Arabidopsis thaliana. Our analysis shows that it would require the analysis of at least a 1000 line bi-parental population to begin to estimate how many polymorphic loci control metabolic variation within Arabidopsis. Understanding the base number of loci that are actually involved in determining variation in metabolic systems is fundamental to developing systems models that are truly reflective of how metabolism is modulated within a living organism.A central goal of systems biology is to develop models that are both predictive and accurately describe the biological system. One complexity to this endeavor is that it is possible to develop models that appear predictive even if they use far fewer components than the biological system itself uses for the same process. This problem also occurs in quantitative genetics where it is often possible to describe the variation in a system using fewer genes than are actually variable due to the complications of linkage between causal polymorphisms and population structure. Thus, there is a crucial need to begin an empirical investigation into the true number of components that are used by biological systems to determine a phenotypic outcome. In this study, we use a meta-analysis of directly comparable metabolomics quantitative studies using quantitative trait locus mapping and genome wide association mapping to show that it is currently not possible to estimate how many genetic loci are truly polymorphic within  A central goal of systems biology is to develop models that are both predictive and accurately describe the biological system. Complicating this endeavor is the observation that it is possible to develop highly accurate models that use far fewer components than the biological system itself uses for the same process. The absence of components means that the models typically are not predictive when moving into new areas  other than the explicit conditions in which the model was developed. This is because in these different areas there are new and previously unrecognized components that need to be included to make the model accurate. Thus, while it is possible to create highly accurate models, these models ability to be predictive into new untested conditions is frequently hindered. Solving this requires developing a base understanding of how large a true molecular network is within a biological system to ensure that the models are of similar scale.This reduction conundrum also occurs in quantitative genetics where it is often possible to accurately describe the variation in a system using far fewer genetic loci than may actually be causing the phenotypic variation. This potential arises from the fact that most genetic populations are smaller than required to allow all independent components varying within the population to behave independently . This laA key approach to study the number of loci causing variation in a phenotype is the use of structured mapping populations. Modeling studies often test how the size of a mapping population and its recombination design impact the ability to find quantitative trait loci (QTLs). However, these modeling studies are typically built on the assumption that existing populations have largely discovered what is available to be discovered within a specific population. This arises because studies often bootstrap the analysis by taking a subset of the population and then testing how many of the final QTLs were found . These aArabidopsis thaliana to begin studying what is necessary to estimate the number of loci that affect a trait in a species. We show that the different RIL populations have similar genetic architecture suggesting that they can be treated as a randomized sample of the species. Using multiple populations of differing sizes measured for the same phenotypes with the same experimental and statistical approaches, we show that the number of QTL identified increases with population size but that it is currently impossible to tell if this relationship is linear or log-linear. Separating these two models is essential to developing future populations but will require a bi-parental RIL population of at least 1000 lines. Interestingly, the new QTLs identified with the increasing population sizes were not of small effect but instead they were of similar effect to the QTLs found in smaller populations. In contrast, most models assume that as more QTL are found they are of smaller and smaller effect. This means that we may be vastly underestimating the genetic potential present within any single RIL population. If we are underestimating a simple bi-parental population than there is an even larger issue of underestimation with more complex multi-parent or genome wide association (GWA) mapping population. New empirical and modeling studies taking into account these meta-analysis observations will be needed to design optimal mapping populations for future analysis.In the report, we conduct a meta-analysis of metabolite QTL mapping studies within simple bi-parental recombinant inbred line (RIL) populations of Arabidopsis RIL populations were not included either because they used different experimental designs that prevented the ability to compare the results or the appropriate data were not available -12 × Ei-2, and Kas × Tsu RIL populations . The larger Kas × Tsu population was slightly skewed toward metabolites with larger population variance while the smaller Bay × Sha population had a slight enrichment in metabolites with lower genetic variance .Heritability showed that the overall genetic architecture affecting the metabolome is similar between the two populations but that the affected metabolites are different. We further tested this using the genetic variance present in each metabolite across the RILs. To do this, we compared the genetic CV for all 258 metabolites across the two populations. As with heritability, the overall distribution of metabolic variance was very similar between the two populations . In contrast, the metabolites specifically variable in the Kas × Tsu population are more associated with stress responses like Shikimate, putrescine, SA, and isonicotinic acid or lipid metabolism . Interestingly, the known compounds that displayed elevated genetic variance in both populations are key energy balance compounds like asparagine, pyruvate, glucose-6-phosphate, fructose-6-phosphate and galactose-6-phosphate . Typically, these central energy flux components are considered constrained in their function which should limit their diversity but this does not appear to be the case in Arabidopsis. The lack of correlation amongst specific metabolites for heritability or genetic CV between the two populations argues that each population has specific genetic polymorphisms that alter distinct metabolites in each population. These genetic polymorphisms are largely not shared between the two populations . Thus, while the specific genetic variation in each RIL population affects different metabolites, the overall genetic architecture (the distribution of heritabilities and CV) of each population is similar. The fact that the overall genetic architecture of the RIL populations is comparable suggests that we can treat diverse RIL populations in Arabidopsis as random sample of the potential genetic diversity within the species.Within the Bay × Sha population the highly variable metabolites were enriched in sugars and amine metabolic processes . The Kas × Tsu population had an average of 1.22 ± 0.02 QTLs per metabolite in comparison to 0.79 ± 0.1 QTLs per metabolite found in the Bay × Sha RIL . The main difference between the two populations was the number of metabolites with at least one detected QTL; Kas × Tsu had a QTL detected for 75% of metabolites while for Bay × Sha this was only 44%. There was also an increase in the number of metabolites with two or more QTLs . Interestingly, 316 lines represents about a 50% increase in the number of lines which is similar to the 50% increase in QTL detected suggesting the potential for a linear relationship between the number of lines present in a population to the number of metabolite QTL detected.To begin testing how an increase in the number of lines between the two populations increases the ability to identify QTLs, we compared the number of QTLs identified for the 258 common metabolites. For both populations we had previously used the same composite interval mapping (CIM) approach to identify and call significant QTLs. In combination with the similar range of heritability’s and genetic variances, this identical statistical approach allows us to conduct a direct comparison where the only major difference in the two populations is line number . There wArabidopsis populations . This increase in QTL identification with population size was found for both aliphatic and indolic glucosinolates . Within these populations, the aliphatic and indolic glucosinolates are controlled by different causal loci suggesting that they are behaving as independent measures of the relationship between power to identify QTL and population size within these populations . Thus, the existing data cannot differentiate between a linear and log-linear relationship of QTL number detected to population size. This is even though the large population is considered to be sufficient to fully sample the potential QTL in a population . Even though both the aliphatic and indolic glucosinolates had different specific regression estimates the two traits generated the same estimate that it would require minimally between 950 and 1000 individuals in a single bi-parental RIL population to test which regression model more accurately approximates the relationship between population size and the power to identify new QTLs .When analyzing the relationship between QTL number found and population size for the glucosinolate phenotypes we found a log-linear relationship between the two parameters as has often been found in modeling studies (r2) explained because the additive effect of an individual locus is not dependent upon the total population variance. In contrast, the r2 per locus is determined both by the effect of the locus and the total population variance . Thus, the new QTL found as population size increased were not of smaller effect but instead of similar effect size to those found with smaller population sizes.A common assumption in QTL mapping is that small RIL populations will identify larger effect loci and the larger populations simply add smaller effect loci . Using ovariance . Thus, aFigure 9). Comparing the genetic CV showed that the RIL populations could capture a majority of the genetic variance controlling metabolite variation within Arabidopsis . Additionally, both RIL populations identified variance not present in the accessions as they had at least 14 metabolites showing twice the genetic variance found in the accessions . In contrast, there were only five metabolites showing elevated variation in the accessions that was not captured in the two RIL populations . Thus, while RILs have lower genetic diversity at individual loci than the accessions, this does not limit the associated phenotypic diversity.A common concern affecting RIL populations is that there are only two alleles per locus and thus might be limited in their genetic variation relative to species-wide diversity . A propoObtaining an accurate estimate of how many QTLs exist within a single population is a key parameter for any quantitative modeling study. However, there is no empirical understanding for how this parameter, QTLs per population, should be set within modeling studies. This leads to a massive range of values in these modeling studies complicating any true comparison between these modeling studies . Even thArabidopsis populations to begin an empirical assessment of how many QTLs are present in a given mapping population. As expected, including more lines in a bi-parental did lead to more identified QTLs. However, the relationship between line number and QTL number equally fit a linear and log-linear relationship even with 411 lines . Extending these linear and log-linear relationships showed that it would require 950 or more lines for even a simple bi-parental population before we could tell which relationship accurately describes the true number of metabolite QTL per population . Accurately testing which model best describes QTL number per a bi-parental population is essential given that most multi-parent populations are stopping at 1000 lines under the assumption that this is sufficient to identify all the QTLs (Arabidopsis to tests if the QTL identification power of a population is a linear or log-linear relationship to its size.Thus, we conducted a meta-analysis of existing metabolite QTL mapping within simple bi-parental the QTLs . HoweverThe above analysis is focused on metabolite related QTLs and as such may not be broadly reflective of all traits. For example, expression linked traits show higher average heritability than the metabolomic traits but lower heritability than the glucosinolate related traits used in this study . Most phArabidopsis populations (Arabidopsis QTLs (In contrast to most quantitative theory, our analysis found that larger population sizes identified QTLs with a similar distribution of effect sizes as smaller populations , 1998. Aulations . Anotherulations . Then upulations . Combinasis QTLs . Thus, tArabidopsis RIL populations shows that it is not possible to accurately estimate how many QTLs are present in a single population with two alleles per locus. This is in contrast to the myriad of modeling studies that make explicit assumptions about this variable. Future work will be required to extend these populations to larger sizes to provide a direct and empirical estimate of how many QTLs may exist in a population. Conducting these experiments will then provide a more firm foundation for extensions of similar estimates into multi-parent populations and further into the entire species. It is only then that we will have a true view of how many naturally variable genes causally affect variation within the plant metabolome.Our meta-analysis of QTL analysis using metabolite phenotypes across multiple 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.


Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations of this highly endangered species of European Community interest. In an era of rapid habitat loss and climate change, one of the key goals in species preservation and conservation genetics is to deepen our understanding of environmental forces responsible for shaping genetic diversity [In the past centuries, habitat modifications induced by climate change and human activity led to the extinction of many plant species or severely confined their geographic ranges , 2. Manyiversity .Species protection programs targeting large areas should focus on the most valuable populations to deliver tangible effects. The initiated actions require a thorough understanding of species biology and ecology as well as knowledge of the levels and distribution of genetic diversity . GeneticDetermining the populations of the highest conservation value is not that simple. Knowing the level of genetic diversity, the question arises; what genetic criteria should be used to define the priorities? Allelic richness is a straightforward measure of genetic diversity, that is commonly used in studies based on molecular markers that aim at selecting populations for conservation , 14, 15.The distribution of genetic diversity also plays an important role in species conservation –22. UndeIn rare and endangered species most of the most threatened populations are peripherals . NonetheUnderstanding the patterns and processes associated with geographical variation across species ranges is also motivated by conservation concerns. Geographically peripheral populations are often rare representatives of relatively widespread species within political jurisdictions . WhetherPulsatilla patens (L.) Mill. (Ranunculaceae) is one of such species. It is regarded as a rare and endangered species across Europe [P. patens populations has been dramatically reduced in the past 50 years, and the present geographic range is highly fragmented [ex situ garden population had to be established to maintain the plants in the wild [P. patens localities and populations from the European continent has prompted the European Commission to initiate active measures aiming to protect this critically endangered species. Pulsatilla patens is protected under Annex I of the Bern Convention [P. patens. There can be both natural and antropogenic reasons behind. It is believed that one of the reason for the disappearance of the populations of Pulsatilla patens is presumably to a great extent related to changes in land use, especially in forestry practices where efficient wildfire prevention and termination of cattle grazing in forests has lead to the formation of a continuous moss layer or a strongly grass-dominated vegetation, which severely hinders the regeneration of this species [s Europe , 37–46. s Europe , 47 in tagmented , 48–53. agmented , all at the wild . The rapnvention , and it nvention . It is d species  Several  species , and unf species .P. patens should be able to maintain higher levels of genetic diversity within a population and lower levels of genetic diversity between populations due to its specific breeding system, which promotes cross-pollination via insect pollination, and a long life-cycle. The present study was undertaken to determine whether habitat fragmentation and decreasing population size have influenced the genetic structure and diversity of P. patens. An analysis of genetic diversity will expand our knowledge of the species' reproductive strategy and provide valuable information for protecting and managing P. patens populations.Small populations can be expected to demonstrate signs of genetic depauperation due to genetic drift. In comparison with annual and self-pollinating plant species, Pulsatilla patens, a critically endangered species in most European countries. Microsatellite markers were used to determine: (1) the distribution of genetic variation across regions, populations and individuals, (2) whether analyzed populations of P. patens, which are small and influenced by stochastic processes are characterized by lower levels of genetic diversity, and (3) P. patens populations with priority conservation value in central Europe.The aim of this study was to evaluate the distribution of genetic diversity in east Central European populations of 2 leaf-segment was done with the consent of the local nature conservation authority in the presence of a representative person.The Polish populations were collected by MS with the permission given by General Directorate of Environmental Protection in Poland and the directors of Biebrza, Białowieża and Wigry National Park. The remaining populations were collected by GS with the assistance of L. Bartha (Cluj-Napoca), B. Lesku (Debrecen), B.A. Lukács (Debrecen), M. Russin (Kiev), I. Sramkóné Gáspár (Debrecen), R. Šuvada (Rožnava), A Szabó (Cluj-Napoca), V. Virók (Aggtelek). These plants were mostly collected from protected areas, and the sampling of a 1–2 cmPulsatilla patens (L.) Mill. (Ranunculaceae) is a polytypic species with several taxa classified under this name either at the rank of subspecies or as species of subsection Patentes Aichele and Schwechler [P. patens sensu lato (s.l.–in a broad sense) is a lowland species with a circumpolar geographic range [P. patens sensu stricto (s.s.–in a strict sense), occupies the western part of the area, and extends from Romania on the south to S Finland on the north, and from Germany on the west to W Siberia on the east, where it is replaced further to the east by P. flavescens (Zucc.) Juz. and P. multifida (Pritzel) Juz. [From a taxonomic point of view, hwechler . P. pateic range , and it el) Juz. , 58.Pulsatilla patens s. s. is an early flowering hemicryptophyte [The European species, ptophyte  which prptophyte . Despiteptophyte , 59. Nevptophyte .As can be implied from the long life-span of the species, the generation time and yearly flowering vigor and fruit-set depend strongly on environmental and climatic conditions , 54, 61.Pulsatilla patens populations: i) increasing, where relatively many rosettes are found in the smallest size classes, the proportion of seedlings is high and the proportions of generative plants is ca. 10%; ii) stable, where the proportion of rosettes in various life-cycle stages remain almost stable; iii) decreasing, where vegetative adults account for the vast majority (96%) of all rosettes and seedlings are usually completely absent. Most of the analyzed populations on the northern part of the area  represented this third type.Based on vegetative and reproductive characteristics, Kalliovirta et al.  distinguP. patens s.s. have been noted in Estonia, Latvia, Czech Republic, Slovakia, Hungary and Romania [P. patens is protected in European countries where it is regarded a critically endangered species. The species is listed in Annex II and Annex IV of the Habitats Directive of the European Union [P. patens is found mostly in pine forests.In the western part of the European distribution, the highest number of localities have been reported in Poland, and only isolated localities of  Romania , 48, 61. Romania . P. patean Union . The plaP. patens were sampled in central and eastern Europe: Poland, Belarus, Slovakia, Hungary, Romania, Ukraine, and Russia. . For populations P21–29, DNA was extracted following a modified CTAB-protocol detailed elsewhere [Total genomic DNA was extracted from 25 mg of silica-dried leaf material using the DNeasylsewhere . Six miclsewhere . ElectroMicro-Checker v.2.2.3 was used to test the genotyping of microsatellite data, identify genotyping and typographic errors and null alleles  using thA), number of unique alleles, effective number of alleles (Ne), average observed heterozygosity (Ho), average expected heterozygosity (He), sample-size weighed expected heterozygosity (UHe), fixation index (FIS). All calculations were performed in GenAIEx v.6.5 [The following genetic diversity parameters were calculated to determine the level of genetic variation within a population: mean number of alleles per locus  and private allelic richness (pRs) within each population were computed by the rarefaction method implemented in HP-Rare v.1.1 [N individuals selected randomly from a sample of N individuals in each population.Allelic richness  and linkage disequilibrium between loci were tested in FSTAT v.2.9.3 . SignifiFST  and relaFST [RST . Null al package , 75. Unc package , whereas package .FST values was also used to perform a hierarchical analysis of molecular variance (AMOVA) [FST/(1-FST) with geographic distance (km) in a Mantel test with 9999 permutations as implemented in GenAIEx. Bottleneck events were tested using two methods described by Cornuet and Luikart [FST distance matrix was carried out in GenAIEx. The Bayesian algorithm implemented in Structure v 2.3.3 [A genetic distance matrix of pairwise  (AMOVA)  in Arleq (AMOVA) . Signifi (AMOVA) , 80 were Luikart  and Garz Luikart . Infinit Luikart , 83. Pri v 2.3.3  was used v 2.3.3  to correLikelihood and delta K values were visualized in Structure Harvester software . Twenty The number of groups were chosen after the Structure output files were analyzed in R software version 3.2.2 . SimilarPul04 to 0.3598 in locus Pul01  was 4.1±1.4 (mean±sd), the observed heterozygosity (Ho) was 0.05±0.04, whereas expected heterozygosity was 0.56 ±0.13. Similarly, the measure of allelic richness (Rs) also indicated genetically poor populations (3.25±0.67). In connection, the inbreeding coefficient (FIS) was considerably high, 0.92±0.06.The highest number of alleles (15) was amplified by primers Ho, which was higher in the Southern group. The average number of alleles and expected heterozygosity values were actually higher in the Baltic populations, whereas the Southern populations were characterized by higher observed heterozygosity values and a smaller extent of inbreeding (FIS)  . NeverthHo) was only 0.04±0.03, and it ranged from 0.000 to 0.119 across the studied Baltic populations (He = 0.58±0.15) and ranged from 0.137 to 0.748 in the examined populations. A very small percentage of heterozygous individuals in the analyzed populations was confirmed by a high inbreeding coefficient (FIS = 0.93±0.07). The highest values of FIS were observed in four Polish populations , whereas in the remaining populations FIS statistics also indicates high values of inbreeding ranging from 0.70 (P7–Białowieża) to 0.988  , He  and population size.The populations analyzed differed considerably in the evaluated genetic diversity parameters. SSR loci revealed moderate genetic diversity in the analyzed populations. The highest average number of alleles per locus was detected in population P8–Kopytkowo, NE Poland (8.1±1.108), whereas the lowest number of alleles was found in population P7–Białowieża, NE Poland (1.5±0.224). The studied populations were characterized by very low percentage of heterozygous individuals. The mean value of observed heterozygosity (ulations . Observe Poland) . Allelic Poland) . PrivateNe) was 3.66±0.29, observed heterozygosity (Ho) was still low 0.08± 0.03, and ranged from 0.025  to 0.125 (P25–Bátorliget). As for the group mean expected heterozygosity (He) we had 0.54±0.03, and the lowest value was exhibited by P23–Primovce, N Slovakia (He = 0.482), whereas the highest value was displayed by P28 –Frumoasa, (Ho = 0.565). As mentioned above, slightly less levels of inbreeding was characteristic for the Southern populations (FIS = 0.89±0.04); the lowest value (FIS = 0.84) was found in P25–Bátorliget, whereas the highest (FIS = 0.96) in P28–Frumoasa.On the southern part of the distribution the genetic diversity values were more similar between the populations; the mean value of average number of alleles per locus  between pairs of 29 analyzed populations were heterogeneous. It is ranged from 0.011 to 0.596 (mean FST = 0.219±0.041). The highest level of genetic differentiation was observed between populations P7  and P17 , whereas the lowest level of variation was observed in P2  and P15 . Utilizing an ENA correction, the overall genetic differentiation and pairwise FST were slightly lower (FST = 0.189), indicating that null alleles only had a small effect on genetic differentiation  and lower variation within populations (74%) with FST = 0.25 (The results of AMOVA point to higher levels of variation within populations (77%) than between populations (23%). Genetic variation between Polish populations of T = 0.25 .P. patens inferred in Structure revealed that K = 3 was the most probable number of clusters for both the admixture and non-admixture models . The remaining two clusters were formed by the Baltic populations. The individuals from populations P1, P3, P4, P6 and P15 were characterized by admixed genotypes, and their Q values were indicative of membership in two clusters, cluster 1 (red) and cluster 2 (green), rather than a single cluster where Q>0.70. The majority of individuals from populations P9, P11, P13, P14, P17, P18 and P20 belonged to the first cluster, whereas most individuals from P2, P5, P7, P8, P10, P12 belonged to the second cluster  occupied an intermediate position. The second axis, which accounted for 20.36% of molecular variance, clearly separated the southern populations of P. patens  based on Nei’s unbiased genetic distance  generall. patens .We found no correlation between genetic distance and geographic distance as indicated by a Mantel-test  .Pulsatilla patens and was characterized by the most homogeneous gene pool, was devoid of 29 alleles that were found in populations from the entire geographic range sampled. These populations also had a different pool of locally common alleles which were characterized by low frequency within populations.The differences between the three genetic groups could be attributed to the presence of unique alleles as well as differences in the frequency of the remaining alleles. The Southern group, which comprised isolated populations of Pulsatilla patens are characterized by low levels of genetic diversity within populations. Very low levels of observed heterozygosity (Ho = 0.0547±0.04) in the analyzed populations point to high heterozygote deficiency and very high levels of inbreeding depression (FIS = 0.917±0.06).The results of our study indicate that east central European populations of P. patens, which is confirmed by recent reports of the species' continued disappearance [P. patens populations could reduce plant fitness and promote a further drop in abundance.Numerous studies of rare and endangered plant species demonstrated that inbreeding can result from a reduction in population size which lowers genetic diversity and leads to inbreeding depression –95. Thispearance , 49–53. pearance , 97, inbPulsatilla patens may be more likely to reflect the populations accumulated history of the size fluctuation than be reflect to present population size. Despite the above, the size of P. patens populations has been dramatically reduced in the past 50 years, and the present geographic range is highly fragmented [FIS.In general, levels of genetic diversity and high fixation index in the present day populations of agmented , 48–51. agmented –51, 53. Pulsatilla patens on the edge of the distribution range of this species and evolutionary processes that usually accompany peripheral populations [P. patens has led to the isolation of the population and also prevented the efficient flow of genes. Although genetic differentiation as measured by FST between the studied population showed an average value (FST = 0.22), but noted that among the study populations were such for which this parameter has reached very high values.One of the reasons of the low level of genetic differentiation and very high fixation index can be the location of most of the studied populations of ulations , 24. FraFIS could be attributed to self-pollination in P. patens populations. Although the analyzed species is xenogamous, selfing can occur but probably this is not the real cause of the significantly lowered genetic diversity. Most of Europe is suffering from the general decline of pollinator insects (mostly bees), a phenomenon called pollination crises [P. patens occurs and, probably more importantly, from the spring season when it flowers, one could expect an elevated rate of self-pollination due to the presumable lack of effective pollinators. Nevertheless, a recent study focusing on Hungarian orchids did not find evidence of pollination crisis in Hungary [P. patens.High values of n crises –100. Alt Hungary , but theP. patens comprising several individuals were characterized by the lowest levels of genetic diversity in comparison with the largest populations. Similar correlations were reported in populations of P. vulgaris [P. patens which was a common species in the past, but is now rapidly declining due to habitat changes partly caused by human activity. Habitat fragmentation, can be one of the reasons that lead to strong genetic drift. Besides anthropogenic factors, we have to consider the current climate change as a reason for decline of this otherwise cold-adapted species.As for the two main groups of samples , there where slight and statistically non-significant differences in genetic diversity measures between the two groups. The only remarkable difference is the slightly lower levels of inbreeding in the Southern group, which might be connected to slightly higher number of pollinators at the lower latitude. In other respect, the lack of statistically significant differences between population genetic indicators hint at a similar rate of declining populations. Positive correlations were observed between most genetic diversity parameters and population size, what confirm earlier studies , 9, 102.vulgaris . Leimu eThe second reason for the presence of a positive correlation between population size and genetic diversity is the fact that plant fitness differentiates populations based on variations in habitat quality , 105. InFST for the studied populations was 0.23, and it is comparable with the values reported for cross-pollinating and long-lived perennial species (FST = 0.22) [Most genetic diversity (77%) was observed within populations, whereas 23% of genetic variation existed between the evaluated populations. The breeding system is one of the key factors determining the distribution of genetic variation in plant populations , 110. In = 0.22) .FST = 0.596) were noted between populations P7 (Białowieża) and P17 (Parciaki). Population P7 was generally characterized by significant genetic distinctness with mean FST of 0.488 (ENA correction)  [P. patens populations were coming from the edge of the distribution, therefore, it can be assumed that gene flow between these populations is hampered. The observed differences were not correlated with geographic distance, however, and random differences in FST values could point to habitat fragmentation and the presence of barriers to gene flow between populations. In P. patens, gene flow could also be obstructed by the method of pollination and seed dispersal. P. patens is an insect pollinated species. Pollen-mediated gene flow is generally limited because most pollinators travel less than 20 km [P. patens, the manner of seed dispersal may also inhibit effective gene flow; in spite of having hairy achaenes adapted to wind-dispersal, most seeds land in the close vicinity of parent plants, and only dispersed on a longer distance by epizoochory [P. vulgaris populations [Despite generally moderate differences between the evaluated populations, significant genetic variations were observed in selected population pairs. The highest differences (rection) . The pre = 1.22) . Couvet  = 1.22)  demonstr = 1.22) . The majan 20 km , and thean 20 km . Plant pulations .P. patens populations due to the absence of flowering individuals or their inability to produce fruit. The above could have inhibited gene flow and exacerbated genetic differences. The observed genetic differences between selected population pairs could also be correlated with the small size of those populations. A limited number of individuals in a population  inhibits effective gene flow. Genetic differences could also be attributed to genetic drift which is often noted in small populations [The considerable genetic differences between selected population pairs could also be attributed to the absence of generative reproduction and thus increased genetic drift , 117. Inulations –120. GenP. patens consists of three clusters comprising several populations each. The most isolated populations  formed a separate group with a comparatively homogeneous gene pool. Baltic populations were more-or-less divided into two genetic groups which were not fully consistent with their geographic distribution. The easternmost populations of P. patens in large forests , harboring the largest populations of the studied species, formed the same group (cluster 2). The remaining populations, including numerous small populations, belonged to the most heterogeneous group 3. This group represents populations occupying the geographic margin of the species range where habitat fragmentation processes take place over much longer periods of time than in eastern populations.The results of Structure and PCoA analyses suggest that the genetic structure of P. patens we may prefer the first explanation  as high values of inbreeding and low levels of migration between populations were reported above, thus we can exclude efficient genetic exchange between the populations studied. Secondly, the split between the Baltic and the Southern group of populations coincide with the border between two biogeographic regions, boreal and steppic biomes in Europe [The observed genetic differences between the three groups were relatively small, but statistically significant. The absence of differences or minor differences between populations, in particular isolated populations, could be explained by two hypotheses. The first hypothesis claims that the distribution of genetic diversity within and between populations reflects historical gene flow processes which led to the