Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop  sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments.Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian ( Differences in dispersal behavior between males and females can have a profound effect on population dynamics. Male-biased dispersal (MBD) has often been described in mammals Trichechus manatus) and Amazonian (T. inunguis) manatees have revealed seasonal and water level dependent migration Studies that include telemetry and field observations on West Indian  indicated high levels of structuring between T. manatus populations, showing three main haplotype clusters T. inunguis in Brazil, with high genetic variability and possible demographic expansion Previous studies using maternally inherited mtDNA D-loop, and 15 microsatellite loci. These results have implications in assessing population migratory limits, which could help to define effective conservation units and enhance global and local management action plans to effectively protect manatee populations of both species.In this study we tested the MBD hypothesis for riverine West Indian manatees in Colombia, where populations are distributed mainly in rivers and swamps The sample collection methodology for this project was approved by Universidad de los Andes, Faculty of Sciences Ethics Comitee and was done following the Caribbean Stranding Network protocols, avoiding animal suffering.Samples were collected as part of Colombia’s West Indian and Amazonian manatees National Management Plan. They were provided by Omacha Foundation , and were collected by employees from Colombian Regional Environmental Authorities  during manatee rehabilitation and releasing programs over the past 20 years. Peruvian samples were collected by the PRODUCE - DIREPRO  and were analyzed at the Peruvian Amazon Research Institute (IIAP) in Iquitos. Regional Environmental Authorities in Colombia and Perú do not require to obtain collection permits for particular projects ), as these institutions belong to the National Environmental Systems of the Ministry of Environment .Fecal samples were collected over the water column from wild individuals. Bone samples were obtained from manatee carcasses illegally killed by local fisherman, and seized by regional authorities. Blood and tissue samples were collected from live rescued individuals; blood was obtained from the anterior pectoral flipper, using a 1.5-inch needle, while tissue was collected from the tail fin using approved protocols.T. inunguis and 58 for T. manatus.Skin, blood and fecal samples were collected from wild and captive manatees. Bone, skin, and muscle samples were collected from carcasses. Skin, muscle and feces were preserved in 70% ethanol, and blood samples in EDTA lysis buffer . A total of 97 samples were collected from 39 for n = 6), (II) Peruvian Amazon River (n = 7), (III) Ucayali River (n = 13), (IV) Marañón River (n = 9), and (V) Napo River (n = 5) (n = 17), (II) Northern Magdalena River (n = 19), (III) San Jorge River (n = 10), (IV) Magdalena River’s Ciénaga de Paredes (n = 7), (V) Meta River (n = 2), and (VI) Orinoco River (n = 3)  Colombian Amazon River ( (n = 5) . West In (n = 3) . NortherD-loop hypervariable portion of the mtDNA CR was amplified by the polymerase chain reaction (PCR), using two pairs of primers, depending on DNA quality. Good quality samples were amplified with CR4 and CR5 primer pair 2, 200 µM dNTP, 2 units BSA, 0.5 µM of each primer, and 1 U Biolase DNA polymerase (Bioline USA). PCR amplification conditions were: 94°C for 2 min, 34 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 40 s, with a final extension period of 72°C for 10 min. Low quality samples (faeces and bones) were amplified using LTMCR01 and HDDCR01 primer pair and previously published PCR conditions A 410bp TmaKB60, TmaA02, TmaE01, TmaE02, TmaE07, TmaE08, TmaE11, TmaE26, TmaH13, TmaJ02, TmaK01, TmaF14, TmaM79, TmaSC5, TmaSC13) Fragment analysis of 15 polymorphic microsatellite loci  T. inunguis sequences were analyzed using two approaches: the first included 410bp sequences from Colombian and Peruvian rivers, and the second reduced the length of sequences to 361bp, to include sequences and geographic information of Brazilian rivers  obtained from Cantanhede et al. 2005. The number of haplotypes (h), nucleotide (π), and haplotype (Hd) diversity indices for each population 410bp AN), levels of polymorphism shown by microsatellite loci, departures from Hardy-Weinberg equilibrium (HWE) , and linkage disequilibrium (LD) (permutations 10000). P values were adjusted using Bonferroni correction for multiple comparisons.ARLEQUIN was used to estimate the number of alleles per locus  from 1 to 10, were run without an a priori population assignment, using the LOCPRIOR prior to improve the detection of weak population structure KSTRUCTURE version 2.3.3 D-loop and microsatellite loci were estimated using Wright’s fixation index ST,F inbreeding coefficient ISF, and molecular variance (AMOVA) STF pairwise distance matrix for mtDNA and microsatellite loci was performed using a Mantel Test under Spearman (non-parametric) method, with 100000 permutations using the VEGAN package in R Among and within population subdivision for T. inunguis, and five for T. manatus according to possible geographic barriers:Migration rates among populations were estimated using a Bayesian inference coalescent approach in MIGRATE-N version 3.2.17 T. inunguis, M0: No migration among populations, M1: One single panmictic population, M2: Full symmetric migration among all populations, M3: [Colombian Amazon – Peruvian Amazon], [Peruvian Amazon – Ucayali], [Ucayali – Marañón], [Peruvian Amazon – Napo], [Peruvian Amazon – Marañón].T. manatus, M0: No migration among populations, M1: One single panmictic population, M2: Full symmetric migration among all populations, M3: [Sinú – Northern Magdalena – San Jorge – Magdalena’s Ciénaga de Parades], [Meta – Orinoco], M4: [Sinú – San Jorge], [Sinú – Northern Magdalena], [Northern Magdalena – San Jorge], [Northern Magdalena – Magdalena’s Ciénaga de Parades], [Meta – Orinoco].A total of three independent analyses with chain length of 100000000 were performed for each migration model to ensure sampling from the stationary distribution. Convergence of the MCMC was assessed through visual inspection of the all parameter traces. In all cases, every parameter had an effective sample size of at least 300. The best-fitting migration model for each species was chosen based on the Bayes factors of the harmonic mean of the likelihood  (G03) , haplotype Ti08 was redundant to haplotype H18, haplotypes Ti09 and T01 were redundant to haplotype H05, and haplotype S04 was redundant to haplotype H12. Average π among T. inunguis individuals when using 410bp sequences was 0.472% (standard deviation (SD) 0.055%), and overall Hd was 0.885 (SD 0.035), showing the highest diversity in the Colombian Amazon population (π was 0.718% (SD 0.113%), the overall Hd was 0.903 (SD 0.019), and the highest diversity was found in the Pará River. T. manatus sequences were more diverse than those of T. inunguis when using 410bp sequences, with an average π of 3.018% (SD 0.315%), and overall Hd of 0.76 (SD 0.052), and less diverse when compared with 361bp sequences that included Brazilian Rivers. The Sinú River’s West Indian manatees had the highest mitochondrial diversity (Sixteen 410bp 01-Ti12) , and nin2) (G03) . New happulation . When usiversity .et al. 2005, and Vianna et al. 2006, one diverse Median Joining Network (MJN) cluster was obtained for T. inunguis D-loop haplotypes using both 361bp and 410bp approaches, with all haplotypes being closely related among them  lower than expected heterozygosity (EH) per locus per population .All 15 microsatellite loci had between 5 and 12 alleles per locus for pulation . AllelicT. manatus, and 8 loci in T. inunguis  in at least one population. Differences in levels of population structure were found for each allele bootstrap replicate. Moreover allele randomization revealed higher heterozygote frequencies in comparison with observed values on every locus per population, due to homozygote excess.We found evidence for presence of null alleles in all loci in K, and variance among runs, was K = 2 in both species. Neither of the clusters obtained were consistent with sampled populations. The two clusters shown for T. manatus were not correlated with the two D-loop clusters, showing not only differences in levels of structure among microsatellites loci and mitochondrial D-loop, but also in their evolutionary trajectory.Populations were not monophyletic in any of the microsatellite loci genealogies trees in either species. The amount of population clusters according to STRUCTURE after using ad hoc statistic DeltaISF for T. manatus and T. inunguis were significant (P<0.05), indicating high levels of inbreeding within populations at 0.456 and 0.282, respectively. Differences among populations using 410bp D-loop sequences demonstrated structure between Peruvian and Colombian T. inunguis manatees , being lower but significant with microsatellite data  (Ti10) with Perú, while the rest of its haplotypes are unique for this population, contrasting with Peruvian populations which share more than one haplotype among them (Estimates of P<0.001) . The strong them .D-loop sequences, population structure was found displaying two main groupings, a Peruvian population including all Peruvian tributaries to the Amazon River and a Colombian and Brazilian population that included the Colombian portion of the Amazon River and all other rivers and tributaries of Brazil .When including 361bp T. manatus structure levels were even higher, revealing five different populations: Sinú River, Northern Magdalena River, San Jorge River, Magdalena River’s Ciénaga de Paredes, and Meta and Orinoco rivers , or four groups if excluding Meta and Orinoco due to low sampling sizes  (In Colombian : n = 3) . HoweverSTF distance matrixes for either species .The Mantel test presented no correlation between pairwise T. inunguis (M2), and among geographically isolated populations of T. manatus (M3), and rejected the model of no migration (M0) for both species , tucuxi  caiman (Caiman crocodilus), and the pirarucu (Arapaima gigas), among others A complex cluster of connected haplotypes for D-loop analysis  or shared ancestral polymorphisms. Our results suggest there are more likely explanations T. manatus and T. inunguis, the null allele hypothesis is rejected and the possibilities of other factors that explain low OH cannot be dismissed. Null migration among populations was the least supported migration model dismissing the possibility of ancestral polymorphisms as a force generating lack of structure among populations  since 1982 D-loop should be taken into account by current reintroduction programs, being important to release animals on their place of origin, even if males are dispersing among populations, as previously proposed by Vianna et al. 2006. High D-loop haplotype diversity of South American riverine manatees reveals that river dynamics could be key in maintaining population’s genetic diversity.Population structure detected in riverine West Indian and Amazonian manatees when using maternally inherited T. manatus between these two populations, to warrant male gene flow and reducing the chances for inbreeding.Conservation strategies in South American manatees should protect riverine populations not only locally, but also on a wider scale, given this is critical in maintaining connectivity among populations. This includes connectivity between coastal wetland systems between the Magdalena and Sinú rivers in the Caribbean coast of Colombia so as to encourage the use of aquatic ecological corridors by male R Script S1R script used for microsatellite allele randomization.(R)Click here for additional data file.R Script S2R script used for microsatellite locus bootstrapping.(R)Click here for additional data file.R Script S3Assessment of population monophyly on each genealogy.(R)Click here for additional data file.


