Tunisian local cattle populations are at risk of extinction as they were massively crossed with imported breeds. Preservation of indigenous livestock populations is important because each of them comprises a unique set of genes resulting from a local environment-driven selection that occurred over hundreds of years. The diversity and genetic structure of Tunisian local cattle populations are poorly understood. However, such information is crucial to the conservation and sustainable use of genetic resources.In addition, comparing the genomic structure of population sets from different parts of the world could help yield insight into their origin and history.In the present study, we provide a detailed assessment of the population structure of the three Tunisian local cattle populations using various methods, and we highlight their origin and history by investigating approximately ~38,000 SNPs in a broad panel of 878 individuals from 37 worldwide cattle breeds representative of African, European and indicine populations.Our study revealed a low level of divergence and high genetic diversity in Tunisian local cattle reflecting low levels of genetic drift. A Comparison with the worldwide cattle panel pinpointed the admixed origin of the genome of the three Tunisian populations with the two main European and African ancestries. Our results were in agreement with previous historical and archaeological reports about the past gene flow that existed between North African and South European breeds, in particular with Iberian cattle. We also detected a low-level indicine introgression in the three Tunisian populations and we inferred that indicine ancestry was inherited via African ancestors.Our results represent the first study providing genetic evidence about the origin and history of Tunisian local cattle. The information provided by the fine-scale genetic characterization of our study will enhance the establishment of a national conservation strategy for these populations. These results may enable the identification of genetic variants involved in adaptation to harsh environmental conditions.The online version of this article (doi:10.1186/s12864-015-1638-6) contains supplementary material, which is available to authorized users. During the last century, an erosion of cattle genetic resources was observed in many countries around the world as a result of a massive replacement of low-productive local breeds with highly productive ones. According to the FAO (Food and Agriculture Organisation), there are currently more than 1300 taurine and indicine breeds in the world. Among these, many local cattle breeds are already extinct (16 %), endangered (16 %) or have an as-yet unknown risk status (30 %), and they may disappear before they are fully studied or their characteristics are recorded , 3.The emergence of high-throughput SNP genotyping facilities coupled with the gradual reduction of genotyping costs may help elucidate the genetic diversity and structure of endangered populations. Such information is crucial for the conservation and sustainable use of genetic resources. Moreover, comparing the genomic structure of population sets from different parts of the world provides a new glimpse into their origin and history.Local cattle in Tunisia belong to three main populations according to morphological criteria: the Blonde du Cap Bon (BLCAP), the Brune de l’Atlas Grise (BRATG), and the Brune de l’Atlas Fauve (BRATF). Today, these three populations are endangered by massive crossing with imported breeds to improve their production performance , 5. IndiThe aim of the present study was to provide a detailed assessment of the genetic structure of Tunisian local cattle populations and to trace back their origin and history using medium density SNP chips.For this purpose, we first genotyped 50 Tunisian individuals from the three populations using the Illumina BovineSNP50 BeadChip, and we subsequently combined our genotyping data with publicly available data from 34 other populations (bovine Hapmap dataset) representative of African (AFT), European (EUT) and zebu (ZEB) cattle. The results of our study provide a detailed assessment of the genetic diversity and structure of Tunisian local cattle populations, shedding light on the origin and history of Tunisian local populations through their genetic relationship with the bovine Hapmap dataset populations; these insights into population structure and genetic diversity in Tunisian local populations are further elucidated by relating our results to previous reports about the domestication and dispersal of cattle in Africa and Europe.The genetic diversity of Tunisian breeds measured by expected heterozygosity (Hs) using all available BovineSNP50 BeadChip SNPs was similar to that observed for European breeds and broadly equivalent to 0.32. Indicine populations of Indian origin  had the lowest Hs rates, ranging from 0.16 to 0.2, while African populations had slightly higher Hs values ranging from 0.19 to 0.24  but slightly positive for BRATG and BRATF  indicating the absence of any cryptic population structure in our Brune de l’Atlas samples  but Fis 95 % CI computed with 5000 randomly chosen SNPs and 100,000 bootstraps was slightly positive  to locate the Tunisian individuals compared to the other populations of the study. PCA grouped individuals in clusters according to their populations of origin Fig. . The firThe predefined group DAPC (see methods) gave similar results to those obtained with PCA, i.e., a first indicine/taurine axis and a second AFT/EUT axis  ancestry (41 % EUT and 10 % ZEB) followed by BRATF with 43 % of AFT (49 % of EUT and 8 % of ZEB ancestries) and by BRATG with 32 % of AFT (60 % of EUT and 8 % of ZEB ancestries). Among all African taurines only SHK showed more indicine (57 %) than African (39 %) ancestry.Increasing the number of clusters to k = 5 resulted in the main subgroups of European breeds. The first group included Brown Swiss (BRU and BSW) and South European breeds  while the second one can be viewed as North European breeds . On the other hand, Tunisian populations showed more Southern than Northern European ancestry. Of the three populations, BRATG showed the highest Southern European admixture level (~45 %) while BLCAP showed the lowest (~26 %).When k was set to 7, the two Brown Swiss populations, BRU and BSW, formed a distinct cluster while two subdivisions appeared among African taurines that might be interpreted as representative of Longhorn  and shorthorn (LAG). Interestingly, both BRATF and BRATG populations showed a substantial level of admixture with Brown Swiss (~11 % for BRATF and ~17 % for BRATG) and up to 25 % of admixture has been observed with some animals. African ancestry within Tunisian populations and OUL (a North African breed), is mainly from Longhorn .At k = 10, ZBO and ZFU were placed together in a separate cluster. Tunisian populations appear to have inherited their indicine ancestry via African ancestors since they shared almost all of their indicine ancestry with the ZBO/ZFU cluster.Finally, fixing k to 37 (the number of populations used in this study) revealed that almost 85 % of the BLCAP genome was shared with 35 % and 26 % of the BRATF and BRATG genomes, respectively.To get more insight into the genetic relationship between the different populations of the study, we estimated the Fst for pairwise comparisons of all populations, using both Genepop software  and BRATF/BRATG (0.0375) but higher levels of differentiation between BLCAP and BRATG (0.0638). Tunisian populations had moderate Fst values  with almost all South European breeds and presented high levels of differentiation  with North European breeds. We also observed moderate Fst values between Brune de l’Atlas populations and the BRU breed.Although, high Fst values were observed between Tunisian populations and African cattle, these were clearly lower with African Longhorn than African shorthorn populations (LAG). Among the three Tunisian populations, BRATF had the lowest Fst values with both African and European populations, while BLCAP tends to have lower levels of differentiation with African populations than BRATG. This trend was reversed when comparing pairwise Fst values of these two breeds with South European breeds .To provide additional insight into the origin and evolutionary history of Tunisian populations, we constructed a NeighborNet graph based on Nei genetic distance Fig. . Breeds Finally, we used the Treemix software to model both population splits and gene flow between a subset of 13 populations. We first constructed a phylogenetic tree where no migration events were allowed Fig. . Among TAll the three phylogenetic trees showed BRATG sister to Brown Swiss (BRU) and identified high levels of introgression from the common ancestor of these two populations into BRATF (~45 %). Additionally, Treemix runs showed an important level of African cattle introgression into BRATG and OUL populations  and evidence of gene flow between BRATF and BLCAP (~34 %). Finally, low levels of indicine introgression were also identified into BRATF and BRATG . These results were in agreement with unsupervised hierarchical clustering results.Significantly negative f3 statistics see  showed eOur results show that gene diversity of the three Tunisian populations, measured by heterozygosity, was among the highest within the populations of the study and was similar to those observed in European breeds. This is more likely due to the combined effect of their European ancestry and their admixed origin. Indeed, all other hybrid populations with a European ancestry showed a similar range of genetic diversity.We attributed low Hs values (relative to European breeds) within indicine breeds to ascertainment bias in the SNP discovery of the BovineSNP50 BeadChip. Indeed, in contrast to our findings, other studies showed that more diversity is observed within indicine breeds than within taurine breeds. For instance, the Bovine HapMap Consortium reported that nucleotide diversity in Brahman cattle is more than twice that observed within Holstein and Angus breeds , 8. By aLow pairwise Fst values between Tunisian populations, shared ancestry detected between them in unsupervised hierarchical clustering when setting k to 37, and reticulations observed between Tunisian populations on the phylogenetic tree Fig. , all sugIn this study, we presented consistent evidence of an admixed origin of Tunisian indigenous cattle with both African and European ancestries. Our analyses showed that these two ancestries are present in varying proportions between the three Tunisian populations. The difference is more pronounced between BLCAP and BRATG. At k = 3, the BLACP population tended to have a balanced proportion of African and European ancestries while BRATG had a more pronounced European influence. This hypothesis was corroborated by the results of pairwise population Fst, PCA, DAPC, and the phylogenetic analysis.Increasing the number of inferred genetic clusters to five showed that European ancestry in Tunisian breeds arises both from North and South Europe, with the proportion of the latter being double of the former. This trend is supported by pairwise Fst results (lower differentiation between Tunisian populations/South