fragmentation of larger populations , 122. Thn Europe , thus imn Europe , and if n Europe , 124. FoP. patens localities led to a dramatic decrease in the size of Pulsatilla patens populations in east Central Europe. The results of our study have confirmed parallel decreasing levels of genetic diversity in the analyzed populations. Low levels of genetic diversity and high levels of inbreeding within populations occupying the western part of the species geographic range could have important implications for the conservation of P. patens. The approach for selecting priority populations for conservation presented here based on neutral molecular markers which are useful for discerning gen flow and evidence of historic events. Populations with high diversity of neutral markers, and high allelic diversity can be considered suitable candidates for high adaptive variation as well [Habitat fragmentation and the decline of  as well  and this as well , 14, 15.P. patens, occupying area greater than 100m2. In the Baltic group of populations, which represented cluster 1 and 2, this populations are: Kopna Góra (P4), Kopytkowo (P8), Zabiele (P15)- cluster 1 and Kolimagi (P14)- cluster 2. Taking into account the biological structure of the population it must be emphasized that in this populations the number of flowering and fruit-producing individuals were the higher. In this populations we also observed the highest percentage of seedlings. Seedling recruitment could be correlated with habitat type, because the above populations were reported from xerothermic grasslands. The remaining Baltic populations were found in pine forests, in sun-exposed locations, generally along forest paths. Therefore, conservation actions have to focus on these large populations of the species in Poland.Our genetic analysis confirms that the highest allelic richness and highest number of locally common alleles are founded in the highest population of P. patens to get a clearer picture on this phenomenon. Interestingly, slightly lower levels of inbreeding were observed in the Southern group, with actually the lowest value of FIS detected in an ex situ conservation garden population (P25–Bátorliget). Unfortunately, we cannot exclude the possibility that this is an artifact, as the manager of this garden cannot fully exclude the possibility of incidentally including seeds from Transylvanian populations (B. Lesku ex verb.).As for the Southern group of populations, presented the third genetic group most populations seems to be equally large in terms of actual number of individuals. The highest genetic diversity, taking into account allelic richness and highest locally common alleles was detected in populations P-28 (Frumoasa). Unfortunately, we did not collect data on reproductive success in that part of the area, but we observed the presence of viable seeds of the plants in several Transylvanian populations. Nevertheless, the generally low levels of allelic richness and heterozygosity clearly warrants for declining population genetic characteristics of the populations studied, just in line with the reported decrease of the plant in the region . EspeciaP. patens populations studied, the low levels of genetic diversity and high values of the inbreeding coefficient throughout the studied range will compromise the species' ability to adapt to environmental change. To combat with the extinction of this highly endangered species, our data suggest that conservation efforts should focus on the largest populations of P. patens, representing the three genetic groups identified in our structural analysis. As practical guidance, we can suggest the usage of honeybees to try to facilitate outcrossing during the spring period at these selected populations.Despite the slight differences in the characteristic population genetic traits of Himantoglossum adriaticum, an endangered orchid species of EU conservation importance, when the plants were situated near a honeybee apiary. Probably, the way how conservation can improve the chance of outcrossing in existing P. patens population, and thus halt the inbreeding depression, is placing bee families nearby them in the spring period.The species, amongst other factors, may suffers from the lack effective pollinators as indicated by high levels of inbreeding. Recently, Biró et al.  reportedS1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


P < 0.001) pairwise FST values of each pair of breeds. Exceptionally, FST values calculated for Honamli and Hair breeds were statistically nonsignificant (P > 0.05). Heterozygosity values ranged between 0.62 and 0.73. According to the structure and assignment test, Angora and Yayladag goats were assigned to the breed they belong to, while other breeds were assigned to two or more different groups. Because this study for the first time presented genetic data on the Yayladag goat, results of structure analysis and assigned test suggest that further analyses are needed using additional and different molecular markers.The objective of this study was to determine the intra- and intergenetic diversities of eight different goat populations in Turkey including Hair, Angora, Kilis, Yayladag, Shami, Honamli, Saanen, and Alpine. A total of 244 DNA samples were genotyped using 11 microsatellites loci. The genetic differentiation between breeds was considerable as a result of the statistically significant ( While goat breeding has decreased between 1991 and 2009, the number of Hair goats continuously and significantly increased between 2009 and 2014 in Turkey. Among goat populations, Hair goat is the predominant (98%) type raised in Turkey , 4.The earliest phylogenetic analysis of goat populations in Turkey comprises protein and enzyme polymorphism –7. HowevDetermination of genetic structure and genetic characterization of animals is the first step in developing gene sources protection strategies. Therefore, the present study aimed to investigate genetic diversity among some goat populations in Turkey using microsatellite markers. Also, this study is the first report regarding Yayladag goat population.n = 32), Yayladag (n = 32), Shami (n = 32), Honamli (n = 32), Saanen (n = 28), Hair (n = 32), Angora (n = 43), and Alpine (n = 13) goat populations, were drawn into tubes containing K3-EDTA. DNA isolation was performed using standard phenol/chloroform method [In the present study, a total of 244 blood samples, which were obtained from Kilis (m method . The stu++ free PCR buffer (Fermentas), 200 μM dNTP (Fermentas), 1.5 mM MgCl++, 0.375 unit Taq polymerase (Fermentas), 5 pmol of each primary pair (μL in volume.Genomic DNAs were amplified by Polymerase Chain Reaction (PCR) using 11 microsatellite markers , which wary pair , and 50–PCRs were performed at two steps using MJ Research PTC-200 Thermal Cycler. After a complete denaturation at 95°C for 2 minutes, step I consisted of denaturation at 94°C for 45 seconds for five cycles, annealing at 59°C for 45 seconds, and elongation at 72°C for 30 seconds. Step II consisted of a total of 30 cycles, each including 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 20 seconds. Finally, a complete adenylation was enabled by keeping the samples at 72°C for 10 minutes.The resulting PCR products were loaded onto a Beckman Coulter CEQ–8000 Genetic Analysis System and allele genotypes were identified eluting by capillary electrophoresis.F-statistics, factorial correspondence analysis (FCA), phylogenetic trees (NJT), and structure analysis were determined using GenAlEx6 [Number of alleles (Na), expected (He) and observed (Ho) heterozygosity levels, GenAlEx6 , PopulatGenAlEx6 , TreeWieGenAlEx6 , GENETIXGenAlEx6 , and StrGenAlEx6  package Among general population parameters, number of observed alleles Na; , expecteIn the present study, a total of 160 different alleles were observed and the mean Na was found to be 14.55. The highest number of alleles  was observed in BM6444, whereas the lowest number of alleles  was observed in McM527 and ILSTS087 markers.It was determined that the mean Ho level changed between 0.188 and 0.969, whereas the mean He levels were observed between 0.427 and 0.917 . Mean HoF-statistics was used and FST values were between 0.005 and 0.125  calculated for all loci was found to be statistically important (P < 0.001).In order to evaluate genetic variation between populations, Wright's nd 0.125 . Total FDA genetic distance values of Nei, it was observed that the populations have been clustered in three main groups  method using n groups . Among tFactorial correspondence analysis (FCA) was performed to determine the genetic relation between the individuals used in the study and each individual was placed on 3-dimensional plane according to their genotypes . IndividK = 4 analyses revealed that all populations differed from each other and that Angora, Alpin, and Yayladag populations in particular are more pure breed compared to the others.Structure test was performed to identify which population or populations the study subjects belong to as well as group them . K = 4 aThe mean number of alleles observed in the study (14.55) and the number of alleles at each loci (10–33) were close to the findings of the other study, which was also conducted in Turkish goat populations using 20 microsatellite markers . Three lThe mean Ho and He values  found in the present study were higher than those found in goat genetic characterization studies conducted in different countries using different numbers of microsatellites –31. MoreIn a genetic study conducted using 13 microsatellites, 6 indigenous Iranian goat populations were separated into two main groups based on phylogenetic tree and FCA analysis . MitochoIn the present study, it was observed that Honamli and Hair goat populations grouped together on the NJT. This suggests that there is high relationship between Honamli and Hair goats and that gene flow is possible as they are raised in close geographical regions. In addition, FCA graph demonstrated that Honamli and Hair goats grouped together in line with these findings. Assignment test was performed to identify the populations using genotypic data and it was observed that animals were generally (93%) assigned in their own populations. It was determined that 8 different populations, which were used also in the structure test, were discriminated from each other and that Angora, Alpine, and Yayladag were more pure breed compared to other populations. Results of assignment and structure analyses revealed that microsatellites used in the study are very useful in discriminating the populations.There is no data or information in Turkey about Yayladag goat population used in the present study. However, previous research , 38 indiIn conclusion, microsatellites are quite reliable markers to be used in the studies to investigate genetic variety and genetic structures of populations and to determine whether the subjects belong to the claimed populations. In addition, there is need for further studies using different markers system to obtain additional data on Yayladag goat.


