Plasmodium vivax (PvTRAP) exhibits sequence heterogeneity among isolates, an issue relevant to vaccine development. To gain insights into variation in the complete PvTRAP sequences of parasites in Thailand, 114 vivax malaria patients were recruited in 2006–2007 from 4 major endemic provinces bordering Myanmar , Cambodia  and Malaysia . In total, 26 amino acid substitutions were detected and 9 of which were novel, resulting in 44 distinct haplotypes. Haplotype and nucleotide diversities were lowest in southern P. vivax population while higher levels of diversities were observed in other populations. Evidences of positive selection on PvTRAP were demonstrated in domains II and IV and purifying selection in domains I, II and VI. Genetic differentiation was significant between each population except that between populations bordering Myanmar where transmigration was common. Regression analysis of pairwise linearized Fst and geographic distance suggests that P. vivax populations in Thailand have been isolated by distance. Sequence diversity of PvTRAP seems to be temporally stable over one decade in Tak province based on comparison of isolates collected in 1996 (n = 36) and 2006–2007. Besides natural selection, evidences of intragenic recombination have been supported in this study that could maintain and further generate diversity in this locus. It remains to be investigated whether amino acid substitutions in PvTRAP could influence host immune responses although several predicted variant T cell epitopes drastically altered the epitope scores. Knowledge on geographic diversity in PvTRAP constitutes an important basis for vaccine design provided that vaccination largely confers variant-specific immunity.Thrombospondin-related adhesive protein (TRAP) of malaria parasites is essential for sporozoite motility and invasions into mosquito’s salivary gland and vertebrate’s hepatocyte; thereby, it is a promising target for pre-erythrocytic vaccine. TRAP of  Plasmodium falciparum is the most pernicious and prevalent species, the significance of P. vivax should not be underappreciated because it can cause chronic relapsing illness due to reactivation of hypnozoites and it can potentially lead to severe complications similar to those caused by P. falciparumP. vivax are of particular concern if they would be wide-spreading as chloroquine-resistant P. falciparumP. vivax and P. falciparum co-circulate in several endemic areas outside Africa where co-infections of both species are not uncommon In low- and middle-income countries in tropical areas, malaria remains one of the leading ten causes of morbidity and mortality, resulting in an estimated economic loss of nearly 40 million disability-adjusted life-years (DALYs) P. falciparum has resulted in limited clinical efficacy in field studies To date, malarial circumsporozoite (CS) protein is a prime candidate for pre-erythrocytic vaccine development P. falciparum (PfTRAP) and of P. vivax (PvTRAP) from clinical isolates revealed microheterogeneity of sequence that has been maintained by positive selective pressure P. falciparumTRAP has been shown to mediate gliding motility and invasion processes of malarial sporozoites into vertebrate’s hepatocyte and mosquito’s salivary gland P. vivax and P. falciparum have been circulating in Thailand with almost comparable prevalence since the past 2 decades. However, regional prevalence of P. vivax relative to P. falciparum seems to differ across major endemic areas of Thailand P. vivax populations in this country exhibited spatial variation in the extent of sequence diversity of major malaria vaccine candidate loci Both The study protocol was approved by the Institutional Review Board on Human Research of Faculty of Medicine, Chulalongkorn University (IRB303/56). Written informed consent was obtained from all participants.P. vivax-infected patients in northwestern , southwestern , eastern  and southern  Thailand during 2006 and 2007. Venous blood samples were taken from each patient and were preserved in EDTA anticoagulant. These isolates have been previously determined to contain single clone infection based on sequence analysis of the merozoite surface protein-1 gene (Pvmsp-1) as described previously Blood samples were obtained from 114 PvTRAP sequence was amplified by nested PCR using a forward outer primer  and a reverse outer primer . The inner pairs of primers were PvTRAP-F1  and PvTRAP-R1 . PCR amplification was performed in a total volume of 30 µl of the reaction mixture containing template DNA, 2.5 mM MgCl2, 300 mM each deoxynucleoside triphosphate, 3 µl of 10x ExTaq PCR buffer, 0.3 µM of each primer and 1.25 units of ExTaq DNA polymerase . Thermal cycling profile for primary PCR included the preamplification denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 2 min, and a final extension at 72°C for 5 min. Nested PCR was done using the same thermal profile except that amplification was done for 25 cycles. DNA amplification was carried out by using a GeneAmp 9700 PCR thermal cycler . ExTaq DNA polymerase reportedly possesses efficient 5′→3′ exonuclease activity to increase fidelity and no strand displacement . The PCR product was examined by electrophoresis in a 1% agarose gel and was purified by using QIAquick PCR purification kit . The PvTRAP sequences were determined directly and bi-directionally from PCR-purified templates and were performed on an ABI3100 Genetic Analyzer using the Big Dye Terminator v3.1 Cycle Sequencing Kit . Whenever singleton substitution occurred, sequence was re-determined using PCR products from two independent amplifications from the same DNA template. Sequences in this study have been deposited in the GenBank Database under the accession numbers KJ807186 to KJ807299.DNA was prepared from blood samples using QIAamp kit . The complete PvTRAP sequence of the Salvador I strain as reference. The PvTRAP sequences of our previous Thai and Brazilian isolates previously published PvTRAP sequences were aligned with the TRAP sequences of P. knowlesi (PkTRAP) and P. cynomolgi (PcyTRAP) . Haplotype diversity (h) and its sampling variance were computed according to equations 8.4 and 8.12 but replacing 2n by n Sequence alignment with the Clustal X program dS) and nonsynonymous substitutions per nonsynonymous site (dN) were computed by using Nei and Gojobori’s method dS and dN was set at 5%.The rates of synonymous substitutions per synonymous site  and the differences between the number of singletons and the average number of nucleotide differences between pairs of sequences (F* test) PvTRAP, a sliding window analysis of Tajima’s D across the coding sequences was done using a window size of 250 bp and a step size of 10 bp Tests for departure from neutral evolution based on intraspecific comparisons were done by Tajima’s D and F tests, and McDonald-Kreitman tests. Fu and Li’s D statistics measure the differences between the number of mutations in external branches of the genealogy and the number of mutations while their F statistic compares the differences between the number of mutations in external branches of the genealogy and the average number of nucleotide differences between pairs of sequences Evidence of departure from neutrality based on interspecific comparsion included Fu and Li’s PvTRAP gene. The single-likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), random effects likelihood (REL) and mixed effects model of evolution (MEME) methods implemented in the HyPhy package a priori distribution of rates across sites whereas REL model first fits a distribution of rates across sites and then infers the substitution rate for individual sites. MEM algorithm detects codons under episodic positive selection unmasked by the abundance of purifying selection along the lineages p values<0.1 and Bayes Factor>1000 for REL followed the default values available on the Datamonkey Web Server Tests of departure from neutrality at specific codons were performed based on estimation of the global ratio of the rate of non-synonymous to synonymous substitutions (dN/dS) across the FST identical to the weighted average F-statistic over loci, θw Fst was calculated based on Fst/(1-Fst) to address the relationship between the magnitude of population differentiation and distance between endemic areas The population genetic structure was analyzed by molecular variance approach (AMOVA) using the Arlequin 3.5 software which is similar to the Weir and Cockerham’s method but it takes into account the number of mutations between haplotypes The genetic structure was also determined by STRUCTURE 2.3.4 program that deploys the Bayesian model–based clustering approach PvTRAP haplotypes was constructed by using the Maximum Likelihood method based on the model with the lowest Bayesian Information Criterion (BIC) scores The phylogenetic tree of the Amino acid properties were characterized based on polarity and charge. Polarity of each residue is categorized as polar  or nonpolar (the remainder). Charge property includes positive , negative (D and E) or neutral (the remainder). Prediction of HLA-I binding peptides was based on the method taken into account proteasomal C terminal cleavage and transporter associated with antigen processing (TAP) transport efficiency. The predicted scores were determined by the NetCTL program PvTRAP sequences, 109 isolates contained 1668 bp and 5 isolates from Tak province whose sequences were identical had 1677 bp. Sequence alignment with the Salvador I strain revealed 36 nucleotide substitutions within 33 codons, resulting in 26 nonsynonymous and 7 synonymous changes. Comparing with previous data from Brazilian, Thai Of 114 complete PvTRAP haplotypes in southern Thailand  was rather skewed towards few haplotypes as shown by a relatively lower value of haplotype diversity (h = 0.653±0.063) whereas parasite populations from Tak, Prachuap Khirikhan and Chanthaburi provinces had significantly higher haplotype diversity values, suggesting a more even distribution of haplotype frequencies in these latter endemic regions  to 0.00272±0.00032 (isolates from Prachuap Khirikhan province). The magnitudes of nucleotide diversity in this locus did not significantly differ among isolates from Tak, Prachuap Khirikhan and Chanthaburi provinces but these were significantly greater than that from Yala and Narathiwat provinces (p<10−4) (The distribution of  regions . It is n regions . The nuc(p<10−4) .STF values did not deviate significantly from zero, implying that they shared a common origin or a high genetic admixture. However, P. vivax populations from Tak and Prachuap Khirikhan provinces displayed the STF values that were significantly greater than zero when compared with populations from Chanthaburi province (p<10−5) as well as that from Yala and Narathiwat provinces (p<10−5). A significant departure from neutrality was also noted when isolates from Yala and Narathiwat provinces were compared with those from Chanthaburi province (p<10−5), implying genetic differentiation between populations with minimum or absence of gene flow between these areas. Furthermore, the Fst values between Tak isolates in the present study (collected in 2006–2007) and those collected in 1996 P. vivax population in this area . Likewise, positive correlation was found when geographic linear distance between endemic areas was considered , implying that the majority of P. vivax populations in Thailand were genetically isolated by distance  except P. vivax isolates from Yala and Narathiwat provinces . Closer looks into the rate of nucleotide substitutions in each domain of PvTRAP has revealed that Nd significantly outnumbered Sd in domains II and IV of isolates from Tak, Prachuap Khirikhan and Chanthaburi provinces. Although no significant difference in Sd and Nd for the entire coding region of the PvTRAP gene was observed when all parasite populations were considered, Nd in domain IV was obviously greater than Sd , reflecting a signature of positive or balancing selection. Consistent results were obtained when the TRAP gene of P. knowlesi was used as the outgroup sequence  for P. vivax populations in Thailand except those from Yala and Narathiwat provinces (0.05<p<0.1). Although these recombination breakpoints occur at different positions, i.e. between nucleotides 1008 and 1009, 1170 and 1171, and 1173 and 1174 for Chanthaburi, Prachuap Khirikhan and Tak provinces, respectively, it is noteworthy that all were located in domain IV of the PvTRAP gene. Meanwhile, estimation of the parameter Rm reveals 3 and 5 recombination sites in parasite populations from Tak and Prachuap Khirikhan provinces whereas Chanthaburi isolates and southern Thai P. vivax populations had equal number of potential recombination sites (Rm = 2). Locations of potential recombination sites for each population are listed in PvTRAP locus.Evidence of recombination in the PvTRAP haplotypes from worldwide origins  reveals that the majority of Brazilian isolates were placed in a separate lineage while