Ambrosia artemisiifolia, a North American native that is invasive in China. This species has been found to possess a strong self-incompatibility system and have high outcrossing rates in North America and we examined whether there has been an evolutionary shift towards the dependence on selfing in the introduced range. Specifically, we estimated outcrossing rates in one native and five invasive populations and compared levels of genetic diversity between North America and China. Based on six microsatellite loci we found that, like the native North American population, all five Chinese populations possessed a completely outcrossing mating system. The estimates of paternity correlations were low, ranging from 0.028–0.122, which suggests that populations possessed ∼8–36 pollen donor parents contributing to each maternal plant in the invasive populations. High levels of genetic diversity for both native and invasive populations were found with the unbiased estimate of gene diversity ranging from 0.262–0.289 for both geographic ranges based on AFLP markers. Our results demonstrate that there has been no evolutionary shift from outcrossing to selfing during A. artemisiifolia's invasion of China. Furthermore, high levels of genetic variation in North America and China indicate that there has been no erosion of genetic variance due to a bottleneck during the introduction process. We suggest that the successful invasion of A. artemisiifolia into Asia was facilitated by repeated introductions from multiple source populations in the native range creating a diverse gene pool within Chinese populations.The mating system plays a key role during the process of plant invasion. Contemporary evolution of uniparental reproduction  can relieve the challenges of mate limitation in colonizing populations by providing reproductive assurance. Here we examined aspects of the genetics of colonization in  The main challenge for populations of self-incompatible outcrossing plants is the mate acquisition phase of sexual reproduction. This part of the life cycle can be especially tenuous for invasive species because it depends on the number and distribution of genetically compatible genotypes. A biological invasion typically originates with the transport of individuals from the species' native to the introduced range. Since long-distance introduction involves a subsampling of the native range gene pool Smaller and more isolated populations of self-incompatible wind-pollinated plants often experience increased pollen limitation Ambrosia artemisiifolia, Bromus tectorum, Clidemia hirta) the level of genetic diversity is actually greater in the introduced ranges Founder events and genetic bottlenecks are generally thought to result in a reduction in the genetic diversity when compared to the total diversity present in the native range Ambrosia artemisiifolia, a plant that has invaded China. This wind-pollinated, monoecious annual is reported to possess a strong self-incompatibility mechanism and exhibit high outcrossing rates in its native North American range The goal of the present study was to document the mating system and levels of genetic variation in No specific permits were required for the described field studies. The sampling location is not privately-owned or protected in any way, and the field studies did not involve endangered or protected species.Ambrosia artemisiifolia L. (Asteraceae) is an aggressive annual weed of agricultural fields and disturbed sites that is native to North America and has become invasive in Europe and Eurasia Ambrosia artemisiifolia was introduced into China in 1930s and now occurs across a large range from Guangdong Province in the south to Heilongjiang Province in the north, and has invaded a variety of plant communities The common ragweed, 2, 0.5 µl 10 mM each fluorescence-labeled forward and reverse primers, 1 U Taq DNA polymerase , 16.8 µl H2O and 1 µl (ca. 10–20 ng) genomic DNA. PCR amplifications were performed in a Bio-Rad thermal cycler as follows: an initial denaturation step at 94°C for 5 min followed by 30 cycles of 30 s at 94°C, 45 s at 50°C and 45 s at 72°C, with a final extension period of 8 min at 72°C. The fluorescence-labeled PCR products were denatured and analyzed on an ABI 3100 genetic analyzer, using the ROX 500 as an internal size standard. The genotypes were scored using GENEMAPPER software version 3.7 (Applied Biosystems).The mating system (selfing rate) was determined using microsatellite markers for one native North American population (LC) and five populations  sampled over a large latitudinal gradient in eastern China . For eacst), multilocus outcrossing rate (mt), outcrossing rate between related individuals (mt−st), inbreeding coefficient of maternal parents (F), and the correlation of paternity or proportion of full sibs among outcrossed progeny (pr) for all six populations using the expectation-maximization method in the MLTRWIN program (version 3.4) We estimated the single-locus outcrossing rate . The selective PCR amplifications were performed with a touchdown cycling process programmed with the following temperature profile: an initial denaturation step 94°C for 2 min, followed by 13 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min with a reduction of the annealing temperature at each cycle by 0.7°C; the annealing temperature was maintained at 56°C for the remaining 23 cycles. Four pairs of primers  were used to amplify the DNA of 254 plants from four American populations and six Chinese populations.AFLP fingerprinting was performed as described by Vos et al. The selective PCR products were denatured and separated on an ABI 3100 genetic analyzer (Applied Biosystems) with a ROX 500 as internal size standard. Fragment sizes were assessed using GENEMAPPER software version 3.7 (Applied Biosystems). Allele size determinations were performed twice manually to reduce scoring errors. The multilocus AFLP profiles were scored as present or absent to create binary matrices.jH, analogous to eH) to estimate the within-population diversity; and the degree of genetic differentiation (stF) among populations within each of the continents.Based on the observed frequencies of AFLP fragments, allelic frequencies at each locus were estimated using Bayesian method with non-uniform prior distribution proposed by Zhivotovsky A. artemisiifolia populations displayed high outcrossing rates (Both Chinese and North American ng rates . The mulng rates .mt−st) were low for Chinese populations (range: 0.065±0.028 to 0.179±0.025) and North American population (0.063±0.020). These values were significantly greater than zero, suggesting some degree of biparental inbreeding in both Chinese and North American A. artemisiifolia populations. The maternal inbreeding coefficients (F) were less than 0.008±0.020 and did not differ from zero for all populations, indicating obligate outcrossing. The correlations of paternity (pr) were 0.028–0.122 in Chinese populations, suggesting that the populations consisted of a large number of pollen donor parents. However, there was a higher number of pollen donor parents in the North American population  were polymorphic at the 5% level. North American and Chinese populations shared 320 out of the 322 bands. Only two bands were private between continents: one band occurred in four North American populations but was absent in China while another occurred in three Chinese populations but was not found in North American populations.The four pairs of FAM-stF values indicate that there is a greater degree of genetic structure in the introduced Chinese range compared to North America. Approximately 94% of the genetic variation exists within North American populations compared to 87% in the invasive Chinese range  were low suggesting that all of the populations possessed a high number of pollen donor parents contributing to each maternal plant. Moreover, the maternal inbreeding coefficients (F) were nearly equal to zero for all the populations, indicating the absence of inbreeding. Combined with the uniformly high outcrossing rates in North America reported by Friedman and Barrett A. artemisiifolia's Asian invasion. Moreover, our results suggest that the positive fixation indices FIS reported in European populations of A. artemisiifoliaThe plant mating system is subject to environmental and genetic influences that operate at both ecological and evolutionary time scales Ambrosia artemisiifolia has also invaded Europe where populations contain similar levels of allelic diversity and heterozygosity as in North America Since colonizing populations often establish with only a subset of the genotypes present in the native range, we expect less genetic variation to exist in introduced populations when compared to those in the native range A. artemisiifolia would be a selfer throughout its invasive range in Europe and Asia. However, it is apparent that the species has a highly outcrossed mating system in both its native North American range One of the goals of research on biological invasions is to understand the ecological and genetic factors that contribute to successful establishment and subsequent geographic spread. It is generally believed that owing to being limited to a single bout of reproduction, annual species should be primarily selfing and not possess a self-incompatibility system


Alouatta belzebul in eastern Amazonia. The analysis included two populations sampled in 1984, representing both margins of the Tocantins river, and three populations sampled 18 years later. Minimal differences in the diversity levels between present-day (Ho = 0.62-0.69 and AR = 6.07-7.21) and pre-flooding (Ho = 0.60-0.62 and A R = 6.27-6.77) populations indicated there was no significant loss of genetic variability, possibly because of successful management strategies applied during the flooding. The changes observed were limited to shifts in the composition of alleles, which presumably reflect the admixture of subpopulations during flooding. Given this, there were significant differences in the Rst values (p = 0.05) in all but one between-site comparison. Both present-day and original populations showed a deficit of heterozygotes, which suggests that this may be typical of the species, at least at a local level, perhaps because of specific ecological characteristics. The relatively large number of private alleles recorded in all populations may be a consequence of the Wahlund effect resulting from population admixture or a process of expansion rather than the loss of rare alleles through genetic drift. Additionally, the levels of genetic variability observed in this study were higher than those reported for other species of Neotropical primates, suggesting good fitness levels in these A. belzebul populations. Regular genetic monitoring of remnant populations, especially on islands, should nevertheless be an integral component of long-term management strategies. We genotyped 15 microsatellite loci in order to evaluate the effects of habitat fragmentation, caused by flooding of the Tucuruí reservoir, on the genetic structure of  Alouatta belzebul) is found in the southeastern Amazon basin and northern Atlantic Forest  at Tucuruí on the Tocantins river resulted in the creation of a 2500 kmg forest . Howlersg forest , and curg forest , althougSamples collected from some of the animals rescued during the flooding are still available, and the primary aims of the present study were to investigate the genetic structure of the original populations they represent, using molecular techniques not available in the 1980s, and to assess the long-term effects of the flooding on the structure of present-day populations. The