Sus cebifrons) as a case study to test the use of genomic information as a tool in conservation management. Two captive populations of S. cebifrons exist, which originated from two different Philippine islands. We found some evidence for a recent split between the two island populations; however all individuals that were sequenced show a similar demographic history. Evidence for both past and recent inbreeding indicated that the founders were at least to some extent related. Together with this, the low level of nucleotide diversity compared to other Sus species potentially poses a threat to the viability of the captive populations. In conclusion, genomic techniques answered some important questions about this critically endangered mammal and can be a valuable toolset to inform future conservation management in other species as well.The list of threatened and endangered species is growing rapidly, due to various anthropogenic causes. Many endangered species are present in captivity and actively managed in breeding programs in which often little is known about the founder individuals. Recent developments in genetic research techniques have made it possible to sequence and study whole genomes. In this study we used the critically endangered Visayan warty pig ( In situ, that is, within the range of the species, most conservation actions focus on habitat protection (protected areas), law enforcement (for reducing threats), and sometimes translocations or reintroductions [ ex situ management, that is, outside the range of the species, such as in zoos or conservation centres, actions are mostly focused on keeping the population viable  and as similar to the wild ancestor populations as possible .Despite the successes achieved with these approaches, there are also several challenges, for example, in prioritizing species for conservation. In trying to solve this problem, the International Union for the Conservation of Nature (IUCN) in 1994 initiated a scientific approach to categorise endangerment of species: the Red List of Threatened Species. This comprehensive list is currently a leading reference for governments, NGOs, and research institutions to decide on how to spend valuable resources for species conservation . If a sp ex-situ management is mostly based on a pedigree, which requires information on relatedness between individuals. Especially for the individuals in the founder generation, however, information on relatedness is often missing. This causes breeding programs to make the potentially dangerous assumption that all founders are unrelated individuals [Nowadays, both as a legacy from earlier times and as a recent conservation measure, many threatened and endangered species are present in captivity . These pividuals . If thisividuals .The development of genomic techniques has opened up many new opportunities for both breeding and population management and has caused a revolution in the field of commercial animal breeding , 9. The The main improvement of applying NGS, with respect to genetic methods such as microsatellites and SNP-arrays, is the enormous increase in loci that can be studied . This al in situ and ex situ.The aim of this study is to apply currently available genomic techniques on whole genome sequencing data of a critically endangered mammal to test their application in species conservation and (captive) population management. It is expected that the outcomes of this study can be of direct use for conservation management of the species bothSus cebifrons) was used. S. cebifrons is endemic to the Philippines and mainly lives on the islands of Negros and Panay  (Sus scrofa) [ in situ conservation measures [ex situ). San Diego zoo received a breeding group from a conservation centre on Panay and in 2004 another group of 8 founders  was moved from conservation centres on Negros to Rotterdam zoo in the Netherlands to found a European captive population [As a case study, the critically endangered Visayan warty pig  . On Negr scrofa) . Despitemeasures , 18 the Demographic information on the captive populations was gathered from studbook files kept by regional  studbook coordinators and analysed in the software program PMx . From th S. cebifrons and other individuals, respectively (http://www.ebi.ac.uk/ena/). Seven complete whole genome sequences of Sus cebifrons individuals were available, of which two came from Rotterdam zoo and five from San Diego zoo  using tion 10.2  and variion 10.2 ). Only vion 10.2 ). Unless S. cebifrons and between S. cebifrons and other species/populations within the genus Sus, a phylogenetic analysis was carried out. Variants were called for individuals using ANGSD . Pairwiof 1e−6 , 28, andof 1e−6 .K  to 2 because the individuals came from two different islands, but tested for different K-values  by calculating cross-validation errors.To obtain insight in whether or not the individuals from the two different islands were admixed, we used the software Admixture . Only bi−8 [To derive an estimate of the historic effective population size of the population and possibly gather evidence for a population substructure, a Pairwise Sequential Markovian Coalescence (PSMC) model was used . This so−8 . Sus species for comparison .Demographic history of the individuals was studied by analysing regions of homozygosity (ROHs). ROHs can be informative for the level of inbreeding of a population; long ROHs are indicative for recent consanguineous matings while short(er) ROHs indicate more distant inbreeding as ROHs will break down over time due to recombination and mutation . ROH abu Sus scrofa and S. cebifrons were retained. Furthermore, the minor allele count for variants to be considered was 2, to remove spurious allele calls as much as possible. Subsequently, variants were annotated using VEP based on Ensembl v83.Besides nucleotide diversity outside ROHs, all filtered variants were analysed with the Ensemble Variant Effect Predictor tool (VEP ). This tF) was calculated for the sampled individuals . These results both indicate that a substructure is present, at least to some extent.The pedigree of the captive populations for both breeding programs was extracted from the studbook files . Sus cebifrons individuals. This average was lower for Negros individuals than for Panay individuals, 55 ± 29 and 142 ± 31, respectively . However, most ROHs in all Sus cebifrons individuals fell within the shortest length category of 0.2–0.5 Mb. Although logic predicts the longest category would cover the largest proportion of the genome, a high number of short ROHs could easily cover a proportion of the genome larger than a few long ROHs. This is also shown by individuals of other Sus species . The large proportion of coverage by long ROHS is an indication of recent inbreeding [For the demographic analyses we also identified regions of homozygosity (ROHs) and nucleotide diversity. On average we found 117 ± 51 (average ± sd) ROHs in theectively . The avecriteria . In mostbreeding . Sus cebifrons individuals (Nucleotide diversity outside the ROHs did not differ between the islands and was on average 12 SNPs per bin (10 kb) in allividuals  and seemividuals . The verFor variant effect prediction, 4679012 variants were retained. Of these, 38321 were exonic, 11532 were nonsynonymous but classified as tolerated according to SIFT predictions, and only 3884 were predicted to be not-tolerated . Only a  Sus cebifrons. This was visible in the results of the Admixture analysis and in the presence of island-specific variation. However, the PSMC analysis showed a similar demographic history for all individuals, regardless of their source population, suggesting one population of origin for the sampled individuals. This indicates that the present structure only arose recently in evolutionary terms . This hypothesis is supported by the relatively recent split in the phylogeny, suggesting that the split between European wild boar populations predated the split between the Sus cebifrons populations on both Philippine islands. For comparison, the split between European and Asian wild boar took place about 1 million years ago but the populations are considered one species [The genomic analyses showed that, at least to some extent, a substructure is present between the two island populations of species . FurtherThe extremely low nucleotide diversity found in the analysed individuals, as compared to other species, is probably a result of the bottleneck visible in the PSMC result. The number of short ROHs present in the genomes is indicative of past inbreeding. The data also showed long ROHs that are signs of inbreeding in recent generations. Because both captive populations in the EU and US are under strict management to minimize inbreeding, it is plausible that some relatedness was already present in the founders of the two captive populations. The small population sizes present at the islands are indicative of this as well. In addition, it is not clear whether the founders were a representative sample of the island populations. It is clear from our results that the assumption of founder-unrelatedness was violated.In the captive populations in the US and EU, numbers have increased rapidly since the founder generation. However, because reproduction was not equally successful for all individuals, the amount of genetic diversity present now is not the maximum that could have been retained. Furthermore, the extremely low nucleotide diversity and the signs of recent inbreeding (long ROHs) found in the current generation potentially threaten the viability of the captive populations. If populations are to be kept separately in the future, inbreeding in each of the populations has to be limited. However, with regard to the problems mentioned above, based on the similarity in demographic history of the individuals, it might be prudent to decide to merge the two breeding programs in order to increase the viability of the total captive population and the probability of reintroduction. By doing this the genetic diversity, and with that the potential for adaptation, will increase. Moreover, given the extremely shallow genetic divergence between the islands, problems of outbreeding depression are not expected.A decision for merging captive populations cannot be based on the present study alone. Further research should focus on deleterious load present in both captive populations. Purging in these naturally small populations may have removed variants that are deleterious in homozygous state . The ratAlthough it is not the focus of this study, the same genomic methods as described here can be used to select individuals for breeding . Individ in situ or ex situ, even with the small sample sizes generally available in conservation settings. In situ the identification of a substructure can lead to reassessing priorities for conservation. Identifying hybrids  can givividuals  and idenividuals . To do tAnother way of using genomic information without sequencing all individuals is to model the population and simulate what will happen with the genetic material under certain management strategies. There are already software programs available that can do just this. Examples are the PMx 2000 software , which mAn example of a measure that could be of interest for these modelling efforts is the number and length of ROHs. It has already been found that the selection of individuals for participating in breeding programs based on ROHs gave the best results in maintaining diversity without losing much fitness, as compared to optimal contributions and including inbred matings (purging of deleterious variation) .Although genomic data can already provide extremely useful information for the development of (conservation) management strategies, we need to understand more about the genomic measures and what they actually tell us about a population before they can be applied on a large scale. For example, about ROHs: how does the recombination landscape of the genome affect the distribution of ROHs over time? And how exactly does ROH-breakdown affect nucleotide diversity? Are these characteristics species-specific or is it possible to derive some general “rules-of-thumb”? With these uncertainties in mind, the incorporation of genomic analyses in conservation management may seem to be something of the distant future. Not least because the translation of academic knowledge to conservation practice is often slow . HoweverGenomic techniques represent a promising new toolset in the field of conservation biology. In this study genomic data analyses answered several questions regarding the captive population of the critically endangered Visayan warty pig. We found evidence for a recent split between the two island populations. However, with the current level of inbreeding, the viability of the total captive population and the probability of reintroduction might increase by merging the two captive populations. With the current rate of development, and associated lower costs, it is expected that genomic techniques will be feasible for broad application in conservation biology in the near future.Supplementary Material for Nuijten et al. 2016 including a description of all species (other than Sus cebifrons) used in this study (Table S1), an overview of the cross-validation errors for the Admixture analysis (Table S2), a presentation of shared and island specific variants found in Sus cebifrons individuals (Table S3) and the number of variants in homozygous or heterozygous state, both for synonymous, non-synonymous and non-synonymous, not-tolerated variants (Table S4). Figures include the results of a PSMC analysis for the other species included in this study for comparison , an overview of the coverage of regions of homozygosity both for Sus cebifrons  and for individuals of the other species  and the distribution of nucleotide diversity in Sus cebifrons individuals .


