Heleobia ascotanensis, a freshwater gastropod restricted to the Ascotán saltpan in the Chilean Altiplano. The current distribution of the species is limited to twelve isolated or partially isolated springs that were affected by transitions between humid and arid periods during last glaciations. The genetic analysis of 322 specimens showed that H. ascotanensis is subdivided into three genetically divergent populations, with low and moderate degrees of historical gene flow among them and incipient morphological differentiation as a consequence of genetic and geographical isolation. Molecular analyses revealed different demographic histories among populations which seem to respond independently to climatic events, probably due to an environmental imposition and idiosyncratic strategies developed to cope with water availability. The results of this study and co-distributed taxa support the hypothesis that contemporary and historical events have influenced microevolutionary differentiation of these snails, although there is a need to complement further information to predict genetic or morphological divergence at microgeographic scale.In the present study, we focus on the phylogeographic pattern, demographic history and morphological differentiation of  Contras50–1850) . With th50–1850) .Taxa distributed in patchy aquatic systems show highly geographically structured populations, probably because appropriate habitats are often surrounded by inappropriate environments, therefore gene flow usually depends on the dispersal capabilities of the species . The genHeleobia Stimpson, 1865 inhabit a variety of brackish and freshwater ecosystems in the Andean Altiplano . Ultimately, we proposed that the microevolutionary differentiation would be similar to the patterns described in co-distributed taxa, but not identical, reflecting an idiosyncratic response to the same historical events.In the present study, we examined the phylogeographic and demographic history of 2), whose main water body (18 km2) is generated by a series of springs located mainly in the eastern area of the system  between 21.2–21.4°S and 68.1–68.2°W at 3,760 m a.s.l. The hydrological system extends around 33 km long by 16 km wide at its maximum range, representing a surface which is mainly covered by evaporitic deposits  were sampled, and in April 2013 Spring 1 was visited. Also, in April 2013 we collected additional specimens from Spring 11 and Spring 12, these individuals share the same genetic pool (haplotypes) and morphometric features as the specimens collected from the corresponding springs the year before, therefore they were considered as the same population. A minimum of 60 snails were collected by hand or using a small sieve (1 mm mesh) from sediment or macrophytes  A.W. Hill and Ruppia filifolia (Phil.)) in each spring. Sampled specimens were preserved in absolute ethanol for posterior morphological and genetic analyses. Gonzalo A. Collado was authorized to collect Heleobia snails in Chile .The specimens of Genomic DNA was extracted from specimens using the cetyl trimethyl ammonium bromide (CTAB) method . Partialπ) in DnaSP  and using the Dirichlet model. Analyses were run using 108 MCMC iterations and collecting data at every 100th iteration , number of haplotypes (K), haplotypic diversity (H) and nucleotide diversity  as implemented in BEAST2  on XSEDEtebrates . The firtebrates .FST) and geographical distances matrix between springs. We estimated the magnitude and direction of historic gene flow among spring populations grouped according to the results of the genetic structure analysis (haplogroups and genetic clusters), using MIGRATE-n v3.2.7 (M) among the studied populations were performed by grouping the individuals according to haplogroups and genetic clusters . For the analysis, a Brownian motion model was run to estimate M, where M = m∕μ . The parameters (Φ)  and M followed an exponential distribution , heating was set with four temperatures  with a static scheme. We ran 10 short chains with a total of 10,000 genealogy samples and three long chains with 1,000,000 samples, following a burn-in of 1,000 samples; three independent runs were performed.The PAST3 software  was usedn v3.2.7  on CIPREHeleobia populations from the Ascotán saltpan using outline analyses based on the elliptic Fourier analysis (EFA). To avoid issues associated with developmental size changes, we exclude juveniles from the analysis. Since we were unable to determine which specimens had reached maturity from shell characters by themselves, we used size as a proxy, analysing specimens in the upper third of the size distribution. Samples used in morphometric analyses (N = 322) corresponded to the same samples used in the molecular analysis. Since genetic analyses results were partially discordant, three different datasets were analysed: (i) the first dataset contained individuals organized by its spring of origin; (ii) the second dataset contained individuals sorted by haplogroups and; (iii) the third dataset contained individuals pooled by genetic clusters defined by GENELAND analysis.We studied shell shape variation of Regarding the morphological study, the image of the shell of at least 23 snails per spring  was takeMF447901–MF448222). The best-fitting model of nucleotide substitution, as determined bModelTest, was the General Time Reversible model plus invariable sites (GTR+I), with I = 0.81. The mtDNA COI sequences of Heleobia showed a pattern of moderate haplotype diversity (H = 0.810), but high nucleotide diversity (π = 0.0074). At the spring level, Spring 11 presented the highest value of haplotype diversity and the highest value of nucleotide diversity was found in Spring 4. Of the remaining springs, the lowest value of haplotype diversity was detected in Spring 8 and the lowest value for nucleotide diversity in Spring 12 , and the closest springs showed lower values of differentiation when compared to remote springs  and for all populations grouped by haplogroups . Signals of population expansion were detected in two populations grouped by haplogroups  that did not refute the demographic model of spatial expansion for the species , and probably due to extremely low genetic diversity in Haplogroup 2, none demographic event was detected. The time of expansion for the Haplogroup 3 (Spring 11) was estimated to have started in the Late Pleistocene (c. 30 kya) through the Last Glacial Maximum  until the Early Holocene (c. 10 kya), after this population expansion the effective population size (Ne) remained constant (c. 10 kya) . Probably due to the extremely low genetic diversity in Haplogroup 2 (Spring 2 to Spring 6), demographic analyses were unable to detect a demographic event in this group. The analysis showed negative and statistically significant values of Fu’s FS neutrality test at the global level  .r = 0.6233, P <  0.005). Estimations of past migration rates  indicated that Haplogroup 2 showed higher immigration rates, followed by Haplogroup 3, whereas Haplogroup 1 showed lower rates  was mainly related with shell width while PC2 (5.01%) corresponded to changes in the whorls suture depth . Only inH. ascotanensis has a high phylogeographic structure, even when its distribution encompass only a few kilometres. Using diverse analysis to examine spatial and temporal patterns of mtDNA sequence variation and morphological differentiation, a complex pattern of historical and demographic events was uncovered in this endemic snail. Three main findings were revealed from the present study: (i) a pronounced mtDNA sequence differentiation at small spatial scale; (ii) complex historical and demographic influences on sequence divergence and diversity within and among springs and; (iii) an incipient morphological differentiation coupled to genetic divergence.The results of the current study revealed that The haplotype network and genetic clusters defined by GENELAND analysis indicate that there are three genetic entities inhabiting the Ascotán saltpan, although the composition of the groups was partially discordant. Both analyses defined specimens from Spring 11 as a different population (Haplogroup 3 and Cluster III), a result congruent with the physiographic arrangement of the saltpan since Spring 11 is the southernmost spring in the system and currently is completely isolated. However, there was an inconsistency in the northern springs’ classification, the haplotype network defined a haplogroup widely distributed in the saltpan (Haplogroup 1), covering all springs except Spring 11. Spatially nested in Haplogroup 1 was defined Haplogroup 2, which mainly grouped samples from Spring 2 to Spring 6. According to the haplotype network, haplogroups 1 and 2 were the most genetically distant groups . In contrast, GENELAND analysis defined Cluster I as the individuals from Spring 1, Spring 7 to Spring 10, and Spring 12; and Cluster II contained individuals from Spring 2 to Spring 6. The Cluster II probably reflects an influence of geographical proximity among springs in the result, since the analysis classified the most genetic distant individuals into the same population. This was also evidenced by the AMOVAs analyses, which showed that the genetic variance was better explained sorting samples by haplogroups than genetic clusters.in situ differentiation); but considering that the saltpan was a continuous hydrological system in the past, and that afterwards the passing of humid periods to a dry season the system was divided into isolated or partially isolated springs suggest that this is a less feasible scenario. The range expansion considered in the first hypothesis may be due to dispersal mediated by high rainfalls seasons during SASM  showed a constant population expansion and growth since c. 0.30 kya until c. 0.10 kya, whereas northern populations show signals of recent demographic expansion  and recent demographic stability (Haplogroup 2). Although the exact mechanisms that account for this are unclear, the constant population expansion denoted by BSP analyses in Haplogroup 3 could be related with the last three major humid periods reported in the Altiplano associated with paleolakes Sajsi (c. 21–25 kya), Tauca (c. 14–18 kya) and Coipasa (c. 11–13 kya)  . Since S–13 kya) . Under t–13 kya)  or an inconditio sine qua non to the saltpan formation. Probably the dynamic change in water availability impose a pressure that promote the development of strategies to respond to a variable environment, and the contrasting demographic histories and genetic diversification suggest that the strategies could be different among the populations of H. ascotanensis; similar environmental changes have caused comparable evolutionary responses in analogous hydrological-desert systems (Estimates of the population expansion in Haplogroup 3 (Spring 11) corresponded approximately to the transit through three major humid periods , suggesting that these snails persisted isolated in the saltpan during the LGM, since they have unique haplotypes not present in other springs. The results indicate that the transition from humid to arid periods had apparently little effect on the population size in Haplogroup 3, which is an important finding considering that these strictly aquatic organisms need to cope with an increase in salinity and dehydration, which are  systems .H. ascotanensis, the morphological differentiation showed by Spring 11 could be induced by genetic differentiation, possibly caused by geographical isolation and adaptive responses to dynamic environmental conditions, as has been reported in the gastropod Potamopyrgus antipodarum   . This arH. ascotanensis compared to other taxa co-distributed in the