Haemonchus contortus in the Punjab, Pakistan. This region provides an opportunity to compare H. contortus populations that have been subjected to a prolonged period of frequent benzimidazole drug treatments on government farms with parasite populations that have been exposed to little or no drug treatment in neighbouring pastoral herds.The impact of drug selection pressure on the overall genetic diversity of parasitic nematode populations in the field is poorly understood. In this study, we address this issue for the small ruminant parasite H. contortus worms were collected from the abomasa of small ruminants from three government farms frequently using benzimidazole drugs, and closed to animal movement, for over 30 years and also from from eighteen pastoral herds subject to minimal drug selection. The frequency of three known benzimidazole resistance associated mutations was determined in each parasite population. For the seven parasite populations in which resistance mutations were found, the diversity, geographical distribution and phylogenetic relationships of isotype-1 β-tubulin benzimidazole resistance haplotypes were determined. In addition, the genetic diversity of the parasite populations on the three government farms were compared with those from four pastoral herds.Adult AC) resistance mutation was present at a very high frequency in H. contortus populations from government herds, but not from pastoral herds, consistent with their respective drug selection histories. Population genetic analysis, using a panel of microsatellite markers, revealed that there was little genetic differentiation among the parasite populations with no significant difference in the overall genetic diversity between government and pastoral herds. In addition, sequence analysis of the isotype-1 β-tubulin locus revealed multiple F200Y (TAC) haplotypes demonstrating soft selective sweeps even in government herds with little or no contemporary parasite migration.The F200Y  contains supplementary material, which is available to authorized users. Haemonchus contortus is one of the most economically important helminth parasites of small ruminants worldwide and is an important model for anthelmintic resistance research [Anthelmintic resistance in parasitic nematodes is a global threat to sustainable livestock production . Howeverresearch . This paresearch , 4.H. contortus. The F200Y (TTC to TAC) mutation has been found in every country examined to date and is often present at high frequency [TC to TAC) and E198A (GAA to GCA) have also been reported in multiple countries but have a more variable occurrence and are generally present at lower frequency than the F200Y (TTC to TAC) mutation [CA) mutation present in multiple populations in southern India was derived from a single origin [Benzimidazole drugs have been used to control livestock parasites for over 40 years and resistance to this drug class is at an advanced stage in many parts of the world , 6. We hrequency –9. We harequency , 10. Themutation –7, 11. WH. contortus in a variety of different countries to provide insights into its origin and spread [H. contortus populations have moderate to high levels of benzimidazole resistance [H.contortus worms collected from abattoirs in the Punjab and North West Frontier provinces revealed just a 7.86 % frequency of the F200Y (TAC) mutation [We are investigating the population genetics of benzimidazole resistance in d spread , 10, 12.sistance –7. In thsistance , 14. Fecsistance . The seqAC) benzimidazole resistance mutation is present at very low frequencies in eighteen H. contortus populations sourced from sheep and goats from rural locations in the Punjab Province of Pakistan. In contrast, this mutation is present at very high frequencies in three government farms that have used frequent and regular anthelmintic treatments for over 30 years. In spite of this difference in drug selection, population genetic analysis, using a panel of microsatellite markers, revealed no significant difference in the overall genetic diversity between the government and pastoral herds. In addition, phylogenetic analysis of isotype-1 β-tubulin haplotypes reveals multiple F200Y(TAC) haplotypes are present in H. contortus on the closed government herds suggesting the presence of soft selective sweeps in the absence of significant contemporary parasite migration.In the present paper, we show that the F200Y , one from the Sargodha abattoir (Pop24S), one from the Jahangirabad government farm (Pop3S) and one from the Okara government farm (Pop1S). Sixteen of these H. contortus populations were collected from goats comprising six from the Lahore abattoir , three from the Okara abattoir , two from the Sahiwal abattoir , four from the Gujranwala abattoir  and one from the Layyah government farm (Pop2G)  was established in 1980 from angora goats sourced from Turkey which were crossed with locally sourced goats of the “hairy breed”. This herd has been treated with anthelmintics (levamisole HCL and oxfendazole) approximately every 3 months since its establishment and has been closed to animal movement since that time. The Okara (Pop1S) and Jahangirabad (Pop3S) government farms were established using local sheep breeds (lohi and kajli) in 1985 and 1989 respectively. Both these herds have been treated alternately with albendazole and oxfendazole approximately every 3 months since their establishment. There has been some historical movement of sheep between the Okara and Jahangirabad farms but these herds have otherwise been closed to animal movement since they were established.Adult worms were fixed in 70 % ethanol immediately following removal from the host abomasum. The heads of individual worms were dissected and lysed in single 0.2 μl tubes containing 50 μl of proteinase K lysis buffer and stored at -80 °C as previously described . One micH. contortus in these populations as previously reported [H. contortus worms were identified per population . Individual H. contortus DNA lysates were pooled for each population for isotype-1 β-tubulin genotyping.A combination of gross morphological identification and genotyping of the SNP at position 24 of the ITS-2 rDNA was used to confirm the identity of reported . A minimH. contortus populations  and a previously published primer pair was used; forward primer (HcPYRF: 5′-GAC GCA TTC ACT TGG AGG AG-3′) and reverse primer (HcPYRR: 5′-Biotin-CAT AGG TTG GAT TTG TGA GTT-3′) [A 328 bp fragment, spanning exons 4 and 5 and intervening intron of the isotype-1 β-tubulin gene was PCR amplified from pooled DNA lysates for each of the 21  GTT-3′) . The theAC), E198A (GCA) and F200Y (TAC) isotype-1 β-tubulin SNPs in the amplicons derived from each parasite population were determined by pyrosequence genotyping using the PryoMark ID system using the allele quantification (AQ) mode . Previously published sequencing primers were used for the F167Y (TAC), E198A (GCA) and F200Y (TAC) mutations; Hcsq167: 5′-ATA GAA TTA TGG CTT CGT-3′, Hcsq198: 5′-ACT GGT AGA GAA CAC CG-3′ and Hcsq200: 5′- TAG AGA ACA CCG ATG AAA CAT-3′, respectively [Following PCR amplification, the relative frequency of the F167Y  was detected . Primers HcPYRF: 5′-GAC GCA TTC ACT TGG AGG AG-3′ and HcPYRR: 5′-CAT AGG TTG GAT TTGTGA GTT-3′ were again used but with the following PCR reaction conditions; 5X Phusion HF reaction buffer, 2 mM MgSO4, 200uM dNTPs, 0.2uM forward and reverse primers and 1U of Phusion high fidelity DNA polymerase . The thermo-cycling parameters of isotype-1 β-tubulin consisted of an initial 98 °C for 30 s followed by 35 cycles of 98 °C for 10 s, 59 °C for 30 s and 72 °C for 1 min with a single final extension cycle of 72 °C for 5 min. Amplicons were cloned into PJET 1.2/BLUNT vector  and sequenced using previously described standard procedures [H. contortus isotype-1 β-tubulin sequence (Acc. No. X67489) and edited using Geneious Pro 5.4 software [The same 328 bp isotype-1 β-tubulin fragment described above was also amplified, cloned and sequenced from the pooled lysates from the seven software . A previHaemonchus placei sequence was used to root the H. contortus network.Networks based on genetic distance were computed using the neighbour-net method employed in SplitsTree4  to produH. contortus microsatellites were screened to produce a panel of eight markers that consistently amplified from the Pakistan populations under study (data not shown) [Previously described t shown) , 22, 23.ST) values were calculated from the multi-locus microsatellite genotype data, by random permutation in Arlequin 3.11 [H. contortus , allele richness and estimation of inbreeding coefficient (FIS) for each locus were calculated using Arlequin 3.1  and allele richness (Ac) were also calculated per locus for each population with correction for unequal sample size using POPTREE2 [Fixation index  and the relative frequencies of the three currently known benzimidazole resistance-associated SNPs F167Y (TAC), E198A (GCA) and F200Y (TAC) was determined by pyrosequence genotyping  SNP was found at high frequency (81–100 %) in all three parasite populations from government farms. In contrast, it was not detected in 14 out of the 18 pastoral populations and only detected at low frequency (2–6 %) in the remaining four  and E198A (GCA) SNPs were not detected in any of the populations.A 328 bp fragment of the isotype-1 β-tubulin gene was PCR amplified from 21 Haemonchus contortus showed a high level of overall genetic diversity in all populations with a mean number of alleles per locus of 7.250 ± 1.208 (range 2–17) and expected heterozygosity (He) across all loci ranging between 0.646 and 0.707  and expected heterozygosity . There were no statistically significant differences in the overall genetic diversity, as measured by corrected allelic richness or expected heterozygosity, between any of the seven populations  and four from sheep . H. contortus between the seven locations. This was also reflected by the very low pairwise FST estimates with only two out of 21 possible pairwise comparisons showing significant values  resistance mutation was detected; three populations from government farms  and four populations from rural locations . Following bioinformatic filtering (see Methods), a total of 21 different isotype-1 β-tubulin haplotypes (GenBank accession numbers KR269920–KR269940) were identified in the 89 sequences generated from the seven populations. Six out of the