Agave inaequidens is used to produce mescal, and a management gradient from gathered wild, silvicultural and cultivated plantations has been documented. We expected different levels of genetic diversity and structure associated with management. Through 10 nuclear microsatellite loci we compared population genetics parameters and found that A. inaequidens exhibits high levels of genetic diversity (He=0.707) and moderate genetic structure (FST=0.112) with no differences among wild and managed populations. Bayesian analysis indicated that genetic clusters best fit with the corresponding habitats where populations grow. Natural mechanisms of gene flow and movement of agave propagules among populations by people explain these patterns. Agave inaequidens Koch is used in central Mexico to produce mescal, and a management gradient from gathered wild and silvicultural populations, as well as cultivated plantations, has been documented. Significant morphological differences were reported among wild and managed populations, and a high phenotypic variation in cultivated populations composed of plants from different populations. We evaluated levels of genetic diversity and structure associated with management, hypothesizing that high morphological variation would be accompanied by high genetic diversity in populations with high gene flow and low genetic structure among managed and unmanaged populations. Wild, silvicultural and cultivated populations were studied, collecting tissue of 19–30 plants per population. Through 10 nuclear microsatellite loci, we compared population genetic parameters. We analysed partition of variation associated with management categories to estimate gene flow among populations. Agave inaequidens exhibits high levels of genetic diversity (He = 0.707) and moderate genetic structure (FST = 0.112). No differences were found in levels of genetic diversity among wild (He = 0.704), silviculturally managed (He = 0.733) and cultivated (He = 0.698) populations. Bayesian analysis indicated that five genetic clusters best fit the data, with genetic groups corresponding to habitats where populations grow rather than to management. Migration rates ranged from zero between two populations to markedly high among others (M = 0.73–35.25). Natural mechanisms of gene flow and the dynamic management of agave propagules among populations favour gene flow and the maintenance of high levels of variation within all populations. The slight differentiation associated with management indicates that domestication is in an incipient stage.Domestication is a continuous evolutionary process guided by humans. This process leads to divergence in characteristics such as behaviour, morphology or genetics, between wild and managed populations. Agaves have been important resources for Mesoamerican peoples since prehistory. Some species are domesticated and others vary in degree of domestication.  Domestication is a gradual and continuous process through which plants undergo phenotypic and genetic changes, mainly resulting from artificial selection favouring organisms with features desirable, necessary or interesting to humans . Since Din situ management of wild plants in forests, forest patches and agroecosystems, which includes several types of interaction: letting stand, encouraging growth and special care and protection of more favourable plants. These interactions in some cases involve artificial selection, resulting in documentable domestication processes  old, with >160 species, ∼75 % of them occurring in Mexico, where it has shown a high adaptive radiation in diverse ecosystems  are prepared with fermented cooked stems. Other important uses are as a source of fibre, and as fodder. The activity that has gained the highest economic importance in recent decades is the production of mescal, spirit drinks produced from the central corm of the plant. For the production of this beverage, the use of 53 taxa has been recorded in Mexico  and one variety that is predominately found in commercial monoculture plantations (A. tequilana var. azul). Using inter simple sequence repeat markers, A. angustifolia have genetic diversity levels similar to those recorded in wild populations. In contrast, genetic diversity in A. tequilana was markedly low. These trends can be explained as a result of the differences between traditional management and commercial monocultures, and the predominant asexual method used to propagate agave crops. In southeast Arizona, A. parryi Engelm. and A. p. var. huachucensis (Baker) Little were cultivated since prehistory, as sources of food and fibre. A number of relict populations from ancient cultivated areas remain in modern landscapes. Only four Mexican lia Haw. . Using iA. angustifolia in southern Jalisco, it is clear that traditional management allows the occurrence of high genetic diversity levels in the crop lands, similar or even higher than in the wild, through the constant let standing or encouraging of plants from wild populations already growing in the crop land, or introducing them to these areas. A similar pattern was also reported for several columnar cacti species , by transplanting saplings taken from their wild populations and others produced in seedbeds or nurseries, but this practice is relatively new, no more than 30 years old  evaluate the levels of genetic structure and divergence between wild, silvicultural managed and cultivated populations of A. inaequidens and (iii) estimate gene flow among populations under different management practices, in order to identify possible sources of the gene pools.Our study aimed to (i) assess levels of genetic diversity in populations of Agave inaequidens is recognized with the common name of ‘maguey alto’, which makes reference to its large size, or ‘maguey bruto’, which makes reference to the caustic property of its tissue, because of the presence of saponins and other secondary metabolites that can cause dermatitis  Multiplex kit at a final volume of 5 µL, including 2.5 µL of Master Mix , 1 mM of each primer, 1.5 µL sterilized water and 0.5–1.0 µL of 50–100 ng µL−1 DNA. For all microsatellite loci, amplifications were carried out with a EscoSwiftMaxPro thermal-cycler, using the following protocol: initial heat activation for 15 min at 95 °C, and then 35 cycles of denaturing at 95 °C for 1 min, annealing at 60 °C for 1 min 30 s for all microsatellite loci and extension at 72 °C for 1 min. We included a final step of extension at 72 °C for 7 min. PCR products were mixed with formamide and the size standard Gene Scan LIZ-500 (Applied Biosystems) and denatured for 5 min at 95 °C. PCR products were analysed in a 3130xl Genetic Analyser (Applied Biosystems) sequencer. The resulting electropherograms were analysed using the program Peak Scanner (Applied Biosystems).Polymerase chain reaction (PCR) was conducted using the QIAGEN , effective number of alleles (Ne) per locus, expected heterozygosity (He), unbiased expected heterozygosity (uHe) and observed heterozygosity (Ho) were estimated using GenAlEx  was calculated with the program FreeNA using the excluding null alleles method assuming null alleles, with 10 000 bootstrap repetitions (FIS) was calculated correcting for null alleles with the INEst program (DC) of D), we constructed a dendrogram through the unweighted pair group method with arithmetic mean (UPGMA) method with 1000 bootstrap replicates of the original matrix with TFPGA 1.3  was determined varying K from 1 to 17, with 10 runs for each K value, in order to determine the maximum value of the posterior probability [ln P(K)]. Every run consisted of 5.0 × 104 burn-in and 106 periods of MCMC repetitions after the burn-in. We used the admixture model with correlated allelic frequencies without prior information. The number of subpopulations was additionally estimated based on the approach of The program STRUCTURE version 2.3.4  paired in both directions, and Theta  was estimated through MIGRATE-N (µ). From the values of M and Θ, we estimated the gene flow or number of migrants per population (Nm). The effective population size (Ne) per population was estimated through an average mutation rate for microsatellites , but identical results were found in the rest of the analysis when either of these loci was removed.In total, we successfully amplified 10 microsatellite loci. No evidence for large-allele dropout was detected in any locus using the software Micro-Checker. Null alleles seemed to be present in 14 populations for loci 6 and 7 .The FST was 0.112 (CI: 0.0783–0.151), indicating moderate genetic structure among populations. The mean FIS across loci was significantly different from zero , indicating inbreeding.The global FST/(1 − FST) on ln of geographic distance  and explained 11 % of the variation in FST/(1 − FST); the association between pairwise in geographic distances and pairwise FST/(1 − FST) values was significant .The UPGMA dendrogram based on genetic distances among populations Fig.  did not nce Fig.  was posiK revealed that the most probable number of genetic groups was K = 5 . When AMOVA was conducted using the STRUCTURE-defined populations, results indicated that most of the variation occurs among groups  and within populations , whereas differentiation among populations  was moderate but significant.The AMOVA carried out to test whether populations had hierarchical genetic structure according to the management type categories was not significant (M) among pairs of populations ranged from 0.703 to 35.25 migrants per generation. The effective population size ranged from 36 in CSAH2 to 709 individuals in the managed population M1 [seeSupporting Information—Appendix S1]. The gene flow or number of migrants (Nm) was below one per generation in all cases considering CSAH2 as the receiver population, and the highest value was 29 migrants from managed populations M2 to M1. In general, the wild populations SICU and SPIE and the cultivated population CROC1 had the highest number of migrants to other populations [seeSupporting Information—Appendix S2].The migration rate , showing estimates comparable with those recorded for the wild populations of A. potatorum , only comparable with the rosetophyllous silversword (0.56 species per my) , the genus  per my) . Often, Agave species are polyploid. We do not discard the possibility that A. inaequidens populations have different ploidy levels, similar to what has been recorded in A. cupreata , it has been similarly reported that the low levels of genetic diversity are due to asexual propagation  and CONACYT (CB-2013-01-221800).This research was funded by C.J.F., main author, involved in the study design, conducting of field and laboratory work, literature review and general data collection, systematization and analysis, wrote the first draft and concluded the final version of this paper. A.C., main coordinator-supervisor of the research project, contributed with original data and the designing of all the researches providing the information for the current analysis; participated in systematization and analysis of data and reviewed several drafts of the manuscript and concluded the final version this paper. P.C.-G., J.M.N. and A.G.-R. contributed to designing and following progress of the research and fieldwork and data analyses. All authors contributed to writing, reading and approved the final manuscript.None declared.The following additional information is available in the online version of this article –Appendix S1. Migration rates (M = m/µ) paired in both directions and parameter Theta (Θ = 4Neµ) for 16 populations of A. inaequidens. + = receiving population. Top row of the table shows donor populations, and first column to the left shows populations that receive migrants.Appendix S2. Effective population size (Ne) and the number of migrants (Nm) for 16 populations of Agave inaequidens. + = receiving population. In the table, the donor population is shown in the top of the table and the population that receives is shown in the left of the table.