Ascotán saltpan indicates that species show a deep genetic structure, but the times of divergence and the organization of population is slightly different  and O. ascotanensis (killifish) present a common pattern in which Spring 11 is genetically divergent from other springs, a result probably related with the geographical isolation of this system. On the other hand, genetic structure in northern springs are idiosyncratic of each taxon, implying that each specie (or population) respond independently to historical changes and that the particular habitat itself is not a sufficient predictor of the genetic differentiation.Finally, our results lend strong support to the hypothesis that species (and populations) respond differently to the same environmental and historical circumstances. Ecological conditions and interactions, and environmental impositions (such as aridity) influence the genetic pool or structure in different taxa, but biological properties  may led to large differences in phylogeographic history . On the ifferent . ConsideHeleobia from the Southern Altiplano. We present evidence of a high genetic structure in H. ascotanensis. Molecular analyses detected three main haplogroups well differentiated and related with the saltpan physiography. Gene flow and demographic analyses showed different connectivity and histories among populations, indicating that each haplogroup responded independently to the fragmentation of the aquatic system occurred during humid-arid transitions that characterized the Plio-Pleistocene period. Morphometric analyses detected a subtle differentiation, and this variation is concomitant with genetic divergence found in Spring 11, the southernmost spring. The genetic structure found in H. ascotanensis is similar to the pattern detected in co-distributed taxa; however, temporal and spatial differences among species populations indicate that each taxon (or population) responded differently to the same historic and contemporary climatic changes. Further work in H. ascotanensis should focus on increasing analysed loci , a strategy that would be helpful for evaluating in detail the mode and tempo of the genetic differentiation detected in this species.In this study, we provide the first phylogeographical investigation on a species of the genus 10.7717/peerj.5802/supp-1Figure S1Shell outlines changes associated with Principal Component 1 (PC1 = 78.64%) and Principal Component 2 (PC2 = 5.01%).Click here for additional data file.10.7717/peerj.5802/supp-2Figure S2Principal component (PC) analysis sorting individuals by (A) springs; (B) haplogroups (median-joining network); and (C) genetic clusters . PC1 and PC2 explained 78.64% and 5.04% of the variance, respectively.Click here for additional data file.10.7717/peerj.5802/supp-3Table S1Pairwise FST values from mtDNA (COI) analysis are below the diagonal and significance values are above the diagonal.Click here for additional data file.10.7717/peerj.5802/supp-4Table S2Results of the non-parametric multivariate analysis of variance (PERMANOVA) and linear discriminant analysis (LDA) grouping individuals under three classifications schemes . For LDA analysis, results are expressed as the percentage of correct classification.Click here for additional data file.10.7717/peerj.5802/supp-5Supplemental Information 1Fasta formatted.Click here for additional data file.


The genetic patterns of these protogynous, insect-pollinated tree species occurring in fragmented, highly-disturbed landscapes were investigated. Datasets containing a total of 340 individuals were tested for their genetic structure and degree of inbreeding. Analyses for genetic structure depicted structuring between species, i.e. strong species integrity. Within the species, all but one population pair were considered moderate to highly differentiated, i.e. no indication of extensive gene flow between populations. No overall correlation was observed between genetic and geographic distance of the pairwise species’ populations. In contrast to the pronounced genetic structure, there was no evidence of inbreeding within the populations, suggesting mechanisms favouring cross pollination and/or selection for more genetically diverse, heterozygous offspring. In conclusion, the data illustrate that the Neotropical Magnolias in the context of a fragmented landscape still have ample gene flow within populations, yet little gene flow between populations.Conserving tree populations safeguards forests since they represent key elements of the ecosystem. The genetic characteristics underlying the evolutionary success of the tree growth form: high genetic diversity, extensive gene flow and strong species integrity, contribute to their survival in terms of adaptability. However, different biological and landscape contexts challenge these characteristics. This study employs 63  Conservation genetics utilises a representative sample of DNA and organisms to quantify and study genetic diversity to preserve species as dynamic entities capable of coping with environmental change  occurring at tropical latitudes, hereafter named Neotropical Magnolias. Magnolia trees provide an interesting case-study with bisexual, protogynous flowers, specialised beetle pollination with tepal movement, variable flowering phenology and seed dispersal by animals  employ the SSR markers for genetic species delimitation between Caribbean Magnolia species; (3) search for patterns of extensive gene flow between Caribbean Magnolia (sub)species and populations; and (4) test for signs of inbreeding within the Neotropical Magnolia populations.This study aims to (1) provide Magnolia from the Caribbean and Mexico, is given in Fig. ex situ collection of M. dealbata.Sample information of the 17 different taxa  and 17 populations included in this study are given in Table S) are given in Table For the 17 populations included in the full genetic analyses, Average Pairwise Distance between individuals (APD), Maximum distance between consecutive individuals (Max), Spatial extent of the populations (SpE) and number of sampled individuals per populations (NAll 347 leaf samples were dried in silica gel and their DNA was isolated using a modified cetyltrimethylammonium bromide (CTAB) (Doyle and Doyle Magnolia species: Magnolia lacandonica (MA39), M. mayae (MA40), M. dealbata (MA41), and M. cubensis subsp. acunae (MA42). The development of the enriched microsatellite library was outsourced to Allgenetics®  where enrichment was performed using the Nextera XT DNA kit probes  with the following motifs: AGG, ACG, AAG, AAC, ACAC and ATCT. The library was sequenced on an Illumina MiSeq® platform.Primer pairs were developed to amplify sequences containing SSR repeats based on four Neotropical From the 4 × 500 predetermined SSR primer pairs provided by Allgenetics®, 176 were selected for further testing: 49 developed from MA39-reads, 20 developed from MA40-reads, 20 developed from MA41-reads and 87 developed from MA42-reads. Selection of the 176 SSR markers was carried out randomly, respecting the characteristics specified in Guichoux et al. . The for2 (Thermo Fisher Scientific), 0.125 µM dNTP, 5U of TrueStart Hot Start DNA polymerase (Thermo Fisher Scientific), and 0.4 mg/ml BSA  per reaction. PCR products were run on a 1% agarose gel, stained with ethidium bromide and visualised under UV-light. Every (sub)species × primer combination was scored. Amplification scores of the 63 published SSR markers are given in the Supplementary Table All 176 markers were screened for amplification success on the 17 taxa, each represented by one randomly selected sample. PCRs were performed on a total volume of 13 µL under the following conditions: 2 min at 95 °C; 35 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 90 s; 72 °C for 6 min. The Master Mix contained 0.2 µM forward primer, 0.2 µM reverse primer, 5 ng/ml DNA (suspended in 1 × TE buffer), 1 × TrueStart Taq Buffer (Thermo Fisher Scientific), 1.5 µM MgClMagnolia species, comprising four individuals per predefined population. The individuals for the test-multiplexes were selected to be spatially spread throughout the populations and have 260/230 and 260/280 OD  ratios approximating 2. The (sub)species × primer combinations were scored: 63 were considered polymorphic and unambiguous SSR markers in at least one of the ten tested taxa . The results were analysed in Geneious v.8.1.9 species, five different supraspecific (i.e. above species level) datasets were instated. Dataset 1 comprises 340 individuals representing 17 populations, genotyped for all their polymorphic and monomorphic loci  level using nine species-datasets (i.e. the taxon-datasets of the two M. cubensis subspecies were joined) and 17 population-datasets.To assess the utility of the SSR markers for genetic species delimitation between closely located Caribbean A first batch of analyses was conducted in STRUCTURE v.2.3.4  on datasets 1, 2 and 3 were executed in R using the package adegenet Jombart . In the ST values  using 1000 bootstrap replicates as a confidence measure.Pairwise FST values using 9999 permutations. Coordinates of one individual were taken as a representative of its population. Species geographic distance was averaged over the populations of the species.Mantel tests on the supraspecific level were performed in GenAlEx v.6.5  for each locus and population was calculated in FSTAT. Tests to detect significant deviations from Hardy-Weinberg proportions (HWP) were calculated in GENEPOP, performing 2-tailed exact tests for each locus in each population. Complete enumeration was performed whenever possible (Louis and Dempster e), and observed heterozygosity (Ho).To test for inbreeding within the Caribbean Overall, 82–92% of the primer pairs amplified, of which 53–67% were scored to be a single amplification product (Supplementary Table M. cubensis (MA42_028), M. domingensis (MA39_199), M. ekmanii , M. hamorii , M. lacandonica (MA39_182), M. pallescens , M. portoricensis (MA42_481) and M. splendens .Results of detection and frequency of null alleles per marker × population combination are given in Supplementary Table Magnolia domingensis and M. lacandonica showed a number of SSR markers with associated alleles that were higher than expected for the number of pairwise tests executed. The other eight taxa fell within their confidence intervals of false positives, whereby one significantly associated pair of SSR markers was detected in M. pallescens (MA40_045 × MA42_472).Associated alleles per marker × species combination are given in Supplementary Table ST values range from 0.216 to 0.618 for dataset 1, 0.166 to 0.472 for dataset 2 and 0.130 to 0.308 for dataset 3 . Mantel tests on the supraspecific pairwise distances were significant for dataset 1 (p = 0.000), but not for dataset 2 (p = 0.080) and dataset 3 (p = 0.256). See Supplementary Figure Supraspecific ΔK and Mean LnK plots are depicted in Supplementary Figure ST values can be found in Table ST values are depicted in Supplementary Figure p = 0.084, dataset 3: p = 0.080): see Supplementary Figure Infraspecific ΔK and Mean LnK plots are depicted in Supplementary Figure p = 0.05). TOR showed significant deviation from HWP for 7/29 loci .Detailed results on the population statistics calculated on the ten taxon-datasets are listed per marker, population and subset in Supplementary Table The data on marker development show an attrition of usable SSR primer pairs during post-sequencing marker development, which is a general issue in SSR development  + M. pallescens; however, in Figs. . domingensis + M. pallescens) + M. hamorii is put forward. Although native to the same island as the three