Paramuricea clavata ), with particular emphasis on the processes of contemporary dispersal, genetic drift, and colonization of a new population. Isolation by distance and genetic discontinuities were found, and three genetic clusters were detected; each submitted to variations in the relative impact of drift and gene flow. No founder effect was found in the new population. The interplay of ecology and evolution revealed that drift is strongly impacting the smallest, most isolated populations, where partial mortality of individuals was highest. Moreover, the eco-evolutionary analyses entailed important conservation implications for P. clavata. Our study supports the inclusion of habitat-forming organisms in the design of MPAs and highlights the need to account for genetic drift in the development of MPAs. Moreover, it reinforces the importance of integrating genetic and demographic data in marine conservation.The integration of ecological and evolutionary data is highly valuable for conservation planning. However, it has been rarely used in the marine realm, where the adequate design of marine protected areas (MPAs) is urgently needed. Here, we examined the interacting processes underlying the patterns of genetic structure and demographic strucuture of a highly vulnerable Mediterranean habitat-forming species (i.e.  Marine protected areas (MPAs) are fundamental tools for the conservation of marine biodiversity . HoweverHomogenizing genetic connectivity and disruptive genetic drift are two of the main evolutionary processes contributing to the patterns of neutral spatial genetic structure of populations . ConnectThe importance of these evolutionary processes at contemporary timescales calls for a better understanding of their interactions with ecological characteristics of the populations (e.g. demographic traits). The value of the combination of ecological and evolutionary data for the conservation of biodiversity has been acknowledged in recent times , 18. IndPrevious demographic e.g. –30) and  and 30])Paramuricea clavata, a sessile and long-lived marine habitat-forming organism of the Mediterranean coralligenous assemblages [The present study is a first step towards the characterization over a contemporary timescale of the interplay between the demographic traits and the evolutionary processes that underlie the demographic and genetic patterns of the Mediterranean red gorgonian, emblages . Particuemblages , 38, whiemblages , 39, 40.The main aim of this study was to combine evolutionary and demographic data of a habitat-forming species, for the first time in the marine realm, in order to enhance the decision-making capacities regarding the design of MPAs. This aim was achieved by delving into the populations' evolutionary processes and demographic traits and by exploring the interactions between them. In particular, we (1) examined the patterns of demography and spatial genetic structure (SGS), (2) assessed the underlying processes, with emphasis on contemporary dispersal and genetic drift, (3) characterized the founding of a new population, and (4) explored the interaction of the ecological and genetic processes, shaping these populations. In this study, we highlighted the necessity of accounting for genetic drift in the development of marine conservation measures. We also suggested new perspectives on the functioning and management of low-dispersal marine habitat-forming species.P. clavata found a significant spatial genetic structure at large scales (∼5 to 3000 Km), produced by a combination between genetic clusters and isolation by distance (IBD) [The Mediterranean red gorgonian is a highly vulnerable species from the coralligenous assemblages, one of the richest and most threatened Mediterranean communities , 41. Thice (IBD) . At locace (IBD) .P. clavata only occur in six small islets, above 60 m depth. Samples were taken at two depths, at five of the islets , indicates that it has beenrecently founded. A small apical fragment of 30–50 colonies per site and depth , where the possibility of creating a MPA has been discussed. There, populations of e islets , except n = 329; , was colTotal genomic DNA was extracted using a salting out procedure, following . Individ2), the mean partial mortality (i.e. mean extent of injury), the size frequency distribution and the skewness coefficient. Skewness is a statistical measure of a distribution's symmetry; if it is significant, the distribution is asymmetric. Positive skewness indicates the prevalence of small size classes, while negative skewness points to the dominance of large size classes. All parameters were estimated following [Demographic patterns were assessed for each population by estimating the population density  and the unbiased expected heterozygosity of Nei (He) [a) with Fstat v.2.9.3.2 [r) and private allelic richness (Ap), using the rarefaction method implemented in HP-Rare [Genetic diversity was explored by calculating (1) the observed heterozygosity (HNei (He)  with GenNei (He) , (2) the.2.9.3.2  and, (3) HP-Rare  with a mST [ST´s (population-specific FST) [ST is based on the F-model [Global and pairwise population genetic differentiation was measured with the θ estimator of FST , computeST . Signifific FST) , 62 withfic FST) . Since t F-model , 65, whi F-model , this paST/(1-FST) and the logarithm of pairwise geographical distances, as recommended in a 2D model [http://earth.google.com) and the significance of the regression was tested with a Mantel test (n = 2000). A Bayesian clustering analysis was made with Structure v.2.3.4 [5 iterations and a burn-in period of 5x104. To determine the K that best captured the structure of the sample, we plotted the log probability of the data (LnP(D)) [The spatial genetic structure was analyzed by testing for isolation by distance (IBD) and genetic clustering. IBD was tested with Genepop, performing a linear regression between pairwise F2D model . Pairwis v.2.3.4 , 67 to i v.2.3.4 , and the v.2.3.4  and rece v.2.3.4  options.(LnP(D))  as a fun(LnP(D)) , and vis(LnP(D)) . To expl(LnP(D))  with 1006 iterations, a burn-in period of 25x105 and a sampling frequency of 2000. Convergence was achieved in all runs and the results were averaged across the five runs. The mixing parameters were adjusted to achieve acceptance rates of 20–40%, following [Contemporary connectivity is one of the main evolutionary processes shaping the spatial genetic structure of populations . In thisollowing . Geneclaollowing  was usedollowing , the Bayollowing  and the ollowing   of each population. Ne was estimated with the single-sample Bayesian computation method implemented in ONeSAMP 1.2 [The strength of genetic drift in each population was determined by estimating the contemporary effective population size  were computed with 2MOD . The pror), private allelic richness (Ap) and number of alleles (Na) were grouped as genetic factors; recent migration rates, first generation migrants and kinship constituted the group of connectivity factors; and the skewness coefficient, density and partial mortality were grouped as demographic factors. Using these groups of factors, we assessed the interplay of demography and genetics at three different levels. First, the generalized linear model of Geste v.2.0 was used to determine the effect of each group of factors on the genetic structuring  of populations . Spearman correlation coefficients for non-parametric variables, with a two-tailed significance test, were used and computed with SPSS 15.0 for Windows®.To explore the interaction between the evolutionary processes and the demographic traits, three groups of factors were conceived: allelic richness (Aons (see ). Then, The significance of all the multiple tests performed in this study was adjusted with the false discovery rate (FDR) method .2 in Escull de Tramuntana and 57.6 col/m2 in Cap Vermell Deep (mean ± SD = 19.2 ± 16.3). Partial mortality varied between 2.2% and 25.2% (13.5% ± 6.8%), being Es Vedrà Deep the population exhibiting more injuries. See The size frequency distribution varied in all populations . Larger o) varied from 0.694 to 0.818 with a mean value of 0.754, while expected heterozygosity (He) ranged between 0.713 and 0.773 and presented a mean value of 0.746. Escull de Tramuntana displayed the highest number of alleles (10.14), while Es Vedrà Deep presented the lowest (7.71). The mean values of allelic richness (Ar), private allelic richness (Ap) and number of alleles (Na) per population were 7.97, 0.38 and 9, respectively. Es Vedrà Deep showed the lowest Ar and Ap , while Na Bosc Deep presented the highest Ar (8.80) and Es Vaixell Shallow the highest Ap (0.80). See Observed heterozygosity . Pairwise FST´s ranged from −0.002 to 0.077 and all but four pairwise comparisons  were significant after FDR correction (Na Bosc Deep (NBD) vs. Escull de Tramuntana, NBD vs. Na Bosc Shallow, NBD vs. Es Vaixell Deep, and Cap Vermell Deep vs. Cap Vermell Shallow; see STs showed that the populations from Es Vedrà were the most differentiated from the others, as they displayed the highest local FST values  (ST of 0.011 and 95%HPDI = 0.002–0.024).Global population differentiation was low, but significant (F4–0.113) , Table 22 = 0.72; P = 0.001) (A significant pattern of isolation by distance (IBD) was detected (R= 0.001) . The Bay= 0.001) : Cap Ver= 0.001) ; and Es = 0.001) . The AMO= 0.001) .The analysis of migration rates and first generation migrants among clusters revealed a higher intra-cluster migration than inter-cluster migration in the three genetic groups . The more , in accordance with their high local FST values (see above). On the contrary, Escull de Tramuntana exhibited the highest Ne (135), in accordance with the higher gene flow occurring at this islet  confirming the results of the connectivity analyses. In the Es Vedrà cluster, the support for the drift model in the tests for migration-drift equilibrium was 93.6%, while the Cap Vermell and the Ses Bledes clusters were in gene flow-drift equilibrium, with a support of 79.9% and 95%, respectively. Moreover, the populations from the Es Vedrà cluster showed the lowest Nslet see .r), kinship and partial mortality, from the genetic, connectivity and demographic factors, respectively , private allelic richness (Ap) and number of alleles (Na), from the genetic factors and with first generation migrants and recent migration rates, from the connectivity factors. Local FST was also strongly and positively associated to kinship. Effective population size (Ne) was strongly and positively correlated with number of alleles (Na) from the genetic factors, and negatively associated with partial mortality from the demographic factors. The correlations between groups of factors revealed an interaction between the genetic and the demographic groups, through the negative association of number of alleles (Na) with partial mortality and between the connectivity and the genetic groups of factors through the positive association of migration rates with allelic richness (Ar) and number of alleles (Na). Interactions were also observed between the demographic and the connectivity groups through the negative relation of partial mortality and migration rates . At the smallest scale, juveniles and small colonies prevailed in the shallower populations, whereas large colonies dominated in the deeper populations indicating a more mature stage of development [P. clavata are “demographically