Lilium regale E.H. Wilson is endemic to a narrow geographic area in the Minjiang River valley in southwestern China, and is considered an important germplasm for breeding commercially valuable lily varieties, due to its vigorous growth, resistance to diseases and tolerance for low moisture. We analyzed the genetic diversity of eight populations of L. regale sampled across the entire natural distribution range of the species using Inter-Simple Sequence Repeat markers. The genetic diversity (expected heterozygosity= 0.3356) was higher than those reported for other narrowly distributed endemic plants. The levels of inbreeding (Fst = 0.1897) were low, and most of the genetic variability was found to be within (80.91%) than amongpopulations (19.09%). An indirect estimate of historical levels of gene flow (Nm =1.0678) indicated high levels of gene flow among populations. The eight analyzed populations clustered into three genetically distinct groups. Based on these results, we recommend conservation of large populations representing these three genetically distinct groups. Lilium spp.) are perennial herbaceous monocots of the family Liliaceae and are commercially important as ornamental plants throughout the world. In addition, lily bulbs are regularly consumed as food in Asia [L. longiflorum Thunb., L.browniiF.E.Br. ex Miellez var. viridulum Baker, L. pensylvanicum Ker Gawl. And L. pumilum DC, have been used traditionally in China as sedative, anti-inflammatory and antitussive agents(cough suppressants)[Lilium spp. and is regularly used to treat lung ailments in China [Lilies ,3. The cin China .Lilium is centered around China with almost half of the species being distributed within China [Lilium regale Wilson is endemic to southwestern China, with the distribution limited to an area of approximately170 km2 in the valley region within the upper reach of the Minjiang River [L. regale blooms annually from May through June, with conspicuous white trumpet-shaped flowers. Since the introduction of L. regale to North America during the period from 1907 to 1911 by Wilson [The native range of in China .Lilium rng River ,7.L. regy Wilson ,due to iy Wilson ,11, it hy Wilson ,includiny Wilson .L. regale are subject to severe anthropogenic and natural disturbances including land use changes, landslides and earthquakes [L. regale is a commercially important endemic species restricted to a narrow geographic area in which severe environmental disturbances are likely to occur, conservation and management plans to protect wild populations are urgently needed. The long-term survival of the species depends on the maintenance of sufficient genetic variability to respond and adapt to new selective pressures under changing environmental conditions [The native habitats of thquakes , which tnditions ,15,16. Anditions ,18,19.In general, species with a narrow range of distribution are considered to have lower levels of genetic diversity than their more widespread counterparts, as they undergo genetic drift, inbreeding and low levels of gene flow resulting from historically small and fragmented populations . AlthougLilium species has been assessed using molecular techniques[L. martugon L.), one of the most common lily species in Europe [L. longiflorum Thunb. var. formosanum Baker)distributed in wide altitudinal ranges from the lowlands to high mountains of Taiwan[L. brownii in southern China[L.maculatum var. bukosanense, the Miyamasukashi-yuri lily, which is endemic to Japan[L. philadelphicum in the midwestern United States [L. japonicum Thunb. var. Abeanum(Honda) Kitam, one of the rarest plants in Japan [To date, the genetic diversity of only six echniques,31,32,33n Europe , the Taiof Taiwan, and L. ern China. A chlorL. regale populations across its entire natural range. ISSR markers represent mostly noncoding regions of the genome flanked by SSRs. Several genetic studies of natural populations using ISSR markers have demonstrated their high variability, reproducibility [L. regale distributed in the Minjiang River valley of China using ISSR markers to assess the levels and distribution of genetic diversity within and among natural populations.In the present study, we used Inter Simple Sequence Repeat (ISSR) data to analyze genetic diversity of cibility ,34,35,36cibility ,38 inclucibility ,39,40,41cibility ,22,40,41L.Regale is not an endangered or protected species and therefore no specific permission is currently required for academic studies. Although L. regale individuals are abundant in the area, they are not prominent because of their small size relative to the dominant vegetation, which consists of several shrub species including Caryopteris incana,Ceratostigma minus,Lespedeza Formosa and Artemisia annua[L. regale plants can be easily detected.The Minjiang River valley is relatively dry compared to humid surroundings in southwestern China, making this a unique habitat for plants. sia annua.ThereforL.regale within its established distribution region [We first surveyed for n region ,7,8 by dLeaf samples from19–23 individuals located aLilium-specific ISSR primers from Masumi Yamagishi[The leaf tissue (100mg) from each sampled individual was ground to powder in liquid nitrogen, and total genomic DNA was extracted following the CTAB-based procedure of Armaleo et al.. The extYamagishi,which we2,0.6μM of each primer, 0.5UTaq-polymerase(Takara),2μl of 10×PCR buffer and double-distilled water. The thermal cycling steps consisted of an initial denaturation at 94°C for 5 minutes, followed by 45 cycles of denaturation at 94°C for 40 seconds, annealing at 52°C for 45 seconds and extension at 72°C for 90 seconds, followed by a final extension at 72°C for 8 minutes. PCR amplification was performed in a PTC-200 DNA thermocycler . The amplification products were separated on 1.5% agarose gels containing 0.5 mg/ml ethidium bromide in 1× Tris-borate-EDTA buffer. The gels were examined under ultraviolet light and photographed using a Kodak EDAS 200 digital documentation system. All samples were analyzed at least twice with each primer to assess the reproducibility of ISSR-based genotyping.The genotyping of PCR reactions was performed in 20-μl reaction volumes containing 20 ngtemplate DNA,0.4mMdNTPs, 1.25 mM MgClH′) [pi2-qi2, where p is the frequency of visual alleles and q is the frequency of null alleles /2 and p = 1-q).MI was calculated as MI = PIC × number of polymorphic loci. Shannon’s index (H′) was calculated by the formula H′ = -pilnpi. We calculated standard genetic parameters, including the average effective number of alleles (Ne), Nei’s gene diversity (h)[I)[He), observed heterozygosity(Ho), inbreeding coefficient (Fis)and linkage disequilibrium across sampled loci and performed an analysis of molecular variance (AMOVA) to examine the partitioning of genetic variability within and among populations using the Arlequin software (version 3.5)[The ISSR banding pattern of each individual was scored as the presence or absence of a given band to construct a binary data matrix (1 and 0) consisting of 172 individuals. The informativeness of markers and diversity was evaluated using marker index (MI) , ShannonH′)  and the H′) ,48. The rsity (h), Shannonty (h)[I)and Nei’sty (h)[I) and genety (h)[I),using thty (h)[I).We also sion 3.5). The unwsion 3.5) was conssion 3.5).hs)as a measure of genetic diversity and θI and θB as measures of population differentiation using the Hickory program (version 1.0)[Nm) was estimated using the formula Nm = (1—Fst) / 4Fst[K) based on the multi-locus genotypic data. The admixture model and correlated allele frequencies were used for each run with a burn-in period of 10,000 and 100,000 Markov chain Monte Carlo replications. The optimal K value, which indicates the number of genetically distinct clusters in the data, was determined from 10 replicate runs for each value of K[K was based on the change in the log probability of the data between successive K values. Structure Harvester version 6.0 [We calculated the Bayesian estimate of panmictic heterozygosity (sion 1.0), which usion 1.0),56,57,58sion 1.0).The gene) / 4Fst.Bayesian) / 4Fst, which palue of K. The valsion 6.0  was usedsion 6.0 .We confiFst with a geographic distance matrix.We performed the Mantel test  in the Ir2 = 0.909, P<0.05).The highest H′ was observed with primer 3A07(0.3858) and the lowest with 3A42 (0.2779).The 10 selected primers generated a total of 180 unambiguous and reproducible bands with sizes ranging from 220 to 2000 bp, of which 176 (97.7%) were polymorphic. The number of bands per primer ranged between 10 (primer 3A53) and 22 (primer 3A21), with an average of 18 bands per primer . Among th and I, with the highest values of h(0.2018) and I(0.3180) in FHB and lowest values (h = 0.1419 and I = 0.2413) in WZT. The SDG, XV and YMF populations showed moderate values of h and I, and the values were lower for the TF,SEG and SP populations. The overall average values of h and I were 0.1989 and 0.3339, respectively. The values of He ranged from 0.2850 in WZT to 0.3473 in FHB. The YMF, XV and SDG populations showed relatively high values of He, TF showed moderate values and SP and SEG showed relatively low values. The highest value of Ho occurred in the FHB population, followed by YMF and TF. The lowest value of Ho was found in SEG. The Bayesian estimates of the panmictic heterozygosity[The average observed number of alleles per population ranged from 1.672 in YMF to 1.811 in TF. FHB,WZT and SDG showed relatively high levels of observed allelic diversity, whereas populations SEG, XV and SP showed low levels of observed allelic diversity. The meaozygosityranged frThe Hardy-Weinberg equilibrium test based on Markov chain Monte Carlo iterations revealed significant departures from Hardy-Weinberg equilibrium at five and six loci in populations XV and SEG, respectively. Similar departures from Hardy-Weinberg equilibrium were observed at one to three loci in the remaining populations, suggesting a varying degree of inbreeding or selection in populations.Fst = 0.1909, P = 0.00). The results of the AMOVA indicated that most (80.91%) genetic variation was within populations, with a moderate amount (19.09%) among populations.Overall, the population differentiation was moderate to high  and the average θB was 0.1849 . The θB value was similar to, but slightly higher than, the Gstvalue (0.1619) estimated using the POPGENE software, while θI was similar to the Fst value (0.1909) calculated with the Arlequin software. The gene flow estimate (Nm) was 1.0678 and suggests a moderate level of migration among populations. The pairwise Nei’s genetic distances (DS) among populations ranged from 0.0201 between SDG in the Minjiang River valley and TF in the Zagunao River valley to 0.0636 between the WZT and SP populations. The pairwise Fst values ranged from 0.1024 between SDG and TF to 0.2581 between XV and WZT  or population differentiation parameters .There were no significant correlations between the geographic and genetic distances tested  were 58.37%,29.80% and10.02%.Theprincipal component analysis also clustered the eight populations into three distinct groups, but these were different from the three groups identified by UPGMA. In PCA, Group i consisted only of population TF, Group ii consisted of FHB, XV and SP, and Group iii consisted of YMF, SEG,SDG and WZT.The Baye at three, suggest at three revealedL.regale. Although the details of the mating system of L.regale are not well known, plants grown ex situin conservation sites have been observed to produce seeds through self-pollination[L. regale are winged, semi-circular, very thin and light (~5mg) [L. regale.The PIC of the ISSR primers ranged from 0.1597 to 0.2783, and the MI ranged from 2.0988 to 5.5660,which is lower than the SSR primers for this