Fst showed high genetic difference between Betwil populations and the rest with Fst ranging from 0.1492 to 0.2447. We found that there is large number of gene exchanges within the Sudanese indigenous chicken (Nm = 4.622).The objective of this study was to assess the genetic relationships and diversity and to estimate the amount of gene flow among the five chicken populations from Sudan and South Sudan and commercial strain of egg line White Leghorn chickens. The chicken populations were genotyped using mtDNA D-loop as a molecular marker. PCR product of the mtDNA D-loop segment was 600 bp and 14 haplotypes were identified. The neighbor-joining phylogenetic tree indicated that the indigenous Sudanese chickens can be grouped into two clades, IV and IIIa only. Median joining networks analysis showed that haplotype LBB49 has the highest frequency. The hierarchal analysis of molecular variance (AMOVA) showed that genetic variation within the population was 88.6% and the differentiation among the population was 11.4%. When the populations was redefined into two geographical zones, rich and poor Savanna, the results were fractioned into three genetic variations: between individuals within population 95.5%, between populations within the group 0.75%, and genetic variation between groups 3.75%. The pair wise  Bos taurus and Bos indicus cattle in two separate locations [ Macropus rufus [Chicken genetic resources comprise a wide range of breeds and populations including red jungle fowl, native and fancy breeds, middle level food producers, industrial stocks, and specialized lines. Sudan possesses potential genetic resources of local poultry, even though most of them have not been genetically and phenotypically characterized. Some efforts had been exerted by the Sudanese nationals , 2 and iocations . Also D-ocations , horses ocations , and goaocations  and in Aus rufus . Study vus rufus , while vus rufus , 12. Advus rufus  probablyus rufus . The undus rufus . Therefon = 21) from El Dilling Locality in South Kordofan State, and Large Beladi of Bhari  was collected from Khartoum North Locality. Large Beladi of Abu-Neama  and Bare Neck  were both collected from Abu-Neama Locality in Sinnar State. The fifth population was  from Malakal Locality; this sample was retrieved from International livestock Research Institute (ILRI) data base. In addition to a commercial strain of egg line White Leghorn type (COML n = 9) was genotyped concurrently with the Sudanese chickens.The study was conducted in four states of Sudan, chosen because they have only indigenous chicken and no exotic breeds have been introduced. A total of 81 blood samples were collected from five chicken populations including Betwil, , and 1 μL Genomic DNA. The mtDNA D-loop (HV1) region was amplified using specific primers based on the partial chicken mitochondrial genome GenBank accession number (AB098668) and complete chicken mitochondrial genome GenBank accession number (NC 001323) . Direct sequence of HV1 segment of D-loop region was performed using two internal primers CR-for and CR-rev as shown in μL comprising 20 ng of purified PCR product as template DNA and 3.2 pmol of primer.m Method . PCR was 001323) . PCR ampThe mtDNA sequence for the first nucleotides (600 bp) of D-loop region  was used Gallus were included, one from wild jungle fowl Gallus gallus gallus and the other from Gallus gallus bankiva GenBank accession (number AB007720 and AB007718), respectively, and seven Clade I, II, IIIa, IIIb, IIIc, IIId, and IV reference sequences that correspond to different clades determined previously in Asian samples [The evolutionary relationships of indigenous Sudanese chicken haplotypes were established using the molecular evolutionary genetics analysis (MEGA) version 3.0 . Genetic samples  (Table 2S), number of haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (π) as explained by Nei [π) is defined as π = n/(n − 1)Σxixjπij or π = Σπij/nc where n is the number of DNA sequences examined, xi and xj are the frequencies of the ith and jth type of DNA sequences, respectively, in the sample, πij is the proportion of nucleotides in the respective types of DNA sequences, and nc is the total number of sequence comparisons [h, is defined according to the formula of Nei [h = 2n(1 − Σxi2)/(2n − 1), where xi is the frequency of haplotype and n is the sample size. The degree of genetic differentiation among the population was estimated using gene or haplotype frequencies. Population genetic structure was investigated by Fst significance test [Nst [Haplotype diversity was illustrated using network analysis implemented by NETWORK 4.1.0.8 . The DNAd by Nei . The anad by Nei . Alignmeparisons . Averagea of Nei , h = 2n using Clustal-X 1.83. Two units of invariant tetradecamer 5′-AACTATGAATGGTT-3′ were detected at positions 267 to 280 and 328 to 341. In the first unit there were two transitions observed G/A for SUD 71 and T/A for LB41 at position 268 and 272, respectively, while in the second unit of the tetradecamer T/C transition was observed for SUD 40 and SUD 13 at position 330. In addition to that the following domains and motif were observed, at the 5′ end of the D-loop. An interrupted thymine string (AATTTTATTTTTT) was observed and found to be conserved in all the individuals studied. There was also an interrupted poly-C sequence (5′-CCCCCCCTTTCCCCCCC-3′) which is widely conserved and downstream to this there is conserved sequence known as poly-G (5′-AGGGGGGGT-3′). Two conserved 5′-TACAT-3′ and 5′-TATAT-3′ were also found in all individuals. There are six TATAT motifs and two TACAT found within the 397 bases of the D-loop and were also conserved. The first 166 base pairs adjacent to tRNA Glu were found to be highly conserved in all individuals except for one substitution a T/G transversions in SUD 40 at position 33 (data not shown).The nucleotide substitutions found in the 14 variable haplotypes comprised one G/T and two C/A transversions and the rest were all transitions of which six were A/G substitutions and ten were C/T substitutions. This demonstrates a strong bias towards transition. The C/T substitutions are more common than A/G substitution . Gallus  were retrieved from GenBank and used as out groups, and seven clade reference haplotypes  were also included. The dendrogram revealed that 11 haplotypes identified in the Sudanese indigenous chicken were placed into two clusters with the domestic chicken Gallus gallus gallus. This indicates a very close relationship between the Sudanese indigenous chickens and Gallus gallus gallus, while they are relatively genetically distanced from Gallus gallus bankiva. Alignment with the reference lineage haplotypes from Asia showedthat all Sudanese indigenous chicken were grouped into clade IV  from the South Sudan  showed uniqueness. It fell into a different clade (IIIa) marked with red color while two haplotypes (SUD13 and SUD40) are both sharing clade 1V with other haplotypes from the Sudan region marked with yellow color H = 6) was found in Large Beladi Abu-Neama populations from Sinnar State, followed by Beladi Malakal chicken from Upper Nile State with (H = 5). Betwil, Beladi Bahri, and Bare-Neck had equal number of haplotypes (H = 4) and are regarded as the lowest haplotype number. The gene haplotype diversity (Hd) was high in Betwil population (Hd = 0.724) followed by Beladi Malakal while it was lower in Beladi Bahri and Bare Neck population (Hd = 0.455). However, the average overall haplotype diversity was approximately (0.577) for the 81 chicken haplotypes. The average nucleotide diversity detected for 81 D-loop sequences of the indigenous Sudanese chicken population was estimated to be 0.00282 substitutions per site. However, the highest nucleotide diversity was found in Malakal population (0.00603 = π) followed by Betwil population (0.00259 = π) and Beladi Neama ( = π0.00179) while Beladi Bahri and Bare recorded lowest nucleotide diversity (0.00126 = π)  .Fst and Nst were computed using DnSP version 4.0 [Fst and Nst were similar (0.098), while Nm was 4.622 for both approaches. This indicates that 9.8% of maternal genetic differentiation estimated in Sudanese indigenous chicken resulted from variation among populations while 90.2% was due to contribution by genetic divergences among individuals within populations. The highest genetic differentiation between populations observed was between Betwil population and the rest of the populations with Nst value ranging from 0.1493 to 0.2450 while the rest of populations showed a relatively little maternal genetic subdivisions sion 4.0 ; the aveMaternal genetic differentiation within population and among population within the Sudanese chicken was quantified using hierarchal analysis of molecular variance AMOVA on Kimura-2-parameter distance considering Sudanese populations as one single group. The genetic variation within population was 88.6% and the genetic differentiation among the populations was 11.4%. When Sudanese chicken population was once more subdivided into two geographical groups: rich and poor Savanna, the resulting variation was partitioned into three fractions. The variation between individuals within populations was 95.5%, between populations within groups was 0.75%, and the genetic variation between groups was 3.75% . Gallus gallus gallus. However, this result indicates the close genetic relationship between Sudanese indigenous chicken and genus Gallus. On the other hand, at the 5′ end of the D-loop HVI domain an interrupted thymine string (AATTTTATTTTTT), an interrupted poly-C (5′-CCCCCCCTTTCCCCCCC-3′) and poly-G (5′-AGGGGGGGT-3′) were widely conserved in all the individuals. These conserved features have been described across many avian species other than Galliform. They include Struthioniformes, Falconiformes, and Sphenisciformes [The complete alignment revealed a very high variability in the mtDNA D-loop region between 167–391 bases; this variation constitutes 23.5% of the 81 sequences. This rate is extremely high compared to the native chicken breeds 5.54–7.05% , 12. Simciformes . Howeverciformes . The conciformes . The preAll Sudanese chicken population from the Northern states fell into two clusters. However, when a second phylogenetic tree was reconstructed including populations from the Upper Nile State  in South Sudan, the dendrogram constructed placed the Sudanese indigenous chicken into three clusters and two different clades meaning that Sudanese chicken came from two different maternal lineages out of the seven clades. Despite the fact that some populations have more than one haplotype yet they fell in the same maternal lineage, that is, all Sudanese different haplotypes fall in Clade IV except haplotype SUD 71 which fell in Clade IIIa, this result may suggest that these populations shared the same maternal ancestor and that their descendants have accumulated mutations to become distinct lineages.