twenty one haplotypes encoded the F200Y(TAC) resistance polymorphism. Summary statistics of the isotype-1 β-tubulin fragment diversity are shown in Additional file The 328 bp isotype-1 β-tubulin fragment was PCR amplified, cloned and sequenced from pooled DNA from each of the seven parasite populations in which the F200Y (TAC) resistance polymorphism was present on haplotypes located in four different parts of the network. Each of these resistance haplotypes were more closely related to one or more susceptible haplotypes than to any of the other resistance haplotypes suggesting they were independently derived. A maximum likelihood (ML) tree was also constructed and showed the same phylogenetic relationships  and one of the parasite populations from a pastoral location (Pop27G) each contained multiple resistance haplotypes. The three remaining parasite populations from rural locations  contained just a single resistance haplotype  resistance mutation was present at high frequencies on all three government farms but was absent, or present a very low frequency, in all the pastoral parasite populations sampled. This study thus provides further clear evidence associating the F200Y (TAC) mutation with benzimidazole resistance in H. contortus; the only three farms out of the 21 examined which had a history of intensive benzimidazole use are also the only ones with a high frequency of this mutation. It is noteworthy that the F167Y (TAC) and E198A (GCA) mutations were not detected in any of the H. contortus populations, including the three government farms subject to intense benzimidazole selection pressure over many years. This is consistent with previous studies from different regions the world that have shown the F200Y (TAC) mutation to be essentially ubiquitous in drug selected populations whereas the other two mutations occur more sporadically. For example, in our recent work in southern India the F167Y (TAC) mutation was apparently absent and the E198A (GCA) mutation, although present at multiple locations, appears to have been derived from a single origin in the region [AC) mutation was present on multiple haplotypes on each of the government farms; Hr12/Hr22/Hr23 for Layyah (Pop2G), Hr22/Hr31/Hr23 for Jahangirabad (Pop3S) and HR22/Hr31 for Okara (Pop1S). These resistance haplotypes are phylogenetically distant to each other in the haplotype network, being more closely related to one or more susceptible haplotypes than to any of the other resistant haplotypes. This suggests that they were derived from independent origins with the possible exception of haplotypes Hr22 and Hr31 since these are more closely related  mutations occur commonly and repeatedly whereas the F167Y (TAC) and E198A (GCA) mutations are much rarer, possibly due to differences in fitness costs between the different mutations.As expected from the known patterns of drug use, we found that the F200Y  mutations on multiple divergent haplotypes on each government farm suggests that a soft selective sweep has occurred at the isotype-1 β-tubulin locus in each population  benzimidazole resistance mutation was present at very high frequencies in H. contortus populations from three government farms, but not from eighteen pastoral herds, in the Punjab region of Pakistan, consistent with known differences in drug treatment history. Multiple F200Y(TAC) haplotypes were present in H. contortus populations from each of the “closed” government herds suggesting soft selective sweeps have occurred even in the absence of significant contemporary parasite migration. The selection for a high frequency of benzimidazole resistance alleles on government farms did not lead to a loss in overall genetic diversity, based on microsatellite marker genotyping. This observation has a number of important implications. Firstly, the retention of high genetic diversity in drug resistance parasite populations suggests they are likely to retain adaptive capacity to other selective pressures including treatment with other drug classes. Secondly, genetic changes associated with drug selection, such as loss of polymorphism and linkage disequilibrium, may be largely confined to the regions of the genome surrounding the loci under selection.The F200Y(T


Hirundo rustica as model taxon to investigate patterns of genetic variability in males and in females of a migratory species showing sex-biased dispersal. We collected blood samples (n = 186) over the period 2006 to 2011 from adults (H. r. rustica subspecies) nesting in the same breeding site at either high  or low  latitude across Europe. We amplified the Chromo Helicase DNA gene in all birds in order to warrant a sex-balanced sample size . We investigated both uniparental  and biparental (microsatellite DNA: 10 loci) genetic systems. The mtDNA provided evidence for demographic expansion yet no significant partition of the genetic variability was disclosed. Nevertheless, a comparatively distant Russian population investigated in another study, whose sequences were included in the present dataset, significantly diverged from all other ones. Different to previous studies, microsatellites highlighted remarkable genetic structure among the studied populations, and pointed to the occurrence of differences between male and female barn swallows. We produced evidence for non-random patterns of gene flow among barn swallow populations probably mediated by female natal dispersal, and we found significant variability in the philopatry of males of different populations. Our data emphasize the importance of taking into account the sex of sampled individuals in order to obtain reliable inferences on species characterized by different patterns of dispersal between males and females.Dispersal affects the distribution, dynamics and genetic structure of natural populations, and can be significantly different between sexes. However, literature records dealing with the dispersal of migratory birds are scarce, as migratory behaviour can notably complicate the study of dispersal. We used the barn swallow  Distribution, dynamics and genetic structure of natural populations can be severely affected by dispersal versus nuclear DNA) represented the most suitable approach to infer discrepancy between the dispersal pattern of males and females. Indeed, differences in the genetic picture drawn by mitochondrial and nuclear markers are expected when sex-biased dispersal occurs et al.The estimate of the dispersal rate in natural populations is often incomplete because it usually requires direct methods with intensive, large-scale and long-term demographic studies Hirundo rustica is a polytypic passerine bird widely distributed throughout most of the northern hemisphere H. rustica populations has never been proved by using either mitochondrial or microsatellite DNA markers The barn swallow In this work we aim at: (i) analysing the genetic variability of European barn swallow populations over a wide sampling area by means of markers from both uniparental  and biparental (microsatellite DNA) genetic systems; (ii) testing the consequences of sex-biased dispersal on the population genetic structure by comparing patterns of variation in a well balanced sample of males and females. Overall, the barn swallow represents an excellent model among migratory species to investigate patterns of genetic variation in males and females. While lack of genetic structure is expected for the whole sample size, we predict the occurrence of different genetic pattern between male and female barn swallows The barn swallow is not an endangered species in all trapping areas of this study. Samples were obtained in the same place  in different years. Adults were trapped with mist-nets. Samples (one blood droplet) were collected by means of wing venipuncture . Birds were not sedated and did not suffer any injury: all of them were released 10 min after blood collection.We report here below the coordinates of the sampling localities see also  togethern = 186) were collected in subsequent years (2006–2011) from nesting adults (H. r. rustica subspecies) at the same breeding site in six areas across Europe (n = 32); Tullynisk, Ireland ; Orti-Bottagone Nature Reserve, Italy ; Itzehoe, Germany ; Polis, Cyprus ; Lake Ladoga, Russia . One blood droplet was collected on filter paper  from each bird by wing venipuncture. Each swallow was ringed and sexed according to a PCR-based assay (see below).Blood samples  following the manufacturer's instructions. DNA content and purity were determined with an Eppendorf BioPhotometer .5'-GAGAAACTGTGCAAAACA-3') and H1272 (5'-TCCAGAATATCTTCTGCTCC-3') previously tested in other bird species Chromo Helicase DNA (CHD) gene of ZZ  and ZW  sexual chromosomes was amplified with primers L1237 (n = 60), and we included in the alignment  was amplified using primers L5216 and H6313 dnasp v. 5.1 dnaalignment v. 1.3.3.1  and the Median Joining method of network v. 4.5.1.0 (2004–2009 Fluxus Technology). We used arlequin v. 3.5.1 h), the nucleotide diversity (π) and the mean number of pairwise differences (k); (ii) investigate the partition of the mtDNA diversity  among and within the populations using the PhiST analogous to Wright's F-statistics (1000 permutations) We used dnasp and different statistics as described in n = 60+16 = 76), (ii) only the males , and (iii) only the females . Ramirez-Soriano et al. FS of Fu R2 statistic FS and R2 statistics was investigated by examining the null distribution of 5000 coalescence simulations using dnasp. Only significant negative FS and positive R2 values were retained as evidence of population expansion DR2 statistic has a greater power than the Tajima's D or FS to detect population expansion when the sample size is small (∼10). Furthermore, the McDonald-Kreitman test dnasp was conducted for the entire dataset to investigate the deviation from an equal ratio of non-synonymous (Ka) to synonymous (Ks) fixed substitutions. Specifically for this test we used two US H. r. erythrogaster samples as outgroup .Inferences of historical demography were obtained using arlequin . The more ragged the shape of the distribution the closer was the population to a stationary model of constant size over a long period  The Mismatch Distribution (MD) of mtDNA pairwise differences was also examined using n = 186) were investigated at 10 loci of the microsatellite DNA  reported in genescan (Applied Biosystems). For the statistical analyses