species[llination.The spect (~5mg) . Both poLilium, the genetic diversity of L. regale was moderately high. Higher genetic diversity in L. regale is likely to be attributable to its potential mixed mating system and long-range pollen and seed dispersal. The genetic diversity of L. regale is higher than the average genetic diversity reported for rare and common plant species at the population  and species  levels [L. regale were higher than those reported for narrowly endemic plant taxa [The evolutionary potential of a species largely depends on its level of genetic variability, and, for plants, the overall level of genetic variability in a population is often correlated with the geographical distribution range of the species,27,69.Co) levels . Overallant taxa ,70,71.L. regale observed in the present study  fall roughly in between those reported for selfed and outcrossed plants, further suggesting a mixed mating system in L. regale. Similarly, the self-pollination of L. regale [exsitu conditions indicates that L. regale is self-compatible. However, the observed Gst value (0.1619) of L. regale was considerably lower than the Gst value (0.510) reported for generally self-pollinated plant species [L.regale flowers suggests that they may attract animals as pollinators. In addition, strong and frequent wind in the Minjiang River valley [L.regale also suggest wind pollination. Thus, it is possible that a combination of animal and wind pollination mechanisms function in L. regale. This is further supported by the fact that the Gst value of L.regale falls between those of typically wind-pollinated (Gst = 0.100) and typically animal-pollinated (Gst = 0.216) plants [The mating system can greatly influence genetic diversity both within and among populations. In general, most of the genetic diversity in self-pollinated plants is distributed among populations, whereas in outcrossed plants, most of the genetic diversity is distributed within populations. The val. regale  under ex species . Further) plants .L. regale is an endemic species with a limited distribution range, the levels of inbreeding in L. regale were lower than the values reported for rare (Fis = 0.175) and common (Fis = 0.184) plant species [Fst = 0.212) and common (Fst = 0.198) species. The inbreeding value in L. regale was also lower than the range reported for the congeneric species L. japonicum Thunb. var. abeanum(Honda) Kitam (Fis = 0.042–0.385)[L. regale (Nm = 1.0678) that we observed may account for the low levels of inbreeding. The light, thin seeds of L. regale with wind velocity of 1.4–4m/s[L. regale seeds and pollen commonly disperse across the rivers.Plants with narrow distribution ranges are prone to high levels of inbreeding, leading to loss of genetic variation and differentiation of populations,73,74. A species , and mea42–0.385). Geneflo 1.4–4m/s. HoweverLilium grow in the forest understory in shaded and moist conditions, L. regale grows in dry habitats in high sunlight in the Minjiang River valley. The high level of outcrossing due to wind-mediated pollination and seed dispersal may increase the genetic diversity in L.regale, allowing it to escape inbreeding depression and to respond to selection pressure under these harsh environmental conditions. Thus, the endemic and narrowly distributed L.regale maintains relatively high genetic diversity and gene flow through its adaptations for the environmental conditions in the Minjiang River valley.Although most species of the genus L.regale was within populations rather than among populations. The UPGMA, principal component and STRUCTURE analyses clustered the populations into three groups. However, the three groups determined by the three approaches consisted of different populations, with SEG,SDG,WZT and TF being the most differentially represented, which might be are result of the mixed ancestry of these populations. None of the clusters corresponded to the geographical distribution of populations, except for YMF and FHB.The results of the AMOVA indicated that most of the variation in L. regale was lower than the corresponding value of the self-incompatible species L. brownii (24.67%) [L. longiflorum Thunb. var. formosanum Baker [L. regale.The among-population genetic variation of (24.67%) but was hum Baker ,again suL.regale populations showed approximately2.3% linkage disequilibrium, and several of the populations showed significant departure from Hardy-Weinberg predictions, suggesting some clonal reproduction. The high number of ramets per genet will result in a large numbers of viable pollen and seeds, increasing the chances of successful establishment and survival of progeny derived from that genet. Moreover, for species with both asexual and sexual reproduction, efficient clonal propagation may result in a reduction in sexual reproduction[L. regale.Typically, clonal propagation is expected to give rise to higher levels of linkage disequilibrium, differentiation between populations, and heterozygosity than Hardy-Weinberg expectations for sexual reproduction ,78. The roduction,80. As aroduction,82. TherL.regale, suggesting that the high genetic diversity in this species is a consequence of recruitment of individuals through sexual reproduction.Clonal plants may improve fitness in stressful environments by increasing resource acquisition through the development of a large rooting system, which may involve the physical integration of ramets, and redL. regale shows relatively high genetic diversity, mostly within populations, and low levels of inbreeding compared with other narrowly distributed endemic plants. Strong winds prevalent in the river valley and a possible mixed mating system in this species may play important roles in maintaining a high level of gene flow among populations through pollen and seed dispersal. The fact that there were three genetically distinct groups of populations of L.regale should be taken into consideration in planning conservation and management programs for this species.Despite being an endemic plant species confined to a relatively small geographical region in southwest China, Lilium regale may serve as an important germplasm for the improvement of many lily varieties, and conservation strategies to protect native populations and prevent genetic erosion are urgently needed. For example, because L.regale grows in relatively dry environments, it likely carries alleles that confer tolerance to drought conditions that are predicted to become widespread under climate change scenarios[scenarios.L. regale populations. First, measures should be taken to minimize anthropogenic impact on native populations of L. regale. The natural habitat of L. regale is subject to heavy grazing and farming activities. The thin, dry soil of the habitat is prone to erosion leading to the loss of many populations and reducing the sizes of remaining populations[L. regale should focus on maintaining local adaptation. Thus, populations representing diverse habitats that include altitudinal or soil moisture extremes should be conserved.Several measures must be implemented for the conservation and protection of pulations. Second,pulations. We recopulations. Third, Further analyses using more robust and more variable markers such as microsatellites or whole genome sequence-based single-nucleotide polymorphisms should be conducted to estimate the levels of self-pollination, consanguineous mating and outcrossing, and genome-wide associations with specific traits of ecological significance. Mating system studies combined with analyses of seed dispersal, germination and viability are needed to test some of the hypotheses resulting from the present study. Capitalizing on recent advances in conservation genomics techniques, including sequencing of whole genomes , many po


Populations occurring in similar habitats and displaying similar phenotypes are increasingly used to explore parallel evolution at the molecular level. This generally ignores the possibility that parallel evolution can be mimicked by the fragmentation of an ancestral population followed by genetic exchange with ecologically different populations. Here we demonstrate such an ecological vicariance scenario in multiple stream populations of threespine stickleback fish divergent from a single adjacent lake population. On the basis of demographic and population genomic analyses, we infer the initial spread of a stream-adapted ancestor followed by the emergence of a lake-adapted population, that selective sweeps have occurred mainly in the lake population, that adaptive lake–stream divergence is maintained in the face of gene flow from the lake into the streams, and that this divergence involves major inversion polymorphisms also important to marine-freshwater stickleback divergence. Overall, our study highlights the need for a robust understanding of the demographic and selective history in evolutionary investigations. Threespine stickleback fish are adapted to lake and stream habitats in Central Europe. Here, the authors show colonization of a lake basin by a stream-adapted ancestor, followed by the emergence of a lake-adapted population in the face of gene flow across lake–stream boundaries. Parallel  adapted to lake and stream habitats within the Lake Constance basin in Central Europe.Distinguishing parallel divergence from ecological vicariance scenarios is thus crucial when attempting to explore how deterministically selection acts at the genomic level during evolution. While this distinction is not possible based on phylogenetic relationships at neutral markers18142 the third largest lake in Central Europe—and multiple adjoining stream-resident populations inhabiting the lake's tributaries2122212321This stickleback system comprises a large and genetically well-mixed population residing in Lake Constance—with 571 kmAlthough the Lake Constance stickleback system has certainly formed postglacially  obtained through restriction site-associated (RAD) sequencingOur investigation focuses on four stickleback populations, including the panmictic  Lake Con23Next, we compared population-specific allele frequency spectra and found that across almost all minor allele frequency (MAF) classes, the lake exhibited the lowest and GRA the highest number of polymorphisms, with BOH and NID being intermediate . The lakFST) for each of the three lake–stream pairings revealed an increase in population differentiation from 0.005 in the lake–BOH comparison to 0.013 and 0.061 in the lake–NID and lake–GRA comparisons, whereas no stream–stream population comparison yielded baseline FST higher than 0.056 . In a rooted phylogeny, the lake population emerged as a distal branch nested within the more basal stream fish  to the lake–NID (r=0.15) and the lake–GRA pairing (r=0.12) .Finally, we quantified linkage disequilibrium (LD) between all pairwise combinations of SNPs within all chromosomes in each population and found that strong allelic associations between SNPs occurred only over a scale of 1 kb or less; beyond this distance, LD was much weaker . The peaFST) between a stream and the adjoining lake population, and not in any of the comparisons between the stream populations separated by dozens of kilometres of lake habitat. Fourth, the lake population proves to be phylogenetically derived from stream fish. All these observations can be brought in line by the biogeographically plausible perspective that the Lake Constance basin was initially colonized by ancestral stream-adapted stickleback. This colonization gave rise to multiple stream-resident populations isolated from each other by the adjoining, ecologically different lake habitat—that is, an ecological vicariance scenario. Subsequently, the lake fish started to adapt to their novel habitat and thereby experienced strong genome-wide