Spirochaeta across three distant biogeographic provinces of the Southern Ocean; maritime Antarctica, sub-Antarctic Islands, and Patagonia. Each targeted Spirochaeta operational taxonomic units were characterized by a substantial intrapopulation microdiversity, and significant genetic differentiation and phylogeographic structure among the three provinces. Gene flow estimations among Spirochaeta populations support the role of the Antarctic Polar Front as a biogeographic barrier to bacterial dispersal between Antarctic and sub-Antarctic provinces. Conversely, the Antarctic Circumpolar Current appears as the main driver of gene flow, connecting sub-Antarctic Islands with Patagonia and maritime Antarctica. Additionally, historical processes  govern up to 86% of the spatial turnover among Spirochaeta populations. Overall, our approach bridges the gap between microbial and macrobial ecology by revealing strong congruency with macroorganisms distribution patterns at the populational level, shaped by the same oceanographic structures and ecological processes.Most of the microbial biogeographic patterns in the oceans have been depicted at the whole community level, leaving out finer taxonomic resolution  that is crucial to conduct intra-population phylogeographic study, as commonly done for macroorganisms. Here, we present a new approach to unravel the bacterial phylogeographic patterns combining community-wide survey by 16S rRNA gene metabarcoding and intra-species resolution through the oligotyping method, allowing robust estimations of genetic and phylogeographic indices, and migration parameters. As a proof-of-concept, we focused on the bacterial genus  Biogeography has traditionally investigated the geographic distribution of macroorganisms in the Eukaryota domain. Yet, the last two decades have witnessed a growing number of studies focused on the biogeography of microorganisms, and repeatedly reporting non-random community assemblages of various prokaryotic microorganisms . Unlike To date, most of the microbial biogeographic patterns have been depicted at the whole community level, taking advantage of the extensive survey capacity provided by the next-generation sequencing (NGS) technologies . NeverthThe oceans have been considered among the most challenging environments to test hypotheses about microbial biogeography, mainly due to the speculated transport of organisms over large distances by marine currents and the absence of perceivable marine barriers impeding potential dispersal events . HoweverDesulfotomaculum in Arctic marine sediments, Targeting the intraspecific microdiversity using NGS data requires specific computational methods to discriminate the stochastic noise caused by random sequencing errors from those associated with biologically significant diversity . For thiIn the present proof-of-concept study, we aim to elucidate the evolutionary processes driving microbial biogeography across different provinces of the SO by combining (1) community-wide surveying provided by the high-throughput sequencing of the 16S rRNA gene, (2) intra-species microdiversity resolution obtained through the oligotyping method implemented in the MED pipeline, and (3) phylogeographic analysis as traditionally developed for macroorganisms as models. Considering the SO as an outstanding idoneous frame, we investigated the geographic distribution of genetic diversity of marine bacterial taxa across three biogeographic provinces: maritime Antarctica , sub-Antarctic Islands of the Indian Ocean (Kerguelen archipelago), and southern South America (Patagonia). We selected sites separated by the APF, i.e., maritime Antarctica and Patagonia, and others connected through the ACC, i.e., Patagonia and Kerguelen archipelago.Abatus irregular sea urchins. The Abatus genus is distributed across the SO and gathers various sibling species homologous in ecology and habitat, such as Abatus cavernosus in southern South America, Abatus cordatus in Kerguelen Islands, and Abatus agassizii in maritime Antarctica . Spirochaeta bacteria are recognized as the most prevalent and abundant genus in the Abatus gut tissue (1). Thus, due to its ease of detection and ubiquity across biogeographic provinces, Spirochaeta represents an illustrative model to validate our methodology and explore marine bacteria’s spatial genetic patterns, from genus to population level. We hypothesized that the strong biogeographic barrier between South America and maritime Antarctica classically observed in the literature for macroorganisms  also affects the fine-scale genetic structure and the phylogeographic patterns within Spirochaeta OTUs. Besides, the ACC-mediated connectivity among sub-Antarctic provinces should be reflected by a greater genetic homogeneity of Spirochaeta populations between South American sites and the Kerguelen Islands, rather than with maritime Antarctica. Alternatively, the potential high dispersal capacity of Spirochaeta taxa may result in the absence of genetic and phylogeographic structure across the SO.As the contribution of geography to biological diversity patterns  is stronger on habitat-specialists  , and emptarctica . Since ttarctica . Within l levels . Workingl levels . As a mot tissue . Moreovet tissue  and, to Abatus individuals were sampled from four localities across the SO, including two sites in Patagonia, southern South America , one site in Kerguelen Islands , and one site in the West Antarctic Peninsula   following ® Kit  following the manufacturer’s recommendations.Adult nd, KGI) . Marine Abatus gut tissue samples. Briefly, extracted genomic DNA was used as the template for PCR amplification using the primers 515F 5′-GTGYCAGCMGCCGCGGTA-3′ and 926R 5′-CCCCGYCAATTCMTTTRAGT-3′ , and converted into a presence/absence matrix to detect the shared or exclusive OTUs across the four selected sites, discarding abundance variations resulting from short-term environmental conditions. Bray–Curtis and Unweighted Unifrac distances were calculated from the OTU presence/absence matrix and used to perform a non-metric multidimensional scaling (NMDS) with the metaMDS function of the ade4 package , using adonis and pairwise.adonis functions implemented in vegan and pairwiseAdonis R packages, respectively  on the Illumina MiSeq platform at the UWBC DNA Sequencing Facility . Reads of 16S rRNA were processed using the open-source software Mothur v1.44.0. Briefly, 3′ and 5′ reads were paired and trimmed according to their length and quality as described in  package . The NMDectively .Spirochaeta OTUs present in the four localities was retained for further analysis . Additionally, each identified oligotype had to have a default minimum relative abundance of 2% in the OTU sequences dataset  within the 16S rRNA gene sequences from ositions , 2015. T dataset . Accumulge iNext  in r v3.ge iNext . Pie chak), the oligotype diversity (H), the number of discriminant sites (S) and the pairwise difference between sequences (Φ) were estimated individually for each Spirochaeta OTU using the packages pegas (st and Φst) among Spirochaeta populations was analyzed using the software arlequin v3.5.2  and deterministic  processes, on Bray) among sites  or dispersal limitation plus drift (RCBray > +0.95). On the contrary, RCBray values > −0.95 and < +0.95 are indicative of ecological drift  approach  among Spirochaeta populations was calculated by multiplying the maximum likelihood estimates (MLE) of the mutation parameter (θ) by the migration parameter (M), both estimated through the Lamarc program. We present the mean and standard deviation of the estimated Nm values obtained from the 10 runs for each Spirochaeta OTU.The amount and direction of gene flow among  v2.1.10 . A totalSpirochaeta genus were retrieved, representing a total of 10 OTUs.A total of 4,184,226 raw reads was generated from the 91 samples of external sediments and gut tissues. Once processed, 2,542,038 cleaned sequences distributed into 727 OTUs were obtained from the external sediment and gut tissue samples (details provided in Spirochaeta OTU composition between the Patagonian sites (PAT1 and PAT2)  . Conversian ones .Spirochaeta OTUs . These three selected co-distributed OTUs are good targets to constitute a metapopulation, which is a meaningful ecological unit of distinct local populations separated by gaps in habitats and interconnected to some extent via dispersal events of individuals  ranged from 0.0681 (OTU40 in KER) to 0.8036 (OTU7 in PAT1) across localities  and phylogeographic (Φst) structures between the two closest localities  were extremely to moderately low. In the case of the OTU40, the genetic diversity and frequencies of Spirochaeta oligotypes were fully homogenous between PAT1 and PAT2, as indicated by the non-significant values of Fst and Φst comparisons  . All in all, these results indicate the existence of both genetically and geographically differentiated Spirochaeta populations across the three biogeographic provinces sampled.Independently of the OTU considered, the genetic  . Consistigotypes . Two exc and Φst . The SnnWithin the 48 oligotypes identified in the OTU6, 11 (∼23%) were exclusive to one of the three provinces, and more than half were exclusive to Kerguelen Islands . MaritimIn the case of OTU7, the percentage of exclusive oligotypes was almost the same as the OTU6, with 21 (∼22%) oligotypes exclusive to one of the provinces . The domFor OTU40, we recorded a predominant group of oligotypes specific to the Patagonian sites representing 65% of the oligotypes identified within the OTU40 . A clearAll the analyzed OTUs showed high genetic similarities between the analyzed Patagonian populations (PAT1 and PAT2). Gene flow analyses identified a bidirectional pattern from PAT2 to PAT1  and from PAT1 to PAT2 (Nm > 14) . The conSpirochaeta OTUs, neutral ecological processes were essential in shaping the population composition turnover in Abatus gut membrane. According to the quantitative parsing of ecological processes, the composition of Spirochaeta population was mostly driven by ecological drift, ranging from 50% (OTU40) to 74% (OTU6) of turnover, followed by dispersal limitation, ranging from 12% (OTU6) to 20% (OTU40) of turnover, and homogenizing selection, ranging from 9% (OTU6) to 19% (OTU40) of turnover  did not exceed 24%.The MMRR approach was used to disentangle the relative effect of geographic distance environmental abiotic differences on the ectively . In contfor OTU6 . Whateveposition , and theSpirochaeta genus, and co-distributed across Patagonia, Kerguelen Islands, and maritime Antarctic provinces of the SO. Through this innovative approach, we identified numerous oligotypes within each of the Spirochaeta OTUs. These oligotypes, corresponding to Spirochaeta sub-taxa, were characterized by contrasting geographic distribution and high levels of 16S rRNA gene similarity (>97%). Taking advantage of the populational taxonomic resolution provided by the oligotype definition, we depicted the Spirochaeta biogeographic patterns across the analyzed provinces in the SO, using various tools adapted from population genetics classically applied in phylogeographic study of macroorganisms’ models. Despite its low substitution rate  or abio events) , known t events) .Spirochaeta taxa across the SO. Our previous study of the Abatus gut microbiota showed that non-neutral processes drove the bacterial community at the OTU level in the host gut tissue , rather suggesting that Spirochaeta may benefit from a priority effect during Abatus early-life, and that the subsequent interactions may deterministically constrain gut colonization success  and the intricate association between rocesses . In strorocesses . Under tulations . Contras success . Moreoverogramma  and materpuratus . The preed state . The preSynechococcus or the Vibrio populations, demonstrate