we used either the whole sample size (n = 186) or males (n = 92) and females (n = 94) separately.All samples  micro-checker v. 2.2.3 rlequin, fstat v. 2.9.3 genepop v. 3.4 HE) and observed (HO) heterozygosity; (iii) infer deviations from both Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LE) ; (iv) estimate gene flow  via the private allele method of Slatkin amova; (vi) infer the degree of genetic differentiation among populations by estimating the average FST distance values. Bonferroni correction FST distance values were plotted on the first two axes of a Principal Component Analysis (PCA) using statistica 5.0/W .The discriminatory power of the whole set of STR loci was evaluated with structure v. 2.3.4 K (unknown) clusters of origin of the sampled individuals and to simultaneously assign them to each cluster. We assumed correlated allele frequencies and we used a prior population information option to take the sampling locality into account 6 Markov Chain of Monte-Carlo iterations, following a burn-in period of 105 iterations, and were replicated five times per each K-value (1 to 12). The number of clusters that best fitted to the data was chosen using the formula of Evanno et al. i = 0.80) as in Bayesian clustering analysis was performed with fstat was used to calculate five different parameters: FIS, FST, relatedness (R), mean (mAIC) and variance (vAIC) of the assignment index (AIC) within each sex mAIC and high vAIC values are interpreted for the dispersing sex. While FST and R are expected to be larger in the philopatric than in the dispersing sex, the opposite occurs for FIS. Nevertheless, the power of these statistics depends on dispersal rates, bias intensity, sampling design and the number of loci In order to test for possible differences in the dispersal rate between males and females, The CHD gene was amplified in all barn swallows: 92 birds were identified as males  while 94 as females .h), average number of pairwise differences (k) and nucleotide diversity (π). The highest number of haplotypes was found in CYP and MED. The Median Joining network showed that all barn swallow populations were genetically admixed with no noticeable divergence among haplotypes . Only MED significantly diverged from all other populations . Finally, when all the estimated parameters were computed by including in the analysis males and females separately, all results matched those produced using the entire dataset (data not shown).The alignment of 76 (60+16) ND2 sequences produced 31 haplotypes (H1-H31: GenBank accession codes HF548562-HF548592) including 27 polymorphic sites. Estimates of all parameters are summarized in plotypes . In partP = 0.022), the statistics we used  could not exclude the occurrence of a demographic expansion for the entire sample size (n = 76) (r = 0.088) curve obtained for the Mismatch Distribution (MD) of the pairwise differences suggested a rapid population growth. When the demographic parameters were computed using males (n = 27) and females (n = 33) (GenBank entries excluded) separately, males showed exactly the same trend as the whole dataset , while for the females a demographic expansion was suggested by all statistics . The McDonald-Kreitman test was not significant for the whole sample size .With the sole exception of the SSD test ((n = 76) . Indeed,n = 186) the STR panel was powerful in discriminating individuals  showed an excess of homozygotes for most of the allele-size classes, thus pointing to the possible presence of null alleles (data not shown). The total number of alleles at each locus ranged between 6 and 25 , with a mean of 13.4 alleles per locus . Both the number of alleles and gene diversity of each locus pointed to a very high degree of genetic variability. Allelic richness ranged between 7.9 and 9.9 . However, we found that no locus deviated from HWE in all populations, and when the Bonferroni's correction was taken into account only three loci  deviated from HWE in some populations yet not in all .The average values of FST = 0.014, P<0.001). In the PCA plot reported in versus CYP and RUS, Fst = 0.026 and 0.027, respectively: P<0.001). Non significant Fst distance values were found among ITA, GER, CYP and RUS . While ITA showed the highest number of barn swallows with admixed genotype . When we excluded from the Bayesian clustering analysis the STR loci showing null alleles and deviating from HWE , barn swallows were assigned to two genetic groups and results matched those obtained when the entire set of loci was taken into account (data not shown).In HO and HE across all loci and populations were relatively homogeneous and showed deficiency of heterozygotes in both male and female barn swallows  and only a very few were assigned to the cluster II or showed admixed genotype .Bayesian clustering analysis performed in males and females separately suggested the occurrence of two groups K = 2, . For eacgenotype . When weFIS and vAIC values than males  and R values  were significantly different and suggested female-biased dispersal. This finding was supported further by higher values of FIS in the females than in the males as well as by the negative value of mAIC in the females ; however, these differences were not statistically significant . On the contrary, studies focusing on other vertebrate species with sex-biased dispersal and taking into account the sex of individuals in the genetic analysis are known There are a few studies focusing on migratory bird species and relying on the use of molecular DNA markers , with the SSD statistic (P = 0.022) being an exception. However, although all tests we employed may be sensitive to unknown structure within populations, P = 0.063). Overall, we felt confident in considering that such a deviation from neutrality was very likely due to demographic changes rather than selective processes, as the McDonald-Kreitman test was not significant in the entire dataset The mtDNA analysis did not suggest any significant structuring of the genetic variability , as it met al. H. r. rustica and H. r. transitiva (Middle East). However, we feel confident that this result was due to the divergence of MED, the easternmost and northernmost population in our sampling scheme. Indeed, when MED was excluded from the analysis the mtDNA genetic structure promptly disappeared  found by Dor appeared .Grus canadensis), a deficiency of heterozygotes may be common in migratory species when a given population consists of local and immigrant individuals with different origin. Overall, departures from HWE would seem also a deficit intrinsic to the barn swallow, which, however, do not necessarily affect the result of the genetic analysis. Indeed, Bayesian procedure does not require perfect equilibria to cluster individuals, yet it attempts to minimize such departures within groups et al. structure could reliably determine the correct number of clusters also for FST values as low as 0.01; hence, we did not exclude any STR locus from our analysis. Nevertheless, we also showed that the output of structure did not change when Hir4, Hir7 and Hir24 loci were ruled out , pointed to the strong divergence between SPA and all other populations . Admixed genotypes were more frequent and gene flow level higher in the females than in the males . This, in turn, suggested that gene flow among populations could be partially influenced by the axis of migration. However, the divergence between ITA and GER also suggests that dispersal does not strictly occur along a North-South axis. On the other hand, the genetic differentiation between eastern and western populations resembles what has been observed for other passerine species showing a clear migratory divide in central Europe et al. H. rustica. This analysis, which was carried out on a large dataset of ringing recoveries, produced weight for the existence of two main clusters: one includes birds breeding in south-west Europe and wintering in central Africa, the other comprises birds breeding in northern and eastern Europe and wintering in southern Africa. The genetic relationships disclosed in the present study, on one side, between RUS and CYP and, on the other, between SPA, GER and IRE, fit to the groups described by et al. When the clustering of the females was considered the occurrence of a significant latitudinal component could not be ruled out . On the contrary, when the SPA males were excluded, we disclosed slight differences among the studied populations that pointed to the occurrence of three groups: (IRE + GER), (ITA + CYP) and RUS . This pavAIC seems the most powerful test, followed by mAIC and FST, which work better with less than 20 loci vAIC was higher in the females than in the males, thus pointing to the occurrence of female-biased dispersal. When the tests were performed and SPA was separated from all other populations, R and FST values were significantly lower in the females than in the males, mAIC value was negative , cluster II (white) or which showed admixed genotypes (grey). Threshold value for assignment to each cluster was Qi = 0.80.(DOC)Click here for additional data file.Table S1The sample size (n = 186) of this study and the mtDNA sequences downloaded from the GenBank (n = 16). Population (Pop), country, region, locality , type of tissue, sample size for STR/mtDNA analysis, and the number of ND2 mtDNA haplotypes are given. The number of male (M) and female (F) individuals genotyped with mitochondrial and STR markers was given (unavailable on line for MED population). GenBank accession codes for KRD and MED populations are reported in (DOC)Click here for additional data file.Table S2Estimates of mtDNA genetic diversity (averagez± SD) as computed for each population and for the whole dataset. Sites: number of segregating sites.(DOC)Click here for additional data file.Table S3ST) as computed among all populations; *, Average mtDNA pairwise distance values Click here for additional data file.Table S4I and QII) as inferred by structure with Proportion of estimated membership of all populations to each cluster (QK = 2 . Computations were performed using: (i) all individuals (n = 186), (ii) males (n = 92) and (iii) females (n = 94). The number of specimens assigned to the cluster I (nI) and II (nII) was reported as well as the number of individuals showing admixed genotype (nmix). The threshold value for admixed assignment was Qi = 0.80.(DOC)Click here for additional data file.