selection. This selection should not only have reduced genetic variation in the lake relative to the streams, but also have driven relatively elevated LD within the lake, predictions clearly borne out by our analyses.In combination, the above analyses resolve the demographic and selective history of stickleback in the Lake Constance basin. First, the demography is inconsistent with the view that the populations originate from a recent introduction of (presumably few) founder individuals, and instead supports an earlier postglacial and extensive natural colonization, presumably via the Danube drainageA key implication of this ecological vicariance scenario . None of these extreme SNPs showed fixed allelic differences between the habitats, but nearly so: FST ranged from 0.94 to 0.75—remarkably high values given the low baseline differentiation  than in the corresponding stream population  of the Lake Constance population being orders of magnitude larger than the stream populations, introgression occurs primarily from the lake into the streams. Nevertheless, many loci resist gene flow and maintain substantial differentiation from the lake121516a priori to be important to lake–stream divergence. We started at the phenotypic level by establishing that lake individuals were mostly completely plated, whereas plating was relatively reduced in all stream populations, most clearly so in NID and GRA  (21FST=0.78) occurred immediately downstream of the Ectodysplasin (Eda) gene. This locus is known as major determinant of lateral plating37cis-regulatory polymorphism having been identified 1 kb downstream of the coding regionFST>0.38.Our analyses of localized signatures of selection within the genome focused on regions likely important to adaptation to the lake and stream habitats, yet it is unknown what phenotypes the polymorphisms in these regions influence. For the extent of lateral plating, however, it was possible to take an alternative route and to investigate the molecular signatures produced by selection on a trait known P<0.001) . This ag0.001) 21. The peaEda locus. We thus predicted molecular footprints of selection at this locus. To evaluate this prediction, we inspected all three lake–stream FST scans for the magnitude of differentiation around Eda (FST=0.40), just 5.7 kb downstream of Eda. However, in this particular comparison, the most divergent SNP near Eda ranked only within the upper 3.5 percentile of the genome-wide FST distribution (lake–BOH and lake–NID comparisons: 8.8 and 2.3 percentile). Similarly, the highest absolute Rsb value around Eda (1.17) also emerged from the lake–GRA comparison but fell only within the upper 23 percentile of the genome-wide Rsb distribution. Hence, thousands of SNPs displayed a stronger deviation from selective neutrality than the Eda locus. Accordingly, subjecting the lake–GRA pairing to a standard selection outlier detection analysis  occur at substantial frequencies, haplotype decay was slower around the allele associated with complete plating  fell within the top 0.06 per cent of the genome-wide distribution.More nuanced insights into the evolution of lateral plating were obtained by analysing haplotype structure around the  plating , top. Mo plating , bottom. plating . To fullegabases . Unexpecased BSA , top: thEda locus highlight our limited ability to elucidate the genetic basis of adaptive population divergence based on genetic markers when selective sweeps are incomplete. Neither the magnitude of differentiation (FST) nor haplotype structure (Rsb) among populations allowed the major plate locus to emerge as an obvious selection candidate—despite substantial evolution in the associated ecologically important phenotype, and despite an extensive selective sweep visible when comparing haplotype structure among individuals grouped by phenotype. Given that stronger signatures than those around Eda are numerous in our data sets, we conclude that hundreds of genomic regions must be involved in the adaptive divergence into lake and stream habitats. We further propose that lateral plate evolution in the Lake Constance basin is governed by at least one other locus besides Eda. Inspecting the newly detected region on ChrIV indeed produces a strong candidate gene, Col23a1 . Like Eda, this gene encodes a transmembrane collagen involved in the development of the epidermisEda occur in close proximity (c. 1.4 Mb apart) in a low-recombination chromosome region12Together, the investigations at the Our genetic data indicate that lake–stream divergence in the Lake Constance basin has occurred in the face of gene flow. Genetic polymorphisms predicted by theory to resist homogenizing gene flow and to diverge between populations particularly well are chromosomal inversions434446FST relative to baseline differentiation , 22 (44%) map to the ChrXXI inversion, including the top value observed overall (FST=0.338) (All inversion haplotypes occurring within Lake Constance further coincide with haplotypes predominant in marine stickleback. This suggests the presence of shared selective features between the ocean and large lakes—possibly mediated by a pelagic lifestyle in both habitats (see ref. T=0.338) . This suT=0.338) , indicatTo summarize, a main goal of our study was to dissect the demographic and selective history of adaptive diversification in lake and stream stickleback populations within a single lake basin. Combining demographic inference with broad scale and localized analyses of genetic differentiation and diversity, linkage disequilibrium and haplotype structure within the genome allows us to reject a standard scenario of parallel divergence of multiple stream populations from a shared ancestral lake population . InsteadFurthermore, our finding of highly heterogeneous genomic divergence conflicts with the recent theoretical prediction that adaptive divergence in the face of gene flow involving selection on extensive standing variation should produce genome-wide reproductive isolation and therefore limit heterogeneity in genome divergenceFinally, our study adds molecular evidence to the idea that chromosomal inversions promote adaptive divergence by acting as loci of large effectN=12, and Unteruhldingen, Germany, N=13; for geographic details see ref. N=22), Nideraach  and Grasbeuren  localities in ref. Specimens from the Lake Constance population were sampled at two localities , and used to generate RAD DNA libraries essentially following the protocol described in ref. DNA was extracted from stickleback fin and muscle tissue using either a MagNA Pure LC278 extraction robot  with the tissue Isolation Kit II, or the DNeasy Blood & Tissue Kit . After an RNase treatment, the extracts were standardized to 18 ng μlhttp://www.novocraft.com/products/novoalign; sequencing and alignment statistics are provided in Each library was single-end sequenced with 100 cycles on a separate Illumina HighSeq2000 lane. Raw sequence reads were parsed by individual barcodes and aligned to the improved assemblyLoci passing the above filters were subjected to consensus genotyping using a refinement of our earlier haplotype-based algorithmfastsimcoal2.1 , migration rates between the lake and each stream population (but not among stream populations) and the SNP mutation rate. The simulation was run in 80 replicates, each including 40 estimation loops with 100,000 coalescent simulations. To determine the best parameter estimates, we selected the 10 most likely replicate runs  and used this subset to calculate the mean for all parameters, along with their 95% confidence intervals . Because the lake population turned out to be particularly strongly influenced by selection, we explored an analogous model in which just the two stream populations most divergent from the lake (NID and GRA) split from an ancestor under gene flow. The joint SFS, the simulation template files, the parameter estimation files, and the command line settings used to run fastsimcoal2.1 are provided as Using the observed joint SFS, we then performed simulations with phangorn . Using only such ‘loner SNPs' avoided potential bias in the estimation of genetic diversity due to pseudo-SNPs caused by micro-indels. We further ignored those loner SNPs displaying a minor allele count <2 across all individuals pooled, thereby avoiding sequencing artifacts. We thus obtained a total of 62,332 genome-wide loner SNPs. As a first measure of diversity, we determined for each population the proportion of the total loner SNPs actually being polymorphic. To obtain a second diversity measure, we screened all loner SNPs for the presence of three alleles across all individuals pooled . On average, one out of 169 loner SNPs proved tri-allelic . We then determined for each population the proportion of the total tri-allelic loner SNPs actually displaying all three alleles.R2) between pairs of SNPs. From the raw SNP matrix, we excluded SNPs that were tri-allelic or had >25% missing genotypes, and individuals with >75% missing diploid genotype calls. The remaining SNPs were filtered for two different MAF ranges (0.05–0.275 and 0.275–0.5). Only a single randomly chosen SNP was retained if multiple SNPs passed these thresholds for a pair of sister RAD loci . The final number of SNPs was 16,088 and 18,787 for the former and latter MAF range . We then ran PLINK , we pooled the two MAF range specific PLINK outputs (one generated for the 0.05–0.275 and one for the 0.275–0.5 MAF range) before binning. Setting a MAF range of 0.05–0.5 right at the filtering step of the raw SNP matrix produced very similar results. To investigate more localized LD along chromosomes, we considered only R2 values between SNPs>2 kb but<50 kb apart . We determined the physical midpoints for all SNP pairs, binned the respective R2 values in non-overlapping 200-kb windows along the genome, and calculated average R2 for each window and population. Different window sizes  yielded similar results supporting identical conclusions. To visualize localized differences in LD along the genome between the lake and GRA populations, we subtracted for each window the GRA R2 value from its lake counterpart, yielding a metric referred to as 'Delta R2'. We further calculated the correlation of R2 values between the lake and each stream population, using the above windows as data points. The magnitude of this correlation was evaluated against its empirical random distribution generated by permuting the R2 data over the windows 10,000 times.We quantified LD within each population using the squared correlation coefficient . Applying these stringent filters, we obtained 55,476, 57,119 and 60,052 genome-wide FST values for the BOH–lake, NID-lake and GRA-lake comparisons. To obtain regions suited for a detailed characterization of signatures of selection, we chose the 25 autosomal SNPs displaying the highest FST values across the three FST data sets combined . To ensure that each of these differentiation extremes represented an independent genomic region, SNPs were ignored if they were closer than 200 kb to a SNP already accepted as extreme.Scans for genomic regions exhibiting strong differentiation were performed for each lake–stream combination.  