Native American populations from the Brazilian Amazon have a low genetic diversity and a different genetic profile when compared to people from other continents. Despite this, few studies have been conducted in this group, and there is no description of their genetic data in the various currently existent international databases. The characterization of the genomic profile of a population not only has an impact in studies of population genetics, but also helps to advance diagnostic and therapeutic response studies, leading to the optimization of clinical applicability. Genetic variations in DNA repair genes have been associated with the modulation of susceptibility to various pathologies, as well as in their prognosis and therapy. This is the first study to investigate DNA repair genes in Amerindians from the Brazilian Amazon region. We investigated 13 important DNA repair genes in the exome of 63 Native Americans, comparing our results with those found in 5 continental populations, whose data are available in the Genome Aggregation Database. Our results showed that 57 variants already described in literature were differentially distributed in the Amerindian populations in relation to the continental populations, 7 of which have significant clinical relevance. In addition, 9 new variants were described, suggesting that they are unique to these populations. Our study reinforces the understanding that the Amazonian Native American population presents a unique genetic profile, and our findings may collaborate with the creation of public policies that optimize the quality of life of these groups as well as the Brazilian population, which presents a high degree of interethnic mixing with Amerindian groups. Genome integrity depends not only on faithful replication, but essentially on proper repair of the data in DNA throughout its synthesis and processing. It is estimated that approximately 10,000 DNA bases are chemically modified every day in each cell . These mGiven the importance of their functions, several studies have associated genes of DNA repair pathways and susceptibility to diseases such as xeroderma pigmentosum ,13, cockDespite the amount of information available in literature regarding imbalances in gene expression of DNA repair genes, there are no studies reporting the frequency of mutations in these genes in Amerindian populations, especially in the Amazon. However, studies of precision medicine have demonstrated the importance of screening not only genetically homogeneous populations, such as the European one, but also Amerindian people distributed worldwide. It has been shown that populations with a high prevalence of Amerindian genomic ancestry have an increased predisposition to develop acute lymphoblastic leukemia, stomach cancer, and tuberculosis ,23,24,25Thus, genomic studies on DNA repair genes in Amerindian populations may provide information on their molecular profile and may potentially discover new variants. The findings from molecular epidemiology studies may also promote the investigation of clinical implications regarding the molecular markers described, allowing them to be used as diagnostic and treatment tools for the Amerindian population, as well as for the admixed populations with marked Amerindian ancestry, such as the Brazilian one . Finallyhttp://conselho.saude.gov.br/comissoes-cns/conep/) and by the Research Ethics Committee of the Tropical Medicine Center of the Federal University of Pará (CAE: 20654313.6.0000.5172). All individuals and community leaders signed an informed consent form.The study was approved by the National Research Ethics Committee .https://gnomad.broadinstitute.org/; accessed on 15 February 2022), a public catalog of human variation and genotype data. This sample included 8128 individuals from Africa (AFR), 56,885 from Europe (EUR non-finish), 17,296 from the Americas (AMR), 9197 from East Asia (EAS), and 15,308 from South Asia (SAS).The Amerindian population data were compared with representatives of five continental populations obtained from the Genome Aggregation Database  and SureSelect Human All Exon V6  kits, following the manufacturer’s recommendations. The sequencing reactions were performed on the NextSeq 500® platform  using the NextSeq 500 High-Output v2 Kit 300 cycle kit .Libraries were prepared using the Nextera Rapid Capture Exome  and filtered to eliminate low-quality reads . Then, the sequences were aligned with the reference genome (GRCh38) using the BWA v.0.7 tool .Reads in FASTQ format were analyzed for quality . The alignment had to be processed to remove duplicate readings , mapping quality recalibration and local realignment (GATK v.3.2—https://www.broadinstitute.org/gatk/). Finally, the result was processed in search of variants  of the reference genome. The allelic variants were annotated in the ViVa®  software. Markers in PCLO gene were also selected in coverage, where the readout should be high-coverage, with a minimum of 10 reads .After alignment, the generated file was indexed and sorted (SAMtools v.1.2—p ≤ 0.05).All statistical analyses were performed using the R Studio v.3.5.1 program , including the discriminant analysis of principal components (DAPC). Significant differences in allele frequencies between populations were analyzed by Fisher’s exact test. The false discovery rate (FDR) proposed by Benjamini and Hochberg  was usedhttp://hannonlab.cshl.edu/fastx_toolkit/; accessed on 25 March 2022); (II) the predicted impact should be “modifier”, “moderate”, or “high” according to the software SNPeff ; (c) the difference in allelic frequency of the variants between NAT populations and continental populations should be significant .We analyzed 13 gene components of 4 different DNA repair pathways in humans (base excision repair (BER), nucleotide excision repair (NER), homologous recombination repair and mismatch repair). A total of 432 variants were found, to which the following selection criteria were applied: (I) the read should be high-coverage, with a minimum of 10 reads ; rs7689099 (NEIL3); rs2294913 (TOP3A); rs3212038, rs1799796, rs861531, and rs861537 (XRCC3); rs6151734 and rs1105524 (MSH3); rs3765682 (MSH4); rs907187, rs2293464, and rs1805404 (PARP1). Overall, for the other SNVs presented, the Amazonian indigenous population differed from two or three of the investigated world populations.Finally, For all missense variants, we used two other impact prediction tools (SIFT and PolyPhen). The study of genetic variations in repair genes has aided the understanding of various pathological mechanisms, describing risks associated with both individual and population genotypes based on the exposure of cells to xenobiotics that lead to genomic instability ,35,36,37The past process of global colonization is known to have played an important role in defining current patterns of genetic diversity, and it partially explains geographic variation in susceptibility to certain complex diseases ,39. ThusTherefore, we analyzed the complete exome of 13 DNA repair genes never previously investigated in Amerindian populations, representative of tribes from the Brazilian Amazon. Our description of frequencies and our DAPC analysis demonstrated that the African, South Asiatic, and Amazonian Native American populations were positioned at opposite extremes, being the most genetically distinct regarding the investigated variants, corroborating with findings on the history of human populations . Our resp = 0.005), as well as a worse progression-free survival [The MSH3 gene is part the MMR system, whose function is to repair DNA after cross-linking chains and recognize and correct base–base mismatches and insertion/deletion loops generated during DNA replication and homologous recombination ,52. The survival . This posurvival ,56 and csurvival .Finally, we found nine new variants, among which three are INDEL-like, and one—which causes a reading matrix change—has an estimated high clinical impact. This mutation is located in the TOP3A gene, responsible for homologous recombination-mediated repair of double-stranded DNA during DNA synthesis, an essential component in the mitochondrial DNA replication process . The remThis is the first study to investigate DNA repair genes in Amerindian populations from the Brazilian Amazon, which are genetically unique and not yet described in any of the available databases on human genetic variability. Knowledge of different patterns in human genetic diversity is important in many areas of medical genetics, and it can be used as a tool to maximize understanding regarding susceptibility, diagnosis, prognosis, and therapeutic management for Native American populations, as well as for populations with high Amerindian ancestry, such as the Brazilian one.Our study reinforces the understanding that the Amazonian Native American population presents a unique genetic profile. The characterization of repair genes in this populations is an important tool for future studies regarding their association with complex diseases in these populations and also in ones with a high degree of admixture with these groups. Furthermore, our data may contribute to the creation of public policies that optimize the quality of life of Amerindian populations investigated here.