Biomphalaria acts as the intermediate host of Schistosoma mansoni, a globally important human parasite. Understanding the population structure of intermediate host species can elucidate transmission dynamics and assist in developing appropriate control methods.The freshwater snail Biomphalaria choanomphala collected around the shoreline of Lake Victoria in Uganda, Kenya and Tanzania, where S. mansoni is hyper-endemic. Molecular markers were utilized to estimate the degree to which snail populations are genetically differentiated from one another.We examined levels of population genetic structure and diversity in 29 populations of Biomphalaria choanomphala.High levels of snail genetic diversity were found coupled with evidence of geographically-determined population structure but low levels of local inbreeding. The data are consistent with an effect of schistosome infection on population structure of intermediate host snails, but other factors, such as habitat and historical demographic changes, could also be important determinants of the degree of population genetic structure in The low stratification of populations and high genetic diversity indicates potentially less local compatibility with intermediate snail populations than previously theorized, and highlights the importance of coordinated parasite control strategies across the region.The online version of this article (doi:10.1186/s13071-014-0524-4) contains supplementary material, which is available to authorized users. Biomphalaria freshwater snails of the family Planorbidae, which are the obligatory intermediate hosts of the globally important parasitic trematode of humans, Schistosoma mansoni, have been the subject of considerable study.Historically, the nature and dynamics of host-parasite relationships has attracted much scientific attention. Parasites of human populations are an area of particular research interest, given the medical interest in controlling their transmission. As such, the genetic diversity and population structure of hermaphroditic Biomphalaria population reduces the overall parasite transmission rate whereas reduced host genetic diversity appears to benefit S. mansoni through an overall increase in transmission rate [Biomphalaria reduces snail fitness, but resistance to infection is itself also associated with reduced offspring production, as often seen in other planorbids [Biomphalaria population structure would result in a tightly coupled system whereby parasites and snails co-adapt, resulting in localized compatibility and comparable population structures, but also driving local high diversity [Schistosoma could equally be implicated in driving diversity in such a system, as Biomphalaria are known to serve as intermediate hosts for a number of nematode and trematode worms [It is known that increased genetic diversity of the host ion rate -5. It haanorbids . As suchiversity . Of courde worms ,9.S. mansoni are, however, not the only external factor known to influence their host. Selection driven by environmental factors, such as seasonality or habitat type, has also been shown to affect freshwater snail population differentiation [Parasites such as ntiation -12. Therntiation .B. glabrata and B. pfeifferi are considered two of the most important intermediate host snail species of S. mansoni in the New and Old World, respectively, and most previous work on snail-schistosome population structure has focused on these species [Biomphalaria species [Biomphalaria choanomphala from Lake Victoria, an important lacustrine environment in East Africa, and a well-known regional hot spot for transmission of S. mansoni [Biomphalaria choanomphala in this hyper-endemic region for schistosomiasis, at a large scale, to better understand factors that may contribute to parasite transmission. species . There a species , and few mansoni -19. The Biomphalaria choanomphala in Lake Victoria was inferred from genetic variation in two mitochondrial genes (cytochrome oxidase sub-unit one (COI) and 16S ribosomal RNA (16S)), and four bi-parentally inherited nuclear microsatellite loci.The degree of population differentiation of Biomphalaria found. Sites were selected to ensure an even geographical spread along the lakeshore, in all three countries and in both marsh and lake habitats; five localities were specifically included because they incorporated both habitat types in close proximity. All sites showed signs of human activity near the shoreline. In order to compare sites of similar population density, all sites selected had a high abundance of Biomphalaria, defined as 30 or more individuals collected, which accounted for the paucity of sites along the eastern shoreline of the lake in Tanzania, where Biomphalaria densities were generally low.Snails were collected from 29 sites around the perimeter of Lake Victoria, using hand held scoops Figure . SamplinB. choanomphala or B. sudanica, based on shell morphology, as these are the species thought primarily to inhabit Lake Victoria [Biomphalaria in Lake Victoria can be considered a single species of which ecophenotypy of shell form gives rise to the previously considered taxonomic distinctions [B. choanomphala.Snails were putatively identified as Victoria . Howeverinctions , and thuSchistosoma mansoni or other parasite cercariae [All collected snails  were exposed, as a group, to sunlight for 2–4 hours to check for infection with ercariae . A randoBiomphalaria choanomphala under examination here. Mitochondrial (COI and 16S) amplifications were carried out in 25 μl total volume, with 2.5 μl MgCl2 (20 mM concentration), 2.5 μl 5 × buffer, 2.5 μl pre-mixed dNTPs (20 mM concentration), 1 μl each of forward and reverse primer (10 pmol concentration) and one unit of TAQ per reaction. Microsatellite reactions were amplified in 12.5 μl total volume, with half of the volumes stated above, and with forward primer concentration of 50 pmol and reverse (fluorescently labeled) primer concentration of 20 pmol. Cycling conditions in all cases followed published methods [Genomic DNA was extracted from between 10 and 12 individuals from each of the selected 29 populations, using a standard CTAB extraction  with a f methods -25.96 Cleanup kits on a vacuum manifold  as per manufacturer’s instructions, using pure water for washing and re-suspension. Product concentration was quantified on a Nanodrop ND-1000 Spectrophotometer , and sequencing reactions were performed on mitochondrial purified PCR products using an Applied Biosystems Big Dye Kit (version 1.1) and run on an Applied Biosystems 3730 DNA Analyzer . Microsatellites were diluted 1:10 in HiDiformamide  and analyzed using an ABI3730 automated sequencer, using a GeneScan 500 LIZ size standard .Positively amplified products were purified using Millipore PCRB. choanomphala populations surveyed from Lake Victoria [The overall framework of the population genetics analyses were designed to examine the population structure, diversity, demography, phylogeography and spatial correlations of the Victoria .http://www.ebi.ac.uk/Tools/msa/muscle/) and edited visually in MacClade v 4 [per population was recorded. The 16S data contained many gap-rich regions, for which it was impossible to be assured of homology; any region with a gap was therefore removed from the alignment before calculating genetic distances amongst sequences. There was only one indel region in the COI alignment, of a single codon repeat, in three haplotypes, which was therefore easily alignable and retained for haplotype analysis.COI and 16S mitochondrial sequences were aligned using MUSCLE on the web , F-statistics, mismatch analysis, Tajima and Fu’s tests for neutrality and a Mantel z-test for correlation by distance. Each AMOVA was conducted using “country” as the primary grouping, to investigate the level of variation partitioned between each country, versus between populations within each country and within each population. “Country” was deemed to be a potentially important geographical factor given that each country in the region undertakes different levels of schistosomiasis control initiatives, thus potentially influencing the level of parasitism experienced by local snails. All tests were executed in Arlequin v 3.2 .A distance-based, neighbor-joining tree was created in Paup* 4.0  for the ST and tests for Hardy-Weinberg equilibrium  and linkage disequilibrium amongst loci were also done using Genepop. The loci were also tested for evidence of previous bottlenecking events by comparing the allelic diversity against the observed and expected heterozygosity, using the program Bottleneck [The number and proportion of private microsatellite alleles per population was calculated using the web-based version of Genepop . Calculattleneck .Biomphalaria choanomphala were analyzed from the selected 29 populations. Both the mitochondrial markers and the microsatellites revealed high levels of genetic variation overall, but also significant population structuring throughout the lake as described below.In total, over 300 The COI and 16S sequences were very genetically variable with 127/308 (CO1) and 181/300(16S) unique haplotypes/total number of sequences respectively. Haplotypes were not highly divergent (0.002-0.058 pairwise distance for COI haplotypes and 0.000-0.047 for 16S). GenBank accession numbers for these datasets are HM769132-HM769258 for COI and HM768950-HM768980 and HM768982-HM769131 for 16S. When unalignable regions with gaps were removed from the analysis of the 16S sequences, the number of unique haplotypes was reduced from 181 to 64. Many common haplotypes were spread throughout the lake, but others were locally restricted and there was high variation in the haplotype richness of the sites: overall, intra-site haplotype diversity estimates varied from 0.200-0.982 for COI and 0.378-1.000 for 16S, where zero indicates complete homogeneity and one indicates maximum diversity.Figure The phylogenetic trees for COI and 16S Figure  were conThe majority of the variation, for both the COI and the 16S data, was explained at the population level based on the analysis of molecular variance (AMOVA), which sought to determine how diversity was partitioned among and between the different sites. For these data, all three levels of structure  were statistically significantly differentiated (see Table ST) values were calculated between each of the populations in turn, the majority demonstrated significant levels of differentiation, for both types of molecular marker. The range in values was 0.000-0.921 for COI and 0.000-0.845 for 16S. For the 16S data, all but one of the non-significant FST values were between sites in the same country, once again demonstrating geographically defined structuring of the populations for this gene. All but one paired site showed significant differentiation between the populations from lake versus marsh habitat, indicating even small-scale geographical segregation. Full tables of the pairwise FST values and their significance can be found in the supplementary information  from assumptions of normality when the COI data was analyzed. However, this population also had a significant result for the sum of squares mismatch analysis (p <0.001), suggesting that demographic change, such as population expansion, may have