Consistent with refs FST-based search for evidence of positive selection was complemented with haplotype-based statistics proving particularly powerful to detect incomplete selective sweeps32fastPHASE . HaFST scan by treating the phenotypic groups as populations, but otherwise following all conventions described above for the population-based FST calculations.To screen the genome for loci influencing the lateral plate phenotype, we performed a BSA by assigning 24 completely plated stream individuals to one phenotypic group, and 24 low-plated stream individuals to another group , and a third pool with all completely plated lake individuals (N=19). We calculated and plotted EHHS for each pool around the SNP exhibiting the highest FST value in the above bulk segregant genome scan . Finally, we subtracted iHS values from the completely plated stream individuals from the corresponding values in the low-plated stream individuals ('Delta iHS') across chromosome IV . Delta iHS was then averaged and plotted in non-overlapping 100-kb windows .For the Our approach to detecting inversions was based on the expectation that the two inversion types (collinear and inverted), representing two isolated populations, differ in their magnitude of divergence from the reference genome. This should cause differential read alignment success across inverted genomic regions. Inversions should thus be revealed by a physically extended distortion of the relative RAD locus sequence coverage between two populations if these populations differ in the frequency of the inversion types . The samTo confirm that the above sequence coverage method reliably detects inversions, we used RAD loci near an expected inversion breakpoint in two of the three emerging candidate regions to design PCR primer pairs across the breakpoint boundaries. These primer pairs were expected to yield a PCR product only for the inversion type occurring in the streams. Ten to 13 individuals representing a given inversion type were subjected to long-range PCR and inspected for the presence or absence of amplification  around the three detected inversions. For this, we screened each of the four population samples separately for polymorphisms with >50% available genotype calls  and calculated haplotype diversity  and the MAF at each SNP. RAD loci were allowed to contribute a single SNP only, keeping the one with the highest diversity when multiple SNPs occurred on the same locus (drawing a SNP at random or averaging diversity estimates of multiple SNPs per RAD locus yielded very similar results). Diversity was visualized using R's implementation of LOESS . The MAF frequency distribution within the inversions was plotted for the lake and for the stream population displaying the strongest inversion frequency shift from the lake. For this population, we also plotted the genome-wide MAF distribution.R2 statistic implemented in mcld . Patterns of LD around the inversions were visualized using the LDheatmap . For the largest inversion (located on ChrXXI), we randomly subsampled the resulting SNP panel to a total of 173 SNPs to reduce complexity. Haplotype reconstruction used PHASE 2.1 , using a minimal node size of two haplotypes for display (-n option). To construct haplotype networks including individuals from across the stickleback's geographic range, we randomly selected 20 SNPs from the Lake Constance-specific haplotype genealogies, and inferred the genotypes at these SNPs for a total of 11 freshwater and 10 marine stickleback specimensTo construct haplotype genealogies for the inversions using individuals from the Lake Constance basin only, we first extracted the SNPs in each inversion, . Next, we excluded SNPs with a MAF<0.05 and with >25% missing genotypes. Different MAF ranges  led to identical conclusions. Individuals with >75% missing 2.1 ref. , optimiz0.0 ref. , using tFST-based lake–stream genome scan.The Lake Constance-specific haplotype networks allowed us to unambiguously infer diploid genotypes at all three inversions for our main study individuals. Of these individuals, 33 had already been RAD sequenced previously using the Sbf1 restriction enzymeN=15 and 20 for the stream and lake inversion type, defined according to FST-based differentiation scan. Additionally, using the same groups, we determined the number and location of loner SNPs specific to each inversion type, or shared between the types, within and around the ChrI inversion. Analogous analyses for the ChrXI and ChrXXI inversions were not possible because here individuals homozygous for the stream inversion type were too rare  and PRJNA284945 (Sbf1 data from the Lake Geneva region).How to cite this article: Roesti, M. et al. The genomics of ecological vicariance in threespine stickleback fish. Nat. Commun. 6:8767 doi: 10.1038/ncomms9767 (2015).Supplementary Figures 1-15, Supplementary Tables 1-2 and Supplementary ReferencesThe observed BOH-Lake joint site frequency spectrum (SFS) input file for the 'full model' including all study populationsThe observed NID-Lake joint site frequency spectrum (SFS) input file for the 'full model' including all study populationsThe observed NID-BOH joint site frequency spectrum (SFS) input file for the 'full model' including all study populationsThe observed GRA-Lake joint site frequency spectrum (SFS) input file for the 'full model' including all study populationsThe observed GRA-BOH joint site frequency spectrum (SFS) input file for the 'full model' including all study populationsThe observed GRA-NID joint site frequency spectrum (SFS) input file for the 'full model' including all study populationsFastsimcoal parameter estimation file for the 'full model' including all study populationsFastsimcoal simulation template file for the 'full model' including all study populationsThe observed GRA-NID joint site frequency spectrum (SFS) input file for the 'GRA_NID model' including only the GRA and NID populationsFastsimcoal parameter estimation file for the 'GRA_NID model' including only the GRA and NID populationsFastsimcoal simulation template file for the 'GRA_NID model' including only the GRA and NID populationsREADME file for the fastsimcoal analyses, including command lines and details on the input files


Autosomal short tandem repeats (STRs) have become the most informative molecular markers because they are highly polymorphic and multiallelic. The objective of the present work is to study the genotypic polymorphisms, allele frequencies and forensic parameters at highly polymorphic STR loci (TPOX) among four Muslim Populations of North India: Pathan, Ansari, Saifi and Mansoori.obs and Hexp) as well as forensic and paternity indices including matching probability (MP), power of discrimination (PD), power of exclusion (PE) and polymorphism information content (PIC) were calculated using the Power Stats v.1.2 software for TPOX locus of the studied population. The POPGENE (v.32) statistical package was used for analyzing allele frequency.The DNA was extracted from blood samples by using the standard phenol-chloroform extraction method and then amplified by PCR using specific primers for TPOX locus. The PCR products were separated by electrophoresis on denaturing 6% polyacrylamide gel and silver staining was done to resolve and observe different alleles. The observed and expected heterozygosity (Hexp = 0.867). The polymorphism information content (PIC), has shown that this marker is highly informative for Mansoori (0.67) and Ansari (0.63) population group.The 6 to 10 different alleles were detected in the studied populations with 8 allele repeats are most common in all four groups of sub-population. The maximum expected heterozygosity was found in Mansoori population (HST = 16.37%). A dendrogram was constructed using the Neighbor-joining (NJ) clustering method. The dendrogram shows the low genetic distance between Pathan and Ansari population groups. According to the statistical parameters, the combined analysis of this TPOX STRs locus is a powerful tool for forensic personal identification, paternity testing and population genetic studies in North Indian Muslim Populations.In conclusion the interpopulation differentiation has been found significantly to differ from each other (F


Triops newberryi and Triops longicaudatus “short”, with different reproductive modes. Reproduction in the tadpole shrimp can occur clonally (parthenogenesis), with self fertilization (hermaphroditism), or through outcrossing of hermaphrodites with males (androdioecy). For all these reproductive modes, population genetic theory predicts decreased genetic diversity and increased population differentiation. Here we use mitochondrial control region (mtCR) sequences and nuclear microsatellite loci to determine if the difference in reproductive mode affects the high genetic structure typical of persistent founder effects. Previous authors indicated that T. newberryi is androdioecious because populations are composed of hermaphrodites and males, and T. longicaudatus “short” is hermaphroditic or parthenogenetic because males are absent. In our data, T. newberryi and T. longicaudatus “short” populations were highly structured genetically over short geographic distances for mtCR sequences and microsatellite loci . Differences between the two Triops species in a number of diversity measures were generally consistent with expectations from population genetic theory regarding reproductive mode; however, three of four comparisons were not statistically significant. We conclude the high genetic differentiation between populations is likely due to founder effects and results suggest both species are composed of selfing hermaphrodites with some level of outcrossing; the presence of males in T. newberryi does not appreciably reduce inbreeding. We cannot exclude the possibility that males in T. newberryi are non-reproductive individuals and the two species have the same mating system.Crustaceans that initially colonize a freshwater temporary pond can strongly bias the subsequent genetic composition of the population, causing nearby populations to be genetically distinct. In addition, these crustaceans have various reproductive modes that can influence genetic differentiation and diversity within and between populations. We report on two species of tadpole shrimp,  Aquatic invertebrates that disperse passively via an encysted embryo use a variety of transport methods to colonize new habitats. Abiotic factors, such as water and wind, FST does not decay for at least 2000 generations in a large population established by a few founders and subsequently experiencing migrant influx.In cyclically parthenogenetic zooplankton, De Meester et al. In addition to founder events, the mode of reproduction can also influence the amount of genetic structure and diversity in large Branchiopods Triops sp.) is a passively dispersing aquatic crustacean that has been said to use several forms of reproductive modes including parthenogenesis, hermaphroditism, androdioecy (a mix between outcrossing and hermaphrodites) and gonochorism  Triops populations, low genetic diversity, deviations from Hardy-Weinberg equilibrium, large inbreeding values (FIS) and large population differentiation have been observed and has been attributed to founder events and the degree of outcrossing between individuals The tadpole shrimp  to female (presence of a brood pouch) ratio within populations; T. l. “short” is comprised of all females and is assumed to reproduce via parthenogenesis or hermaphroditism whereas T. newberryi is thought to be androdioecious, with populations comprised of hermaphrodites that outcross with males Triops showed that T. l. “short” and T. newberryi are not monophyletic, calling into question whether species status is warranted Many of the previous studies have focused on Triops species and determine what factors  influence population differentiation. Secondly, we compare the effect of different presumed reproductive modes and the degree of inbreeding to the genetic diversity and structure of the Triops populations. We hypothesize, based on population genetic theory, that the androdioecious species will have more alleles, higher allelic richness, fewer private alleles, higher observed heterozygosity, lower FIS and FST, and relatively greater genetic variance within as opposed to between populations. The last objective is to evaluate whether the two putative species of