healthy”. At larger scales, the demographic heterogeneity was supported by the high variation of partial mortality and density across all populations.The populations of elopment . The coeelopment  and otheelopment  for examST values were low, but significant. At the more local scale (∼30 m), some pairwise comparisons were significant , while others were not . Furthermore, the variable local FSTs, highlight important variations in the genetic structuring as they indicate how similar each population is compared to the whole set of populations [Corallium rubrum [o), expected heterozygosty (He), number of alleles (Na) and allelic richness (Ar) were similar to those reported for this species at a much larger spatial scale [P. clavata from Ibiza constitute a valuable repository of genetic variation that should be conserved [A highly heterogeneous genetic structure was also observed at the small and large spatial scales. In general, pairwise Fulations . At the ulations  and othem rubrum , 88, andm rubrum . Regardial scale . These honserved .P. clavata were mainly focused on gene flow. Low levels of gene flow between populations [STs with recent migration rates and first generation migrants confirms that contemporary connectivity is a relevant process in the genetic structuring of P. clavata.In accordance with demographic and genetic structure patterns, the underlying processes were also spatially heterogeneous. To our knowledge, the spatial variability of the neutral evolutionary processes (drift and migration) on the patterns of genetic structure has been scarcely documented in marine species ulations  and a shulations  were deme of populations when compared to the census population size . These results contrast with the rather large Ne reported in P. clavata using paternity analyses at a small spatial scale (2 m2) [STs were significantly and negatively related with genetic diversity  and significantly and positively associated with kinship, indicating that the more distinct populations are more prone to the effect of drift and inbreeding [ST, is evolving purely through drift, contrary to the two other clusters that are at migration-drift equilibrium.Genetic drift also had a pronounced impact on the genetic structure, as confirmed by the limited Ne (2 m2) . Neverthe (2 m2) , 91 for e (2 m2)  and compe (2 m2) ) should breeding . AccordiP. clavata remain poorly studied despite their importance in the current environmental context. Our demographic data suggest that Escull de Tramuntana was recently founded, allowing us a first insight into this process. The founding of a new population may be accompanied by a founder effect, which occurs when populations are established from few individuals [e and low local FST. Moreover, it shows a small kinship coefficient, a high genetic diversity, and a larger amount of immigration when compared to other populations, suggesting that it has been founded from immigrants coming from various genetically differentiated populations. This hypothesis is supported by the origin of the immigrants, which are native to the Cap Vermell, Ses Bledes and Es Vedrà clusters, indicating that a founder effect is not occurring in this population. Complementary studies involving other recently founded populations are needed to generalize this lack of founder effect during the colonization process of P. clavata. It would be interesting to conduct a temporal survey on the genetic diversity of Escull de Tramuntana, to test for a putative bottleneck during the future reproduction of the currently non-reproductive colonies.Colonization processes in ividuals . It is cividuals . SurprisThe characterization of the interaction between ecological and evolutionary processes has been widely studied in land and freshwater environments (e.g. ), due toe), number of alleles (Na) and migration rates. These results indicate that the less diverse populations undergoing a larger effect of drift and receiving less migration (i.e. the Es Vedrà populations) are the most affected in terms of injured surface. In P. clavata, partial mortality is caused by multiple stressors, among which environmental disturbances, such as thermal anomalies, play a fundamental role [Fucus serratus were less resilient to high temperatures likely due to the loss of genetic diversity caused by genetic drift.Here, the most striking relation between demography and genetics arose from the significant interaction of partial mortality with effective population size  is remarkable, as it is the opposite of what has been observed in other organisms, such as salmonids and plants, in which high population densities caused reductions in Ne [ST and negatively related to first generation migrants before FDR correction. As these relations (or the lack of them) may help to better understand the interactions of demography and genetics, we believe that additional analyses using more samples should be undertaken.No significant interactions of skewness or density with other factors were observed. The lack of relation between density and effective population size  reinforces the value of integrating genetic and demographic data in the design of conservation measures, (ii) provides a useful approach for the management and conservation of marine habitat-forming species with low dispersal capacities, and, most importantly, (iii) evidences the relevance of accounting for genetic drift in marine conservation planning, given its impact on populations responses to disturbances.Our study provides new insights for the conservation of S1 Dataset(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 Materials and Methods(DOCX)Click here for additional data file.S1 Table(DOCX)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.


Abies balsamea), a transcontinental North American boreal conifer, was inferred using mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) markers. Genetic structure among 107 populations (mtDNA data) and 75 populations (cpDNA data) was analyzed using Bayesian and genetic distance approaches. Population differentiation was high for mtDNA (dispersed by seeds only), but also for cpDNA (dispersed by seeds and pollen), indicating that pollen gene flow is more restricted in balsam fir than in other boreal conifers. Low cpDNA gene flow in balsam fir may relate to low pollen production due to the inherent biology of the species and populations being decimated by recurrent spruce budworm epidemics, and/or to low dispersal of pollen grains due to their peculiar structural properties. Accordingly, a phylogeographic structure was detected using both mtDNA and cpDNA markers and population structure analyses supported the existence of at least five genetically distinct glacial lineages in central and eastern North America. Four of these would originate from glacial refugia located south of the Laurentide ice sheet, while the last one would have persisted in the northern Labrador region. As expected due to reduced pollen-mediated gene flow, congruence between the geographic distribution of mtDNA and cpDNA lineages was higher than in other North American conifers. However, concordance was not complete, reflecting that restricted but nonetheless detectable cpDNA gene flow among glacial lineages occurred during the Holocene. As a result, new cpDNA and mtDNA genome combinations indicative of cytoplasmic genome capture were observed.The phylogeographic structure and postglacial history of balsam fir ( It olaricina . Neverthlaricina ,40. Contlaricina  and shorlaricina –44 and hlaricina . Thus, sThe main objective of this study was to infer the population structure of balsam fir from mtDNA and cpDNA variation and take advantage of the species low pollen production and short pollen dispersal distance to gain insights into large-scale phylogeographic patterns in North America. In balsam fir, highly polymorphic cpDNA microsatellites (or cpSSR) are expected to reveal a stronger population structure than typically observed in other widespread boreal conifers, which should also translate into increased congruence between cpDNA and mtDNA geographical structures. Thus, given the wide distribution of the species from central Canada to the Atlantic Ocean, investigating its phylogeography may bring new insights regarding the existence of several cryptic or controversial glacial refugia in eastern North America.Abies balsamea, Abies lasiocarpa and Abies fraseri) are neither endangered or protected according to the 'Endangered & Threatened Species List' provided by the U.S. Fish & Wildlife Service (U.S.A.) or the 'List of Wildlife Species at Risk' provided by Environment Canada (Canada). However, Fraser fir appears endangered according to the IUCN Red List of Threatened Species. No permit was required to collect tissue in any location sampled in this study. Samples were either collected on public lands or in U.S.A. State Parks or Canadian Provincial Parks after permission to do so was granted by park managers. All seed samples were obtained from collections of the National Tree Seed Centre . Live twigs were collected in a non-destructive and non-disruptive manner, so as to not interfere with the growth and/or health of either sampled species.The three fir species sampled in this study  and following the manufacturer’s instructions. Seeds were dissected to isolate megagametophytes, which cytoplasmic genetic background is representative of mother trees, and total DNA was extracted from megagametophytes with the DNeasy 96 plant kit . Two population sets were analysed. The first group, that contained all populations 107) and all trees sampled (1616), was used to analyze mtDNA population structure (see next section for details regarding the screening of polymorphism). Because cpDNA data provides less insights than mtDNA data regarding the fine scale historical population structure of boreal conifers  and all , cpDNA dAbies lasiocarpa  and three populations of Abies fraseri , two species phylogenetically closely-related to A. balsamea [A. lasiocarpa’s natural range in western Alberta, and from the natural range of A. fraseri, which is endemic to the southern Appalachian Mountains in eastern U.S.A.Two additional subsets included three populations of balsamea –48. TheyA. balsamea from 9 geographically remote populations (see nad 5 gene (nad5-4). Using primers developed by Wu et al., 1998 [A. balsamea individuals sampled from 12 populations distributed throughout the species’ range. In addition, three populations of A. fraseri  and A. lasiocarpa  were also sequenced. These sequences were used to determine the nature of polymorphism and to design new internal primers using OligoPerfect Designer  for further sequencing. These internal primers . Polymerase chain reaction (PCR) conditions consisted of an initial denaturation step (4 min at 94°C), followed by 35 cycles of denaturation (1 min at 94°C), annealing (30 s at 56.3°C), extension (1 min at 72°C), and a final extension (10 min at 72°C). PCR products were sequenced on an ABI-3730xl DNA Analyzer  using the dideoxynucleotide chain termination procedure (Sanger method). Sequence alignment and allele scoring were performed using the CodonCode Aligner version 3.7.1 software .DNA was amplified in a PTC-225 thermal cycler  using 25 to 50 ng of DNA template, 0.1 μM of each primer, 0.1 mM of each dNTP, 1X of reaction buffer, 1.5 mM MgClet al., 1996 [2, 20 μM of fluorescent-labelled M13 primer , and 0.05 unit of Platinum Taq