π) nucleotide substitutions per site; it was higher in Malakal and Betwil populations, while it was lower in Beladi Bahri and Bare neck populations. This low nucleotide diversity in Large Beladi of Bhari and Bare neck populations may indicate loss of gene diversity for these populations which may be attributed to relatively recent population bottleneck. On the other hand high nucleotide diversity in Malakal and Betwil populations may suggest that the populations are more ancient [Total nucleotide diversity among the Sudanese chicken was found to be , [Fst = 0.106 [Nst results, the Sudanese chicken population was first defined into two geographical groups or regions, rich and poor Savanna. The genetic variation between individuals within populations was 95.5% and that occurring between populations within the groups was 0.75%, while the genetic variation between groups accounted for 3.75%. The low genetic variation between the groups that were defined geographically may suggest weak phylogeographic structure of Sudanese chicken and may be an indicator of common maternal origin. When considering Sudanese chicken population as one single population group, the genetic variation within the population accounted for 88.6% of the total variance while the proportion between populations was 11.4%. This indicates that the Sudanese chicken populations are genetically differentiated along geographical localities. Finally the study concludes that the region of mtDNA D-loop HVI which ranges from 167 to 397 has higher variation among Sudanese domestic chicken population.The hierarchical analysis of molecular variance AMOVA and  0.060),  but smal = 0.106 . When us


Aedes albopictus is one of the most significant pathogen vectors of the twenty-first century. Originating from Asia, it has invaded a wide range of eco-climatic regions worldwide. The insect-associated microbiota is now recognized to play a significant role in host biology. While genetic diversity bottlenecks are known to result from biological invasions, the resulting shifts in host-associated microbiota diversity has not been thoroughly investigated. To address this subject, we compared four autochthonous Ae. albopictus populations in Vietnam, the native area of Ae. albopictus, and three populations recently introduced to Metropolitan France, with the aim of documenting whether these populations display differences in host genotype and bacterial microbiota. Population-level genetic diversity (microsatellite markers and COI haplotype) and bacterial diversity (16S rDNA metabarcoding) were compared between field-caught mosquitoes. Bacterial microbiota from the whole insect bodies were largely dominated by Wolbachia pipientis. Targeted analysis of the gut microbiota revealed a greater bacterial diversity in which a fraction was common between French and Vietnamese populations. The genus Dysgonomonas was the most prevalent and abundant across all studied populations. Overall genetic diversities of both hosts and bacterial microbiota were significantly reduced in recently established populations of France compared to the autochthonous populations of Vietnam. These results open up many important avenues of investigation in order to link the process of geographical invasion to shifts in commensal and symbiotic microbiome communities, as such shifts may have dramatic impacts on the biology and/or vector competence of invading hematophagous insects.The Asian tiger mosquito  Aedes albopictus) is of major concern as it is known to be able to carry 26 arboviruses including Dengue and Chikungunya , Portes-Lès-Valence (PLV), and Nice (NC). NC is one of the first invaded sites in France since 2004  using a Bioblock Scientific MM 2000 mill . DNA was then extracted with Qiagen DNeasy Blood and Tissue kit  following the manufacturer's recommendations for both Gram negative and Gram positive bacteria. Assuming that the remaining mosquito bodies (hereafter referred to as carcasses) should be dominated by Wolbachia, they were pooled per population  and 1061R (5′-CRRCACGAGCTGACGAC-3′) as described , 0.06 mg mL−1 of bovine serum albumin , 40 μM of dNTP mix, 200 nM of each primer . Amplifications were carried out on Biorad C1000 thermal cycler  with 5 min at 95°C, followed by 40 cycles at 95°C for 40 s, 54.2°C for 1 min, 72°C for 30 s, with a final extension step of 7 min at 72°C. Forty PCR amplification cycles were necessary to generate an optimal amount of amplicons for Miseq sequencing. The three PCR product replicates from each sample were pooled, purified with Agencourt AMPure XP PCR Purification kit , and quantified using the Quant-iT Picogreen dsDNA Assay Kit .Hypervariable V5-V6 https://www.ebi.ac.uk/ena) under the project accession number PRJEB6896. A total of 9, 222, 165 reads were obtained, paired-end reads were joined with PandaSeq  using STAMP software 2.0.9  by ProfileXpert—ViroScan 3D . All FastQ files were deposited at EMBL European Nucleotide Archive (COI) gene was amplified with CI-J-1632 (5′-TGATCAAATTTATAAT-3′) and CI-N-2191 (5′-GGTAAAATTAAAATATAAACTTC-3′) primers using 45 ng of DNA matrix as described  under the accession numbers LM999972-LM999977.To maximize the quality and quantity of DNA obtained, our previously optimized extraction protocol for individual whole mosquito was used  in Vietnam and from invasive populations at three sites  in France  between wAlbA and wAlbB strains was found among all the populations tested  were sequenced with MiSeq technology. Analysis of a negative control showed the presence of bacterial sequences that probably derived from contamination during laboratory sample handling  (Table p = 0.002), Shannon-Weather α-diversity  and variance of abundance-weighted β-diversity  distances were not significantly different between populations of the two countries.The insect gut is a key organ in insect physiology and immunity. Moreover, previous studies have demonstrated that this organ harbored low concentration of ng Table . Howeverng Table . The gens Figure , althoug4) Table . Similar4) Table . Consequy Figure . Howevert-test, p < 0.05) . Only three OTUs assigned to Dysgonomonas and Aeromonas genera and Enterobacteriaceae family were shown to be significantly associated with French populations. The OTU overlap was calculated after merging and subsampling sequences at the country level , and 34% (n = 503), and 45% (n = 656) specific of France and Vietnam, respectively. Shared OTUs counted for 85% of the overall sequences.Overall, 15 OTUs were shown to be significantly associated with Vietnamese populations (Welch corrected ) Figure , includil Figure . The resAe. albopictus hosts was evaluated by barcoding based on mitochondrial COI gene sequences. A total of 6 haplotype variants were found in individuals from all populations. The genetic heterogeneity was more pronounced between countries  than between sites  (Table The genetic makeup of 3) Table . H_1 was3) Table .Ar) and the heterozygosity (He) at each genomic locus were analyzed for each sampling site. For both these analyses of variation, values for populations from France were significantly lower than those from Vietnam . The bottleneck analysis did not show any significant heterozygosity excess under a two-phase model or a single stepwise model (Table IS) between 0.12 (for PLV) and 0.196 (for HCM) reflecting an excess of homozygotes  with ENA correction for null alleles and 0.145 (CI95% 0.061–0.270) without correction. Consequently, the presence of null alleles did not strongly impact the estimation of differentiation. The structure of the populations was evaluated with the Bayesian method of assignment. The optimal number of clusters selected with the second-order change in likelihood method was K = 2  but a moderate variation among sites within a country   to 30 (Alb-tri 18). Rarefied allele richness of populations from each site was 4.56 for SP, 4.57 for PLV, 5.91 for NC, 6.50 for HCM, 7.47 for VT, 8.25 for BGM, and 8.62 for BD. To test for hypothetical population bottlenecks or expansions, the allelic richness  and no significant correlation with the Cavalli-Sforza Edwards measure of microsatellite genetic distance . However, for all sampling sites, positive correlations were observed between mean bacterial α-diversity (H′) and respectively host Ar     and haplotype structure  Figure  and host) Figure .Ae. albopictus and to test for correlations with host genotype.Mosquitoes may be regarded as holobiont units in which the host and its microbiota may display symbiotic relationships and multitrophic interactions  and low richness (less than three haplotypes per sampling site after rarefaction). Therefore, mitochondrial markers are highly sensitive to admixture in comparison with nuclear markers such as microsatellites. For this reason, it is not suitable to rely on mitochondrial haplotypes when estimating intraspecific genetic diversity in Ae. albopictus.Ae. albopictus microbiota are systematically dominated by Wolbachia sequences  , house flies (Musca domestica), fruit flies (Drosophila spp.), red palm weevils (Rhynchophorus ferrugineus), and sea bass (Dicentrarchus labrax)  and were preferentially associated with the autochthonous populations from Vietnam. These bacterial genera are ubiquitous in the environment  may have erased signs of a past bottleneck. In addition, genetic diversity reductions in France could also be explained by other patterns . Interestingly, the most genetic diversified of these populations was in Nice, a invaded site since 2004   as well as the host immune system  on Funding for this project was provided by grants from EC2CO CNRS and CMIRA Région Rhône-Alpes. This research was also partially funded by ERA-NET BiodivERsA with the national funders ANR-13-EBID-0007-01, FWF I-1437, and DFG KL 2087/6-1 as part of the 2012-2013 BiodivERsA call for research proposals, and was carried out within the framework of GDRI “Biodiversity and Infectious Diseases in Southeast Asia.” To achieve this work, we used the computing facilities of the PRABI cluster. We also gratefully acknowledge the contribution of the DTAMB platform of the FR41 Bio-Environment and Health (University Lyon 1).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.