fine-tuning of their physiology by accumulating microdiversity in functional genes through duplication events, SNPs, and allelic variants , the more they tend to exhibit cosmopolitan oligotypes . It is not unreasonable to infer that a larger population may have more chance to migrate and successfully reach a suitable habitat, while small-size populations may be more likely diluted along the dispersal route with no/too few dispersive particles to establish in the new habitat  a high populational and genetic homogeneity within the Patagonia province, (2) a strong barrier to dispersal between Patagonia and maritime Antarctica due to the APF, resulting in a high differentiation of Spirochaeta populations, and (3) the existence of connectivity between sub-Antarctic provinces of the Kerguelen Islands and Patagonia, and from Kerguelen Islands to the maritime Antarctic, due to the ACC-mediated connectivity. Nevertheless, as connected as these provinces are, the gene flow does not seem to be strong enough to prevent the ongoing intraspecific microdiversification of the Spirochaeta taxa. The microdiversity of Spirochaeta, underlying these biogeographic patterns, is essentially driven by historical processes, such as ecological and genetic drift, and dispersal limitation related to the SO’s oceanographic features. In the future, extending this framework to other localities and taxonomic groups will contribute to the comprehensive understanding of the SO microbiota.Our study highlights the application of V4–V5 16S rRNA gene metabarcoding and oligotyping approach as rapid, robust, and resolutive enough to unravel marine bacterial phylogeographic patterns and detect genetic connectivity among the SO provinces. Taken together, the three https://www.ncbi.nlm.nih.gov/PRJNA658980, https://www.ncbi.nlm.nih.gov/PRJNA590493, and https://www.ncbi.nlm.nih.gov/PRJNA659050.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: EP, JO, and LC designed the study. EP and JO organized the sampling missions. EP, JO, LC, and GS collected samples. GS extracted the DNA, organized sequencing and managed data mining and analyses. NS contributed to the gene-flow and the MMRR analyses and designed the illustrative maps. GS, JO, NS, CG-W, and EP interpreted the results. GS wrote the manuscript. All authors contributed substantially to manuscript revisions and read and approved the final manuscript.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.


The last glacial period (LGP) promoted a loss of genetic diversity in Paleolithic populations of modern humans from diverse regions of the world by range contractions and habitat fragmentation. However, this period also provided some currently submersed lands, such as the Sunda shelf in Southeast Asia (SEA), that could have favored the expansion of our species. Concerning the latter, still little is known about the influence of the lowering sea level on the genetic diversity of current SEA populations. Here, we applied approximate Bayesian computation, based on extensive spatially explicit computer simulations, to evaluate the fitting of mtDNA data from diverse SEA populations with alternative evolutionary scenarios that consider and ignore the LGP and migration through long-distance dispersal (LDD). We found that both the LGP and migration through LDD should be taken into consideration to explain the currently observed genetic diversity in these populations and supported a rapid expansion of first populations throughout SEA. We also found that temporarily available lands caused by the low sea level of the LGP provided additional resources and migration corridors that favored genetic diversity. We conclude that migration through LDD and temporarily available lands during the LGP should be considered to properly understand and model the first expansions of modern humans. Despite intense efforts for understanding the evolutionary history of early modern humans (EMHs), diverse past evolutionary events are still unclear. One of them involves the genetic consequences of environmental conditions that occurred during the first expansion of our species. In this regard, the last glacial period ) produced large ice sheets in the northern hemisphere regions and increased the extension of deserts ,4. In adIn order to understand the consequences of the LGP (and LDD) on the genetic diversity of current SEA populations, here we evaluated the fitting of four alternative evolutionary scenarios, which either consider or ignore the LGP (sea level changes) and LDD, with observed genetic data from a variety of SEA populations. We applied approximate Bayesian computation (ABC), based on extensive spatially explicit computer simulations of demographic and genetic data, to identify the best-fitting evolutionary scenario. Next, for the selected scenario, we estimated diverse population genetics parameters  to understand their role on the observed genetic diversity.In this section, we describe the studied genetic data, the methodology to perform the phylogenetic tree reconstruction and the ABC methods applied to identify the best-fitting evolutionary scenario (considering or ignoring the LGP and LDD) and to estimate the population genetic and demographic parameters.The observed genetic dataset  comprised sequences of the mtDNA hypervariable I region  obtaγ distribution (HKY +I +G). Next, we inferred a maximum likelihood (ML) phylogenetic tree with RAxML-NG [In order to explore the genetic relationships among the studied samples, we performed a traditional phylogenetic tree reconstruction. We used jModelTest2  to identRAxML-NG  under thNONE); (ii) considering the LGP and considering gradual migration ; (iii) ignoring the LGP and considering gradual migration together with LDD ; and (iv) considering the LGP and considering gradual migration together with LDD . Illustrative examples of spatially explicit simulations performed under these evolutionary scenarios are shown in We evaluated the fitting of four alternative evolutionary scenarios  with the observed data using ABC. These scenarios were based on two criteria, considering or ignoring the LGP (concerning sea level variation), and considering or ignoring a fraction of migration events presenting LDD: (i) Ignoring the LGP and considering gradual migration  from current Bangladesh (N) varied according to a uniform prior distribution between 25,000 and 75,000 individuals, which is large enough to ensure estimates falling within the range of the prior distribution [r) was sampled from a uniform prior distribution ranging between 0.4 and 1.0 and the prior distribution of the migration rate  varied between 0.2 and 0.3. The carrying capacity (K) was modeled with a uniform prior between 1000 and 4000  included spatial and temporal variation in the carrying capacity and migration rate. In particular, the carrying capacity and migration rate of submersed demes were set to 0, but when previously submersed demes are exposed (due to sea level decrease), their migration rate (m_temp) and carrying capacity (K_temp) were treated as additional parameters  and we estimated them separately to assess the impact of the temporary lands on the expansion and genetic diversity of SEA populations. The geographic area of temporarily available lands was based on previous studies [μ) sampled from a uniform prior distribution . Evolut studies ,19,44,45 studies  and unde studies . Concernribution  that wasST) between the three populations located at the northwesternmost region of SEA  and the three populations located at the southeasternmost region of SEA ; (ii) decay in the genetic differentiation (based on FST) between the Bago population (the closest population to the origin of the range expansion) and all the other populations with the geographic distance between them; and (iii) the decay in nucleotide diversity  per population with the geographic distance from the origin of the range expansion. The last two SS include the slope of the fitted line obtained from the linear regression between the previously indicated geographic distances and genetic statistics.Summary statistics (SS) from observed and simulated data were computed with Arlequin ver 3.5.2.2 . The appmnlogistic) and the neural networks  methods implemented in the abc library of R [cv4postpr function of the abc library) and considering a tolerance of 1% [postpr function of the abc library [mnlogistic and neuralnet methods and retaining 1% of the simulations  with SS closer to the SS of the observed data.We selected the best-fitting evolutionary scenario with the multinomial logistic regression  using tLGP&LDD evolutionary scenario, which was the evolutionary scenario that best fitted the observed data. The parameters estimation was based on the multiple linear regression method implemented in ABCtoolbox [We estimated the evolutionary parameters of the Ctoolbox . FollowiCtoolbox ,51, we eIn this section, we present the results of the phylogenetic analysis of SEA populations followed by the selection of the best-fitting evolutionary scenario and the population genetics parameters estimation.The reconstructed phylogenetic tree revealed remarkable genetic admixture among SEA groups A and popmnlogistic and neuralnet, see LGP&LDD) best fitted the observed data, showing posterior probabilities of 0.87 and 0.70 under the mnlogistic and neuralnet methods, respectively  of the estimates  from the 100 pseudo-observed data points see , which h−6, with 95% HPDI: 1.051 × 10−7–9.213 × 10−6) agrees with estimates from previous studies [Next, the parameter estimates for the observed data are presented in  studies ,52 and t studies ,17.The influence of the LGP on the first expansions of EMHs has been a subject of debate by scientists, particularly because this environmental process changed the available land and the vegetation distribution . In thisWe found that the colonization of SEA was affected by the sea level variation caused by the LGP and by migration events with LDD , which fThe estimated demographic and genetic parameters generally agreed with estimates from previous studies ,14,23,41Next, we discuss the population genetics consequences derived from using temporarily available lands (lands exposed during the LGP), in comparison with permanent lands. We found that the migration rate estimated from temporarily available lands was similar to the migration rate estimated from permanent lands . This fiAnalyzing SEA as an illustrative example, we show here that past environmental fluctuations affected the first expansion of EMHs and could produce genetic consequences that can be detected (to a certain extent) today. Despite in multiple regions of the world the LGP produced a decline in genetic diversity due to range contractions and habitat fragmentation (caused by the increase of ice sheets and deserts), we found the opposite situation in SEA, where the LGP (through the lowering sea level) and LDD favored the expansion and admixture of modern human populations. Consequently, we believe that studies on the evolution of modern humans should consider, as much as possible, past environmental changes.It is known that the range contractions, range shifts and habitat fragmentation derived from the large ice sheets during the last glacial period overall produced a genetic diversity decline in Paleolithic populations. However, the last glacial period, together with long-distance dispersal, could increase the genetic diversity in some regions of the world, such as Southeast Asia. In particular, the temporarily available lands caused by the lowering sea level allowed active human migration and produced large carrying capacities, likely from shellfishing and beachcombing activities. In general, we conclude that environmental factors should be considered to properly understand and model the evolution of our species.