the majority of Tak isolates collected in 1996, in 2006–2007 and most Prachuap Khirikhan isolates were clustered together. Several isolates from Chanthaburi, Yala and Narathiwat provinces seem to be located in separate branches from the remaining Thai isolates. However, there was no clear lineage for each parasite population because several isolates from different endemic areas shared or was placed in closely related branches. More importantly, all branches in this phylogenetic inference received bootstrap values less than 80%, suggesting no distinct lineage of the  origins .Host adhesion and motility are fundamental properties of malarial sporozoites to establish infection that involves sporozoite-specific proteins, one of which is TRAP. It has been shown that TRAP-deficient sporozoites were severely impaired in their host cell adherence property and gliding motility; thereby, invasion of mosquito’s salivary glands and hepatocytes was interrupted PvTRAP locus among parasite populations in Thailand seems to be comparable among endemic areas except that from southern region  than that from Thai-Cambodia border (Chanthaburi isolates) seems to be in line with Rm in each population. It is likely that transmigration of people could maintain and spread of P. vivax harboring variant PvTRAP haplotypes while recombination could further generate novel alleles. Intriguingly, it seems that reduction in genetic diversity of southern parasite population may not be simply explained by clonal population structure of P. vivax in this region because recombination event could be traced despite the paucity of potential recombination sites observed (Rm = 2). In contrast, several decades of extensive malaria control in this region and a lack of remarkable re-introduction of malaria cases from Malaysia could have resulted in population bottleneck as previously suggested PvTRAP haplotypes may gradually increase the level of genetic diversity in this population.The extent of sequence diversity of the n region . The resPvTRAP locus had significant difference in the rate of nonsynonymous substitutions than that of synonymous substitutions. Evidence of positive or balancing selection was also reaffirmed by the McDonald-Kreitman test. Although departure from neutrality in the entire coding PvTRAP sequence was not apparent by using Tajima’s D and related statistics, sliding window analysis has identified significant negative Tajima’s D values in domains II of P. vivax population from Prachuap Khirikhan and Chanthaburi provinces while significant positive values occurred in domain IV among isolates from Yala and Narathiwat provinces. Differential patterns of departure from neutrality among populations could reflect different evolutionary forces exerted on PvTRAP of each population. Alternatively, intrinsic statistical property of Tajima’s D statistics can be confounded by demographic processes D observed in southern Thai isolates could imply balancing selection or a recent bottleneck because an excess of intermediate frequency alleles as rare alleles are lost from the population due to genetic drift can be found immediately after the bottleneck. Meanwhile, significant negative Tajima’s D value in domains II could imply purifying selection and selective sweep while migration and population growth may be the confounders PvMSP4 and PvMSP5 in which frequency of recombination seems to correlate with endemicity of each area Our analyses have shown that domains II and IV of the PvTRAP locus among P. vivax populations could be deployed for analysis of population structure. Pairwise comparison of populations revealed that South Korean isolates displayed high and significant Fst indices when compared with malaria populations in other endemic areas. Likewise, slightly lower but significant Fst indices were observed when isolates from Brazil were compared with populations elsewhere  having low and non-significant Fst indices. Therefore, diversity in the PvTRAP locus of parasites in Thailand seems to exhibit spatial but not temporal variation. It is noteworthy that regression analysis of linear geographic as well as normal logarithm of geographic distances and pairwise linearized Fst values yielded significant correlation , indicating that P. vivax populations in these 4 endemic areas have been isolated by distance Click here for additional data file.Table S2Characteristics of amino acid substitutions in PvTRAP of worldwide and Thai isolates.(DOC)Click here for additional data file.Table S3Minimum number of recombination events (Rm) and recombination sites in the PvTRAP gene of each parasite population in Thailand.(DOC)Click here for additional data file.


The distribution of genetic variability from the interior towards the periphery of a species’ range is of great interest to evolutionary biologists. Although it has been long presumed that population genetic variation should decrease as a species’ range is approached, results of empirical investigations still remain ambiguous. Knowledge regarding patterns of genetic variability as well as affected factors is particularly not conclusive in plants.Oryza rufipogon Griff. from China, genetic diversity and population structure were studied in five northern & northeastern peripheral and 16 central populations using six microsatellite loci. We found that populations resided at peripheries of the species possessed markedly decreased microsatellite diversity than those located in its center. Population size was observed to be positively correlated with microsatellite diversity. Moreover, there are significantly positive correlations between levels of microsatellite diversity and distances from the northern and northeastern periphery of this species. To investigate genetic structure and heterozygosity variation between generations of O. rufipogon, a total of 2382 progeny seeds from 186 maternal families were further assayed from three peripheral and central populations, respectively. Peripheral populations exhibited significantly lower levels of heterozygosities than central populations for both seed and maternal generations. In comparisons with maternal samples, significantly low observed heterozygosity (HO) and high heterozygote deficit within populations (FIS) values were detected in seed samples from both peripheral and central populations. Significantly lower observed heterozygosity (HO) and higher FIS values were further observed in peripheral populations than those in central populations for seed samples. The results indicate an excess of homozygotes and thus high inbreeding depression in peripheral populations.To determine genetic divergence in peripheral populations of the wild rice O. rufipogon favor inbreeding and possibly selfing, leading to the rapidly decreased effective population size. Genetic drift, reduced gene flow, and possible local selection, consequently lead to lowered gene diversity, accelerated genetic divergence and increased inbreeding depression found in peripheral populations of O. rufipogon. Given these characteristics observed, northern and northeastern peripheral populations deserve relatively different conservation strategies for either germplasm sampling of ex situ conservation or setting in situ reserves for the adaptation to possible environmental changes and the future germplasm utilization of wild rice.Our results together suggest that historical contraction of geographical range, demographic changes, and environmental conditions near the northern and northeastern margins of  The partitioning of genetic variability on both local and large geographical scales, in either animal or plant species, is of great interest to ecologists and conservation biologists. As one moves from the interior towards the periphery of a species’ range, the spatial distribution, dynamics and structure of populations change . PopulatAlthough it has been long presumed that population genetic variation should decrease as a species’ range is approached, results of empirical investigations still remain ambiguous. In addition to the increasing references in animal studies –11, numeOryza rufipogon Griff., is a perennial herbaceous species with a mixed mating system, primarily growing on ditches, ponds, and swamps, or along rivers, streams, and lakes. It is widely distributed in the tropics and subtropics of monsoon Asia [O. rufipogon suggested that the Chinese populations possessed higher levels of genetic diversity than those from other geographical origins [The common wild rice, oon Asia –25. As eoon Asia , the speoon Asia . Worldwi origins . In addi origins , of whic origins . Periphe origins . With thO. rufipogon. Using microsatellite analysis, five peripheral populations were examined, and in comparison, 16 more centrally located populations were selected. We are specifically interested in the following questions: 1) Do the populations located on the periphery of the species range have reduced genetic variability with respect to more centrally located populations? 2) Do peripheral populations have a significant genetic structure that is different from those in the center? 3) Is the genetic diversity of this species influenced by population size? 4) Does inbreeding depression affect genetic structure and make difference between peripheral populations and those located in the center? and 5) What are conservation implications based on the observed patterns of genetic variability in the wild rice species?In this context, the aim of the present study was to investigate the population structure and genetic diversity of the northern and northeastern peripheral populations of O. rufipogon mainly grows in southern China with great abundance in large populations  and northeastern  ranges. For the purpose of sampling populations, it is important to define the periphery and core of the species. Most workers estimated the periphery, or consider peripheral only those populations remotely isolated at the geographical extent of a species’ range. However, there were several studies that gave definitions of periphery [O. rufipogon occurs in Australia, Bangladesh, China, India, Indonesia, Laos, Malaysia, Myanmar, Nepal, Papua New Guinea, Philippines, Sri Lanka, Thailand, and Vietnam [ulations . For exaeriphery . For exa Vietnam  Fig 1)..O. rufipO. rufipogon in China were collected between November and December of 2000  and central  populations from November to December of 2000. More than 25 individuals were randomly sampled in each population. All the seeds were collected from respective maternal plants, and all the seeds from one maternal plant were referred to as a family. For these six natural populations, a total of 2,382 seeds from 186 families were assayed.To study genetic structure and heterozygosity variation between generations of et al. [O. sativa [http://www.gramene.org/microsat/ssr.txt. Microsatellite polymorphisms were analyzed using specific polymerase chain reaction (PCR) conditions as described in Panaud et al. [et al. [O. rufipogon populations.Total DNA from the silica-dried leaves of a single individual or young leaves from a single germinated seed was isolated according to the protocol of Edwards et al. . Six mic. sativa –39. Thesd et al. . Microsa [et al. . To deteRs), the observed heterozygosity (HO), the gene diversity within the sample (HS), and the heterozygote deficit within populations (FIS). To avoid the lower sample sizes in the peripheral group could lead to a lower detection probability of genetic variation, allelic richness for specified sample sizes was computed with the rarefaction method developed by Hurlbert [Genetic parameters overall and for each population were assessed by calculating the allelic richness -based exact test [t = (1- FIS)/(1+ FIS) [t test [Overall estimates for two groups of populations were also obtained by pooling central and peripheral populations, respectively. ion 3.1c  while Rson 2.9.3 . To quanon 2.9.3  estimatoon 2.9.3 . Bootstract test  with FSTact test . A Manteact test  was perf(1+ FIS) . To exam [t test .Rs = 3.4331, HO = 0.2281, and Hs = 0.6766), as expected, detected much higher levels of genetic diversity for the populations studied here  reported previously [Rs = 3.518, HO = 0.217, and Hs = 0.664) were significantly larger than those for the peripheral population group. In addition, estimates of allelic richness at three of six loci significantly higher for the central populations than those for the peripheral populations with the rarefaction method (P < 0.05)  . One norn = 21; Rs: r = 0.3645, P = 0.0038; Hs: r = 0.2553, P = 0.0195)  and from the northeastern margin  .In this study, Pearson Coefficients were used to estimate the correlations between population size, geographical distance and genetic diversity within peripheral and central populations, respectively. Population size was positively correlated with microsatellite diversity ( 0.0195) . Our datF-statistics. The FST estimate among peripheral populations (FST = 0.266) was slightly higher than that