Carcinus maenas L., independently introduced to the northwest Atlantic. Previous investigations based on mitochondrial DNA sequences demonstrated that larval dispersal driven by advective currents could explain observed southward displacement of an admixture zone between the two invasions. Comparison of published mitochondrial results with new nuclear data from nine microsatellite loci, however, reveals striking discordance in their introgression patterns. Specifically, introgression of mitochondrial genomes relative to nuclear background suggests that demographic processes such as sex-biased reproductive dynamics and population size imbalances—and not solely larval dispersal—play an important role in driving the evolution of the genetic cline. In particular, the unpredicted introgression of mitochondrial alleles against the direction of mean larval dispersal in the region is consistent with recent models invoking similar demographic processes to explain movements of genes into invading populations. These observations have important implications for understanding historical shifts in C. maenas range limits, and more generally for inferences of larval dispersal based on genetic data.Biological invasions offer unique opportunities to investigate evolutionary dynamics at the peripheries of expanding populations. Here, we examine genetic patterns associated with admixture between two distinct invasive lineages of the European green crab,  Becauet al.  predictsCarcinus maenas, one of the most notorious global marine invaders, was first introduced to the northwest Atlantic in the early nineteenth century [et al. [While empirical investigations of such dynamics in natural systems are becoming more common ,10,14, t century ,17, wher century ,19. In t century . Using m [et al.  exploredC. maenas populations offers a remarkably tractable system for investigating evolutionary dynamics at population boundaries, the mechanisms of genetic introgression and the demographic processes underlying these phenomena. We expand here on earlier work by introducing analysis of nuclear microsatellite data, revealing unexpected discordance in the temporal dynamics of mitochondrial and nuclear genomes across the C. maenas admixture zone. These patterns suggest an important role for demographic processes other than larval dispersal in shaping the evolution of genetic clines and offer novel support for hypotheses explaining C. maenas range limits in the north Atlantic.The dynamic admixture of two independently introduced 3.3.1c oxidase subunit I (COI) gene sequences used in this study have been described elsewhere [Carcinus [et al. [Crab samples, tissues and mitochondrial cytochrome lsewhere . Figure Carcinus  was used [et al. . Microsa [et al. ,23. Samp3.2α=0.05) in mean diversity between southern, central and northern sample regions were determined using the Tukey–Kramer method to account for unequal sample sizes, implemented in the R statistical programming environment v. 3.0.2 (http://www.R-project.org). Regional boundaries were drawn in the south between Lubec, ME (L) and Chance Harbor, NB (CH) and in the north between Chester, NS (C) and Halifax, NS (H)  . These rK=1 to K=n  found that the optimal number of population clusters was K=2 in all collection years, corresponding to the northern and southern invasion (not shown). To assess individual admixture, subsequent model runs were conducted with K=2 (five runs of 104 generations burn-in followed by 105 generations of data collection) and admixture between the two clusters was estimated using the individual co-ancestry coefficient Q of the highest likelihood model run. All analyses assumed uncorrelated allele frequencies allowing for admixture.To assess nuclear admixture, we adopted the Bayesian inference model implemented in Structure v. 2.3.4  to assig6 iterations with 2.5×105 discarded as burn-in. Clines were based on frequency of southern haplotypes in the case of mitochondrial data and on mean population co-ancestry (Q) in the southern Structure cluster in the case of microsatellite date.Effect of geography on admixture was determined based on changes in allele frequency against along-shore linear distance from the southernmost sampling site at Rye Playland, NY (RP). Distances were calculated with ArcGIS . Genetic clines consistent with neutral assumptions were assessed using a Metropolis–Hastings Markov chain Monte Carlo (MCMC) algorithm implemented in the HZAR R package . The HZATo identify individuals with cytonuclear discordance, we conducted additional Structure runs employing the USEPOPINFO flag to pre-assign individuals to either the northern or the southern mitochondrial haplotype cluster. Crabs identified by Structure as ‘immigrants’ in this analysis suggest individuals in which northern mitochondrial haplotypes have been introduced into southern nuclear backgrounds or vice versa.4.4.1In 2002, genetic diversity was significantly lower in the southern part of the range  south) for both nuclear and mitochondrial loci . By 20074.2Genetic cline analysis reveals shifts in both nuclear and mitochondrial clines over the period between 2002 and 2007 . Cline cAnalysis at the level of individual genotypes reveals the geographical extent of admixed crabs for the three collection periods . In 20005.C. maenas, may be explained in part by underlying demographic processes.The unexpected discordance between patterns of genetic variation at nuclear and mitochondrial loci strongly suggests that larval transport is not the only factor shaping temporal dynamics of genetic variation in this system. Specifically, our analyses indicate that the introgression of mitochondrial genomes from the southern to the northern populations, against the direction of mean larval transport and beyond a previously established range limit for C. maenas in the northwest Atlantic [C. maenas, advective currents would be expected to drive similar shifts in both nuclear and mitochondrial cline centres, consistent with our observations. However, the relatively rapid bi-directional expansion of the mitochondrial cline suggests an important role for demographic processes that, unlike larval dispersal, are expected to have differential influences on nuclear and mitochondrial genomes.An earlier examination of mitochondrial haplotype frequencies of Atlantic  observedp=7/22 in 2007) then the probability of making the observation of zero southern haplotypes at that site in 2002 is pbinom =0.00047.Discordance between genomes is revealed most strikingly by patterns of cytonuclear mismatch in individual crabs. Nuclear admixture between northern and southern genetic clusters remains largely constrained to the primary admixture zone, with some modest admixture in samples from northern New England . In staret al. [Ldiff), which would allow larval dispersal northward and expansion of the southern mitochondrial haplotypes. However, the magnitude of Ldiff was roughly 3.5 times greater than mean larval transport and substantially higher than what would be predicted by known regional current patterns. The authors suggested that variation in larval dispersal may have been shaped by long-distance anthropogenic transport or by differences in currents encountered by larval cohorts spawned at different times.The bidirectional expansion of cytonuclear mismatches is particularly conspicuous given the overall shift in cline centre to the south. Notably, from 2001 to 2007, Pringle et al.  documentet al. ,23) towaA number of recent theoretical and empirical studies reveal that demographic dynamics at expanding range margins may drive surprisingly large stochastic shifts in allele frequency . Rapid pC. maenas mitochondrial haplotypes in the northwest Atlantic. Both southern and northern lineages are invasive in the region, but the former had been established nearly 200 years before the latter and appeared to have reached a stable range limit near Halifax, NS [et al. [Ldiff values more consistent with regional circulation statistics. In addition, the demographic models invoked here may help explain why the southern population failed to expand beyond its northern range limit prior to the second, northern invasion. If substantial northward larval transport is unnecessary to explain the bidirectional spread of mitochondrial haplotypes, then it seems likely that previous range limits were set largely by the lack of sufficient northward dispersal to establish stable populations in northern Nova Scotia, consistent with observations of ephemeral populations north of Halifax prior to the northern invasion [et al. [These demographic processes may be at the heart of the observed spread of ifax, NS ,19. Cont [et al. . Genetic [et al.  proposedC. maenas reproductive behaviour do suggest an important role for female mate choice and male competition [C. maenas and its sibling species C. aestuarii concluded that these drivers best explain the observed introgression of mitochondrial genomes across species boundaries [Although there has been considerable recent interest in the implications of demographic processes for genetic dynamics at range margins, our understanding of these dynamics remains imperfect . Rapid ipetition , and a rundaries . Such feC. maenas shows biased introgression of mitochondrial genomes. This pattern occurs in both directions, but is most dramatic from south to north. Such introgression may be driven by different processes in different areas (or at different times) depending on demographic dynamics. Without additional data, particularly from sample sites within the initial contact zone and closer to the time of first contact, it is difficult to say for certain which models are the most appropriate for fully understanding admixture dynamics in C. maenas. Yet we do believe it is clear that existing knowledge of invasion history and observed genetic patterns are most consistent with selectively neutral drivers of introgression. Cytonuclear discordance has sometimes been attributed to selection on mitochondrial-encoded genes [C. maenas populations that potentially confer selective advantage, including significantly higher physiological tolerance to low temperature stress among northern lineages in the northwest Atlantic [C. maenas. For example, a number of studies have indicated that admixture may promote colonization and population expansion by facilitating response to novel selective pressures [C. maenas expansion patterns in the northwest Atlantic have been attributed entirely to neutral mechanisms [Advective systems are complex, with multiple mechanisms  likely to be shaping the temporal development of genetic clines. In our study system, ed genes , as mitoed genes ,40. Amoned genes . In factAtlantic ,43. Neveressures –46. To dchanisms ,21, but C. maenas in the northwest Atlantic, our results may have more general implications for studies aimed at better appreciating the role of dispersal at population range margins. Some authors have recently attempted to estimate such dispersal by analysing neutral genetic cline decay across dynamic admixture zones [Beyond providing insights into the invasion history of re zones ,10. Thes