Population differentiation is a pervasive process in nature. At present, evolutionary studies on plant population differentiation address key questions by undertaking joint ecological and genetic approaches and employing a combination of molecular and experimental means. In this special issue, we gathered a collection of papers dealing with various ecological and genetic aspects of population differentiation in plants. In particular, this special issue encompasses eight research articles and two reviews covering a wide array of worldwide environments, plant functional types, genetic and genomic approaches, and common garden experiments to quantify molecular and/or quantitative trait differentiation in plant populations. Overall, this special issue stresses the validity of traditional evolutionary studies focused on plant populations, whilst emphasizing the integration of classical biological disciplines and state-of-the-art molecular techniques into a unique toolkit for evolutionary plant research. Several ecological and genetic processes acting in concert account for population differentiation, which is a pervasive process in nature that may eventually lead to speciation. In this special issue, we gathered a collection of papers dealing with various ecological and genetic aspects of population differentiation in plants covering a wide array of worldwide environments, plant functional types, genetic and genomic approaches, and common garden experiments to quantify molecular and/or quantitative trait differentiation in plant populations. Mutation, anisotropic migration, random genetic drift and natural selection shape among- and within-population patterns of genetic diversity at different spatial and temporal scales, which steadily promote population differentiation, and in the end, may lead to speciation. This well-accepted statement identifies the major processes underlying evolutionary change, which is one of the major contributions of the Modern Synthesis that put together natural selection and Mendelian genetics under the umbrella of population genetics almost a century ago  and organized by the guest editors with the sponsorship of ountries , contribountries , the Ibeountries , encompaSolanum pimpinellifolium (Solanaceae) in South America. This allowed the authors to hypothesize about the origin of the species, which remained controversial. In addition, they identified the major ecological factors accounting for demographic behaviour and estimated the genetic offset, that is, whether the current genetic composition is maladaptive to future environments (Brasilianthus carajensis (Melastomataceae) from Amazonian Savannas. The authors not only characterized the population differentiation of a recently discovered species, but also employed genomic data to outline conservation/management actions to prevent the loss of unique genetic variation and maximize species resilience to future environmental change  in SE Spain. 4 grass Panicum hallii (Poaceae) across its distribution in North America (Mexico and USA) also merging RAD sequencing and species distribution models. The authors identified genetic clusters that followed the main ecoregions of the species across its range, and pinpointed South Texas as a major hotspot of genetic diversity with the co-occurrence of all genetic clusters and admixture between the two Panicum varieties of study.Furthermore, Lupinus angustifolius (Fabaceae) to two ecologically meaningful water availability treatments. The common garden experiment tested the effect of drought treatments on plant traits and allowed the authors to identify cogradient variation in drought-related traits, that is, plasticity to drought was in most cases in the same direction as among-population quantitative genetic differentiation (Geum triflorum (Rosaceae). Betula pendula (Betulaceae) in three common garden experiments across a substantial part of the species’ latitudinal range in Europe . In their study, P. hallii varieties  in a common garden experiment in South Texas (USA). In this case, the authors used an experimental approach as a complement to characterize the divergence between the two Panicum varieties of study, finding important differences between them in morphological features, cold stress tolerance and lifespan . On top of architectural and life-history data, gene expression data can also be used to assess genetic differentiation and local adaptation for samples from field populations, common garden experiments and/or reciprocal transplant experiments , understanding evolutionary processes still requires field studies of natural populations. In fact, differentiation is an all-encompassing process that eventually molds organisms’ phenotypic integration and their genetic make-up. Here, we gathered a collection of papers illustrating the relevance of traditional evolutionary studies focused on plant populations, but also highlighting the integration of classical biological disciplines and state-of-the-art molecular techniques into a unique toolkit for evolutionary plant research.A. thaliana; Based on the papers of this special issue, we envisage two promising future research lines to keep making progress in the discipline. First, it is time to bring together ecological genetics/genomics studies combining among- and within-population variation in genetic diversity and quantitative traits at large geographical scales (see We conclude by stressing the current serious challenges menacing the existence of plant biodiversity due to the unprecedented transformation of the planet at the beginning of the Anthropocene. The continuous differentiation process affecting the evolution of plants is probably experiencing dramatic alterations with unpredictable consequences. We need population differentiation studies, like the ones in this special issue, more than ever to comprehend the causes and consequences of rapid environmental changes on plant populations to foresee—and perhaps mitigate—their effects on plant biodiversity’s fate.


White clover is an important temperate legume forage with high nutrition. In the present study, 448 worldwide accessions were evaluated for the genetic variation and polymorphisms using 22 simple sequence repeat (SSR) markers. All the markers were highly informative, a total of 341 scored bands were amplified, out of which 337 (98.83%) were polymorphic. The PIC values ranged from 0.89 to 0.97 with an average of 0.95. For the AMOVA analysis, 98% of the variance was due to differences within the population and the remaining 2% was due to differences among populations. The white clover accessions were divided into different groups or subgroups based on PCoA, UPGMA, and STRUCTURE analyses. The existence of genetic differentiation between the originally natural and introduced areas according to the PCoA analysis of the global white clover accessions. There was a weak correlation between genetic relationships and geographic distribution according to UPGMA and STRUCTURE analyses. The results of the present study will provide the foundation for future breeding programs, genetic improvement, core germplasm collection establishment for white clover. Trifolium repens L.) is a cool-season, allotetraploid (2n=4x=32) perennial legume species . SSRs primers developed in previous studies  were usePi2, and the Pi is the frequency of the i-th allele (Na), the effective number of alleles (Ne), Nei’s (1973) gene diversity (h), and Shannon’s information index (I) were calculated by GenAlEx 6.5 (The amplification bands were scored for the presence (1) or absence (0) and a binary matrix was formed for SSR markers. The total number of bands (TNB), number of polymorphic bands (NPB) and percentage of polymorphic bands (PPB) were calculated. Polymorphic information content (PIC) was calculated using the formula PIC = 1 − ∑ h allele . The numAlEx 6.5  to evaluhttp://taylor0.biology.ucla.edu/structureHarvester/)  and the analysis of molecular variance (AMOVA) were conducted using GenAlEx 6.5 . The unwvester/) .h) and Shannon’s Information index (I). The h is ranged from 0.198 to 0.345 with an average of 0.280, and the I is ranged from 0.339 to 0.520 with an average of 0.437  were polymorphic . The numof 0.437 .The genetic diversity was analyzed for the natural and introduced groups . The perNa ranged from 1.891 in subgroup Asian from the natural group and 1.979 from subgroup Australia from the introduced group. The Ne varies from 1.419 to 1.449, which was recorded in the Mediterranean and Europe from the natural group. The Nei’s gene diversity values for subgroups ranged from 0.265 (Asian from Introduced) to 0.279 . Correspondingly, the highest I was recorded for subgroup European (0.434) and Australia (0.434), and the lowest for subgroup Asian from Introduced (0.413).The genetic diversity index also is calculated in the subgroups . The NPLP < 0.05). Of the total accessions, 98% of the variance was due to differences among the accessions within the groups and the remaining 2% was due to differences between the groups. Of the natural group, 97% of the variance was due to differences among the accessions within the subgroups and the remaining 3% was due to differences among the subgroups. It showed the same result in the AMOVA analysis of the introduced group, 97% differences showed among the accessions and 3% showed among the subgroups.Analysis of molecular variance (AMOVA) was implemented to evaluate variance components among groups and subgroups , which sK) values, the number of optimum groups was four  values, the number of optimum groups was three  values, the number of optimum groups was two  was slightly more evident than that of the introduced population . The high-level genetic diversity partly because the two diploid progenitors of white clover come from very different environments   which was due to the European region was the origin of white clover. The Australian subpopulation had a higher level of genetic diversity  than the other two introduced subpopulations. This suggested the Australia accessions may have more diverse sources, and multiple introductions from different regions resulted in high genetic diversity in Australia.White clover has a high level of genetic heterogeneity within natural and synthetic populations . In thisabitats) , and parGenetic variation between the populations (97%–98%) was higher than that within populations (2%–3%) in the present study. The result is consistent with the previous studies of white clover based on RAPD (73% within population) , AFLP anWhite clover is a successful allotetraploid example of allopolyploidy-facilitated niche expansion, which has facilitated global radiation of the previously confined specialist progenitor genomes . It is cEruca sativa (Vicia faba (Camellia sinensis (In contrast, clear population differentiation with the geographic origin was absent in UPGMA and STRUCTURE analyses, in which no group exclusively included the accessions from a single region. The results were consistent with a sativa , Vicia fcia faba  and Camesinensis . It may sinensis . In the sinensis , human ssinensis , differesinensis  and randsinensis . The givsinensis .White clover spread by natural means to the largest part of the Asiatic mainland . It was The white clover accessions in the present study were divided into different groups or subgroups based on PCoA, UPGMA and STRUCTURE analyses. It could be attributed to the different statistical principles . PCoA caIn conclusion, the findings of the study confirmed that global white clover accessions contained a high level of genetic diversity. And the weak correlation between genetic relationships and geographic distribution of white clover accessions. Our result will provide molecular evidence for breeding improvement, germplasm resources conservation and core germplasm collection establishment for white clover.10.7717/peerj.11325/supp-1Table S1Click here for additional data file.10.7717/peerj.11325/supp-2Table S2Click here for additional data file.10.7717/peerj.11325/supp-3Table S3Click here for additional data file.10.7717/peerj.11325/supp-4Figure S1Click here for additional data file.10.7717/peerj.11325/supp-5Figure S2Click here for additional data file.10.7717/peerj.11325/supp-6Figure S3Click here for additional data file.