biased the neutrality test. No other populations, for either marker, came out as significant in the mismatch analysis.The relationship with geography was elucidated through the significance of the Mantel z-test for both the COI and 16S data , meaning that the genetic and geographical distances between the populations was positively correlated; in other words, sites closer together were more likely to be genetically similar, even if the composition of distinct haplotypes differed per site , linkage disequilibrium and for an excess of heterozygosity relative to allelic diversity, which would be consistent with populations having recently passed through a bottleneck.ST values ranged from 0.000-0.298, but fewer values were significant between populations, compared to the mitochondrial data. The full matrix of pairwise FST values and their significance can be found in the supplementary information . The microsatellite data also supported the mitochondrial evidence for genetic spatial autocorrelation, as the Mantel z-test was significant (p = 0.017).As with the mitochondrial data, based on an AMOVA, the majority of the variation in microsatellite genotype was explained at the population level; values of differentiation at the intragroup and intrapopulation were both highly significant , when tested for evidence of a past bottleneck, showed heterozygote excess , but this value was not significant once the Bonferroni correction for multiple tests was applied.IS values and apparent random mating observed here further supports this hypothesis. The snails collected for this research were examined for infection with schistosomes and other parasites in the field as part of a different study; however, without more accurate and consistent methods such as molecular probes [Biomphalaria remain.The populations studied here were characterized by high intrapopulation diversity as well as high levels of population structure, with low levels of gene flow as inferred from molecular data and negligible inbreeding. High diversity is consistent with what would be expected if parasitism were strongly influencing population structure ,7, and tr probes  for the r probes ) and infr probes ,35. EvenB. choanomphala from the six sites from which there were published data on schistosome population structure [We would also expect parasite populations to adapt to local snails, thus mirroring to some extent their population structure. We used a Mantel test  to compatructure , and foutructure ,37. Defitructure .Biomphalaria in other settings. Such considerations link back to parasitism, and particularly control of schistosomiasis in human populations, in terms of gaining a better understanding of drivers for distribution and abundance of host snails under different conditions. Studies of B. pfeifferi in Madagascar, for example, revealed high levels of interpopulation variation but, in contrast to what we observed here, consistently low levels of intrapopulation genetic differentiation [B. pfeifferi are also known to be frequent in-breeders, which would assist in maintaining a metapopulation with seasonal local extinction events [Given the possibility of parasitism as a driver for population structure in Lake Victoria snail populations, it is important to consider what other factors might be influential in this system, and particularly how these differ from previous studies of ntiation . This wan events .B. choanomphala [Biomphalaria.In contrast, Lake Victoria possesses very different environmental conditions; as a very large, permanent lake, it rarely experiences the kind of environmental perturbations that characterize temporally transient ponds and streams. Such homogeneity of habitat, with associated infrequent extinctions and opportunity for genetic drift, could account for the high levels of intrapopulation diversity that are maintained in Lake Victorian nomphala . This apS. mansoni observed previously in this region counters the hypothesis of highly specialized local parasite compatibility [B. choanomphala haplotypes were widespread around the lake. This suggests that, as seen in B. pfeifferi from elsewhere in Africa, there may be a risk of transmissive genotypes becoming widely successful, if there is selection for traits independent of parasite susceptibility [Although the low population structure in tibility  for bothtibility .S. mansoni experience more intense selection pressure based on frequent mass drug administration campaigns, and given the generally transmissive snail populations, there is a risk that these highly adapted forms of S. mansoni will be able to spread rapidly throughout the region. This emphasizes the need for coordinated control strategies, both at the level of human treatment as well as improved water access and sanitation, throughout lakeshore communities in the region.Another factor to consider is human-mediated changes to parasite populations: all three countries bordering Lake Victoria have initiated efforts to control schistosomiasis in human populations through mass treatment campaigns, although these initiatives are not synchronized and differ in their degree of coverage. The low levels of population structuring seen in the parasite  are likeB choanomphala snails, yet also high population structuring; the high levels of observed inter- and intrapopulation diversity are consistent with parasitism as an influencing factor, but further investigation is needed, utilizing new tools to detect infection and new markers directly associated with resistance or susceptibility, in order to confirm the role of parasitism in driving high diversity in this system. Moreover, other factors, such as environmental and demographical stability may also contribute to the observed population structure. From a public health perspective, population genetic surveys of intermediate hosts should seek to increase the scale of their focus, as parasite transmission is likely influenced by factors, such as human migration and national treatment campaigns, that act on a national or even regional level. Public health initiatives should take such issues of scale into consideration when designing control strategies.In summary, our analysis observed high genetic diversity of


Tanacetum cinerariifolium Trevir. /Sch./ Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.Dalmatian pyrethrum ( DW; frequency down-weighted marker values) as an indicator of the historical processes of a species DW values and genetic diversity Conservation of genetic diversity is essential for any species long-term survival The Balkan Peninsula itself represents a unique ecological and biogeographical phenomenon in Europe possessing the tremendous number of plant and animal species and pronounced richness in endemic taxa as well. This remarkable diversity is the result of the variety of its regions, complex geological history, and interactions between populations, species and ecosystems It is widely acknowledged that geographical and environmental factors can also play significant roles in the modeling of species-specific inter- and intra-population genetic structure. Spatially separated populations may experience isolation by distance (IBD) phenomena in which geographical barriers and physical distances cause restricted pollen and seed dispersal, i.e., gene flow Nevertheless, species are greatly affected by direct or indirect human activities including population fragmentation and overharvesting pressures, all resulting in loss of genetic diversity and the alteration of their population genetic structure Tanacetum cinerariifolium Trevir. /Sch./ Bip.), an insecticidal perennial plant. Dalmatian pyrethrum is an outcrossing diploid (2n = 18) For our investigation we have chosen Dalmatian pyrethrum  Pleistocene climatic oscillations, (ii) geographic (IBD) and (iii) environmental isolation (IBED), and (iiii) human activities through overexploitation and cultivation on the obtained pattern of genetic diversity. AFLP markers have found the widest application in assessing the variability and genetic structure of populations in a variety of plants http://narodne-novine.nn.hr/clanci/sluzbeni/dodatni/403046.pdf). Today, as an endemic species, Dalmatian pyrethrum is threatened by anthropogenic habitat loss and degradation related to factors such as urbanization and habitat conversion; therefore, the need and importance of these species’ genetic resources protection must be recognized even more. The gathered information could be of utmost importance for future activities regarding the conservation and breeding programs of this endemic and economically important species.The goal of this study was to analyze the genetic diversity and structure within and among 20 populations of Dalmatian pyrethrum across their geographical range in Croatia and to determine impact of .T. cinerariifolium were sampled from 20 locations along the Adriatic coast in Croatia . The restriction digestion and adapter ligation, pre-amplification and selective amplifications were performed in a GeneAmp PCR System 9600 . Six primer combinations were selected for amplification; VIC-EcoRI-ACGC+MseI-AGA, NED-EcoRI-AGA+MseI-CAG, VIC-EcoRI-ACG+MseI-CGA, FAM-EcoRI-ACC+MseI-CGA, NED-EcoRI-AGA+MseI-CGA, PET-EcoRI-ATG+MseI-CGA. The amplified fragments were separated by capillary electrophoresis in an ABI3130xl Genetic Analyzer . The amplified DNA fragments in the size range of 50 to 125 bp were scored with GeneMapper 4.0 software  as being present (1) or absent (0) in order to create a binary matrix.The total genomic DNA was isolated from 90 to 110 mg of fresh leaves tissue with DNA-GenElute Plant Genomic DNA Miniprep Kit , according to manufacturer’s instructions. Prior to isolation leaves tissue was quickly frozen in liquid nitrogen (−196°C) and ground to a fine powder with TissueLyzer homogenizer set  at 30 Hz for 1 minute. The DNA concentrations were measured using a Qubit Fluorometer . The AFLP analysis was carried out following the original protocol that was described by Vos et al. The selection of markers for further statistical analysis and the estimation of the error rate per primer combination were performed as suggested by Herrmann et al. (%P), the number of private alleles pr)(N and Shannon’s information index (I) TH) as well as the average intra-population diversity (PH) and the diversity within (P/HTH) and among populations  distances, and a three-way Mantel’s test was conducted between the same distances matrices while considering the geographical distances among populations. Thus, the correlations between the residual environmental and genetic distances among the analyzed populations represent isolation by environmental distance (IBED) pattern. The significance levels were obtained after 10,000 permutations as implemented in NTSYS-pc ver. 2.10s A simple Mantel’s test was used to test the linear correlation between the matrix of environmental and genetic , were obtained for each of the 10 independent runs for each K (from K = 1 to 21). The estimates of  values decreased, while the variances increased substantially and, therefore, the ΔK values were considerably lower (0.10 to 5.80) in comparison to K = 2 and 3. Thus, the hypothesis of having more than three clusters (gene pools) was far from optimal.The results of the STRUCTURE