Triops in southern New Mexico are reproductively isolated in the ponds in which they co-occur.The first objective of this study is to assess the genetic structure of each Triops as they are not listed as a protected species. Triops newberryi and T. l. “short” samples were collected in 2008–2011 at nine temporary ponds in southern New Mexico on public land, the Chihuahuan Desert Rangeland Research Center (CDRRC) and the Jornada Experimental Range (JER), both owned by New Mexico State University: two natural playa lakes , six modified playa lakes , and one man-made flood retention pond (FP-03)  and T. l. “short” (n = 66). Reaction volumes of 25 µl consisted of 10 µl GoTAQ Green Master Mix , 400 pM of each forward and reverse primer and 1 ng/µl of genomic DNA. PCR reactions were run in a Fisher thermocycler  with the following conditions: 94°C for 2 minutes, followed by 35 cycles of 94°C for one minute denaturation, 50°C for one minute annealing, 72°C for one minute elongation and a final extension of 72°C for 15 minutes. PCR products were checked for strength of amplification on a 1% agarose gel. Purification of PCR products was performed with ExoSAP-IT  following manufacturer’s protocol. Purified PCR products were then sequenced by NMSU’s MOLBIO Molecular Analysis Service in both forward and reverse directions (http://mmas.research.nmsu.edu).Extraction of DNA followed a modified version of the HotShot method described by Montero-Pau et al. π), and haplotype diversity (h), for each playa and all samples collectively were obtained by Arlequin v3.5 Sequences were aligned using the assembly function in the program Geneious Pro v5.4.6 The appropriate substitution model (TrN+I) for the data was selected based on the Akaike information criterion (AIC) using Modeltest v3.7 ST values. A sequential Bonferroni analysis ST/(1−ΦST)] were directly compared to log transformed pairwise geographic distance between the playas to test an isolation by distance hypothesis with the IBDWS v3.21 utility on the web that uses a Mantel test for the analysis The program Arlequin v3.5 T. newberryi from six ponds and 156 T. l. “short” samples from six ponds were genotyped for eight loci developed specifically for Triops species found in southern New Mexico , 2.5 mM MgCl2, 0.2 mM DNTPs, 0.04 U/µl Taq-Polymerase  and 2 ng/µl genomic DNA. Microsatellite PCR reaction conditions were 95°C for 3 minutes, followed by 35 cycles of 94°C for 30 seconds denaturation, primer specific annealing temperature for 60 seconds, and 72°C for 60 seconds elongation, before a final extension at 72°C for 3 minutes. Genotyping of all samples was performed on an Automated Laser Flourescence (ALF) II express . Internal and external standards as well as one reference sample (previously sequenced sample) were included on each gel to facilitate consistent scoring across gels. Alleles were scored with the AlleleLinks 1.02 software (Amersham Parmacia Biotech).Genomic DNA for the microsatellite analysis was extracted by the phenol-chloroform protocol s TCB-99 . To guarFST, their significance values, FIS (the inbreeding coefficient), and allelic richness was calculated in FSTAT v2.9.3.2 as well as a two-sided statistical test to compare allelic richness, observed heterozygosity, FIS and FST between species S) in each population was calculated, based on the estimated FIS values, using the equation S = 2FIS/(1+FIS) S if the FIS values do not accurately represent inbreeding, and are instead from genotyping error or population substructure T. newberryi populations, as it would be expected that an increased proportion of males would cause an increase in the rate of outcrossing, therefore lowering selfing estimates. The migration rate (Nm) Triops was calculated in GenAlEx v6.41 FST/(1−FST), was compared to log geographic straight line distance between playas to detect the presence of isolation by distance using the IBDWS v3.21 utility on the web that uses a Mantel test for the analysis The expected and observed heterozygosity of each population and linkage disequilibrium were calculated in Genepop on the Web v4.0.10 To visually determine the genetic structuring between populations within a species, a discriminant analysis of principal components DAPC  was perfT. newberryi mtCR ranged in length from 548 to 551 base pairs long with an overall total of 24 polymorphic sites, 21 substitutions and a transition to transversion ratio of 20∶4 (Genbank accession numbers KJ627793–KJ627799). Summary statistics for each playa and for all playas combined are in The T. newberryi mtCR sequences revealed that most individuals are of two haplotypes; 55 individuals had Haplotype 1 and 46 individuals had Haplotype 2  indicating strong genetic differentiation across all populations of T. newberryi. Estimates of pairwise ΦST values indicated significant structure between sampled playas , except for the comparison between PL-05 and PL-07  (P = 0.65).The overall Φ= 0.009) . AMOVA i= 0.009) . DivergeT. l. “short” samples sequenced, in contrast to T. newberryi, there was only one mtCR haplotype 549 bp in length (Genbank accession number KJ627792). The closest T. newberryi haplotypes to the T. l. “short” haplotype were Haplotypes 5 and 6, which differed by 18 pairwise differences. There were 18 polymorphic sites between the T. l. “short” haplotype and T. newberryi Haplotypes 5 and 6 consisting of 17 substitutions and 1 indel. No connection was found between the T. l. “short” haplotype and the T. newberryi haplotypes in the TCS network at any confidence level.Of the 66 T. newberryi indicated that 70.4% of the variation was within populations compared to 29.6% of the variation among populations (T. l. “short” indicated that 35.2% of the variation was within populations compared to 64.8% of the variation among populations (P = 0.001) when comparing the species overall FST values; T. l. “short” has a significantly greater degree of genetic structure and reduced gene flow than T. newberryi. The overall FST value for T. newberryi was 0.252 and every pairwise FST comparison was significant after Bonferroni correction ; b ; c ; T. l. “short”, PL-05 and PL-33 were separated from the other populations : FP-03 vs. PL-07; FP-03 vs. PL-36; PL-07 vs. PL-36 : PL-03 vs. PL-07; PL-03 vs. PL-08; PL-07 vs. PL-08 , it had four haplotypes. The amount of nucleotide and haplotype diversity also varied from the lowest nucleotide and haplotype diversity in PL-36 at zero to the highest nucleotide diversity of 0.014 in FP-03 and the highest haplotype diversity in PL-07 at 0.625  and the largest occurred between PL-36 and FP-03 (2.34%)  . The ave (2.34%) .T. newberryi microsatellite loci ranged from one to 14 with an average of 3.15 alleles across all loci , while T. l. “short” specific markers were less variable . The largest allelic richness was in PL-07 (2.97) and the smallest was in PL-33 (1.54). Evidence of linkage disequilibrium was found in one playa lake (PL-09) only between loci TCB-99 and TLS-9. Observed heterozygosity was smaller than expected heterozygosity in each population and a significant departure (after Bonferroni correction) from Hardy-Weinberg expectations was observed in all populations .For all loci . Markersulations . The difT. l. “short” and T. newberryi, the individuals were examined for the presence or absence of a brood pouch, indicative of female or male. Every T. l. “short” individual had a brood pouch, congruent with the literature and the proposed parthenogenetic or hermaphroditic reproductive mode T. newberryi as an androdioecious species T. newberryi population. The amount of males per population ranged from one male out of 30 samples  in PL-07 to eight males out of 28 (28.6%) in FP-03 , largest in PL-03 (S = 0.941) and a (geometric) mean for all T. newberryi populations was S = 0.740 , but the FIS and S estimates for these populations were the largest and smallest observed, respectively. Like T. newberryi, inbreeding analysis in T. l. “short” indicated a significantly large FIS value over all populations: 0.547 . The proportion of overall selfing in T. l. “short” populations (geometric mean S = 0.689) was slightly lower than T. newberryi, with the largest proportion of selfing in PL-05 (S = 0.794) and the smallest in PL-08 (S = 0.433)  .T. newberryi populations from T. l. “short” populations, with the exception of one T. l. “short” individual that did not cluster with either species group  and T. l. “short”  and both species of Triops had low estimated migration (Nm) between playa lake populations. Populations of T. newberryi and T. l. “short” were characterized by low diversity, a large degree of inbreeding and high proportions of selfing in all populations. There was no statistically significant difference between any of these measures  between the species, but all interspecific differences except FIS and the proportion of selfing (S) were consistent with population genetic theory. The presumed androdioecious species (T. newberryi) had higher diversity, fewer private alleles, higher heterozygosity, lower FST, and greater within-population genetic variance. Lastly, there appears to be a clear genetic distinction between the putative species in southern New Mexico and no evidence of hybridization despite co-occurrence in some playa lakes.The results indicated that populations of Triops in this study have a high degree of genetic differentiation. For example, PL-03 and PL-05 are separated by a distance of only 1.45 km, however the microsatellite results indicate T. newberryi and T. l. “short” are each genetically distinct across the two ponds. There is no evidence of isolation by distance in either species with mtDNA or microsatellites, indicating factors beyond immigration and contemporary dispersal are influential in structuring Triops populations in southern New Mexico.Despite the potential for dispersal across habitats located in close proximity, playa lake populations of T. cancriformis in Europe Lepidurus packardi in California T. newberryi and T. l. “short” in southern New Mexico populations. The migration estimates between most populations are too small (<1) A survey of FST values) among the T. l. “short” populations. The difference is illustrated in the different shapes of the DAPC plots; the hierarchical island model as seen for T. l. “short” populations is congruent with increased differentiation as compared to the island model for T. newberryi. The differences in the FST values between species could be due to a variety of factors including influence from the mating system (discussed below), different population sizes, a genetic bottleneck, and/or distinct colonization events. For example, the T. l. “short” PL-05 and PL-33 populations cluster together in the DAPC scatterplot, possibly indicative of the same genetic lineage colonizing and successfully reproducing in both of these locations. In addition, T. l. “short” PL-03, PL-07, and PL-08 populations cluster together and have slightly increased Nm values (>1), which may indicate founding lineages and historical connectivity. Zierold et al. T. cancriformis after glacial maximum, but significant differentiation was still observed. This trend observed in populations of Triops is more consistent with founder events being relatively more influential for the genetic structure than contemporary dispersal.Despite both species being highly structured genetically, there was a significantly higher degree of differentiation  that are thought to reproduce either through parthenogenesis or hermaphroditism T. newberryi because there can be some proportion of “males”  in a population and individuals with a brood pouch can reproduce in isolation T. newberryi populations ranging from 3.3%–28.6%. In comparing the results of the two Triops species with different presumed mating