among central populations (FST = 0.233), but the difference was not significant (P = 0.6461)  and central populations , respectively. The partitioning of total genetic variation of O. rufipogon among and within two groups of populations by AMOVA is shown in FIS = 0.755) than central populations (FIS = 0.673). However, the FIS difference between peripheral and central populations was not significant . Nevertheless, these differences were not significant.Genetic variation detected in peripheral and central populations was portioned into within- and between- population components by  0.6461) . The FSTnificant . The est species . PeripheHO = 0.0775, and Hs = 0.4567; maternal populations: mean HO = 0.1488, and Hs = 0.5026) than central populations  . No significant difference was detected for the allelic richness (RS) between peripheral and central populations . For the maternal samples, no significant difference was found for the FIS values between peripheral and central populations. However, peripheral populations (mean FIS = 0.8238) exhibited significantly higher FIS values than those in central populations (mean FIS = 0.6954) for the seed samples (P = 0.0281).For both two generations , peripheral populations exhibited significantly lower levels of heterozygosities (seed populations: mean HO) than seed populations  (P < 0.05) (data not shown). The FIS values were significantly higher in seed populations than in maternal populations (P < 0.05)  . In addi < 0.05) .O. rufipogon. Microsatellite analysis revealed significantly larger genetic variation in central populations than in peripheral populations. The rarefaction method confirmed significantly higher estimates of allelic richness for the central populations than those for the peripheral populations. The pattern is in agreement with theoretical predictions based on the assumption that drift or stable directional selection is major determinants of genetic diversity [O. rufipogon near the Northern limit of distribution is genetic drift that is apt to occur in severely isolated small peripheral populations. The Chinese almanac recorded that this wild rice species had a wider northern peripheral range (38° 5’ N) about 958–1,774 years ago [O. rufipogon contracted its northern range southward and reached the present-day range (28° 14’ N) due to the gradual decrease of global temperature (and other related climatic changes) in the past centuries [We obtained the evidence for decreased microsatellite variation at the periphery of the geographical range in iversity , 51. Theears ago  Fig 1)..O. rufipenturies . Peripheenturies . TherefoO. rufipogon is due to the increased inbreeding within small populations. The positively high FIS values with microsatellite analysis in the whole sample are indicative of an excess of homozygotes related to Hardy-Weinberg expectations and thus inbreeding. As pointed out by Brown [FIS values than those in central populations, we found that a positive correlation between population size and microsatellite diversity was significant in O. rufipogon. The result is expected based on the population genetic theory, which predicts that genetic variation should increase with effective population size [It is also possible that the loss of genetic variation in the peripheral populations of by Brown , small pion size , thus fuion size , 12, 55.O. rufipogon. The finding suggests that gene flow from central to peripheral populations of O. rufipogon may be reduced in proportion to their extent of geographical distance. It is understandable that historical contraction of geographical range in O. rufipogon might have led to directional gene flow from the central to peripheral populations. Reduced gene flow, according to Mayre's [Reduced gene flow promotes inbreeding and genetic drift and thus results in the decreased genetic variation in the peripheral populations. We found the significantly positive correlation between levels of microsatellite diversity and the distance from the northern margin and northeastern margin in  Mayre's  statemen Mayre's , 57–59.O. rufipogon. Nevertheless, genetic drift in small peripheral populations could be important to have promoted the likelihood of its genetic divergence from central populations of O. rufipogon. It could at least cause peripheral populations to have different alleles and/or allele frequencies than central populations as detected by microsatellite loci in this study (data not shown but available upon request). Studies of allele frequencies using enzyme electrophoresis and microsatellite analysis have also detected such differences in many other plant species [Although some researchers reported different genetic structure of peripheral populations from central populations , 16, 60, species , 60, 61,FST statistic [O. rufipogon.Mating system is important to determine patterns of intraspecific variation in gene diversity , and coutatistic , 64. Esttatistic . TherefoO. rufipogon, ecologically peripheral populations are mostly rather small and inbred, thus “homoselection” due to limited ecological niches; central populations, however, are dominated by “heteroselection” due to the most favorable environmental conditions [O. rufipogon [O. rufipogon under environmental changes in a clinal manner. In order to prove natural adaptation is not the only reason for the lowered genetic diversity in peripheral populations, we compare the genetic diversities between peripheral populations   and central populations   which almost distributed in the same latitude (23°–24° 2’ N). The results suggest that the lowered genetic diversity in peripheral populations even if the similar natural environments and climates in populations. Further evidence should be sought to test whether most observed gene variation is of adaptive significance and thus is maintained by some form of balancing selection.Natural selection, in addition to genetic drift, may serve as another primary force determining the increased genetic divergence of peripheral populations from central populations . As discnditions . Moreovenditions . Consideufipogon , for exaO. rufipogon, we detected significantly low observed heterozygosity (HO) and high heterozygote deficit within populations (FIS) values in seed samples in comparisons with maternal samples, indicating an excess of homozygotes. The increased frequencies of homozygotes in seed populations imply that a great number of hetorozygotes had been eliminated during the transformation from adult plants to seeds in O. rufipogon. When an individual self-fertilizes or mates with a relative, the lethal or highly deleterious recessive alleles are often made homozygous, and thus an inbreeding depression occurs within seed populations of such an outcrossing species. The observation may come from inbreeding and thus excessive homozygotes as a result of the rapidly decreased population sizes in the wild rice species [The variation of homozygote frequency between seed populations and maternal or adult populations may be a better parameter to detect inbreeding depression. For both peripheral and central populations of  species .O. rufipogon. There is no significant difference of the FIS values between peripheral and central populations for maternal samples. However, for seed samples, significantly lower observed heterozygosity (HO) and higher FIS values were found in peripheral populations than those in central populations. The results indicate an excess of homozygotes and thus high inbreeding depression in peripheral populations. It is likely that small effective population size promotes mating events among relatives within a peripheral population. As a result, more hetorozygotes had been removed in peripheral populations of O. rufipogon than those in central populations when transforming from adult plants to seeds.Our data further suggest that, comparing with central populations, a serious inbreeding depression might occur within peripheral populations of O. rufipogon represent a significant proportion of the total genetic variation of the species. As a consequence, these populations should be considered in the maintenance of genetic diversity in the species. A reduction in genetic diversity is considered fatal for the future survival of a species and its abilities to adapt to changing environments [O. rufipogon such as those studied here, may be valuable in the evolutionary process and should be taken into consideration with respect to the conservation. Third, our results indicate an excess of homozygotes and thus high inbreeding depression due to lowered effective population size in peripheral populations of O. rufipogon. The evidence that heterozygote advantage might be more pronounced in peripheral populations [Knowledge of levels and partitioning of genetic variability within and among peripheral and central populations obtained here suggest that northern and northeastern peripheral populations of ronments –70. Thisronments . Second,ronments , 73. Thuulations  results O. rufipogon, patterns of genetic variation and population structure have substantial implications for gene conservation strategies. Comparing with continuous central populations, northern and northeastern peripheral populations possess lowered genetic diversity but increased population structure. This finding is further supported by a recent genome-wide study that documented significant correlations between levels of genomic diversity and geographic distributions in Chinese O. rufipogon populations [ex situ conservation or setting in situ reserves. Comparatively speaking, sampling strategy for gene conservation purposes will be relatively more critical for peripheral populations than in central populations. Conserving genetic diversity in northern peripheral populations may require larger reserves for in situ conservation than required in central populations.For protecting the integrity of the whole gene pool of ulations . The strO. rufipogon favor inbreeding and possibly selfing. Genetic drift, reduced gene flow, and possible local selection, consequently lead to lowered gene diversity, accelerated genetic divergence and increased inbreeding depression in peripheral populations of O. rufipogon. These characteristics make these peripheral populations likely to develop local adaptations to extreme environmental changes, and therefore are possible to be of great interest for wild rice germplasm conservation. To meet needs of the future global environmental changes further studies are of potential importance to guide rice breeder to find novel alleles for the rice improvement programs.Historical extinction events, demographic changes, and environmental conditions near the northern and northeastern margins of


Pygoscelis papua) individuals from 14 colonies along the Scotia Arc and Antarctic Peninsula, together with mitochondrial DNA data, we find strong genetic differentiation between colonies north and south of the Polar Front, that coincides geographically with the taxonomic boundary separating the subspecies P. p. papua and P. p. ellsworthii. Using a discrete Bayesian phylogeographic approach, we show that southern Gentoos expanded from a possible glacial refuge in the center of their current range, colonizing regions to the north and south through rare, long‐distance dispersal. Our findings show that this dispersal is important for new colony foundation and range expansion in a seabird species that ordinarily exhibits high levels of natal philopatry, though persistent oceanographic features serve as barriers to movement.Climate change, fisheries' pressure on penguin prey, and direct human disturbance of wildlife have all been implicated in causing large shifts in the abundance and distribution of penguins in the Southern Ocean. Without mark‐recapture studies, understanding how colonies form and, by extension, how ranges shift is challenging. Genetic studies, particularly focused on newly established colonies, provide a snapshot of colonization and can reveal the extent to which shifts in abundance and occupancy result from changes in demographic rates  or migration among suitable patches of habitat. Here, we describe the population structure of a colonial seabird breeding across a large latitudinal range in the Southern Ocean. Using multilocus microsatellite genotype data from 510 Gentoo penguin ( Crucial to the study of evolution is characterizing population differentiation and understanding the mechanisms that disrupt gene flow. Population differentiation is the first step toward reproductive isolation in the classic model of allopatric speciation Mayr  and creaEuphausia superba)  breeds on the Falkland Islands, whilst the Southern Gentoo (P. papua ellsworthii) is found breeding in the Southern Ocean on South Georgia, the South Sandwich Islands, the South Orkney and South Shetland Islands and the Western Antarctic Peninsula  according to the manufacturer's instructions for animal blood. The extracted DNA was suspended in 200 μL of elution Buffer AE and stored