Conandron ramondioides. C. ramondioides is an understory herb occurring in primary forests, which has been grouped into two varieties. We reconstructed the evolutionary and population demography history of C. ramondioides to infer its divergence process. Nuclear and chloroplast DNA sequences were obtained from 21 Conandron populations on both sides of the East China Sea (ECS) to explore its genetic diversity, structure, and population differentiation. Interestingly, the reconstructed phylogeny indicated that the populations should be classified into three clades corresponding to geographical regions: the Japan (Honshu+Shikoku) clade, the Taiwan–Iriomote clade, and the Southeast China clade. Lineage divergence between the Japan clade and the Taiwan–Iriomote and Southeast China clades occured 1.14 MYA (95% HPD: 0.82–3.86), followed by divergence between the Taiwan–Iriomote and Southeast China clades approximately 0.75 MYA (95% HPD: 0.45–1.3). Furthermore, corolla traits  correlated with geographical distributions. Moreover, restricted gene flow was detected among clades. Lastly, the lack of potential dispersal routes across an exposed ECS seafloor during the last glacial maximum suggests that migration among the Conandron clades was unlikely. In summary, the extant Conandron exhibits a disjunct distribution pattern as a result of vicariance rather than long-distance dispersal. We propose that allopatric divergence has occurred in C. ramondioides since the Pleistocene. Our findings highlight the critical influence of species’ biological characteristics on shaping lineage diversification of East Asian relic herb species during climate oscillations since the Quaternary.The evolutionary histories of ornamental plants have been receiving only limited attention. We examined the origin and divergence processes of an East Asian endemic ornamental plant,  Ornamental plants are known for their aesthetic appeal, and their horticultural history can be traced back to ancient Egypt . At leasIt has been proposed that population differentiation is the driving force for plant speciation, which should involve the process of how barriers affect gene flow between previously interbreeding species ,7. East Cercidiphyllum japonicum [Kalopanax septemlobus [Quercus variabilis [Viola orientalis [Platycrater arguta [Dendrobium moniliforme [P. arguta populations over land was unlikely [P. arguta, estimated restricted gene flow between populations distributed on the continent (China) and on islands (Japan) suggests a lack of migration between these populations since their first divergence. Exploring genetic compositions and modeling suitable habitat ranges from past geographical periods can allow researchers to explore the divergence process of target species. However, the role of post-divergence gene flow between continent- and island-distributed populations in shaping lineage divergence has not been sufficiently examined among the species mentioned above.In East Asia, many plant species exhibit continent–island distribution patterns ,13,14,15aponicum , Kalopanriabilis , and Vior arguta  and Dendunlikely . In studConandron ramondioides is not only an East Asian endemic temperate understory herb but also an ornamental plant in the plant market. C. ramondioides is distributed across East China, Taiwan, southern Okinawa, and Japan but is absent in between these locations, thus exhibiting a disjunct distribution pattern. This has been interpreted as the species having migrated from the Asian continent to adjacent islands or fragmented from an ancient mega population in East Asia. Based on morphological traits (lobe length to tube length ratio) of samples obtained from Japan and Taiwan, C. ramondioides has previously been assigned to two varieties: C. ramondioides var. ramondioides and C. ramondioides var. taiwanensis [C. ramondioides is not yet known. Two scenarios have been proposed so far: one is a recent long-distance dispersal scenario, and the other is a vicariance scenario. In the former, island-distributed C. ramondioides populations may have originated from the Asian continent, followed by dispersal to adjacent islands. The vicariance scenario proposes that populations currently distributed on the Asian continent and islands are fragments of an ancient mega population. We were especially interested in whether gene flow between C. ramondioides var. ramondioides and C. ramondioides var. taiwanensis took place during their evolutionary history. We used molecular markers and phylogeographical analysis to verify which scenario is the most likely to have occurred.wanensis . HoweverLEAFY), is subject to stochastic processes. The results of single-gene trees may be over-interpreted if researchers do not consider coalescent stochasticity [In phylogeographical studies, chloroplast DNA sequences (cpDNA) have been used successfully to infer relationships among closely related species or populations of plants ,18,19,20asticity . To prevasticity . Using bC. ramondioides by examining genetic variations obtained from both nrDNA  and cpDNA (including trnL-trnF and trnH-psbA) markers of 21 C. ramondioides populations in East Asia. We aimed (i) to reveal genetic relationships, approximate divergence time, and gene flow between continent- and island-distributed Conandron populations; (ii) to verify whether their taxonomic classification corresponds to their genetic relationships; and (iii) to reveal the potential suitable habitat range via simulation of Conandron during the last glacial maximum (LGM).In this study, we performed a phylogeographic survey of C. ramondioides in East Asia . In summary, haplotype networks reconstructed from four low-copy nuclear markers suggested that haplotypes of C. ramondioides formed three groups corresponding to geographical regions.Among these four haplotype networks, the ITS ribotype network. One group includes haplotypes distributed across Honshu, SE-China, Taiwan, and Iriomote, with rare ribotypes connecting to it. The second group represents ribotypes descending from ribotypes distributed in Honshu–Iriomote, which differ from the former ribotype by four mutational steps  test was run. If Pn/Ps is not significantly different from Dn/Ds, this indicates that CrCYC1 does not deviate from the neutral hypothesis. The results show that Pn/Ps within C. ramondioides (20/5) was significantly higher than Dn/Ds between C. ramondioides and C. umbellifera   . The higectively . SignifiConandron individuals  were used to verify whether internal transcribed spacers (ITS) amplified from Conandron were subjected to concerted evolution. We obtained multiple haplotypes (ranging from three to six) from colony sequences    calculated from these molecular markers of  × 10−3) . A compa × 10−3) .Significant pairwise Fst values are measured among three assigned groups of all markers . For theC. ramondioides, the species phylogeny was reconstructed by using the genetic information from the aforementioned three nuclear markers, including ATG2 intron 1, GroEs intron 1, and LFY intron 1. The reconstructed phylogeny showed C. ramondioides was assigned into five monophyletic clades with moderately to highly statistic support (posterior probability ≥0.75) (C. ramondioides populations formed a monophyletic clade with high statistic support (posterior probability = 1). Lastly, the estimated lineage divergence time between the Honshu–Shikoku clade and the Southeast China–Taiwan–Iriomote clades was dated back to 1.13 (95% CI: 0.26–1.8) million years ago (MYA), while the estimated divergence time between the Southeast-China-distributed population and those distributed in Taiwan and Iriomote occurred 0.75 million years ago (95% CI: 0.37–1).To trace the lineage divergence process of y ≥0.75) . The TaiFor the multi-population IMa2 analysis, we present summary statistics in C. ramondioides was the TI group, then HS group and finally C group  . The lin C group . The estked at 0 . Lastly,p < 0.05, bold numbers). It is regarded that Fu’s Fs is more sensitive for identifying population expansion and past population growth. Therefore, population expansion may have been restricted to Taiwan and Iriomote. Furthermore, population contraction may have been restricted to individual Honshu and Shikoku populations due to most populations and regional groups exhibiting non-significant positive Fu’s Fs values    . Despite Taiwan) .C. ramondioides flowers  of available  flowers . The L/TConandron populations are isolated from each other and disjunctively distributed in East Asia, Conandron shows similar or even higher levels of nuclear and cpDNA haplotype and nucleotide diversity  [Conandron in Taiwan [Conandron. Furthermore, seeds of Conandron lack both hair-like appendages for attachment to seed dispersers and any structures attractive to animal seed consumers. These two morphological characteristics of Conandron seeds suggest that long-distance seed dispersal is unlikely. Moreover, the short stature of Conandron and its understory forest habitat may discourage visits from insect pollinators [Conandron populations via pollen and seeds is probably severely constrained. This pattern may be a consequence of the species’ requirements of a cool and moist environment in temperate deciduous forest habitats at mid-elevation. Due to its biological features , the repeated exposure of the ECS seafloor may not have served as a land bridge allowing migration among Conandron populations since the Quaternary. To further examine the lineage divergence and identify whether post-divergence gene flow ever occurred, construction of a phylogeny and estimated post-divergence gene flow of Conandron were conducted (see below).Although the iversity  than hasgeshoma) ,23,24. Ageshoma)  but alsogeshoma) , are thon Taiwan . This sulinators , reducinConandron populations could be assigned to three groups corresponding to geographical regions: the Honshu + Shikoku group, the Southeast China group, and the Taiwan + Iriomote group , these localities were once connected by an exposed ECS seafloor during