DNA polymerase . Amplifications were performed according to the protocol described in Oetting et al., 1995 [Pt26081, Pt30204, Pt63718 and Pt71936) revealed intraspecific length polymorphism. However, only two of them (Pt30204 and Pt71936) showed consistent amplifications and therefore, were retained for genotyping.Microsatellite markers designed by Vendramin l., 1996  were usel., 1995 . An addiPt30204 and Pt71936) were further sequenced for a subset of 81 individuals , as well as mitotype and chlorotype diversity  were caid data;  with a ‘GSTcp, RSTcp for cpDNA and GSTmt, NSTmt for mtDNA) were then estimated with Permut/cpSSR [GST,NST and RST take into account the relatedness among haplotypes to estimate population differentiation. Thus, a significantly higher value for NST or RST than for GST would be indicative of a phylogeographical structure. Jost’s differentiation index  and Dcp(group)) were also calculated among mtDNA and cpDNA BAPS groups (see below for groups delineation) in order to compare the magnitude of genetic differentiation among mtDNA and cpDNA lineages.Population fixation indices based on allele size and allele identity  was alsiversity . Hence, iversity ,55, suchk-value) so as to maximize the differentiation among groups using k-values and allele frequencies as varying parameters. A logarithm value of maximal likelihood (log (ml) associated with the 10 best partitions visited is estimated. However, the recommended approach to determine the optimal partition [k-value ranging between 2 and 10. The resulting optimal log (ml) values were plotted as a function of the number of clusters and the number of groups corresponding to the inflexion point of the curve was considered optimal  were used as outgroups in order to root the trees. Trees were generated using the software MEGA 4 [Population structure was assessed independently for mtDNA and cpDNA data, using the ‘spatial clustering of groups’ option implemented in the software BAPS 5.4 . BAPS alartition  yielded artition . Therefoartition –58 was e optimal . Additioe MEGA 4 .FCT estimates the level of genetic differentiation among groups of populations presumably representative of genetically distinct lineages, this index reflects historical genetic differentiation due to geographic isolation of populations in glacial refugia. Contrastingly, FSC estimates population differentiation within historical lineages. Thus, this index rather reflects seeds and pollen dispersal abilities over generations and their homogenizing effect on genetic diversity (i.e. mtDNA and cpDNA gene flow) rather than historical divergence among lineages. The statistical significance of differentiation indices was tested using 50 000 permutations. Estimates of population differentiation within mtDNA and cpDNA BAPS groups (FSCmt and FSCcp) were then used to estimate the effective number of migrants per generation  according to Takahata & Palumbi, 1985 [A. balsamea. For the sake of comparison, similar estimates were derived for Picea mariana, Pinus banksiana and Tsuga canadensis from previously published cytoplasmic marker data [A hierarchical analysis of molecular variance (AMOVA) was conducted with Arlequin 3.1  to assesbi, 1985 , and theker data ,7,22.nad5-4 was the only polymorphic locus. This result was expected given the much conserved nature of plant and conifer mtDNA exons and introns [nad5-4 which, once combined, yielded five mitochondrial haplotypes or mitotypes (A. fraseri and A. lasiocarpa). All A. fraseri and A. lasiocarpa individuals harbored mitotype I and II, respectively, while these two mitotypes were also observed in A. balsamea (GenBank accession nos. KJ705284-KJ052288).Out of the 43 mitochondrial regions initially screened,  introns ,65 and l introns . Sequencitotypes . No specf = 0.112), but was geographically restricted to the southeastern part of the species’ natural range. All three other mitotypes  were more locally distributed and less frequent . Both average mitotype diversity (Hmt) and average mitotype number (nhmt) were low with values of 0.168 and 1.6, respectively n followed by (T)n for Pt30204, and (A)n for Pt71936. Although Pt30204 included two variable regions, no evidence of fragment length homoplasy was found in the sequences analyzed.The sequencing of 81 trees confirmed that SSRs two loci . The lenPt30204 and Pt71936, respectively. When considered together, the two loci yielded a total of 86 chlorotypes, from which 11 had a frequency greater than 0.015 , and chlorotype 11 was the less frequent (f = 0.016). Most of the variants (76%) were rare chlorotypes (f < 0.01) and one third of those were private (population-specific). Estimates of average chlorotype diversity (Hcp = 0.773) and average number of chlorotypes (nhcp = 6.7) were much higher than those obtained for mtDNA data  = 0.552). Population differentiation within BAPS mtDNA groups (see below) followed the same trend  among populations within BAPS groups indicated that seed gene flow was low (Nemmt = 1.771). The distribution of mitotypes was well geographically and phylogeographically structured  and differentiation among mtDNA groups was high and significant . MtDNA group #2  = 0.594, respectively). More noticeably, and contrary to expectations, Jost’s estimates revealed that cpDNA population differentiation was higher than mtDNA population differentiation (Dcp = 0.610 > Dmt = 0.202) and that differentiation among BAPS groups was comparable between mtDNA and cpDNA data (D(group) = 0. 552 and 0.594 for mtDNA and cpDNA, respectively). Population differentiation within cpDNA BAPS groups likely representative of different glacial lineages  than in the other sympatric conifers previously surveyed .As expected, cpDNA differentiation among populations and among BAPS cpDNA groups was significant and lower than that estimated with mitotypes  was similar to that of the sympatric and largely distributed boreal species black spruce  harbour reportedly higher mtDNA diversity than A. balsamea [A. balsamea is possibly related to the fixation of mitotype I in a large number of populations of presumably distinct glacial origin. This could indicate that genetic drift during isolation in refugia or early Holocene recolonization depleted mtDNA diversity in some balsam fir glacial lineages.Balsam fir mitotype diversity . Withinbalsamea . Low mtDHcp = 0.773) was in the same range as that observed in A. fraseri , Abies guatemalensis (Hcp = 0.934), Abies hickelii (Hcp = 0.937), and Abies religiosa (Hcp = 0.908). CpSSR diversity in balsam fir was also comparable to that of other North American boreal and temperate conifers: for instance, black spruce , jack pine , Abies and 0.96 ,70), Abip = 0.98 ), Abies ectively ) and in y [Abies : Abies f = 0.727 ).GSTmt = 0.480) was significant but lower than that observed for other widely distributed North American boreal conifers sympatric with balsam fir . However, mtDNA population differentiation was possibly underestimated in balsam fir due to the fact that the mtDNA group #1 included populations of diverse ancestry which shared the same mtDNA background . This inference is all the more likely that the mutation rate of mtDNA in plants and conifers is low [A. balsamea was also lower than that of Mesoamerican firs [MtDNA differentiation among populations .Population differentiation within mtDNA BAPS groups was also significant but lower in a Tables  and 3, il., 2010  for the GSTcp = 0.104) was much higher than that of other boreal conifers with similar ranges such as P. banksiana [T. canadensis [P. glauca , except for P. mariana [P. mariana populations occurring within the sampled range of A. balsamea (i.e. excluding populations from Alberta and westward), differentiation was much lower than that observed for A. balsamea  and that only a few chlorotypes were shared among all populations, Jost’s differentiation index provides a more valid framework to estimate population differentiation [A. balsamea . Low pollen-mediated gene flow is likely the main cause for the detection of a cpDNA phylogeographic structure in A. balsamea , contrary to what was observed in all other sympatric North American conifers surveyed.The estimate , likely iversity –77. Indentiation ,78. Cont mariana  for the nadensis . In thesDmt = 0.202 < Dcp = 0.610). Contrary to GST, Jost’s D is independent of gene diversity [Nemmt = 1.77 and Nemcp = 6.20), differentiation and migration estimates indicate that the homogenizing effect of pollen gene flow is considerably reduced in this species. This is further supported by estimates of differentiation among BAPS mtDNA and cpDNA groups, which were comparable (Dmt(group) = 0.552 and Dcp(group) = 0.594). Hence, balsam fir cpDNA should provide particularly valuable insights into the species’ postglacial history. It should be much more informative than seen in other sympatric conifers previously surveyed.Taken together, these evidences indicate that balsam fir populations are more differentiated and structured than those of any other conifer occurring in the same geographic area for which similar data are available. The most unexpected result, with regard to empirical observations in conifers, lies in the comparison between mtDNA and cpDNA differentiation. Indeed, Jost’s differentiation index showed that cpDNA differentiation was substantially higher than mtDNA differentiation in balsam fir . Sucreported  (Table 3Choristoneura fumiferana (Clemens)) [A. balsamea and P. glauca populations in central and eastern Canada [P. glauca is generally less affected ) , an indin Canada ,84. Budwn Canada , causingn Canada ,86. Thisn Canada , but is n Canada . Major on Canada ,89,90, wn Canada ). Duringted (52% ). Thus, ted (52% ). AlthouA. fraseri  was geographically isolated from the eastern subset  by a large area where populations carried predominantly mitotype II, along with mitotype I (mtDNA group #2). Hence, these two disjunct population subsets do not likely originate from the same glacial refugium despite their similarity in mtDNA composition. This hypothesis is in line with the cpDNA population structure, which showed a clear genetic divergence between populations from northeastern Canada , and western Canada (cpDNA group #1), corresponding approximately to these two disjunct subgroups of mtDNA group #1. Given the very low polymorphism observed in the mtDNA of balsam fir and low mutation rate of plant mtDNA [Overall, five genetically distinct glacial refugia were inferred from mtDNA and cpDNA variation in balsam fir . The fir fraseri . The wesnt mtDNA , it is pnt mtDNA .A. balsamea lineages originating from two geographically distinct glacial refugia located south of the continental glacier. A first lineage , would correspond to the mtDNA lineage originating from the cryptic refugium . These populations, which correspond to mtDNA BAPS group #3 , or frolachians . As evid mariana , 19, whegroup #3 , would fgroup #3 , possiblnadensis , and posistocene . The curn Quebec , a patteanksiana ,20. In lanksiana . Around A. balsamea range due to differential gene flow between cytoplasmic genomes, as illustrated by the observed geographical discordances between mtDNA and cpDNA lineages. Populations from western Canada, Wisconsin and the Upper Michigan Peninsula harboured an mtDNA signature typical of the Western lineage and a cpDNA signature typical of the Central lineage . Converg. [e.g. ). A detag. [e.g. ) and to In addition, despite the generally high