at −20°C.Between 48 and 54 feathers from each colony were selected for extraction based on feather cleanliness and appearance. Tail feathers (retrices) were prioritized over molted body feathers, as they contain a blood supply Williams . For allSpheniscus mendiculus), Magellanic penguins (Spheniscus magellanicus)     , some of which have been tested for cross‐amplification in small numbers of Gentoo penguins , 2 μM of each primer, and 2.5 μL of template DNA. Amplifications involved an initial cycle of 95°C for 15 min, 40 cycles of 94°C for 30 sec, 60°C for 90 sec, and 72°C for 45 sec, followed by a 10‐min extension at 72°C. A volume of 1 μL of diluted PCR product (1:90) was then suspended in 9 μL of HiDi Formamide  with 0.3 μL of GeneScan 500 LIZ size standard, and visualized on an ABI 3100 Genetic Analyzer. All but one marker amplified, although not all did so consistently. Twenty‐nine markers could be scored, with only eight loci being sufficiently variable and reliable for large‐scale analyses : Ech030, Ech036, Ech050, Ech065, Ech071, Ech091 . Forward and reverse sequences were aligned and a consensus multiple sequence alignment was generated in Geneious and exported for further analysis.For mitochondrial DNA, the hypervariable region of the mitochondrial control region (HVR1), also known as Domain I, was amplified using the primers GPPAIR3F and GPPAIR3R, as described in Clucas et al. . Ten newHO and HE, respectively) and number of alleles, were quantified for each colony at each locus using Arlequin v3.5.1.2 . Hierarchical F‐statistics were then calculated to search for genetic structure and find the population grouping that maximized the among‐group variation (FCT) and minimized the variation among populations within groups (FSC)  for rate heterogeneity among sites, as calculated in jModelTest 0.1.1 , and linearized estimates of ΦSTs was calculated using Mantel's test with 10,000 permutations in Arlequin.To test for isolation by distance for mitochondrial data, the correlation between these same geographic distances and pairwise α was allowed to vary. The program was run with a burn‐in of 10,000 iterations, followed by 1,000,000 MCMC steps. Each value of K (number of populations) between 1 and 14 was run 10 times, and significance was calculated from the posterior probabilities ; Falklands (n = 101); King George, South Shetland Islands (n = 41); Port Lockroy, Antarctic Peninsula (n = 37); and Signy Island, South Orkney Islands (n = 42). We modeled island location as a discrete trait using a symmetric substitution model with the Bayesian stochastic search variable selection (BSSVS) procedure, and we reconstructed ancestral states for all ancestors. We set the clock model for the mtDNA data to a strict molecular clock. We used a coalescent tree prior with constant population size and used a normally distributed prior for the mtDNA clock rate with a mean of 0.55 and standard deviation of 0.15, based on previous calculations for the mitochondrial mutation rate in the sister species Pygoscelis adeliae , as we had not sampled all possible source locations for migrants.Finally, we used the microsatellite data to assign individuals to populations to determine whether there were any recent migrants within the populations that we had sampled. Assignment tests were run in Genodive v2.0b27 . All colonies within the Falkland Islands are strongly and consistently differentiated from those south of the Polar Front (FST range = 0.103–0.315), with strong differentiation from Signy and King George Island (FST range = 0.162–0.282) and from the most distant colony, Port Lockroy (FST range = 0.176–0.315). Of those colonies south of the Polar Front, all are strongly differentiated from one another apart from King George and Signy Island, which are not significantly differentiated from one another (FST = 0.008). Relative to the other Southern Gentoos, Bird Island (South Georgia) is genetically closer to, but still retains moderate to strong differentiation from, the Falkland Island colonies (FST range = 0.106–0.200).Pairwise FST = 0.117, P < 0.001). The proportion of variation resulting from differences among groups was maximized at 16.38% (P < 0.001) when populations were placed into four groups: (1) Falkland Island colonies; (2) Bird Island, South Georgia; (3) Signy Island, South Orkneys and King George Island, South Shetlands; and (4) Port Lockroy, Antarctic Peninsula. Explained among‐group variation remained high at 15.66% (P < 0.001) when split into five groups, with Signy Island (South Orkneys) and King George Island (South Shetlands) split into separate groups, and at 15.47% (P < 0.001) when Shallow Harbour  was isolated from the remaining Falkland Island colonies, as part of a five‐group hierarchy, as shown in Table AMOVA indicated the presence of hierarchical population structure across the Scotia Arc , with almost all variation explained by separating Shallow Harbour from the rest of the colonies (FCT = 5.26%), although this was not significant (P = 0.100).Within the Falkland Islands, very weak differentiation was present  and mitochondrial  regions, as might be expected over this geographic range.Mantel's test detected significant isolation by distance in both the microsatellite (K) inferred from STRUCTURE using Evanno's method (2005) is depicted in Table K = 2 , depending on model assumptions. Taking into account the AMOVA results, the plots for individual assignments when K = 4 are displayed in Figure The summary of results for the number of populations  and Southern Gentoos (P. papua ellsworthii), with all Falkland individuals grouping together, as a separate clade from individuals south of the Polar Front. The Southern Gentoos seem to have radiated from a population that was in the vicinity of King George Island in the South Shetland Islands (P = 0.52), or possibly Signy Island in the South Orkney Islands (P = 0.38). From here, migrants appear to have dispersed north to South Georgia (Bird Island), with another portion of migrants moving southward from King George Island (South Shetlands) to the Antarctic Peninsula (Port Lockroy). Both the Bird Island and Port Lockroy individuals cluster to form well‐defined clades on the MCC tree, with the exception of three individuals from Port Lockroy. This suggests that populations in both South Georgia and the Antarctic Peninsula were established by a single or a small number of migration events and that ongoing gene flow has been low. As this tree is based on a single mitochondrial marker, we do not attempt to date the nodes on the tree, as more genetic loci would be necessary to draw reliable conclusions, but 95% highest posterior densities (HPDs) for node heights can be seen in Appendix S2.The maximum clade credibility tree resulting from discrete Bayesian phylogeographic analysis is depicted in Figure Results from the population assignment tests also suggest that ongoing gene flow between those colonies south of the Polar Front has been low. None of the individuals were identified as migrants at Bird Island, King George Island, Signy Island, or Port Lockroy. However within the Falkland Islands, six individuals were identified as recent migrants between the colonies of the Falkland Islands.This study has revealed that both intrinsic and extrinsic factors are important in determining gene flow in Gentoo penguins. The Polar Front, an extrinsic barrier, appears to be the most important determinant of genetic differentiation across the Scotia Arc, rather than a tendency toward natal philopatry and year‐round residency near colonies (intrinsic factors). Using both nuclear and mitochondrial markers, we have shown significant population structure in Gentoo penguins at the regional scale and explored the levels of admixture present at a finer scale within the species. The high degree of population differentiation either side of the Polar Front is consistent with similar evidence from biometric and acoustic analyses    emerges as a distinct population, being most closely related to the population that comprises King George Island (South Shetland Islands) and Signy Island (South Orkney Islands). Port Lockroy on the Western Antarctic Peninsula also emerges as a distinct population, again being most closely related to King George and Signy Island. It also has lower genetic diversity than any of the other populations, which is expected because of its recent establishment (Unpublished data), the maintenance of a founder population at Port Lockroy is suggestive of a single immigration event involving immigrants from outside the immediate vicinity. To have a strong founder signal, a small group of founders from the same population must have established the colony and then internal recruitment, rather than continued immigration, must have been the main driver of population growth. Populations residing along the northern portions of the Antarctic Peninsula, between Port Lockroy and King George Island, were not sampled in this study, and therefore we cannot attest to the strength of this founder effect or a potential ghost gradient that could exist in this unsampled spatial arena. However, the clustering of the majority of the Port Lockroy individuals into a clade on the mitochondrial DNA tree and the lack of detected migrants can rule out regular immigration into the Port Lockroy colony from areas outside of the Antarctic Peninsula. Future fine‐scale sampling of colonies between Port Lockroy and King George Island, as well as at additional newly established populations south of Port Lockroy, may provide additional information on the frequency and history of dispersal at smaller spatial scales.The establishment of new colonies at the southern end of the Gentoo penguin's breeding distribution in concert with recent climate change has been interpreted as the result of local dispersal from range‐edge populations to newly suitable breeding sites just beyond , and therefore a full assessment of mitochondrial diversity within Shallow Harbour is not possible with our data.Shallow Harbour, a small, west‐facing colony located on West Falkland, presents some of the most interesting signals of fine‐scale differentiation. Analytic techniques differed in their ability to find significance in the patterns of allele frequencies in this particular colony relative to the rest of the Falkland Islands. AMOVA did not show increased levels of among‐group variation when Shallow Harbour was separated from the remaining Falkland Island colonies (5 groups vs. 4 groups), although pairwise The population genetic structure of Gentoo penguins in the Scotia Arc coincides with the oceanographic barrier presented by the Polar Front, with additional population genetic structure across the Antarctic Peninsula and on the Falkland Islands. The Polar Front appears to act as an extrinsic barrier to gene flow, even in this highly mobile seabird. We also detected a genetic signal of radiation among Southern Gentoos from King George Island, which has led to a southward founder effect at Port Lockroy. Long‐distance dispersal and colonization events appear to be rare in this species. These patterns indicate that recruitment and survival strongly influence population dynamics and that intrinsic factors such as philopatry and a tendency to remain near the breeding colony year‐round have resulted in population differentiation around the Scotia Arc. Furthermore, our findings highlight how understanding patterns of genetic diversity can help identify the demographic mechanisms influencing recent population trends in Southern Ocean predators in a time of rapid environmental change.None declared.http://dx.doi.org/10.5061/dryad.84c78. The ten new mitochondrial sequences are available from Genbank with accession numbers KU527128 – KU527137. The sequences taken from our previous study can be accessed via Genbank accession numbers KJ646314 – KJ646562.New mitochondrial DNA sequences will be deposited on GenBank prior to publication. Microsatellite genotyping data, along with GPS coordinates for sample sites are available from the Dryad Digital Repository: Appendix S1. Genetic diversity of Gentoo penguins (Pygoscelis papua) at fourteen breeding sites across the Scotia Arc.Appendix S2. Maximum clade credibility tree derived from mtDNA showing the origin and differentiation of Pygoscelis papua lineages north  and south of the Polar Front .Appendix S3. Graph of the mean likelihood of the number of populations L(K) versus the number of populations K, resulting from STRUCTURE Harvester using data from all study colonies (n = 14), under the Admixture model with Correlated allele frequencies, using No Prior Location in STRUCTURE.Appendix S4. Graph of the mean likelihood of the number of populations L(K) versus the number of populations K, resulting from STRUCTURE Harvester using data from all Falkland Island colonies (n = 10), under the Admixture model with Correlated allele frequencies, using No Prior Location in STRUCTURE.Click here for additional data file.


