriacus ,28, and . quinoa ,16. The Polymerase chain reaction (PCR) is commonly employed to assess the genetic diversity of individual amaranth species . IdentifARF6, a key factor in regulating the auxin response. The genetic polymorphism of diverse plant species can be explored using EPIC-PCR primers targeting the ARF6 gene family [ARF function in plants other than model organisms, such as Arabidopsis, is sparse. Comparing genetic polymorphism in auxin response genes across different amaranth species might be useful for studying their genetic traits and evolution [The literature data show that the phytohormone auxin plays a crucial role in regulating growth and development processes in plants subjected to environmental stressors. This regulation occurs via gene expression control, mediated by a family of diverse DNA-binding ARFs (auxin response factors) ,33,34,35e family ; howevervolution .SOD genes in Amaranthaceae species [Plants subjected to abiotic stress simultaneously experience oxidative stress. Various biological activities, molecular functions, and cellular components rely heavily on redox reactions. Defence enzymes are actively involved in reducing ROS (reactive oxygen species). Consequently, agricultural crop stress tolerance is largely determined via ROS concentration and defence enzyme activity, particularly SOD (superoxide dismutase). Different SOD isoforms serve as the primary defence line in plants against ROS-induced damage ,39,40. I species . Univers species .Amaranthaceae [Molecular genetic polymorphism encompasses genetic variation not only in genome coding regions but non-coding regions as well, which contain a large number of mobile elements in eukaryotes. An examination of repetitive elements in the amaranth genome indicated the presence of Copia- and Gypsy-like retrotransposons ,43. Thesnthaceae ,51.SOD and ARF gene families in the Amaranthaceae species, such as A. caudatus, A. deflexus, A. hypochondriacus, A. retroflexus, A. spinosis (epinard), and Ch. Quinoa, as well as conduct a retrotransposon genetic analysis utilising genomic profiling approaches to elaborate knowledge on the role of the above genetic factors in conferring tolerance to abiotic stressors in Amaranthaceae species.This study aimed to investigate the genetic polymorphism of the ARF6 gene group revealed different levels of allele (Na) richness among the plant species analysed. A. deflexus had the highest Na value (1.255) among the species, indicating a greater number of distinct alleles in its population. The effective number of alleles (Ne) was generally higher than Na, indicating that some alleles were more dominant in certain species. Shannon’s Diversity Index (I) ranged from 0.103 (A. spinosis) to 0.266 (A. deflexus), the latter having the highest allele frequency diversity.The investigation of genetic diversity within the SOD genes, Na varied across species. The highest Na value of 1.019 was found in A. deflexus, illustrating its genetic diversity. The effective allele number (Ne) followed the same trends as Na, demonstrating that allele frequencies vary. Shannon’s diversity index (I) varied from 0.048 for A. hypochondriacus to 0.257 for Ch. quinoa.In the context of A. spinosis (epinard), which had the greatest mean allele count (Na = 1.141). The effective allele count (Ne) revealed a range of values, reflecting a variety of allele frequencies. Shannon’s Diversity Index (I) varied from 0.056 for Ch. quinoa to 0.282 for A. spinosis, showing varying degrees of genetic diversity. Thus, accessions of A. deflexus had the largest effective number of alleles (Ne) and percentage of polymorphic loci (%P), indicating potentially substantial genetic diversity (The iPBS profile research indicated a wide range of genetic characteristics among species. The most diverse group was iversity .A. spinosis (epinard) samples exhibit the lowest levels of heterozygosity (He) and unexpected heterozygosity (uHe) among the other species, indicating a potentially low genetic variability of A. spinosis. To assess the overall genetic diversity among all studied Amaranthaceae species, general genetic parameters such as Mean and SE were employed. The percentage of polymorphic loci (NPB) for auxin response genes was found to be 39.7%, indicating a relatively high level of genetic diversity across all examined species.In addition, the ARF6 genes provides insights into the structure of the examined amaranth species by dissecting the dispersion of this characteristic into various hierarchical components. Amaranthaceae species studied.The AMOVA  of the PhiPT value, a measure of genetic differentiation among species, was estimated at 0.253, indicating moderate differences between the species investigated.The remaining 75% of the molecular variance can be attributed to the genetic diversity within the species. The ARF6 genes and DNA regions. A. hypochondriacus, A. spinosis (epinard), and Ch. quinoa are the most genetically similar species in terms of auxin response genes, while A. deflexus exhibits the highest genetic diversity and differs significantly from the above species.Amaranthaceae species.To analyse the significance of the detected polymorphism and to identify the influence of selection on the diversity of gene nucleotide sequences and the number of dispersed repetitions, Tajima’s statistical test for neutrality was used. The data provided in Amaranthaceae and demonstrated the presence of a potential effect of selection on genetic variability in the species tested. The percentage of polymorphism (ps) was reported as 0.96, suggesting a high degree of homozygosity in the iPBS loci and antioxidant system genes studied within the Amaranthaceae species. Additionally, the values of the parameters Θ and π were low, and the value of D was negative, indicating the presence of positive selection phenomena.Tajima’s D statistical test allowed us to examine deviations from the neutral evolutionary model of SOD genes between Amaranthaceae species. On the other hand, differences within the species accounted for 39% of the observed variation.The analysis of variance results  indicateSOD gene families was performed on six Amaranthaceae species, and the results were analysed using a fingerprinting technique (He) and observed heterozygosity (Ho) for all the Amaranthaceae species ranged from 0.101 to 0.149, respectively. These findings suggest that the observed heterozygosity exceeds the expected values, potentially indicating the occurrence of intraspecific crossing.The amplification of echnique . The aveI) and the coefficient of unexpected heterozygosity (uHe), which evaluate the diversity of genotypes within species. Notably, the average values of uHe were 0.038 in A. hypochondriacus and 0.198 in Ch. quinoa, demonstrating varying levels of genetic diversity among the investigated species. Consequently, the heterozygosity (He) and unexpected heterozygosity (uHe) values for A. hypochondriacus samples were significantly lower compared to the other species. These results suggest that