glacial periods, potentially allowing Conandron to disperse into East Asia. However, restricted post-divergence gene flow was measured between the north and south groups and between the C (Southeast China) and TI (Taiwan and Iriomote) groups, suggesting that dispersal among Conandron populations since lineage divergence was unlikely  MYA between the north (Honshu and Shikoku) and the south  groups, while a second diversification event occurred at c. 0.75  MYA between the Southeast China and Taiwan–Iriomote groups . Our estoccurred . Thus, tunlikely . FurtherConandron is a cold-tolerant but drought-intolerant understory herb, the relatively drier paleo-environment conditions on the ECS land bridge (compared with the present) were not favorable for C. ramondioides. The ENM-modeled suitable habitat range may provide further support for this hypothesis  [Quercus variabilis [Cercidiphyllum japonicum [Kalopanax septemlobus [Conandron and other studied herbaceous species do not possess such reproductive traits. Thus, predictions regarding the role of the ECS land bridge have to take into account both the presence of the land bridge and other relevant biological features of each species, especially its reproductive characteristics.Contrary to earlier hypotheses, the exposed ECS floor did not necessarily serve as a land bridge enabling migration of currently isolated populations or species. There is an increasing number of studies suggesting that the ECS land bridge acted as a barrier to some East Asian temperate plants by selectively hampering migration but allowing free migration of some other species. For example, genetic breaks across the ECS have been detected in the populations of several understory fern, herbs, orchids, and shrubs from temperate forests in East Asia  ,24,33,34riabilis , Cercidiaponicum , and Kaltemlobus . ReproduC. ramondioides [C. ramondioides var. ramondioides (distributed in mainland Japan); the other is C. ramondioides var. taiwanensis (distributed in Taiwan and Iriomote). However, the Southeast-China-distributed Conandron is not included in the previous study. Here, our multi-loci phylogeny not only provides molecular support for the taxonomic treatment of Conandron varieties but also offers molecular support in lineage divergence of the Southeast-China-distributed Conandron. There are two lineage divergence events which occurred, leading to the genetic differentiation of Conandron into three distinct groups , two varieties were defined within the ndioides . One is t groups . The firulations . DespiteConandron  than those in ITS , provide evidence to support the first assumption  rather than synonymous substitutions (Ds)  ranged from three to six, suggesting heterogeneity. ITS is one of the most popular molecular markers used for reconstructing plant phylogeny due to rapid concerted evolution within and among nuclear ribosomal DNA component subunits, fast substitution rate, and availability of universal primers [Lespedeza [Carapichea ipecacuanha [Pyrus [Mammillaria [ITS in plants is common. species . We thus primers ,42,43. C primers . Howeverespedeza , Carapiccacuanha , Pyrus [a [Pyrus , and Mammillaria , suggestConandron populations and the coastal-Fujian-distributed Conandron populations  from the geographical regions, which revealed corresponding morphological groups. Our study represents a comprehensive approach to revealing the speciation process of East Asian endemic species by integrating information from molecular evolution, paleo-climate simulation, and morphological trait measurements.In summary, the current disjunctive distribution pattern of C. ramondioides var. ramondioides and twelve populations of C. ramondioides var. taiwanensis , covering most of the species’ distribution range in East Asia  and five nuclear markers  were amplified from all sampled individuals. The first four markers  were sequenced using the Sanger sequencing method [trnL-trnF region were from Taberlet et al. [trnH’ (CGC GCA TGG TGG ATT CAC AAT CC) and the reverse primer ‘psbA’ (GTTATGCATGAA CGT AAT GCT C) were used to amplify the trnH-psbA region [2O, and 1 μL of template at 20 ng/μL. The PCR profile for cpDNA markers amplification was 35 cycles of 30 sec at 94 °C, 30 s at 56 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C, after an initial denaturing for 3 min at 94 °C for both trnL-trnF and trnH-psbA. For nuclear markers, CrCYC1 sequences were amplified using the forward primer “SPF” (5’-AGCAAGACATGCTTTCTGG-3’) and the reverse primer “SPR” (5’-GACATTGGATTCAATATGGTG-3’) [ITS region [CrCYC1 and ITS amplification. The PCR reaction was made up to a total volume of 25 μL, containing 13 μL of 2× Ampliqon master mix Red, 0.25 μL of each primer at 2 μM, 10.5 μL of ddH2O, and 1 μL of template at 20 ng/μL. All PCR products were examined with 1.0% (w/v) agarose gel electrophoresis, and then the band of correct size was used for sequencing. The PCR products were sequenced using an ABI 3700 automatic sequencer . All sequences were examined firstly with a Sequence Scanner v1.0 and then aligned for further analysis. To examine within-individual homogeneity of ITS, we amplified nine individuals  using ITS primer pairs with a proofreading KAPA HiFi PCR kit . These nine PCR products were cloned using a pGEM-T Easy Vector kit , following the manufacturer’s protocols. Finally, five to six clones of each PCR product were sequenced. Sequences were deposited in GenBank under the following Accession Numbers: CrCYC1 (OP631034-OP631413), ITS (OP578698-OP579075), trnL-trnF (OP689756-OP689952) and trnH-psbA (OP689953-OP690149).To evaluate the divergence patterns of g method  in 2016.t et al. . The forA region . The PCRS region . The PCRC. ramondioides in 2018 [We developed specific single-copy nuclear intron markers for  in 2018 . Due to  in 2018 . Out of CrCYC1, ITS, and concatenated cpDNA) were aligned with the MUSCLE algorithm [Sequences of all six markers , Taiwan, and Southeast China were classified into these separate geographical regions for examination of the genetic structure. Iriomote populations were assigned to the Taiwan group since they are geographically close to Taiwan. An analysis of molecular variance (AMOVA) of our genetic data was performed using the program Arlequin ver 3.5 [F-statistics were calculated to estimate the proportion of gene variability found among populations (Fst), among the population within groups (Fsc), and among groups (Fct) of our datasets. If the ECS serves as a barrier, we would expect the highest genetic variation to be attributed to the group level. The statistical significance of all estimated fixation indices was tested with 10,000 permutations [The ECS is considered a barrier inhibiting gene flow for shrubs or herbs ,15,24. T ver 3.5 . F-statiutations .C. ramondioides individuals for each tested marker, we constructed a statistical parsimony network with a 95% connection limit using the R package “Haplotypes” [To assess the genetic relationships of sampled lotypes” . Indels lotypes” .Conandron populations, *BEAST with multi-locus species tree examination in the BEAST v2.4.7 program [To rebuild the relationship among  program ,69 was u program , the HKY program . For the program ,69. The  program .Conandron groups. The geometric mean of Watterson’s theta [−9 × 393 bp for the LFY marker) was used as a reference for the other two loci to rescale the IMa2 parameters.The grouping pattern was inferred from the multi-loci species tree; we further reconstructed the population differentiation process by gene flow with an isolation with migration model, which was implemented in IMa2 . An infi’s theta  per sequConandron groups. Divergence time estimated from a pairwise analysis will show splitting order among groups [5 iterations of pairwise group pairs were discarded as burn-in, followed by 106 iterations for sampling (100 iterations per sample). To confirm whether our analysis exhibited convergence, three independent runs were performed. To determine the statistical significance of gene flow between groups, the Likelihood ratio tests (LRT) were computed [Furthermore, the same upper bound settings were applied to our pairwise dataset of the three g groups ; therefocomputed .CrCYC1 at the codon level. Therefore, the CrCYC1 dataset was subjected to further examination of deviation from neutral evolution using the McDonald–Kreitman test [To evaluate whether the molecular markers conformed with the expectations of neutrality theory, both Tajima’s D  and Fu’sman test  as impleman test .Conandron groups  through time by assessing whether the species had undertaken population expansion or decline during cold periods . An extended Bayesian skyline plot (EBSP) implemented in BEAST v2.4.7 was used, and EBSP uses coalescent-based models to estimate posterior probabilities of effective population size change through time [We reconstructed the effective population size change in ugh time . SubstitConandron was modeled using the maximum entropy method [http://www.gbif.org/, accessed on 1 December 2017), and 19 BIOCLIM variables from the WorldClim data set [Conandron were modeled and then projected to the “last glacial maximum occurrence” layers to show potential suitable habitat ranges of Conandron during the LGM period.To estimate the least-cost path between populations, we first generated a resistance map by using a reciprocal of a suitable map. Resistance-to-suitability mapping is simply a monotonic transformation in which locations with higher suitability exhibit lower resistance. Therefore, the potential suitable habitat range of y method ,83. Firsdata set , includiFinally, the pairwise least-cost path was measured among 21 populations from the resistance map using the R package “gdistance” . The resC. ramondioides populations distributed across Japan from those in Taiwan and Iriomote [C. ramondioides corolla was measured following the method of Kokubugata and Peng [Corolla morphology (lobe length to tube length ratio) has been proposed as a key trait to distinguish Iriomote . Therefoand Peng .