dispersal potential of conifer pollen, the strong structure of the cpDNA diversity observed in balsam fir points to a rare case of restricted pollen gene flow in conifers. It also indicates that fragmentation at the landscape and natural range levels in balsam fir can turn out to bear more genetic consequences than in other Pinaceae, especially when distant populations are considered. Implications for gene conservation in this species and other ones with similar life history and reproductive features should be further studied. From a biogeographic perspective, this study also brought support for the existence of three controversial refugia, in the Driftless area of central United States, in Labrador region, and in the Maritimes-Appalachians region of eastern Canada and northeastern United States. However, regional studies with increased sampling would help deciphering the exact location of these refugia and guide conservation efforts by assessing their relative significance in term of genetic diversity and differential of adaptation, for instance to coastal versus more continental climates.S1 FigArrow shows the inflection point and the partition selected. See the section (TIF)Click here for additional data file.S2 Figk = 7 corresponding to the seven colored tracings on the map). (b) Neighbor-Joining dendrogram based on chord genetic distances among BAPS groups; the color of filled circles matches the color of BAPS groups on the map; putative suture zones are indicated by a square; ellipses correspond to the final grouping presented in (a) Spatial distribution of BAPS initial mtDNA groups Click here for additional data file.S3 Figk = 9 corresponding to the nine colored tracings on the map). (b) Neighbor-Joining dendrogram based on chord genetic distances among BAPS groups; the color of filled circles matches the color of BAPS groups on the map; putative suture zones are indicated by a square; ellipses correspond to the final grouping presented in (a) Spatial distribution of BAPS initial cpDNA groups Click here for additional data file.S1 TableTable S1 contains three sheets corresponding to geographic data, mtDNA-cpDNA haplotype counts and genetic diversity indices, respectively.(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Mustela lutreola, L. 1761) is a critically endangered mustelid, which inhabits several main river drainages in Europe. Here, we assess the genetic variation of existing populations of this species, including new sampling sites and additional molecular markers (newly developed microsatellite loci specific to European mink) as compared to previous studies. Probabilistic analyses were used to examine genetic structure within and between existing populations, and to infer phylogeographic processes and past demography.The European mink  and Southeastern (Romania) European populations showed the highest intraspecific diversity. In contrast, Western European (France and Spain) populations were the least polymorphic, featuring a unique mitochondrial DNA haplotype. The high differentiation values detected between Eastern and Western European populations could be the result of genetic drift in the latter due to population isolation and reduction. Genetic differences among populations were further supported by Bayesian clustering and two main groups were confirmed (Eastern vs. Western Europe) along with two contained subgroups at a more local scale .Genetic data and performed analyses support a historical scenario of stable European mink populations, not affected by Quaternary climate oscillations in the Late Pleistocene, and posterior expansion events following river connections in both North- and Southeastern European populations. This suggests an eastern refuge during glacial maxima . In contrast, Western Europe was colonised more recently following either natural expansions or putative human introductions. Low levels of genetic diversity observed within each studied population suggest recent bottleneck events and stress the urgent need for conservation measures to counteract the demographic decline experienced by the European mink.The online version of this article (doi:10.1186/s12862-015-0427-9) contains supplementary material, which is available to authorized users. Mustela lutreola, L. 1761) is a riparian mustelid that used to occupy most of the main river drainages in Europe. The rapid decline of this species throughout its distribution range [Neovison vison), a direct ecological competitor potentially spreading diseases to which M. lutreola is vulnerable [http://www.iucnredlist.org) [The European mink  . In an eAt present, the European mink consists of scattered isolated populations restricted to three areas: Northeastern Europe , Southeastern Europe , and Western Europe  , 9. To det al. [In the present study, we combined molecular data (both mtDNA and nuclear microsatellites) from Michaux et al.  with newet al.  on all sh = 0.857 ± 0.014, respectively (Table h = 0.862) and a high percentage (92.31 %) of private haplotypes for NE populations (mostly Russia). In contrast, SE populations were characterised by four haplotypes  and low mtDNA variation (π = 0.0019 and h = 0.352). Lowest haplotype diversity was detected for W populations, where all individuals shared the same haplotype, which was not found in Eastern European (NE and SE) populations. Analyses based on the mtDNA dataset II identified rivers North Dvina, West Dvina and Volga (NE populations) as significantly contributing to mean genetic diversity levels  were found among NE, SE, and W populations (mtDNA dataset I), with all comparisons being statistically significant  to 0.742 (Belarus + Estonia vs. W) (data not shown). Similarly, significant genetic differentiation was also detected among the tested river basins  strongly supported genetic differentiation of the European mink, but failed to detect geographic structuring of East (NE + SE) vs. W populations or NE vs. SE populations  low negative Fs and Tajima’s D values  of non-expanding (stable) populations, as was shown by the general pattern of non-significant P > 0.0 low negCoalescent-based reconstructions of the demographic history of the NE and SE populations inferred from BSP analyses revealed histories of long-term stability in effective population size until approximately 5 Kyr for the NE populations, and a recent demographic expansion period in the SE populations during the Last Glacial Maximum, corresponding to a population increase of more than 30 % . Thus all loci were used in further analyses.Microsatellite alleles were obtained from a total of 313 specimens . Thus, microsatellite loci were considered statistically independent.No evidence of linkage disequilibrium was found as none of the corresponding exact tests remained significant after Bonferroni correction  were detected. After Bonferroni correction, only the Spanish population showed significant departures  from HWE for the following loci: MVIS22 (FIS = 0.265), MVIS72 (FIS = 0.274), and MER41 (FIS = 0.422), due to a heterozygote deficit.Microsatellites were polymorphic in all tested populations with the exception of locus MLUT04, which presented a single allele within the Spanish samples. A total of 64 alleles  were obtained. Private alleles were found in both Eastern (NE + SE) and W populations but at different percentages, 52.46 % and 9.38 %, respectively  had the lowest and highest levels of genetic variability, respectively, whereas SE populations showed intermediate values Table . Individk = 4  for an arrangement of populations in k = 4 clusters, corresponding to the lineages identified by STRUCTURE.The Bayesian clustering procedure  obtained= 4 Fig. . Individ= 4 Fig. . Most instimated   and drainage (microsatellite dataset II) levels. The only exception was the comparison between Garonne and Charentes rivers in France , whose populations are geographically close , and the North Dvina and Volga  rivers in France and NE, respectively . However, no genetic structuring was supported when STRUCTURE results  were considered. The genetic variation in the data set was significantly explained by variation among and within populations  and Spanish (SP) populations in relatively recent times. The analyses gave estimations of divergence times among these populations  is one of the most endangered mammals on the continent showing only a few scattered populations restricted to three separated regions. Human pressure  is likely the main underlying cause of the current critical situation of European mink populations. Other factors affecting population viability such as past demographic events and the ecological dependence of the species on riparian habitats might also be responsible for local declines. The present study investigates the population genetics of the European mink based on a better survey of the distribution range of Mustela lutreola and a more representative number of specimens per locality with respect to prior studies [et al. [The European mink  and W European populations, respectively. At a more local scale, Eastern Europe was differentiated into NE and SE populations, and Western Europe into French and Spanish populations. The FCA plotting analysis also supported this pattern. Isolation due to large habitat fragmentation produces strong genetic drift among regions, and this may ultimately result in the significant genetic differences (NE vs. SE vs. France vs. Spain) detected in this study.At a large geographical scale, Bayesian clustering, FPicea abies [Rana arvalis [Ips typographus [M. lutreola was able to survive glacial maxima in Eastern Europe is further supported by the non-significant results of performed neutrality tests. Bayesian skyline plot analysis also indicated a scenario of stable populations, suggesting that the demographic course of European mink was not seriously affected by Quaternary climate oscillations in the Late Pleistocene.However, our Bayesian (ABC) results also indicate that the existence of these genetic units is relatively recent, and that they were historically connected after the last glaciation. In the network reconstructions, Eastern European (NE + SE) populations retained the most abundant missing intermediate haplotypes and showed the highest levels of genetic diversity, supporting the hypothesis that the European mink survived glacial periods across an extensive geographic area covering Eastern Europe around the central Russian Upland , 11 in aea abies , Rana ar arvalis  or Ips tographus ) have inographus , supportographus , 33. TheThe AMOVA results showing no significant geographic structuring among regions strengthen the hypothesis of a rapid post-glacial expansion. According to the refuge theory , 34, rapThe current distribution of European mink is closely linked to river courses, which could be the main force driving the historical and current genetic structuring of the species. We tested such hypothesis, and significant differentiation was detected associated to river drainages. However, this result could be an artifact since inclusion of populations with low numbers of individuals such as the Danube River population may have lead to overestimation in the detection of genetic structure of the species. In fact, AMOVA results on samples clustered according to different rivers were non significant. This suggests that European mink may be capable of dispersal across river drainages over long periods of time, provided that natural conditions