A. hypochondriacus may possess lesser genetic diversity than the other species.SOD gene were most similar between A. hypochondriacus and A. spinosis (epinard), as well as between A. caudatus and Ch. quinoa; conversely, A. deflexus exhibited the greatest genetic diversity, displaying significant differences from the other species.The cluster analysis results b indicatAmaranthaceae species, based on the analysis of SOD gene polymorphism, yielded statistically significant values  between species was determined to be 49%, indicating that approximately half of the genetic variability was attributable to the differences between species; however, the within-species genetic variability level was also significant at 51%, suggesting the influence of mutations, genetic drift, or selection within the studied species.The AMOVA results  indicateNe) and expected heterozygosity (He) ranged from 1.068 to 1.321 and 0.119 to 0.128, respectively. These values indicate a considerable number of alleles and a high degree of genetic variability within the studied species.The analysis of genetic diversity indicators  suggesteA. spinosis (epinard) exhibited the highest number of alleles (1.141) and expected heterozygosity (1.321); conversely, Ch. quinoa displayed the smallest number of alleles (0.328) and expected heterozygosity (1.068). The diversity index, observed heterozygosity, and percentage of polymorphism also followed a similar pattern, with A. spinosis (epinard) demonstrating the highest values and Ch. quinoa displaying the lowest.Meanwhile, A. deflexus and A. spinosis (epinard), also exhibited elevated levels of expected heterozygosity, indicating their greater genetic diversity. Furthermore, A. hypochondriacus and A. caudatus showed similar levels of genetic diversity, suggesting potential shared evolutionary pressures or demographic events. In contrast, other species displayed more variation in their levels of genetic diversity. Overall, accessions of A. spinosis (epinard) showcased the highest genetic diversity, while accessions of Ch. quinoa exhibited the lowest diversity.Amaranth species with higher observed heterozygosity, such as A. retroflexus, A. hypochondriacus, and A. caudatus (A. spinosis (epinard) showed the greatest genetic diversity and differed significantly from the other species.The cluster analysis results based on the retrotransposon analysis indicated that caudatus c exhibitAmaranthaceae species revealed that they are not in a state of Hardy–Weinberg equilibrium. This deviation suggests the presence of natural selection and balancing selection. Balancing selection refers to a mechanism that maintains, increases, or regulates genetic variability without the emergence of new forms or morphophysiological adaptations. In the context of balancing selection, the absence of rare alleles is observed. The evenly distributed D value further confirms the balance between the frequencies of different alleles, as presented in The neutrality test conducted on the Amaranthaceae representatives is a relatively recent evolutionary event [The speciation of ry event , offerinAmaranthaceae species exhibit variations in their level of domestication. While species within this family possess valuable macro- and microelements, as well as biologically active substances with medicinal properties in their grains and green biomass, certain species such as A. spinosis (epinard), A. retroflexus, and A. deflexus are regarded as invasive weeds in numerous regions.Amaranthaceae family exhibit variations in their chromosome numbers. The majority of recorded species have diploid chromosome numbers, such as 2n = 32 for species like A. hypochondriacus, A. cruentus, A. caudatus, and A. retroflexus. Other species, such as A. spinosis and A. deflexus, have a chromosome number of 2n = 34, while Ch. quinoa has a chromosome number of 2n = 36 [Species within the  2n = 36 ,53.Amaranthaceae family, which likely took place between 36.7 and 67.9 million years ago. This WGD event may have been accompanied by subsequent stages of chromosome fusion or loss [These observations suggest the occurrence of a whole genome duplication (WGD) event in the  or loss . OverallNowadays, geneticists have made significant progress by utilizing various research approaches and resources in the field of amaranth research. These include the use of genetic markers and genetic linkage maps ,54,55. MOne such approach is genome profiling based on intermittent repeats, which relies on the distribution of transposable elements, particularly long terminal repeat (LTR) retrotransposons, throughout the genome. PCR using a single primer targeting conserved sequences in LTR retrotransposons is an exemplary technique used for interspersed repeat-based genome profiling. Additionally, the iPBS amplification has proven to be a powerful genome fingerprinting technique that does not require retrotransposon sequence information. Furthermore, the palindromic sequence-targeted (PST) PCR enables genome crawling and profiling, facilitating the initial characterization of intra- and interspecific genetic variability, as well as tracking specific lineages and genotypes .Polymorphism, characterized by the presence of multiple variants  of individual genes within a species, can arise from various factors including mutations, random gene drift, and natural selection . NaturalTo elucidate the nature of polymorphism and evaluate its significance, geneticists have developed various criteria that help assess the presence of selection pressure and identify the type of natural selection occurring. In particular, Tajima’s test is the commonly used statistical test for neutrality, which examines the distribution of allele frequencies of the genes. This test enables a comparison of the average number of nucleotide substitutions and polymorphic sites within a sample .ARF6 genes, for instance, allowed us to assume the occurrence of positive selection, wherein there was a drive to propagate and fix emerging “beneficial” polymorphisms. This suggests that the species may have undergone expansion at that particular time. In such species, it is common to observe a relatively high frequency of rare “beneficial” alleles, which could contribute to stress tolerance in the studied amaranth species [By employing EPIC-PCR, the dominant selection type was determined. The data obtained from the  species .D-criterion of Tajima’s test and other parameters, such as those obtained from SOD genes and iPBS profiling, exhibit a positive value (D > 0), it suggests either a sudden reduction in population size or the influence of disruptive  selection. This may indicate the presence of excessive heterozygosity in the studied species due to gene flow. In such cases, genetic variability is preserved, increased, or regulated without the emergence of new morphophysiological adaptations or life forms, and rare alleles are typically absent. Balancing selection promotes the expansion of adaptive capabilities within species without the emergence of new forms and contributes to the