European breeds than that between Tunisian populations/North European breeds) and by the proximity of Tunisian populations from South European rather than North European populations in the PCA and DAPC. Additionally, we noticed that the difference in the proportions of European ancestry between the three Tunisian populations arises from a South European origin. Previous genetic studies supported by archaeological evidence reported a difference between North and South European cattle populations, attributing it to movement of cattle occurring along two distinct routes from the cattle domestication center in the Middle East. The first migration route, called the Danubian route, was followed by Neolithic farmers as they moved along the Balkans into the plains of central and Northern Europe. The second, called the Mediterranean route, arose as farming spread from the Balkans and Southern Italy to Corsica, Southern France and Spain by 7700 years BP . Based oFrom f3 statistics and the results of unsupervised hierarchical clustering (k = 7), we detected a substantial Brown Swiss influence, both on BRATF and BRATG and to a much lesser extent on BLCAP (only one significant f3 test indicated admixture between BLCAP and Brown Swiss). However, when Brune de l’Atlas populations were analyzed independently, we observed that only 12 % of the animals had more than 30 % of their ancestry originating from Brown Swiss, while more than 77 % of BRATF animals had less than 10 % of Brown Swiss ancestry  and ~75 % of BRATG animals had less than 18 % of Brown Swiss ancestry. These findings led us to conclude that introgression from Brown Swiss occurred several hundreds of generations ago and left few footprints in the Brune de l’Atlas genome.The pronounced signals of Brown Swiss identified in a few Brune de l’Atlas individuals most likely have a much more recent origin . Indeed, due to their morphological similarity to Brown Swiss cattle, Brune de l’Atlas animals were commonly crossed with the Brown Swiss breed .Likewise, from the low levels of genetic differentiation observed between Tunisian populations and the CGU breed, it has been posited that Tunisian populations are closely related to local breeds from the Iberian Peninsula. Indeed, CGU cattle were introduced in the Caribbean islands by Spanish and Portuguese conquerors after the second trip of Columbus in 1493 –14. ManyAdditionally, our results suggest that African Taurine influence on Tunisian local cattle has a Longhorn origin and that the small amount of indicine ancestry found in the three populations mainly has an African origin. Archaeological studies reported that Longhorn Taurines were introduced from domestication centers (western Asia) into Africa through Egypt and the Horn of Africa in about 5000 BC , while zTo our knowledge, our study is the first of its kind aiming to assess the genetic structure of Tunisian indigenous populations and to establish their genetic origin using medium density SNP chips and comparisons with worldwide cattle. In the present study, we show that Tunisian local cattle populations have high genetic diversity, reflecting the absence of strong artificial selection. We also show the existence of a recent introgression of Brown Swiss in some Brune de l’Atlas individuals. Given that our sample collection took place at locations that are quite remote with respect to artificial insemination circuits, we can conclude that the presence of purely local individuals has become rare and thus highlighting the need to implement a national conservation strategy. Finally, as our study provided a comprehensive picture of the genetic structure and origins of Tunisian local cattle populations, it can also be used to investigate genetic variants underlying adaptation traits in these populations.All animal experimentation complied with the Tunisian Veterinary Authorities’ rules. No ethics approval was required by a specific committee, since the selected animals were not animals bred for experimental reasons.Blood samples belonging to 50 Tunisian animals from the three populations were collected from different geographical regions between the North and the center of the country. The BLCAP population is mainly found in the northeast of the country. Thus, most animals were selected from different geographical locations within this region. Similarly, Brune de l’Atlas (BRAT) individuals were sampled from both the northwestern and central parts of Tunisia . Since most of the local animals were massively crossed with imported breeds, every attempt was made to sample purebred individuals. This was done by sampling the animals from areas that are isolated from the artificial insemination circuit. Many of these regions are located in mountainous areas.http://www.illumina.com) resulting in 52,886 autosomal genotyped SNPs.Sampling was carried out by a team from the Livestock and Pasture Office of Tunisia (OEP). Identification of purebred animals was based on specific morphological criteria. Blood samples were collected in EDTA Vacutainer tubes. DNA was extracted using The Wizard® Genomic DNA Purification Kit (Promega) and by phenol-chloroform using a standard protocol . DNA quaWe also included genotyping data from individuals belonging to 34 other cattle breeds (bovine Hapmap dataset) representative of European taurines , African taurines , indicine , and four crossbreed population . Genotyping data for 42,194 SNPs were available on these breeds, from three previous studies –24. The http://pngu.mgh.harvard.edu/~purcell/plink/) for genotyping data quality control. Samples genotyped for less than 90 % of markers were excluded from the analysis. SNPs genotyped for less than 90 % of the animals and those with MAF less than 0.01 were also discarded.We used PLINK ver.1.07  . Similarly, 1115 SNPs were excluded because of low genotyping rates, and 5949 SNPs were also excluded as they were monomorphic (MAF <0.01). An exact test for Hardy-Weinberg Equilibrium (HWE) was then carried out within each population separately on the remaining SNPs using PLINK, which led to 45,691 SNPs retained for further analysis.Genotyping data from Tunisian samples were then merged with the bovine Hapmap dataset. The whole dataset consisted of 878 individuals from 37 populations genotyped for 38,597 SNPs spread over all autosomal chromosomes. Average marker density was one SNP every 65.6 kb  and inbreeding within subpopulations measured by Fis to assess the genetic diversity of each population.In order to check for an effect of ascertainment bias (due to SNP discovery) on genetic diversity parameters, we estimated (Hs) using either all data  or only SNPs with MAF above 0.01 in African and indicine populations. Discarding SNPs whose MAF was below 0.01 is supposed to limit the effect of ascertainment bias.Genetic differentiation between populations was estimated using Genepop 4.0 software  using alPhylogenetic relationships between populations were investigated using Nei genetic distance. NeighborNet graphs were constructed from the estimated genetic distances using the Splitstree software .Patterns of splits and mixtures of a subset of 13 populations were carried out using Treemix  and by sThe genotyping data and the phylogenetic trees supporting the results of this article are available through the Data Dryad digital repository. doi:10.5061/dryad.sj548.


Lolium perenne L.), and freezing tolerance is a complex trait of major agronomical importance in northern and central Europe. Understanding the genetic control of freezing tolerance would aid in the development of cultivars of perennial ryegrass with improved adaptation to frost. The plant material investigated in this study was an experimental synthetic population derived from pair-crosses among five European perennial ryegrass genotypes, representing adaptations to a range of climatic conditions across Europe. A total number of 80 individuals  from the second generation of the two divergently selected populations and an unselected (US) control population were genotyped using 278 genome-wide SNPs derived from perennial ryegrass transcriptome sequences. Our studies investigated the genetic diversity among the three experimental populations by analysis of molecular variance and population structure, and determined that the HF and LF populations are very divergent after selection for freezing tolerance, whereas the HF and US populations are more similar. Linkage disequilibrium (LD) decay varied across the seven chromosomes and the conspicuous pattern of LD between the HF and LF population confirmed their divergence in freezing tolerance. Furthermore, two Fst outlier methods; finite island model (fdist) by LOSITAN and hierarchical structure model using ARLEQUIN, both detected six loci under directional selection. These outlier loci are most probably linked to genes involved in freezing tolerance, cold adaptation, and abiotic stress. These six candidate loci under directional selection for freezing tolerance might be potential marker resources for breeding perennial ryegrass cultivars with improved freezing tolerance.Low temperature is one of the abiotic stresses seriously affecting the growth of perennial ryegrass ( Lolium perenne L.) is an important forage grass species due to its productivity and high forage quality. However, with changing climates, improving its resistance to abiotic stresses is important to sustain grassland production. Frost is one of the abiotic stresses causing serious concern to the growth of perennial ryegrass  the reproductive biology of the organism  and (ii) population history. Inbreeding plant species have high LD-levels due to high levels of homozygosity, with non-random associations of alleles spanning large distances. In worldwide accessions of the inbreeding model plant, nd 10 kb  and in rnd 10 kb . Studiesnd 10 kb , and betnd 10 kb . Howevernd 10 kb .Knowledge of the LD structure of populations can be exploited for different purposes. If the aim is to identify causative polymorphisms underlying phenotypic variation, low LD ensures that the resolution of a marker-trait association is limited to a small section of the chromosome . This type of study must utilize either extremely high marker density (if the markers are randomly chosen) or a set of carefully selected markers which have high likelihood of being close to causative polymorphisms  . ConversUnderstanding the genetic control of freezing tolerance would aid in the development of cultivars of perennial ryegrass with improved adaptation to frost. As part of the EU project ‘GRASP’, 20 heterozygous genotypes  of diverse origin were assembled for identification of single nucleotide polymorphisms (SNPs) in candidate genes for specific traits and development of SNP assays for candidate gene allele tracing and validation in selection experiments . NucleotOur objectives were to: (1) test whether there are genetic variation for freezing tolerance in the synthetic population by studying responses to phenotypic selection; (2) study how divergent selection for freezing tolerance affects levels of allelic diversity, genetic structure and LD; and (3) identify candidate genes responding to selection for freezing tolerance that can be useful in breeding for freezing tolerance.Figure 1). LTS 