are favorable, which still seems to be the case in some parts of the NE region, and likely was throughout Europe during the rapid postglacial expansion of European mink. In particular, field studies corroborate important movement capacity of the European mink during the reproductive period in populations with low densities, when animals are looking for potential mates, moving up to 40–50 km during these periods .At present, however, natural conditions in riverine habitats are not that favorable for survival in much of the distribution of the species, and the significant levels of heterozygosity excess detected together with the results of genetic bottleneck analyses suggest that European mink has suffered recent (human-mediated) genetic bottleneck processes, which might result in local extinctions. This would be consistent with the reported overall demographic decline of the species during the past century . Only inThe origin of the W population of European mink has been the subject of a hot debate –38. Our The ultimate causes of the observed genetic uniformity of the W population could be explained by several working hypotheses. If European mink was not present in the Western region until recently, low genetic diversity could be attributed either to natural migration of European mink into Western Europe from Central Europe or to a putative human-mediated introduction of individuals from Central and/ or Eastern Europe. If the presence of the species in Western Europe is old enough, a rapid decline of the population in France for unknown reasons causing a severe genetic bottleneck followed by restoration of the population could also explain the observed genetic pattern. Alternatively, other more general but less obvious causes influencing also other species than the European mink could be underlying the evolutionary process. Unfortunately, the poor fossil record of European mink in the Western region and the uncertainty associated to estimated divergence times based on genetic data do not allow dating when exactly the species started inhabiting the region. Moreover, the rapid extinction process of European mink from Central Europe precludes any valid conclusions with regard to the feasibility of competing hypotheses. Future studies that may be capable of obtaining genetic data from museum specimens originated in Central Europe, as well as other focused in the population genetic structure of other related species  could help discerning among the above-mentioned hypotheses.Since two-four isolated units of European mink exist, conservation criteria could be targeted at maintaining current genetic differences in these regions by managing them separately. However, our results indicate that throughout most of their evolutionary history, European mink populations were connected, and indeed likely formed a panmictic unit. Thus, the isolation of the units is viewed here as a recent human-induced event. The vast genetic differences detected among the distinct geographic areas are likely the result of the drastic population decline occurring during the past two centuries, although a founder effect cannot be precluded as an alternative source of the marked genetic divergence signature observed in W populations .in-situ actions  would minimize other factors increasing the extinction risk associated with the European mink [Hence, our results suggest that to return to the historical optimum of the species, European mink populations should actually be managed as a single management unit, and that strategies should be taken to promote gene flow among scattered existing populations. Actions to improve genetic connectivity are considered key to counteract inbreeding depression, as well as to preserve evolutionary potential and ensure long-term survival of endangered species –41. Withean mink –45.Based on both mt and nuclear (microsatellite) data, we found that the European mink populations of the Eastern European region (NE and SE) hold the highest genetic diversity. In contrast, the low values of genetic polymorphism and the structure detected in the W populations could be the result of founder events, possibly followed by genetic depauperation. Our results suggest that European mink populations were stable during Late Pleistocene climate oscillations, expanded through river connections after the last glaciation period, and later suffered relative high levels of extinction . Moreover, European mink likely experienced recent bottleneck events throughout Europe, consistent with the reported overall demographic decline of the species during the past centuries. According to the genetic patterns observed in this study, management strategies aimed to halter the ongoing decline of the endangered European mink should promote gene flow among scattered populations in order to improve genetic connectivity.n = 76), Spain (n = 94), Russia (n = 89), Estonia (n = 3), Belarus (n = 28), and Romania (n = 54). The performed research did not involve any experiment with animals and complies with international ethic guidelines. Samples were either stored in 96 % ethanol or frozen at −20 °C. For genetic analyses, each mtDNA and microsatellite data from all individuals were grouped according to i) their regional distribution (datasets I) into Northeastern Europe , Southeastern Europe , and Western Europe  or ii) their river drainage distribution (datasets II) into the Northern Dvina (Severnaya Dvina), Volga and Western Dvina (Zapadnaya Dvina) in Russia, Belarus and Estonia; the Danube in Romania; the Charentes, Garonne and Adour in France, and the Ebro and Cantabrian rivers in Spain  samples were obtained from mink captured for tracking purposes, from specimens in museum collections and from traffic-killed individuals in France  were usMustela lutreola [Mustela erminea  and Neovison vison  [et al. [et al. [A total of 11 polymorphic microsatellite loci were analyzed. Five specific loci  were isolated directly from lutreola , whereas Mvis75) . The PCR [et al.  and Cabr [et al. . Only in2 [e) of the Eastern (NE and SE) European mink populations . The models HKY + G [−1, which was estimated for the closely related M. erminea mtDNA complete cytochrome b and partial control region genes [8 iterations and sampled every 10,000 iterations with 10 % discarded as burn-in. To assess the robustness of parameter estimates, two independent runs were performed with identical settings and combined with LogCombiner [eτ), median estimate, and 95 % highest posterior density (HPD) limits. The generation time used to estimate the Ne was assumed to be one year [Sequences were aligned using default parameters of CLUSTALX v.2  and furt2  tests we2 . Statist2 . Bayesia HKY + G  and F81  HKY + G  were sel HKY + G , 60. Simon genes . A pieceCombiner . Tracer Combiner  was usedone year .Φ-statistics [p-values were adjusted using Bonferroni correction [Genetic structure among populations was determined by estimating pairwise differences between haplotypes. atistics  were estrrection .8 generations, sampled every 1000 generations (10 % of trees were discarded as burn-in). Robustness was assessed through non-parametric bootstrap [8 iterations and sampled every 10,000 iterations with 10 % discarded as burn-in using TreeAnnotator. Different models of coalescence were applied as the tree prior to investigate consistency in the results. MCMC analyses were run for 20 million iterations and sampled every 2000 iterations. The reconstructed trees had the same 50 % majority-rule consensus topology as shown in Fig. All sequences were collapsed into distinct haplotypes using DNASP v.5  and combootstrap  proportiootstrap  was seleootstrap , 60. MCMIntraspecific genetic variation was determined using two different networking approaches. First, mtDNA haplotypes were plotted on a median-joining (MJ) network applying NETWORK v.4.112 . After nA), private alleles, allelic diversity (A), as well as observed (Ho) and expected (He) heterozygosities [P-values were adjusted with Bonferroni procedures that correct for the effect of multiple tests [IS [Genetic diversity was estimated per locus and per population based on total number of alleles , the proportion of falsely significant values (α type I error) was in all cases lower than the intended value of 5 %.The possible existence of null alleles and their frequencies were inferred using the EM algorithm  as implek) was calculated with no prior population information and an admixture model. We performed 10 series of independent runs for K from one to ten populations, setting default values with constant lambda (λ) and the same alpha (α) values for all populations. MCMC consisted of 105 burn-in iterations followed by 106 sampled iterations. Further, the modal value of lambda, ∆k [k. Clusters were depicted using Distruct v.1.1 [Two different Bayesian clustering methods were used to determine the population structure of the European mink. First, we used STRUCTURE v2.2 . The nummbda, ∆k  was alsoct v.1.1 .http://fisher.berkeley.edu/structurama/) was used to verify congruence among results obtained with STRUCTURE, assuming that the number of populations is a random variable that follows a Dirichlet process prior [4 MCMC cycles were run.In addition, STRUCTURAMA v1.0  is maximized. The geographic coordinates for each region were calculated as the centre of the different localities. We tested a range of values of k from 2 to 5, using 100 simulated annealing steps.Finally, a spatial analysis of molecular variance, , and RSel, SMM; ). The siel, SMM;  and Arlerrection . PatternApproximate Bayesian Computation implemented in the DIYABC v.1.0.4  was usedScenario 1 corresponds to a non-differentiation between the NE and SE populations at step 1, followed by an admixture at step 2. This leads to a new lineage corresponding to the FR population, which diverge at step 3 to give the SP population.Scenario 3 is similar to scenario 1 at the exception of step 2, which leads to a lineage corresponding the SP population. This will diverge at step 3 to give the FR population.Scenario 5 is also similar to scenario 1 but without differentiation between FR and SP populations at step 3.Scenario 2, 4, and 6 propose similar divergence as compared to the last three scenarios, respectively, at the exception of a differentiation between NE and SE populations at step 1.Scenarios from 7 to 14 are also characterized by three steps of divergence, but without admixture processes. Each of these scenarios is characterized by different possibilities of divergence leading to the four existing mink populations, from the most ancient lineages corresponding to the NE and SE populations to the most recent ones (SP and FR).The biogeographic analyses were based on the microsatellite data set. We compared fourteen different biogeographic models assuming that NE and SE populations diverged from the same ancestral (source) population or from an admixture of two populations Fig. . BecauseA total of 14 million simulations were run, providing one million simulations for each scenario. The parameters of each model  were considered as random variables drawn from prior distributions .mtDNA sequences: GenBank accession numbers, EU548035 (Mlh1), EU548036 (Mlh2), EU548037 (Mlh3), EU548038 (Mlh4), EU548039 (Mlh5), EU548040 (Mlh6), EU548041 (Mlh7), EU548042 (Mlh8), EU548043 (Mlh9), EU548044 (Mlh10), EU548045 (Mlh11), EU548046 (Mlh12), EU548047 (Mlh13), EU548048 (Mlh14), EU548049 (Mlh15), EU548050 (Mlh16), EU548051 (Mlh17), Data sets used in this study were deposited in TreeBase