maintenance of polymorphism [When the morphism . One mecmorphism .A. spinosis (epinard), A. retroflexus, and A. deflexus demonstrated the highest level of genetic polymorphism, which enhances their adaptability and survival in diverse environmental conditions. For instance, in the case of A. deflexus, the percentage of polymorphic loci based on ARF6 genes was 56.36%, and the observed heterozygosity was 0.171. In contrast, Ch. quinoa exhibited lower values of 21.82% and 0.068, respectively. Similarly, for SOD genes, A. deflexus showed high values of these parameters . iPBS profiling revealed that A. spinosis (epinard) had a higher percentage of polymorphic loci at 53.13% and observed heterozygosity of 0.188, while Ch. quinoa exhibited lower values of 9.38% and 0.039, respectively. These findings align with the relatively low level of outcrossing observed in cultivated amaranths [According to the obtained results, weed species such as maranths ,68. The Amaranthaceae species exhibit varying yet significant levels of genetic diversity in the studied genes and retrotransposons. This genetic diversity is likely to play a crucial role in their survival and resilience to future stress factors; furthermore, it provides valuable insights for future genetic breeding efforts aimed at developing improved cultivars of amaranth.Overall, the studied Amaranthaceae species: A. caudatus, A. deflexus, A. hypochondriacus, A. retroflexus, A. spinosis (epinard), and Ch. quinoa (Tajik variety “Vahdat”). The seed material was obtained from the collections of the Novosibirsk Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences (ICG SB RAS), and the Centre for Genetic Resources of the Tajik Academy of Agricultural Sciences (CGR TAAS). Each species of plants analysed consisted of 25 randomly selected seedlings, with 7–8 shoots per replicate.The analysis involved seedlings of six 3EDTA, and 100 mM HEPES at pH 5.3. For DNA isolation, an equivalent amount of fresh tissue from one of the twenty-five randomly selected seedlings, which were grown under identical conditions, was used for each sample.Genomic DNA was extracted from fresh plant leaves using a modified CTAB acidic extraction buffer treated with RNAse A . The extARF6) and superoxide dismutase (SOD) genes. The sequences were retrieved from Genbank NCBI  and Ensembl Plants . The primer characteristics for EPIC-PCR amplification of the ARF6 family genes are provided in PCR primer pairs were designed in this study utilizing the entire nucleotide sequences of the auxin response factor  and diversity (PIC), enabling their utilization in the analysis of genetic polymorphism within the SOD gene introns, the primers listed in For evaluating the polymorphism of Amaranthaceae species and the significance of these genes in plant adaptation to diverse environmental conditions. The variations observed in amplicon sizes obtained through the primer combinations may indicate the presence of individual differences in SOD genes among different amaranth species.The utilization of the polymorphism informativeness coefficient (PIC) in analysing locus polymorphism enables the evaluation of genotype diversity within a species. Our findings demonstrate a moderate level of PIC for both primer combinations, suggesting a high degree of genetic diversity in the ® Hot Start II PCR  buffer with 1.5 mM MgCl2, 1 µL of 10 mM primer, 0.2 µL of a 10 mM dNTP mixture, and 0.2 µL of 1 U Phire Hot Start Polymerase. The amplification protocol consisted of an initial denaturation step at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 5 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, and a final extension step at 72 °C for 3 min. The amplification was performed using a T100 Thermal Cycler .The PCR reaction was performed in a 25 µL reaction mixture containing 50 ng of DNA, 1 × PhireThe amplified products were subjected to electrophoresis on a 1.5% agarose gel, and the bands were visualized using ethidium bromide staining . The lenThe analysis employed universal Primer Binding Sites (PBS) primers, and their characteristics are provided in ® Hot Start II PCR buffer with 1.5 mM MgCl2, 1 µL of primer (10 mM), 0.2 µL of a mixture of dNTPs (10 mM), and 0.2 µL of 1 U Phire Hot Start Polymerase. The amplification protocol consisted of a preliminary denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 5 s, annealing at 53–55 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 1 min. The PCR was performed using an Applied Biosystems VeritiPro 96-Well Thermal Cycler .The PCR reaction was conducted in a 25 µL reaction mixture, consisting of 50 ng of DNA, 1 × PhireThe amplified products were subjected to electrophoresis on a 1.2% agarose gel, and visualization was achieved by staining with ethidium bromide . To deteThe gels were assessed using the fingerprint method, and a binary matrix was compiled based on the presence (1) or absence (0) of each fragment. The matrix was then used to perform Tajima’s neutrality test using the Molecular Evolutionary Genetics Analysis (MEGA-X) program . Key indSOD allelic variants, ARF6 genes, and the iPBS profiling of genome non-coding regions revealed genetic variability and adaptability in all studied species of the Amaranthaceae family, which are currently actively introduced to agriculture in different regions of the globe. Among the studied species, A. spinosis (epinard) and A. deflexus showed the highest genetic diversity, indicating their greater potential for adaptation to environmental changes and suggesting their presence at the evolutionary base. Species with higher genetic diversity offer a wider range of genetic tools that can be used to enhance the stress tolerance of existing cultivars. These findings are valuable for strategy selection to improve existing Amaranthaceae cultivars via hybridization with wild types.The analysis of


Debaryomyces hansenii isolates from dry-fermented sausages and their application in the meat industry, because it is the most frequently isolated yeast species from these foodstuffs and its probiotic effects for animals as well as its possible probiotic activity for human beings have been demonstrated. Within the functional ability of foodborne yeasts, anti-inflammatory, antioxidant, antimicrobial, antigenotoxic, and immunomodulatory properties have been reported. Similarly, the use of dry-fermented sausages as vehicles for probiotic moulds remains a challenge because the survival and development of moulds in the gastrointestinal tract are still unknown. Nevertheless, some moulds have been isolated from faeces possibly from their spores as a form of resistance. Additionally, their beneficial effects on animals and humans, such as the decrease in lipid content and the anti-inflammatory activity, have been reported, although they seem to be more related to their postbiotic capacity due to the generated bioactive compounds with profunctional attributes than to their role as probiotics. Therefore, further studies providing knowledge useful for generating dry-fermented