Capsicum spp.) production in the tropics and subtropics. Of the three species of Colletotrichum implicated as causal agents of pepper anthracnose, C. truncatum is considered to be the most destructive in agro‐ecosystems worldwide. However, the genetic variation and the migration potential of C. truncatum infecting pepper are not known. Five populations were selected for study and a two‐locus  sequence data set was generated and used in the analyses. Sequences of the ITS region were less informative than β‐tubulin gene sequences based on comparisons of DNA polymorphism indices. Trinidad had the highest genetic diversity and also had the largest effective population size in pairwise comparisons with the other populations. The Trinidad population also demonstrated significant genetic differentiation from the other populations. AMOVA and STRUCTURE analyses both suggested significant genetic variation within populations more so than among populations. A consensus Maximum Likelihood tree based on β‐TUB gene sequences revealed very little intraspecific diversity for all isolates except for Trinidad. Two clades consisting solely of Trinidad isolates may have diverged earlier than the other isolates. There was also evidence of directional migration among the five populations. These findings may have a direct impact on the development of integrated disease management strategies to control C. truncatum infection in pepper.Anthracnose is among the most economically important diseases affecting pepper ( Colletotrichum species are among the most destructive plant pathogens worldwide. Member species cause the disease anthracnose, which affects fruit and plant parts in a range of economically important host species including legumes, fruit trees, and vegetables  production in the tropics and subtropics  Andrus & Moore, C. gloeosporioides (Penz.) Penz. and Sacc., and C. acutatum (Simmonds), have been implicated as important pathogens causing pepper anthracnose, with C. truncatum as the most destructive  Butler and Bisby‐ to determine the contribution of migration events to the observed genetic variation, and (3) to identify the relative contribution of sexual and asexual reproduction to pathogen evolution.The main objectives of the study were (1) to assess the diversity and phylogenetic relationships of N = 88) of C. truncatum were prepared from symptomatic fruit as described by Ramdial and Rampersad . PCR amplification was carried out using the universal primer pair, ITS4/5 . A total of 98 sequences were used in the final two‐locus data set. For each gene: 10 sequences from the Trinidad population; 8 sequences from the USA population; 7 sequences from the India population; 14 sequences from the Thailand population; 12 sequences from the Malaysia population (Table S1A and B). The USA population was specifically selected for the study because the host species for this population did not include Capsicum spp. and was, therefore, used for comparison as a non‐Capsicum host population. Only published sequences were included in this study.Sequences were identified through comparisons of cognate sequences available in the GenBank and EMBL public databases were made using the gapped BLAST algorithm. Additionally, sequences were also included based on the work of Ramdial and Rampersad . Sequenchttp://mafft.cbrc.jp/alignment/server/). Bioedit sequence alignment editor software version 7.2.5 (http://www.mbio.ncsu.edu/bioedit/page2.html) was used to visually inspect and edit the aligned sequences to common nucleotide lengths for β‐TUB and ITS gene regions.Multiple nucleotide sequence alignments were conducted using the online version of the multiple sequence alignment program, MAFFT version 6 (k), and the nucleotide diversity (π) were determined. Watterson's estimator of mutation rate (θ‐W) on a per‐sequence basis was also calculated for each population.DNA polymorphism analyses were performed using DNA Sequence Polymorphism software (DnaSP) version 5.10  was determined using DnaSP and ARLEQUIN. Hierarchical analysis of molecular variance (AMOVA) was carried out to assess the proportion of observed variation that was attributable to within‐population differences versus among‐population differences. Significance was assessed using 9999 permutations of the original data using GenAlEx version 6.3  was carried out to assess the proportion of observed variation that was attributable to within‐population differences versus among‐population differences. The significance of these differences was assessed using 9999 permutations of the original data using GenAlEx version 6.3 , which assisted in selecting specific restriction enzymes that uniquely discriminate the different sequence groups and using NEBCutter V.2  based on the approach by Ramdeen and Rampersad  of every sample  and using Bonferroni correction (P ≤ 0.001). The Index of Association (IA) and rBarD were also calculated using Multilocus 1.3b (Agapow and Burt A) and rBarD are expected to be zero if populations are freely recombining and greater than zero if there is association between alleles . Detection of possible recombination events was also performed using Recombination Detection Program, RDP3.44  or sexually reproducing (where linkage among loci is not expected). The extent of linkage disequilibrium among the five populations was determined by detecting allelic association at different loci ). The rate variation model allowed for some sites to be evolutionarily invariable . The gene tree with the highest log‐likelihood was shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by using subtree‐pruning‐regrafting (SPR) as the branch‐swapping algorithm. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 306 positions in the final data set. The robustness of the trees obtained was evaluated by 1000 bootstrap replications. C. lindemuthianum was used as the out‐group . GARLI performs heuristic phylogenetic searches based on the General Time Reversible (GTR) model of nucleotide substitution with or without gamma‐distributed rate heterogeneity and a proportion of invariant sites. The implementation of this model is equivalent to that applied in PAUP*. The ML GARLI tree topology was compared with that of the ML tree generated by MEGA 6.Phylogeny was inferred using the Maximum Likelihood method based on the General Time Reversible model  based on analysis of combined loci. When individual gene sequences were analyzed, greater genetic variation within populations than among populations was also indicated. The β‐TUB gene sequence analysis indicated that 4% of the observed variation was due to differences among populations and 96% of the variation was due to within‐population differences : Malaysia , India , and USA (#33); group 2 (magenta): Malaysia #15), USA (emia#29), and Trinidad ; group 3 (yellow): all Thailand sequences and all other sequences. For this K = 3 structuring, all members had a coefficient >80 except for only four members whose coefficient was approximately 20  also indicated that all Trinidad population pairings were the most differentiated compared with any other pairwise analyses, regardless of the gene considered.Hudson's statistics  greater than zero, which indicates linkage among loci  to infer migration events that may be occurring among the populations. This approach provides more biologically realistic information, as this type of analysis does not depend on assumptions of populations in equilibrium and of equal size. The direction of gene flow can also be estimated in addition to the rate of migration, taking into account the number of possible migrants involved in the event   Fig. . There wThe ML tree based on the ITS sequences was also generated; however, the branching pattern was entirely polytomic, with very poor resolution and low confidence of estimates of isolate placement (tree not shown). This tree reflected the low level of DNA polymorphism of the sequences in the data set, which resulted in very weak phylogenetic signal.C. truncatum infecting pepper; (2) to determine the contribution of migration events to the observed genetic variation; (3) to identify the relative contributions of sexual and asexual reproduction to pathogen evolution. The findings presented may only pertain to the set of loci analyzed and may not be representative of patterns that apply to the whole fungal genome. The availability of accurately identified sequences for this fungal species for the two loci under study in the GenBank repository was also a limitation.The objectives of the study were (1) to assess the genetic diversity and phylogenetic relationships of The Trinidad population was the most differentiated from all other populations in the study and had the highest nucleotide diversity, which could be explained by (1) secondary contact between divergent lineages or haplotypes resulting in an abundance of and mixing of individuals, (2) high mutation rates, (3) inadvertent sampling of cryptic species, and (4) possible sampling bias for the different populations. There was also evidence of within‐population variation more so than among populations based on AMOVA, and STRUCTURE analyses. High rates of migration and recurrent gene flow as a result of repeated introductions through exchange of infected seed may explain these findings.β‐TUB gene sequences. There are five processes that can produce linkage disequilibrium in a population: (1) selection, (2) nonrandom mating, (3) genetic drift, (4) genetic hitchhiking, and (5) gene flow  belong to the destructivum and not to the truncatum clade . Alternatively, the data may suggest a host jump, which has been described for another Colletotrichum species .There was evidence of asymmetrical migration among populations, which can be explained by historical and/or recent anthropogenic events. We hypothesize two possible mechanisms to explain the asymmetrical movement of the pathogen among the five populations. First, it is proposed that the migration events were recent ones as a result of importation of infected seed. India exports P ≤ 0.05) genetic differentiation from the India population. It is hypothesized that this one‐way migration event to India was an historical one that dated back to the mid‐19th century, as there is no current formalized or direct exportation of any agricultural commodities from Trinidad to India. With the abolition of the Atlantic slave trade in 1833, a system of indentureship or “organized slavery” began in 1845 and ended in 1920 . Compared with the India population, Trinidad may be older, with a higher genetic diversity, and the Trinidad isolates may have diverged earlier. Populations at or near the center of origin of a species have the highest gene diversity  aided in distributing Capsicum throughout the Caribbean basin by, at least, the 16th century  and (B) can be found in the Dryad public archive using the following link below: Table S1. (A). ITS sequences of the five populations used in the study. (B) β‐TUB sequences of the five populations used in the study.Click here for additional data file.