Dominican Magnolias, M. ekmanii is conspicuously differentiated from them, as well as from all other species. There is a hint that EKM is most closely related to the Cuban Magnolias: their pairwise FST calculated on dataset 1 is significantly lower compared to the other EKM pairwise comparisons   is most likely due to a major reduction in population size: a recent bottleneck. This is concluded given that (a) there is genome-wide allelic association for all three populations, in contradicting strengths when compared across populations pairs per species; and (b) the visited locations had a high degree of disturbance. The samples studied of the ROD and LAC populations indicate that they have not been able to recombine their genetic material since the bottleneck. For the YAJ population it cannot be excluded that a high degree of kinship between the samples produced the results. The 20 samples of this population could only be collected at the border of, what is expected to be, a much larger population and include two adults and 18 juveniles. It is recommended to either exclude the population from species-focused analyses, or to recollect a better representation of the population.Magnolia species studied for comparison , in the same extent as the three populations suspected to experience the Wahlund effect. They also have the highest reported values of Ho and He compared with the other Magnolias of this dataset. In the Splendentes-normalized-dataset (dataset 3), the statistics of M. hamorii do not stand out anymore. However, they remain in the higher range of values, now similar to the statistics found for M. cubensis, M. portoricensis and M. splendens. The latter three species also show A- and H-values in the higher range of values in the calculations of their full taxon-datasets.We cannot easily label the observed genetic diversity Table  to be heSecondly, GRA, MAN and ROD report the three lowest A values in their taxon-datasets, and MAN and ROD show lower A and H values than the GRA and BAR populations, respectively. The lower statistics of the GRA and MAN populations confirm that conservation management of Magnolias in the last remaining forests of Haiti is urgent. Interestingly, even though MAN appeared deforested in an equal, or even higher extent than the ROD population, its alleles tested to be independently associated. LD decreases after recombination events at a rate that depends on the recombination frequency and generally takes more than one generation of random mating to restore, even for  unlinked loci Slatkin . Hence, IS) of the 14 populations not suspected to be under the Wahlund effect, do not significantly differ from zero. Taking the reproduction biology of Magnolias into consideration, both arguments in favour and against this result can be listed. No (apparent) inbreeding seems likely given that (1) Magnolia flowers are reported to be protogynous  the reproductive biology of the Magnolia trees  and its limits, shaping the high genetic differentiation between, and high gene flow within populations; (2) the genetic diversity of closely related non-threatened Magnolia species, either in fragmented or continuous landscapes, placing past and future SSR Magnolia studies on threatened populations in perspective; and (3) splitting Magnolia conservation genetic studies according to age, to exclude this potential time-lag and detect whether or not the younger generation of Magnolia trees are genetically depauperate (e.g. Graignic et al. In conclusion, the data showed structuring on three different levels. Firstly, the supraspecific structuring confirms high species integrity with no extensive gene flow between species. Secondly, species sets within islands express lower genetic structuring but no signs of current gene flow, which is interpreted as a more recent shared ancestry. Thirdly, the populations within species also show moderate to strong differentiation, uncorrelated with the distance between the population pairs. The generalisation of extensive gene flow in trees does not withhold in the studied species. Our data support the hypothesis that the generalized concept of extensive gene flow in trees mainly applies to wind pollinated trees or trees that have larger animal vectors such as mammals (Dick et al. Data available from Dryad: 10.5061/dryad.0m625h4.Genbank accession numbers for the 63 original sequences on which the primers were developed range from MH923371 to MH923433.Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7


Ginkgo biloba L.) is one of the oldest living plants and a fascinating example of how people have saved a species from extinction and assisted its resurgence. By resequencing 545 genomes of ginkgo trees sampled from 51 populations across the world, we identify three refugia in China and detect multiple cycles of population expansion and reduction along with glacial admixture between relict populations in the southwestern and southern refugia. We demonstrate multiple anthropogenic introductions of ginkgo from eastern China into different continents. Further analyses reveal bioclimatic variables that have affected the geographic distribution of ginkgo and the role of natural selection in ginkgo’s adaptation and resilience. These investigations provide insights into the evolutionary history of ginkgo trees and valuable genomic resources for further addressing various questions involving living fossil species.As Charles Darwin anticipated, living fossils provide excellent opportunities to study evolutionary questions related to extinction, competition, and adaptation. Ginkgo ( Ginkgo is one of the living fossils from the plant kingdom. Here, authors conduct population genomics analyses to reveal its refugia and demographic history, and provide evidence of multiple anthropogenic introductions of ginkgo from eastern China into different continents. A once-diverse and widespread group of gymnosperms, the ginkgo lineage has survived glaciations as a relic in China, has no current living relatives and has recently been redistributed globally via human-aided introductions5. As one of the best-known and most distinctive trees worldwide, ginkgo has fascinated humans for centuries by its significance in biology and medicine as well as its power as a source of artistic and religious inspiration8.Despite numerous efforts on investigations of living fossils, mysteries have remained for centuries regarding why living fossils appear essentially unchanged , whether living fossils are evolutionary dead ends and what roles humans have played in the survival and spread of living fossils. The ginkgo tree is an enigmatic living fossil, characterized by morphological stasis with almost no morphological change for at least 200 million years11, population dynamics in response to Pleistocene climate change13, the roles that humans have played in the dispersal of ginkgo trees in China15 and when and how ginkgo trees were introduced to Japan/Korea, Europe, and North America6 as well as the potential factors contributing to the persistence and resilience of the species16. Exploration of these questions has been impeded by difficulties in obtaining sufficient samples that represent the entire gene pool of this living fossil and the limited genetic markers available for such an isolated gymnosperm species. Given that the ginkgo draft genome was completed16, genome-wide resequencing of ginkgo trees across the world offers an unprecedented opportunity to reveal the evolutionary history of ginkgo, which would provide further insights into the extinction, adaptation, and resilience of living fossils, and eventually facilitate the conservation and management of rare and endangered species in general.Although many investigations on the evolutionary history of this mysterious tree have been undertaken, numerous uncertainties and controversies remain, including the identification of relict populations and potential refugia5. Specifically, we identify four ancient genetic components and three glacial refugia of ginkgo populations in China. Our demographic analyses detect multiple cycles of population expansion and reduction along with glacial admixture of relict populations in northern China in response to Pleistocene climate change. We also present a scenario on how ginkgo trees have been dispersed out of refugia and out of China based on multiple lines of evidence. Particularly, we characterize the major bioclimatic variables that have shaped the geographic distribution of ginkgo and addressed the potential roles of natural selection in the survival and resilience of ginkgo populations. These investigations lay an important foundation for further studies of the potential mechanism of ginkgo’s survival and resilience.In this study, we resequence 545 ginkgo genomes and uncover the evolutionary history of this living fossil. Taking advantage of nuclear and chloroplast genomic data, we investigated unresolved debates on the evolutionary history of ginkgo17, we obtained 214.33 million raw SNPs and 161.04 million high quality SNPs (Dataset 0) using a strict filtering standard  occurred between the southwestern populations (the SWEST lineage) and the eastern populations (the EAST lineage) plus the southern populations (the SOUTH lineage)  being admixed with three ancient components  Fig. . It is ents Fig. . A neighnts Fig.  and 7, i14. Pairwise FST values calculated based on plastome dataset also revealed deep divergence among the lineages with a large number of identical sequences both within and between lineages  and 2.19–2.41 × 10−3 for the average pairwise diversity within populations (π) was observed for all four lineages of Chinese ginkgo populations  , which reflects the characteristics of admixed origin of ginkgo populations in northern and central China and supports the argument of glacial admixture of relict populations in many species in eastern Asia25.A comparably high level of genetic diversity 26 to assess population size changes and obtained a well-defined demographic history from 20 million to 60,000 years ago .An important question about living fossils is whether their extinction is underwayago Fig. . We foun28, we simulated the demographic fluctuations since 0.4 mya  and SOUTH (71.55%) lineages  was observed for the four descendant lineages   Fig. , consist14) Fig. . No signThe plastome dataset also indicated deep divergence among the three ancient lineages with possible seed-mediated dispersal, given that a large number of identical chloroplast sequences were found among lineages  comprise three ancient genetic components that are only found in the EAST and SOUTH lineages  genetic distance between individuals Fig. . We founope Fig. . The paiope Fig. , suggest15.Interestingly, the pairs with the largest IBS values  only occurred in China, mainly between the EAST and SWEST lineages Fig. , a refle30 , during the last glacial maximum , and the last interglacial (LIG: c. 115–130 kyr BP) with all 19 bioclimatic variables using species distribution modeling (SDM)0–2000, d31 (Supplementary Note P < 0.05) in EAST-g and SWEST-g, respectively, including many terms involving responses to various abiotic and biotic stress  each from the EAST and SWEST lineages, herein named EAST-g and SWEST-g, respectively  based on resequencing of hundreds of samples covering all known localities of the species. Here we used an updated genome assembly using HiC technolog17 to conduct substantial investigations on population genomics and evolutionary history of ginkgo. Such a chromosome-level reference provides the distribution information of SNPs17 and thus represents a valuable resource for addressing numerous questions involving biologic