sausages with improved functionality are fully necessary.The role of indigenous fungi in the appropriate development of sensory properties and the safety of dry-fermented sausages has been widely established. Nonetheless, their applications as probiotic agents have not been elucidated in such products yet, despite their promising functional features. Thus, it should be interesting to evaluate the probiotic potential of native  Dry-fermented meat products are consumed and appreciated over the world and constitute a source of proteins and microelements . AdditioAll these microorganisms have been described to be intentionally added to dry-fermented sausages as starter and/or protective cultures. Before being used, their safety must be checked. Those microorganisms considered under the qualified presumption of safety (QPS) by the European Food Safety Authority  or generFurthermore, once the microorganisms have the QPS/GRAS status or passed all the required safety tests, the previously mentioned four microbial groups could also be used as potential probiotic sources in dry-fermented sausages, because they are naturally adapted to develop in these matrices, reaching high levels ,8,9. NevAmong these microbial groups, LAB have been extensively evaluated for their probiotic potential ,13. In fApart from the probiotic potential, the postbiotic effect can be understood as positive attributes rendered by microbial bioactive compounds, instead of the activity performed directly by the microorganisms in the body . In contThe long-term consumption of dry-fermented meat products has been associated with chronic diseases, such as colorectal cancer, blood vessel blockage, high blood pressure, and arteriosclerotic metabolite promotion ,19. The Therefore, this review aims to collect current knowledge about the fungi native to dry-fermented sausages or compatible with them, provided with possible probiotic ability, as well as the challenges that remain to be performed to maximise their use in the meat industry.The surface of dry-fermented sausages is naturally colonised by yeasts and moulds during their ripening. Some yeasts and moulds can provoke a wide variety of human diseases, such as allergic reactions and fungemia. However, these infections due to the consumption of food with these microorganisms are infrequent . On the Debaryomyces hansenii (teleomorph of Candida famata) the predominant species [Candida zeylanoides and Yarrowia lipolytica have also been isolated to a lesser extent [Pichia, Rhodotorula, Cryptococcus, Saccharomyces, and Trichosporon [6 cfu/g [Dry-fermented sausages constitute an optimal substrate for the growth of yeasts due to their physicochemical characteristics, such as their pH as well as salt- and carbon-rich compound content , being D species ,27,28,29r extent . Apart fhosporon ,25. Norm[6 cfu/g ,31,32,33D. hansenii, promoting colour development through oxygen absorption and proteolytic activity  has been shown to produce the PgAFP protein, which confers to the mould the ability to reduce the growth of toxigenic moulds as well as OTA production [P. nalgiovense may also be applied as a protective culture. Bernáldez et al. [P. nalgiovense to prevent the presence of toxigenic moulds and their mycotoxins in these kinds of products , nutraceutical abilities , and improvement in food quality and safety  have been stated . FurtherSaccharomyces boulardii and Kluyveromyces fragilis B0399 are currently used as probiotics for humans [Despite these features, only r humans ,75. Perhr humans .Saccharomyces and non-Kluyveromyces yeasts from fermented food are also available. Such yeasts include the species D. hansenii and Y. lipolytica, both found in dry-fermented sausages [D. hansenii has been determined, Y. lipolytica can only be applied for production purposes when this implies the absence of its viable cells in the final product and food and feed products based on microbial biomass [D. hansenii strains as both starter and protective cultures has been reported for fermented meat products [D. hansenii isolated from cheeses has been confirmed, although it was strictly strain-dependent, which could lead to different effects [in vitro stresses under microaerobic and/or anaerobic conditions and could have a potential to reach the gut alive  isolated from Greek-style black olives were able to grow at a low pH, and a vast majority of them tolerated the presence of bile salts  concentration and alanine aminotransferase, enhancing the health of broilers supplemented with fermented rice by the mould [Aspergillus awamori added to the diet of broilers decreased the abdominal fat, plasma cholesterol, lipid peroxidation, and glutamic–oxaloacetic transaminase activity [Aspergillus oryzae has also been tested as a probiotic in poultry, although it is known that the mould cannot survive in anaerobic conditions, and consequently its activity is due to its postbiotic action [A. oryzae improved the defence against hypoxia stress by increasing the antioxidative enzymes superoxide dismutase and glutathione peroxidase and the immunomodulation by the decrease in glucose and cortisol [Rhizopus oligosporus decreased the glycated haemoglobin A1 and the triglyceride concentrations of type II diabetic patients when the “tempeh” was encapsulated [Regarding the functional attributes of moulds, species belonging to the boflavin  (Table 5reatment  (Table 5 in mice  (Table 5he mould  (Table 5activity  (Table 5c action . This spc action ,98 (Tablcortisol  (Table 5cortisol  (Table 5psulated  (Table 5Because dry-fermented meat products have been linked to different diseases, strategies to improve their composition or transform them into functional foods deserve to be exploited. Fungi colonising these products can be an excellent tool for achieving this challenge.D. hansenii is the pre-eminent one in dry-fermented meat products. Therefore, its probiotic potential deserves to be further explored to satisfy consumer demand and the competitiveness of this industry. It is worth noting that D. hansenii’s functional properties could vary depending on individual strains.Focusing on yeasts, When referring to moulds, their probiotic effect is rather limited due to the anaerobic environment in the gastrointestinal tract. Then, their proved postbiotic effect could maximise the enhancement of these products. For this microbial group, two possible strategies could be followed. On the one hand, those moulds able to colonise dry-fermented sausages could be directly inoculated on their surface, and their postbiotic production should be evaluated. On the other hand, in those moulds whose growth and physiological requirements do not fit with the dry-fermented sausages, their inclusion after growing in a suitable matrix under the required conditions should be explored.Both strategies based on fungi seek to resolve meat industry challenges and transform their products into healthier ones. Additionally, these tools can be considered within the clean label movement and are highly appreciated by consumers.