isolation of relatively small populations on islands may lead to increased inbreeding and random effects such as genetic drift . These pAlouatta belzebul) from the area of the Tucuruí hydroelectric reservoir  in the Brazilian state of Pará  and were supervised by a veterinarian from the National Primate Center in Belém. All procedures were conducted in compliance with federal animal care legislation. Blood samples of ~ 3 mL were collected from the femoral vein of anesthetized howlers and stored in Falcon tubes containing 300 μL of 0.02 M EDTA. Thirty blood specimens were collected from howlers on the 129-ha Germoplasma Island (LB02), close to the present-day left bank of the Tocantins, and from 22 individuals on the right bank: 15 from Base 4 (RB02) and seven from Cornélio Island RB02B; , which iA. belzebul , whereas the remaining five were specific for Callithrix jacchus . Polymerase chain reactions (PCRs) were done in a final volume of 20 μL, containing 0.5 unit of Taq DNA polymerase, 5 ng of DNA, 25 μM of each dNTP, 50 mM KCl, 10 mM Tris-HCl and 0.4 μM of each primer. Optimal conditions for the 15 primers were determined by varying the temperature of association . The size of the amplified fragments was determined using Allelinks 1.0 software.Fifteen microsatellite loci were analyzed . Ten of palliata  and Cebuociation , number Recent bottleneck events often present genetic characteristics that can be detected in microsatellite data. Typically, allelic diversity is lost more quickly than heterozygosity, and observed heterozygosity is different from that expected based on the number of alleles under mutation-drift equilibrium . Allele R), an index of the number of alleles independent of sample size, was estimated using the To characterize the genetic diversity of each population, allele frequencies, the mean number of alleles (A), observed (Ho) and expected (He) heterozygosities according to Hardy-Weinberg equilibrium, and their means, were calculated using Popgene32  in all populations , althougThe results of the Bottleneck analyses  varied cvs. RB02) to 0.2277 (LB85 vs. RB02B) (vs. RB02 (the same site in different years), all comparisons were significant (p < 0.05), and those involving RB02B generally returned the highest values, as expected from the relative configuration of the sites, i.e., LB02 was more similar to LB85 than RB85, and so on. Despite being the most differentiated population, the Rst values for RB02B were consistent with its location in time and space, with smaller values for the RB populations and the smallest for the more recent RB02.Values of pairwise Rst varied from -0.0109 (RB85 . RB02B) . With thvs. RB02 suggested that there was more differentiation within each population than between them. RB02 was nevertheless more similar to both LB populations than it was to RB02B. This almost certainly reflects the admixture resulting from the release of rescued animals at Base 4, in contrast to Cornélio Island, which has remained isolated since the flooding.Other patterns were more complex, however, in particular the relatively low values recorded for RB02. Indeed, the negative value obtained for RB85 Within-population variation accounted for > 90% of the molecular variance in all combinations of populations, except where RB02B was compared with the remaining populations . In all e.g.Leontopithecus rosalia . Despite these values, there was considerable variation among populations, which may be accounted for primarily by differences in sample size. Indeed, et al. (2004) have shown that mean allele excess may be strongly influenced by sample size and θ-values, and this seems to be the case with our data (not shown).A relatively large number of alleles was recorded per locus compared with other platyrrhines, Unfortunately, the allelic richness parameter has not been used widely in studies of platyrrhines, which reduces its value for interspecific comparisons, although it does permit more conclusive inferences on differences among the populations studied here, without the bias of sample size. A comparison of allelic richness values  indicateA. belzebul populations from Tucuruí, both before and after flooding of the reservoir. Once again, these values are also relatively high when compared with those for other microsatellite studies in platyrrhines: 0.49 (range: 0.34-0.65) in L. rosalia , 0.33 (0.09-0.65) in C. moloch  might be explained by the admixture of different local subpopulations during flooding, although it seems likely that these populations, especially those on islands, have been relatively stable since then. However, the 18 years that separate the two periods represent only two or three howler generations , which mL. rosalia in a fragmented habitat. In particular, the similarity between the left and right bank populations of A. belzebul suggests that the original course of the Tocantins river was not a very effective barrier to dispersal between the region's original populations. This might be expected, given the preference of Amazonian howlers for flooded forest ecosystems , although studies at other sites have indicated a maximum life span of much less than 10 years  may reflect the relative isolation of this island population. It is possible that formation of the island led to the isolation of a relatively differentiated local subset of the right bank population, which was not sampled in 1985, and has been reinforced by its subsequent isolation.A. belzebul show relatively ample genetic variability, with little evidence of significant bottleneck events or loss of variability in comparison with the original, pre-flooding populations. The changes observed were limited to shifts in allelic composition, presumably because of the admixture of local subpopulations during flooding. The phenotypic effects of inbreeding are not apparent at the moment, although the relatively small populations on islands are likely to be susceptible to inbreeding and genetic drift over the long term (Overall, the populations of ong term .The relatively large number of private alleles recorded in all populations  may be ae.g. Various studies (


As a representative of taurine breeds, Hanwoo cattle (n = 40) were also included in the study for reference. Among Ethiopian cattle populations, the proportion of SNPs with minor allele frequencies (MAFs) ≥0.05 ranged from 81.63% in Borana to 85.30% in Ambo, with a mean of 83.96% across all populations. The Hanwoo breed showed the highest proportion of polymorphism, with MAFs ≥0.05, accounting for 95.21% of total SNPs. The mean expected heterozygosity varied from 0.370 in Danakil to 0.410 in Hanwoo. The mean genetic differentiation  in Ethiopian cattle revealed that within individual variation accounted for approximately 99% of the total genetic variation. As expected, FST and Reynold genetic distance were greatest between Hanwoo and Ethiopian cattle populations, with average values of 17.62 and 18.50, respectively. The first and second principal components explained approximately 78.33% of the total variation and supported the clustering of the populations according to their historical origins. At K = 2 and 3, a considerable source of variation among cattle is the clustering of the populations into Hanwoo (taurine) and Ethiopian cattle populations. The low estimate of genetic differentiation (FST) among Ethiopian cattle populations indicated that differentiation among these populations is low, possibly owing to a common historical origin and high gene flow. Genetic distance, phylogenic tree, principal component analysis, and population structure analyses clearly differentiated the cattle population according to their historical origins, and confirmed that Ethiopian cattle populations are genetically distinct from the Hanwoo breed.In total, 166 individuals from five indigenous Ethiopian cattle populations – Ambo ( Bos indicus) and the Near Eastern B. taurus (B. indicus), sanga (zebu × B. taurus), zenga (sanga × zebu), and the humpless B. taurus , Borana (n = 35), Arsi (n = 30), Horro (n = 36), and Danakil (n = 38). As a representative of taurine breed, Hanwoo cattle (n = 40) were also included in the study for reference. The target populations/breeds represent the four main groups of cattle: zebu , sanga (Danakil), zenga . To minimize the sampling of related animals, herdsmen and owners of the animals were contacted. Nasal samples were collected using Performagene LIVESTOCK’s nasal swab DNA collection kit and DNA was extracted from nasal samples according to the manufacturer’s recommendations . Autosomal markers (6987) were used for SNP polymorphism distribution analysis. Markers selected for diversity analysis were required to be located on autosomal chromosomes, have call rates of ≥90%, and have minor allele frequency (MAF) ≥0.05 in all populations included in the study. After application of these selection criteria, 4235 SNPs remained and were included in the analysis.In total, 166 randomly sampled animals representing five indigenous cattle populations of Ethiopia were genotyped for 8,773 SNPs using the Illumina Bovine 8K SNP BeadChip , which wAnalyses of molecular variance (AMOVA) analyses were carried out on three datasets by using the program Arlequin . The firNm), an indirect estimate of gene flow, was calculated using the program GenAlEx 6.41  was carried out to illustrate the relationship among the populations by using Golden Helix SNP Variation Suite version 7 . PCA wasK populations (clusters), which contribute to the genotype of each individual and each is characterized by a set of allele frequencies at each marker locus. The method attempts to assign individuals to populations on the basis of their genotypes, while simultaneously estimating progenitor population allele frequencies. A Monte Carlo Markov chain method was used to estimate allele frequencies in each of the K populations and the degree of admixture for each individual animal. The number of clusters was inferred using five independent runs with 100,000 iterations and a burn-in period of 20,000 following the admixture ancestry model and correlated allele frequencies with K values ranging from two to six. We performed four independent runs for each predefined number of populations (K = 2–6).For the analysis of population structure, a Bayesian model-based analysis was performed with the most commonly used software STRUCTURE 2.3.4 . This soTable  Among Ethiopian cattle populations, the proportion of SNPs with a MAF ≥0.05 ranged from 81.63% in Borana to 85.30% in Ambo, with a mean of 83.96% across all populations. The Hanwoo breed, where MAFs ≥ 0.05 accounted for 95.21% of all SNPs, showed the highest proportion of polymorphism. The mean expected heterozygosity varied from 0.363 (0.130) in Danakil to 0.415 (0.123) in Hanwoo. Similarly, the highest and lowest observed heterozygosity was found in the same 2 breeds . The mean values of observed and expected heterozygosities in Hanwoo were generally higher than those in Ethiopian cattle populations. The majority of polymorphic SNPs were found to be in HWE, both across the whole sample and in each separate population. For Ethiopian cattle populations, only 293 (6.92%) of 4235 SNPs markers were calculated to significantly deviate from HWE (p ≤ 0.05), whereas in Hanwoo, 240 (5.67%) of the markers appeared to be in disequilibrium.Level of polymorphism and genetic variability within the different cattle populations and departures from the HWE are shown in FST), 0.6% (FIT), and -0.3% . FST value for Ethiopian cattle population was generally very low; however, significant (p < 0.001), while most of the population variance could be explained by within individual