Pinus ponderosa populations over 27 years. Mean environmental conditions and their spatial patterns of variation at the seed source populations were characterized based on six basic climate parameters. Despite the small area of origin, there was significant genetic variation in phenotypic plasticity for tree growth among populations. We found a significant negative correlation between phenotypic plasticity and the patch size of environmental heterogeneity at the seed source populations, but not with total environmental spatial variance. These results show that populations exposed to high microhabitat heterogeneity have evolved higher phenotypic plasticity and that the trigger was the grain rather than the total magnitude of spatial heterogeneity. Contrary to theoretical predictions, we also found a positive relationship between population plasticity and summer drought at the seed source, indicating that drought can act as a trigger of plasticity. Finally, we found a negative correlation between the quantitative genetic variance within populations and their phenotypic plasticity, suggesting compensatory adaptive mechanisms for the lack of genetic diversity. These results improve our understanding of the microevolutionary drivers of phenotypic plasticity, a critical process for resilience of long‐lived species under climate change, and support decision‐making in tree genetic improvement programs and seed transfer strategies.Phenotypic plasticity is a main mechanism for organisms to cope with changing environments and broaden their ecological range. Plasticity is genetically based and can evolve under natural selection, such that populations within a species show distinct phenotypic responses to the environment if evolved under different conditions. Understanding how intraspecific variation in phenotypic plasticity arises is critical to assess potential adaptation to ongoing climate change. Theory predicts that plasticity is favored in more favorable but variable environments. Yet, many theoretical predictions about benefits, costs, and selection on plasticity remain untested. To test these predictions, we took advantage of three genetic trials in the northern Rocky Mountains, USA, which assessed 23 closely located  It is the most broadly distributed pine species of the Western Hemisphere  Do populations from more spatially heterogeneous environments show greater phenotypic plasticity? (2) Does the scale of the spatial heterogeneity contribute to explain variation in plasticity? (3) Do populations from environments with lower resource availability show more constrained phenotypic plasticity? And (4) is phenotypic plasticity related to genetic variation at the population level? We hypothesize that the magnitude of phenotypic plasticity in tree growth increases in populations from more heterogeneous and fine‐grained environments, and that populations originating from low resource environments will show limited plasticity, as well as reduced intrapopulation variation due to stronger selective pressures. Therefore, we expect a positive relationship between plasticity and genetic variation within populations.22.1This study was conducted on three ponderosa pine genetic trials, including 23 closely located natural populations from western Montana and Northern Idaho  were established. Additional planting of containerized 2‐year‐old seedlings occurred in 1975 to replace mortality during the first year. We mapped each tree of the three trials using a submetric accuracy GPS device (Trimble Geo HX).Tree growth was measured as tree height at ages 2, 4, 5, 11, 16, 21, and 27, and diameter at breast height  at ages 5, 11, 16, 21, and 27. Positive genetic correlations among ages and between height and diameter were found, and DBH at age 27 was chosen as the best predictor of genetic growth potential in these trials k(j) is the random effect of the family k of the population j, S × Pij and S × F(P)ik(j) are the corresponding random interactions, and εijkl is the random tree effect of the ijklth individual or error term. The within‐site block effect and its interactions were not included in the model since the dependent variable was already free of spatial autocorrelation. Given that the dependent variable met the assumptions of normal distribution, the mixed model was fitted using the mixed procedure of SAS  for phenotypic plasticity in growth across populations was estimated as the ratio of additive genetic variance to total phenotypic variance. The standard error of heritability was estimated by the Delta method based on asymptotic estimates of the variances and covariances of the variance components of the random model  options in the random statement of the mixed procedure of SAS , we followed the likelihood framework proposed by Yang , mean temperature of the warmest month , mean temperature of the coldest month , annual precipitation , summer precipitation , and summer heat‐moisture index [SHMI = (MTWM)/(MSP/1000)]. These variables were selected based on results from prior studies examining growth responses to climate in ponderosa pine /MSP/000]. The2.2.72). The sill or max variance and the patch size of the spatial patterns were then extracted from the theoretical semivariogram models, and used as descriptors of the intensity and scale of climate heterogeneity, respectively.Climate heterogeneity at each seed source location was estimated using geostatistical approaches. Semivariograms were constructed for the six climatic parameters described before considering the 20 km radius from the center of each population , evidencing significant plastic responses to the environmental conditions at each test site. Growth was highest at Condon , intermediate at Little Wolf (9.1 m in height and 17.8 cm in DBH), and lowest at Lubrecht (7.5 m in tree height and 15.5 cm in DBH) , while the Population effect was not significant. Thus, most of the genetic variation in tree growth occurred within populations. By contrast, Family × Site interaction was not significant while Population × Site was (p < 0.01), showing that genetic variation in plasticity was Population driven. Population × Site interaction variance for DBH increased with tree age, and it was up to 28‐fold larger than Family × Site interaction variance at age 27  Figure . Family 3.2p > 0.05), 0.62 ± 0.24 (p < 0.05) and 0.87 ± 0.21 (p < 0.01), respectively, indicating that Lubrecht and Condon are the more dissimilar sites in terms of population rankings.A comprehensive likelihood‐based analysis regarding the relevance and interpretation of the Population × Site interaction is summarized in Table 3.3The best fitting model for population stabilities of tree DBH at age 27 based on the lowest AIC and BIC values favored the Eberhart–Russell model Table . Hence, 3.4sill (maximum variance) at which the data cease to be correlated which determines the patch size of spatial heterogeneity. Patch sizes were widely variable among populations and climate parameters ranging from 8 to 112 km (coefficients of variation across populations varying from 0.37 [for MAT and SHM] to 1.03 [for MSP])  Table , pointin]) Table , which s3.5sill) of the spatial pattern of heterogeneity at the seed source of each population. However, we found that correlations between plasticity and patch size at the seed source were generally negative . These results suggest that more plastic populations originated in more heterogenous environments  in terms of temperature of the coldest month.We did not find a correlation between plasticity and the maximum variance  , the site with more constrained growth.Correlations between phenotypic plasticity and within‐population genetic variation for each population showed a negative general trend at the three test sites Figure  but were4Under a scenario of climate change, phenotypic plasticity is critical for the resilience of populations of long‐lived species where long generation times limit rapid evolutionary change. Here, we provide evidence for genetic variation in the magnitude of plastic responses to environmental variability in closely located populations of a forest tree species. We also show some evidence that fine‐grained spatial heterogeneity and resource availability are related to phenotypic plasticity in tree growth. Overall, our results show differentiation in phenotypic plasticity among plant genotypes. This differentiation was mostly population driven, such that genotypes from the same population showed similar plastic responses across the three test sites but different from other populations suggesting that plastic responses are contingent upon the environment where populations evolved. Our results also provide support to the long‐hypothesized role of environmental heterogeneity on phenotypic plasticity. Populations that have been exposed to higher microhabitat heterogeneity