Glycine max breeding lines and varieties cultivated in West Siberia (103 accessions) and other regions of Russia (72 accessions), and we compare them with the cultivated soybean varieties from other geographical locations. Principal component analysis revealed several genetic clusters with different levels of genetic heterogeneity. Studied accessions are genetically similar to varieties from China, Japan, and the USA and are genetically distant to varieties from South Korea. Admixture analysis revealed four ancestry groups based on genetic ancestry and geographical origin, which are consistent with the regions of cultivation and origin of accessions and correspond to the principal component analysis result. Population statistics, including nucleotide diversity, Tajima’s D, and linkage disequilibrium, are comparatively similar to those observed for studied accessions of a different origin. This study provides essential population and genetic information about the unique collection of breeding lines and varieties cultivated in West Siberia and other Russian regions to foster further evolutionary, genome-wide associations and functional breeding studies.Soybean is a leguminous plant cultivated in many countries and is considered important in the food industry due to the high levels of oil and protein content in the beans. The high demand for soybeans and its products in the industry requires the expansion of cultivation areas. Despite climatic restrictions, West Siberia is gradually expanding its area of soybean cultivation. In this study, we present the first analysis of the population structure and genetic diversity of the 175 soybean  Glycine max) is an important species in the food industry due to the high levels of oil and protein concentration in the beans. It is grown almost worldwide with large soybean production quantities related mainly to North and South America , China, India, and East European countries [Soybean , and discuss genetic similarities and differences between them.In this study, we present the first population structure and genetic diversity analysis—including the principal component analysis (PCA), nucleotide diversity π and θ, and linkage disequilibrium of the 175 soybean Glycine max accessions including 96 breeding lines from West Siberia and 88 Russian and European cultivars, with 1 wild accession, were stored and multiplied in West Siberia by the Siberian Federal Scientific Center of Agro-BioTechnologies of the Russian Academy of Sciences  (The 185 soybean  Russia) . Eleven https://soybase.org/snps/, accessed on 25 September 2023). This version of the reference genome was chosen because the BeadChip array was developed for this version.Genomic DNA was extracted using the CTAB method from 3- to 4-day-old seedlings grown in Petri dishes according to the method described earlier by . The genhttps://soybase.org/snps/, accessed on 25 September 2023). It includes 20,087 G. max and G. soja accessions genotyped with 42,509 SNPs. Only G. max-related datasets were used in our study.An additional dataset of genotyped soybean accessions was retrieved from , availabG. max varieties.The dataset with 179 soybean accessions and 52,041 SNPs was filtered with the following criteria: --geno 0.05 --maf 0.05 --mind 0.05 using Plink (v1.90b6.26 ). Next, The merging procedure between our dataset and the additional dataset was performed using Plink. The merged dataset was subjected to quality control with the same parameters as mentioned above.We performed PCA for 175 studied accessions. In addition, we performed PCA for a joint dataset of 175 accessions and the SoyBase dataset. We also performed PCA for a joint dataset of 175 accessions and varieties from the SoyBase dataset marked with the origin “Russia”. Principal component analysis, kinship analysis, and estimation of heterozygosity were performed using Plink. For admixture analysis, we used Admixture (v.1.3.0 ). At firLinkage disequilibrium values were obtained from the TASSEL tool (v.5.2.84 ) with thAs a result of quality control, 29,724 SNPs and 175 soybean accessions passed through the filtration. For the other dataset, 16,986 samples and 26,741 SNPs passed quality control.In general, SNPs in the analyzed dataset of 175 soybean accessions were distributed equally between chromosomes, considering chromosome length, and there were just a few SNPs located on the “Unknown” chromosome .Allele frequency distribution  had two The distribution of heterozygosity values  shows thThe population genetic structure of studied soybean accessions is described using the PCA . There aKinship analysis supported the results of PCA, showing that two clusters on the left are very genetically similar within themselves—there were 62 pairs of accessions with an IBS (identity-by-state) coefficient higher than 75%.Admixture analysis revealed four clusters of origin for studied soybean accessions .Linkage disequilibrium analysis  showed tG. max dataset SoyBase. Principal component analysis of the merged dataset is shown in We merged our dataset with the publicly available Y axis can be attributed to one cluster and above that—to another.In this study, we have performed a comprehensive population genetics examination of breeding lines and varieties cultivated in West Siberia and other regions of Russia and compared them with the genotypes from other regions. The overall results of admixture analysis are in correspondence with the result from a similar analysis of Russian varieties in . Four clComparison between breeding lines and varieties cultivated in Russia, and varieties from other countries , shows tThe first attempts at soybean cultivation in Russia were made in the early 20th century in the Amur region. For a hundred years, almost all the soybean-sown areas have been concentrated in this region, and the most extensive soybean breeding has been carried out there. As in the USA, where the founders of American cultivars originated from China , in the Attempts to introduce soybeans in Siberia have been made since the early 1920s . The maiThe first Siberian soybean variety SibNIIK-315 was selected by individual selection of the Swedish sample from the Federal Research Center “N.I. Vavilov All-Russian Institute of Plant Genetic Resources” collection. SibNIIK-315 and its line SibNIIK-315_st_9 were included in this study as a standard variety and line, respectively. Interestingly, these samples were separated into different clusters , but theG. max, it was equal to 0.23 [It could be problematic to compare some population genetics parameters that were used to describe soybean varieties cultivated in Russia before because of the different genotyping technology and coverage used . Nevertheless, in comparable studies, it was found that in domesticated soybeans, the mean per-site π was 0.189, and in  to 0.23 , while i to 0.23 , θ for c to 0.23 ,14. In s to 0.23 , and var to 0.23 . Also, w to 0.23 ,40.There is some discordance with the estimated LD decay and half-life in other studies. For all chromosomes, the LD decay was estimated to be 0.33 Mb and LD half-life as 1.2 Mb. For each chromosome separately, LD half-life varies from 0.49 for chromosome 3 to 10.56 for chromosome 14 . Some stThis study has several limitations. First, the dataset does not include wild accessions that could be of special importance as a potential source of genetic diversity for the selection. Second, the analysis was performed on genotypes obtained with SNP-array technology. Obviously, results obtained with WGS could potentially be more robust, descriptive, and comparable with other studies. Future studies will remove these limitations and widen our knowledge about soybean breeding lines and varieties cultivated in Russia.Overall, in this study, we present the first population structure and genetic diversity analysis of the 175 breeding lines and varieties cultivated in Russia, among them 103 accessions cultivated in West Siberia, and compare them with the cultivated soybean varieties from other locations. These results provide information about their genetic similarity and origin that can be further used for selection and in consequent genetic analyses like QTL mapping.