variability. Within-populations inbreeding (FIS) and total inbreeding (FIT) were not found to be significant (p > 0.05). Further analysis of the five Ethiopian cattle populations grouped by breed types showed that within-individual genetic differences accounted for 99.44% of variation, with variation among groups being insignificant, at -0.04% . When Hanwoo cattle were included in the analysis, the mean variance among groups was significant at 17.40% , while 81.51% of the total genetic variation was within populations. Grouping of Ethiopian cattle populations based on their ecological distributions (highland and lowland) revealed an estimated among-group variation of 0.42% (FCT).Analyses of molecular variance were performed to examine the partitioning of genetic variation. Analyses revealed that the five Ethiopian cattle populations had overall fixation indices of 1%  and Reynold genetic distance (0.019) were both found between Horro and Ambo populations. As expected, FST and Reynold genetic distance were the largest between Hanwoo and Ethiopian cattle populations with average values of 17.62 and 18.50, respectively. For all pairs of populations, the FST statistic indicated statistically significant differences (p < 0.01), revealing significantly differentiated populations. The mean number of migrants per generation (Nm) across all Ethiopian cattle populations was estimated at 15. The phylogenetic relationship among the six cattle populations, based on Reynolds’ distance  explaining 71.75% and 6.58%, respectively, of the total variation. On clustering of the populations according to their origin, PC1 and PC2 explained approximately 78.33% of the total variation, whereas the third principal component (PC3) accounted for approximately 6.21% of the variation and somewhat isolated the Borana cattle population from the other Ethiopian cattle. Further, PCA analysis validated the results of the population structure and dendrogram analysis.Figure 4. At K = 2 and 3, a considerable source of variation among cattle is the clustering of the cattle into Hanwoo (taurine) and Ethiopian cattle populations. At K = 2 and 3, with the exception of approximately 12% of the individual animals from the Borana population, which clustered in separate group, there was no clear differentiation among indigenous Ethiopian cattle populations and they did not cluster according to their traditional classifications or geographical distribution. Differentiation within Ethiopian populations was first observed at K = 4 breeds, with over 88% of Ambo, Arsi, and Horro (highland populations) assigned to the same cluster and 77% of the Borana and 92% of Danakil (lowland breeds) sharing the same cluster, which corresponded to the ecological distribution of these populations. Table 5 presents the proportion of the six populations belonging to each of the five clusters. At least 88% of Ambo, Arsi, and Horro cattle were assigned to cluster 4, while 71% of Danakil and 75% of Borana were in clusters 3 and 1, respectively. The results also indicate that 97% Hanwoo fall within cluster 5.A graphic representation of cluster structure analysis is depicted in B. taurus breeds. B. taurus populations have previously been reported to have higher MAFs than B. indicus breeds (FIS= 0.012) and uncontrolled mating practices that are common among the pastoral herds. In the pastoral communal system, animals have more chance to mix both while grazing and at watering points.Most SNPs identified in the Ethiopian and Hanwoo cattle populations exhibited a high degree of polymorphism. Hanwoo cattle displayed the highest, and Borana the lowest, levels of polymorphism. The level of polymorphic SNPs in this study was higher than has previously been reported for taurine breeds . Similars breeds . Genetice) of 0.25 for African cattle and 0.30 for European originated breeds, respectively, based on SNP analysis were reported  and Reynolds’ genetic distance estimates revealed close relationships among Ethiopian cattle populations. The low level of differentiation between the Ethiopian cattle population (FST= 1%) could be attributed to common ancestry, short domestication history, admixture of the population, and lack of selection pressure. The value observed in this study is in good agreement with FST values of 1.3% (FIS) value of -0.003 and total inbreeding (FIT) value of 0.006 determined in this study are higher than values reported for Ethiopian indigenous cattle breeds based on microsatellite analyses  values indicates the relative strength of gene flow and genetic drift. Genetic differentiation will result in substantial differentiation where Nm < 1 but not where Nm > 1 (Nm) was considerably higher than an earlier estimate for Ankole cattle populations  dramatically decreased beyond at the assumed K = 6. At K = 4 and 5, breeds or populations sampled from highland agro-ecology  were grouped together, while lowland breeds (Borana and Danakil) tended to cluster separately at K = 6 and showed some degree of admixture. We found that the allelic frequencies for two of the highland populations were very similar . This was further confirmed by a low FST value for these populations. The clustering of Borana and Danakil separately from other Ethiopian cattle populations could be attributed to their unique genetic compositions, geographical isolation, and ecological differences. The detected signature of admixture of the two breeds and separation from the rest of the groups could be due to the fact that, ancestrally, the two breeds share input from long horn taurus and B. indicus populations. Similarly, Danakil (sanga) is an intermediate type, formed by hybridization of the indigenous humpless cattle with zebu , might be a consequence of the peculiar admixture between the different cattle breeds. Hitherto, these populations have represented a unique genetic resource and unexploited opportunity that warrants initiatives for their sustainable conservation and utilization. The clustering of populations by ecological distribution is an insight suggesting that further investigation of the association between genetic markers and geo-environmental parameters could better enable exploitation of valuable genetic material. Apparent partition of the populations (Hanwoo and Ethiopian) according to their historical origins, and corroboration of the established details of the genetic diversity and composition of Ethiopian cattle populations, suggest that analyses using 4235 SNP markers provided sufficient genetic information to properly assess the genetic structure.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.


Leptinotarsa decemlineata) populations have experienced a rapid range expansion and intense selection by insecticides. By comparing native and invasive beetle populations, we studied the origins of organophosphate (OP) resistance-associated mutations in the acetylcholinesterase 2 (AChE2) gene, and the role of selection and demography on its genetic variability.Invasive pest species offers a unique opportunity to study the effects of genetic architecture, demography and selection on patterns of genetic variability. Invasive Colorado potato beetle , this mutation was found in 100% of Mexican, 95% of US and 71% of European beetle sequences analysed. Only two susceptible haplotypes, genetically very differentiated, were found, one in US and one in Europe. The genetic variability at the AChE2 gene among native populations suggests this mutation is the ancestral state and thus, either a pre-adaptation of the beetle for OP resistance or the AChE2 is not the major gene conferring OP resistance. The long historical association with host plant alkaloids together with recombination may have contributed to the high genetic variation at this locus. The genetic diversity in the AChE2 locus of the European beetles, in turn, strongly reflects founder effects followed by rapid invasion. Our results suggest that despite the long history of insecticide use in this species, demographic events together with pre-invasion history have been strongly influential in shaping the genetic diversity of the AChE2 gene in the invasive beetle populations.Stochastic effects during invasion explain most of the observed patterns of genetic variability at the three genes investigated. The high frequency of the S291G mutation in the  Genetic variability in a given gene is the result of a complex interplay of evolutionary forces such as selection, mutation rate and recombination, and demographic history of the populations including changes in population size, migration and divergence. The evoIn addition to selection by insecticides, populations of invasive pest species are often affected by demographic events. For instance, genetic variation is often reduced by population bottlenecks and allele frequencies can be drastically changed by founder events. SubsequLeptinotarsa decemlineata), an invasive insect pest of potato, is well known for its ability to evolve resistance to insecticides)cticides and speccticides-26. Howecticides). By exaAChE genes . For primer sequences, PCR mixture and thermal cycle programs, see Additional fileAChE2, DP1 and JHE genes, we were able to obtain sequence data from 98, 91 and 69 individuals of the 100 individuals included in the study, respectively. As for JHE, we obtained the JHE-b form of the gene, a shorrding to. BecauseAChE2) and 17 (DP1) individuals, in which the haplotypes could not be inferred reliably by PHASE, were cloned to determine the correct phase of the variable sites. The PCR mix and cycles were as described in Additional fileAChE2 had no M13 tails. The PCR products were purified according to the manufacturer’s instructions using the Qiaquick® PCR purification Kit (QIAGEN) (for AChE2) or GeneJET Gel Extraction Kit (Fermentas) (for DP1). The ligation and transformation were performed according to the manufacturer’s instructions using Qiagen PCR Cloning Kit (QIAGEN) and Qiagen EZ competent cells (for AChE2) or CloneJET PCR Cloning Kit (Fermentas) with NovaBlue Singles cells (Novagen) (for DP1). The PCR mixes used to amplify the cloned inserts were as described in Additional fileAChE2 and pJET1.2 for DP1. The PCR cycle programs to amplify cloned inserts are described in Additional fileHaplotypes were inferred from genotypic data using the program PHASE 2.1.1 which isW). The mekey.org,. Haplotykey.org,. The parkey.org,] run frokey.org,.ΦST values for each locus were performed with ARLEQUIN (number of permutations 10000). To test whether genetic distance correlated with geographical distance among European populations, and whether population pair-wise ΦST values correlated among populations with genes, a Mantel test was conducted with the program FSTAT 2.9.3[ΦST and Reynold’s distance values for European populations were obtained with ARLEQUIN.We performed a hierarchical analysis of molecular variance (AMOVA) computedAT 2.9.3 using 10Tests of selective neutrality were analysed with the program DNASP to assess whether the observed frequency distribution of nucleotide polymorphism departs from neutral expectations. Neutrality tests performed included Tajima’s D, Fu and The authors declare that they have no competing interests.SP, LL, AL, JM and AG designed the study. LL, JM, YHC, VI and AG collected the samples. SP and AG carried out the experimental work, performed the analyses and drafted the manuscript. LL, AL, JM, YHC and VI participated in the draft of the manuscript. All authors contributed and approved to the final version of the manuscript.Summary statistics for the Colorado potato beetle populations pooled according to geographical regions.Click here for fileFrequencies (%) of observed patterns of non-synonymous mutations of AChE2 variants in North American and European Colorado potato beetle populations.Click here for fileGraphical presentation of genetic diversity indices  and neutrality tests  for Mexican, US, and European Colorado potato beetle populations.Click here for filePairwise genetic differentiation(ΦST) for (a) DP1 and (b) JHE-b loci between European and North American Colorado potato beetle populations.Click here for fileMethodology used in the amplification of AChE2, DP1 and JHE-b genes.Click here for file