The SNc, together with the ventral tegmental area (VTA), play a key role modulating motor responses through the basal ganglia. In contrast to the large amount of existing literature addressing the mammalian dopaminergic system, comparatively little is known in other vertebrate groups. However, in the last several years, numerous studies have been carried out in basal vertebrates, allowing a better understanding of the evolution of the dopaminergic system, especially the SNc/VTA. We provide an overview of existing research in basal vertebrates, mainly focusing on lampreys, belonging to the oldest group of extant vertebrates. The lamprey dopaminergic system and its role in modulating motor responses have been characterized in significant detail, both anatomically and functionally, providing the basis for understanding the evolution of the SNc/VTA in vertebrates. When considered alongside results from other early vertebrates, data in lampreys show that the key role of the SNc/VTA dopaminergic neurons modulating motor responses through the basal ganglia was already well developed early in vertebrate evolution.Dopamine is likely the most studied modulatory neurotransmitter, in great part due to characteristic motor deficits in Parkinson’s disease that arise after the degeneration of the dopaminergic neurons in the substantia nigra  This is reflected in the severe motor deficits that appear when this dopaminergic system is compromised, as in Parkinson’s disease. Studies in lampreys and other vertebrates have shown that this system has an ancient origin, and is largely conserved through vertebrate evolution, which is reflected in the fact that motor deficits such as those seen in Parkinson’s disease appear in all vertebrate groups when SNc/VTA neurons are depleted lication c 23]..1-like f1-like family has been found in these animals [2-like family have been reported [Lampreys are thought to have diverged after the first WGD , but som animals ,29, wherreported ,22,30.1-like family shows a high degree of similarity to the D1A subtype, a broad expression pattern in the brain [1A receptor. Within the D2-like family, one receptor shows clear phylogenetic similarities with the D2 subtype, with its brain-wide expression pattern and modulatory role in the striatum also supporting this homology [2-like family receptor basal to the D4 sequences of other vertebrates, and its restricted expression pattern also supports this identity [1A and D2 receptor sequences, their conserved expression areas, and that they are present in all vertebrate groups, it seems likely that these two subtypes carry out the more conserved functions, such as mediating the excitability changes in the striatum (see below). Consequently, they likely represent the ancestral D1-like and D2-like receptors from which the other subtypes arose through genomic duplications.The receptor belonging to the Dhe brain , and itshe brain  and optihe brain , which ahomology ,28,30. Pidentity . Given tAlthough the basal ganglia are not the focus of this review, it is impossible to discuss the role of dopamine modulating movement without the basal ganglia framework. This group of subcortical nuclei plays a key role in selecting behaviors, motor control, and learning ,31, and pars reticulata (SNr) and the globus pallidus interna (GPi) [1 dopamine receptor, and dopamine increases the excitability of these neurons that directly target the output nuclei (SNr and GPi) through the direct pathway [2 receptor, and its excitability is decreased by dopamine [externa (GPe), which in turn targets the excitatory subthalamic nucleus (STN) [In lampreys, as in mammals, the output of the basal ganglia is represented by the substantia nigra na (GPi) ,33,34   for a reus (STN) ,40. The The high degree of similarities between the lamprey’s forebrain and that of mammals is not restricted to the basal ganglia. Recently, a pallial region has been identified as a homologue of the dorsal pallium and thus the precursor of the mammalian neocortex ,42,43,441 or D2 receptors form separate populations in the lamprey that target the output nuclei, either directly or indirectly , described in the lamprey, salamander, and rat ,83,84,85Interestingly, the same dopaminergic neurons have been shown to send axonal branches to both the striatum and tectum/SC, both in lampreys and mammals, and thus both structures become modulated in parallel ,41,89. DAltogether, one may conclude that the SNc/VTA modulation of motor responses is more refined than previously thought. Moreover, given the large degree of conservation of SNc/VTA neurons, as well as the presence of their direct pathways to motor centers across different vertebrates, it seems logical that direct modulation may have a role in motor deficits observed in Parkinson’s disease ,85. The Although the focus of studies on the SNc/VTA has been dopamine-related mechanisms, these nuclei are formed by heterogeneous populations that include several neurotransmitter phenotypes. Single-cell RNA sequencing has revealed that mesodiencephalic neurons can be divided into seven subgroups, five of which express dopaminergic markers, and two express glutamate and GABAergic markers . GABA, gSurprisingly, dopamine neurons in the lamprey have been found to differentially co-release these two neurotransmitters. As previously described, the same dopaminergic neurons have been found to target the striatum and tectum simultaneously ,41. HoweAs discussed above, the available data strongly support that the diencephalic dopaminergic population of the lamprey is the homologue of the mammalian SNc/VTA. However, this homology has been somewhat controversial, as the SNc/VTA has been classically considered mesencephalic. Nevertheless, later studies in mammals have shown that SNc and VTA neurons originate in diencephalic, mesencephalic, and isthmic domains ,112,113.A segmental approach therefore clarifies the diencephalic position of the lamprey population, while the multimeric nature of the SNc/VTA explains how, from an ancestral dopaminergic population that appeared in early vertebrates, several scenarios with small differences have evolved. The diencephalic portion of the SNc/VTA innervating the striatum is present in all vertebrate groups, indicating that it was the earliest to evolve and remains largely conserved. The presence of a mesencephalic population in elasmobranchs indicates that this component appeared early after the cyclostome–gnathostome split, and the lack of mesencephalic dopaminergic neurons in teleosts suggests that this component was lost secondarily in this lineage . LungfisIn mammals, the striatum is divided into a ventral striatum, also known as the nucleus accumbens, and a dorsal striatum. The nucleus accumbens receives inputs from limbic areas and the hippocampus, and its dopaminergic innervation arises in the VTA, via the so-called mesolimbic pathway . The dorWe have so far discussed the nigrostriatal pathway and referred to the lamprey dopaminergic population as the homologue to the SNc/VTA. The lack of segregation between the dorsal and ventral striatum in these animals, as well as the absence of proven mesocortical and mesolimbic pathways, constrains a clear separation between SNc and VTA. In terms of projections, the lamprey dopaminergic population in the posterior tuberculum could therefore be considered a homologue of the SNc. However, apart from the three pathways mentioned above, the overall SNc and VTA connectome is quite similar in mammals , and furWe can conclude that the SNc/VTA, together with its key role in modulating the basal ganglia, originated early in the vertebrate phylogenetic lineage and has retained a high degree of conservation. It is consequently clear that the appearance of the basal ganglia and their modulation from the SNc/VTA signified a big leap in evolution, providing new advantageous tools for survival. While lampreys command a limited behavioral repertoire compared to mammals, primarily limited to locomotion, steering, foraging, or control of body position, they nevertheless need to compete in a complex aquatic environment. Thus, it is not surprising that the basal ganglia and dopaminergic system were well-developed already in basal vertebrates, providing the basic machinery to signal salient events and evaluate actions, ultimately contributing to behavioral selection, motor control, and learning.