Tara Oceans metagenomic data together with a reference-free computational method.Plankton seascape genomics studies have revealed different trends from large-scale weak differentiation to microscale structures. Previous studies have underlined the influence of the environment and seascape on species differentiation and adaptation. However, these studies have generally focused on a few single species, sparse molecular markers, or local scales. Here, we investigated the genomic differentiation of plankton at the macro-scale in a holistic approach using FST-based genomic differentiation of 113 marine planktonic taxa occurring in the North and South Atlantic Oceans, Southern Ocean, and Mediterranean Sea. These taxa belong to various taxonomic clades spanning Metazoa, Chromista, Chlorophyta, Bacteria, and viruses. Globally, population genetic connectivity was significantly higher within oceanic basins and lower in bacteria and unicellular eukaryotes than in zooplankton. Using mixed linear models, we tested six abiotic factors influencing connectivity, including Lagrangian travel time, as proxies of oceanic current effects. We found that oceanic currents were the main population genetic connectivity drivers, together with temperature and salinity. Finally, we classified the 113 taxa into parameter-driven groups and showed that plankton taxa belonging to the same taxonomic rank such as phylum, class or order presented genomic differentiation driven by different environmental factors.We reconstructed the Our results validate the isolation-by-current hypothesis for a non-negligible proportion of taxa and highlight the role of other physicochemical parameters in large-scale plankton genetic connectivity. The reference-free approach used in this study offers a new systematic framework to analyse the population genomics of non-model and undocumented marine organisms from a large-scale and holistic point of view.The online version contains supplementary material available at 10.1186/s12862-023-02160-8. Marine species from epipelagic plankton are drifting organisms that are abundant in the global ocean, play an active role in Earth’s biogeochemical cycles, and form a complex trophic web with high taxonomic diversity based on fish resources –9. UndeMacrocystis pyrifera [Two major forces can affect gene flow between planktonic populations: abiotic factors, including marine currents, and biotic factors. First, as planktonic species are transported passively and continuously by marine currents, we could expect that the “isolation-by-current” shapes the genetic structure of populations. Conversely, cosmopolitan, panmictic and/or unstructured species have been reported multiple times in Copepoda , 22–26, pyrifera . Given pyrifera  to bettepyrifera . In thispyrifera . Succespyrifera –41  andpyrifera , 36.Simultaneously, changing environmental conditions may lead to selective pressure that counteracts the effect of dispersion induced by marine currents, leading to higher differentiation. Some good examples are temperature-driven genetic structures from bacteria to cnidaria , 30, 42 Tara Oceans data provide a unique dataset from many locations in all oceans worldwide, enabling global approaches to investigate plankton [Advances in environmental genomics realized by shotgun sequencing offer a new perspective for the population genomics of marine plankton species based on metagenomic data. Diversity in ocean microorganisms can now be better-understood, thanks to ambitious expeditions , 48. Parplankton –52, but plankton , 53–56. plankton .Tara Oceans metagenomic data generated 18 million SNPs and a proof of concept of their utility and robustness for population genomics has been demonstrated on the epilagic copepod Oithona nana using its genome as a reference to relocating SNPs [An alternative method for studying plankton population genomics has been proposed based on single nucleotide polymorphisms (SNPs) calling directly from metagenomic data using an assembly-free strategy . The lating SNPs . SNPs caing SNPs . This apTara Oceans expeditions in the Mediterranean Sea, Atlantic Ocean, and Southern Ocean. We clustered the 18e6 SNPs into 113 taxa that may correspond to complexes of closely related species. Minor allele frequencies of each species were used to estimate genomic differentiation using pairwise FST. The genomic distances were modelled with environmental parameters, including Lagrangian travel times between sampling sites [Here, we propose to study plankton connectivity from a holistic point of view, using metagenomic data extracted from samples gathered during ng sites  to estim6 SNPs called using a reference-free approach generated from 114 metagenomic samples collected from 35 Tara stations , Haptophyta (4), Mamiellales (3), Collodaria (2), Ciliophora (2), Cryptophyta (1), and Pelagomonadaceae (1). Only four taxa presented very poor assignment (unclassified or eukaryotes) and one virus. In Mamiellales, two species were identified as Bathyccocus prasinos and were related to previously observed results from Tara Oceans , being higher in the 180–2000 µm and lower in 0.8–5 µm and  .The taxa were then clustered into eight groups using k-means based on their t-SNE coordinates Fig. B. We ideFST (0.35 to 0.84) and revealed very low connectivity between their populations  shows population genomic differentiation linked to local marine barriers, with the population from TARA_148 being more isolated from the others  Fig. A. Anotheons Fig. B. Partic−1 against 0.031–0.106 µmol.L−1)   population from TARA_70 was more isolated from the other NAO populations and the TARA_70 site is also characterized by a higher phosphate concentration  from the “Salinity” cluster, with temperature being also an important explaining factor Fig. G. It wasTara Oceans data [Prochlorococcus and Synechococcus), and constitute most viral populations in GOV 2.0 [Synechococcus genus, were detected in the same locations in the Mediterranean Sea, with clear FST unimodal distributions . Thus, we showed how population genomic analyses at different trophic levels are important to decipher the connectivity of plankton and can be complementary to the traditional metabarcoding approach that fails to quantify the connectivity and intra-species structure patterns. The next step would be to better capture the relative effects of evolutionary forces acting on plankton genomes, such as genetic drift and selection. Haplotype data could resolve this question, but in the framework of metagenomics, the latter remains a technical and computational challenge.Global warming and its impact on ocean climate are expected to have a significant impact on marine plankton biogeography by restructuring plankton assemblages . In this6 SNPs produced in a previous study [Tara Oceans sampling sites corresponding to four distinct size fractions  from the water surface layer, for a total of 114 samples , Northern Atlantic Ocean , Southern Atlantic Ocean , and Southern Ocean . The full protocols for sampling, extraction and sequencing have been detailed in previous studies [https://github.com/ropensci/rnaturalearth).We used a set of 18eus study . SNPs we studies , 89. AllTo identify taxon specific SNPs, we used metaVaR version v0.2 . We disc–5). The taxonomic ID and bit scores of each match were maintained. A fuzzy Lowest Common Ancestor (LCA) (see https://github.com/institut-de-genomique/fuzzy-lca-module) method was used to assign a taxonomy to each sequence using bitscore as a weight with -r 0.67  and -ftdp options. The highest phylogenetic rank was retained as the best assignment