Araucaria angustifolia in comparison with populations in the main area of the species continuous natural distribution (southern Brazil), was tested. The 673 AFLPs markers revealed a high level of genetic diversity for the species (Ht = 0.27), despite anthropogenic influence throughout the last century, and a decrease of H in isolated populations of southeastern Brazil (H = 0.16), thereby indicating the tendency for higher genetic diversity in remnant populations of continuous forests in southern Brazil, when compared to natural isolated populations in the southeastern region. A strong differentiation among southern and southeastern populations was detected (AMOVA variance ranged from 10%-15%). From Bayesian analysis, it is suggested that the nine populations tested form five “genetic clusters” (K = 5). Five of these populations, located in the northernmost limit of distribution of the species, represent three “genetic clusters”. These results are in agreement with the pattern of geographic distribution of the studied populations.Habitat fragmentation and a decrease in population size may lead to a loss in population genetic diversity. For the first time, the reduction in genetic diversity in the northernmost limit of natural occurence (southeastern Brazil) of  Deforestation is one of the main factors leading to forest fragmentation, or in other words, the complete transformation of a large area from a continuum of habitats to a large number of small patches of vegetation isolated one from the other . It is iThe fragmentation of forests into small patches is responsible for the diminishing habitat-area .Araucaria angustifolia (Bertol.) Kuntze, also known as the Brazilian pine, is a dioecious wind-pollinated species whose seeds are dispersed mainly by authocory. A. angustifolia is one of the most important trees in its natural range of distribution, due to its economical, social and ecological relevance. As a result of the high quality of its timber, the wood is used for construction in general, furniture making and the production of long-fibre cellulose . Studies based on the “Bioclim” algorithm , between latitudes 24° and 30° S. The species is also sparsely spread throughout other states in Brazil, such as Minas Gerais, São Paulo and Rio de Janeiro, as isolated, relict populations, between latitudes 18 and 24° S and at higher altitudes (1200 m). It also occurs as a small extant population in the Province of Missiones, in Argentine  Figure . Howeverlgorithm , as mentA. angustifolia in southeastern Brazil. The main goal of the present study was to evaluate the reduction in genetic diversity of five A. angustifolia populations in southeastern Brazil, when compared with other populations from the south (the descendants of continuous forests).It is possible that the depletion of wide areas of araucaria forests may have led to a decrease in genetic diversity, to the point of interfering in its use for conservation and exploitation of its genetic resources. At present, a large number of approaches have been undertaken by using various markers, all of which point to the fact that, notwithstanding araucaria forests having undergone drastic reduction in areas of natural distribution, a considerable level of genetic diversity has still been maintained  and Mangueirinha, in the state of Paraná (PR) - and individuals from five populations in southeastern Brazil - Itatiaia, Rio de Janeiro (RJ-1), Teresópolis, Rio de Janeiro  and Liberdade, in the state of Minas Gerais (MG) - were sampled, comprising a total of 190 individuals . A 3100 Genetic Analyzer ABI Prism 377 (Applied Biosystem®) automatic sequencer was used to obtain the electropherograms. Fluorescent AFLP patterns were scored with a Gene Mapper 3.5 (Applied Biosystem©). The AFLP profiles of each individual were scored for band presence (1) or absence (0) to create binary matrices. Population allele frequency data were estimated from AFLPs loci, assuming Hardy-Weinberg equilibrium as described by Needles and cambium were extracted according to the CTAB method  with modThe percentage of polymorphic loci estimates were calculated for each primer-enzyme combination (PEC) and for the six PECs together, based on 99% criteria, and using Population Genetic Analysis (TFPGA) software .Ht) and the number of polymorphic loci (S) at 5% level were assessed using Arlequin  individually, and subsequently, due to the small sample size, we analyzed the three as one. Gene diversity for each population, total heterozygosity , as described by Genetic variation within and among groups and populations was tested by a non-hierarchical analysis of molecular variance AMOVA,  and the K). Analyses were carried out based on non-admixture and allele frequency correlated models, with 50.000 Markov Chain Monte Carlo (MCMC) steps and 10.000 burn-in periods. All the possible models for A. angustifolia from K = 2 to K = 12 were tried, and twelve replicates were run for each K. The most favourable K was chosen according to that suggested by Bayesian analysis with Structure Software  and the lowest with PEC EcoRI-CT/MseI-GACC in RJ-2 (30.92%). On following the same pattern, the percentage of polymorphic loci for all six primer-enzyme combinations, when considered together, yielded higher values in the southern populations and lower ones in southeastern populations RJ-2, RJ-3 and RJ-4 , together with the MG population, presented the lowest estimates of gene diversity (0.15-0.17). The total set of nine populations used in this analysis yielded a value for total heterozygozity of 0.27. When 26 individual were considered per population, the “frequency-down-weighted marker values” (DW) ranged from 82.54 (RS-3) to 97.51 (RJ#) . On considering 10 randomly chosen individuals per population, DW ranged from 67.31 in population RS-2 to 84.00 in population RJ#  was indicated from Bayesian analysis The average non-admixture proportion for each population of A. angustifolia among the five different clusters found by means of this analysis appears in According to the approach suggested by A. angustifolia were investigated in order to define to what extent forest fragmentation and natural isolation can interfere in genetic diversity. As a result, accumulated data indicate higher genetic diversity in remnant populations of A. angustifolia in continuous forests in southern Brazil than in, isolated, relict populations in the southeast  indicate warmer but still dry weather until around 2600 C14 yr BP. Under these conditions, A. angustifolia possibly survived in humid refugia. This dry period was followed by a more appropriate climate, suitable for the expansion of A. angustifolia to the highlands  it is supposed that araucaria forests were restricted to protected valleys and wetter coastal slopes, besides bordering rivers and forming gallery forests  and thus are protected by Brazilian law, but there are still other relict populations left that need to be further studied and protected.Genetic drift and inbreeding may influence small isolated plant populations by changing patterns of gene diversity and fitness. Both effects have implications for conservation. It is known that the loss of genetic variation may decrease the potential to persist in the face of biotic and abiotic environmental changes . It may


Littorina saxatilis , exhibits extreme morphological variation between and within geographic regions and has become a model for studies of local adaptation; yet a comprehensive analysis of the species' phylogeography is lacking. Here, we examine phylogeographic patterns of the species' populations in the North Atlantic and one remote Mediterranean population using sequence variation in a fragment of the mitochondrial cytochrome b gene (607 bp). We found that, as opposed to many other rocky intertidal species, L. saxatilis has likely had a long and continuous history in the Northwest Atlantic, including survival during the last glacial maximum (LGM), possibly in two refugia. In the Northeast Atlantic, several areas likely harboured refugial populations that recolonized different parts of this region after glacial retreat, resulting in strong population structure. However, the outlying monomorphic Venetian population is likely a recent anthropogenic introduction from northern Europe and not a remnant of an earlier wider distribution in the Mediterranean Sea. Overall, our detailed phylogeography of L. saxatilis adds an important piece to the understanding of Pleistocene history in North Atlantic marine biota as well as being the first study to describe the species' evolutionary history in its natural range. The latter contribution is noteworthy because the snail has recently become an important model species for understanding evolutionary processes of speciation; thus our work provides integral information for such endeavours.The North Atlantic intertidal gastropod,  Recent biogeographic histories of arctic and north-temperate species demonstrate a strong influence of climate variation during glacial periods of the Pleistocene Epoch In the Northwest Atlantic (NWA), the southern margin of the ice sheet was near Long Island during the LGM Littorina saxatilis is highly abundant and widely distributed across the North Atlantic, thus presenting a good model species to explore phylogeographic patterns in the region. Littorina saxatilis exists in a myriad of habitats, including exposed cliffs, rocky shore boulders and macroalgae, as well as salt marshes and mudflats, and is naturally distributed in populations throughout the North Atlantic: from the Barents Sea to Portugal in NEA, North Atlantic islands , and from Baffin Island to Delaware Bay in NWA L. saxatilis lived possibly as far south as Gibraltar and the coast of Morocco; alternatively, they are the result of more recent introductions The rough periwinkle snail L. saxatilis exhibits extreme variation in shell morphology, and distinct ecotypes can be found in many locations L. saxatilis possesses an ovoviviparous reproductive strategy , and adult snails have limited home-range dispersal  L. saxatilis survived the LGM in refugia in the NWA vs. extinction and later re-colonization from Europe; (2) phylogenetic connections in present-day NEA populations originate from one common refugium vs. colonisations from several refugia; and (3) the Venetian population (one of the two cryptogenic populations in the Mediterranean) is a relict population vs. a recent anthropogenic introduction. To determine the relationship of our model organism among other North Atlantic rocky intertidal species with broad distributions, we also compared our results for L. saxatilis with existing phylogeographic evidence of other North Atlantic species.In the present study, we reconstruct the phylogeography of We obtained 34 samples covering most of the species' natural range, including a cryptogenic Mediterranean sample from Venice, Italy .