systems, T. l. “short” had fewer alleles, decreased allelic richness, and smaller HE and HO than T. newberryi . With mitochondrial genes, Vanschoenwinkel et al. T. newberryi and T. l. “short” and Macdonald et al. Triops species. Despite some males within T. newberryi populations, the FIS values were large for both species of Triops in southern New Mexico indicating an overall high degree of inbreeding. There was also no correlation between the fraction of males in each population and the FIS values of T. newberryi, as would be expected if populations with more males experience more outcrossing, and therefore, a smaller inbreeding coefficient.Inconsistent with the theory for a selfing species is the inbreeding coefficient and proportion of selfing, as T. longicaudatus populations and androdioecious populations of T. newberryi in the southwestern United States, but inbreeding values were not given. Zierold et al. FIS values, heterozygosity, or allelic richness between T. cancriformis populations that had some males compared to populations with no males. Velonà et al. T. cancriformis that contained males and are presumed to be outcrossing. While the genetic diversity may have differed between androdioecious and selfing populations, the inbreeding coefficient, as in this current study, was not able to distinguish between those populations that have some outcrossing compared to parthenogenetic populations Using allozyme data, Sassaman et al. T. l. “short” and T. newberryi. The aforementioned evolutionary history and/or demographic differences between the species may explain why T. l. “short” has decreased genetic diversity compared to T. newberryi despite both species experiencing the same level of inbreeding. The inbreeding coefficients and selfing rate estimates are inconsistent with what would be predicted if T. newberryi populations were composed of hermaphrodites that can outcross with males (androdioecy). This might also suggest that although T. newberryi has slightly greater genetic diversity, the role of androdioecy does not alleviate the effects of inbreeding with regard to loss of alleles with genetic drift. In a confirmed androdioecious clam shrimp T. newberryi. If there is an insufficient level of outcrossing to decrease the estimate of inbreeding, then do males have a reproductive role within a population? We cannot exclude the possibility that individuals lacking a brood pouch are non-reproductive and do not contribute genetic material to subsequent generations. If this is true, then T. newberryi and T. l. “short” populations could be composed of hermaphrodites that can outcross with other hermaphrodites and both species could have the same mating system. Outcrossing in both species is supported by the large values of FIS and selfing rates that are not near unity in value, which would indicate complete self fertilization as expected in a pure hermaphroditic scenario. Further clarification of the mating system will require a more direct method, such as a progeny array, to determine the reproductive mode of the Triops species within southern New Mexico. Work is also needed to clarify how adaptable the species are to their respective environments and if these differences contribute to the genetic diversity and structure of T. newberryi and T. l. “short” populations.Based on population genetic theory, there should be strong differences in the diversity and structure of a self fertilizing species compared to a species that experiences some outcrossing T. l. “short” and T. newberryi co-occur in Southern New Mexico in this study. In a global phylogeny of the Notostraca, Vanschoenwinkel et al. T. longicaudatus “short” and T. newberryi were not monophyletic and should be considered conspecifics. It is of interest to know if the species hybridize when they co-occur or if they are reproductively isolated We presently have analyzed three ponds in which T. l. “short” and T. newberryi population clusters  and androdioecious species (T. newberryi), the results indicate similar levels of inbreeding occurring in both species. We conclude both species could be composed of hermaphrodites that can outcross with other hermaphrodites and androdioecy for T. newberryi remains unconfirmed. There are clear differences genetically between T. l. “short” and T. newberryi and little evidence to suggest hybridization between the species, which supports T. l. “short” and T. newberryi being distinct species as suggested by Macdonald et al. The population genetic structure of File S1Supporting Tables. Table S1. GPS coordinates of each sampled playa location. Table S2. AMOVA results. Table S3. Triops newberryi mitochondrial control region genetic distances.(DOCX)Click here for additional data file.


The geographical and ethnic distributions of the polymorphic methylenetetrahydrofolate reductase (MTHFR) mutations (C677T and A1298C) and methionine synthase reductase (MTRR) mutation (A66G) remain heterogeneous in China. The goal of this study was to estimate the pooled frequencies of the alleles and associated genotypes of these gene polymorphisms among healthy populations in Mainland China.We systematically reviewed published epidemiological studies on the distributions of 3 genetic variants in Chinese healthy populations living in Mainland China through a meta-analysis. The relevant electronic databases were searched. All of the raw data of the eligible citations were extracted. The frequency estimates were stratified by geography, ethnicity and sex.Pvalues≤0.001). The frequencies of the 1298C, 1298CC, 66G and 66GG genotypes decreased along the south-central-north direction across the country .Sixty-six studies were identified with a total of 92277 study participants. The meta-analysis revealed that the frequencies of the MTHFR C677T, A1298C, and MTRR A66G gene polymorphisms varied significantly between different ethnic groups and along geographical gradients. The frequencies of the 677T allele and 677TT genotype increased along the southern-central-northern direction across Mainland China (all Our meta-analysis strongly indicates significant geographical and ethnic variations in the frequencies of the C677T, A1298C, and A66G gene polymorphisms in the folate metabolism pathway among Chinese populations. The 1298CC genotype exhibited similar variability with frequencies of 7% (5%-9%) in the minority groups and 4% (3%-5%) in the Chinese Han .In contrast to C677T, the distribution of the A1298C polymorphism by ethnicity demonstrated the reverse trend: the 1298C allele was much more common among the minority groups  than the Chinese Han  compared with 25% (24%-26%) among the Chinese Han group  and 4% (4%-5%), respectively. Among the 43 articles that reported on the MTRR A66G polymorphism, 42 studies included 84416 females. The 66G allele and 66GG genotype frequencies in females were 26% (25%-27%) and 7% (6%-7%), respectively.P values were ≤0.001, I2 = 100.0).Tables Methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C) and methionine synthase reductase (MTRR) mutations (A66G) cause mild hyperhomocysteinemia and low folate level and are associated with several disorders. The geographical and ethnic distributions of these alleles and the associated genotypes are important to study worldwide.The frequencies of the MTHFR C677T and A1298C and MTRR A66G polymorphism in 68 epidemiological studies covering 23 provinces in Mainland China were pooled and investigated in the present study. Currently, there is a lack of national data regarding the prevalences of gene polymorphisms in the folate metabolism pathway in healthy general populations in China. We documented distinctive geographical and ethnic variations in the frequencies of the C677T and A1298C polymorphisms of the MTHFR gene and the A66G polymorphisms of the MTRR gene among nation-wide samples in China.Worldwide data have revealed that significant heterogeneities in the frequencies of the T allele and TT homozygosity exist in every population and even with racial groups. One investigations conducted in Texas reported that the frequency of the 677T was lowest among African-Americans (11.9%), followed by in Caucasians (32.7%) and Ashkenazi Jews (47.7%), and the highest frequency exists among the Hispanic population (47.9%) . In the 12 [12 supplementation, which are associated with low levels of homocysteinemia. Across all 23 of the studied provinces, we observed increases in the 677T allele and 677TT genotype frequencies in the southern-central-northern direction across Mainland China. Because high 677T allele and 677TT genotype frequencies were observed in the northern populations, we assumed that the folic acid intakes are greater in the northern populations than in the southern populations; however, the opposite pattern has been observed in nutritional studies. One such nutritional investigation revealed that the geometric mean of the blood folate concentration is lower in the northern populations than the southern populations [Population genetic comparisons provide an appropriate method for picturing geographical and ethnic variations and can suggest that environmental factors may exert selective pressures on genetic mutations. A north-to-south increase in the frequency of the 677T allele has been observed in Europe . North-t12 . The populations .Worldwide epidemiological data have revealed that the frequency of A1298C homozygosity varies from continent to continent. The frequencies of the 1298C allele range from 18% to 70% in East Asia, 17% to 44% in Asia, 24% to 40% in Europe, 0% to 15% in South America and 14.7% in North America . The preBased on all 8 of the investigated minority ethnic populations , the minority ethnic populations seemed to carry greater 1298C allele frequencies than the Chinese Han population. Notably, the frequency of the 1298C allele has been reported to vary between different ethnic populations worldwide, and the lowest frequency has been found in Indians (10%)  followedAlthough A1298C homozygotes do not exhibit elevated blood homocysteinemialevels, many investigations have revealed that compound heterozygotes for C677T/ A1298C may be at risk for hyperhomocysteinemia and low folate levels, which can contribute to many disorders, such as neutral tube defects .Because lifestyle and environmental factors, such as folate supplementation, vary across different ethnic populations and may influence the frequencies of the C677T and A1298C alleles, these factors cannot be ruled out when considering the influences of environmental-genetic interactions on the distributions of MTHFR gene polymorphisms.Our pooled data revealed that the frequencies of the 66G allele and 66GG genotype exhibited variations across geographical gradients and ethnic populations. Globally, the distributions of the MTRR 66G allele and 66GG genotype frequencies also exhibit geographical and ethnic variations. For example, the frequencies of the 66G allele have been reported to be 58% in the Yadav, 62% in the Scheduled Castes, and 71% in the rural Sunni Muslim population in Uttar Pradesh in India ,97. Our In conclusion, our meta-analysis revealed significant geographical variations in the frequencies of the MTHFR C677T and A1298C and MTRR A66G polymorphisms in the folate metabolism pathway between different ethnic populations in China. Our findings provide an overall picture of these three genetic polymorphisms in the folate metabolism pathway among the general populations in Mainland China, and these evidence-based genomic data should be integrated into medical and public health practices.S1 File(TXT)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(TXT)Click here for additional data file.S4 File(TXT)Click here for additional data file.S5 File(TXT)Click here for additional data file.S6 File(TXT)Click here for additional data file.