3 and LTS 4 are the Falster  and Veyo  genotypes, respectively, grandparents of the Danish VrnA mapping population  in all pairwise combinations, and mixing equal amounts of seeds from each cross to establish the Syn1 population . The 300 genotypes were cloned in several ramets; some ramets were used for freezing tests and some were vernalized during autumn/winter and used to establish the divergent selections and the random mating, non-selected Syn3 population by intercrossing the following summer in pollen-proof isolation greenhouse chambers. Freezing tests of Syn2, C1+ and C1- were conducted as described by 1+ and C1- populations were tested using four ramets and the LTS genotypes by testing 12 ramets of each genotype. A selection intensity of 10% was used with 30 genotypes selected out of 300 for each round of recombination in both directions, creating the first generation high (C1+) and low (C1-), and the second generation high (C2+) and low (C2-) freezing tolerance populations. In order to quantify the effect of genetic drift, 100 randomly selected genotypes among the 300 C0 genotypes were intercrossed to make Syn3 seeds, from which 100 randomly selected individuals was selected among 300 individuals and recombined to make Syn4 . Twenty-four, 29 and 27 genotypes were randomly selected from the second generation high (C2+), low (C2-), and US Syn4 population, respectively, and used for SNP genotyping . In the following presentation, the C2+ selection is termed high-frost (HF) tolerance, the C2- is termed low-frost (LF) tolerance and Syn4 is termed US .The Syn2 generation was produced from Syn1 by open pollination in isolation. Three hundred randomly selected individual plants from Syn2, hereafter termed CFor SNP genotyping, genomic DNA was extracted from the leaves of a total number of 80 genotypes representing the three populations. About 100 mg of leaf tissues were crushed with a pestle and mortar in liquid nitrogen and used for extraction of genomic DNA using a Qiagen DNeasy Plant Mini Kit (Qiagen Cat. no. 69106) following the manufacturer’s instructions . The DNA quality was assessed and normalized using the Nanodrop ND-1000 Spectrophotometer .L. perenne genic SNP markers distributed across the seven linkage groups  were selected from the 768 Illumina GoldenGate assay based SNP markers . These Illumina GoldenGate assay based SNPs were converted to Sequenom MassARRAY iPLEX platform (Two hundred and seventy-eight  markers . The dis markers  (Supplemplatform  SNPs thaplatform . The dif® Assay Design software for the 278 SNPs across 12 multiplex panels set with the following parameters: amplicon length (bp): min:80, optimum:100, max:120; PCR primer length (bp): min:16, optimum:20, max:25; extension primer length (bp): min:16, max:28; hybridization Tm (°C): min:45, max:65. PCR reactions were performed using Sequenom iPLEX gold reagent kits following standard procedures (Supplementary Tabel S1). Approximately 20 ng of genomic DNA was amplified using a pool of 278 pairs of PCR primers under cycling conditions at 95°C for 15 min, 46 × , and final extensions at 72°C for 3 min.High-throughput genotyping was performed using the Sequenom MassARRAY iPLEX platform  at the Centre for Integrative Genetics (CIGENE), Norwegian University of Life Sciences, Norway. Briefly, multiplex assays were designed using the MASSARRAYe), and observed heterozygosity (Ho) using the ARLEQUIN software 3.5.1.3  <5% were removed. SNP genotyping data obtained after filtering was used for calculating expected  to vary from 1 to 10. Once inferring the most likely K, we implemented more stringent parameters with a length of burn-in and MCMC (Markov Chain Monte Carlo) of 200,000 each with ten independent simulations of K varying from 1 to 5. The best K was determined by the log likelihood of the data (LnP(D)) in the STRUCTURE output and an ad hoc statistic ΔK based on the second-order rate of change in LnP(D) between successive K values  between the pairs of loci. The loci were considered to be in significant LD when P < 0.001, the rest of r2 values was not considered as informative. The pattern and distribution of intra-chromosomal LD was visualized and studied from LD plots generated for each chromosome by HAPLOVIEW software version 4.2. To investigate the average LD decay in the whole genome among the panel, significant intra-chromosomal r2 values were plotted against the genetic distance between markers.Genome-wide LD analysis was performed in each of the three population groups  by pairwise comparisons among the SNP markers distributed across seven linkage groups using the HAPLOVIEW software version 4.2  with thestF values and an estimate of P-value for each locus. This method detects genes under selection based on the distributions of heterozygosity and stF  annotation using the Blast2GO software tool version 3.0  and high (C1+) freezing tolerance are presented in Table 1. Visual scoring of regrowth is not comparable across experiments since it is dependent on experimental conditions, seasons, and conditions during cold acclimation of the plant material. The LTS genotypes were all tested in the same freezing test but independent of the other populations. The freezing scores for the C1- and C1+ populations can be compared since they were obtained in the same freezing test, while the freezing scores of the Syn2 population was obtained in an earlier, independent test and is not comparable with the others. Therefore, it is not possible to use the selection differentials and the selection responses to calculate realized heritability. The broad sense heritabilities (repeatabilities) presented were calculated using the between ramet variation as estimate of the environmental variation, and the variation of genotype means as the genotypic variation (Table 1). The results show that there is a large variation for freezing tolerance in the base population, ranging from 0.33 to 6.47 , actually very similar to the range among all LTS genotypes (0–6.38). However, the mean value in the Syn2 population was low (3.23). This is expected in view of the rather low freezing tolerance among the five LTS genotypes used to establish the Syn2 population; the four genotypes that was successfully phenotyped had an average freezing score of 4.21. Freezing tolerance is a complex trait with low heritability, and proper replication is required in freezing tests. This effect is evident in the present study. Broad sense heritabilities were 0.18 and 0.25 in the C1+ and C1- populations, respectively, with four ramets of each genotype tested, while it increase to 0.45 in the test of the LTS genotypes which were replicated 12 times each (Table 1). Despite relatively low heritability of the freezing tolerance, the responses to one generation of selection were clear as evident from the different mean freezing scores of the C1- and C1+ populations (Table 1). However, the distributions of the freezing scores in the two populations was nearly completely overlapping .The results of phenotyping the LTS genotypes, the synthetic base population (Syn2) and the divergently selected populations for low (CL. perenne genic SNP markers distributed across the seven linkage groups  in 80 genotypes of HF (24), LF (29), and US (27) . Eleven SNPs failed in genotyping and 27 SNPs with missing data >5% were removed from the dataset. Of the remaining 231 SNPs, 181 SNPs (78.3%), 162 SNPs (70.1%), and 180 SNPs (77.9 %) were polymorphic across the HF, LF and US populations, respectively. Observed heterozygosity was slightly lower than expected heterozygosity in all the populations, indicating either the occurrence of null alleles or a slight level of inbreeding (Table 2). The FIS (inbreeding coefficient) is negative in the high frost population, implying a considerable degree of outbreeding, whereas positive FIS in the low and US populations showed considerable degree of inbreeding. The AMOVA for the three populations revealed that 91.6% (P < 0.014) of the genetic variation is found within individuals, whereas 8.67% (P < 0.0001) of the genetic variation is found among populations . In addition, AMOVA was separately conducted between HF and LF, HF and US, and LF and US populations in order to study genetic variation between populations. 13.14% (P < 0.0001) of the genetic variation was found between HF and LF, only 3.08 % (P < 0.0048) between HF and US, and 8.67% (P < 0.0001) between LF and US (Table 3).High-throughput genotyping was performed by Sequenom MassARRAY iPLEX using 278 D) value for each given K (number of subpopulations) increased from 1 to 2 and gradually decreased between 3 and 4 and slight increase in 5, showing evidence of maximum at K = 2 . The second order likelihood, ΔK was calculated and observed the maximum ΔK value at K = 2. Both Pritchard’s and Evanno’s methods confirmed the K-value as 2 , however, we plotted the barplots from K = 1 to 5, as LnP(D) value was slightly higher at K = 5. The barplots showed higher proportion of presumably alleles for freezing tolerant (blue) and lower proportion of presumably alleles for low freezing tolerant (orange) in HF and US populations, whereas, high proportion of orange and low proportion of blue was observed in LF population. This pattern was observed consistently from K = 2 to 5 .To infer the population structure from multilocus genotype data among the three populations, we implemented the linkage model in the STRUCTURE software. Overall, this model retains the main elements of the admixture model and reports the overall ancestry for each individual, taking account of the linkage. However, the model is not designed to deal with background LD between very tightly linked markers . Hence, Figure 4).The PCoA based on the dissimilarity matrix clearly separates the HF and LF populations, whereas the US population overlaps nearly completely with the HF population. The first and second principal coordinates explained 23.36 and 8.45% of the molecular variance  were calculated after removing the low frequency alleles (<0.05). The r2 values for the linked loci were plotted against the genetic distance to observe the LD decay . Slow LD decay was observed on chromosomes 4 and 7, exceeding 90 cM, whereas rapid LD decay was observed in chromosome 5, below the significant threshold (r2 ≥ 0.03) within 0.5 cM. However, the pattern of LD decay varied between HF, LF, and US across the chromosomes. LF has low decay in chromosomes 2, 4, and 6 with significant threshold (r2 ≥ 0.10), compared to HF and US. In addition, we also observed the LD around the important frost candidate genes LpCBFVb (Chr.1), LpPHYC (Chr. 4), LpCBF6 (Chr.5), and Lp6FT (Chr.7) between HF, LF, and US populations . No LD was observed at LpCBFVb for HF, whereas very high LD was observed in LF with several tightly linked markers, and moderate LD was observed in US with few tightly linked markers. LD at LpPHYC was almost similar in the three populations. LD at LpCBF6 was high in US and LF, but low in HF. At Lp6FT, very high LD was observed in HF and US, where all five SNP markers are in high LD, but no LD was observed in LF .A random subset of markers spanning across all seven chromosomes  of Figure 6). Putative balancing selection was detected at 15 loci . Directional selection was detected for 20 