Φsc = 0.07, p = 0.07); therefore, we pooled ecotype data for all further analyses. Detailed analysis of genetic variation between sympatric ecotypes of L. saxatilis is the focus of another on-going study and will not be discussed further here. Furthermore, in four areas , L. saxatilis lives in sympatry with two closely related and morphologically similar species, L. compressa and L. arcana. In these areas, we only sampled mature, brooding females, ensuring accurate identification since L. saxatilis is the only ovoviviparous species in its genus.In three of our populations  and at Macrogen Inc. .DNA was extracted from the snail's foot using a BioSprint 96 DNA Blood kit  or a standard CTAB method. We amplified a 669 bp fragment of the mitochondrial cytochrome Sequencing was performed in both forward and reverse directions and sequences were assembled and manually inspected for ambiguities in Geneious 4.8.4 and Sequencher 4.8. All sequences were trimmed to a final length of 607 bp. Sequences were aligned without gaps using the ClustalW algorithm b sequence from L. fabalis  divergence time between L. fabalis and the “saxatilis” species complex, including L. saxatilis, L. compressa and L. arcana, that occurred between 4–3.5 Ma (the colonization of the North Atlantic by the common Pacific ancestor after the opening of the Bering strait) and 2 Ma . The constructed priors were normally distributed with mean = 3 Ma and SD = 0.5 Ma (so that 95% of prior distribution lays between 2 and 4 Ma) for the first calibration point and log-normally distributed with mean = 0.5 Ma and SD = 0.5 Ma for the second calibration point .For haplotype clades, identified in the phylogenetic analyses, times to most recent common ancestor (TMRCA) were obtained by the Bayesian Markov chain Monte Carlo method, implemented in BEAST v.1.5.2 ariation . Our datWe implemented the same model of sequence evolution as in the Bayesian phylogenetic analyses and a relaxed molecular clock with uncorrelated rates among lineages. To choose the coalescence model with the best fit to the data, we tested four possible models: constant population size, exponential growth, population expansion, and Bayesian skyline. We then compared model likelihoods by calculating Bayes Factors . Marginal likelihoods were estimated as harmonic means of the sampled likelihoods in Tracer v. 1.5 Littorina in the cytochrome b gene has been estimated to be 7.6% per site per Ma In addition, we used the molecular clock to calculate the mean age of multiple tip haplotypes, which form star-like phylogenies in the tree. These haplotypes were separated from their central haplotype by one transition in the third codon position; the rate of this class of mutations for the genus h and nucleotide diversity π were estimated using Arlequin 3.01 ΦST), and tested significance of differentiation in Arlequin. Hierarchical analysis of molecular variance (AMOVA) was used to estimate variation among two (“NWA” and “NEA+ISL”) or three regions .Haplotype diversity a priori division of populations into regions, we explored spatial relationships among populations using spatial analysis of molecular variance (SAMOVA) in SAMOVA 1.0 a-priori divisions. Based on Φ-statistics and a simulated annealing algorithm, this method divides all populations into k groups, maximizing variation among groups and minimizing variation among populations within groups. We tested divisions from 2 to 15 groups and ran 100 initial conditions to ensure that the algorithm converged. For population divisions found at each k level, the significance of the Φ-statistic between groups was assessed by non-parametric randomization tests using 10 000 permutations. The grouping of populations explaining the largest proportion of total variation was chosen by examining the pattern of changes in Φ-statistics at different k levels.In addition to a priori and identified SAMOVA groups were tested by calculating the Mantel r correlation between the linearized ΦST values = ΦST/(1−ΦST) and log-transformed geographic distances between samples ; significance was assessed in Arlequin.Isolation-by-distance patterns in population structure for both our rP-values indicate deviation of the data from the expansion model). In addition, Tajima's and Fu's neutrality tests were conducted in Arlequin to provide support for possible population expansion, detected by the mismatch analysis. In the absence of selection, significant negative values of Tajima's D and Fu's F statistics indicate historical population growth. For the population groups, for which a sudden expansion could not be rejected, time of the start of expansion (t in generations) was calculated from the expansion parameter τ using the formula t = τ/2u, where u = 2μk . Based on mutation rates of 2 and 4% per site per Ma u is 24.3–48.6 Ma; t in generations was converted to time in years (T) using generation time estimates of 0.5–1 year In populations that have recently undergone rapid expansion, the distribution of pairwise genetic differences between sequences (mismatch distribution) is expected to be unimodal 1, θ2, and ancestral θA), and migration rates (m1/μ and m2/μ) between two populations assumed to have shared a common ancestor. We performed 2–3 replicate runs of each comparison, which included 30 chains of at least 2 million steps per chain after an initial burn-in period of 100 000 steps; we ended runs when ESS (effective sample size) values were >50 and posterior density parameter curves were stable. Divergence estimates were rescaled to time since separation in years using substitution rates of 2 and 4% per site per Ma as was discussed earlier for LittorinaPopulation divergence estimates were performed using the Isolation with Migration-analytical (IMa) program . Most substitutions (52) were silent and only six were non-synonymous. We found a total of 73 haplotypes . This model was subsequently used in Bayesian tree reconstructions. In the Bayesian consensus tree, rooted with an L. fabalis sequence, haplotype 26, representing a likely introgression from L. arcana, had a basal position in respect to other L. saxatilis haplotypes  and the mean TMRCA for L. saxatilis and L. arcana was within the range of initial calibration intervals from fossil and allozyme divergence  ranged from 50 to 95 ka; however, all 95% HPD intervals include possibilities for post-glacial expansion of 13–17 kya (The Bayesian skyline model (BSL), which allows several changes of population size through the species' history, had decisively stronger support than the constant population size model (BF = 613295) and the exponential growth model (BF = 10967), and it had substantially stronger support than the population expansion model  were dated even closer to the LGM: with a mean of 29–34 ka (1–76 ka) from the coalescent estimation in BEAST; when based on the mutation rate of 7.6% per site per Ma  or within NEA, excluding the Mediterranean monomorphic sample . Private haplotypes  were found in many populations from all clades on the gene tree  between populations of L. saxatilis, and 458 of 561 total pair-wise comparisons were statistically significant after sequential Bonferroni correction  corresponding roughly to their geographical location along the coastline, northeast to southwest. This also coincided with the relative frequencies we observed in mitochondrial clades A and B: the former being more common in the south and decreasing in frequency north with the latter showing the opposite cline . InteresThe NEA and ISL populations were divided into four groups IV–VII, . Group IP>0.05) except for SAMOVA group IV. Interestingly, this one group showed significant correlation between pairwise genetic differentiation and geographic distances  and included populations from Newfoundland, Iceland, Faeroes, Ireland-2 and three of the most northern NEA populations ; θNWA = 45 (HPD 26–66)] and migration rates, estimated as m/μ, between the two regions were low, with no significant difference in directionality (mNEA→NWA = 0.22 (HPD 0.01–0.41) versus mNWA→NEA = 0.14 (HPD 0.01–0.27)]. Mean divergence estimates (t)  ranged from 91–182 (HPD 24–320) kya. Comparisons of NEA+ISL versus NWA regions for all the parameters produced results very similar to those above (IMa results (with 90% highest posterior density (HPD) intervals) for various regional and subregional comparisons are summarized in se above .NEA(S)→ISL = 3.66 (HPD 0.01–7.74); mISL→NWA(S) = 3.29 (HPD 0.01–6.67)] were an order of magnitude higher than migration rates between NWA and NEA (shown above). However, there was no significant directionality in migration rates between ISL and any of the compared regions. Divergence times between ISL and the compared regions were significantly lower  than for NEA-NWA and included the post-glacial time period , NWA(S)], demonstrated an effective population size for ISL that was significantly lower than all compared regions. In addition, migration rates between ISL and NEA, NEA(S) and NWA(S) ; however, migration rates from both North American regions to Europe were relatively low (mCAN→NEA(MAIN) = 0.16 (HPD 0.01–0.37); mUSSOUTH→NEA(MAIN) = 0.13 (HPD 0.01–0.28)].Third, comparisons between mainland Europe [NEA(MAIN)] and two groups of NWA populations—a) Maritime Canada (CAN) and b) Southern United States (USSOUTH)—revealed significantly higher migration from NEA(MAIN) to CAN than NEA(MAIN) to USSOUTH . Migration was significantly higher from NEA(MAIN) to BI than from BI to NEA(MAIN) [mNEA(MAIN)→BI = 1.91 (HPD 0.17–3.53) versus mBI→NEA(MAIN) = 0.74 (HPD 0.01–1.64)], and generally low between NEA(MAIN) and SPAIN [(mNEA(MAIN)→SPAIN = 0.28 (HPD 0.01–0.63) versus mSPAIN→NEA(MAIN) = 0.10 (HPD 0.01–0.23)]. Estimated divergence time for NEA(MAIN)-BI was also significantly lower than NEA(MAIN)-SPAIN [tNEA(MAIN)-BI = 26–51 (HPD 7–231) kya versus tNEA(MAIN)-SPAIN = 46–92 (HPD 15–498) kya]  and the British Isles (BI) and Spanish (SPAIN) populations revealed an effective population size for NEA(MAIN) that was significantly higher than SPAIN but significantly lower than BI [θ98) kya] .VENICE = 0.10–0.21 (HPD 0.02–1.57)], which was also significantly lower than both European groupings. We found high migration rates (the rates between EUROPE(S)-VENICE being in fact higher than for all other comparisons) and no significant directionality in migration. Divergence estimates were significantly lower for EUROPE(S)-VENICE than NEA+ISL-VENICE [tEUROPE(S)-VENICE = 1–3 (HPD 0.62–5) ka versus tNEA(ISL)-VENICE = 3–5 (HPD 0.40–10) ka] as well as all other comparisons, notably NEA(MAIN)-BI and NEA(MAIN)-SPAIN  and two European groupings—a) NEA+ISL and b) European populations grouped with VENICE in SAMOVA [EUROPE(S)]—showed a very low effective population size for the Venice population [θN)-SPAIN .Our extensive dataset of 778 mitochondrial sequences from numerous populations on both sides of the Atlantic as well as 3 North Atlantic islands revealed considerable genetic structure across all regions. In addition, genetic diversity was high on both sides of the Atlantic, signifying  long-term divergence between the two coasts. Notably, the few connections we did observe between the two regions were across North Atlantic islands. Below, we discuss these results in detail, focusing on the two regions and their connections as well as evidence for the existence of glacial refugia in each region.L. saxatilis were comprised of two mitochondrial lineages, A and B, with divergence times (estimated as TMRCA for the clades) of 110–640 ka, of which lineage B appears to be the oldest of all L. saxatilis mitochondrial lineages and, in our dataset, is found just in North America. Both the A and B lineages possess multiple alleles, forming star-shaped phylogenies (b mutation rate (33 ka) and mismatch distribution analyses  was shared with NEA populations. Moreover, the level of NWA diversity was comparable to NEA (32 versus 35 endemic haplotypes). In