analysis are reported in K = 2 to 3 based on the run with the highest [ln P(X|K)], and populations were assigned to a particular gene pool . The results were in accordance with the results that were obtained using the distance-based method. Representative populations of each gene pool that were identified in the STRUCTURE analysis were located in proximity of each other. At K = 2, the representative populations of gene pool A were mainly those from the northern Adriatic, while the populations from the middle and southern Adriatic were representative of a separate gene pool B. The most admixed populations were 9 and 11 with almost equal proportions of ancestry from the two gene pools  of each individual in each of the gene pools were calculated for ne pools . At K =  Biokovo .The BAPS mixture with or without spatially informative priors resulted in the congruent assignment of individuals to two clusters. The best partitions received log marginal likelihoods of −51646.2626 at P = 1  and −51793.7943 at P = 1 . The results were consistent with the results of the STRUCTURE analysis .r = 0.236; MantelP = 0.012), suggesting that 5.6% of the genetic differentiation between populations could be explained by geographical distances  were detected, mostly among eleven temperature-related variables (BIO01–BIO11) and among eight precipitation-related variables (BIO12–BIO19). The strong negative correlations (r<−0.70) were found in 10 cases, usually between temperature-related and precipitation-related variables.As expected, 19 environmental variables were highly intercorrelated. Out of 171 pairwise correlations, 107 (62.57%) were significant at P<0.05 . In 49 cA Principal Component Analysis based on the correlation matrix revealed that first three principal components had an eigenvalue >1 and explained more than 90% of the total variation . Eight tr = 0.376; MantelP<0.001) than between the genetic and geographical distances  suggesting that isolation by environmental distance (IBED) explains 12.3% of the observed genetic pattern .A greater correlation was found between the genetic and environmental distances  of isolation occurs. Three genetically and geographically distinct and well-defined groups of Dalmatian pyrethrum populations therefore support the “refugia within refugia” model that was already confirmed in numerous studies in the Balkan Peninsula DW were found in group of northern Adriatic populations from Kvarner bay. The genetic structure of these populations, which are grouped together within the same cluster as revealed by the STRUCTURE analysis, suggests their ancient origin and significant isolation through a recent period of evolution Podarcis melisellensis)Vipera ammodytes)Edraianthus tenuifoliusDW decrease. Overall, the detected pattern is not in concordance with the general predictions that the highest levels of diversity can be detected among southern regions of species distribution range DW. An exception is the population 19 located in the southern parts of the species’ range. Because it exhibits high levels of gene diversity accompanied by low levels of private alleles and DW, this population is likely the zone of secondary contact between the divergent lineages and is anthropogenic in origin. Additionally, some populations show a quite admixed pattern comprising large fractions of different gene pools . The specific structure of these populations could also easily be a consequence of the admixture of divergent lineages through secondary contact after postglacial colonization, however, more likely explanation of the obtained results could lie in the cultivation practices of Dalmatian pyrethrum, which began in 1850s near Dubrovnik  and rapidly expanded along the Dalmatian coastal region and the islands DW. Moreover, extensive seed exchange between different populations caused admixture as observed in pops. 8, 9 and 11. Thus, this phylogeographical study could represent typical example of humans’ shaping the genetic structure of investigated plant species. The genetic impoverishment as a result of either overexploitation of wild resources or cultivation practices has also been demonstrated for American ginseng (Panax quinquefolius L.) Rheum tanguticumScutellaria baicalensisEucommia ulmoidesMoringa peregrinaThe results of our study show genetic differentiation of into two  to threeinto two  distinctEH = 0.092–0.096), which were also identified as a separate gene pool by the Bayesian cluster analysis. The Mt. Biokovo populations  were sampled in geographically and ecologically marginal areas of the species distribution, and it is generally expected that in such peripheral populations, the amounts of diversity may be reduced The lowest values of gene diversity were detected in the populations from Mt. Biokovo  (Fraxinus angustifolia Vahl) due to contrasting ecological conditions of the sampling sites. Similarly, strong fine-scale spatial genetic structure associated with habitat factors was detected in continuous population of Biscutella laevigataCanis lupus) genetic differentiation among local populations was correlated to contrasting climatic conditions Evidence that both geography and environmental heterogeneity contributed significantly to the obtained patterns of genetic divergence was found. The IBD analysis showed positive and significant correlations, which could in principal be the result of successive postglacial colonization from the refugia per se.In our study the detected IBED pattern explained more than twice the genetic divergence than did the IBD pattern . Therefore it is clear that the revealed genetic differentiation in Dalmatian pyrethrum is more strongly influenced by the bioclimatic conditions of the sampling sites rather than by geographic distances Tanacetum cinerariifolium Trevir. /Sch./ Bip.) populations from Croatia. Overall, our results show that, as a thermophytic plant, Dalmatian pyrethrum could have survived the Quaternary climatic oscillation along the eastern Adriatic coast by tracking suitable habitats, i.e., microrefugia. Northern Adriatic populations, which were identified as a separate cluster by the STRUCTURE analysis, are characterized by high levels of gene diversity and DW and should be treated as conservation priority sites. Human influence through overexploitation and cultivation has resulted in the low genetic variability of southern Adriatic populations. The results also provide evidence that both IBD and especially IBED contributed to the genetic differentiation among some populations.The aim of this study was to analyze the genetic diversity and structure of natural Dalmatian pyrethrum  using software Structure and assuming K = 2, (B) using Structure and assuming K = 3; (C) using BAPS without spatially informative prior, and (D) using BAPS with spatially informative prior. Each individual plant is represented by a single vertical line divided into colors. Each color represents one cluster, and the length of the colored segment shows the individual’s estimated proportion of membership in that cluster. White lines separate populations that are labelled below the figure (1–20).(TIF)Click here for additional data file.Table S1Correlations coefficients  and its significance  among 19 environmental variables at 20 sampling sites of T. cinerariifolium in Croatia.(DOCX)Click here for additional data file.


Tabebuia aurea to understand the effects of Quaternary climate change on its current spatial patterns of genetic diversity. We sampled 285 individuals from 21 populations throughout Brazilian savannas and sequenced all individuals for three chloroplast intergenic spacers and ITS nrDNA. We analyzed data using a multi-model inference framework by coupling the relaxed random walk model (RRW), ecological niche modeling (ENM) and statistical phylogeography. The most recent common ancestor of T. aurea lineages dated from ~4.0 ± 2.5 Ma. T. aurea lineages cyclically dispersed from the West toward the Central-West Brazil, and from the Southeast toward the East and Northeast Brazil, following the paleodistribution dynamics shown by the ENMs through the last glacial cycle. A historical refugium through time may have allowed dispersal of lineages among populations of Central Brazil, overlapping with population expansion during interglacial periods and the diversification of new lineages. Range and population expansion through the Quaternary were, respectively, the most frequent prediction from ENMs and the most likely demographic scenario from coalescent simulations. Consistent phylogeographic patterns among multiple modeling inferences indicate a promising approach, allowing us to understand how cyclical climate changes through the Quaternary drove complex population dynamics and the current patterns of species distribution and genetic diversity.Understanding the dispersal routes of Neotropical savanna tree species is an essential step to unravel the effects of past climate change on genetic patterns, species distribution and population demography. Here we reconstruct the demographic history and dispersal dynamics of the Neotropical savanna tree species  Spatial displacements in species distributions due to Quaternary climate changes have played an important role in shaping the genetic diversity of many species across space  with fossil data and paleoclimatic simulations in an ecological and biogeographical context , which is widely distributed through the Neotropics, occurring in seasonal savannas and wet-savanna grasslands , carpenter bees (Xylocopa spp.) and Centris spp., and its winged seeds are wind dispersed.A multi-model inference approach is particularly useful to investigate the complex dynamics and current patterns of genetic diversity in tree species with a long generation time and life span. This is the case of s Figure . LocallyDuring the Last Glacial Maximum (LGM), for instance, the climate was drier in most of South America, leading to a retraction in geographical range of many arboreal savanna taxa and expansion of grasslands , populations in these places are barely found . The Northeast Brazil is dominated by a xeromorphic steppe vegetation with seasonally dry forest and some island of savanna where T. aurea may occur but the high level of anthropogenic disturbance had removed most natural vegetation. We also sampled and sequenced individuals of Tabebuia impetiginosa, Tabebuia chrysotrica, and Cybistax antisyphillitica to include as outgroups in coalescence analyses  of a Figure . Distances Table .psbA-trnH, trnC-ycf6, and trnS-trnG : Consensus sequences were obtained using the software SeqScape v2.6  and aligned with the software ClustalΩ  diversity were estimated for each population and overall populations following Nei (FST and tested whether genetic differentiation across T. aurea populations (linearized FST) is correlated with geographical distance (logarithm) among populations using the Mantel test. Analyses were performed using the software ArlequinVer 3.11 and statistical significance was tested by a non-parametric permutation test .To investigate genetic diversity and structure of populations, chloroplast and nuclear ITS were analyzed separately. Nucleotide (π) and haplotype . Thus, recombination was neglected in all coalescent analyses. To set the priors, evolutionary model