loci by LOSITAN and 6 loci by ARLEQUIN . Although footprints of directional selection were differently identified for some loci with these two outlier methods, six loci were indicated to be under directional selection by both hierarchical methods employed in ARLEQUIN and LOSITAN. Putative functions of these loci are described in Table 4.Signatures of directional and balancing selection were identified at 41 loci among the three populations using the programs LOSITAN and ARLEQUIN (Table 2). These results are consistent with the genetic diversity studies of the same LTS genotypes for WSC content , LF (C2-), and US (Syn4) populations for freezing tolerance, we see effect of selection already in the first generation of high (C1+) and low (C1-) populations  and we expect that there has been further differentiation in the following generation. The effect on genetic differentiation was largest in the LF population (negative selection). The LF population is almost entirely separated from HF and US populations in the PCO , and in the Structure analysis with K = 5 we see that the population is dominated by one of the five parental genotypes . One of the parental genotypes, LTS16, has markedly lower freezing tolerance than the others (Table 1) and this genotype most likely contributes more to the LF population than the other parental genotypes do. Individuals with higher freezing tolerance in C0 and C1- probably have more of the genomes from the other, more freezing tolerant parents, and they will be selected against. Moreover, contributions from LTS16 is probably selected against in the HF population, making the HF and LF populations highly differentiated (Table 3). Remarkably, in spite of phenotypic divergence there is little genotypic divergence between the HF and US populations. Only 3.08% of the variation is found between the groups (in contrast to the 8.675% found between LF and US) and there is nearly complete overlap between the HF and US in the PCO plot . The parental genotypes LTS03, LTS11 and LTS15 all have medium levels of freezing tolerance, however, they vary in their genetic background and probably harbors variation for freezing tolerance resulting in approximately equal contribution  to the HF population. Consequently, divergence from US will be minimal. This is opposite to what was found in Although we did not test the HF , similar to the previous studies on the gibberelic acid insensitive gene (GAI) region in a collection of 47 Eurasian ryegrass ecotypes  is located  a QTL for freezing tolerance . Similar interchromosomal heterogeneity in LD was observed in rice  by LOSITAN and hierarchical structure model using Arlequin to detect true positive loci under selection . stF outlier models to weed out false positive loci in conifers., tomato  and comm, tomato  for idenstF values that displayed differentiation higher than the 99% limit of the confidence intervals . These six loci, i.e., PTA_817_C1, PTA_1219_C2, PTA_475_C1, PTA_1433_C1, PTA_450_C1, and PTA_1254_C1 may be directly under selection. Annotation of these SNP sequences revealed the putative functions of all these six candidate loci (Table 4). PTA_817_C1 was identified as an elongation factor G-2, chloroplastic which might be involved in freezing tolerance. A previous transcriptome study of barley leaves revealed that the induction of several elongation factors and the chloroplast function in the leaf were the key differences between a frost tolerant and a frost-susceptible cultivar  under positive selection based on cultivar . Two canAtRab7, a gene involved in regulation of vesicle trafficking, increased endocytosis in roots, as well as salt and osmotic stress tolerance  control population are most similar. LD decay varied across the seven chromosomes and patterns of LD between the HF and the LF population are signs of divergence relative to freezing tolerance. Finally, six candidate loci detected independently by two stF outlier methods were candidates for loci being under directional selection for frost and might be potential marker resources for breeding perennial ryegrass cultivars with improved freezing tolerance.The selection scheme employed here by establishing an experimental synthetic population from five diverse http://datadryad.org/resource/doi:10.5061/dryad.sd1dt).Single nucleotide polymorphisms genotyping raw data and processed data are deposited in DRYAD Digital Repository along with the input files for running Arlequin and Structure softwares (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Austronesian expansion, one of the last major human migrations, influenced regions as distant as tropical Asia, Remote Oceania and Madagascar, off the east coast of Africa. The identity of the Asian groups that settled Madagascar is particularly mysterious. While language connects Madagascar to the Ma’anyan of southern Borneo, haploid genetic data are more ambiguous. Here, we screened genome-wide diversity in 211 individuals from the Ma’anyan and surrounding groups in southern Borneo. Surprisingly, the Ma’anyan are characterized by a distinct, high frequency genomic component that is not found in Malagasy. This novel genetic layer occurs at low levels across Island Southeast Asia and hints at a more complex model for the Austronesian expansion in this region. In contrast, Malagasy show genomic links to a range of Island Southeast Asian groups, particularly from southern Borneo, but do not have a clear genetic connection with the Ma’anyan despite the obvious linguistic association. The Austronesian expansion was a major human migration in Southeast Asia, triggered by the spread of agricultural populations approximately 5,000 years ago1224Despite clear evidence, based on biological67891113141116171819201617182225Several genetic studies have sought to detect Indonesian genetic connections in the Malagasy genome 6789Here, we perform that genome-wide analysis in the Ma’anyan and other groups from southern Borneo to determine the genetic background and potential Asian sources of the Malagasy. Using Illumina HumanOmniExpress Bead Chips, we genotyped over 700,000 genomic markers in 169 Ma’anyan individuals, together with a further 42 individuals from Dayak ethnic groups across southern Borneo. The aims of this study were dual: i) to examine the genetic diversity of populations in southeastern Borneo (focusing on the Ma’anyan and other indigenous Dayak groups), and thereby determine their place in the wider genetic diversity of Island Southeast Asia; and ii) to identify whether the clear linguistic relationship between the Ma’anyan and Malagasy is also reflected in a shared genetic inheritance.ST values calculated on the low density dataset . To characterize the Ma’anyan and SK-Dayak gene pool within an Asian context, we focused our analyses on Island Southeast Asian, East Asian and Mainland Southeast Asian populations . In a Pr dataset .This unique genetic placement of the Ma’anyan is supported by admixture estimates, also performed on the low density dataset , especia27An f3-statistics analysis reveals more clearly that the Ma’anyan is not an admixed population . Defininx) was explored, where x represents all populations in turn in the low density dataset. The highest value was obtained when x was the Igorot from the Philippines or non-Ma’anyan Borneo populations  analysis performed with Refined IBD on the high density dataset. The Ma’anyan share more haplotypes with surrounding Borneo populations and the Igorot than with Mainland Southeast Asian groups  . When fiWe performed PCA using the low density dataset, finding that the first two components described 54.6% of the observed variance . The firExplicit admixture analysis on the low density dataset confirms this assessment, showing that the three Malagasy populations have ~70% African ancestry (red) versus ~30% Asian ancestry   , thus suTogether, these analyses confirm that Malagasy are a mixture of African and Island Southeast Asian populations, as suggested by much previous research678930A genome-wide analysis of 211 individuals from Borneo reveals the unique genetic diversity of the Ma’anyan, opening an unexpected viewpoint into Southeast Asian prehistory. Our data reveal that the Ma’anyan are characterized by a specific genomic component that differentiates them from other Island Southeast Asian groups . This do24Recent studies have identified at least three broad genomic classes that dominate the gene pool of Southeast Asian individuals: Papuan ancestry, Mainland Southeast Asian ancestry, and Austronesian ancestry33333533343733343539Despite strong linguistic affinities111617111617Hikayat Banjar  describes an old Malay settlement in southern Borneo, further inland than today’s south Borneo coastline, that acted as a trading outpost of Malay Kingdom – such as the important Hindu kingdom of Srivijaya, which was dominant from the 7–13th centuries AD252411Other anthropological data may shed new light on the complex history of the Ma’anyan, perhaps reconciling this discrepancy between the linguistic and genetic data. Prior to their migration to Madagascar around 1,400-1,000 years ago, proto-Malagasy people had probably already developed a derived language that differed from Ma’anyan11Our study shows that the Ma’anyan have genetic diversity that is unique in Southeast Asia, complicating existing scenarios of dispersal during the Austronesian expansion. Surprisingly, this component clearly shows that the Ma’anyan are not the primary source population of the Malagasy, as has long been supposed based on their common linguistic origin. The Asian parental population of the Malagasy instead appears to lie among the ethnic groups of the South East region of Borneo, potentially represented by the Banjar, or more generally, by the South Kalimantan Dayak people. This discrepancy between linguistic and genetic evidence may reflect the complex history of the south Borneo region, and more focused study of its peoples is needed to explore this hypothesis further.A total of 211 DNA samples were analysed from two groups in Borneo: The Ma’anyan ethnic group , and the South Kalimantan Dayak, which comprises a mixed assemblage of diverse Dayak ethnic groups  . The samTwo datasets were compiled from previous published data to fulfil key analytical criteria: i) the low density dataset has wide geographical coverage, but includes relatively few SNPs; while ii) the high density dataset has greatly increased SNP density, but includes fewer populations. This approach, which is necessitated by the wide range of DNA genotyping chip technologies used by the scientific community , allows −6) were excluded; iii) samples with an overall call rate <0.99 and individual SNPs with missing rates >0.05 across all samples in each population were excluded; and iv) variants in high linkage disequilibrium  were also removed for the low density dataset.Filtering and quality controls were performed using PLINK v1.9et al., unpublished data), Indonesian populations including the Lebbo’ and BajoThe final low density dataset contained 9,743 SNPs in 1,817 individuals from 73 populations, after excluding 7 Ma’anyan and 2 South Kalimantan Dayak individuals for reasons of low data quality. This low density dataset includes East and Southeast Asian populations .