likely through recent evolutionary time showed higher phenotypic plasticity and thus may be better equipped to respond to changing climates than populations from more homogenous environments. Importantly, we found that the patch size of environmental heterogeneity but not the total variance among patches was the driver of phenotypic plasticity, suggesting that the grain of spatial heterogeneity rather than the total magnitude drive evolution of plasticity in highly heterogeneous forest ecosystems. Moreover, trees from populations on drier sites were more plastic than trees from populations on mesic sites, providing evidence that reduced water availability, a common stress, triggers the evolution of phenotypic plasticity. Finally, phenotypic plasticity was negatively linked to quantitative genetic variation in tree growth, suggesting compensatory mechanisms of adaptation for the lack of genetic diversity and that phenotypic plasticity does not prevent depletion of genetic variance due to natural selection. Overall, our study highlights the need to consider within species population differentiation in phenotypic plasticity in order to provide insights into species' range shifts under climate change and into seed transfer strategies in assisted migration programs.4.1Tree growth depends on resource availability and strongly influences individual success among competing individuals, mainly during early seedling establishment when density‐dependent mortality in trees typically occurs  were the ones showing higher magnitudes of plasticity, while total variation among patches was not related to plasticity. Therefore, we also provide rare evidence that the grain of spatial heterogeneity rather than the total magnitude can be a more relevant trigger of phenotypic plasticity. In fine‐grained environments, the progeny has more chances to experience a range of environmental variation within the dispersal breath of the mother tree, and selection should favor adaptive phenotypic plasticity. Adaptive plasticity enables dispersing seedlings to maximize fitness by appropriately matching their phenotype to the environment where they are established. By contrast, in coarse‐grained environments, dispersed seeds are more likely to experience a homogeneous environment similar to that of their mother tree, and selection for plasticity should be relatively weaker or should favor canalized trait expression if costs of plasticity are sufficiently strong , our results show greater plasticity in populations from dryer and hotter environments. However, the degree to which resource availability enhances or prevents phenotypic plasticity is highly controversial. On the one hand, theory predicts phenotypic plasticity to be more advantageous under high resource availability . These programs have traditionally considered genetic variation in plasticity as a troublesome source of noise which hinders selection of genotypes fitted to a wide range of environments. Based on our results, populations exhibiting higher plasticity can be expected to outperform less plastic populations in more variable environments  as descriptors of local environments would increase the ability to detect environmental cues triggering phenotypic plasticity. Moreover, growth is a complex process and additional fitness related traits  should be studied to gain a deeper understanding of adaptive plastic responses under environmental change.The authors declare that there is no conflict of interest.Appendix S1Click here for additional data file.


Toona ciliata is an important timber species but is recognized as an endangered species at level II in China. Its genetic conservation is of increasing concern. Provenance trials and other breeding programs were conducted to develop seed transfer rules and multiplications. Here, we investigated twenty-nine populations sampled across the natural distribution of the T. ciliata complex using mtDNA and nrDNA ITS  markers. Haplotype diversity was h = 0.190 ± 0.202 and nucleotide diversity was π = 0.000383 ± 0.000536 for mtDNA marker. Nucleotide diversity for ITS sequences was 0.00837 ± 0.000783. Haplotypes exhibited phylogeographic structure in spatial distribution. The extent of genetic differentiation was significant (stF = 0.6994 ± 0.0079 for ITS and 0.8870 ± 0.0077 for mtDNA marker). Isolation by distance (IBD) and by elevation (IBE) occurred among populations. Phylogenetic relationships from mtDNA marker indicated three genetically distinct regions, each without IBD effects. Compared with pollen flow, seed flow was strongly impeded in the western region, but extensive in the central region, and less impeded in the eastern region. Most populations did not exhibit expansion, with only a few populations showing expansion after bottleneck effects. We discussed a strategy of region-based genetic conservation and proposed to conserve multiple populations in the western and eastern regions and a few populations in the central region. T. ciliata is a deciduous or semi-deciduous tree in the Toona genus of the Meliaceae family and an important timber species in southern China [T. ciliata has high-quality wood, with a tough and straight texture, beautiful patterns, dark reddish-brown heartwood, and light sapwood, and is also known as Chinese mahogany [T. ciliata is an ideal material for art processes and antique furniture in China and has a significant economic value [T. ciliata is currently recognized as an endangered species at level II because of over exploitation and low natural regeneration [rn China . It is nmahogany ,3,4. T. ic value . Howeverneration ,7. Its gneration . Other bneration ,10.T. ciliata is taxonomically classified into five varieties, namely T. ciliata var. ciliata, T. ciliata var. yunnanensis, T. ciliata var. pubescens, T. ciliata var. sublaxiflora, and T. ciliata var. henryi [T. ciliata complex. Previous studies of population genetic structure were mostly conducted in terms of the T. ciliata complex rather than a single variety, showing significant population differentiation using either the sequence-related amplified polymorphism (SRAP)   or the s = 0.35) . There w complex . T. cili markers . NeverthT. ciliata complex also include its reproductive system which was shown to be a predominantly outcrossing system, with selfing and inbreeding [T. ciliata was divergent from other species of the same family at a wide range of evolutionary times (7.4–48.4 Mya) [T. ciliata was divergent from its close species T. sinensis of the same genus at about 6–25 Mya using the whole genome sequences [T. ciliata in the Meliaceae family and the significance of conserving T. ciliata from a broader perspective.Studies pertinent to genetic conservation of breeding . Inbreedbreeding . In addi8.4 Mya) ,17,18,19equences ,21. ThesT. ciliata complex, with emphasis on inferring gene flow underlying the observed phylogenetic pattern, and to discuss genetic conservation of the species. Previous studies with SRAP and SSR markers were mainly confined to population structure of T. ciliata complex and did not infer the evolutionary processes underlying the observed pattern [T. ciliata complex using both nuclear and organelle genetic markers. For nuclear genomes, we selected a ribosomal internal transcribed spacer (ITS) marker that has been applied to population genetic analysis of plant species [T. ciliata complex. A combination of cytonuclear markers helps to infer the relative rate of pollen to seed flow among natural populations [The purposes of this study were to investigate phylogeographic variation across range-wide populations of  pattern ,13. This species ,23 and p species . Its ove species . For orgulations ,26.T. ciliata complex are unknown in South China, potential historical changes in populations size could yield different extents of genetic variation, as implied by postglacial colonization of some plant species [T. ciliata complex. This information was then combined with population structure and gene flow to develop a strategy for genetic conservation.In addition, we investigated the potential historical range expansion or contraction, which could provide additional information on genetic conservation. Although postglacial impacts on the distribution of  species ,28,29,30 species . ThroughT. ciliata complex in China (w/v) agarose gel electrophoresis. All quantified DNA samples were stored at −20 °C for polymerase chain reaction (PCR) amplification.Leaf samples were collected from 29 populations in 11 provinces, covering the range-wide distribution of in China . Parent For the mitochondrial genome, we tested twenty pairs of primers, with fourteen pairs from the literature ,33,34 anFor the nuclear genome, we selected ITS fragment as marker , which c2 and 400 μM each dNTP) and 9.5 μL ddH2O. The amplifications were performed in Dongsheng Thermal Cycler . The PCR