Lanius meridionalis koenigi) in the Canary Islands, where it is an endemic subspecies and an important secondary seed disperser. We show that the Canarian subspecies is polyphyletic with L. meridionalis elegans from North Africa and that shrikes have colonized the Canary Islands from North Africa multiple times. Substantial differences in genetic diversity exist across islands, which are most likely the product of a combination of historical colonization events and recent bottlenecks. The Eastern Canary Islands had the highest overall levels of genetic diversity and have probably been most recently and/or frequently colonized from Africa. Recent or ongoing bottlenecks were detected in three of the islands and are consistent with anecdotal evidence of population declines due to human disturbance. These findings are troubling given the shrike's key ecological role in the Canary Islands, and further research is needed to understand the community-level consequences of declines in shrike populations. Finally, we found moderate genetic differentiation among populations, which largely reflected the shrike's bottleneck history; however, a significant pattern of isolation-by-distance indicated that some gene flow occurs between islands. This study is a useful first step toward understanding how secondary seed dispersal operates over broad spatial scales.Studying the population history and demography of organisms with important ecological roles can aid understanding of evolutionary processes at the community level and inform conservation. We screened genetic variation (mtDNA and microsatellite) across the populations of the southern grey shrike ( For over a century evolutionary, biologists have studied island flora and fauna as a way of gaining insight into otherwise intractable ecological and evolutionary processes. Island archipelagos are simplified and replicated study systems that vary in their size, isolation, and ecology, thus providing “natural laboratories” for evolutionary research  islands is secondary seed dispersal, or diplochory, whereby a seed is eaten by a frugivorous species , which is in turn eaten and dispersed by a carnivorous species . Numerous cases of secondary seed dispersal have been documented on islands  were taken by brachial venipuncture and stored at room temperature in 1-mL screw-capped vials filled with 800 μL absolute ethanol. All birds were released after sampling.The Canarian subspecies of the southern grey shrike occupies four main islands in the Canarian archipelago , three islets off the north coast of Lanzarote , and one islet off the north of Fuerteventura  gene was amplified in at least 11 individuals per population using the primers L-15035 and H-15985 , 0.5 μL (0.01 mmol/L) each primer, 2.5 μL H2O, and 1.5 μL DNA. Reaction conditions were as described by Klassert et al.  of primers, 1.5 μL sequencing buffer, and 6.35 μL H20. Sequencing reaction conditions were as follows: 94°C for 2 min, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min, with a final extension of 60°C for 1 min. Final PCR products were sequenced using an ABI Prism 3730 sequencer.A 809-bp fragment of the mtDNA cytochrome t et al. . SequencAll individuals were genotyped at eight microsatellite loci identified as polymorphic in the southern grey shrike (Mundy and Woodruff π) with their standard deviations were calculated in DnaSP 5.0  and northern Africa . We also included sequences from African, European, and Asian populations of L. m. uncinatus,L. somalicus,L. dorsalis,L. excubitor sibiricus, and L. e. homeyeri . Analyses were carried out using constant size population priors, the Hasegawa-Kishino-Yano nucleotide substitution model and a strict clock method that assumed a rate of 2.1% sequence divergence per million years  equilibrium for each locus and population, and linkage disequilibrium between all pairwise combinations of loci, were tested using GENEPOP (Raymond and Rousset K) to vary between 1 and 6. For each run, a burn-in of 10,000 steps with 500,000 MCMC repetitions was used. A total of four independent runs were completed for each value of K, and we compared average log probability of data for each value of K to determine the most likely number of genetic clusters. In addition, we calculated the statistic ΔK, which is based on the rate of change in the log probability of data between successive K values  were grouped with low nodal support as a terminal lineage within this clade (Appendix S3). Estimates of divergence time suggested that cyt-b sequences from L. m. koenigi and L. m. elegans shared a single common ancestor 284,000 years ago .Both the Bayesian inference and maximum likelihood tree showed an identical, well-supported topology (Appendix S3). Phylogenetic analyses grouped the Canary Island shrikes with the North African subspecies with high nodal support. P > 0.05), and no loci were found to be in linkage disequilibrium. Null allele frequencies were <0.1 at all eight loci. Differences in genetic diversity across populations were marginally nonsignificant when heterozygosity was considered  and marginally significant for allelic richness . Heterozygosity was highest in Fuerteventura, Gran Canaria, and Lanzarote  clusters, both of which corresponded roughly to geographic distribution (Appendix S4). Assuming K = 2, one cluster represented individuals from Tenerife (both Teide and coast), one from the other populations, whereas assuming K = 4 yielded the Tenerife cluster, plus one cluster corresponding to Gran Canaria, one to Fuerteventura, and one to Lanzarote and La Graciosa combined (Appendix S4). As with the PCA, STRUCTURE did not separate the shrike populations into clear genetic groups, instead suggesting either admixture or a lack of resolution from the microsatellite loci (Appendix S4).A PCA of the microsatellite loci indicated moderate differentiation among populations, with birds from Tenerife coast, Tenerife Teide, and Gran Canaria most divergent from the other populations Fig.A. BayesiFST and DEST at the microsatellite loci were highly correlated , and thus, only FST was used in subsequent analyses. All pairwise comparisons of differentiation based on microsatellite markers were significant, with highest levels of structure found between Tenerife and the other populations .Pairwise values of ons Table. A positThe Canary Islands have proved excellent models for studying biogeography, especially with regard to colonization, dispersal, and population history , blue tits (Cyanistes teneriffae), and common chaffinches (Fringilla coelebs) (Illera et al. We found pronounced differences in genetic variation among populations, which are most likely the product of a combination of historical and recent colonization events, bottlenecks, and gene flow. While the general pattern of higher genetic variation in the Eastern islands held true for both mtDNA and microsatellite markers, subtle differences in variation across marker types exist Table, Fig.2. M ratios suggesting a bottleneck in Tenerife coast and Tenerife Teide, while tests for heterozygosity excess suggested bottlenecks in Gran Canaria and Lanzarote (TableM ratios but not heterozygosity excess is expected when a bottleneck is older, more severe, and/or the population has recovered, whereas a signal of heterozygosity excess but not reduced M ratios is expected when the bottleneck is weaker and more recent (Williamson-Natesan Specific tests for recent bottlenecks using microsatellites yielded surprising results, with ote Table. Interesote Table. DetectiThere are increasingly complex and efficient approaches to directly estimate patterns of migration and test hypotheses of colonization history from genetic data (e.g., Beerli & Felsenstein, Given the patterns of structure observed here, and the evidence for multiple colonization from the African mainland, it is clear that there is potential for shrikes to act as secondary seed dispersers both from Africa to the Canary Islands, and among the Canary Islands. It certainly seems likely that shrikes play a greater role in interisland seed dispersal than primary seed dispersers such as lizards or frugivorous bird species, which tend to be more differentiated from the mainland and exhibit less gene flow among islands (Suárez et al.