Much of the data resolution of the haploid non-recombining Y chromosome (NRY) haplogroup O in East Asia are still rudimentary and could be an explanatory factor for current debates on the settlement history of Island Southeast Asia (ISEA). Here, 81 slowly evolving markers (mostly SNPs) and 17 Y-chromosomal short tandem repeats were used to achieve higher level molecular resolution. Our aim is to investigate if the distribution of NRY DNA variation in Taiwan and ISEA is consistent with a single pre-Neolithic expansion scenario from Southeast China to all ISEA, or if it better fits an expansion model from Taiwan (the OOT model), or whether a more complex history of settlement and dispersals throughout ISEA should be envisioned.We examined DNA samples from 1658 individuals from Vietnam, Thailand, Fujian, Taiwan , the Philippines and Indonesia. While haplogroups O1a*-M119, O1a1*-P203, O1a2-M50 and O3a2-P201 follow a decreasing cline from Taiwan towards Western Indonesia, O2a1-M95/M88, O3a*-M324, O3a1c-IMS-JST002611 and O3a2c1a-M133 decline northward from Western Indonesia towards Taiwan. Compared to the Taiwan plain tribe minority groups the Taiwanese Austronesian speaking groups show little genetic paternal contribution from Han. They are also characterized by low Y-chromosome diversity, thus testifying for fast drift in these populations. However, in contrast to data provided from other regions of the genome, Y-chromosome gene diversity in Taiwan mountain tribes significantly increases from North to South.The geographic distribution and the diversity accumulated in the O1a*-M119, O1a1*-P203, O1a2-M50 and O3a2-P201 haplogroups on one hand, and in the O2a1-M95/M88, O3a*-M324, O3a1c-IMS-JST002611 and O3a2c1a-M133 haplogroups on the other, support a pincer model of dispersals and gene flow from the mainland to the islands which likely started during the late upper Paleolithic, 18,000 to 15,000 years ago. The branches of the pincer contributed separately to the paternal gene pool of the Philippines and conjointly to the gene pools of Madagascar and the Solomon Islands. The North to South increase in diversity found for Taiwanese Austronesian speaking groups contrasts with observations based on mitochondrial DNA, thus hinting to a differentiated demographic history of men and women in these populations. The Taiwanese population comprises three groups of inhabitants. Taiwan Han (TwHan), presently the most numerous group, are descendants mainly of immigrants who came to Taiwan ~400 years ago and include Minnan and Hakka, as well as Han Chinese who immigrated more recently from all over China. The Taiwan mountain tribes Aborigines (TwMtA) include 12 of the 14 officially recognized ethnic groups of the island and represent about 2% of its total population. Finally, a minority group generally described as the Taiwan plain tribe Aborigines (TwPlt) or Pingpu, who are believed to be heavily sinicized and mixed with the Taiwanese Han [TwMtA, Filipinos and Indonesian people speak languages that belong to the Austronesian languages family . Ten priIn addition to linguistics, scholars have also examined evidence from archaeology and genetics to determine the original homeland of Austronesian speakers. The most consistent and generally accepted view based on archaeological evidence  suggestsRecent advances in molecular genetics using human mitochondrial DNA (mtDNA) complete genome sequencing, and genotyping of the haploid non-recombining Y chromosome (NRY) have generated a large amount of genetic data from populations in continental Asia and ISEA -14. UsinDespite all contentions, the use of maternally transmitted genetic markers from mtDNA and paternally transmitted markers such as single nucleotide polymorphisms (Y-SNPs) and microsatellites (Y-STRs) of the non-recombining portion of the Y chromosome (NRY) have now become powerful tools to describe independently distinct genetic patterns within and between populations and to retrace their movements across regions. These methods have not only become complementary to each other, but because of the lack of recombination, they are also very informative in tracing human prehistory, temporally and spatially .Several studies have retraced the earliest expansion Y-chromosome macro-haplogroup O1-M175 in East Asia and ISEA -31. It hOur results support a northeastward dispersal from Southeast Asia (SEA) to Taiwan and Island Southeast Asia and corroborate the hypothesis of separate migration routes to Indonesia from SEA and Indochina ,18,25-27Whole blood or saliva specimens were collected from 1660 unrelated male individuals from 35 ethnic groups in ISEA, Taiwan, the east coast of China and Indochina (Vietnam and Thailand) .DNA was extracted from blood or saliva using QIAmp DNA kit (Qiagen inc. USA) and ORAgene DNA self-collection kit (DNA Genotek Inc. Canada) respectively, with minor modifications to the procedure recommended by the manufacturer.Comparative data with other populations of south China, ISEA and Oceania were taken from the literature and comprised Yueh/Daic-speaking populations ,30,36-38Using the PCR-SSP method, a combination of 81 markers, the majority of which are slowly evolving SNPs , and to 2, 0.1 mM each of the four deoxyribonucleotide triphosphates, 0.2 μM each of forward/reverse primers [Exo I) enzymes  following the conditions recommended by the manufacturer (37°C for 30 min and 80°C for 15 min respectively). Sequencing was performed on both strands using the DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems) according to manufacturer recommendations. Purification on a G50 sephadex column was performed before the final run on an automated DNA sequencer (ABI Model 377).PCR was performed in 10 μl reactions containing 0.5 U AmpliTaq Gold polymerase (Applied Biosystems), 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2 mM MgCl primers  and 50–1 primers . Prior tAll samples were further subjected to genotyping with 17 microsatellites  and analyzed using Y-filer kit (Applied Biosystems). In brief, PCR products were mixed with GeneScan 500LIZ (Applied Biosystems) as internal size standard and analyzed by capillary electrophoresis with an ABI Prism 310 genetic analyzer (Applied Biosystems) in the mode of standard fragment analysis protocol. Genetyper 2.5.2 (Applied Biosystems) was used for allele scoring. For all statistical and network analyses, we used data from DYS389II by subtracting DYS389I from DYS389II .h, and its standard error were calculated using the formulas given by Nei [The Y-chomosome SNP dataset was used to obtain frequency distributions of haplogroups (clades and sub-clades) in the population samples by mere counting, and the unbiased gene diversity index, n by Nei .Contour maps of interpolated spatial frequency variations of the most relevant clades in East Asia were constructed by applying the Kriging algorithm in Surfer 8.0 (Golden software). Similarly, the internal diversity of each relevant Y-SNP clade was estimated on the basis of its STR variation by computing the average variance in repeat size over STR loci (the rho statistic), following the method of Zhivotovsky et al. , and theDYS389 I, DYS389 II, DYS390, DYS391, DYS392, and DYS393). Age estimates of STR variation of haplogroups comprised less than 10 individuals were also calculated but results are to be considered with caution.Thirteen Y-STR loci  were used to estimate the age of the variation within each SNP haplogroup following the modified coalescence method of Zhivotovsky et al. -50, assuGene contribution estimates between populations were inferred by two methods; firstly the coalescent approach of Admix version 2.0, which infers contributions from parental populations according to STR haplotype frequencies considering each haplotype as an allele of the same locus ,52, and Both the SNP and STR datasets were used to perform AMOVA analyses of population structure using the Arlequin 3.5.1.2 software . MultidiMedian joining (MJ) networks of Y-STR haplotypes  for relevant SNP haplogroups and sub-haplogroups of the O clade were constructed from reduced median joining networks using the NETWORK 4.1.0.6 software . We usedThe frequency distribution of Y-chromosome SNP haplogroups detected in Taiwan, ISEA and Indochina is shown in Figure The most prominent clade in Taiwan, O-M175 as a whole, represents almost 90% of Y chromosomes among the TwHan, about 95% among the TwPlt and more than 99% among the TwMtA  and in the Taiwanese Minnan and Hakka (13% and 21%), as well as among the TwPlt (12%), whereas it occurs at an extremely low frequency in TwMtA , in the Philippines (0.8%) and in Indonesia (2%) Figure . Accordi* included O3*, O3a*, O3a1*, O3a1b, O3a1c, O3a2*, O3a2a, O3a2c*, O3a3 and O3a4 [Other commonly observed haplogroups in the O3 series are the paragroups of the O3a2*-P201 branch , in Taiwan  and the Philippines, but was more frequent in Indonesia 8.54%) , in the Philippines (from 8% to 50%) and in western Indonesia , while it is rather uncommon in Indochina and among the Han , with the notable exception of estimates for the Han groups  due to the high frequency of haplogroup O1a1*-P203  analysis of pairwise Reynolds genetic distances obtained from high definition Y-SNP haplogroup frequencies in our dataset is shown in Figure In a second MDS analysis, we included earlier published data  are reported in Table Results between Admix and LS Table  correlatAnalysis of molecular variance (AMOVA) was performed first using Y-SNP haplogroups and then Y-STRs haplotypes Table . The TwMIn contrast to TwMtA, high SNP variation between individuals within populations is found for Western Indonesia (94.30%), the Philippines (98.10%) and TwPlt (96.24%), and the levels of differentiation among populations within groups are correspondingly low, ranging from non-significant for Filipino populations to less that 6% among western Indonesians, even though populations in Western Indonesia and the Philippines are broadly dispersed over many isolated regions of ISEA. On another other hand, the high variance seen within TwPlt populations (96.24%) was expected as they are heavily admixed groups.When testing genetic differentiation among four separate geographical groups, namely TwMtA, Philippines, Western Indonesia and Han (representing mainland China), or three distinct language family assortments , we observe that the variation between groups is large  and did not show much difference between geographic or linguistic groupings. This pattern remains the same when including Indochina as a fifth geographic region or as a fourth linguistic group.Very little changes from the Y-SNP results were observed when using Y-STRs to perform AMOVA computations on the individual groups of populations, with the variance due to differences between individuals within populations being extremely high for TwPlt, Philippines and Western Indonesia (above 96%) and much lower for TwMtA (68%). However, with Y-STRs we observe that the component of the variance due to differences between groups of populations, although significant, is always lower than that due to differences among populations within these groups, thus indicating that, contrarily to Y-SNPs, Y-STRs fail to detect a population structure associated with geography or linguistics.Our data, obtained through the genotyping of 81 high Y-SNP definition markers to determine the fine distribution of Y-chromosome haplogroups O in ISEA, revealed a high level of population structure in the region including Taiwan, the Philippines and Indonesia, mainly defined by variable distributions of haplogroups belonging to the O clade ,27,70. WThe O clade, to which belong 95% of Y-chromosomes in Taiwan, reflects an ancestral relationship to the early modern human settlement in East Asia -30 with The Y-SNP gene diversity among TwMtA was found generally low . This increase is concomitant with a tendency for decreasing frequency of haplogroup O1a1* − P203 and increasing Y-STR diversity in this haplogroup, but both these patterns are statistically not significant , and was above 87% in all northern and central mountain tribes . Conversely, O1a1*-P203 rarely exceeds 20% in the Philippines and Indonesia  among some Daic-speaking groups from southern China and Hainan, it was suggested that its expansion throughout ISEA followed an OOT model of migration reaching sequentially, Taiwan, the Philippines and Indonesia ,65. Our % to 25% A remarkable result emerging from our dataset is the high frequency of the O2 clade, more specifically of haplogroup O2a1a-M88, found in the Bunun  and its MJ network are consistent with repeated introductions of this haplogroup by gene flow from mainland Southeast Asia