amplification for ITS and mtDNA primers was conducted as follows: preheating at 95 °C for 3 min, 32 cycles at 95 °C for 1 min, annealing at 50 °C for 1 min, and elongation at 72 °C for 1.5 min, followed by a final extension at 72 °C for 30 s. The amplified DNA fragments were subsequently sent to Sangon Biotech (Shanghai) for Sanger sequencing. All sequence data obtained by the company were checked with Chromatogram Explorer 3.2 software. The sequences with high quality and no mixed peak signals were used for downstream analyses.The PCR amplification was carried out in a 25 μL reaction volume that contained 1μL template DNA, 1 μL of each forward and reverse primer, 12.5 μL 2×ES Taq Mastermix , nucleotide diversity (We used DNAsp v5  to estim(θ=4Neμ) ,39,40. T(θ=4Neμ)  was usedPopulation genetic differentiation was measured using several indices, including Fst , Nst [43Fst [Nst , Gst [40Gst [ϕst . Both Gsuin v3.0  and DNAsuin v3.0  to estimIsolation by distance (IBD) was tested through regression analysis of distance ,48:(1)Fsb from zero indicates the presence of IBD effects. Note that the geographical distance between two populations was calculated using their longitude and latitude coordinates  = 1/(1 + 2Nem) and stF(diploid) = 1/(1 + 4Nem), respectively, where m is the migration rate and eN is the effective population size. MtDNA marker is maternally inherited and only seed flow contributes to its gene flow. The nrDNA ITS marker is biparentally inherited and both seed and pollen flow contribute to its gene flow. According to Ennos [sm is the rate of seed flow and pm is the rate of pollen flow. From Ennos [We assessed the extent of gene flow of nd model , Fst forto Ennos  and Hu ato Ennos , populatom Ennos , the relp/msm, let To estimate the standard deviation of the point estimate of u et al. . TherefoThe variance The covariance p/msm is estimated byFrom Lynch and Walsh , the varThe Jacknife method was applied to estimate variances of th FSTAT , which wcox1−nad1 and 26S−rRNA−tRNA−Leu were successfully screened for generating polymorphisms among individuals. Sequences of these two fragments were aligned and concatenated to produce a length of 1242 bp for downstream analyses  indicated that significant genetic differentiation occurred among populations . Estimatp < 0.05). Likewise, the Population differentiation based on sequence divergence  for the concatenated mtDNA sequences, and p-value = 1.57 × 10−9 for ITS sequences. These results indicated that significant IBD effects occurred among range-wide populations of T. ciliata complex.T. ciliata complex  or within the combined western and central regions . This supported that the natural distribution of T. ciliata complex could be divided into three genetically differential groups in terms of mtDNA marker.Regression analyses indicated that IBD effects were insignificant within each of three regions. However, significant IBD effects occurred among populations within the combined eastern and central regions , between the central and western regions for mtDNA marker  but not for ITS marker , and between the eastern and western regions . Differentiation among three regions was significant .Analysis of genetic differentiation indicated that there was significant difference between the eastern and central regions , but significant effects in the combined central and western region, p-value = 0.0007).Phylogenetic relationship from ITS sequences indicated that two clusters could be grouped among twenty-nine populations . One clup-value = 0.00022 for the mtDNA marker; p-value = 0.0094 for the ITS marker). However, correlation between r=0.0176, p-value = 0.9566) and central  regions, but significant in the eastern region  for the mtDNA marker. Similarly, correlation between r = 0.0198, p-value = 0.8554) but significant in eastern region  for the ITS marker.A significantly positive correlation occurred between T. ciliata complex.Based on population genetic differentiation derived from both ITS and mtDNA markers and Equations (4) and (9), we calculated the relative rate of pollen to seed flow . Pollen D and Fu’s F values were negative in most populations  while nine populations had significant Fu’s F values . Only three populations exhibited significantly negative values of both Tajima’s D and Fu’s F values, including Xilin (XL) in Guangxi Province, Luodian (LD), and Wangmo (WM) in Guizhou Province. The three populations were in the central region and potentially underwent an expansion after bottleneck effects. In general, the whole population-based test did not show a significant departure from neutrality when the significance level was adjusted by Bonferroni correction . p-value = 0.84) and Harpending’s raggedness index (Rag) was 0.013 . Other estimates of parameters were p-value = 0.94), Rag = 0.020 , p-value = 0.53), Rag = 0.023 , T. ciliata complex potentially underwent a weak expansion in some local regions but did not exhibit a substantially global expansion.Analysis of mismatch distribution indicated that populations with significant Tajima’s y expand  althoughT. ciliata complex. Both marker analyses indicated the presence of phylogeographic structure  , Tamarix0.00217)  and Achy0.00188) , but sma= 0.316) , Medicagld pool) , and Fag= 0.833) . Our resT. ciliata complex. The results from nrDNA ITS marker consolidate previous findings derived from nuclear SRAP and SSR markers [T. ciliata complex. However, the results from mtDNA markers suggest three genetically differential regions. These three regions have three levels of elevations that increase from the eastern region (617.34 ± 264.19 m) to the central region (732.37 ± 250.87 m) and to the western region (1407.67 ± 321.81 m). Although IBD effects were significant across 29 populations, they were absent within each of three regions in terms of mtDNA markers. This suggests that geographical distance did not significantly impede seed dispersal within each region, but globally impeded seed dispersal in the natural distribution of T. ciliata complex.Analysis of genetic structure indicated that significant and substantial genetic variation occurred among populations within  markers ,13 whereT. ciliata. Its predominantly outcrossing system enhances pollen flow [T. ciliata. Generally, pollen dispersal can travel a longer distance than seed dispersal for predominantly outcrossing species [The difference in genetic structure between mtDNA and nrDNA markers is related to the effects of pollen flow on genetic variation of nrDNA markers and to the reproductive ecology of len flow . Pollen  species . Gene fl species ,26, popuntiation . When seIn the western region, barriers to seed dispersal could be strong due to the Hengduan Mountains (7556 m above sea level at the highest peak). Populations investigated in this study cover both the north  and south (SM and PW) regions of the Hengduan Mountains. Populations BS and YR are almost in the middle region of the Hengduan Mountains. Barrier to seed flow is expected to be greater than that to pollen flow although elevation did not significantly affect population genetic differentiation. This is because all populations except PW are located at comparable elevations (above 1300 m). As a result, the ratio of pollen to seed flow was much greater than 1  .In the eastern region, IBD effects were insignificant for both mtDNA and nrDNA ITS markers, implying that barriers to both seed and pollen flow were weak. Most investigated populations except WY are located at elevations below 1000 m. As expected, the ratio of pollen to seed flow was not much greater than 1, implying the presence of a certain level of seed flow among populations. The negative correlation between Machilus pauhoi [IBD effects were significant at the global level, implying limited seed and pollen flow. In addition, significant correlation between s pauhoi , where hD and Li and Fu’s F imply that most populations did not undergo recent expansion. Only three populations  had significantly negative values of Tajima’s D and Fu and Li’s F. Analysis of mismatch distribution supported that these populations potentially had expansion after bottleneck effects. These results imply that T. ciliata complex could undergo recent expansion in some local regions. One caution is that we only used nrDNA ITS sequences to detect bottleneck effects, which could limit the power of disentangling selection and demography due to their similar footprints in DNA polymorphism. The demographic change causes the entire genome variation while selection brings about regional variations along genomes. Thus, further analysis with multiple genes could help to elucidate these two processes [Analyses of Tajima’s rocesses .T. ciliata would not substantially shift from 2000 to 2050 year, relative to the adaptive regions in 1950–2000 year [T. ciliata, followed by seasonal precipitation. Based