Knowledge of the genetic structure and diversity of germplasm collections is crucial for sustainable genetic improvement through hybridization programs and rapid adaptation to changing breeding objectives. The objective of this study was to determine the genetic diversity and population structure of 281 International Institute of Tropical Agriculture (IITA) soybean accessions using diversity array technology (DArT) and single nucleotide polymorphism (SNP) markers for the efficient utilization of these accessions. From the results, the SNP and DArT markers were well distributed across the 20 soybean chromosomes. The cluster and principal component analyses revealed the genetic diversity among the 281 accessions by grouping them into two stratifications, a grouping that was also evident from the population structure analysis, which divided the 281 accessions into two distinct groups. The analysis of molecular variance revealed that 97% and 98% of the genetic variances using SNP and DArT markers, respectively, were within the population. Genetic diversity indices such as Shannon’s diversity index, diversity and unbiased diversity revealed the diversity among the different populations of the soybean accessions. The SNP and DArT markers used provided similar information on the structure, diversity and polymorphism of the accessions, which indicates the applicability of the DArT marker in genetic diversity studies. Our study provides information about the genetic structure and diversity of the IITA soybean accessions that will allow for the efficient utilization of these accessions in soybean improvement programs, especially in Africa. Glycine max L. Merrill) is an important legume crop, providing approximately 68% of the world’s supply of protein-rich meals and serving as an excellent source of vegetable oil and renewable fuel. The average worldwide yields of soybean increased from 2183 kg ha−1 in 1994 to 2769 kg ha−1 in 2019, with an annual increase of 26 kg ha−1 [Soybean  markers are more reliable in estimating crop diversities . RecentlRecently, soybean germplasms from Africa have been reported to have a narrow genetic base, based on phenotypic evidence . HoweverA total of 19,505 SNPs and 16,116 DArT markers were generated for the 281 soybean accessions. After the first stage of filtering, which excluded unmapped markers, 18,270 SNPs and 14,051 DArT markers were obtained. The second stage filtering returned 7244 SNPs and 4443 DArT markers. The SNPs were distributed across the 20 soybean chromosomes a, with GA cluster analysis of the SNP and DArT markers revealed two groups, with each group having sub-groups A, compriFst) was 0.23 and 0.40, while the expected heterozygosity was 0.45 and 0.33 for groups Q1 and Q2, respectively, (The model-based (Bayesian) analysis of the SNP markers estimated two groups (Q1 and Q2) A, corresctively,  indicatiThe model-based analysis of the DArT markers also estimated two groups (Q1 and Q2) C, which p < 0.01), suggesting a high level of difference, and the number of migrants (Nm) was 18.02 (p < 0.01), while the Nm was 19.54  based on SNP markers revealed that 97.00% of the genetic variance was within the population and 3.00% was among populations a. The Phas 18.02 . The DAras 18.02 b. The Phas 19.54 .Based on SNP markers, Shannon’s diversity index was used to quantify the genetic diversity of the 281 soybean accessions. The Shannon’s diversity index ranged from 0.13 in population 4 (Chile) to 0.52 in populations 5 and 13  . The lowBased on the DArT markers, Shannon’s diversity index ranged from 0.09 in population 11 (Thailand) to 0.64 in populations 10 and 13 , followed by 0.62 in population 5 (China) . The lowStudies of genetic diversity of the IITA soybean accessions are important for conservation and utilization. These studies will assist soybean breeders in the development of suitable procedures in attaining wide diversity in soybean breeding programs in Africa . AnalyseIn the present study, 19,505 SNPs and 16,116 DArT markers were analyzed and filtered to remove redundant markers that may not be functional. After filtering, 7244 polymorphic SNPs and 4443 DArT SNPs were retained and distributed across the 20 chromosomes of the soybean genome. Redundant markers are mostly not useful for specific genetic analyses, as reported previously ,26. FurtFrom the SNP marker distribution, chromosome 12 had the least number of polymorphic SNP and DArT markers. Fewer polymorphic markers on chromosomes have been reported to be an indication of lower genetic diversity ,28; thusFst values of 0.23 (SNP Q1), 0.40 (SNP Q2) and 0.32 (DArT Q1) showed the existence of genetic diversity within these sub-populations, and parental materials can be selected to develop variable populations for breeding programs. Wright [Fst value of 0.25 is an indication of good differentiation between population groups, from 0.15 to 0.25 is an indication of moderate differentiation and the differentiation is negligible if the Fst value is 0.05 or less. The similar grouping of some accessions from different countries indicated their close relatedness through common ancestry but may also be attributed to improper documentation during germplasm exchange; thus, the same accession may be recorded with different origins [The cluster and PC analyses identified two groups that were similar to the model-based population structure analysis for the 281 accessions. The highest level of structure observed (k = 2) was similar to the structure reported by . This si. Wright , suggest origins . HybridiCamelina sativa [The AMOVA revealed that 97% and 98% of all the genetic variances using SNP and DArT markers were within the population, and only 3% were attributed to population structure (variance among populations). Previous studies on soybeans  and othea sativa , wheat [a sativa , Banglada sativa  and ricea sativa  attributp = 0.001, for SNP and DArT markers, respectively). These significant differences may be as a result of the low genetic exchange and gene flow which was evident from the low value of the number of migrants. A low value for the number of migrants has been described as evidence of significant differences among populations [The PhiPT value from the AMOVA showed that there were significant differences between the groups .Genotyping of the soybean accessions was performed using the DArT-based GBS platform of SEQART AFRICA. Briefly, genomic DNA was extracted from young, fresh soybean leaves using the NucleoMag Plant genomic DNA extraction kit . The quality and quantity of DNA in each sample were checked via 0.8% escribed . Samplesescribed  and the The SNP characteristics  were generated by the DArT GBS platform. The filtering of the SNPs was performed in two stages; the first stage filtering removed all SNPs that were unmapped to any of the 20 soybean chromosomes, while the second stage filtering removed the redundant markers. The redundant markers were markers with missing rates of more than 10%, MAF below 5% and PIC < 10%. The genome summary plugin in the TASSEL v.5.2.37 software  was usedCluster and principal component analyses of the 281 tropical soybean accessions were performed in TASSEL v.5.2.37 software  using thThe structure of the populations was assessed using a model-based (Bayesian) analysis in STRUCTURE v2.3.4 . The k-vAnalysis of molecular variance (AMOVA) was conducted using the results of population structures to determine the variation within and among the populations using GenAlEx 6.5 . GeneticThe allelic pattern analysis across the population was performed using GenAlEx 6.5 . The ShaThe use of different marker types in evaluating the genetic diversity of crops provides opportunities for a comparison of the marker types and gives a more reliable estimate of the diversity present. In the present study, the estimated genetic diversities were comparable between SNP and DArT markers. The few inconsistencies observed may be a result of the characteristics of the markers. The SNP and DArT markers provided similar evidence regarding the genetic diversity and population structure of the accessions. The DArT GBS platform produced high-density markers that were distributed across the 20 chromosomes of the soybean genome. Both markers revealed two population structures for the 281 soybean accessions. Greater genetic diversity existed within the population than among populations; thus, these populations could be useful in breeding programs aimed at recombining favourable alleles within adapted gene pools for the improvement of grain yield, stress resistance and seed quality traits. Additionally, the introgression of novel genes from diverse sources using advanced genomic techniques such as CRISPR/Cas9 technology, RNA interference (RNAi) and overexpression could be explored to develop more diverse populations. Our study provides an understanding of the genetic structure and diversity of the IITA soybean accessions, which will allow for the efficient utilization of these accessions in soybean improvement programs, especially in Africa.Our next objectives are to identify promising genotypes and to conduct genome-wide association studies for seed quality traits, yield and biotic and abiotic stresses.