into the islands Figure . As alreIt has been proposed that, after the initial colonization approximately 50 Kya of SEA and Indochina by modern humans bringing along haplogroups C, M and S, the origin and spread of most haplogroups seen today in east Asia, Taiwan and ISEA could be retraced according to four stages (A to D) of a paternal demographic model . Stages The MJ networks associated with the O1 clade are the only ones in which Y-STR haplotypes of TwMtA are observed among the f0 founder nodes Figure . Along wPopulations that first went south from Southeast Asia along or from the Indochinese peninsula, Malaysia, western Indonesia  and the Philippines represent the southern branch of the pincer model. This dispersal would include haplogroups O1a1*-P203, O2a1-M95/M88, O3a*-M324, O3a1a-M121, O3a1c*-IMS-JST002611, O3a2*-P201 , O3a2a-M159, O3a2b*-M7, O3a2c*-P164 and O3a2c1a-M133 ,67,78. FThe tips of the two branches of the pincer model may have, at times, reached and crossed over in the Philippines. Such bidirectional dispersals have been previously proposed on the basis of specific patterns of mtDNA diversity, namely the northward spread of mtDNA haplogroup E from the Philippines to Taiwan through gene flow  and the The pincer model described here applies mostly to western ISEA. The two branches rejoin further east through ISEA where significant eastward decreasing frequency clines of most haplogroups of the O clade have been observed, although with resurgence of some derived haplogroups of the O3 clade in the Bismark archipelago and Polynesia ,81. MostO1a*”, which was further resolved here as O1a1*-P203 [Our results are in agreement with the multidirectional gene flow out of SEA described by Li et al. for haplogroup “a1*-P203 ,27. Altha1*-P203 -34. Indea1*-P203 ,83, rathConsistent with the first stage (A) of Karafet et al. , we founMore generally, the presence of haplogroups C, K* or F* in Western Indonesia may be associated with the Paleolithic colonization of ISEA, whereas that of haplogroups R and H is likely linked to more recent migrations from Arabs, Indians (or Eurasians) and need to be determined further. Lastly, haplogroups D, N, P* and Q-M242 may be associated with isolated migrations from central Asia ,14,50,84We confirm stage B of Karafet et al.  coalesceStage C describes later migrations/gene flow, between approximately 8 to 6 Kya ago, corresponding to the early Neolithic Austronesian expansion and that brought not only O1a1*-P203, O1a2-M50/M110 and O3a2*-P208  but alsoIn addition to O3a2b*-M7 in stage D of Karafet et al. , we propMadagascar and the Solomon islands may share a common paternal molecular past. While haplogroup O1a2-M50 is commonly seen in Taiwan and moderately distributed throughout western ISEA, O2a1-M95 is rare all over the Philippines , common We also found that all nodes leading to the Solomon Islands in the O2a1-M95/M88 MJ network include Y-STR haplotypes also observed in Madagascar Figure . In otheLastly, we found that the Taiwan plain tribes share more Y-STR haplotypes with Taiwanese Han (12%) than with TwMtA 3%)  in pooled samples from Taiwan compared to the Philippines, Indonesia and mainland Southeast Asia. Figure S3. Variation of diversity measures according to latitude in TwMtA. The three plots display the estimated values and linear regression of, respectively, gene diversity , frequency of haplogroup O1a1*-P203 , and STR diversity of haplogroup O1a1*-P203 , on latitude. Pearson’s linear correlation coefficient and its statistical significance are given in the respective three captions. Figure S4. MDS plot (stress 0.203) using our data and literature data from Additional file 1: Table S1 (over 6000 chromosomes). Haplogroup frequencies were adjusted to 20 basal haplogroups (low definition SNP). See Additional file 1: Table S1 for correspondence of numbers and populations. Table S1. Asian population data from this study and from previously published studies. Data used for MDS analysis shown in Additional file 1: Figure S3. Table S2. Frequency distributions and gene diversity of Y-SNP haplogroups in populations from Taiwan, Island Southeast Asia and Indochina. Table S3. Age (in 1000 years) and Standard Error (SE) of Y-STR variation within Haplogroups (7 STRs). Table S4. Y-SNP and Y-STR raw data.Frequency distributions of O clades  in samples from Taiwan and two neighboring populations . Click here for file


Centaurea solstitialis L., (yellow starthistle). We used these data to test hypotheses about the invasion pathways of the species that were based on historical and geographical records, and we make inferences about historical relationships among populations and demographic processes following invasion. We confirm that the center of diversity and the native range of the species is likely the eastern Mediterranean region in the vicinity of Turkey. From this region, the species likely proceeded to colonize other parts of Europe and Asia via a slow, stepwise range expansion. Spanish populations were the primary source of seed to invade South America via human-mediated events, as was evident from historical records, but populations from the eastern Mediterranean region were also important. North American populations were largely derived from South America, but had secondary contributors. We suggest that the introduction history of non-native populations from disparate parts of the native range have allowed not just one, but multiple opportunities first in South America then again in North America for the creation of novel genotypes via intraspecific hybridization. We propose that multiple intraspecific hybridization events may have created especially potent conditions for the selection of a noxious invader, and may explain differences in genetic patterns among North and South America populations, inferred differences in demographic processes, as well as morphological differences previously reported from common garden experiments.The natural history of introduced species is often unclear due to a lack of historical records. Even when historical information is readily available, important factors of the invasions such as genetic bottlenecks, hybridization, historical relationships among populations and adaptive changes are left unknown. In this study, we developed a set of nuclear, simple sequence repeat markers and used these to characterize the genetic diversity and population structure among native (Eurasian) and non-native (North and South American) populations of  Inferring processes and historical relationships from contemporary patterns is one of the primary goals of population genetics, and that information can help shed light on important environmental issues such as invasive species management. In particular, the invasion history and the evolutionary processes that contribute to the success of a species may be postulated by understanding the genetic diversity and similarities among populations from the native and non-native ranges. One evolutionary process that population genetics routinely illuminates is intraspecific hybridization, which along with other factors such as altered selective regimes in new habitats, may have significant impacts on the competitive capabilities and performance of invasive species Centaurea solstitialis L. (yellow starthistle). This rangeland weed of the Asteraceae is relatively innocuous The emergence of superior competitors after repeated opportunities for intraspecific hybridization resulting from the globalization of plant species would therefore seem to be an expected outcome. Through globalization, individuals from vastly different parts of the native range are repeatedly moved to new habitats, eliminating former geographic barriers to reproduction, and enabling individuals from disparate populations to interbreed. Increasing attention to the role of hybridization and admixture in colonizing species has highlighted the importance of heterosis and the increased evolutionary potential of admixed populations in initiating non-native invasions C. solstitialis in non-native regions is remarkably well understood. Maddox C. solstitialis seeds in mission buildings constructed prior to 1824, but did find seeds from those constructed after that date. Robbins et al. 1951  allows us to hypothesize whether populations from geographically distant regions share a common history, while characterizing the portion of overall genetic diversity that is partitioned within and among different regions (AMOVA) allows us to detect evidence of population substructure. Together, these data elucidate possible critical differences in invasion dynamics within North and South America and provide a sketch of the global invasion history of the species.Here, we seek to extend these genetic studies at greater sampling depth by looking at seven microsatellite  loci designed from an EST database. We sampled 520 individuals from multiple populations from the non-native region, including California, U.S.A., Argentina, and Chile, as well as multiple populations from the putative native region, including those in Spain, Turkey, the Republic of Georgia, Armenia, Romania, Hungary, and Uzbekistan, encompassing the most ambitious collection of populations of this global invader to date. The scope of our collections and the hypervariability of the SSRs, allows us to compare population genetic patterns at multiple levels. Because the goal of this work was to conduct a global survey of the species, samples from local populations were small. The assayed loci from our specimens allow us to compare levels of diversity within and among regions of the world to assess the relative frequency and source of dispersal events in certain regions. That is, comparing the proportion of alleles that are unique to certain regions  and calculating genetic distances  and identified all ESTs with 2 bp and 3 bp SSRs with ten or more repeats and all 4 bp SSRs with five or more repeats using the program MiSA We screened the EST database of the Composite Genome Project . We used different amplification protocols, all featuring variations of the following protocol: 5 minutes denaturation at 94°C, 35 cycles of 94°C for 30 seconds, optimized annealing temperature for 30 seconds . We used Peak Scanner software for analysis (ABI). Peaks were assigned numbers by Peak Scanner based on the 400HD ROX size ladder which approximated the length of the amplicon and each allele call was confirmed individually. A subset of individuals was re-sampled at some loci for confirmation.To control for sample size variation, we scored private alleles and calculated allele richness (As) using a rarefaction method in HP-Rare 2 tests and a likelihood ratio test for unknown gametic phase in Arlequin v. 3.1.1 We examined linkage disequilibrium via χC. solstitialis and characterized as part of the Composite Genome Project, we identified all 2 bp and 3 bp SSRs with ten or more repeats (101 and 64 ESTs respectively) and all 4 bp SSRs with five or more repeats (187 ESTs); these data are available at http://www.genetics.umb.edu/. We designed primers for 34 of the longest SSRs with sufficient 5′ and 3′ flanking space. Eighteen were polymorphic within a sample subset, but because the SSRs were isolated from an EST database, it was common to find unexpected introns in the final amplicon that made scoring difficult. We confirmed the presence of introns by sequencing 2 values were not significant for nearly all pairs of loci in all populations. Linkage disequilibrium was detected for some pairs of loci in some populations using a likelihood ratio test in Arlequin v. 3.1.1, but this was not consistent among populations and was likely caused by historical founding events in invaded regions or consanguineous matings and unknown local population structure (data available at http://www.genetics.umb.edu/). Average observed heterozygosity (Ho) per population for each marker ranged from 0.42–0.76, and did not differ significantly from expected heterozygosity (He) in 34 of the 39 populations , also had high numbers of alleles, ranging from As = 3.40–3.68, 3.30–3.92, and 3.82–4.10 alleles per locus respectively. Private alleles, or alleles that are unique to particular populations, were found in 12 populations from California, Argentina, Chile, Spain, Turkey, Armenia, Romania, and Uzbekistan  within populations was generally high with values averaging 0.65 and ranging from 0.31–0.78. There were no differences in average heterozygosity across all loci among native or non-native populations  or among the populations from Eurasia, North America, and South America .Wright's Fixation Index ST, Nei's genetic distance, and Shannon's diversity index for