addition, coalescent estimates of effective population sizes were not lower for NWA than for NEA , and meaL. saxatilis, especially since the frequency of clade B is highest in northeast NWA populations  is deeply diverged from all alleles found in NEA while the other (A) is closely related to European alleles, strongly suggesting that the dispersal of these lineages across the Atlantic took place at different time periods. At present, the frequencies of these lineages in NWA populations appear to show a strong latitudinal gradient: the A lineage is more frequent in southern populations while the B lineage is more frequent in northern populations . This result may potentially reflect survival of the lineages in two different NWA refugia, one northern and one southern, followed by a post-glacial expansion along the North American coast. The oldest clade B may have evolved within NWA or it could have been present in NEA in the past. As suggested for other marine intertidal species ulations .L. saxatilis in the NWA, which could have been within the southern-most area of the species' present range where the frequency of lineage A is highest , and also where haplotype 8, which is basal for clade A and dated to 40–420 ka, is located . Even though habitat south of Long Island lack rocky shores, these areas may still have served as possible refugial locations for intertidal species able to utilize soft sediment habitats L. saxatilis is also known to live in mud-flat and salt-marsh habitats throughout its range Clade A, on the other hand, is shared between the eastern and western Atlantic, and this suggests a dispersal event (probably in the Late Pleistocene) for the species across the Atlantic. However, even though western and eastern haplotypes in this clade are phylogenetically closely related, most of the NWA haplotypes are neither shared nor descended from European haplotypes , as woulL. saxatilis across the North Atlantic via Atlantic islands. Likewise, these data corroborate earlier evidence showing connections across northern North Atlantic populations in parasite diversity analyses of L. saxatilisWhile the majority of haplotypes in clade A are predominantly found in the US South, it is notable that two subclades (A1 and A2) include haplotypes present in the northern NWA, specifically in Atlantic Canadian populations at Newfoundland and Cape Breton Island  and mean divergence estimates between Venice and NEA collectively, as well as a subset of NEA grouped together with Venice in SAMOVA see , ranged L. saxatilis had high nucleotide diversity and private haplotypes, making identification of possible refugia challenging. This suggests that after glacial melt, L. saxatilis re-colonized habitats very rapidly - in fact, our results show that virtually all populations of the snail underwent rapid increases in population size after the LGM. Thus, despite its lack of pelagic larvae, L. saxatilis is actually a very effective coloniser, supporting the hypothesis that establishment of new populations in remote habitats may be facilitated for those species dispersing via egg clutches or brooding females rather than by pelagic larvae The most characteristic signature of glacial refugia is high genetic diversity and the presence of private haplotypes/alleles, in contrast to recently colonized areas that have lower diversity and/or only common haplotypes L. saxatilis has been hypothesized to occur via rafting or birds L. saxatilisL. saxatilis that we observed , since only older, interior haplotypes were found to be shared among populations. Their derived tip-haplotypes were population-specific, indicating that migration occurred some time in the past and pre-dated the species' population expansion. Even so, we did find one disjunct connection (haplotype 69) between a US population in the lower part of the species' range  with Limerick, Ireland in Europe, which appeared unusual given that that all the other instances of haplotypes shared between NWA and NEA (n = 5) were found in NWA populations from Maritime Canada. Thus, haplotype 69 could potentially represent an unnatural connection via anthropogenic transport; however, further investigations are required to be certain.Natural long-distance dispersal in Littorina saxatilis appears to have existed in the Northwest Atlantic for a very long time, and according to our dating analyses, its presence in the Northwest Atlantic could have been for as long a period of time as in the Northeast Atlantic. The snail also appears to have had two major dispersal events in the Northwest Atlantic: 1) early in the species' North Atlantic history and 2) later in its history, possibly during the Eemian interglacial. Glacial refugia for the species along the North American coast were likely located in Maritime Canada and possibly close to the southern edge of the species' present-day distribution. In the Northeast Atlantic, our data suggest the existence of several refugia that were probably located on the Galician coast, Iceland/Faeroes, and possibly southwest Ireland and the English Channel. We also determined that the Venetian population in the Mediterranean Sea is the result of a recent, likely anthropogenic introduction. Post-glacial origin from different refugial populations appears to be a key factor in the large-scale population structure of L saxatilis, with little gene flow between the regions at present, except for the noteworthy stepping-stone connection we observed across northern populations in NEA and NWA and North Atlantic islands. Overall, our detailed phylogeography of L. saxatilis adds an important piece to the understanding of Pleistocene history in North Atlantic marine biota as well as to resolving possible locations within the North Atlantic for glacial refugia during the LGM. Finally, our investigation provides essential information for studies of adaptive variation and the origin of ecotype populations of L. saxatilis.Figure S1Rarefaction analyses of mitochondrial haplotype diversity with sampling effort in Littorina saxatilis.(DOC)Click here for additional data file.Table S1Sampling localities, coordinates, sample size and measures of genetic diversity within sampled populations of Littorina saxatilis.(DOC)Click here for additional data file.Table S2Summary of polymorphic sites and unique haplotypes in the cytochrome b fragment in Littorina saxatilis.(DOC)Click here for additional data file.Table S3Cytochrome-b haplotype accession numbers and their occurrences in sampled populations of Littorina saxatilis.(DOC)Click here for additional data file.Table S4Pairwise ΦST and tests for significance of differentiation between populations of Littorina saxatilis.(XLS)Click here for additional data file.Table S5Probability calculations of pairwise comparisons for IMa parameter estimates.(XLS)Click here for additional data file.


Environmental gradients caused by altitudinal gradients may affect genetic variation within and among plant populations and inbreeding within populations. Populations in the upper range periphery of a species may be important source populations for range shifts to higher altitude in response to climate change. In this study we investigate patterns of population genetic variation at upper peripheral and lower more central altitudes in three common plant species of semi-dry grasslands in montane landscapes.Briza media, Trifolium montanum and Ranunculus bulbosus genetic diversity, inbreeding and genetic relatedness of individuals within populations and genetic differentiation among populations was characterized using AFLP markers. Populations were sampled in the Swiss Alps at 1800 (upper periphery of the study organisms) and at 1200 m a.s.l. Genetic diversity was not affected by altitude and only in B. media inbreeding was greater at higher altitudes. Genetic differentiation was slightly greater among populations at higher altitudes in B. media and individuals within populations were more related to each other compared to individuals in lower altitude populations. A similar but less strong pattern of differentiation and relatedness was observed in T. montanum, while in R. bulbosus there was no effect of altitude. Estimations of population size and isolation of populations were similar, both at higher and lower altitudes.In Our results suggest that altitude does not affect genetic diversity in the grassland species under study. Genetic differentiation of populations increased only slightly at higher elevation, probably due to extensive (historic) gene flow among altitudes. Potentially pre-adapted genes might therefore spread easily across altitudes. Our study indicates that populations at the upper periphery are not genetically depauperate or isolated and thus may be important source populations for migration under climate change. Altitudinal gradients comprise an assemblage of environmental, especially climatic, variables which influence the distribution of plant species Ne) in upper peripheral populations compared with lower altitude populations closer to the range centre, a so-called Abundant Centre distribution The potential for altitudinal climatic gradients to influence the distribution of genetic variation within and among plant populations is highly complex and variable among species While population size is a major determinant of genetic variation in populations with Abundant Centre distribution, similar genetic patterns might arise independent of population size. For example, upper peripheral populations could show reduced genetic diversity, increased inbreeding and genetic differentiation compared to lower more central populations, because of limited pollen or seed mediated gene flow from higher altitudes Species characteristics, especially the pollen dispersal mode, influence patterns of gene flow and consequently patterns of genetic variation in alpine environments The species rich semi-dry grassland is an important and relatively common habitat type along altitudinal gradients in the European Alps At montane and submontane altitudes, semi-dry grassland patches are often separated by areas of forest which protect the area below them from avalanches, landslides and rock falls . These protection forests have increased in size over recent decades Briza media and the two insect-pollinated herbs Trifolium montanum and Ranunculus bulbosus. Anthropogenic land cover change may undermine putative Abundant Centre demographic patterns, as outlined above. Therefore, our expectation was that population size does not follow an Abundant Centre distribution pattern along our studied altitudinal gradients. To test this expectation we estimated population size and geographic isolation.In this paper we investigate differential patterns of genetic diversity, inbreeding and population genetic structure in three common plant species of semi-dry grasslands in population pairs at 1200 and 1800 m a.s.l. in the Swiss Alps using the amplified fragment length polymorphism (AFLP) technique. Our study species were the wind-pollinated grass Specifically, we investigated patterns of population genetic diversity in the context of the altitudinal gradient and pollen dispersal mode using the following hypotheses as a heuristic framework: (i) Neutral genetic diversity is lower and inbreeding is higher in upper peripheral populations compared with populations at lower elevations. (ii) Upper peripheral populations are more differentiated and have increased individual relatedness within populations compared to populations at lower elevation. (iii) Altitude has a smaller effect on the above mentioned population genetic traits in the wind-pollinated species compared to the insect-pollinated