diversity, population genetics, and evolutionary history. To evaluate the applicability of SNPs called based on draft genome where no position information is available, we used the SNPs called based on the draft genome16 to perform various analyses. As demonstrated by ADMIXTURE analyses  of ginkgo  genetic distances of nuclear genomes  with various hypotheses such as ecological conservatism35. Our SDM analysis indicated the habitat persistence in the potential refugia of ginkgo trees  is another famous living fossil that was believed to have been extinct for several million years and re-discovered in the 1940s in several isolated areas in central and southern China38. Similar to ginkgo, dawn redwood has successfully expanded its distribution to approximately 50 countries via decades of human efforts, even over a much wider range than the fossils indicated24. Living gymnosperms that were considered as ancient and living fossils are convincing examples of successful plant lineages, in which many extant species occupy diverse habitats and survive dramatic environmental changes by adaptive shifts40. For example, in a study on the cycad lineage, Nagalingum et al.41 found that cycads underwent global rediversification beginning in the late Miocene, indicating that the species diversity of today’s cycads arose from a relatively recent radiation, less than 12 mya. Ginkgo’s resilience, along with other similar cases such as that in dawn redwood24 and cycads41 as well as some other gymnosperms39 supports the argument that intuitively frangible living fossils may possess the ability to survive successfully and even flourish if they are reintroduced to suitable habitats8, particularly when aided by humans. Together, the results of our study provide not only a comprehensive evolutionary framework for ginkgo research and conservation but also insights into the evolutionary history and conservation of other living fossil species.Whether living fossils and relict species are undergoing extinction is an intriguing question. As an example, the giant panda has long been regarded as an evolutionary dead-end but proved to be a successful species highly adapted to environmental changes42 and obtained clean sequencing reads with an average depth of up to 6.1-fold, ranging from 4- to 10-fold for each sample  toolkit to conduct read mapping and SNP calling, with the clean reads of FASTQ files used for alignment, sorting, duplicate removal, and variant calling. About 99% of the samples were aligned to the reference genome with a mapping ratio of more than 85% , with the majority of the SNPs showing high quality scores and low missing rates  from K = 2 to 10. PCA was conducted to study the relatedness and clustering of populations or samples. The top 10 PCs of the variance-standardized relationship matrix were extracted using PLINK (ver. 1.90)47 with the–pca parameter. To quantify the relatedness between individuals, the pairwise identity-by-state (IBS) genetic distance matrix of all 545 samples was calculated using PLINK48 (ver. 1.90) with the parameter–distance 1-ibs. We constructed a neighbor-joining (NJ) phylogenetic tree using MEGA (ver. 4.0)49 based on the distance matrix. A haplotype network was constructed for the plastome dataset using PopART50 based on codon-based alignments from chloroplast genomes.We performed routine analyses of population genetic structure and phylogenetic relationships of ginkgo with both the resequencing and plastome datasets  4 and 5.51 based on 8 samples from four lineages with a high sequencing depth (~30-fold)  was constructed from posterior probabilities of sample allele frequencies using easySFS.py (“https://github.com/isaacovercast/easySFS). Each model was run 100 times with 100,000 simulations for the calculation of the composite likelihood, and 10–40 expectation-conditional maximization (ECM) cycles  genetic distance calculated by PLINK (ver. 1.9), we used a histogram to display the IBS distribution. Pairs with short genetic distances (IBS < 0.03 or IBS < 0.07) may reflect recent dispersal or introduction.PSMC was used to infer historical dynamics of effective population size and the divergence timing of ginkgo lineages56 to explore the impacts of climate on the species distribution and to detect major climatic variables that contribute to the species distribution (Supplementary Note HE and FST)57 and the other in the likelihood-based program SweeD (version 3.1)59. We performed gene ontology (GO) enrichment analysis for the candidate genes in EAST-g and SWEST-g using a perl script60.We performed species distribution modeling (SDM) using the software MAXENT (v3.3.1)Further information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Source Data file


Poa annua L. is an example of a plant characterized by abundant, worldwide distribution from polar to equatorial regions. Due to its high plasticity and extraordinary expansiveness, P. annua is considered an invasive species capable of occupying and surviving in a wide range of habitats including pioneer zones, areas intensively transformed by human activities, remote subarctic meadows and even the Antarctic Peninsula region.P. annua populations representing contrasting environments from the worldwide range of this species. The electrophoretic patterns of polymerase chain reaction products obtained for each individual were used to estimate the genetic diversity and differentiation between populations.In the present study, we evaluated the utility of inter-primer binding site (iPBS) markers for assessing the genetic variation of P. annua populations characterized by their different climatic conditions. According to the analysis of molecular variance, the greatest genetic variation was recorded among populations, whereas 41.75% was observed between individuals within populations. The results of principal coordinates analysis (PCoA) and model-based clustering analysis showed a clear subdivision of analyzed populations. According to PCoA, populations from Siberia and the Kola Peninsula were the most different from each other and showed the lowest genetic variability. The application of STRUCTURE software confirmed the unique character of the population from the Kola Peninsula.iPBS genotyping revealed a pattern of genetic variation differentiating the six studied SF statistic and analysis of mismatch distribution, it was not followed by significant traces of a bottleneck or a founder effect. For the Antarctic population, the observed level of genetic variation was surprisingly high, despite the observed significant traces of bottleneck/founder effect following demographic expansion, and was similar to that observed in populations from Poland and the Balkans. For the Antarctic population, the multiple introduction events from different sources are considered to be responsible for such an observation. Moreover, the results of STRUCTURE and PCoA showed that the P. annua from Antarctica has the highest genetic similarity to populations from Europe.The lowest variability of the Siberia population suggested that it was subjected to genetic drift. However, although demographic expansion was indicated by negative values of Fu’s The observed polymorphism should be considered as a consequence of the joint influence of external abiotic stress and the selection process. Environmental changes, due to their ability to induce transposon activation, lead to the acceleration of evolutionary processes through the production of genetic variability. Vascular plants developed complex mechanisms to respond and adapt to recurring biotic and abiotic stresses . There aPoa annua L.) representing the large Poaceae family consisting of 780 genera and around 12,000 species  enables the development of a number of multiplex DNA-based marker systems , which aP. annua populations collected from different environmental conditions: from the temperate climate of Central Europe, through the warm but seasonally dry southern part of the Old Continent, up to the harsh climate of western Siberia, northern Europe and maritime Antarctic. Furthermore, we investigated whether the selected method could be useful in establishing the link between assessed genetic variability of studied populations and their geographic origin and associated climate conditions.In the present study, we evaluated the utility of iPBS markers for assessing the genetic variation of six P. annua populations originating from different climatic conditions  in only one or both replicates. Amplification was performed as described by A total of 20 iPBS primers were tested according to the procedure described by NB), percentage of polymorphic bands (P), Shannon’s information index (I) and expected heterozygosity (HE).All bands that could be reliably read across all individuals were scored as either present (1) or absent (0) across genotypes and treated as single dominant loci. Based on the binary matrix obtained , the folK), achieving linkage equilibrium within clusters  was used to test the mutation-drift vs bottleneck hypothesis  was performed with Arlequin 3.5. For this analysis, the iPBS data was treated as haplotypic, comprising of a combination of alleles at one or several loci . The sig. 1.2.02  software. 1.2.02  for eachpothesis . The sigP. annua samples using eight iPBS primers identified 147 bands. The highest number of bands (25) was found in the iPBS2229 primer, whereas the lowest number (14) was scored for iPBS2231. The average number of bands per primer was 18.37. Out of all identified loci, 64 (43.5%) were polymorphic  and AL (19.05%), whereas the lowest number of polymorphic bands was observed for the SI population (6.12%) . The higK, the second-order rate of change of the likelihood function with respect to K, has a maximum at K = 2 . D did not show any deviation from 0, while Fu’s SF was negative and significant for all populations  which allow it to expand its distribution and to adapt to broad array of climatic conditions  and Poland (Olsztyn) revealed both qualitative and quantitative differences  appeared to share a considerably high degree of similarity (as shown by PCoA results). However, although the AL population departed slightly from PO and AM populations along the first coordinate, STRUCTURE failed to detect any genetic structure among them. The observed data structuring is characteristic of the populations from the central part of the species distribution, where gene flow, varying in rate and direction, may counteract the effects of putative demographic and selection processes.P. annua from the KO population deserves special attention. As with population SI, it is characterized by a lower-than-average level of genetic variation, expressed in the number of polymorphic loci and expected heterozygosity  or the purification of a selection against deleterious alleles, hampers the evolution rate. As a consequence, balanced selection selects the optimal compromise among these constraints. The wide geographic range of P. annua is a result of the outstanding phenotypic and physiological plasticity of this species, which leads to its high ecotypic differentiation in response to various limiting environmental factors in each habitat. However, the answer to the question of whether the ecological breadth of the species is a consequence of such a specialist strategy or is possible due to the existence of plastic “multi-purpose” genotypes (Inter-primer binding site markers revealed an interesting pattern of genetic variation among studied populations of enotypes  seems to10.7717/peerj.6888/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.6888/supp-2Supplemental Information 2Click here for additional data file.