on the MaxEnt model, Zhang et al. [T. ciliata var. ciliata would potentially expand as climate changes in the future. However, Zhang et al. [T. ciliata var. pubescens would shrink in Yunnan Province. These results suggest that the natural distribution of T. ciliata complex would not substantially shift in the future.Previous simulations with the model of maximum entropy (MaxEnt) using 19 climate variables predicted that the most adaptive regions for 000 year . Future 000 year ,65. Hu [000 year  showed tg et al.  predicteg et al.  predicteThe phylogeographic patterns derived from both nrDNA and mtDNA markers provide insight into genetic conservation of this endangered species. In general, we need to consider multiple regions because of the significant IBD effects at the global scale and substantial genetic differentiation among regions. In both western and eastern regions, multiple populations deserve conservation because a certain level of population genetic differentiation occurred for both nuclear and organelle markers within each region. Those populations with higher haplotype diversity deserve protection. However, only a couple of populations could be appropriate for conservation in the central region. This is because population genetic differentiation was small for both nuclear and mitochondrial markers. The haplotype diversity within populations was low as well. Gene flow was extensive within the central region.T. ciliata complex [Division into three regions is probably supported from the results in a provenance trial of  complex . This ex complex ,69.T. ciliata complex, which was obtained in a separate study with different samples even in the same location [Our results indicate that pollen flow was more frequent than seed flow in the western and central regions but was slightly greater than seed flow in the eastern region. This could be related to the pattern of mating systems of location . A compllocation . Partiallocation . As disclocation , practicT. ciliata is an endangered tree species in China due to over cutting and its low natural regeneration. Currently, an important concern is its genetic conservation. In this study, we used both mtDNA and nrDNA ITS markers to elucidate the phylogeographic pattern of T. ciliata complex. We investigated twenty-nine populations that covered the range-wide distribution of T. ciliata complex in China. Specific conclusions could be drawn below: (1) Haplotypes of mtDNA markers were not randomly distributed in space and phylogeographic structure existed. (2) Population genetic differentiation was significant and most genetic variation occurred among populations. Isolations by distance (IBD) and by elevation (IBE) were present at the global scale. (3) Phylogenetic relationship derived from mtDNA markers indicated three genetically distinct regions each without significant IBD effects. The results from nrDNA ITS sequences showed two regions, consolidating previous findings using nrDNA SRAP and SSR markers. (4) The haplotype diversity was the largest in the western region, followed by the eastern region and the central region. Nucleotide diversity was the smallest in the central region. (5) The rate of pollen to seed flow was large in the western region but small in the central region. The rate of pollen to seed flow was slightly greater than 1 in the eastern region. (6) Most populations did not undergo expansion, with only a few populations showing expansion after bottleneck effects. Based on these phylogeographic patterns, we discussed a strategy of region-based genetic conservation and proposed to conserve multiple populations in the western and eastern regions and a few populations in the central region.


Bipolaris oryzae and Exserohilum rostratum are the main fungal species isolated from BSR infected tissues and they are frequently observed in the same field. However, we are lacking information on the genetic diversity and population structure of these fungi in Burkina Faso. The mode of reproduction is also unknown. The genetic diversity of isolates of B. oryzae (n=61) and E. rostratum (n=151), collected from major rice-growing areas of Burkina Faso, was estimated using genotyping-by-sequencing (GBS). The mean values for nucleotide diversity (π) were 1.9 x10-4 for B. oryzae and 4.8 x10-4 for E. rostratum. There is no genetic differentiation between the geographical populations of each species. The analysis of molecular variance revealed that 89% and 94% of the genetic variances were within the populations of B. oryzae and E. rostratum, respectively. For each species, four genetic clusters were identified by two clustering methods (DAPC and sNMF). The distribution of these genetic groups was independent of the geographical origin of the isolates. Evidence of recombination was detected in the populations of B. oryzae and E. rostratum. For B. oryzae balanced mating type ratios were supporting sexual reproduction. For E. rostratum overrepresentation of MAT1-2 isolates (79%) suggested a predominant asexual reproduction. This study provides important information on the biology and genetics of the two major fungi causing brown spot disease of rice in Burkina Faso.In recent years, Brown spot disease of rice (BSR) has been observed on leaves and seeds of rice in all rice-growing areas of Burkina Faso.  Rice is the second most important source of food after corn in Sub-Saharan Africa SSA, . HoweverAmong diseases, Brown spot of rice (BSR) is becoming a threat in SSA. This disease is expanding, and is currently estimated to be responsible for the loss of 3.3% of the regional rice production . BSR occIn Burkina Faso, the importance of BSR in rice nurseries and fields was already highlighted in 1962 . An outbBipolaris oryzae, B. bicolor, B. indica, B. victoriae, B. zeicola (Exserohilum rostratum (Bipolaris oryzae (syn. Cochliobolus miyabeanus) and Exserohilum rostratum (syn. Setosphaera rostrata), are the two species most frequently isolated from BSR infected tissues   . Most sttructure . Sexual ilibrium . Bipolariability . Investiplotypes .B. oryzae starts with the deposition of asexual spores (conidia), on aerial parts of rice. Conidia produce a germ tube that adhere to the plant surface thanks to a mucilaginous substance. Then, an appressorium is formed that allows direct penetration of cells  was released recently. The description of the complete circular mitochondrial genome of E. rostratum (ZM170581) isolated from maize followed (E. rostratum strains (BioSamples: SAMN14931704 and SAMN30522240) isolated from rice have also been sequenced recently (https://www.ncbi.nlm.nih.gov/biosample/). For B. oryzae a reference genomes are available from a strain isolated from cultivated rice   . These dlustris) .B. oryzae in rice fields in Burkina Faso. Our knowledge of E. rostratum in Burkina Faso is limited since no population study was performed to date. The objectives of this study were to: (i) assess the genetic diversity, (ii) describe the genetic structure, and (iii) determine the mode of reproduction of B. oryzae and E. rostratum populations from major rice-growing areas of Burkina Faso.However, there is a lack of information on the genetic structure and diversity, as well as the mode of reproduction of Oryza sativa) showing symptoms of BSR were collected in 2018 and 2019 in different rice fields from Bagré, Bama, Banfora, Banzon and Sourou  and incubated in the oven at 65°C for 2 h. The tubes were then centrifuged for 3 min at 8,000 rpm and approximately 900 µL of the supernatant was transferred to a new 2 mL tube. One mL of chloroform isoamilalcohol  was placed in each tube and mixed by inversion before being centrifuged at 10,000 rpm for 10 min. After centrifugation, 700 µL of the supernatant was transferred to 1.5 mL tubes. Six hundred µL of isopropanol cooled to -20°C and 210 µL of 5M NaCl were added and mixed by inversion. The tubes were kept at -20°C overnight. They were then centrifuged at 4°C for 30 min at 13,000 rpm and the supernatant was removed by inverting the tubes. Five hundred µL of 70° ethanol was added to each tube and centrifuged at 4°C for 5 min at 13,000 rpm. Ethanol was removed by inverting the tubes and draining on absorbent paper. The tubes were dried for 1 h in the fume cupboard. DNA was re-suspended in 100 µL of sterile milliQ water. The quality and quantity of the extracted DNA was assessed by electrophoresis in 1% (w/v) agarose gels and spectrophotometry (Nanodrop™ 2000).Genomic DNA was extracted from fresh mycelium with modifications to the cetyl trimethyl ammonium bromide (CTAB) method described by ApeKI (5 U) at 75°C for 2 h. The restriction digestion of DNA was followed by ligation with adapters. The adapters included different barcodes for tagging individual samples and common adapters. The ligation was performed using T4 DNA ligase at 22°C for 30 min