Tomicus yunnanensis, is a recently-discovered, aggressive pest of the Yunnan pine stands in southwestern China. Despite many bionomics studies and massive controlling efforts, research on its population genetics is extremely limited. The present study, aimed at investigating the origin and dispersal of this important forestry pest, analyzed the population genetic structure and demographic history using a mitochondrial cox1 gene fragment. Our results showed that T. yunnanensis most likely originated from the Central-Yunnan Altiplano, and the divergence time analysis placed the origin approximately 0.72 million-years ago. Host separation and specialization might have caused the speciation of T. yunnanensis. Genetic structure analyses identified two population groups, with six populations near the origin area forming one group and the remaining six populations from western and eastern Yunnan and southwestern Sichuan comprising the other. Divergence time analysis placed the split of the two groups at approximately 0.60 million-years ago, and haplotype phylogenetic tree, network, as well as migration rate suggested that populations of the latter group were established via a small number of individuals from the former one. Migration analysis also showed a certain degree of recent expansion from southwestern Sichuan to eastern Yunnan. Our findings implied that T. yunnanensis underwent both historical expansion and recent dispersal. The historical expansion may relate to the oscillation of regional climate due to glacial and interglacial periods in the Pleistocene, while human-mediated transportation of pine-wood material might have assisted the relocation and establishment of this pest in novel habitats.The Yunnan shoot borer,  Tomicus yunnanensis Kirkendall & Faccoli (Coleoptera: Curculionidae: Scolytinae), is one of the most aggressive pest species in genus Tomicus, which has caused serious annual damage in up to 20,000 ha of the Yunnan pine, Pinus yunnanensis Franchet, since the major outbreak in southwestern China in the 1980s The Yunnan shoot borer, T. yunnanensis has been confused with T. piniperda Linnaeus due to morphological resemblance, but genetic comparison between the southwestern Chinese population and the Eurasian T. piniperda suggested that the former should be treated as a previously undescribed Tomicus species et al. (2008) analyzed the morphological characters of all known Tomicus species and described this beetle for the first time For the past two decades, P. yunnanensis, is the only known host of T. yunnanensis to date T. yunnanensis has brought severe damage to the environment in this region T. yunnanensis is endemic to southwestern China, and its distribution range is highly overlapped with the Yunnan pine The Yunnan pine, T. yunnanensis and the formation of its current distribution pattern are two interesting questions underpinning the infestation process of this pest. In an attempt to answer these questions, the present study used mitochondrial DNA data to analyze the genetic structure, population relationships, and demographic history of T. yunnanensis. The present study will elucidate the origin of the species, and understand its historical expansion routes. Meanwhile, the findings may also benefit the quarantine procedures to prevent further expansion of this pest.The origin of Tomicus yunnanensis (10 populations from Yunnan and two from Sichuan), with the sample size of the population from Yanshan being the lowest  and that of the population from Xiangyun being the greatest . The geographical coordinates and altitudes of sampling sites were recorded with a Garmin eTrex Vista GPS handset   ; Fig. 1.Pinus yunnanensis in 2007 to 2012 and instantly preserved in 95% ethanol. In order to avoid sampling closely related individuals, for instance individuals sharing the same parents, at least five pine trees 100 m apart were chosen to collect the beetles, and the samples used in the present study were randomly taken from the mixed sample pool of each population.Adult beetles were captured from egg galleries on trunks of T. armandii Li & Zhang and an individual of T. piniperda Linnaeus were chosen as outgroup due to their close phylogenetic lineage to T. yunnanensis . All samples were carefully identified under a Nikon SMZ1500 stereoscope  following Kirkendall et al. (2008) et al. (2010) An individual of No specific permits were required in the present study for collection of this native forest insect. The authors confirm that the sampling sites were not privately owned or protected. This study did not involve any endangered or protected species.In order to prevent contamination by parasitoids and fungi, each sample was washed twice with double distilled water, and the antennae, elytra, and abdomen were removed Wolbachia and the nuclear copies of mtDNA fragments (Numts) are the most common problems in phylogenetic research using mtDNA markers Numts in PCR, and a Wolbachia search was also carried out to detect possible contamination (see below). Extraction protocol for genomic DNA followed the phenol-chloroform method described by Hu et al. (2013) µL dilutions were used as templates in polymerase chain reactions (PCR).Endosymbiotons such as cox1 gene was amplified by PCR on a Biometra T-Professional Standard thermocycler . The thermal profile consisted of an initial denaturation at 94°C for 3 min; followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 47°C for 1 min, and elongation at 72°C for 2 min; then a final elongation at 72°C for 10 min.For all samples, a ≈800 bp fragment of the  µL volume system using the TaKaRa Ex Taq kit , which contained 2.5 µL of 10× PCR buffer, 2.5 µL of MgCl2 (25 mmol/L), 4.0 µL of dNTP mixture (2.5 mmol/L each), 0.5 µL of both the forward and reverse primers , 0.25 µL of Ex Taq polymerase (5 U/µL), and 1.0 µL of DNA template. The primers used to amplify the fragment were Cl-J-2183 (5′-CAA CAT TTA TTT TGA TTT TTT GG-3′) and T2-N-3014 (5′-TCC AAT GCA CTA ATC TGC CAT ATT A-3′) The PCR reaction was applied in a 25Wolbachia genes in the PCR products, a strict Wolbachia search was also carried out. Another set of PCR was applied to all samples for the Wolbachia surface protein (wsp) gene using primers wsp 81F (5′-TGG TCC AAT AAG TGA TGA AGA AAC-3′) and wsp 691R (5′-AAA AAT TAA ACG CTA CTC CA-3′) wsp gene. The genomic DNA extracted from the abdomen of a Wolbachia infected female Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) and a tetracycline-treated female S. furcifera were used as positive and negative controls respectively.In order to finally exclude the possibility of obtaining cox1 gene were purified using TaKaRa Agarose Gel Purification Kit (version 2.0)  and sequenced by Sangon Biological Engineering Technology & Services Co., Ltd. . Sequencing reactions were carried out in both forward and reverse directions on an ABI Prism 3730xl automatic sequencer .The PCR products of Numts (nuclear copies of mtDNA fragments). Also, a search for nonsynonymous mutations, in-frame stop codons, and indels was carried out to further minimize the existence of cryptic NumtsRaw sequences were proofread and aligned using Clustal W Fst values, haplotype diversity (H) and nucleotide diversity (π) of each population, the degree of gene flow among populations (Nm) were calculated in Arlequin 3.11 Fst values were tested by 1,000 iterations for statistical significance, and the optimal gamma shape used in Arlequin 3.11 was estimated by jModelTest 0.1 Genetic distances and standard errors between populations were calculated with Kimura's two-parameter (K2P) model T. yunnanensis. To analyze the genetic structure of the 12 populations of T. yunnanensis, the spatial analysis of molecular variance (SAMOVA) was performed in SAMOVA 1.0 K) to the dataset. The optimal K value was selected when the FCT value in the training results reached the plateau and no single population was assigned to any group. In the present study, the range of K was 2–6 in the training. Once the optimal K value was obtained, the result of SAMOVA analysis was tested by the analysis of molecular variance (AMOVA) Nm value between the population groups were calculated using MEGA 5.1 and Arlequin 3.11. A median-joining haplotype network was constructed using Network 4.6 (Fluxus Engineering Ltd.) and categorized by population groups determined by the SAMOVA analysis.A multidimensional scaling (MDS) plot tMRCA (time to most recent common ancestor) in BEAST 1.7.5 tMRCA inference was calibrated at 1.03% per million years for Polyphaga A phylogenetic tree of haplotypes was reconstructed using the Bayesian Inference (BI) method using MrBayes 3.2.2 T. yunnanensis, the mismatch distributions DsFIn an attempt to analyze the demographic history of Nm values can be finally used to determine the extent of genetic exchange between populations.The software Migrate 3.5.1 was used to assess the degree of genetic exchange between populations by estimating the migration rates from one population to the another using both of the Bayesian inference and the maximum likelihood methods Fst against the pairwise geographic distances (as well as the logarithm transformation) between populations. The approximate spherical distances between populations were measured between coordinates of each sampling locality in Google Earth. The test of IBD was performed using IBDWS 3.23 Fst against the geographic distance and its logarithm transformation.The isolation-by-distance (IBD) is an effective tool for detecting the correlation between genetic and geographic distance. A null hypothesis was tested using the pairwise genetic differentiation Wolbachia search detected only eight infected samples scattered in five populations, and no sign of correlation between the infection rate and the molecular diversity indices was found , 28 informative parsimony sites (3.9%), and 13 singleton