Aporrectodea icterica) sampled in northern France to explore how historical species range changes, microevolutionary processes and human activities interact in shaping genetic variation at a regional scale. Because combining markers with distinct modes of inheritance can provide extra, complementary information on gene flow, we compared the patterns of genetic structure revealed using nuclear (7 microsatellite loci) and mitochondrial markers (COI). Both types of markers indicated low genetic polymorphism compared to other earthworm species, a result that can be attributed to ancient bottlenecks, for instance due to species isolation in southern refugia during the ice ages with subsequent expansion toward northern Europe. Historical events can also be responsible for the existence of two divergent, but randomly interbreeding mitochondrial lineages within all study populations. In addition, the comparison of observed heterozygosity among microsatellite loci and heterozygosity expected under mutation-drift equilibrium suggested a recent decrease in effective size in some populations that could be due to contemporary events such as habitat fragmentation. The absence of relationship between geographic and genetic distances estimated from microsatellite allele frequency data also suggested that dispersal is haphazard and that human activities favour passive dispersal among geographically distant populations.Despite the fundamental role that soil invertebrates (e.g. earthworms) play in soil ecosystems, the magnitude of their spatial genetic variation is still largely unknown and only a few studies have investigated the population genetic structure of these organisms. Here, we investigated the genetic structure of seven populations of a common endogeic earthworm ( In particular, there is little information on the amount and spatial distribution of genetic variation in earthworm populations. Few studies have simultaneously investigated the influence of historical events, such as glacial periods, and contemporary processes, such habitat fragmentation, on the genetic diversity of these species. In their review on the genetic structure of soil invertebrate populations, Costa et al. Aporrectodea icterica is an abundant species commonly found in agricultural soils A. icterica is also believed to feed at least partly on leaf litter A. icterica populations revealed the existence of two mitochondrial lineages with divergence values ranging from 10% to 11% A. icterica, nuclear analysis indicates that the two lineages interbreed Our model earthworm species, A. icterica, it was suggested that lineages came into contact and mixed during recolonisation, during the warmer interglacial periods Deep mitochondrial divergences within morphospecies can be attributed to population isolation within distinct periglacial refugia 2), comparing the spatial regional patterns obtained using mitochondrial (mtDNA) and nuclear (nDNA) molecular markers. We discuss the role of evolutionary forces including genetic drift and contemporary gene flow, large-scale landscape changes  and anthropogenic effects in structuring the genetic diversity and in the differentiation of populations.Here, we focus on the genetic structure of seven earthworm populations sampled at a regional scale . Six populations were located in >6 year-old pastures , within a distance of 3 to 10 km from the city of Yvetot . Each proprietor gave his agreement for sampling to J. Mathieu who should be contacted for future permissions. These pastures are grazed by dairy cattle from mid-March to mid-September with a stocking rate of 2–6 animal units ha−1 depending on the season, and spread with cow manure each year. Plant cover consisted mainly in Festuca elator L., Phleum pratense, Trifolium repens L., and Lolium. The seventh population was located 35 km away from Yvetot (IR), near the University of Rouen in a location for which no specific permission was required  wash and DNA precipitation with sodium acetate (3 M) and ethanol.cytochrome c oxidase subunit I mitochondrial gene (COI) was amplified and sequenced using the universal primers LCO1490 and HCO2198 A fragment of the h) and nucleotide diversity (π) were estimated using DNAsp v. 5.10 software FsR2 statistic R2 is appropriate for small sample sizes R2 and Fs were evaluated by comparing their observed values with their null distribution, generated by 10 000 random replicates using the empirical population sample size and observed number of segregating sites implemented by DnaSP v. 5.10. COI sequences were aligned manually using BioEdit v. 7.0.5.3 To describe the phylogenetic relationships between haplotypes, a statistical parsimony network was constructed using TCS v. 1.21 A. icterica populations at the mitochondrial level, the overall genetic differentiation between populations was first estimated by calculating the global Φst in Arlequin v. 3.5.1.2 software ct value, corresponding to the optimal number of groups, was selected.To investigate the regional structure of a +8°C, 10 cycles in which the temperature was decreased by 1°C per cycle, 1 min at 72°C, 25 cycles of 35 s at 94°C (except for PB10D and C4 for which 30 cycles were done), 45 s at Ta −2°C, 1 min at 72°C, with a final elongation step of 10 min 72°C. Each amplification mixture (15 µl) contained 10 ng/µl DNA, 1X reaction buffer (GoTaq Flexi buffer 5X), 2.5 mM of MgCl2 (except for Ai56 and Ai68 for which 1.5 mM was used), 0.5 µM dNTPs, 0.25 µM of each primer and 0.5 units of GoTaq Flexi DNA polymerase . PCR products were loaded on an ABI 310 sequencer along with the LIZ500 size standard; alleles were scored using Genescan software .Individuals were genotyped using seven microsatellite loci: Ai45, Ai56, Ai68, PB10D, 2PE40, 2PE70, C4 Nall) and unbiased expected heterozygosity (He) using Genepop v. 4 software Fis was calculated using Genepop v. 4 In each population, the genetic diversity was analysed by computing allelic frequencies, number of alleles (We tested for deviation from mutation-drift equilibrium in the study populations using the approach detailed in Cornuet & Luikart http://www2.unil.ch/popgen/softwares/pcagen.htm). The significance of each principal component was assessed from 1000 permutations. We also used the software STRUCTURE v. 2.3.1 To investigate the genetic structure among populations, a G-test of allelic differentiation was carried out using Genepop v. 4 To estimate recent migration rates and test for significant cases of assignment to populations other than the population of origin (i.e. first-generation dispersers) we used the Bayesian method Fst/(1−Fst) to compute a linear relationship following the recommendations of Rousset To test for the null hypothesis of independence between genetic and geographic distances, the logarithm of Euclidian geographic distances were plotted against Departures from random cytonuclear associations were tested using the CNDm programme h) ranged from 0.135 to 0.742 and nucleotide diversity (π) ranged from 0.00864 to 0.05973 ((11)≤1) while I25, I27 and IR showed relatively high values of haplotypic richness (2.5 to 3). None of the Fu's Fs and R2 values were significant.The amplified COI fragment contained a homopolymer poly-C. In most of the reactions, the sequence became mixed after the poly-C, most probably because of polymerase stutter. Sequences were thus truncated (fragment length <200 bp). Consensus sequences shorter than 374 bp were excluded of the analysis. Over the whole mtDNA data set (134 sequences), we detected 15 haplotypes defined by 34 parsimony informative sites (9%) among 44 variable sites (12%). Within populations, haplotype diversity . In terms of regional mitochondrial structure, the SAMOVA showed that the highest significant value (Φct = 0.409) was obtained when the populations were split into two groups  than the other populations (3.14<A<3.84 and 0.497<He<0.515). Depending on the population, the Hardy-Weinberg expectations (HWE) test showed a significant deviation for some of the loci . However, null alleles were suspected for several loci . No pattern of isolation by distance was observed among the six Yvetot populations . Clustering analysis  clearly Contemporary gene flow was detected at this regional scale with 64 individuals (29%) identified as first-generation dispersers. Among these 64 individuals, 30 belonged to Cluster 1, 25 belonged to Cluster 2 and the 9 remaining migrants belonged to the I19 population . Most ofThe test for overall non-random association between microsatellite alleles and mitochondrial lineages revealed significant cytonuclear disequilibrium after Bonferroni correction for four of the most polymorphic microsatellite loci . Three aThe test was also carried out within each population that showed both lineages, with the rarest representing at least 30% . The association between allele 129 at the Ai56 locus and L1 was suggested in the IR population, but was not significant after Bonferroni correction (p = 0.037). In addition, there was a trend for an association between allele 178 of the PB10 locus and L2 (p = 0.092) in the I20 population.A. icterica populations using microsatellite markers and COI sequences was surprisingly low (A) ranged from 2.57 to 4.14, but values of 5 to 17 alleles have been reported in other earthworm species .A relatively high level of genetic differentiation was revealed among localities, regardless of the marker used. Interestingly, no relationship between genetic and linear geographic distances was observed at this regional scale (populations separated by less than 13 km in the Yvetot area), corroborating other earthworm population genetics studies  mitochondrial lineages were observed within studied populations of ed to L2 . In RoueA. icterica, our results indicate that the two divergent lineages were randomly interbreeding with respect to mtDNA haplotypes over a relatively restricted geographical area. Deep mtDNA divergence despite clear interbreeding can reflect long periods of geographical isolation followed by secondary contact favouring gene flow, homogenising the nuclear genome over time.Divergent sympatric mitochondrial lineages often reveal the existence of cryptic species, particularly when divergence is confirmed in the nuclear compartment of the genome and/or when reproductive isolation between lineages has been demonstrated A. icterica lineages originated from distinct refugia and that they merged during post-glaciation recolonisation. MtDNA divergence may thus be the result of neutral differences within the species, representing a historical mark of divergent lineages that have remerged A. icterica has small effective population size and has undergone serial population bottlenecks during the process of post-Pleistocene recolonisation in northern Europe, further accentuated by recent bottlenecks due to habitat fragmentation. We therefore assume that A. icterica lineages have come into contact too recently for lineage sorting to be completed. Furthermore, human activities could be, at least in part, responsible for the merging of two divergent lineages. For instance, the contribution of historical human activities to the current pattern of spatial genetic structure was documented for numerous plant species  the importance of historical events for explaining their current genetic variation and (ii) the weak relationship between genetic and geographic distances suggesting the importance of passive dispersal in structuring earthworm populations. Nevertheless, some uncertainties persist such as the underlying cause of the mito-nuclear discordance and the respective roles of active