and the ligase was inactivated by holding at 65°C for 10 min. Two pools were created. To assess the reproducibility of GBS, each pool contained 6 randomly selected and duplicated isolates. Ligation products were purified with the QIAquick purification kit from QIAgen. The library was enriched by PCR to 50 µL of reaction solution including 25 µL of 2X Taq Master mix NEB, 19 µL of water, 2 µL of DNA and 2 μL each of the forward and reverse primers (10 μM). Amplification was done according to the following program: 98 ˚C for 30 s, followed by 18 cycles of 98 ˚C for 10 s, 65 ˚C for 30 s, 72 ˚C for 30 s, ending at 72 ˚C for 5 min. PCR products were purified with QIAgen’s QIAquick Purification Kit before being evaluated with BioAnalyzer 2100 (Agilent Technologies) for fragment size distribution. The GBS libraries were sequenced on an Illumina HiSeq 4000 producing 150 bp paired-end reads.The preparation of GBS libraries was carried out in collaboration with the genotyping platform of the UMR AGAP/CIRAD according to the method described by https://github.com/sravel/RattleSNP) was used to process the data. The workflow performs demultiplexing, sequence mapping to the reference genome and variant calling. The sequences of B. oryzae isolates were mapped onto the genome of strain ATCC 44560 (E. rostratum onto the genome of BF9006 sequenced by CIRAD (https://www.ncbi.nlm.nih.gov/biosample/SAMN30522240/). Prior to variant calling, strains with a mapping rate of less than 75% to the reference genome, a library size of less than 100,000 reads and a sequencing depth of less than 3 x were considered low quality data and excluded from the dataset. SNPs from strains meeting the criteria (61 isolates for B. oryzae and 151 isolates of E. rostratum) were filtered for missing rate (–max-missing 0.70), minor allele frequency (–maf 0.05), depth (–minDP 2) and site quality (–minQ 30) using VCFtools version 01.16   and sNMFsNMF of the LEA R software package was used to estimate the number of discrete genetic clusters (K) and admixture coefficients . The optST) was calculated using the ‘genet.dist’ function in the hierfstat package of R   were perage of R , followiage of R .bitwise.dis function of the R poppr package  values were calculated with VCFtools across the genome with 10 kb windows .2) over a 10 kb window using the PopLDdecay program  and clonality was tested with the PHI test from genotyping data. Pairwise LD between loci was calculated based on allelic frequency correlations  and E. rostratum (n=151) collected in Burkina Faso, was estimated using genetic markers (SNPs) obtained by genotyping-by-sequencing. After alignment to the B. oryzae ATCC 44560 reference genome composed of 619 scaffolds and filtering, 28,907 SNPs were identified in the B. oryzae samples (61 isolates). Sequences of E. rostratum isolates (n=151) aligned to the BF9006 isolate reference genome (377 scaffolds) yielded 102,464 SNPs.The genetic diversity of isolates of B. oryzae and E. rostratum populations from Burkina Faso, the isolates were clustered by DAPC according to their multilocus genotypes. Inference of ancestry was also performed with sNMF.To study the B. oryzae, the bayesian information criterion (BIC) of DAPC suggested the best clustering for four genetically distinct populations  of DAPC suggested the best clustering for four genetically distinct populations  and 7% (11/151) of the isolates in each sample of B. oryzae and E. rostratum, respectively  between isolates from different rice-growing areas ranged from 2.20 to 3.04 with an average value of 2.45. These indices are influenced by population size and do not allow comparison between different localities. The Simpson index corrected for sample size ranged from 0.999 to 1  indicating a high level of genotypic diversity within each population. The regularity index (E.5) was equal to 1 for all populations.Genotypic and gene diversity indexes are presented in -4 to 2.4 x10-4 with an average of 1.9 x10-4. The Banzon population also showed the lowest value of nucleotide diversity (1.3 x10-4). The Sourou population showed the highest diversity (2.4 x10-4). It was followed by the populations of Bama, Bagré and Banfora.In terms of genetic diversity, nucleotide diversity values ranged from 1.3 x10E. rostratum, the genotypic diversity (H) between isolates from different rice-growing areas ranged from 1.61 to 4.23 with an average value of 2.98. The Simpson index corrected for sample size ranged from 0.999 to 1  indicating high genotypic diversity within each population. The regularity index (E.5) was equal to 1 for all populations. The values of nucleotide diversity varied between 5.7 x10-4 and 9.8 x10-4. The average value was 4.8 x10-4. The isolates from Bama showed the highest value of nucleotide diversity (9.8 x10-4), while the lowest value was observed in Bagré (5.7 x10-4).For B. oryzae populations from Burkina Faso, Linkage Disequilibrium (LD) pattern showed a rapid decay to less than 1 kb in all populations  produced pseudothecia containing ascospores. Concerning E. rostratum, out of 364 crosses made, no pseudothecia were observed.To test the sexual fertility of B. oryzae isolated from rice in Burkina Faso and the first study of E. rostratum populations. It is also the first study using genotyping-by-sequencing based analysis to characterize populations of B. oryzae and E. rostratum.Brown spot of rice (BSR) is an emerging disease in Burkina Faso. Although it was already described, the incidence of the disease increased very recently to reach 80% of farmer’s fields from the western part of the country . KnowledB. oryzae and E. rostratum populations were sampled in five distinct rice-growing areas of Burkina Faso with an average distance of 365 km between sites . For each fungal species, the five geographic populations did not display a significant genetic differentiation. The absence of genetic differentiation among B. oyzae geographical populations was already observed in Bangladesh . So, a 1:1 segregation of mating types is expected after sexual reproduction. All these genetic and biological characteristics are consistent with sexual reproduction. However, sexual reproduction of B. oryzae was seldom observed in the crosses we performed in vitro and pseudothecia  have never been observed in the field. Timing sexual reproduction phenomena remains problematic and it is difficult to know if they are still ongoing or if they occurred in the past . These results are similar to those of E. rostratum isolates from rice in Malaysia. The linkage disequilibrium and homoplasy tests did not reject the hypothesis of recombination within the E. rostratum populations of Burkina Faso. However, sexual reproduction of E. rostratum was not observed in the crosses performed in vitro and pseudothecia have never been observed in the field. In addition, the strong bias toward one mating type (79%) suggested that actual E. rostratum populations are predominantly asexual. In addition, we identified 10 isolates of E. rostratum, i.e. 6% of our sample that carried both the MAT1-1 and MAT1-2 idiomorphs. Such isolates with two mating types were also identified within E. turcicum populations from maize in China  repository, accession number PRJEB56783.KK: Collection of samples, Data production, Data analysis, Writing and proofreading AK: Collection of samples, Writing and proofreading. HA: Data production, Writing and proofreading. JM: Data production, Writing and proofreading. SG: Data production, Writing and proofreading. LB: Data production, Writing and proofreading. LC: Data production, Writing and proofreading. SR: Data analysis, Writing and proofreading. FC: Data analysis, Writing and proofreading. MB: Collection of samples, Writing and proofreading. CT: Collection of samples, Writing and proofreading. M-HL: Writing and proofreading. DT: Data analysis, Writing and proofreading. All authors contributed to the article and approved the submitted version.www.pathobios.com; twitter.com/PathoBios). We are very grateful to the fieldworkers in Burkina Faso notably Kader Guigma, Yacouba Kone, Compaoré Abraham Terry Cedric and Hébié Bakary. We thank the technical support structures  and the rice farmers from the study areas for their kind collaboration during sampling rice leaves and seeds. We also thank Pierre Mournet and Julien Frouin for their help in the development of the GBS technique at the UMR AGAP/CIRAD genotyping platform. Part of the experiments were supported by Agropolis Fondation (Labex Agro:ANR 10 LABX 0001 01) under RiPaBIOME project (ID 1702 011).We thank the RICE CRP and the Islamic Development Bank for their financial support. This work was facilitated by the “International joint Laboratory LMI PathoBios: Observatory of plant pathogens in West Africa: biodiversity and biosafety” (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.