species.P- values (PBonf) of >0.05; estimates in B. media which tended to have larger inhabited semi-dry grassland patches within 400 m radius at 1800 compared with 1200 m a.s.l. (PBonf = 0.081). Average July temperatures were, for all populations, appr. 4°C lower at 1800 than at 1200 m a.s.l.Estimators for population size  and distance to nearest neighbouring habitat patch did not differ among the two altitudinal levels in pair-wise Wilcoxon tests (within species Bonferroni corrected He), percentage of polymorphic loci (PPL) and Bayesian cluster diversity (BCD) did not differ between low (1200 m a.s.l.) and high altitude populations (1800 m a.s.l.) in all three species  did not differ among altitudes in T. montanum and R. bulbosus (P>0.130), while in B. media high altitude populations showed larger fAFLP-values compared to low altitude populations (P = 0.020). This difference became marginally significant (P = 0.064) after setting the fAFLP -values with low confidence to “0” (i.e. in 13 out of 20 cases the estimates did not significantly differ from 0).The three genetic diversity measures, expected heterozygosity  were 0.099 (±0.004), 0.118 (±0.006) and 0.071 (±0.003) for B. media, T. montanum and R. bulbosus, respectively. All pairwise FST.overall values differed significantly from zero except the “Prae12” and “Prae18” pair in T. montanum. We evaluated four aspects of population differentiation at the individual and population level, using frequentistic and Bayesian statistics: (i) A Mantel test indicated isolation by distance relationships (P<0.001) with Mantel-r of 0.36, 0.50 and 0.55 in B. media, T. montanum and R. bulbosus. (ii) A hierarchical AMOVA showed that in B. media, T. montanum and R. bulbosus 8.1%, 12.0% and 7.7% of the variation, respectively, was explained by differences among locations , while 5.9%, 5.4% and 4.3%, respectively, was explained by differences within location . The rest was explained by differences within populations (>83%). (iii) A Principal Coordinates Analyses (PCoA) based on Nei’s standard genetic distances separated distinct grouping concurrent with the geographic distribution of the populations  and to the populations within the same altitude  was calculated. In B. media high altitude populations were more differentiated than low altitude populations . This was similar in T. montanum, however overall differentiation was only marginal . In R. bulbosus genetic differentiation was equal between altitudes   was similar to the estimates among high as well as among low altitude populations. In R. bulbosus FST estimates among high and low altitude populations within the same location differed from the other three groups of pair-wise FST-values.Genetic differentiation of high and low altitude populations was summarized in two ways. First, differentiation of a given population to all other populations (i.e. ctively) . Second,ctively) . Based oB. media (P = 0.004). In T. montanum rc tended to be larger at high altitude (P = 0.064) and there was no difference among altitudes in R. bulbosus  was greater in high compared to low altitude populations in km2) than between 1100 and 1300 m a.s.l. (31km2) in Switzerland Lavandula latifolia declined from low central to high peripheral altitudes, following an Abundant Centre pattern Our population size estimates suggest that populations at high altitudes, which are in the upper range periphery, are of similar size and have similar plant densities as those at lower altitudes closer to the range centre. The total area of semi-dry grassland between 1700 and 1900 m a.s.l. is larger  between the two altitudes.He-values in our study were in the medium range found in AFLP studies of herbal species Stipa capillataVaccinium stamineumOne interpretation of the absence of any altitudinal effect on genetic diversity is that there is no Abundant Centre effect. This could be explained by anthropogenic drivers of habitat distribution and patch sizes such as reforestation and agricultural intensification. These processes can have a major impact on population sizes and thus intraspecific genetic diversity in pre-alpine grasslands, potentially counteracting effects of natural gradients on population genetic diversity. The similarly large population sizes at both altitudes appear to contain relatively large gene pools. Indeed, the Campanula rotundifoliaArabis serrataThe rate of gene flow among populations at different altitudes could mitigate the potential effects of altitude on genetic diversity. Particularly, pollen flow B. media high altitude populations were slightly more inbred compared to low populations. This is somewhat counterintuitive, since one would expect that increased inbreeding leads to a reduction in genetic diversity T. montanum and R. bulbosus inbreeding did not differ among altitudes, which is similar to findings for the alpine grass Poa hiemataRutidosis leiolepisT. montanum, as indicated by its overall correlation with the average relatedness per population. Populations with higher individual densities tended to be more inbred (P = 0.061). The limited seed dispersal by gravity may lead to the aggregation of related individuals at short distances. This coupled with frequent pollen dispersal over short distances could increase inbreeding values.In B. media and T. montanum. Likewise, relatedness within populations was increased at higher elevation in these two species. Furthermore the two high altitude populations “vaet_18” and “chat_18” in T. montanum nearly exclusively consisted of individuals belonging to one of two rare Bayesian clusters each , Trifolium montanum (Fabaceae) and Ranunculus bulbosus (Ranunculaceae) are mainly found in Mesobromion erecti grasslands below timber line and rely on continuous management Briza media as well tolerates more moist and acidic situations T. montanum also occurs at forest edges R. bulbosus . All species are predominantly outcrossing Briza media is wind-pollinated, but also reproduces clonally R. bulbosus is pollinated by generalist insects T. montanum is predominantly pollinated by bumblebees and bees B. media, T. montanum and R. bulbosus 1194±15 and 1781±13 m a.s.l.; 1190±13 and 1786±15 m a.s.l.; 1196±22 and 1761±20 m a.s.l respectively. For B. media and T. montanum these paired samples were collected from ten locations and for R. bulbosus from nine locations   and sampled material immediately dried with silica gel.The Swiss inventory of semi-dry and dry grassland (referred to as semi-dry grassland throughout the manuscript) areas of national conservational importance ions see . We ende . In B. m25m2), 2 (appr. 3–15 per25m2) and 3 (appr. >15 per25m2). Second, area of inhabited grassland patch was calculated for each population (Arc GIS 9.3.1) relying on a GIS vector model of dry grassland patches based on the TWW-inventory We characterized size and isolation of populations by estimating plant density as well as area and spatial isolation of inhabited semi-dry grassland patches. First, we estimated densities of flowering plants in the field at three density levels: 1 (appr. 1–2 perwww.meteoschweiz.admin.ch) and interpolated across altitude with Daymet In order to test for temperature differences in high (1800 m a.s.l.) compared to low altitude populations (1200 m a.s.l.) we determined average July temperatures based on temperature records from nearby climate stations /CTAG; ACA (VIC)/CATG; AAC (NED)/CTGA; ATG (PET)/CTAG, for T. montanum: AGG (FAM)/CTA; ACC (VIC)/CTA; AAC (NED)/CAA; ATG (PET)/CTG and for R. bulbosus: AGG (FAM)/CGTA; ACC (VIC)/CAGT; AAG (NED)/CTAG; ATG (PET)/CTAG. AFLP analyses followed a modified version of the AFLP plant mapping protocol of Vos et al. N = 565, 578 and 512 for B. media, T. montanum and R. bulbosus respectively, At least twenty AFLP primer combinations were screened per species for highest number of polymorphic loci and highest reproducibility of AFLP fragments. In each species four primer combinations were chosen to characterize genetic variation. The fluorescence labelled  EcoRI- and non-labelled MseI-primers were for B. media, T. montanum and R. bulbosus, respectively. In order to diminish bias in estimation of population genetic parameters, AFLP datasets were pruned from loci with allele frequency of >1– (3/N), where N is the total number of sampled individuals B. media, T. montanum and R. bulbosus .Peaks of AFLP fragments between 50 and 500 bp were binned in Genemapper 3.7 (ABI) and peak heights were exported from un-normalized electropherograms for further semi-automatic genotyping with AFLPScore 1.4 B. media an identity analysis was carried out in Cervus 3.0.3 B. media showed that all samples did belong to different genets.Initially the three datasets were scanned for AFLP loci under differential selection with outlier loci analysis in BayeScan 2.01 He) with AFLP-SURV 1.0 He was calculated based on allelic frequencies following Zhivotovsky fAFLP) into account (see below) To assess neutral genetic diversity of populations we estimated expected heterozygosity (fAFLP) were obtained on an individual basis from FAFLPcalc fAFLP-values differed from zero by calculating bonferroni corrected 95% confidence intervals (CI) on the population level. If CIs included zero, the respective fAFLP -estimate was set to zero for further analyses.For each species, estimations of F-values  FST-values between populations of the respective altitude were averaged .As measure for overall genetic differentiation pairwise We further used four approaches for describing population differentiation:FST.overall values and log-transformed geographic distances. Significance was estimated via 10,000 permutations. (ii) Partitioning of genetic variation among locations, among altitudes and within populations was determined with a hierarchical analysis of molecular variance (AMOVA) using GenAlex and the Bayesian cluster analyses using BAPS. For the AMOVA the populations were grouped by locations and 999 permutations were done for significance testing. (iii) A multivariate principle coordinates analysis (PCoA) based on pairwise Nei’s standard genetic distances We tested for spatial limitation of gene flow resulting in an isolation by distance pattern and carried out a Mantel-Test We further assessed the relatedness within populations by calculating Hardy’s among-individual relatedness coefficient (rc) in SPAGeDi 1.3 fAFLP and rc. Bonferroni corrections were applied whenever multiple tests were performed. If not otherwise stated, the analyses were carried out in R To assess the differences between low and high altitude populations in the measures described above, Wilcoxon tests were carried out accounting for the paired sampling of the populations (i.e. paired tests). To evaluate the impact of inbreeding within populations on the genetic similarity within populations Spearman rank correlation test was done between Figure S1PCoA plots of Nei’s Distances among populations in a) Briza media, b) Trifolium montanum and c) Ranunculus bulbosus. Populations within the same location are connected by dashed lines.(TIF)Click here for additional data file.Figure S22] of semi dry grasslands between 100 and 2000 m a.s.l. in Switzerland, based on the Swiss national inventory of dry grasslands (TWW).Estimates of total area , percentage of polymorphic loci (PPL), expected heterozygosity (He), average pairwise FST per population , average inbreeding coefficient per population (fAFLP), and average relatedness coefficient per population (rc) for all sampled populations of Briza media, Trifolium montanum and Ranunculus bulbosus.(DOCX)Click here for additional data file.Text S1AFLP genotyping protocol.(DOCX)Click here for additional data file.