Acacia woodmaniorum.Understanding patterns of pollen dispersal and variation in mating systems provides insights into the evolutionary potential of plant species and how historically rare species with small disjunct populations persist over long time frames. This study aims to quantify the role of pollen dispersal and the mating system in maintaining contemporary levels of connectivity and facilitating persistence of small populations of the historically rare A. woodmaniorum were genotyped with nine polymorphic microsatellite markers. A low number of fathers contributed to seed within single pods; therefore, sampling to remove bias of correlated paternity was implemented for further analysis. Pollen immigration and mating system parameters were then assessed in eight populations of varying size and degree of isolation.Progeny arrays of tm = 0·975) and a lack of apparent inbreeding suggested that a self-incompatibility mechanism is operating. Population parameters, including size and degree of geographic disjunction, were not useful predictors of pollen dispersal or components of the mating system.Pollen immigration into small disjunct populations was extensive (mean minimum estimate 40 % and mean maximum estimate 57 % of progeny) and dispersal occurred over large distances (≤1870m). Pollen immigration resulted in large effective population sizes and was sufficient to ensure adaptive and inbreeding connectivity in small disjunct populations. High outcrossing (mean A. woodmaniorum. It is proposed that maintenance of genetic connectivity through habitat and pollinator conservation will be a key factor in the persistence of this and other historically rare species with similar extensive long-distance pollen dispersal and highly outcrossed mating systems.Extensive long-distance pollen dispersal and a highly outcrossed mating system are likely to play a key role in maintaining genetic diversity and limiting negative genetic effects of inbreeding and drift in small disjunct populations of  Patterns of pollen-mediated gene flow and variation in the mating system directly influence levels of genetic diversity, levels of genetic connectivity and genetic structure, and are key to the evolutionary potential of plant populations  small effective population size, and geographically disjunct and patchily distributed populations with geographically restricted ranges . In accoAcacia woodmaniorum sect. Alatae is one historically rare edaphic endemic of WA's BIF outcrops that has a distribution suitable for investigation of the impacts of small disjunct populations on patterns of pollen dispersal and mating system parameters. Populations occur over two relatively large series of BIF ranges, and a number of small populations are located on small BIF outcrops at varying distances from the main ranges. The taxon was only recently described  as it is rich in historically rare plant species with typically small population size and disjunct and patchy population distributions. Within this landscape, banded iron formation (BIF) inselbergs act as ecological islands that support an especially large number of edaphically endemic and historically rare species. escribed , and litA. woodmaniorum has been historically restricted, with long-term persistence of some small and disjunct populations along with large populations in the main range. Evidence from indirect F-statistic-based estimates has indicated that seed and/or pollen dispersal in A. woodmaniorum is sufficient to maintain a moderate degree of genetic diversity with some broad genetic structure among populations  How extensive is pollen immigration into spatially disjunct, small populations? (2) Over what distances does pollen dispersal occur? (3) What are the parameters of the mating system, including rates of outcrossing, inbreeding and correlated paternity within pods? (4) Is pollen immigration, outcrossing rate or the level of inbreeding in A. woodmaniorum affected by population size or the degree of isolation?Patterns of phylogeographic and genetic structure suggest that the distribution of Acacia woodmaniorum (Maslin and Buscomb) sect. Alatae (Benth.) is a sprawling, prickly, woody shrub 1–2 m tall and up to 2 m wide. Its small, globular, racemes are comprised of many light golden flowers. Pods are dark brown, narrowly oblong, curved, and sometimes twisted and 10–45 mm long. The pods bear small, hard coated seeds 3–4 mm long and dark greyish brown to black in colour, that are released following rapid dehiscence of the pods. Intensive surveillance over the species range, which covers <40 km2 in WA, has mapped approx. 25 000 known plants. These are highly substrate specific to the skeletal soils of the steep slopes, rock crevices and gullies of low-altitude BIF outcrops and can be broken down into two main geological and geographic regions  woodland with other Acacia, Allocasuarina, Melaleuca and eucalypt species, and surrounding low hills by Senna, Eremophila and Acacia shrubs  across the species range at 15 nuclear microsatellite loci and collected detailed spatial co-ordinates with a differential Global Positioning Satellite system for each sampled plant  within each selected population. However, pod production and seed-set was low in idelines . NecessaAcacia species, the number of pollen grains in the polyad is correlated with the number of ovules in the ovary so that a single polyad pollinating a stigma is capable of fertilizing all ovules within a flower  at a site within the main range , using the sibling pair method in the MLTR v 3.4 program  was determined for each population using CERVUS  per population ranged from two to six Table . Given ttm), the single locus outcrossing rate (ts), the apparent level of selfing due to biparental inbreeding (tm – ts), the correlation of selfing among maternal plants (rs) and the multilocus correlated paternity (rpm). Parameters were estimated using the expectation–maximization algorithm, with pollen and ovule gene frequencies estimated separately and standard errors obtained with 1000 bootstrap replicates, with families as the re-sampling units, and used to assess whether values were significantly different from one or from zero. Wright's fixation index was estimated for the parental (Fpar) and progeny (Fprog) generations using GenAlEx, and the effective number of pollen donors per maternal tree was estimated as Ne = 1/rpm. Regression analysis was conducted for the outcrossing rate and the degree of biparental inbreeding, and for the outcrossing rate and population parameters.The use of nine highly variable microsatellite loci ensures that progeny array sizes as small as two are informative in relation to mating system estimates see . We usedA. woodmaniorum progeny cohorts, with a total of 60 alleles detected in 525 seedlings. A total of 98·77 % of loci were polymorphic, the mean number of alleles per locus was 4·123 (±0·194), the mean number of effective alleles per locus was 2·070 (±0·094), and expected and observed heterozygosities were 0·445 (±0·022) and 0·490 (±0·029), respectively. Progeny allele frequencies per population are provided in Supplementary Data Table S1.Population size was significantly positively correlated with degree of population isolation and with degree of isolation from large populations. The degree of population isolation was also significantly correlated with the degree of isolation from a large population Table . The ninrpm = 0·492 (±0·080), indicating that an average of two fathers sire all seed within pods.Multilocus correlated paternity within pods calculated using the sibling pair method in MLTR was high, The probability that the set of loci will exclude an unrelated candidate male parent from paternity of an arbitrary progeny when the genotype of the mother is known varied for the eight populations and averaged 0·944 Table . Rates oNeither the minimum nor the maximum pollen immigration rate (CERVUS) was significantly correlated with any population parameter Table . MinimumR2 = 0·0372, d.f. = 7, P = 0·6472), degree of isolation  or isolation from a large population . Correlation of outcrossing or selfing among maternal plants (rs) and mean biparental inbreeding (tm – ts), were low for all populations. There was no statistically significant correlation between the degree of biparental inbreeding and population parameters of size , degree of isolation  or isolation from a large population . Multilocus correlated paternity was generally low and not statistically significantly correlated with population parameters of size , degree of isolation  or isolation from a large population . Estimated numbers of pollen donors among pods within maternal trees (Ne) varied  and occurred over large distances (≤1870m), confirming previous FST-based estimates of high levels of gene flow across the species' range, including between small disjunct populations  dispersal associated with prevailing wind conditions, indicating that generalist insects carrying pollen loads may be conveyed over long distances via thermal updrafts . This observation suggests that poor pod-and seed-set in A. woodmaniorum was not solely a result of adverse temporal environmental conditions and, combined with high levels of abortion of developing pods and seed , may indicate post-zygotic seed abortion or the effect of inbreeding depression following self-pollination or when pollination occurs between related individuals.Despite its rarity, short range and persistence in small populations, mpatible . Self-inA. woodmaniorum are not mate limited due to extensive pollen dispersal , due to its highly restricted distribution and the prospective mineral exploration and active extraction activities that cover its range. Future anthropogenic disturbance is also likely in this landscape, and loss of populations may impact gene flow patterns and thus influence population persistence. A number of conservation measures that aim to alleviate negative genetic and demographic impacts of reduced connectivity can be employed for the long-term conservation of recently fragmented species and those for which further or future population fragmentation is envisaged. Maintenance of gene flow can be achieved by the direct augmentation of populations or establishment of populations at previous or new sites with germplasm sourced from a number of different populations. Genetic augmentation is likely to improve mate availability and reproductive output, but must also take into account the likelihood of any fitness reduction via outbreeding depression in the resulting progeny (A. woodmaniorum given the habitat specificity and limited geographic range, and this, in combination with a highly outcrossed mating system, suggests that outbreeding depression is unlikely to be an issue. Levels of genetic diversity (Maintenance of genetic connectivity through significant pollen-mediated gene flow over extensive dispersal distances, and high levels of outcrossing, are important features of Acacia woodmaniorumSupplementary data are available online at www.aob.oxfordjournals.org and consist of Table S1: Allele frequencies of nine nuclear microsatellite loci in progeny cohorts from nine populations of .