selection for both chloroplast and ITS regions was performed using Akaike Information Criterion implemented in the software jModelTest2 , the exponential growth rate  and t is time in mutational unit), and the immigration rate among all population pairs . Estimations were based on Bayesian model using the Markov Chain Monte Carlo (MCMC) approach implemented in Lamarc 2.1.9 software  through time to better understand changes in population size, combining data from different partitions. We used the substitution models reported above and the relaxed molecular clock model  for both chloroplast and ITS. Mutation rates for both chloroplast and ITS regions were the same used for a taxonomic related species Tabebuia impetiginosa  analysis . For the diffusion process, we used the Symmetric Substitution Model that uses a standard continuous-time Markov chain (CTMC) in which the transition rates between locations are reversible. Priors for sequence evolution were the same as described above. For tree priors, we used the Coalescent GMRF Bayesian Skyride model . MCMC conditions and number of runs also remained unchanged. The independent runs were analyzed using Tracer 1.6 and results were considered when ESS ≥ 200. We also ran an empty alignment (sampling only from priors) to verify the sensitivity of results to the given priors. The analysis showed that our data is informative because posterior values  were different from those obtained from empty alignment.We obtained a coalescent tree based on Bayesian coalescent analysis implemented in the software BEAST 1.8.0 to estimate time to most recent common ancestor (TMRCA). For this analysis, we included the outgroup sequences from T. aurea across Neotropics from the online databases GBIF  and Species Link (http://splink.cria.org.br/), which were mapped in a grid of cells of 0.5° × 0.5° (longitude × latitude), corresponding to 55 km at the equator, encompassing the entire Neotropics (Table We obtained 237 occurrence records of cs Table .www.ecoclimate.org), along with subsoil pH , mid-Holocene (6 ka) and Last Glacial Maximum  from four coupled Atmosphere-Ocean General Circulation Models (AOGCM): CCSM4, CNRM-CM5, MIROC-ESM, and MRI-CGCM3 , selected by factorial analysis with Varimax rotation from 19 bioclimatic variables obtained in the EcoClimate database  as a response variable to disentangle the effects of climate change on species distribution through the time from predictive uncertainties in the potential distribution due to modeling components . For this, the ENM and AOGCM components were nested into the time component, but crossed by a two-way factorial design within each time period  for each time period, which were used to support alternative demographic scenarios. By keeping constant the ENM and AOGCM across time periods, we computed the range shift across the last glacial cycle , and classified the 52 predictive maps according to three general demographic scenarios: (i) “Range Stability,” no difference in range size through time; (ii) “Range Expansion,” range size was lower at LGM than in present-day; (iii) “Range Retraction,” range size was larger at LGM than in present-day. Map classification for coalescent simulation was performed only for the time slice from LGM to present-day due to the TMRCA for the analyzed genome regions .Along with the three hypotheses supported by ENMs (see above), a fourth biogeographic hypothesis of “Multiple Refugia” was derived from paleoclimatic .The demographic scenarios were simulated based on coalescent analysis Kingman,  implemenT. aurea effective population sizes  to t1750 generations ago (at 21 ka), with sizes Nt = {ln(N1/N0)/t}. At t0, all demes had the same population size N0 and reached N1750 according to our theoretical expectation for each demographic scenario  and Akaike Information Criterion (AIC) were estimated for each comparison. The log-likelihood was estimated as the product of the height of the empirical frequency distribution at the observed value of diversity by the maximum height of the distribution (see BayeSSC website www.stanford.edu/group/hadlylab/ssc/index.html). AIC was transformed into AIC weight of evidence (AICw), given by exp[−0.5(AIC − AICmin)] . One-tailed probability  and nuclear ITS . Although pairwise genetic differentiation was significant among all pairs of populations for cpDNA, differentiation for nuclear ITS was mainly due to populations CHG, SDO, and SEC  or no association between the geographical distance and the genetic differentiation among pairs of populations .The combined data of chloroplast intergenic spacers generated 2330 bp and the nrDNA ITS, 567 bp  and potentially indicates a demographic expansion or departure from neutrality.The Extended Bayesian Skyline Plot also showed increasing population growth through the last 1.2 Ma, especially in the last 100 ka Figure . Fu' tesT. aurea lineages, one in the west (CAC) and other in the southeast Brazil (SCA)  occurred toward populations in northern Brazil, corresponding to the spatial displacement in suitable climatic conditions as showed by ENMs (see below). The mean rate of dispersal was 1.17 km/yr , and the Bayes factor showed that most links among localities are well-supported (BF > 8.0).The phylogeographic reconstruction of lineage dispersal showed the two most probable ancestral locations for ) Figure . These l) Figure . However) Figure . Most diT. aurea was potentially distributed across central and northeast Brazil through the last glaciation  as the most likely general scenario among alternative demographic hypotheses, matching our theoretical prediction, ENMs and extended Bayesian Skyline Plot results. Because favorable climatic conditions for in situ diversification of many haplotypes in SAF . Although our findings show possible connections among SAF and other populations , we hypothesize that the relative isolation may have precluded the dispersal of lineages from SAF to other populations. It is important to note that events of long distance dispersal, such as from SCA to SAF, are most likely due to stepping-stone dispersal and would be detected only with continuous spatial sampling. However, continuously sampling in large scale is hindered by the high savanna fragmentation in Brazil. Besides, estimates of the mean rate of dispersal equal to 1.17 km/yr using RRW is congruent with the pollen dispersal distance reported for T. aurea  have high genetic diversity. We hypothesize that these populations, at the edge of historical refugium may represent “stable rear edges,” i.e., relict populations that have persisted in suitable local habitats across the Quaternary glaciation cycles (see Petit et al., T. aurea was also supported for other Brazilian savanna species such as Caryocar brasiliense (Collevatti et al., Dipteryx alata (Collevatti et al., Mauritia flexuosa (Lima et al., Finally, the scenario of range and demographic expansion through time for T. aurea may be the outcome of population expansion through warming periods of the Quaternary, with recurrent events of spatial displacements along the cyclic events of glaciations. We are aware of the differences in the time scale of predictions from different approaches, given that the RRW predicts spatial displacements occurring in a deeper time than ENMs. However, all approaches converge to consistent patterns: general dynamics of T. aurea following suitable climatic conditions toward the northern regions during warming periods in the early Quaternary, with retraction of populations during glacial phases, and new dispersal events at the middle and end of the Quaternary. Such consistent patterns from different methods and analyses indicate that our multi-model inference approach is dynamic and flexible, allowing the inclusion of new components to directly infer processes affecting phylogeographic patterns through time, and promises advances in hypothesis testing in phylogeography.In conclusion, our findings suggest that the pattern of genetic diversity in RC and ML conceived the overall study. SR generated the genetic data. RC, LT, and ML analyzed the data and wrote the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drimys confertifolia, endemic to the two major islands of the Juan Fernández Archipelago, Chile, to determine genetic consequences of anagenesis, to examine relationships among populations of D. confertifolia and the continental species D. winteri and D. andina, and to test probable migration routes between the major islands. Population genetic analyses were conducted using AFLPs and nuclear microsatellites of 421 individuals from 42 populations from the Juan Fernández islands and the continent. Drimys confertifolia shows a wide genetic variation within populations on both islands, and values of genetic diversity within populations are similar to those found within populations of the continental progenitor. The genetic results are compatible with the hypothesis of high levels of genetic variation accumulating within anagenetically derived species in oceanic islands, and with the concept of little or no geographical partitioning of this variation over the landscape. Analysis of the probability of migration within the archipelago confirms colonization from the older island, Robinson Crusoe, to the younger island Alejandro Selkirk.A common mode of speciation in oceanic islands is by anagenesis, wherein an immigrant arrives and through time transforms by mutation, recombination, and drift into a morphologically and genetically distinct species, with the new species accumulating a high level of genetic diversity. We investigate speciation in The online version of this article (doi:10.1007/s10265-014-0666-7) contains supplementary material, which is available to authorized users. Patterns and processes of speciation in oceanic islands have long captured the attention of evolutionary biologists  and Echium (Boraginaceae) in the Canary Islands  in the Juan Fernández Archipelago , Schiedea (Caryophyllaceae), Cyanea, Lobelia and Trematolobelia (Lobeliaceae) in the Hawaiian Islands  in the Galápagos Islands . The genus contains seven species distributed in Central and South America , grow in continental Chile, with the others disjunct in Brazil and northwestern South America (with extensions northward into Mexico). Drimysconfertifolia is endemic to the Juan Fernández Archipelago, common on Robinson Crusoe Island, and with a patchy distribution on Alejandro Selkirk Island. These three species comprise a closely related complex, as evidenced by very similar ITS sequences  establish the most probable migration route(s) of the endemic species between islands; and (3) assess genetic consequences of island anagenetic speciation.The objectives of this paper are to: (1) determine genetic relationships and variation within and among populations of Drimys confertifolia Phil., “Canelo” , Fagara mayu (Rutaceae), and Juania australis (Arecaceae) , Dicksonia externa (Dicksoniaceae), Histiopteris incisa (Dennstaedtiaceae), and Lophosoria quadripinnata (Dicksoniaceae)  R.A.Rodr. et Quez. This species  and var. chilensis (DC.) A.Gray (“Canelo”). The former variety is a tree to 17 m, and is found in the subantarctic forests in the extreme southern portion of Chile (45°44′–55°58′ S)  and from 15 populations on Alejandro