Papaver somniferum) is one of the world’s oldest medicinal plants and a versatile model system to study secondary metabolism. However, our knowledge of its genetic diversity is limited, restricting utilization of the available germplasm for research and crop improvement. We used genotyping-by-sequencing to investigate the extent of genetic diversity and population structure in a collection of poppy germplasm consisting of 91 accessions originating in 30 countries of Europe, North Africa, America, and Asia. We identified five genetically distinct subpopulations using discriminate analysis of principal components and STRUCTURE analysis. Most accessions obtained from the same country were grouped together within subpopulations, likely a consequence of the restriction on movement of poppy germplasm. Alkaloid profiles of accessions were highly diverse, with morphine being dominant. Phylogenetic analysis identified genetic groups that were largely consistent with the subpopulations detected and that could be differentiated broadly based on traits such as number of branches and seed weight. These accessions and the associated genotypic data are valuable resources for further genetic diversity analysis, which could include definition of poppy core sets to facilitate genebank management and use of the diversity for genetic improvement of this valuable crop.Opium poppy ( Papaver somniferum L.) is one of the oldest cultivated plant species. Archaeological evidence shows that poppy has been cultivated and used for thousands of years, dating back to the earliest Neolithic ages4. However, its origin and domestication history has remained unclear until recently. Domestication traits are poorly defined, making it difficult to distinguish domesticated and wild forms especially in archaeological records5. Several lines of evidence, based mainly on archaeological data and geographical distribution of cultivated and wild species, suggest the Mediterranean as the centre of poppy origin and domestication6. Changes in capsule and seed sizes and capsule indehiscence, which is the retention of seed in the capsules, are believed to be amongst poppy domestication-related traits7.Opium poppy  including morphine, codeine, thebaine, papaverine and noscapine for the pharmaceutical industry and for the clandestine production of heroin. Poppy seeds are also used in the food industry for baking and extraction of edible oil, whilst the plant is grown for ornamental purposes in some countries due to its attractive flowers11. Poppy cultivars used in food applications are required to contain no or negligible amounts of alkaloids12. The availability of commercial cultivars with specific alkaloid profiles is also vital to meet the needs of the pharmaceutical industry and subsequent consumers12. Obtaining codeine and thebaine from morphine-free plants can also contribute to preventing illicit production of the morphine-derived heroin.Currently, poppy is widely cultivated as both a licit and illicit crop in Asia, Europe, Oceania and South America17. Germplasm collections of varying sizes exist in some of these countries19. Additionally, a substantial number of poppy genetic resources are currently maintained as seeds in global genebanks. The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) genebank in Germany has over 1100 accessions of poppy that were collected worldwide20. A collection of similar size is maintained at the Institute of Protection of Biodiversity and Biological Safety in the Slovak University of Agriculture21. Germplasm collections provide the genetic and phenotypic diversity used in crop breeding and development. They are also vital resources for research aimed at dissecting the genetic and molecular basis of essential plant processes including secondary/specialized metabolism. However, these resources are underexploited in poppy partly because the genetic diversity of the available germplasm has not been studied in detail. Additionally, the legal opium poppy industry is strictly regulated under international law, restricting the movement of germplasm between countries. Most available reports of opium poppy genetic diversity are based on studies that either assessed small collections of germplasm from a single country or used a limited number of classical DNA markers such as amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNAs (RAPDs) and simple sequence repeat markers (SSRs)23.Considerable poppy genetic diversity has been reported in several countries including India, Turkey, Czech Republic, and Australia25. Recent advances in next generation sequencing have greatly reduced the cost of genome sequencing, allowing the generation of very large numbers of molecular markers. However, reduced representation sequencing (RRS) is still widely used when analyzing large number of samples in species with large genomes, such as opium poppy. Genotyping-by-sequencing (GBS) is a low-cost and fast RRS method for SNP discovery and mapping, which reduces the complexity of genomes by generating smaller fragments via restriction digestion26. A draft of the P. somniferum genome that captured 94.8% of the estimated genome size, with 81.6% of the sequences assigned to individual chromosomes, has been sequenced recently28. Given the genome is an estimated 2.8 Gb and comprised of 70% repetitive elements, RRS methods are an appropriate, cost-effective method for poppy diversity analyses. Such studies could facilitate the development of poppy core sets, enabling the mapping and isolation of genes or genomic regions associated with traits of interest.Molecular markers have been instrumental for the study of genetic diversity and population structure of germplasm collections. Such studies generate information key to both germplasm conservation and use of the resources for genetic improvement of crops. As the most abundant types of sequence variation in plant genomes, single nucleotide polymorphisms (SNPs) are suitable for several applications that require high-density and genome-wide markers including genetic diversity and population structure analyses, QTL mapping and map-based cloningPapaver species, P. somniferum L. subsp. somniferum (P. somniferum hereafter) and P. somniferum L. subsp. setigerum (DC.) Arcang. (P. setigerum hereafter). P. setigerum is commonly thought to be the direct ancestor of P. somniferum and historically has also been used for alkaloid production29. The accessions originated from diverse geographic regions that encompassed 30 countries of Europe, North Africa, America, and Asia. We provide a genome-wide assessment of the genetic diversity and population structure of the accessions using GBS. The data generated here provides a resource that will allow detailed analysis of poppy germplasm and the definition of poppy core set for effective management of poppy genetic resources. It may also facilitate the genetic improvement of opium poppy through the generation of mapping populations, the identification of useful traits and development molecular markers for marker-assisted selection.In this work, we applied GBS analysis to characterize 91 poppy accessions obtained from the IPK genebank in Germany. The accessions consisted of two 30. Although some RE combinations are often used in plants, the optimal combinations need to be determined for the genome of each species31. To select the optimal enzyme combination for opium poppy, we prepared eight double digested libraries from a pool of 3 representative samples using the enzyme combinations PstI/MspI, PstI/MseI, PstI/NlaIII, PstI/HpyCH4IV, EcoRI/MspI, EcoRI/MseI, EcoRI/NlaIII and EcoRI/HpyCH4IV. We then compared the absence of visible repeat regions within the size selection area (280–375 bp) and level of amplifications to select EcoRI/NlaIII as the optimal combination for opium poppy GBS library preparation , and the two-enzyme GBS protocol uses a combination of a rare- and common-cutting REsion Fig. .EcoRI/NlaIII-based GBS libraries for 91 accessions was sequenced to generate a total of 103,802,122 raw 150-bp single end reads  but included 3 P. setigerum accessions (Table 32. Four clusters plus a single accession (PAP 400) far from all others were identified  with chromosomes smaller in size compared with the diploid (2n = 22) P. somniferum35. PAP 400 had a slightly smaller genome size than the diploid P. somniferum . Five clusters (hereafter termed subpopulations) were inferred at the lowest Bayesian information criterion (BIC) score  on the DAPC plot illustrates the extensive genetic difference between P. somniferum and P. setigerum.Understanding the genetic structure of populations is useful for germplasm conservation and plant breeding. To infer population structure, we analysed the 90 ore Fig. a. To undore Fig. b. Subpop37. Based on ΔK values, the most optimum K value detected was four to eight  that ranged from 0.235 to 0.627, suggesting either low levels of allele sharing or differences in allele frequencies between subpopulations , calculated from the 131,039 SNP dataset. This further confirmed the genetic distinctiveness of P. setigerum. The percentage of polymorphic loci varied from 3.93 (SP3) to 25.05% (SP5). The level of observed heterozygosity (HO) was highest for SP1 (0.166) and lowest for SP2 (0.009). HO was lower than the expected heterozygosity (HE) for all subpopulations except SP1 (P. setigerum), indicating high levels of inbreeding in P. somniferum. This finding was supported by the higher inbreeding coefficients (FIS) for the P. somniferum subpopulations (0.061 to 0.377). FIS was negative for SP1, possibly a consequence of an excess of the observed heterozygotes. The highest nucleotide diversity (π) was observed in SP1 (0.219) and the lowest in SP3 (0.040). The genetic variations were both due to differences between (41.6%) and within (44.8%) subpopulations, determined using analysis of molecular variance  and minor (papaverine and opripavine) alkaloids in dry capsules of the 90 accessions that reached full maturity  and 1.416 (PAP 784) g/100 g DW. Codeine and thebaine contents ranged from 0.002 (PAP 795) to 0.342 (PAP 151) and 0.000 to 0.336 (PAP 719) g/100 g DW, respectively , ranging from 32.3 to 96%  Fig. b. CodeinP. setigerum accessions were clearly separated from all others, while accessions of subpopulation 2 had the most diverse and distinct alkaloid composition among the P. somniferum accessions. Accessions with the highest codeine proportions, including PAP 150 (53.9%), PAP 151 (52.0%), PAP 152 (50.4%), PAP 200 (42.2%), PAP 739 (35.6%), PAP 149 (33.8%) and PAP 354 (28.7%), were clearly separate from the others  and accessions in Groups 2 to 5, representing accessions of the DAPC SP1 to SP4, were generally highly branching and produced lighter seeds   Fig. c. Seed sgy Figs. , S5. TheP. somniferum and P. setigerum. This optimized protocol provides a rapid and cost-effective method for genotyping of unlimited number of accessions.The poppy germplasm currently available in several genebanks is a rich potential source of useful alleles. Accurate genotyping of this germplasm is crucial to dissect the available genetic diversity. This is an important step for germplasm conservation and can also facilitate the identification and deployment of promising genotypes/alleles for genetic improvement of the crop. With this in mind, we optimized a GBS protocol for poppy and applied GBS-based analysis of SNP markers for the assessment of genetic relationships in diverse poppy accessions from P. somniferum genome is a draft with only 81.6% of the sequences assigned to individual chromosomes27. However, we were able to identify significantly more markers when using it as a reference for both P. somniferum and P. setigerum SNP calling than when using a reference-free GBS analysis method  and P. somniferum (diploid) was a potential limitation of our analyses, but GBS has previously been implemented on species with different ploidy levels51. However, accurate identification of SNPs from polyploids is still a challenge that requires improvement of existing