Sitobion avenae from areas north and south of the Qinling Mountains, and characterized their molecular variation at seven microsatellite loci and quantitative variation at nine life-history traits. Our results demonstrated that southern populations had slightly longer developmental times of nymphs but much higher lifetime fecundity, compared to northern populations. Of the nine tested quantitative characters, eight differed significantly among populations within regions, as well as between northern and southern regions. Genetic differentiation in neutral markers was likely to have been caused by founder events and drift. Increased subdivision for quantitative characters was found in northern populations, but reduced in southern populations. This phenomenon was not found for molecular characters, suggesting the decoupling between molecular and quantitative variation. The pattern of relationships between FST and QST indicated divergent selection and suggested that local adaptation play a role in the differentiation of life-history traits in tested S. avenae populations, particularly in those traits closely related to reproduction. The main role of natural selection over genetic drift was also supported by strong structural differences in G-matrices among S. avenae populations. However, cluster analyses did not result in two groups corresponding to northern and southern regions. Genetic differentiation between northern and southern populations in neutral markers was low, indicating considerable gene flow between them. The relationship between molecular and quantitative variation, as well as its implications for differentiation and evolution of S. avenae populations, was discussed.Quantitative trait differences are often assumed to be correlated with molecular variation, but the relationship is not certain, and empirical evidence is still scarce. To address this issue, we sampled six populations of the cereal aphid  Barriers to gene flow presented by mountain ranges can have significant consequences for ecology and evolution of various organisms, and even lead to the isolation of populations. The isolation of populations may in turn cause allopatric speciation in different environments where reproductive isolation between populations could occur gradually and incidentally as a result of mutation and drift, and indirect effects of local selections  –2. The QSignificant geographic variation in life-history traits  can occur among populations of a species separated by environmental barriers like the Qinling Mountains . DetermiSitobion avenae (Fab.)), a serious pest on cereal crops 0.5 The Flury hierarchical method was utilized to compare the resulting G matrices in the software CPCrand [Our bioassays use clonal genotypes, and this design allows us to estimate the total variance of a particular quantitative trait (ariance) . Broad-sariance) . Geneticariance) . The gen CPCrand . Based o CPCrand ). Briefl CPCrand ). The st CPCrand , 17.QST values were evaluated using genetic variances and defined as QST = VGB /(VGB + 2VGW), where VGB is the genetic component of the variance between population means and VGW is the average genetic variance within populations [QST was estimated between each pair of populations, as well as between northern and southern regions. Differentiation among populations within a region was evaluated similarly as QSR. Pairwise population QST values  were also computed for the composite life-history trait, which was the first component obtained from the PCA performed on abovementioned quantitative traits. Following Chapuis et al. [QST was considered different from corresponding FST if their respective 95% confidence intervals did not overlap. Another method of evaluating the variation in genetic variances for comparison among characters and populations is the coefficient of genetic variance  [ulations . QST was et al. , QST waenotype) .Over 40 clones per population were randomly selected and genotyped at seven microsatellite loci  following the procedures described in Simon et al. .FST (θ) [FIS (f) [HE) [FST (hereafter referred to as FSR), FIS, HE, Ho (observed heterozygosity), and RS  were also estimated and their differences between regions were tested by performing 1000 permutations of alleles among individuals. The abovementioned F-statistics and related tests were conducted in FSTAT version 2.9.3 [Using identified microsatellite loci, population differentiation was assessed by calculating pairwise FST (θ)  between FIS (f)  were estf) [HE)  for eachon 2.9.3 .We also analyzed the microsatellite data with an individual based principal component analysis (PCA) using the program PCA-GEN version 1.2.1 . This anNorthern populations showed significantly reduced developmental time for the first, second and third instar nymphs, but not for the fourth instar nymphs . The tot2clone). However, the coefficient of genetic variance (CVG) and mean broad sense heritability (H2) for the populations in the northern region were both significantly higher than those in the southern region.Genetic difference between northern and southern populations was shown in S. avenae populations from both regions (Significant genetic correlations were found between life-history traits for  regions . SignifiP < 0.01), and between those for Longting and Mianxian from the southern region , in other words, matrices shared only one of the nine possible principal components. However, unrelated structures were found for all other comparisons . So, G-matrices comparison showed significant structural differences among populations within regions, as well as between regions. The strong structural differences identified can be interpreted as a result of divergent selection among populations.G-matrix comparisons by Flury’s method and jump-up approach  showed significant differences between paired matrices of life-history traits . The CPCP > 0.05). The differences in allelic richness (RS), observed heterozygosity (Ho), and inbreeding coefficient (FIS) between the two regions were not significant  or Mianxian (FST = 0.1083). Substantial molecular variation was also found between population Jinshui and population Longting (FST = 0.1197) or Mianxian (FST = 0.1228).Pairwise Mianxian . MoleculQST values varied from 0.0094 for population Luochuan vs. population Tongchuan to 0.6215 for population Fuping vs. population Luochuan. In terms of quantitative traits, population Fuping was significantly different from population Luochuan (QST = 0.6215) or Tongchuan (QST = 0.5554). Population Luochuan from north of the mountains differed substantially from all of the populations from south with pairwise QST values ranging from 0.3181 to 0.5936, and similar substantial variation was found between population Tongchuan and any population from south (pairwise QST varied from 0.2624 to 0.524). However, differentiation in quantitative traits was low between population Fuping of the northern region and any population of the southern region (pairwise QST varied from 0.0102 to 0. 0808). Quantitative trait differentiation between populations from south of the mountains was also found to be low with QST values varying from 0.0377 to 0.0612. Of all the pairwise comparisons, eight of them showed significantly higher QST than corresponding FST.Pairwise QST values of the developmental times of first to fourth instar nymphs and the total developmental time of nymphs were not significantly higher than corresponding FST values .The T values . HoweverQSR) was significantly higher for northern populations than that for southern population, but there were no significant differences in molecular subdivision  between northern and southern regions  in the north was significantly higher than molecular subdivision among populations (FSR) in the same region. FSR appeared to be higher than QSR in the south, but no significant differences were found between the two parameters.The magnitude of subdivision in quantitative traits  and then phenotyped [QST and FST.Many studies use molecular markers as a surrogate for adaptive genetic variation , and verenotyped , allowinQSR and CVG), but not in molecular characters. The decoupling of molecular and quantitative variation was also evident in PCA analyses where clustering patterns from molecular and quantitative trait data were distinct. The conversion of non-additive variation underlying quantitative traits to additive variation could occur due to isolation and drift following bottlenecks, and quantitative variation can be increased as a result [S. avenae lives on highly seasonal and sometimes unpredictable habitats [FST = 0.018), indicating populations from the two regions shared the majority of identified genotypes. Therefore, high dispersal ability of S. avenae and the resulting gene flow can be an important factor in retarding the molecular differentiation between populations from both sides of the Qinling Mountains. The different pattern for molecular and quantitative traits might also be simply explained by differences in mutation rates, because mutation rates for quantitative characters are typically several orders of magnitude higher than those for molecular characters [In our study, northern and southern populations showed significantly different quantitative traits and G-matrices, so high quantitative variation was evident between populations from the two regions. However, microsatellite data did not show the expected variation between both types of populations. In addition, populations within the northern region had increased subdivision in quantitative traits  differed significantly between northern and southern regions, as well as among populations within a region. However, northern populations showed a higher DT4 than southern populations in our previous study [ differedFST ranging from 0 to 0.1228  have been reported in response to changes in selective pressure [FST , indicating divergent selection among S. avenae populations. There were over half pairwise population comparisons in the QST–FST analysis for the composite life-history trait that yielded higher QST values than their corresponding FST estimates , providing additional evidence of divergent selection. Populations of S. avenae that are better adapted to seasonal and ephemeral habitats are expected to have higher fitness under our experimental conditions (e.g. higher lifetime fecundity). In this study and [S. avenae. Another way to test for local adaptation is to perform reciprocal transfer of individuals in wild populations, and it is logistically demanding and extremely difficult to carry out such experiments [S. avenae.The role genetic drift plays in genetic differentiation of cumented –35, but pressure . Howevertudy and , southereriments ,36. HoweS. avenae is not unexpected in our study, because the separation of Qinling Mountains might cause the isolation of its populations. In addition, the pattern of natural selection can vary greatly between areas north and south to the Qinling Mountains because of different environmental conditions . The mountains cause harsh winter weather conditions in the north and mild ones in the south. The differences in winter weather conditions can have significant consequences for S. avenae’s life-history, for example, the reproductive mode of S. avenae clones in Romania (harsh winter) was different from that in France (mild winter) [The occurrence of local adaptation in  winter) . Thus, l winter) , naturalS. avenae individuals seemed to be able to disperse across the Qinling Mountains because cluster analyses using both quantitative trait  indicated a significant amount of gene flow between them. Therefore, the Qinling Mountains did not result in the isolation of S. avenae populations in our study area, and allopatric speciation for S. avenae appears to be highly unlikely.Despite the highly likely occurrence of local adaptation in northern and southern populations, tudy and ) and micD. noxia . On the Sitobion avenae presents a good model to study how differentiation can evolve among populations of small organisms with high dispersal ability, which is crucial to understand speciation and the match between environment and phenotype [S. avenae populations. Temperature is a particularly likely candidate for the cause of differences between populations identified in our study. Because development is closely tied to temperature, natural selection might cause individuals from colder, northern climates to develop more rapidly than southern ones when raised under common laboratory conditions. The structure of G-matrix for S. avenae’s quantitative traits also has implications for its population differentiation and evolution. Interestingly, quite a few negative covariances  were found between quantitative traits measured in our study. These trade-offs  may play a role in slowing the evolution of habitat specific S. avenae genotypes and thus slowing the differentiation of S. avenae populations in our study. The G-matrix structure of different quantitative traits for aphid genotypes have complex relationships with factors like genotype specialization, trade-offs, and genotype-by-environment interactions [S. aveane populations represented a rare phenomenon, because empirical studies have generally supported the stability of G-matrices for populations of the same species [S. avenae, as well as in its evolutionary potential.henotype . Furtherractions . The ide species . Further