all pairs of populations (data available at http://www.genetics.umb.edu/) and for populations grouped by country . However, for Wright's FST values, a population from Hungary (Hun1) and one from Uzbekistan were most distinct (FST  = 0.404), while both Nei's genetic distance and Shannon diversity indices identified a different pair, one population from Romania (Rom4) and another from Spain (Spa1)  as most distinct (http://www.genetics.umb.edu/). Regardless of the distance statistic used, the PCA showed three groups of populations. Populations from Hungary and Romania formed one distinct group, populations from California, Argentina, Chile, and Spain formed a second, and populations from Turkey, the Republic of Georgia, and Armenia formed a third , but did detect significant differentiation among those in California and those in Argentina  and in Chile . Thus, Californian populations appear distinct from South American populations even though alfalfa records and FST values suggest they have a shared history.Differences among populations from the non-native regions  were also detected. AMOVA analysis C. solstitialis with a wider geographical scope than previous studies. Many global invaders, including C. solstitialis, exhibit remarkable differentiation between their native and non-native populations The present work provides a more comprehensive assessment of the genetic diversity, population structure, and relationships among native and non-native populations of the global invader Centaurea solstitialis is a relatively recent introduction to North and South America, but it has quickly established itself as one of the most aggressive invaders in large areas of its new range The Eurasian native C. solstitialis, however, appear to possess extensive genetic diversity in their non-native range; this result was first reported by Sun o in non-native regions than native regions, we found no significant difference in Ho nor He between these regions nor among the continental groups of populations . We did, however, find great heterogeneity for gene diversity (He), allelic richness (As) and private allele values among populations in Eurasia. Populations from the eastern Mediterranean region and particularly the populations in Turkey had the highest levels of gene diversity (average of populations He = 0.75), highest rarified allelic richness (average of populations As = 4.1) and, along with Armenia, the highest number of private alleles (4 and 5 respectively). The populations of this eastern Mediterranean region  cluster together in our STRUCTURE analysis  than in any of the American regions . Pairwise comparisons for genetic differentiation between American populations and multiple Eurasian countries were often low. Indeed, Nei's D and FST values averaged across populations were lower in the American and Turkish comparisons than for the American and Spanish ones , the populations of California are significantly differentiated from both . There are also differences in population structure within these non-native regions. The FST values among populations in California are all low, but tend to be higher among populations in both Chile and Argentina. Low substructure in California was also noted by Sun C. solstitialis probably expanded its range rapidly from diverse founding populations. In contrast, the South American region and particularly populations from Argentina show more substructure and more differentiation among populations, possibly suggesting a less rapid range expansion. This less rapid range expansion could be due to dispersal barriers such as climatic differences and altered selective pressures in Argentina. Eriksen et al. Further dissection within STRUCTURE (K = 4) and close inspection of the PCA help to reveal the final stage of global expansion examined in this study. Californian populations are clearly distinct from those in South America, and while the AMOVA shows no significant difference between populations in Chile and those of Argentina , north to central Europe (Romania and Hungary) and west to Spain. Each of these regions evolved independently and retained the signatures  of bottlenecks within introduced populations. The initial expansion of this species out of the core Mediterranean region may have involved human-mediated events, however we suggest that more natural dispersal could be responsible for the observed patterns in Eurasia. The clearly human-mediated invasion to the Americans likely involved more rapid and multi-step processes with the initial influx of seed coming from Spain to South America and from there to California, U.S.A. These initial events were likely followed by additional introductions to South America and also to California from the eastern Mediterranean center of diversity increasing the genetic diversity in these non-native regions beyond that of the initial introductions from Spain. Certain individuals from California, Argentina, and Chile, have high posterior probabilities of being from Eurasia , but popC. solstitialis populations have experienced a history of introductions from multiple sources of the native range, and this melting pot of rearranged genomes was, along with new introductions from native regions, a major source of populations that have now colonized North America. Thus, admixture events have occurred multiple times between diverse populations and subspecies in the non-native regions, and may have created especially potent conditions for the selection of a serious invader. Several studies have used common garden experiments to document morphological differences between native and non-native populations that may indicate evidence for adaptive morphological changes between native and non-native populations of C. solstitialisMultiple introductions of an invasive plant can create a “melting pot” of genetic diversity derived from a wide range of habitats and geographically isolated populations from the native region


Myricaria germanica, a threatened riparian shrub which is capable of both wind and water dispersal. Our data led us to reject the ‘one catchment – one gene pool’ hypothesis as we found support for two gene pools, rather than four as expected in a study area including four catchments. This result also implies that in the history of the studied populations, dispersal across catchments has occurred. Two contemporary catchment-crossing migration events were detected, albeit between spatially proximate catchments. Allelic richness and inbreeding coefficients differed substantially between gene pools. There was significant isolation by distance, and our data confirmed the one-dimensional stepping-stone model of gene flow. Contemporary migration was bidirectional within the studied catchments, implying that dispersal vectors other than water are important for M. germanica.One of the major distinctions of riparian habitats is their linearity. In linear habitats, gene flow is predicted to follow a one-dimensional stepping stone model, characterized by bidirectional gene flow between neighboring populations. Here, we studied the genetic structure of  Riparian habitats host a rich assemblage of specialist plant species confined to floodplains One of the important characteristics distinguishing riparian from other habitats is their linearity. Linear habitats may function as corridors, facilitating rapid movement of individuals and genes across a landscape In plants, quantifying migration is a notoriously difficult task, because it is often not possible to observe the dispersal of propagules directly The various ways for migration to occur in plant populations allow us to test an explicit hypothesis on gene flow. Our first hypothesis states that gene flow is mainly directed downstream, as expected if hydrochory is the most important dispersal mode. Our alternative hypothesis is that gene flow should be bidirectional as predicted in a riparian species dispersed mainly by a combination of vectors including water, wind, and animals. We approached testing the null hypothesis by making use of migrate-n, a powerful software that allows the quantification of bidirectional migration rates and population sizes in a coalescent framework using Markov Chain Monte Carlo computing, and by quantifying contemporary migration with assignment tests using the software GeneClass2, which can be used to identify and assign first-generation migrants to source populations. In migrate-n, apart from testing upstream vs. downstream stepping-stone migration models, we tested whether populations in a catchment were consistent with a single panmictic population. Last but not least, we tested whether there was statistical support for an island model .Our second hypothesis relates to the spatial distribution of gene pools in multiple catchments. We hypothesize that in a plant species dispersed via water, there should be genetic divergence between populations from different catchments because the crossing of catchments would not be feasible with this dispersal vector. Hence, if the populations of a riparian plant in multiple catchments have remained isolated over many generations, each catchment should be populated by its unique gene pool (‘one catchment – one gene pool’ hypothesis). Alternatively, gene flow by other vectors than water would lead to the spatial distribution of gene pools across multiple catchments.FIS) provides important insight into the mating system. Genetic diversity of populations may be influenced by mating system. If local population size has remained small over several generations, selfing leads to an increased frequency of homozygous individuals and may lead to a loss of rare alleles over time due to random sampling effects. Here, we tested specifically whether there was i) a relationship between ISF and affiliation to gene pool, and ii) whether high-elevation sites exhibited a different level of inbreeding than low-elevation sites.Our third objective was to explore patterns of genetic diversity across space and between genetic clusters. We hypothesized that genetic diversity should be related to elevation, with highest diversity in downstream sites as a consequence of seed dispersal with water. Moreover, in agreement with population genetic theory, larger populations should harbor more genetic diversity than smaller populations and downstream populations could be larger owing to the immigration of individuals from upstream sites. Finally, we analyzed the mating system using population-specific inbreeding coefficients 10 of 10 of geographic distance should be about −1.0, if gene flow occurs mainly in one dimension in space, as expected in linear habitats Our fourth hypothesis concerned isolation by distance Myricaria germanica (Tamaricaceae) is a threatened riparian shrub growing on gravel banks along rivers. In Central Europe, this character plant of riparian vegetation Myricaria is able to self M. germanica, and to determine which model of migration fits this species best in catchments of major rivers in Switzerland.Here, we used population genetic analyses of a large dataset of microsatellite genotypes to understand regional patterns of gene flow and genetic diversity in t-test assuming unequal variance among groups .Analysis of contemporary migration based on first-generation migrants using the software GeneClass2 revealed a number of first-generation migrants within the Inn and Rhine catchments, with migration being directed both upstream and downstream . The numModel selection based on natural logarithmic Bayes Factors in analysis of recent migration with Migrate-n gave support for migration being directed downstream in a stepping-stone fashion in the Rhine catchment . For theOur data exhibited a high amount of genetic differentiation between populations. Analysis of molecular variance revealed significant genetic structure due to the grouping of sites by river  than within clusters . Within Cluster 1, FST values ranged from 0.01 to 0.98; within Cluster 2, they ranged from 0 to 0.44.In agreement with the results from Bayesian analysis of population structure and population graphs, analysis of pairwise Properties of the collecting sites including their allelic richness and inbreeding coefficients are given in A clear geographic trend in allelic richness was visible in our data: Populations located in the Engadine, the valley of the river Inn in southeastern Switzerland had lower allelic richness than populations from other regions . These sM. germanica had lower genetic diversity than low or middle altitude populations , indicating variation in mating system across sites (FIS = 0.15 to 0.48) or outcrossing mating system (FIS = 0.07). Sites belonging to Cluster 