Selkirk Island (Nos. 17–31) , two populations of D. winteri var. winteri (Nos. 41 and 42), and eight populations of D. winteri var. chilensis (Nos. 33–40) were collected ; MseI–CTC/EcoRI–ACA (FAM); MseI–CAC/EcoRI–ATG (VIC); MseI–CAG/EcoRI–AAG (VIC); and MseI–CTC/EcoRI–AGC (NED). A total of 421 individuals was analyzed, 279 from D. confertifolia, 16 from D. andina, 104 from D. winteri var. chilensis, and 22 from D. winteri var. winteri . The range for allele call was 150–510 base pairs. Samples with size calibration below 90 % were manually adjusted. An automatic panel editor was generated for each selective primer combination ] where pi is the frequency of the ith band in the respective population based on all AFLP bands recorded) were performed using FAMD ver. 1.25  was used.With respect to genetic divergence parameters and population structure, the number of private bands (NPB) was calculated with FAMD ver. 1.25 , expected proportion of heterozygotes (HE), number of alleles per locus (NA), and inbreeding coefficient (FIS), were calculated using FSTAT 2.9.3.2 (Goudet NPA), and number of locally common alleles (NLCA). Allelic richness was calculated for populations that contained seven or more individuals using the rarefaction method  as per the model ranking method  to the models of unidirectional migration from Alejandro Selkirk to Robinson Crusoe Island (M1) and vice versa (M2). In order to standardize population sizes, the 16 populations of D. confertifolia in Robinson Crusoe Island and 15 populations in Alejandro Selkirk Island were pooled each and randomly sampled for each replicate . Runs were carried out under a Brownian model multiple times with varying parameter settings to achieve convergence, and for the final MCMC parameters were one long chain with 200,000 recorded steps and 100-step increment with a burn-in of 10,000. Uniform priors  were placed for both theta  and M . Ten replicates of single long Markov chains were implemented using different random number seeds. The MIGRATE analysis was performed at the University of Oslo Bioportal .The direction of migration between island populations of Drimys, we found a total of 583 fragments, of which 574 are polymorphic , in D. andina 383 fragments , and D. winteri 485 fragments  are: 153/81/126/for primers MseI–CTG/EcoRI–ACA (FAM); 120/76/102 for MseI–CAC/EcoRI–ATG (VIC); 112/72/80 for MseI–CTC/EcoRI–ACA (FAM); 104/58/76 for MseI–CAG/EcoRI–AAG (VIC); and 77/42/54 for MseI–CTC/EcoRI–AGC (NED). All individuals had unique AFLP phenotypes. The reproducibility of the bands was 95 %.With each pair of primers, the numbers of fragments in each species  after Bonferroni correction. The frequency of null alleles across populations and loci was estimated using Micro-Checker, resulting in the highest frequency of 0.188 (AWL0W), with an average frequency of 0.080 in all of the eight markers. Significant LD was not found between any pairwise combination of loci in all populations within each species (P < 0.05) after Bonferroni correction.All microsatellite markers, except for one marker AOES3, were successfully genotyped in 281 individuals of Drimys analyzed are shown in Table D. confertifolia, from Robinson Crusoe/Alejandro Selkirk Islands, are for percentage of polymorphic bands (PPB) 66.8/58.8; for total number of bands (TNB) 433.5/414.7; for Shannon Diversity Index (SDI) 103.98/90.53; and for average genetic diversity over loci (AGDOL) 0.24/0.22. All estimates reveal a similar pattern and are highly correlated with r ranging from 0.897 to 0.956 . This pattern holds when those correlations on the single islands were tested. Comparisons of genetic diversity between populations on the northern side of Robinson Crusoe Island (1–7) with populations on the southern side (9–16) show significant differences in the parameters TNB, SDI, and AGDOL. In Alejandro Selkirk Island, the northern populations  are not significantly different in all genetic diversity parameters from the southern populations (28–31). Overall the genetic diversity within populations is higher on Robinson Crusoe Island (average SDI 103.98 ± 6.88) than on Alejandro Selkirk Island (average SDI 90.53 ± 10.99).The AFLP measures of genetic diversity in populations of D. confertifolia, 0.203 in D. andina, and 0.208 in Drimys winteri. In D. confertifolia a higher value was found on Robinson Crusoe Island (0.259) compared to Alejandro Selkirk Island (0.234)  was 0.276 in Drimys are shown in Table D. confertifolia from Robinson Crusoe/Alejandro Selkirk Islands for number of alleles per locus (NA) are 4.89/3.73; for allelic richness (AR) are 4.24/3.30; and the expected proportion of heterozygotes (HE) are 0.62/0.47. All estimates are correlated; HE was highly correlated with NA (r = 0.868) , and AR (r = 0.634) . When analyzing this parameter by island, only the pair NA and HE is correlated (r = 0.810)  in Robinson Crusoe Island; no correlations were found between these three parameters in Alejandro Selkirk island. Positive FIS values (P < 0.05) after Bonferroni correction were significant in 11 populations of D. confertifolia and in four populations of D. winteri.In microsatellites, the values of genetic diversity parameters for all species of NA, AR, and HE, among northern  and southern populations (28–31).When dividing the populations of Robinson Crusoe Island into northern (1–7) and southern (9–16) sections, they show no significant difference. The same analysis holds for Alejandro Selkirk Island, revealing no significant differences between the parameters HE) within species was 0.675 in D. confertifolia, 0.733 in D. winteri, and 0.501 in D. andina  and rarity index (RI) in Robinson Crusoe/Alejandro Selkirk Islands are 0.40/0.14 and 1.50/1.76 respectively. These values are not correlated, (r = 0.065) . The same lack of correlation occurs when analyzing Robinson Crusoe and Alejandro Selkirk Islands separately. Comparison between northern populations with southern populations on Robinson Crusoe Island reveals significant differences only for the Rarity Index. The same analysis in Alejandro Selkirk Island reveals no significant differences in both measures of divergence.For AFLPs, the values of genetic divergence are shown in Table FST values between D. confertifolia on the two islands and continental D. andina and D. winteri reveal the highest differentiation (FST = 0.357) between D. winteri and D. confertifolia of Alejandro Selkirk island, and the lowest between D. confertifolia from Robinson Crusoe and Alejandro Selkirk (FST = 0.186) .Pairwise FST = 0.57 betweeDrimys species are shown in Table D. confertifolia the number of private alleles (NPA) and number of locally common alleles (NLCA) in Robinson Crusoe/Alejandro Selkirk Islands are 0.04/0.02 and 0.75/0.50, respectively. The differences between the values of this parameter are not correlated (r = 0.361) . The same tendency is seen when we analyzed separately each of the islands. We also compare the NPA between the two island populations of D. confertifolia. The NPA of Robinson Crusoe populations is 3.125 (±0.875), and that of Alejandro Selkirk is 0.75 (±0.366). Geographical division between northern  and southern populations (28–31) in Alejandro Selkirk Island showed no significant differences. The same lack of divergence between northern and southern populations also occurs on Robinson Crusoe Island.For microsatellites, the parameters for estimating genetic divergence within FST between D. confertifolia in Robinson Crusoe and Alejandro Selkirk Island and the other species of Drimys correspond to the pair D. confertifolia (Alejandro Selkirk island)–D. andina (FST = 0.283), and the lowest to D. confertifolia (Robinson Crusoe)–D. winteri (FST = 0.060) .The highest values of D. confertifolia are shown in Table Results of the analysis of molecular variance (AMOVA) at different hierarchical levels in D. confertifolia, D. andina, and D. winteri is shown in Fig. D. confertifolia forming two groups, the first clearly differentiated and corresponding to Alejandro Selkirk Island, and the second to the populations of Robinson Crusoe Island. No clear separation between populations is visible, with only a weak divergence of populations 23, 30, 31 on Alejandro Selkirk Island and population 13 on Robinson Crusoe Island. The cluster of D. winteri does not show a separation between variety winteri and var. chilensis. No geographical partitioning is observed in all species.The NeighbourNet tree based on AFLP data using all individuals of Drimysconfertifolia forms two groups, each restricted to a different island. The populations from Alejandro Selkirk Island are linked more closely to D.winteri than to the other populations on Robinson Crusoe Island. No geographical division is found in D. winteri, and no division between D. winteri var. winteri and D. winteri var. chilensis is seen. Drimysandina is connected to D. winteri.The Neighbour-Joining tree using microsatellite data at the population level is shown in Fig. D. confertifolia using the Bayesian approach STRUCTURE 2.3.3 )  are more likely than the null hypothesis of no gene flow (M0) between the two islands  than the other model involving arrival first to Alejandro Selkirk and then migration to Robinson Crusoe Island (M1).Results of the MIGRATE analyses Table  showed cds Table . The LBFDrimys, some taxonomic changes have been suggested during past decades. Drimys confertifolia was early regarded as a variety of D.winteri , all individuals of this species formed a single group, positioned within the complex of D. confertifolia. The same trend was shown by Jara-Arancio et al. , whereas D. winteri var. chilensis occurs through most of the country . Our molecular results, however, do not support these varietal concepts within D. winteri (var. winteri and var. chilensis); the varieties are neither clearly differentiated nor geographically separated  occurs from the populations of the coastal refugia of Fray Jorge and Talinay .At the variety level, Smith  and Rodrted Fig. . Ruiz etted Fig.  did not ted Fig. , using RDrimys of Chile, D. confertifolia has been shown, based on cp DNA and ITS sequences , rather than the reverse, for two reasons. First, the islands are geologically much younger (1–4 myr) than the continent , perhaps by transporting fruits internally  has been estimated by Donoso  from Japan, in comparison with the anagenetic derivative in Ullung Island, D. takesimana, showed a higher genetic diversity (AFLPs) in the endemic island species than in the one from Japan. Takayama et al. , in comparison with the progenitor W. coraeensis var. coraeensis, an incomplete anagenetic speciation process and a lower level of genetic diversity in the variety that occurs on the island. This is perhaps due to more recent immigration and less time available for accumulation of differentiating genetic variation. The other notable component of anagenetic speciation is that no geographical structure was observed in genetic variation among populations distributed over the island landscape. All indicators, the low proportion of variation among populations within the island (AMOVA: 5.79 %)  Table , the Manlia Fig. . In addiSupplementary material 1 (DOC 154 kb)Supplementary material 2 (DOC 36 kb)Below is the link to the electronic supplementary material.