software packages or development of new ones52.The current od Table . This rens Table . The P. Whole genome sequencing (WGS) is an ideal tool to capture complete information about genome variability including SNP variants. However, the RRS approaches such as GBS are practical and cost-effective methods for simultaneous SNP discovery and genotyping of species with large and complex genomes or when analysing large number of samples. Furthermore, for species like opium poppy where the available germplasm is yet to be fully characterized, GBS can be applied to characterize a large collection from which a core set could be selected to represent the genetic diversity of the entire collection. WGS can then be applied to the accessions in the core collection for further, deep analysis. This is a critical step to efficiently manage the genebank collection and promote its use. The accessions characterized in our study and the associated GBS data can significantly contribute towards this goal.Papaver species, particularly that of the cultivated P. somniferum and the wild species P. setigerum, is still debated46. Some treat the two as separate species, while others consider P. setigerum as a subspecies of P. somniferum based on morphology and alkaloid profiles46. Our findings confirmed the genetic separation of the accessions from the two named groups  and P. setigerum tetraploid (2n = 44) . Both natural and induced mutants with altered alkaloid profiles have been instrumental to elucidate the molecular mechanisms underlying differences in alkaloid contents in poppy63. Consequently, future genetic diversity studies need to take into consideration intra-accession variability. Based on our preliminary observations, we also suggest that traits such as seed weight, capsule size and dehiscence, and branching be considered in studies of the domestication and phylogenetic relationships of opium poppy and related species. Data generated from such studies would also enable the development a poppy core set, which is an important step towards the selection of suitable parents and development of mapping populations or genetic panels for elucidating the genetic architecture of traits of agronomic and pharmaceutical importance.Our study demonstrates the utility of GBS for genetic analyses in opium poppy. Many hundreds of poppy germplasm accessions are available from genebanks worldwide. These are an immense potential resource if fully exploited. Application of GBS to the entire poppy germplasm collection, complemented by the currently available draft reference genome sequence of poppy, could drive further studies to unravel the extent of genetic diversity in the species. Although poppy is largely self-pollinating, a considerable degree of outcrossing has been reportedPapaver accessions from diverse geographical origins were obtained from the global poppy germplasm collection maintained at Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Genbank in Germany  and P. setigerum (3 accessions)  Fig. . Seeds whttps://www.rapidtables.com/web/color/RGB_Color.html). The corresponding approximate colours were determined from Hex colour codes using HTML CSS Color Picker (https://www.htmlcsscolor.com/hex/).At full plant maturity data was recorded on number of branches (main stem plus side branches), 1000-seed weight, seed colour and capsule and seed characteristics. Images of capsules were taken under normal room light using a SM-G950U1 digital camera. The seed images were captured using a Leica M205 FA stereomicroscope with a digital camera Leica DFC 420 and Leica Application Suite Software  with 10% light and 50% zoom in condition. To measure seed weight, we first removed debris then counted 1000 clean seeds using a Contador seed counter fitted with feed container no. 3 for fine seeds . To determine seed colour, images of the seeds were taken using a Cannon EOS-600D camera together with a ColorChecker passport (X-rite) for camera calibration and colour correction. The original Red, Green, Blue (RGB) colour channels of seed digital images were calibrated in Lightroom Classic CC (Adobe Creative Cloud). The RGB values were then converted to Hex colour codes using RGB Color code chart  buffer (pH 8.0) containing 1% polyvinylpyrrolidone, 0.9% L-ascorbic acid, and 2% 2-mercaptoethanol to remove polysaccharides and phenolic compounds. DNA quality was checked by agarose gel electrophoresis and quantified with a NanoDrop spectrophotometer ND-1000 version . For GBS library construction, the double digest RAD-seq (ddRAD) based library preparation protocol was used65. The protocol included the following steps: DNA digestion with two restriction enzymes, ligation of barcoded adapters compatible with restriction sites overhang, size selection of pooled digested-ligated fragments using Blue Pippin, and amplification of library via PCR using indexed primers. For protocol optimization, eight double digested libraries were prepared from a pool of 3 representative samples  using the restriction enzyme combinations PstI/MspI, PstI/MseI, PstI/NlaIII, PstI/HpyCH4IV, EcoRI/MspI, EcoRI/MseI, EcoRI/NlaIII and EcoRI/HpyCH4IV. The library generated using EcoRI and NlaIII was sequenced on the Illumina NextSeq500 platform  with the standard protocol for single-end reads in 150-cycle mid-output mode at the Australian Genome Research Facility .For DNA extraction, ~ 50–100 mg fresh young leaves were harvested in liquid nitrogen and ground to a fine powder using a TissueLyser II system (Qiagen). Genomic DNA was extracted using the CTAB (cetyl trimethylammonium bromide) method66. After trimming barcode sequences, all trimmed reads (150 bp) were checked for quality, and low-quality reads  and “no RadTag” reads were removed. The filtered reads were aligned to the draft poppy genome sequence retrieved from NCBI (GCA_003573695.1_ASM357369v1) using BWA v0.7.17 and SAMtools v1.968. SNP calling was performed using the refmap.pl pipeline of STACKS v2.41. All the 91 accessions were treated as a single population. SNP loci that were mapped with reads from less than 90% of the accessions sequenced (--min-samples-per -pop 0.9) were excluded from further analysis. The same parameter was applied for the non-reference-based SNP calling using denovo.pl pipeline of STACKS. The density and distribution of the filtered SNPs across poppy chromosomes were determined using BEDTools v2.26.0 and SnpEff v4.1l70. To visualize SNP density, the “MVP.report” function with 1 Mb non-overlapping window from rMVP package was performed in R studio73.Raw sequence data were demultiplexed and sorted using the “process_radtags” function in STACKS v2.41 with the default parameters: “--inline-index” for barcode option and “--renz_1 EcoRI --renz_2 NlaIII” for enzymes option32. Second, for elucidation of the genetic structure and identification of the optimum number of subgroups, we removed the outlier PAP 400 and applied the Bayesian information criterion (BIC) analysis to the remaining 90 accessions. BIC analysis as a nonparametric method was performed with the adegenet package v2.1.3 in R studio based on 131,039 SNP dataset75. The best number of subgroups/subpopulations was determined as the K-means corresponding to the lowest BIC score using the “find.clusters” function. To determine the relationship between the subpopulations, we carried out discriminant analysis of principal components (DAPC). A cross validation function “Xval.dapc” was used to determine the optimal number of PCs to be retained.The population structure of the accessions was assessed using four methods. First, the accessions were clustered using identity-by-state (IBS) to determine the relationship between accessions based on the proportion of shared alleles between pairs of individuals in PLINK v1.0737. With the assumption that SNPs at the same RAD locus are linked, --write_single_snp flag (in STACKS) was applied to ensure that only one SNP per RAD locus was used for STRUCTURE analysis. To determine the most likely number of subpopulations (clusters), four independent runs with 500,000 iterations and a 150,000-step burn-in period were performed for each K from 1 to 10. The output was obtained by structureHarvester v0.6.93 using the maximum estimated log-likelihood [log(P(X|K)] model and the highest ΔK in Evanno method77. After determining the most probable K values, ten runs of 500,000 iterations followed by a 150,000 step burn-in were performed using STRUCTURE for each K. Additionally, for each optimal K, CLUMPP was used to generate individual and population Q matrices from the membership coefficient matrices of the ten replicates obtained from STRUCTURE78. Bar plots were generated using DISTRUCT software79. Forth, principal component analysis (PCA) was conducted using PLINK v1.07 and plotted using ggplot2 package in R studio80.Third, to investigate the population structure in detail, the admixture within the accessions was determined using Bayesian clustering based on a Bayesian Markov Chain Monte Carlo model (MCMC) implemented in STRUCTURE v2.3.4HO and HE), nucleotide diversity (π) and inbreeding coefficient (FIS) were calculated for the five subpopulations using the “populations” function in STACKS v2.4166. The genetic differentiation between the subpopulations was calculated based on pairwise population differentiation (FST) values from GENODIVE v3.0581. Significance levels (α = 0.05) of the FST values were determined by running 999 permutations and assessing this against a Bonferroni-adjusted P-value to account for multiple testing. The correlation matrix was visualized using the corrplot package in R studio82. To determine the distribution of genetic variation, analysis of molecular variance (AMOVA) was performed using GENODIVE v3.0581. Significance level was tested using 999 permutations.Common measures of genetic diversity including private allele number (AP), percentage of polymorphic loci (%Poly), observed and expected heterozygosity  from the “populations” function in STACKS v2.41 to the SplitTree4 software40. The split network was created with the “uncorrectedP” method, which ignores ambiguous sites, and visualized using “Neightbornet” network. One thousand bootstrap values were used.To explore the phylogenetic relationships between 38. Only accessions that produced capsules samples sufficient for analysis were included. Alkaloids were also analysed in four accessions that had failed to germinate during the first trial and subsequently lacked GBS data , which has genome size of ~ 900 Mb84, were simultaneously isolated from leaves, stained and analysed with poppy nuclei as an internal reference standard. Nuclei were isolated with the Galbraith lysis buffer using a modified protocol from Gutzat and Scheid as follows: (1) Chopping fresh young leaf tissue (0.5 g) using double-sided razor blades in 2 mL ice-cold lysis buffer, (2) filtering the homogenate through a 40 µm nylon mesh, (3) adding 2.5 µL of RNase (10 mg/mL) to 500 µL filtered homogenate, then incubating on ice for 10 min, (4) centrifugation at 400 g for 3 min, removing the supernatant and resuspending the pellet gently in 1 mL lysis buffer, then incubating on ice for 15 min, and (5) filtering the homogenate again through a 40 µm nylon mesh86. For nuclei staining, 25 µL PI (1 mg/ml) was added into 500 µL nuclei solution to get a final concentration of 50 µg/mL. Samples were screened on a CytoFLEX S flow cytometer . The PerCP-A fluorescence intensity of G1 and G2 phase cells of internal standard and samples was used to estimate genome size and ploidy level of the samples.The genome size and ploidy level of selected accessions was estimated using flow cytometry analysis of propidium iodide (PI)-stained nuclei isolated from poppy leaves. Nuclei of tomato (Seeds of all poppy accessions were obtained from a public seedbank and transferred in accordance with international legislation. All experimental research in this study was conducted in compliance with the relevant institutional, national, and international guidelines and legislation.Supplementary Information.