Abrothrix olivacea brachiotis, one of the most common species of small mammals and an inhabitant of these biodiverse forests. Sequencing data showed that along with the recovery of high haplotype variation for this species, there was a low nucleotide diversity between haplotypes, showing no genetic differences between the Chiloé Island and continental populations in southern Chile or through any other geographic barrier in the study area. However, microsatellite data exhibited some level of population structuring. The most evident clusterings were those of the Chiloé Island and that of North Patagonia. These findings are corroborated by a barrier analysis that showed a genetic barrier in the latter areas, whereas the Chacao Channel was not a significant barrier for this rodent. Overall, the genetic variability and structure of A. o. brachiotis was concordant with historical factors, such as the Last Glacial Maximum and the presence of geographic elements that isolate populations.The temperate rainforests of southern Chile, a recognized biodiversity hotspot, were significantly affected by Pleistocene glacial cycles in their southern portion and have been severely disrupted mainly due to recent human activities. Additionally, the landscape is characterized by a series of potential barriers to gene flow, such as the Chacao Channel, Cordillera de Piuche in Chiloé and both the Ancud and the Corcovado gulfs. We used mitochondrial DNA sequences and microsatellite data across several populations to evaluate the genetic variability and structure of the sigmodontine rodent  The temperate rainforest of South America, mostly in Chile, is one of the largest areas of forests in the southern hemisphere and is rich in endemic species . The regHistorical processes have modified the landscape, creating geographic barriers among populations and changes in population sizes. Among these barriers, there are the Piuche mountain range in Chiloé Island and the Gulf of Ancud and the Gulf of Corcovado that separates the island from North Patagonia. Alternatively, the Pleistocene glacial cycles affected an important area of the landscape in the Americas, particularly the southern cone of South America. Historical records show that the temperate rainforests of the Cordillera de la Costa in Chile played a central role in maintaining higher endemism, species and genetic diversity. Because the southern portion of South America was severely affected by the Pleistocene glacial cycles , the geoAbrothrix olivacea (the “olive field mouse”), one of the most abundant and characteristic rodents of this biome. The species ranges from 18 to 54°S  southward to Aysén (45°S), including the Chiloé Island and southern archipelagoes nearby  are one of the most important inhabitants of the Chilean temperate rainforests, including  to 54°S , encompa to 54°S , includis nearby . The hom2,530 m2 , which i forests . This su forests . The latix manni , the pygcaudatus , and theliroides . The oppudu puda , Darwin’fulvipes , and eves pictus . Leopardpresent; . The latThe information regarding genetic patterns of variability and structuring in the temperate rainforest of Chile has been mainly sourced by mitochondrial sequence analysis. The use of more variable markers such as microsatellites will provide valuable and sensitive data from a genetic scope . With thAbrothrix olivacea brachiotis along with its distribution in the temperate rainforests of southern Chile. In addition, we evaluated the potential effects of existing geographic barriers in the geographic distribution of A. o. brachiotis, such as the Chacao Channel, the Piuché Mountains in Chiloé Island, and the border of the LMG. The ecological and evolutionary features of A. olivacea populations, coupled with the hotspot of biodiversity where this species lives, provides a useful model for examining historical and ecological processes that shape the genetic variability and population structure of A. o. brachiotis.Using microsatellite and mitochondrial sequence data along several populations from both continental and island (Chiloé) areas of southern Chile, we evaluated the genetic variability and structuring of Abrothrix olivacea brachiotis, between Los Rios (40°S) and Aysen (45°S) regions in the temperate rainforest ecoregion , Departamento de Ecología, Pontificia Universidad Católica de Chile, Santiago, Chile, and in the Museum of Southwestern Biology (MSB), Department of Biology, University of New Mexico, Albuquerque, New Mexico, USA. The remainder of the samples were the result of field campaigns in which standard Sherman traps were used  with oats and vanilla as bait. Fieldwork protocols followed the standard bioethical and biosafety protocols outlined by the American Society of Mammologists  or ethanol preserved tissues  using the Wizard Genomic DNA Purification Kit  or the Phenol-Chloroform protocol (modified from MH714355 to MH714446). In addition, another 15 sequences of the subspecies Abrothrix olivacea brachiotis were obtained from GenBank (Accession numbers: AY840064 to AY840078). We thus completed a total of 107 sequences that were aligned using the Clustal W program (Through the polymerase chain reaction (PCR), we amplified the complete hypervariable domain I (HVI) of the mtDNA control region, using primers and protocols outlined in  program  implemen program . All samA total of 28 microsatellite primer pairs were specially designed for this study through the company Genetic Marker Services Laboratories . Following the manufacturer’s instructions, we standardized only those that presented good quality in the report given by the company. A subset of 16 SSR was screened for polymorphisms and then tested for genotyping problems in Microchecker . After t® v4.0; Applied Biosystems®, Foster City, CA, USA). In the standardization process, at least 8 samples of different localities were repeated to ensure consistency in the genotyping process. Additionally, the dataset was analyzed with the software Microchecker  diversity using popART 1.7 , which is not significant, meaning that populations exhibited neutrality.The haplotype gene diversity (Hd) was 0.965 (variance 0.00007), the nucleotide diversity (Pi) was 0.01070, and the Tajima index calculated over the mitochondrial data showed a value of −1.84  was recovered that included the two latter localities of Mininco and Rio Blanco. Other haplotypes  was higher in the continent than in Chiloé Island and North-Patagonian populations. The same K = 5) with the highest probability  were assigned to clusters “orange” and “blue.” We also show analysis with K = 4, exhibiting the same pattern, with coastal south temperate forest populations forming a not well defined cluster, two clusters in Chiloé Island, and another grouping the populations of North Patagonia.bability . From noclusters , identifK = 5 clusters with the highest probability, K = 4 has greater biological sense. Following this finding, we estimated the effective size of K = 4 obtained with STRUCTURE . The biggest effective size was in the continent with 683.3, followed by Senda Darwin with 274.9; the smallest cluster was Cucao with 54 individuals. In the mainland, the top limit of the confidence interval is infinite  that shares haplotypes with the continent, and the North Patagonia haplogroup that included populations of Mininco and Alto Rio Ibañez (ARI). The last haplogroup had one shared haplotype between Valdivia  and Mininco, and few mutational steps with other Valdivian haplotypes. In summary, the mitochondrial analysis showed low genetic structuring for the subspecies brachiotis and the nonoccurrence of exclusive haplotypes for any of the localities and recovered shared haplotypes with North continental localities. The overall mtDNA network had low nucleotide diversity, meaning that besides the high number of haplotypes (high haplotype diversity), there were little differences between them.Using both mitochondrial and microsatellite markers, we evaluated the structuring and variability across the distribution of The Microsatellite Genetic diversity indices showed that across all populations sampled (11), there were no significant differences and Ho versus He was similar with the exception of La Picada, although this may be due to small sample size. The pairwise Fst showed that only close localities did not show significant differences, such as Mininco with Rio Simpson in North Patagonia. This finding is consistent with the pattern of isolation by distance found in the data. Additionally, the La Picada population, when compared with other localities, did not show  significant differences, most likely due to small sample size.A. o. brachiotis, we did not find a heterozygosity deficit in any of the localities, as there were no significant differences when comparing mainland and island populations. Thus, it seems that despite recent intense forest fragmentation and