variation sites (1.8%). No indel and in-frame stop codon were detected. Based on current available analytical tools, no sign of Numts was detected. The nucleotide composition showed a high A-T bias of nucleotide usage comprising 69.3% of the total average nucleotide composition.The proofread of all chromatograms did not find any double peaks. The as found ; Table 2cox1 sequence were defined based on polymorphic sites and designated in numeral order, among which 14 haplotypes (24.6%) were shared by at least two populations and the remaining 43 haplotypes (75.4%) were locally private. All haplotypes and outgroup were deposited in GenBank under the accession numbers JX448430–JX448477 (H1–H47 and T. armandii) and KC986943–KC986953 (H48–H57 and T. piniperda).Fifty-seven haplotypes of the T. yunnanensis, the number of haplotypes varied greatly, with three haplotypes being the least in the population from Yanshan and 14 being the most abundant in the population from Xiangyun  of the 12 populations of T. yunnanensis was 0.378–0.949, with the diversity index of the Yanshan population being the lowest  and that of the populations from Nanhua, Ninglang, and Xiangyun all exceeded 0.94 (π) of the 12 populations varied from 0.00139 to 0.00704, with the index of the Yanshan population being the lowest and that of the Luliang population being the greatest (Rshr) also varied among the 12 populations, with that of the population from Nanhua being the lowest and that of the population from Yanshan being the highest  distances, FCT  = 0.322, P<0.01), 13.35% among populations within groups , and 54.49% within populations . For the two groups, genetic variation among populations within Group 2 (24.81%) was greater than that within Group 1 (14.34%) , Nei's average number of differences was 7.718 (P<0.001), and the Nm value was 0.955. The multidimensional scaling (MDS) plot also divided the 12 populations of T. yunnanensis into two groups, with populations from Anning, Luliang, Mengzi, Shilin, Yanshan, and Yuxi forming one group (Group 1) and that from Huili, Nanhua, Ninglang, Xichang, Xiangyun, and Zhanyi comprising the other (Group 2)  and Huili, Nanhua, Ninglang, Xichang, Xiangyun, and Zhanyi forming the other (Group 2) . The AMO(14.34%) . BetweenGroup 2) .tMRCA inferred the divergence time between outgroups and clade a as approximately 0.72±0.11 million years before present (Ma BP), and that between clade a and clade b as approximately 0.60±0.10 Ma BP.The BI phylogenetic tree of the 57 haplotypes and two outgroups showed two somewhat clear clades. Clade a directly connected to the outgroups, was predominately occupied by haplotypes found in populations in Group 1 (marked in red), and clade b mostly represents haplotypes derived from populations in Group 2 (marked in green). There were seven haplotypes in the BI tree contained individuals from both of the two population groups (marked in blue) ; Fig. S2The median-joining haplotype network was clearly structured, even with three loops and five median vectors . The netD values showed positive values in four populations from Anning, Huili, Luliang, and Yuxi, and negative values in the remaining eight populations as well as the global dataset and the populations in both groups. However, among all the negative values, only that of population from Yanshan was flagged a 0.05-level significance  (Tajima's  P<0.05) .sF values showed positive values in seven populations of T. yunnanensis, from Anning, Huili, Mengzi, Shilin, Yanshan, Yuxi, and Zhanyi. The values showed negative deviation in populations from Luliang, Nanhua, Ninglang, Xichang, and Xiangyun, and significant negative values were detected in populations from Ninglang  and Xiangyun . Neutrality tests showed significantly large negative values in the global dataset of the 12 populations  and that of populations in Groups 2 , while the test did not produce significant value of populations in Group 1   .P<0.001), and all raggedness indices were not significant in our analysis (Most of the sum squared deviation (SSD) were not significant except for that of the population from Mengzi  of Group 2 was greater than that of Group 1 at all confident intervals (CI), and the migration rate (M) from Group 1 to Group 2 was all greater than that from Group 2 to Group 1 at all CI  and the geographic distances was significantly positive , and the correlation between Fst and the logarithm transformed geographic distances was also significantly positive . However, no significant positive correlation was found when testing IBD within each of the two population groups using either the geographic distance  or the logarithm transformed geographic distance .Isolation-by-distance (IBD) was observed for the 12 populations of nanensis . The corT. yunnanensis is currently confined in the Central-Yunnan Altiplano and the adjacent areas of western Yunnan, southwestern Sichuan, and western Guizhou Pinus yunnanensis, diverged in 4–3 Ma BP T. yunnanensis diverged from its closest ancestor, T. piniperda, in 0.72±0.11 Ma BP  and nucleotide diversity (π) of these populations were greater; also Fu's sF values and the mismatch distribution identified a constant growth pattern over time for these populations  (Rprv) of populations in Group 2 were evidently greater than that of Group 1 while the nucleotide diversity (π) were much poorer ( P<0.01) ; Fig. 1Bh poorer . TherefoT. piniperda and distributed near the root of the phylogenetic tree and the haplotype network  in the populations from Huili, Nanhua, Ninglang, Xichang, and Xiangyun all came from the populations in Group 1, while four haplotypes  were completely shared by the five above-mentioned populations   ; Fig. 4.T. yunnanensis, which would likely reduce the genetic diversity, while the warmer climate of interglacial periods could release the stress and allow T. yunnanensis to expand quickly, which would likely produce a great number of private haplotypes due to the founder's effect. Our tMRCA analysis inferred the divergence of Group 1 and Group 2 in approximately 0.60±0.10 Ma BP, when the northern portion of LRGR was experiencing an interglacial period and the climate was warmer, which allowed T. yunnanensis to expand into the area, however, T. yunnanensis underwent multiple cold and warm climate oscillations due to glacial and interglacial cycles in that area after the entry. Hence, the authors speculated that the five populations of Group 2 have experienced multiple selective processes as a result of regional climate oscillation, during which the loss of ancestral haplotypes and the generation of private haplotypes occurred.The five populations are all located in the northern portion of the longitudinal range-gorge region (LRGR) of West Yunnan. Compared to the area of Central and East Yunnan, that particular area has experienced cycles of glacial and interglacial changes in approximately 0.70–0.13 Ma BP T. yunnanensis from Huili to Zhanyi should be facilitated by other factors, instead of natural dispersal via the flight of the beetles. Like other borers, T. yunnanensis lives in the trunk of its host, which could be easily relocated with untreated wood material T. yunnanensis.The population from Zhanyi in Group 2 was different from the other five populations; despite its greater ratio of Group 2 component of the shared haplotypes, the ratio of private haplotypes was considerably lower (only one) ; Table 2T. yunnanensis was originated in central Yunnan in more than 0.7 Ma BP, and expanded into northern, western Yunnan and Sichuan in approximately 0.6 Ma BP. Our analysis implied that regional climate oscillation in the Pleistocene might be responsible for the natural expansion of this beetle, it also the main driving force for shaping the different genetic profiles of these populations. However, the divergence time calculation was solely based on estimated molecular clock, thus the results should be applied with discretion. Moreover, our analysis also identified possible contemporary human-mediated relocation of the beetle from Sichuan to Yunnan.The present research suggested that T. yunnanensis could be relocated to novel habitats and go through subsequent establishment. The present research inferred that the expansion of T. yunnanensis from central Yunnan to other localities in Yunnan and Sichuan occurred during an interglacial period took place and the regional climate was evidently warmer. Providing its capability of enduring and adapting to climate oscillation, T. yunnanensis may expand its range further driven by the warming of global climate change.Like many other pine borers, T. yunnanensis is currently confined to the distribution range of the Yunnan pine due to the restriction of host range T. yunnanensis has not been reported to date. Hence, quarantine precautions should be established in advance just to prevent the beetle from spreading into these areas.Fortunately, the occurrence of Figure S1Gel images of Wolbachia search for 12 populations of T. yunnanensis. M: the TaKaRa DL2000 DNA marker, NC: negative control, PC: positive control.(TIF)Click here for additional data file.Figure S2BI phylogenetic tree of 57 haplotypes of T. yunnanensis and two outgroups.(TIF)Click here for additional data file.Table S1The Kimura two-parameter (K2P) distances  and the Nm values  between the 12 populations of T. yunnanensis.(DOC)Click here for additional data file.Table S2The pairwise Fst between populations , Nei's average number of differences within population , and Nei's average number of differences between populations .(DOC)Click here for additional data file.Table S3Migration rates (migrate from row to column) between the 12 populations of T. yunnanensis via maximum likelihood method with 10 short chains consisted of 1,000,000 replications and three long chains consisted of 100,000,000 replications.(DOC)Click here for additional data file.