The distribution of genetic diversity across a species distribution range is rarely homogeneous, as the genetic structure among populations is related to the degree of isolation among them, such as isolation by distance, isolation by barrier, and isolation by environment.Jenynsia lineata is a small viviparous fish that inhabits a wide range of habitats in South America. To decipher the isolation processes that drive population structuring in J. lineata, we analyzed 221 sequences of the mitochondrial cytochrome c oxidase I gene (COI), from 19 localities. Then, we examined the influence of the three most common types of isolation in order to explain the genetic variation found in this species.J. lineata were altitude (isolation by environment) and type of basin (isolation by barrier).Our results revealed a marked structuration, with three groups: (a) La Plata/Desaguadero Rivers , (b) Central Argentina, and (c) Northern Argentina. A distance‐based redundancy analysis, including the explanatory variables geographical distances, altitude, latitude, and basin, was able to explain up to 65% of the genetic structure. A variance partitioning analysis showed that the two most important variables underlying the structuration in Our results show that in this species, the processes of population diversification are complex and are not limited to a single mechanism. The processes that play a prominent role in this study could explain the high rate of diversity that characterizes freshwater fish species. And these processes in turn are the basis for possible speciation events. The processes of population diversification are complex and are not limited to a single mechanism. Population structuring may lead to population reproductive isolation and ultimately to speciation. The population structure can be expressed as different patterns. The three most common patterns of structuration are as follows: (a) by geographical distance ; (b) bySerrasalmus rhombeus  which inhabits both uplands, up to 2,300 MASL (Meters Above Sea Level), and lowlands across Argentina, Brazil, and Uruguay , or they infiltrate the ground until they disappear (arheic rivers). Such endorheic or arheic rivers are frequent from the northwestern Argentinian Pampa up to southern Bolivia and they are the result of dry periods  and examine the possible underlying processes. Particularly for IBB, we test whether the events of fragmentation–reconnection of basins have played a role in the population structuration of J. lineata.The wide distribution of 22.1https://www.conicet.gov.ar/wp‐content/uploads/OCR‐RD‐20050701‐1047.pdf) and its biological sampling guide (https://proyectosinv.conicet.gov.ar/solicitud‐colecta‐cientifica/). Also, fish handling during sampling was performed following guidelines of the UFAW Handbook on the Care and Management of Laboratory Animals (http://www.ufaw.org.uk). Fish were anesthetized and killed using water containing a lethal dose of eugenol (clove oil).In Argentina, fish were collected with the permission of the local authorities. The study was approved by the National Council of Scientific and Technical Research of Argentina (exp. 7879/14) and it is a requirement of this institution to follow the guidelines of its "Comité de Etica" , Gambusia holbrooki Girard 1859 and Phalloceros caudimaculatus (Hensel 1868). A 598‐base pair (bp) of the mitochondrial cytochrome c oxidase I gene (COI) was amplified by polymerase chain reaction (PCR) from each fish sample. The following primers were used for COI amplification: SILCOI‐D and SILCOI‐R  using MEGA 7.0.26 ) and Tajima's D were calculated in Arlequin 3.5.2.2  using SAMOVA 1.0  to unravel the variables explaining the genetic differentiation of z et al. . We usedThe geographical distance between each pair of sampling sites : For this analysis, we used the geographical distance calculated by considering the course of the rivers instead of using the Euclidean distance extracted from the geographical coordinates. To transform the matrix of geographical distance into vectors, we applied a Principal Coordinates of Neighbour Matrices (PCNM) by using the package PCNM : We incorporated a dummy variable in the model according to the type of basin of each sampling site: The sites were categorized either as “0” if exorheic basins—have connection with the main La Plata Basin or the sea—, or “1” if endorheic basins—without connection—.Hydrographic system (representing possible IBB): The sampling sites were classified in 8 systems: (a) Mar Chiquita, (b) La Plata, (c) Río Quinto, (d) Salar Ambargasta, (e) Río Colorado‐Negro, (f) Salar de Pipanaco, (g) Sierras de San Luis, and (i) Este Uruguay; these were taken as factors since it was necessary to give them a numerical value for the analysis.J. lineata is present from lowlands to up to 2,300 MASL, we used altitude data inferred for each sampling site in our model. Values were taken from Google Earth Pro. However, contrary to the haplotype network reconstruction where we classify the localities into groups of altitudes, in which we used five categories of altitude  group 1 from 0 to 99 MASL, (b) group 2 from 100 to 299 MASL, (c) group 3 from 300 to 499 MASL, (d) group 4 from 500 to 699 MASL, and (e) group 5 from 700 MASL), for the db‐RDA we used this variable as a continuous variable. Altitude : As Elevational gradients may be challenging for most species and can be considered as IBE for many reasons: (a) The number of species increases downstream with a marked difference in species' richness between upland and lowland areas : For The gradients across latitudes in the La Plata Basin imply changes in community composition and climatic variation. Therefore, we added latitude in our model as a proxy for environmental heterogeneity. This variable was included in the model as decimals, measured according to the geographical coordinates of each site's south latitude. The latitudinal gradients of species richness for fishes generally corroborated the paradigm of latitudinal diversity gradient (LDG)  and a chi‐square  variables to test for the independence of the variables. Then, to identify the variables that explain part of the genetic structure, we first ran a db‐RDA on the full model  using the function “capscale” of the package vegan  indicated a low genetic differentiation in es Table . The higp = 0.129 and 0.457, respectively).We tested the correlation between both geographic matrices  with the genetic distance matrix. In both cases, the Mantel test results were not significant  best reflects the genetic structure of this species in the region. The FCT values did not increase substantially with an increasing number of groups . These results agree with the three major haplogroups observed in the network. The first group includes the nine localities that are in the province of Buenos Aires, Desaguadero River and the locality located in Uruguay, all leading to the Atlantic and forming part of the exorheic system basins. Surprisingly, this group is also composed of two endorheic adjacent rivers. The second group includes the two endorheic localities in the center of Argentina and other four nearby localities, some of which only recently became exorheic by anthropogenic actions  the geographical distance between localities as indicated by the first six axes of a PCNM on the geographical distance matrix where each axis was considered as separate variables (representing IBD); (b) the basin and (c) system type (both representing IBB), as categorical variables; (d) the altitude expressed as meters above sea level; and (e) latitude expressed by decimal coordinates (both representing IBE).J. lineata, we proceeded as follows: First, the full model was considered with all the variables, but it was not significant (p =.182). We also ran a db‐RDA on nested models to identify the best model (AIC = −132.68). However, the best model only considered one variable (third axis of PCNM) and it was not significant (p =.091).To select the set of variables that best explains the population structuration of p  and contained three variables out of the initial 9: the geographical distance expressed as the third axes of the PCNM analysis (variable 1), basin type (variable 2) and altitude (variable 3). The variance partitioning analysis was performed only on the variables that were included in the optimal model, and we grouped them according to the pattern of isolation they generate: IBD, IBB, or IBE. The results showed that the variable driving IBE  explained 25% of the variance of the genetic structure, whereas the variable IBB (basin type) explained 22%. On the other hand, corroborating the Mantel test results, geographical distance (IBD) alone did not explain (p nonsignificant) the genetic variation  where we did not detect any IBD pattern in this species.A higher genetic differentiation is expected for populations that are geographically far away from each other because their gene flow is reduced as compared to populations that are geographically close (IBD) Wright, . The deg4.2J. lineata as a case study was its great tolerance to environmental factors. This species is found in freshwaters and marine environments covering a wide distribution range  to see whether they play an important role in the structuration of populations of this species. As explained before, features as altitudinal or latitudinal clines are important landscape characteristics that affect the proportion of suitable habitats, influence migration patterns and ultimately genetic divergence of populations . Moreover, the analyses of variance partitioning allowed us to unravel the relative role of these variables and the importance of their interactions. Hence, we showed that IBB and IBE processes have explained almost 50% of the population structuration in J. lineata. The power of this methodologies was corroborated here that it can easily be implemented for any other species.Most population studies focus on how a single mechanism could affect structuration or speciation in a group of organisms. Our results show that the process of fish population diversification in the La Plata Basin is complex and not limited to a single process. Using a general method, we demonstrated that interactions among several processes have had an impact on the population structuring of The impact of the basin fragmentation phenomenon (as IBB pattern) on the diversity of freshwater fishes is expected to be significant and also general for all freshwater organisms. The fact that the basin fragmentation–reconnection hypothesis states a periodic repetition of the events, it may well represent a major process by which population structuring or new species are periodically created and may spread all over the basin during river reconnection periods. Nonetheless, further research is needed to disentangle this challenging issue. We expect to find in future studies that this hypothesis will be corroborated in other fish species and freshwater organisms.The impact found of the phenomenon of fragmentation of the basin highlights the importance of the recent anthropic modifications that alter the natural runoff of the rivers. Dams and the constructions of artificial aquatic channels cause obvious disturbances in rivers and are likely to modify the gene flow previously attributed to natural features. For example, the Quinto River, which was originally endorheic, was affected in recent years as it has reactivated a superficial connection between this river and the Salado River through the artificial channel Arturo Jauretche (Ministry of Infrastructure of the Province of Buenos Aires 2015). Due to this, it can no longer be considered as strictly endorheic.During the last decade in South America, it was necessary to focus studies at the species level or higher taxonomic level ; formal analysis ; resources ; software ; writing–original draft . Luiz Jardim de Queiroz: Data curation ; formal analysis ; methodology ; software ; writing–original draft . Sergio Bogan: Data curation ; formal analysis ; resources ; writing–review and editing . Ariel Paracampo: Data curation ; formal analysis ; writing–review and editing . Paula Posadas: Project administration ; writing–review and editing . Gustavo M. Somoza: Project administration ; writing–review and editing . Juan Montoya‐Burgos: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; project administration ; resources ; supervision ; writing–original draft ; writing–review and editing . Yamila P. Cardoso: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision ; visualization ; writing–original draft ; writing–review and editing .Supplementary MaterialClick here for additional data file.