for each sequence. For the second method, the sequences were mapped using the blastn algorithm implemented in diamond on MATOU, a unigen catalog based on Tara Oceans metatranscriptomic data [Taxonomic assignment of each species was performed using three different methods , multiple comparison Wilcoxon tests were performed between groups. To estimate the genetic connectivity between and within basins, we regrouped Tara stations based on their locations  and computed the mean FST between and within basins.To investigate the genomic differentiation of each taxon, ulations . Two FSTTara stations back and forth. We used the public database of the Global Drifter Program (GDP), managed by the National Oceanographic and Atmospheric Administration (NOAA) (https://www.aoml.noaa.gov/phod/gdp/), which contains information on drifters ranging from February 15, 1979, to September 31, 2019. We extracted the data for both drogued and undrogued drifters  to maximize the information. No drifters have been observed to exit the Mediterranean Sea through the Strait of Gibraltar. Therefore, to avoid missing data, we arbitrarily added 100 years to the travel times of pathways out of the Mediterranean Sea over the Strait of Gibraltar and added one year to the pathways going into the Mediterranean Sea, based on previous models of surface water [To estimate Lagrangian transport, we used a method based on drifter data  to compuce water , 96. We Tara stations were extracted from the World Ocean Atlas public database (https://www.nodc.noaa.gov/OC5/woa13/woa13data.html) for the period 2006–2013 on a 1° × 1° grid, covering the dates of Tara Oceans expeditions. The following parameters were obtained: temperature (°C), salinity (unitless), silicate (µmol.L−1), phosphate (µmol.L−1), and nitrate (µmol.L−1) . For each pairwise FST matrix, the corresponding matrix of the minimum Lagrangian travel time is retrieved. Temperature, salinity, silicate, phosphate, and nitrate measurements were extracted for all the stations where the plankton species were present, and a Euclidean distance was computed between the stations for each of these parameters. The LMM was then applied to pairwise FST values using the five environmental distances and Lagrangian travel times after scaling, adding a variance of 1 for each explicative variable. We considered these parameters as the independent variables. As a result, an estimate of the contribution of each parameter to the total variance of the pairwise FST was obtained. Additionally, a fixed effect and proportion of unexplained variance were retrieved. After FST variance decomposition, two principal component analyses (PCA) were performed. The first was performed on the variance explained by the six variables and the unexplained part of the variance over the 113 species. From this PCA, the unexplained FST variance  of the variables to the components were extracted. PCAs were performed using the FactoMineR v2.3 R package [To estimate the relative contribution of environmental parameters and Lagrangian travel time to the variance of the genetic connectivity, a LMM was applied using the R package MM4LMM . The mo package .p < 0.05).To identify taxa sharing similar environmental parameters that drive their genetic connectivity, the variance explained by each factor was used with dimensional reduction through t-distributed stochastic neighbour embedding (t-SNE) using the Rtsne R package  with a pAdditional file 1.Additional file 2: Supplementary Figure S1. Occurrence of species. Species are noted “MVS” for metavariant species. Supplementary Figure S2. Distributions of pairwise-FST by species. Supplementary Figure S3. Lagrangian estimates matrices. Supplementary Figure S4. Lagrangian trajectories for stations of Southern Ocean. Supplementary Figure S5. SNPs clustering with metaVaR. Supplementary Figure S6. Overview of the taxonomic assignment procedure. Supplementary Figure S7. Environmental parameters maps. Supplementary Figure S8. Principal component analysis of the contribution of environmental parameters to the genomic differentiation of plankton species. Supplementary Table S2. Species assigned to Bathycoccus.


Rusa unicolor swinhoei is an economically important animal in Taiwan, owing to the market demand for its velvet antler. In this study, we investigated the genetic diversity and structure of Formosan sambar deer using microsatellites. The observed genetic diversity was low, which is likely attributable to inbreeding and bottleneck effects. Moreover, this study revealed two distinct genetic groups within the captive populations and found no significant population genetic structure among the captive populations in Taiwan. These findings have the potential to improve breeding management and contribute to the mitigation of inbreeding, thereby promoting the productive potential of Formosan sambar deer.Rusa unicolor swinhoei) are of great economic significance in Taiwan, resulting in a substantial increase in deer farming to meet the high demand for velvet antlers. Inbreeding depression and reduced genetic variability can lead to the deterioration of captive populations. In this study, 239 Formosan sambar deer were genotyped using 13 microsatellites to analyze their genetic diversity and population genetic structure. Our results indicate a high-resolution power of these microsatellites in individual discrimination and parentage analysis. However, captive populations exhibit a low level of genetic diversity, likely because of inbreeding and bottleneck effects. Both principal coordinate analysis (PCoA) and STRUCTURE analyses revealed two distinct and segregated genetic groups within the captive populations and indicated no clear population genetic structure among the captive populations. Introducing new genetic material from the wild through translocation offers a potential solution for mitigating the impact of inbreeding and enhancing genetic diversity. The comprehensive information obtained from these genetic analyses is crucial for the development of effective breeding strategies aimed at preserving and enhancing Formosan sambar deer populations.Formosan sambar deer ( Rusa unicolor is a large ungulate distributed throughout Southeast and South Asia, including South China, India, Nepal, Cambodia, Vietnam, Taiwan, and Malaysia  [The power of discrimination (PD) was assessed for each microsatellite: PD = 1 − ΣPinotype i . We comp− PD13)] . Additio− PD13)] . The ord− PD13)] .IS), which represents the level of inbreeding within a population, and genetic differentiation (Fst) and Nei’s genetic distance between populations. PowerMarker v3.25 was used to assess the polymorphic information content (PIC) for each marker [Genetic diversity parameters, including alleles per locus (A), observed heterozygosity (Ho), and expected heterozygosity (He) were evaluated for each locus and population using the GenAlEx 6.5 software . Additioh marker . The Neih marker . Statisth marker ,37 to exh marker , infinith marker , and twoh marker  in Bottlh marker .ML-RELATE calculates relatedness and relationships using maximum-likelihood estimation . Maximumhttps://taylor0.biology.ucla.edu/structureHarvester/) (accessed on 10 July 2023) to estimate the ΔK (delta K) values for each genetic group and infer the optimal number of K (genetic clusters) [Principal coordinate analysis (PCoA) is a multivariate statistical technique used to visualize and explore the similarities or dissimilarities between samples based on a distance or dissimilarity matrix. In this study, PCoA was performed using the GenAlex 6.5 software to identify and illustrate the genetic clusters within the dataset . In addilusters) .In the present study, a total of 83 alleles were identified among 239 Formosan sambar deer screened through 13 microsatellite loci, and all loci exhibited polymorphisms. The average discrimination power (PD) was 0.421 and ranged from 0.0041–0.7850. Ca18 and Ca71 exhibited the highest and lowest discrimination powers, respectively. The probabilities of identity for unrelated individuals (PI) and siblings (PIsibs) were evaluated for each locus and ranged from 0.073 to 0.992 and 0.375 to 0.991, respectively. Ca18 and Ca71 showed the lowest and highest values for PI and PIsibs, respectively .