Inga vera subsp. affinis as a model species, we genotyped five natural and five planted populations in the Atlantic forest of northeastern Brazil at polymorphic microsatellite loci. We studied the breeding system and population structure to test how much genetic diversity is retained in planted relative to natural populations. We then genotyped seedlings from these populations to test whether genetic diversity in planted populations is restored by outcrossing to natural populations of I. vera. The breeding system of natural I. vera populations was confirmed to be highly outcrossing , with populations showing weak population substructure (STF = 0.028). Genetic diversity in planted populations was 50% less than that of natural populations . However, seedlings from planted populations showed a 30% higher allelic richness relative to their parents . Understanding the processes and interactions that shape this system are necessary to provide ecologically sensible goals and successfully restore hyper-fragmented habitats. Future restoration plans for I. vera must consider the genetic diversity of planted populations and the potential for gene flow between natural populations in the landscape, in order to preserve ecological interactions (i.e. pollination), and promote opportunities for outcrossing.Planting of native trees for habitat restoration is a widespread practice, but the consequences for the retention and transmission of genetic diversity in planted and natural populations are unclear. Using  The long term survival of a species is critically influenced by the maintenance of genetic variation within populations. Habitat loss and fragmentation, and reduced population sizes, decrease genetic diversity within populations, and may increase the levels of inbreeding and population substructure of plant species 2In tropical forests, habitat loss and fragmentation are a ubiquitous consequence of human population growth and the expansion of agricultural areas Inga, with a high density of individuals. Unfortunately, genetic diversity is neither explicitly measured nor considered in some habitat restoration projects The reported consequences of habitat loss and fragmentation on the reproduction of flowering plants include the reduced range of reproductive attributes of communities, deprived plant-pollinator interactions and decreased gene flow between populations Inga present brush type-flowers, crepuscular or nocturnal anthesis, and secrete large amounts of nectar Inga spp. Species of I. vera, and how plantations influence the transmission of genetic diversity from adults to seeds in a fragmented landscape. We test the hypotheses that (i) adult individuals in planted populations have reduced genetic diversity relative to adults from natural populations and (ii) outcrossing between planted and natural populations increases genetic diversity in seedlings from planted populations relative to their parents. Microsatellite markers revealed remarkable differences in genetic diversity among adults and seedlings of planted relative to natural populations. Additionally, plantations of Inga seem to be connected with the natural remnants by pollen flow.The aim of this study is to determine the level of genetic diversity in planted relative to natural populations of Inga vera or Inga edulis, trees of which are planted in single species clumps at a density ranging from 20 to 240 (159±89.8) trees per hectare. These monospecific patches are physically isolated from natural areas, and since seed dispersal in I. vera is most likely by primates, which are unlikely to cross non-forest areas, there is little chance that any adult Inga trees observed in plantations could have arrived via seed dispersal.We conducted this study in Usina Serra Grande (USGA), a private property located on the Borborema Plateau, Alagoas state, northeastern Brazil . Approval to conduct fieldwork was kindly granted by the landowner (L.A. Bezerra) and one of the property managers (C. Bakker). The total area is 24,000 ha, of which ca. 9,000 ha represents Atlantic forest remnants of different shapes and sizes. These remnants are scattered in an agricultural matrix, dominated by sugar cane monoculture and pastures Inga), Sapotaceae (13 spp.), Lauraceae (11 spp.) and Sapindaceae (8 spp.) The vegetation in the area is classified as moist tropical forest under the Holdridge system, with a dry season from October to February and a rainy season from April to September . The richest plant families, in terms of tree species, are Leguminosae  is distributed from Central America to Uruguay I. vera flowers are pollinated by hawkmoths (sphingophilous), they are also visited by bats and hummingbirds I. vera flower synchronously and massively at the same period each year I. vera is an excellent model species to investigate how planted stands of trees affect gene flow across disturbed landscapes.I. vera: five planted populations in the restored monospecific stands, and five natural populations in Atlantic forest fragments (vs planted) and two generations (adults vs seedlings), we collected young leaves from individuals and stored them in silica gel for DNA extraction.We randomly selected 10 populations of ragments . We defiragments . To ensuPithecelobium elegans and I. edulis2, 3 mM MgCl2, 160 µM of dNTPs, 40 µM of Taq DNA polymerase (Fermentas). We used the following PCR profile: 95°C for 2 min followed by 30 cycles of 95°C for 15 s, 56 or 62°C for 30 s, 72°C for 30 s; 72°C for 15 min  protocol PCR fragments were run on an ABI3730 genetic analyzer (Applied Biosystems). Alleles were scored manually using Peak Scanner 1.0 (Applied Biosystems) and binned according to their motif length with Flexibin We tested for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium for each population using FSTAT v.2.9.3.2 I. vera trees between planted and natural populations to test our first hypothesis that genetic diversity is lower in planted than in natural populations. The average number of alleles per locus (A), allelic richness rarefied to the minimum sample size of diploid individuals (RA) OH and SH) We compared the genetic diversity of A, RA, OH and SH) between adults and seedlings of each population. Differences between adults and seedlings were tested using a hierarchical analysis with 1,000 permutations implemented in FSTAT, followed by Bonferroni correction.The transmission of genetic diversity in planted and natural populations were assessed by comparing estimates of genetic diversity . The presence of these unique alleles would be consistent with pollen-mediated gene flow from natural to planted populations. By considering unique alleles found in all 50 plants in natural populations against the 50 plants in planted populations, we hope to avoid the upward bias of estimates of private alleles that would be expected given the small sample sizes of individuals per population .ISF) I. vera. We used 10 adults and 10 seedlings (i.e. one seedling per adult tree) in each population for inferring ISF. Due to the sensitivity of ISF to small sample sizes, we also calculated the mean across natural and planted stands. Calculations were performed in FSTAT, and significance levels were assessed with 100,000 permutations.We calculated the inbreeding coefficient  analysis of natural and planted populations. To estimate the outcrossing rate, we used 10 families per population. Families comprised five seedlings from different fruits per tree (i.e. one seedling per fruit). The maternal parent was also genotyped. We used the Bayesian method implemented in BORICE t. We choose a chain of 100,000 steps with a burn-in of the first 10,000 steps to reach a stable posterior distribution of t values. The t value per population was obtained after five replicate runs.The breeding system of STFP<0.05).Two approaches were used to investigate the substructuring of populations. First we calculated the average Inga 08. We included this population in subsequent analyses following correction for null alleles, although this had little influence on our results. No evidence for linkage disequilibrium was detected for any planted or natural population, and all populations were in Hardy-Weinberg equilibrium for at least two loci. Measures of genetic diversity  were consistent between loci , ranging from 3.42 to 4.26 (38A = 7.599) relative to adults of natural populations  in planted (SH = 0.696) than in natural populations  than adults (31) from planted populations. Furthermore, all alleles absent in the adults of planted populations, but present in their seedlings, were found in adults of natural populations. The average allelic richness per population in seedlings from natural stands was 7.06 (population range 6.37 to 7.95), while an average allelic richness of 4.91 (population range 3.96 to 5.66) was found in planted populations . The gen38A = 10.468) than in adults  was higher (P = 0.027) than that of adults (SH = 0.696) in planted populations  between adults and seedlings of natural populations , with population values ranging from −0.106 to 0.019. The inbreeding coefficient of seedlings from natural populations, based on one seedling per maternal tree, differed significantly from zero . In accordance with the low values of ISF, we detected a high outcrossing rate in natural populations , ranging from 0.83 to 0.98, as would be expected in species with an outcrossing breeding system. Outcrossing rate in planted populations was also high .The population inbreeding coefficient of adults in natural populations was not different from zero . There was no difference between the inbreeding coefficient of adults from planted and natural populations , and significantly different from zero (P<0.05), and higher than ISF of seedlings from natural populations  compared to adults of natural populations  of planted populations.The clustering of We found evidence suggesting that planted populations might be outcrossing with natural populations. We detected an increase of 30% in allelic richness in seedlings compared with adults of planted populations, while no change was detected in natural populations. The presence of alleles only found in adults from surrounding natural forest fragments (and not in planted stands) provides some support for pollen flow from natural stands enhancing levels of diversity in planted populations. We also detected an increase in expected heterozygosity from adults to seedlings in planted populations, without a change in natural populations, as found for allelic richness. We do, however, recognize that our experimental design, intended to sample multiple populations and seedlings with low coverage of individuals, is unlikely to detect the full range of allelic diversity in a given population, and therefore cannot comprehensively prove outcrossing to natural populations.Pinus canariensis, which is a wind pollinated tree Pinus canariensis plantations surrounded by larger natural stands. Our results are also consistent with genetic recovery in planted populations of trees, but in this case in an angiosperm tree species that is insect pollinated in a hyper-fragmented area. This shows that while the planting of trees generally results in populations with lower allelic richness I. vera, genetic diversity can, to some extent, be restored by receiving pollen from natural populations.Gene flow from natural to planted stands has been detected for Inga species, including I. vera, demonstrated that all species were obligate outcrossers (have a xenogamic mating system) and are dependent on cross pollination to set fruits and seeds I. vera reported here are consistent with obligatory xenogamy. Furthermore, we found high genetic diversity  and outcrossing rate values in adults and seedlings from natural populations, as would be expected in an obligate xenogamic tree species with large effective population sizes. Similar patterns of low inbreeding values and high genetic diversity have also been found in I. edulis from Peruvian Amazon forest Inga species.Previous pollination experiments with nine I. vera is likely to indicate high levels of gene flow between populations. Weak population genetic structure may be a consequence of the pollination system of I. vera and also confirms the occurrence of outcrossing in the populations that we studied. The majority of Inga species, including I. vera, can be considered hawkmoth pollinated, despite occasional vistitation by bat and hummingbirds during the day Inga species are ensured by the cross pollination associated with vectors that can move long distances I. vera.The low genetic structuring detected among populations of I. vera. Habitat fragmentation can alter reproductive strategies in plant communities Curiously, there is an increase in inbreeding of seedlings relative to adults in both natural and planted populations despite the outcrossing breeding system and low population substructuring in adults of I. vera in both natural and planted populations.Despite their large foraging area, hawkmoth species are negatively affected by habitat disturbance and may become locally extinct, or have reduced foraging routes in hyper-fragmented habitats Inga in which the main pollinators are typically hawkmoths, bats and hummingbirds Many habitat restoration plans have been implemented in the Atlantic forest during the last 30 years I. vera in the northeastern Atlantic forest I. vera which are typical of other IngaI. vera with a high density of individuals can increase the availability of floral resource such as nectar and pollen, and therefore favour the attraction of pollinators The genetic recovery found in this study may also be attributed to the highly synchronous flowering among populations of Inga vera reveal that low genetic diversity of planted populations can be recovered by cross-pollination with natural stands, particularly given the outcrossing nature of the study species. Recovery of genetic diversity will, however, be hampered if the genetic diversity of natural stands is reduced by habitat degradation, or if very large stands are planted using a narrow genetic founding stock. Future restoration plans in the Atlantic rain forest should consider the potential of pollen dispersal across large spatial scales, and make an effort to ensure high genetic diversity in planted populations, in order to facilitate successful restoration in this hyper-fragmented ecosystem.Our population genetic data for