degradation . The only population that exhibited a lower number of alleles was La Picada (4.33). When populations were grouped into four clusters , the difference was even smaller (8.70–10.53). Senda Darwin in Chiloé had the lowest allelic richness and the continental groups had the highest. This fact may be due to the recent separation of this population (Senda Darwin) from the continent. High continental allelic richness may explain the different sources of alleles: more than one refuge represents the genetic diversity of this cluster. In fact, this cluster also exhibits the highest number of private alleles. In the case of radation , the habzygosity .A. o. brachiotis’s geographic range recovered five genetic clusters with STRUCTURE, with those results having the highest probability through the Evanno method. Two of these clusters corresponded to populations from the mainland, two others corresponded to populations from Chiloé Island, and a single cluster was restricted to the southernmost limit of the subspecies range in the North Patagonian forests (Aysen region). The clusters recovered in the continent , and the major town of Castro  towards the east of the Coastal Cordillera. Other additional barriers that might have isolated the Cucao area from the rest of the island are historical events, such as the glacial border of the LGM during the Pleistocene that advanced throughout the island. This glacial border left a refuge area in northwest Chiloé where a portion of the National Park is located  and with Geneland, which show clusters that are separated by the aforementioned barriers. The Barrier analysis also demonstrated that the Chacao Channel would not be a genetic barrier for A. o. brachiotis, and these results are consistent with those of Geneland  would not constitute a genetic barrier for this rodent. The genetic structuring observed would respond to the geographic and long dated isolation of populations, as observed by the presence of Cordillera de Piuché that isolated populations in north Chiloé (Senda Darwin) with respect to those of Parque Nacional Chiloé (Cucao). In addition, populations from the latter geographic area are located in what was a glacial refuge during the Pleistocene. North Patagonian populations, on the other hand, may constitute a genetic clustering due to their isolation with respect to Chiloé populations and the geographic distance from the northern range of this subspecies. This isolation might be enhanced by geographic barriers, such as both the gulfs of Ancud and Corcovado and the mountain range of the Andes.In this study, we obtained a subtle structuring of populations at the northern portion of the continental range of 10.7717/peerj.6955/supp-1Table S1List of specimens analyzed and geographic coordinates, with also the genetic marker used Click here for additional data file.10.7717/peerj.6955/supp-2Table S2Click here for additional data file.10.7717/peerj.6955/supp-3Table S3p values below diagonal. ( ∗ = p > 0.01)Pairwise comparison between population above diagonal and Click here for additional data file.10.7717/peerj.6955/supp-4Dataset S1List of alleles of 187 individuals for 12 loci, each line represent one individual and every two column is a different lociClick here for additional data file.


IGH) is poorly known and mostly characterized only by serological methods. Moreover, this genomic region is not well-covered in genomic databases and genome-wide association studies due to particularities that impose technical difficulties for its analysis. Therefore, the IGH gene has never been systematically sequenced across populations. Here, we deliver an unprecedented and comprehensive characterization of the diversity of the IGHG1, IGHG2, and IGHG3 gene segments, which encode the constant region of the most abundant circulating immunoglobulins: IgG1, IgG2, and IgG3, respectively. We used Sanger sequencing to analyze 357 individuals from seven different Brazilian populations, including five Amerindian, one Japanese-descendant and one Euro-descendant population samples. We discovered 28 novel IGHG alleles and provided evidence that some of them may have been originated by gene conversion between common alleles of different gene segments. The rate of synonymous substitutions was significantly higher than the rate of the non-synonymous substitutions for IGHG1 and IGHG2 , consistent with purifying selection. Fay and Wu's test showed significant negative values for most populations (p < 0.001), which indicates that positive selection in an adjacent position may be shaping IGHG variation by hitchhiking of variants in the vicinity, possibly the regions that encode the Ig variable regions. This study shows that the variation in the IGH gene is largely underestimated. Therefore, exploring its nucleotide diversity in populations may provide valuable information for comprehension of its evolution, its impact on diseases and vaccine research.Even though immunoglobulins are critical for immune responses and human survival, the diversity of the immunoglobulin heavy chain gene ( Immunoglobulins (Ig) are glycoproteins produced exclusively by activated B-lymphocytes and plasma cells that mediate humoral response against pathogens. Each B-cell clone presents an antigen-specific membrane-bound immunoglobulin that, together with CD79A and CD79B molecules, comprise the B-cell receptor (BCR). After stimulation by antigens, B-cells secrete immunoglobulins (antibodies) with the same antigen-binding sites than the membrane-bound molecules.H) and three or four constant domains . Each light chain exhibits one variable domain (VL) and one constant domain (CL). The variable region (VH and VL) is responsible for antigen recognition and binding while the constant regions (CH and CL) primarily mediate the Ig effector functions, which includes complement activation and Fc Receptor binding  is located in chromosome region 14q32 and consists of four groups of gene segments: the variable heavy (IGHV), diversity heavy (IGHD), joining heavy (IGHJ), and constant heavy (IGHC). The IGHC group includes IGHM, IGHD, IGHG3, IGHG1, IGHEP1, IGHA1, IGHGP, IGHG2, IGHG4, IGHGE, and IGHA2 gene segments and pseudogenes. Immunoglobulin light chains are encoded by two different genes: lambda (IGL) at 22q11 and kappa (IGK) at 2p11.2 , IgA (IgA1 and IgA2), and IgE. The human humoral immune response is mainly mediated by Ig gamma (IgG), which is subdivided into four subclasses, IgG1, IgG2, IgG3, and IgG4, ordered by decreasing abundance in peripheral blood  and exon H, which encodes the hinge between the CH1 and CH2 domains  on the Ig constant domain that provide binding sites for antibodies . CertainIGHG gene segments has been characterized , Guarani Ñandeva , Guarani Mbya , Kaingang from Ivaí , and Kaingang from Rio das Cobras ; and two are urban populations: Japanese-descendants  and Euro-descendants from Curitiba . Their detailed geographic location and sample sizes are found in This study was approved by the Brazilian National Human Research Ethics Committee (CONEP), protocol number CAAE 02727412.4.0000.0096, in accordance to the Brazilian Federal laws. We analyzed a total of 357 individuals from seven Brazilian populations, of which five are Amerindian: Guarani Kaiowa . The Guarani speak a Tupi-Guarani language, which belongs to the Tupi language family. The Kaingang speak Jê, which belongs to the Macro-Jê language stock. Analyzing mtDNA segments and the proposed time of origin of Tupi-Guarani and Jê linguistic families, Marrero and colleagues (The Amerindians samples were collected between late 1980s and early 1990s. According to public data from the Brazilian Institute of Geography and Statistics (IBGE), there are approximately 900,000 Amerindians individuals in Brazil, distributed across 693 official indigenous lands . The population here referred as CTBA only included Euro-descendant individuals. Therefore, we excluded all individuals with known miscegenation with Amerindian and/or other non-European ancestries. Paraná State also hosts the second largest Japanese community in Brazil, one of the largest outside Japan. The Japanese migration started in the twentieth century with the Treaty of Friendship, Commerce and Navigation between Brazil and Japan. All Japanese-descendent individuals of this study (BrJAP) were born in Brazil, with either both parents or all four grandparents born in Japan. They reported no history of admixture with non-Japanese ethnicities.The two urban samples were from Curitiba, the capital of Paraná State and the 5th largest city in Brazil. As a result of the Brazilian history of European colonization 500 years ago, and especially the more recent European migrations since the 19Genomic DNA was extracted from peripheral blood samples by standard salting-out  or by thIGHG alleles and designed primers to amplify each segment specifically. To define the best set of primers