2 exhibited high inbreeding coefficients  and geographic distance . Gene flow as estimated by the log10 of 10 of geographic distance d according to the equation log10(10(d)  . The slolog10(d) . Based oM. germanica was bidirectional, whereas historic gene flow was directed downstream in the Rhine catchment. Our data rejected the ‘one catchment-one gene pool’ hypothesis, as a single genetic cluster was distributed across four catchments and another one across two. Population graph analysis showed that there were no connections between the two clusters found, highlighting their genetic isolation. Sites situated in the southeast of Switzerland were characterized by low diversity, high inbreeding coefficients, and were differentiated from all remaining sites, belonging to a separate genetic cluster. Last but not least, our data showed significant isolation by distance, supporting a one-dimensional stepping-stone model.Our data showed that contemporary gene flow in M. germanica. Few other studies have found evidence for contemporary dispersal between catchments in riparian plant populations.Contemporary gene flow, as estimated from analysis of first generation migrants based on assignment tests, took place mainly within catchments, with two exceptions where gene movement was detected between catchments in sites that were spatially proximate . Hence, dispersal between catchments is possible in M. germanica. Also isolation by distance analysis supported bidirectional migration (see below). While migration events directed downstream are most likely to have arisen from hydrochory of seeds in combination with wind dispersal, upstream migration must have taken place either by wind or animals. It is unlikely that long-distance pollen dispersal events would have been reported as migrants, as the method only allowed detecting individuals with both gene copies in the source population.Moreover, contemporary gene flow occurred both in upstream and downstream direction in Myricaria laxiflora, unidirectional gene flow downstream and considerable genetic differentiation between populations has been reported Sparganium emersum, an accumulation of genetic diversity in downstream populations was found, together with high differentiation between populations pointing towards source-sink population dynamics M. laxiflora, which showed evidence for linear, unidirectional migration via hydrochory Several studies have found support for either bidirectional gene flow or a source/sink scenario Contemporary migration was bidirectional. In contrast,historic gene flow was directed downstream in the largest catchment, Rhine. Contemporary and historic directionalities of gene flow may differ for several reasons. Historic gene flow reflects the main directionality of gene flow over a long time, and support for the directionality downstream does not mean that there have never been any events in the other direction. Individuals dispersed by the vectors wind/animals could have lower reproductive success in the populations they are dispersed to, and then their genes may not be traced in historic signal. Moreover, we can not rule out that the importance of individual dispersal vectors may have changed over time. For example, it could well be that some dispersal events represent recent human-aided dispersal in the framework of conservation translocations, which would lead to a discrepancy among contemporary and historic directionalities.FST values and AMOVA that is typical for selfing plant populations. Since the downstream models of gene flow did not converge in these two cases, we can not fully exclude the possibility that model selection in Migrate-n based on Bayes Factors inferred an erroneous model.Along three catchments, model selection in Migrate-n based on Bayes Factors provided evidence of panmixia. For the Inn catchment, this result seems plausible as genetic differentiation between sites was generally low. For Rhone and Maggia, however, the result of panmixia is in conflict with the strong population subdivision evident from Our historic gene flow analysis highlights the importance of water and wind in seed dispersal for the Rhine catchment. Several other studies have emphasized the importance of seed dispersal via hydrochory in riparian and aquatic plants M. germanica do not form a single continuous population. We found substantial population subdivision between and within rivers; this result is similar to what was found in other studies of riparian and aquatic shrubs or herbs M. germanica, hence their capability for long-distance seed dispersal by wind should be greater M. germanica can likely be explained by frequent selfing. In selfing plant species, pollen dispersal is low, leading to population structure unless seed dispersal is highly efficient. Moreover, selfing reduces effective population size, thus increasing drift and leading to higher degrees of population subdivision It is obvious from Bayesian analysis of population structure, analysis of molecular variance, population graphs, and from the contemporary pattern of migration that the sites sampled for Rhododendron ripense corresponded to Pleistocene river systems Our data rejected the one-catchment, one gene pool hypothesis, under which we would have expected four genetic clusters to occur, each in one catchment. Instead, there were only two clusters, and the same cluster occurred in multiple catchments. None of the studies we examined for riparian and aquatic plant populations found support for the one-catchment, one gene pool hypothesis. Several studies reported multiple gene pools of riparian and aquatic plants in a single catchment M. germanica implies that there must have been catchment-crossing dispersal events at some time in the history of Cluster 1, founding populations in different catchments. Moreover, there must have been at least one historic dispersal event among catchments for Cluster 2 which also spans across two catchments in southeastern Switzerland.The spatial distribution of a single gene pool across multiple catchments in The arguably most striking pattern with respect to genetic diversity found in the data was the vast discrepancy in genetic diversity between Clusters 1 and 2. Cluster 2 sites located in the Engadine valley in southeastern Switzerland exhibited a far lower diversity than all remaining sites, with the notable exception of two .Myricaria investigated with population genetic approaches to date, M. laxiflora, was determined to be predominantly selfing in a study that used amplified fragment-length polymorphisms (AFLPs) to investigate genetic variability in populations of the Yangtze River in China M. germanica has a mixed mating system, with frequent selfing. When doing hand-pollinations to make crosses of plants from different catchments (Inn vs. Rhine), a number of the progeny turned out to be selfed, rather than out-crossed .One result of interest is the vast discrepancy of inbreeding coefficients across sites, indicating geographic variation in mating system. A high level of inbreeding was inferred for sites belonging to Cluster 2 (Engadine). The only other species of FIS. Low genetic diversity could also result from recent changes in population size, e.g. bottlenecks or founder events after the colonization of new habitat patches Contrary to the theoretical expectation FIS. Our linear models indicated that this effect is likely due to the confounding effect of the highly inbred, low-diversity Engadine sites being located at high elevations.We found significant relationships with elevation in allelic richness and FST) followed a linear relationship with geographic distance as determined by a Mantel test. Most of the prior studies of riparian and aquatic plants did not find isolation by distance Ainsliaea faurieana, isolation by distance was only found when several catchments were analyzed in combination, but not within a single river We found statistical support for isolation by distance in the studied sites; genetic differentiation , M. germanica grows on gravel banks along rivers from the colline to the subalpine altitudinal zone (500–2100 m). Being a pioneer species on gravel bars, M. germanica forms patchy populations, with frequent colonizations of new patches and extinction of existing populations after disturbance by flooding. When rivers are channelized in a way that suitable habitat, in particular sites with intermediate disturbance frequencies are lacking, the species can go locally extinct. Thus, M. germanica has faced a severe decline in many of the major rivers of Europe in the past decades. Once a rather common species on the Swiss Plateau, the species is now restricted to a few sites in this region. In Switzerland, M. germanica is most common in the Southeast, i.e. the Cantons of Grison and Ticino . PCR, fragment analyses, and genotyping of 20 nuclear microsatellites were performed as described in FIS and pairwise FST values were calculated with Arlequin version 3.5 Gene diversity and allelic richness were calculated using FSTAT version 2.93; to map the values, allelic richness was averaged over the 20 nuclear microsatellites. Population-specific inbreeding coefficients F-statistics in the AMOVA were based on the number of different alleles, and significance of variance components was tested with 1000 permutations We performed analysis of molecular variance in Arlequin. The In order to depict the genetic relationships between sites, we calculated population graphs in R using the package ‘gstudio’ home, the likelihood of drawing an individual's genotype from the site where it was sampled, given the observed allele frequencies of all sites To estimate contemporary migration patterns, we used an assignment method allowing to detect first-generation migrants implemented in the software GeneClass2 version 2.0 M. germanica, we used the coalescent-based software Migrate-n version 3.26 i) constant population size through time or random fluctuation around an average size; ii) individuals are randomly mating within populations; iii) the mutation rate is constant through time and is the same in all parts of the genealogy; iv) the immigration rate is constant through time; and v) the studied populations exhibit a recent divergence and not an old split, so they exchange material through gene flow th step for a total of 200,000,000 genealogies after a burn-in of 200,000 steps in the chain. A Brownian motion model was used which approximates the stepwise mutation model commonly used for microsatellite data, but converges faster than the standard stepwise mutation model. Populations were randomly resampled to 100 individuals to speed up convergence. Starting parameters for population size θ and migration rates were inferred from FST values; mutation rate modifiers were deduced from the data using Watterson's θ. We performed a series of preliminary runs to explore run conditions and prior distributions for the data from each catchment. Bayesian estimation of migration rate and population sizes were run with one long chain and static heating . The models compared were a full model with all migration rates and population sizes, a stepping-stone model with bidirectional gene flow between neighboring populations, a model where gene flow was directed downstream (but not restricted to neighboring populations), a stepping-stone model where migration occurred only downstream, and a panmictic model. Model selection was performed based on model probabilities and natural log Bayes factors, calculated as LBF = 2(lnmLm1-lnmLm2), with lnmLm1 and lnmLm2 being the log marginal likelihoods of model 1 and 2, respectively M. germanica. We assumed a rate of 10−3−2 to 10−5To test specific models about the directionality of gene flow and to obtain Bayesian estimates of effective population sizes and bidirectional rates of migration in M. germanica, we calculated linear regression models of allelic richness and FIS (response variables), affiliation of population to clusters resulting from Bayesian analysis of population structure with software Structure, log10-transformed population size, and elevation in R using the function ‘lm’ To identify the factors explaining genetic diversity in FST values (FST/(1−FST) and Euclidean geographic distance and Kendall's rank correlation with 999 permutations.Pairwise estimates of genetic distance are not statistically independent; thus, in this case, significance testing of genetic vs. geographic distance through linear regression is not reliable 10 of the gene flow estimate 10-transformed geographic distance If the genetic data follow a stepping stone model, gene flow should decrease with distance. More specifically, the log