−7 and 1.871 × 10−3, respectively  ranged from 2.000  to 15.000 (Ca18), with a mean value of 6.385 alleles. The polymorphism information content (PIC) values ranged from 0.004 (Ca71) to 0.759 (Ca18), with a mean value of 0.388 . Of the IS) ranged from to −0.002 (Ca71) to 0.983 (Ca30). These results indicated that there was a heterozygote deficiency observed at all loci, except for loci Cu02, Cu09, Cu10, and Ca71 (p < 0.05), except for Cu02, Cu09, and Ca71.The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.004 (Ca71) to 0.706 (Ca67) and 0.004 (Ca71) to 0.785 (Ca18), respectively. The mean Ho and He values were 0.299 and 0.421, respectively. The inbreeding coefficient (Fand Ca71 . All micp = 0.001) and TPM (p = 0.008), but not under IAM (p = 0.685). Furthermore, this test conducted on captive populations of Formosan sambar deer revealed evidence of a bottleneck.There was a significant excess of heterozygosity in the entire captive population of Taiwan relative to the mutation-drift expectation under both SMM (IS)  varied from 2.231 (NTO and YUN2) to 3.923 (TNN4 and TNN5) in 15 captive populations. The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.222 to 0.346 and 0.299 to 0.449, respectively, with mean values of 0.291 and 0.375, respectively . PopulatIS) . These f2 = 0.0001, p = 0.469)  .A principal coordinate analysis (PCoA) is also presented to summarize the individual relationships. The first component accounted for 29.36% of the total genetic variability, whereas the second and third components accounted for 18.49% and 14.60% of the variability, respectively . The PCoSTRUCTURE clustering revealed a comparable genetic structure . Based oThe 13 microsatellites used in this study possessed high levels of polymorphisms and resolution, thus making them suitable for parental analysis. Maximum likelihood estimates from the ML-RELATE program were used for the 239 captive individuals. In total, 475 (1.670%) PO, 1035 (3.639%) FS, 3809 (13.394%) HS, and 23,122 (81.297%) U were identified among the 28, 441 pairs . Among the within-population pairs, we identified 81 (0.285%) PO, 222 (0.781%) FS, 425 (1.494%) HS and 1749 (6.150%) U relationships. For inter-population pairs, we found 394 (1.385%) PO, 813 (2.859%) FS, 3384 (11.899%) HS, and 21,373 (75.147%) U relationships. There were more inter-population pairs  with kinships than within-farm pairs  .In Taiwan, the primary focus of deer farms is the extraction of deer velvet antlers, which is the main source of income. However, owing to the absence of pedigree records and the failure of farmers to follow scientific mating plans in their herds, there is a risk of inbreeding accumulation and a subsequent decrease in production potential within these farms. Microsatellites are powerful tools for paternity testing in the management of captive animal populations. This enabled accurate parentage determination and assisted in maintaining genetic diversity . In this−7, and 1.871 × 10−3, respectively (−3 to 10−4) and PIsibs (<2 × 10−2) for wildlife forensic applications [PD, PI, and PIsibs are the three essential parameters used in individual discrimination of parentage determination based on microsatellite data. The cumulative PD, PI, and PIsibs values based on all 13 polymorphic microsatellite loci were 0.999, 4.589 × 10ectively . The cumications ,52. ThesGenetic diversity serves as the foundation for animal breeding because high genetic diversity is essential for meeting future human needs and preserving a rich gene pool, particularly in the face of climate change . A high C. elaphus montanus in European  [C. elaphus scoticus in England  [C. nippon in Japan  [C. nippon hortulorum in China  [C. nippon pseudaxis in Vietnam  [Hydropotes inermis inermis in China  [C. nippon in Japan  [The frequent inbreeding of individuals within a population can lead to decreased genetic diversity, resulting in reduced adaptability to the environment . Allele = 0.870) , C. elap= 0.801) , C. nipp= 0.610) , and C. = 0.662) , and C. = 0.656) , our stuIS values (p < 0.05 in SMM and TPM modes). These findings suggest that captive populations have experienced a reduction in genetic diversity and have undergone a substantial decrease in effective population size. The presence of a bottleneck effect indicates that the population has experienced a severe reduction in numbers, which can lead to the loss of genetic variation and increased vulnerability to genetic drift [Vicugna vicugna) and found that translocation from the wild played a crucial role in effectively restoring genetic diversity in the captive populations. Our study highlights the importance of implementing effective genetic management strategies to prevent inbreeding and mitigate the effects of bottlenecks, thereby ensuring the long-term health and sustainability of captive populations of the Formosan sambar deer.The reduced genetic diversity observed in the Formosan sambar deer may be attributed to inbreeding and the bottleneck effect, both of which can have substantial consequences on captive animal populations. In the present study, we observed that all captive populations displayed a deficiency in heterozygosity with positive FS values . Furtheric drift . Anello ic drift  conductePopulation genetic structure, however, refers to the way genetic variation is distributed within and among populations. This structure is influenced by factors such as migration, genetic drift, and natural selection ,65. UndeThe Formosan sambar deer has been an economically important animal in Taiwan since 1963. Unfortunately, the original sources of the captive populations remain unknown. Captive populations were divided into two major groups based on the PCoA . SimilarHowever, we also found that each genetic group comprised individuals from all captive populations . These aIn Taiwan, when a deer farm successfully breeds outstanding individuals with high velvet antler production, other farms may purchase their offspring or semen to improve herd productivity. Consequently, there may be frequent gene flow between different deer farms. According to the parentage analysis, there were more inter-population pairs  with parent–offspring kinships than within-farm kinships  . InitialThe use of microsatellites in this study offers valuable insights into a wide range of ecological and evolutionary enquiries. Our findings indicated that captive populations of Formosan sambar deer exhibited lower levels of genetic diversity, which can be attributed to the effects of inbreeding and a bottleneck effect. To mitigate the adverse effects of inbreeding and to minimize the loss of genetic diversity, it is recommended to implement an action plan for captive populations that involves introducing new genetic material from wild populations. This study has the potential to enhance breeding management practices and aid in reducing the negative impacts of inbreeding on the production potential of Formosan sambar deer.