we used the following approaches: (i) we ruled out unspecific amplification by verifying that all amplicons did not exhibit any variant that was specific of other segments; (ii) we certified that the genotype distribution of all single nucleotide variable sites, in each amplicon, were in accordance to Hardy-Weinberg equilibrium (p > 0.05).We aligned all previously known IGHG1, IGHG2, and IGHG3 as follows: 1X Buffer (Invitrogen); 0.2 mM dNTP (Life Technologies); 1.5 mM MgCl2 ; 0.3 μM of each primer; 0.05 U/μL Taq polymerase Platinum® ; and 2 ng/μL genomic DNA. The segments were amplified in a Mastercycler ep Gradient S thermocycler , with a first step at 94°C for 2 min and 10 cycles of 94°C for 15 s, TmA °C for 15 s and 72°C for 60 s, followed by 25 cycles of 94°C for 15 s, TmB °C for 15 s and 72°C for 60 s, with a final extension step of 72°C for 60 s  was performed for Sequencing was performed using Big Dye® Terminator Cycle Sequencing Standard v3.1 , according to manufacturer's instructions. The sequencing reactions were performed in a Mastercycler ep Gradient S thermocycler  with a first step at 95°C for 60 s and 25 cycles of 95°C for 10 s, 50°C for 5 s, and 60°C for 4 min, followed by capillary electrophoresis in a 3500xl Genetic Analyzer Sequencer .After sequencing, the alleles were identified according to the known alleles described at IMGT database  . IMGT daThe nucleotide sequence of each individual was aligned with consensus sequences with Mutation Surveyor® DNA Variant Analysis Software v5.0.1 (Softgenetics), and their variable sites were annotated. Alleles that were different from the ones listed in the IMGT database were considered novel and were subsequently confirmed by sequencing and/or molecular cloning as described below.IGHG1*07, IGHG1*08, IGHG2*09, IGHG2*13, IGHG3*21, IGHG3*22, IGHG3*26) were confirmed by direct re-sequencing from a different PCR product. Novel alleles observed in heterozygosis without phasing ambiguities due to the presence of only one heterozygous position  were also confirmed by re-sequencing. The new variants with ambiguous phasing  were confirmed by molecular cloning. In this case, the segments were re-amplified and ligated into a PTZ57R/T vector  with terminal deoxynucleotidyl transferase (TdT) enzyme. Afterwards, recombinant plasmids were obtained and purified from multiple transformed colonies and sequenced as described above. Novel alleles were verified based on sequences from at least two independent colonies containing each allele.The novel alleles that were observed in homozygosis  algorithm  with NetST , 26 withST . PrincipST  for grapD .Neutrality tests were performed using the Tajima's D , Fu and D, and F  and Fay and F (H  in DnaSPand F (H . HomologIGHG3 single nucleotide variant at the position chr14:106235856 (GRCh37.p13 primary assembly) in the CTBA population. This new variant was submitted to the dbSNP database (rs155533833 (NC_000014.8:g.106235856G>A).Within all three gene segments in the seven populations analyzed, we found a total of 49 exonic variable sites, of which 26 were non-synonymous substitutions. Based on the Grantham scale , which rdatabase  under reIGHG alleles have been found in our study: nine in IGHG1 , IGHG1*08 in BrJAP , IGHG2*08 in BrJAP , IGHG2*09 in GRC , IGHG3*21 in BrJAP , and IGHG3*22 in GRC .Interestingly, some new alleles of all gene segments were observed at high frequency , which encodes the Gm haplotype “C” Gm21,26,27,28;17,1;(.), the most frequent lgG allotype haplotype in our populations , that is encoded in our data by the allele haplotype IGHG3*11,IGHG2*03,IGHG2*03, by IGHG3*11,IGHG1*14,IGHG2*03, or by IGHG3*11,IGHG1*03,IGHG2*08. In order to simplify the interpretation of the data, Gm haplotype identifiers (from A to M) were used as suggested by Lefranc et al. , with low to moderate FST values (0.02–0.10) . More conspicuous differences were found between the Japanese-descendant and Euro-descendant populations compared to each other, and between each of these two populations compared to the Amerindian populations, with FST values ranging from 0.11 to 0.52, indicating moderate to high genetic differentiation.ulations . A small02–0.10) . AllelicThe principal component analysis (PCA) grouping was consistent with ancestry and geography . Amerindp >1).Genotypic distributions for all gene segments were in accordance with Hardy-Weinberg equilibrium in all population samples . In contrast, more variable sites (21 and 24) were observed for the other two Guarani populations. In addition, many variants that were in LD in GND were not observed in LD in GRC.Linkage disequilibrium (LD) patterns differed among populations . Interesrs1071803) and the G2m23 allotype (rs8009156) were in strong LD in all Amerindian populations , as well as in CTBA , and BrJAP  in which fewer SNPs were observed in strong LD.The G1m3 allotype , could have been generated by gene conversion between the most frequent IGHG2 allele  and the most frequent IGHG1 allele . In addition, gene conversion between the frequent IGHG2*03 and IGHG3*14 alleles  could explain the origin of allele IGHG2*09 (f = 0.14).Alignment of all the known alleles of the segments . For exaD, Fu and Li's D and F were non-significant for most populations. However, Fay and Wu's test resulted in significant negative values for most populations, which may indicate positive selection at an adjacent site   , consistIGHG gene segments in populations of different ancestries. Before this study, only 30 IGHG alleles have been described for IGHG1, IGHG2, and IGHG3 together (n = 57) and seven in one population sample of Euro-descendants (n = 51). It is interesting that even in Amerindian populations, which exhibited a limited diversity, seven new alleles were found. This is clear evidence that the diversity of IGHG is far from being fully described and possibly a much larger number of novel alleles will be discovered as more populations are interrogated. We focused on the segments that code for the most abundant Ig in serum. Considering the homology and high sequence similarity, a different strategy would be needed for the precise characterization of IGHG4 due to the high frequency of duplications observed for this gene segment , exhibited a lower frequency in Guarani Ñandeva , and was absent in Guarani Kaiowa (GKW). These three populations share a more recent common ancestor and the differences observed can be explained by its demographic history and genetic drift. Demographic factors played a major role in shaping the diversity of other genes important for immune responses in these same Amerindian populations  and was observed at lower frequency in Amerindians: GND (f = 0.094), GRC (f = 0.010), KIV (f = 0.038), KRC (f = 0.020), being absent in GKW. This allele corresponds to the allotype G3m5,10,11,13,14,26,27 which has been previously shown to be highly frequent in Europeans but absent in non-admixed Amerindians , but absent in the other populations. According to the nucleotide sequence, it encodes the haplotype Gm5,10,11,13,14,26,27, whose frequency was previously reported as 15.2% in a study with Japanese families   was obsers1071803) and G2m(.) (rs8009156) are in LD  and are present in the allotype haplotypes C and D, reported as the most common in Japanese populations  indicates that gene conversion between alleles IGHG2*13 and IGHG2*03 originated the IGHG2*07 allele. Moreover, we suggest that the novel alleles IGHG1*11 and IGHG2*09 resulted from gene conversion between two frequent alleles of different gene segments. Overall, our data point to a major role of recombination and gene conversion originating new IGHG alleles, which is consistent with the tandem positioning and high sequence similarity of these segments, which favor unequal crossing-over , and similar allele frequencies , much earlier than the separation of the Kaingang populations that was estimated at of 207 ybp  and IGHG2 (p = 0.03). We observed that synonymous (dS) substitution rates were higher than non-synonymous (dN) substitution rates. It was previously demonstrated that Gm1 allotypes have a different impact on the IgG1 ability to bind the Fc gamma receptor (FcγR)-like proteins from viruses. Antibodies with G1m1,2,17 allotype exhibit lower affinity to the viral FcγR-like protein of the human cytomegalovirus (HCMV), which decreases susceptibility to this infection  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Brazilian National Human Research Ethics Committee (CONEP).DA designed the study. VC-S, DA, LV, RD, and HI performed DNA sequencing and genotyping. VC-S analyzed the data. RW, VC-S, RD, HI, and LV performed molecular cloning and validation of novel alleles. MP-E, DA, DM, and RW contributed with reagents. VC-S, DA, MP-E, DM, and MB drafted the manuscript. All authors significantly contributed with ideas and critically reviewed this 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.


Soil microbial diversity is often studied from the perspective of community composition, but less is known about genetic heterogeneity within species. The relative impacts of clonal interference, gene-specific selection, and recombination in many abundant but rarely cultivated soil microbes remain unknown. Here we track genome-wide population genetic variation for 19 highly abundant bacterial species sampled from across a grassland meadow. Genomic inferences about population structure are made using the millions of sequencing reads that are assembled de novo into consensus genomes from metagenomes, as each read pair describes a short genomic sequence from a cell in each population. Genomic nucleotide identity of assembled genomes was significantly associated with local geography for over half of the populations studied, and for a majority of populations within-sample nucleotide diversity could often be as high as meadow-wide nucleotide diversity. Genes involved in metabolite biosynthesis and extracellular transport were characterized by elevated nucleotide diversity in multiple species. Microbial populations displayed varying degrees of homologous recombination and recombinant variants were often detected at 7–36% of loci genome-wide. Within multiple populations we identified genes with unusually high spatial differentiation of alleles, fewer recombinant events, elevated ratios of nonsynonymous to synonymous variants, and lower nucleotide diversity, suggesting recent selective sweeps for gene variants. Taken together, these results indicate that recombination and gene-specific selection commonly shape genetic variation in several understudied soil bacterial lineages. Acidobacteria, Verrucomicrobia, and Gemmatimonadetes phyla, are underrepresented in or nearly absent from culture collections and genomic databases, even at the level of class or phylum [Soil microbial communities play key biogeochemical roles in terrestrial ecosystems . Within r phylum , 5, 6. Fr phylum –11. Sequr phylum , 13.Cyanobacteria approach panmixia, where recombination is so frequent that individual cells are unlinked random mixtures of alleles [Vibrio, recombination is high but large blocks of alleles important for ecological niche differentiation are co-inherited and may remain linked due to selection [Streptomyces flavogriseus isolates were also found to approach a freely recombining panmixia [Myxoccocus xanthus isolates recovered from a series of soil samples were distinct but highly clonal, implying recombination between strains was low [Acidobacteria, Gemmatimonadetes, and Verrucomicrobia [Homologous recombination can vary dramatically in its importance relative to the other processes for different microbial species. Analyses of reference genomes have shown that homologous recombination frequently occurs in bacteria populations, both globally and locally –16. For  alleles . In otheelection , 19. In panmixia . In cont was low . Howevermicrobia , conjugamicrobia , or the microbia .Chlorobium population in a freshwater lake [When recombination rates are low or selection is extremely strong, several clonal strains compete until one or more beneficial alleles is highly selected for, resulting in a single clonal genotype increasing in abundance or even sweeping to fixation. However, when recombination unlinks gene variants within a population, beneficial alleles can sweep through a population independent of genomic context in a selective sweep  with genter lake , while mter lake , 28. In 3, a cation exchange capacity between 17 and 19 meq per 100 g soil, C:N ratios from 10–12, and total C concentrations of 10 mg g−1 up to 18 mg g−1 [Previously, we conducted a large scale genome-resolved metagenomics study of soils from a grassland meadow in the Angelo Coast Range Reserve in northern California that established a dataset of 896 phylogenetically diverse microbial genomes dereplicated by species, and reported on community composition . The soi8 mg g−1 . The gra8 mg g−1 . The mea8 mg g−1 , inverte8 mg g−1 , fungal 8 mg g−1 , soil or8 mg g−1 , metabolThe sampling scheme, local soil characteristics, and study design that were utilized in this analysis have been previously described . Previou).The 10,538 genome bins previously described obtained from the study site were dereplicated using dRep  with theS2Open reading frames were predicted using Prodigal  and annoPossible contamination was further removed from representative genomes using the assembled replicate genomes for each species population. The pan-genomic analysis pipeline Roary  was run r2 cutoff to those at a 98% ANIr2 cutoff and found a strong correlation , where A, C, G, T and the observed proportions of each respective nucleotide. This is equivalent to the definition from [For all population diversity metrics, we used reproducible custom python scripts (available under Code Availability) that calculated metrics, each explained below, from all filtered cross-sample read mappings. For each representative genome in our set of 19, we analyzed its data in samples that passed a cutoff of at least 50% of the genome being covered with at least 5× coverage. Five hundred eighty-six out of 1140 sample genome comparisons (19 genomes × 60 samples) passed this minimum requirement. Base pairs of reads with Phred scores less than 30 were not used in SNP or linkage analyses. Nucleotide diversity was calculated as the expected frequency of a difference between two sequencing reads at a position, equation pi=1 −  of 5% were called as SNPs. Because meadow-wide coverages were at least 224× (and ranged up to 908×) the MAF cutoff alone would likely be a stringent cutoff for Phred 30 error rates. SNPs were assigned as synonymous or nonsynonymous using a custom BioPython script and the gene calls annotated by Prodigal.SNPs were called on the combined meadow-wide population set of filtered reads. We constructed a simple null model based on an error rate of 0.01% (the Phred 30 cutoff) and simulated simple sampling with replacement to construct estimated rates of erroneous SNP counts at genomic positions of varying coverages. Positions with an alternative allele that occurred with counts that had a false positive rate of ~10R2 and D′ linkages were calculated using formulas described by [FST, , was calculated on sites segregating across both blocks being compared  using the Hudson method [FST, and genes which had coverages in a block outside of the range of two standard deviations were excluded from the analysis. A ratio of averages was then used to determine mean FST for each gene. A two-sample Wilcoxon test was used to determine if average linkage of highly differentiated loci differed from the genomic average for each species, and two-sample t-tests in R [Linkage was calculated within mapped reads for all pairs of segregating sites that were spanned by at least 30 read pairs with high quality base pair data. ribed by . The reln method  as recomn method , as impln method . A site sts in R  were usests in R  method.Chloroflexi, Acidobacteria, Verrucomicrobia, and Candidatus Rokubacteria, which are known to be abundant globally in soils [Candidatus ANGP1 . Based on measurement of the relative DNA abundance of each population across the entire meadow, these bacteria are some of the most abundant species in the soil, although no individual species contributed >1% of the DNA in a sample . Further, we tested if genomes obtained from the same soil depth were more similar than those collected from different depths. We found that the genetic variation of genomes from 12 of the 19 populations were significantly associated with sampling plot, and that genetic variation within 5 of the 19 populations were significantly associated with sampling depth   Fig. . Short cr2 metric of linkage disequilibrium decay as the genomic distance between two polymorphisms increased  are selected against [r2N) having higher r2 than synonymous (r2S)  suggests an increase in the ratio of beneficial to slightly deleterious nonsynonymous SNPs as diversity increases.In the less genetically diverse populations, there was a noticeable higher nts Fig. . This ha against . In more2S) Fig. . One recorrhoeae ; for sixn 1 Fig. , also inr2 is often used as a signal to identify the presence or absence of recombination, r2 values <1 can occur with or without recombination. For example, three of four possible haplotypes (pairs of variants) for two biallelic sites could occur due to lineage divergence prior to mutation occurring at one of the sites. D′, an alternative metric of linkage equilibrium, is only <1 if all possible combinations of a pair of biallelic sites are observed [D′ for a population linearly correlated with mean r2 , at 7% to 36% of all site pairs in each population , purely neutral growth and processes cannot explain the observed differences in nucleotide diversity between these populations. Similarly, we also did not observe a significant relationship between diversity and abundance within species for 14 of the 19 populations . In all species except the least diverse population (an Acidobacterium), nonsynonymous variants also had consistently higher values of D′ than synonymous variants. Further, as genome-wide D’ decreased (more recombination), the degree to which nonsynonymous variants were more linked than synonymous (D’N/D’S) increased , consistent with dispersal of most alleles between blocks  selective pressures acting on that locus [FST, we scanned over a moving 5 gene window and tested if that region had a mean FST greater than 2.5 standard deviations above the genomic mean. We removed genes with either coverage 2 standard deviations above or below the mean coverage in either block from this analysis (Supplementary Table FST values, we extended successful windows until the mean FST fell below this cutoff. This test at first identified 48 loci of elevated FST within some microbial genomes, despite those genomes having low average FST (Fig. FST or strong changes in nucleotide diversity, our stringent criteria narrowed that set down to 8 high FST loci with significantly increased rates of linkage compared with the genomic background (Fig. 5c; Fig. When specific loci are characterized by significantly higher at locus . To idenFST Fig. . To furtFST Fig. . We notiVerrucomicrobia population also showed evidence of a selective sweep occurring at a putative hopene biosynthesis operon, often involved in regulating membrane stability. Within a Deltaproteobacteria population, a highly differentiated locus encoded for numerous genes involved in two-component systems and histidine kinases, potentially related to environmental sensing and response. Taken together, these multiple genomic signals suggest that gene-specific selection partially drives differences in population genetic structure across meadow soils.Some loci with evidence of recent differential selection across the meadow contained transporter genes Fig. , which cDeveloping a cohesive picture of the genetic structures of soil bacterial populations is crucial for understanding the evolution and distribution of genes that can play critical ecosystem and niche-specific functions. Doing so has been difficult because the most abundant soil bacteria are difficult to cultivate, and when cultivation is possible, it is uncertain if microbial isolates are truly random samples of a population, considering the intense selective pressure put on cells during the isolation process. Here, we show that recent advances in sequencing technologies and software tools enable the monitoring of heterogeneity within genomically defined bacterial populations needed to address these questions in soil. Compared with a previously published study on freshwater lake microbial populations , we obseThe results of this study suggest that recombination and gene-specific selection are important modes of evolution across the most abundant soil microbes, and are capable of structuring populations at a scale of meters in soils. Even within a single meadow, our data demonstrate that there are likely thousands of combinatorial genetic mixtures for each species, with recombination resulting in no easily measurable numbers of irreducible strain-like lineages. We were able to calculate the relative rate of recombination to mutation for half of our species studied, and comparing these values to those reported for other species in the literature places them among other known recombinogenic species. Thus, the dynamics of dominant soil bacterial populations may be partially described using ‘quasi-sexual’ models , 17. AltSupplementary Figure LegendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary Figure S8Supplementary Figure S9Supplementary Figure S10Supplementary Tables S1-S6