Cellana toreuma along the China coast in order to acsertain impacts of historic events, ocean currents and especially freshwater discharge from the Yangtze River on the connectivity of intertidal species with limited larval dispersal capability.Genetic data were used to measure the phylogeographic distribution of the limpet, C. toreuma (n = 418), ranging from the Yellow Sea (YS), East China Sea (ECS) and South China Sea (SCS), were determined from partial mitochondrial cytochrome c oxidase subunit I gene. Genetic diversity and divergence based on haplotype frequencies were analyzed using CONTRIB, and AMOVA was used to examine genetic population structure. Historic demographic expansions were evaluated from both neutrality tests and mismatch distribution tests. Among the 30 haplotypes identified, a dominant haplotype No. 1 (H1) existed in all the populations, and a relatively abundant private haplotype (H2) in YS. Pairwise FST values between YS and the other two groups were relatively high and the percentage of variation among groups was 10.9%.Genetic variation in 15 populations of C. toreuma.The high nucleotide and gene diversity in the YS, with large pairwise genetic distances and relatively high percentages of variation among groups, suggests that this group was relatively isolated from ECS and SCS. This is likely driven by historic events, ocean currents, and demographic expansion. We propose that freshwater discharge from the Yangtze River, which may act as physical barrier limiting the southward dispersal of larvae from northern populations, is especially important in determining the separation of the YS group from the rest of the Chinese populations of  Understanding the forces which contribute to the biogeographic distributions of marine organisms has long been of interest to biologists in their attempts to interpret present day patterns of marine biodiversity Fossil and pollens records have shown that historical climate change is a pivotal factor affecting the abundance and distribution of species As many intertidal species have planktonic larvae which can be carried in the water column before settlement for days or weeks Patella rustica along the coast through occasional larval transport Limpets are important keystone grazers on rocky shores Hexechamaesipho pilsbryi , for example, has been shown to be strongly influenced by the Kuroshio Current 11 m3The distribution patterns of intertidal species along the coast of China are poorly studied but are likely to be influenced by past historic events as well as present day environmental conditions. The Bo, Yellow and East China Sea along the continental shelf of China, for example, are shallow seas and when global sea levels fell during the Quaternary glaciations, these three seas are thought to have become partly continuous landmasses . Sea surCellana toreuma  is widely distributed along the China coast C. toreuma from Qingdao in the north, to Weizhoudao in the south of China were used to investigate the importance of historic events, the role of ocean currents and Yangtze River discharge in affecting the present day genetic connectivity of intertidal, rocky shores species along the China coast.The limpet ellana toreuma from 15 rocky shore localities which represented populations from different marginal seas were collected in 2009–2010 within a geographic range of ∼2,600 km along the coastline of China  of protease K was added to the homogenate, which was then incubated in a water bath at 55°C. After 1∼3 h incubation, 400 µl NaCl (6 M) was added and the homogenate was centrifuged at 12,000 g for 30 min. An equal volume of isopropanol was added to the supernatant and then the mixture was centrifuged at 12000 g for 15 min. The precipitate was washed using 70% ethanol twice and then dissolved in ultrapure water. A 598 nucleotide fragment of cytochrome oxidase subunit I mtDNA (COI) was amplified and sequenced using the following primers: LCO1490F5′- GGT CAA CAA ATC ATA AAG ATA TTG G -3′, HCO2198R 5′- TAA ACT TCA GGG TGA CCA AAA AAT CA -3′ All individuals were stored in absolute ethanol until DNA extraction. Genomic DNA was extracted from foot muscle tissue using methods as described in previous studies lustal X1.81 ST values, and haplotype frequencies) were performed using Arlequin 3.5 ontrib 1.02 was used to standardized samples sizes, and haplotype frequencies were then calculated to estimate gene diversity and divergence of populations and groups Sequences of each individual were aligned with Crlequin, was used to examine population genetic structure. Historical demographic expansions were examined by Tajima's D test Fs rlequin.Analysis of molecular variation (AMOVA) ega 5.0 ST were calculated against geographic distance between all locations. The strength and significance of the relationship between genetic differentiation and geographic distances were assessed with Mantel tests using Ibd 1.53 ST values were plotted against geographic distance using Prismv5.0 .Phylogenetic trees of haplotypes were constructed using MCellana toreuma (GenBank access numbers JQ313140-JQ313557). Among all the individuals, 30 nucleotide sites were polymorphic. The genetic variation of COI mtDNA in the 15 populations of Cellana toreuma along the China coast was low, whether measured as gene diversity  or nucleotide diversity . The percentage of H1 in all populations ranged from 67–97%. In populations from the East China Sea, percentages of H1 in all the samples were >85% (86%–97%). All other haplotypes differed from H1 by only one or two mutations. In the two populations from the Yellow Sea (QD and DGD), however, the percentages of haplotype No. 2 (H2) were both 23%, indicating H2 was an important private haplotype in these populations , were low . The absolute gene differentiation (DGst) calculated using CONTRIB was highest in the YS group (0.115) as compared to the two other groups , genetic differences among populations within groups (FSC) and genetic differences among populations . However, such a relationship between genetic variation and geographic difference was not found among populations from the East China or South China Sea groups (P = 0.192), with only ∼1% of the variation in genetic differentiation being explained by geographic distance  between FFs were used to test for neutrality, and the results in different locations were variable , especially in the East China Sea populations. The unimodal mismatch distribution was also in accordance with an expected sudden population expansion C. toreuma populations along the China coast had experienced a large-scale population expansion.A dominate haplotype (H1) was found to be ubiquitously distributed in all the studied populations of Cellana toreuma in northern China and can explain the post-LGM recolonization from southern to northern China after the LGM. The genetic connectivity of C. toreuma along the China coast can, therefore, be explained by a demographic expansion which may have been mediated by transport of larvae via the Kuroshio Current, China Coastal Current and the Yellow Sea Warm Current , such as salt marshes, which might act as possible barriers restricting the dispersal of C. toreuma larvae. In this region, however, there are intermittent islands, dams and harbours, which may provide suitable hard substrates for colonization by C. toreuma and act as stepping-stones for colonization, as has been described for rocky intertidal invertebrates in south-eastern Australia There was only one mutational step between H1 and H2, and the genetic distance between the two haplotypes was very low, indicating the isolation of H2 was a relatively contemporary event. In southern Australia, a clear phylogeographic break in the distribution of the barnacle, C. toreuma. Reproduction in C. toreuma usually occurs in summer. In north Zhejiang, China, C. toreuma reproduces from June to September C. toreuma may cause a disconnection between the Yellow Sea populations and the southern populations of this limpet. This may, therefore, isolate the unique haplotype (H2) in the Yellow Sea group as larvae with this haplotype would be unable to disperse south to the East China Sea and subsequently South China Sea populations of C. toreuma.The relatively higher nucleotide and gene diversities in the Yellow Sea group are, therefore, most likely related to population disjunctions, due to the impact of ocean currents and/or Yangtze River discharge. This explanation seems plausible as pelagic larval dispersal will be the major source of contemporary gene flow and hence distribution of Cellena toreuma is different from that seen in the alga, Sargassum horneriS. horneri populations have good genetic connectivity with no apparent physical barriers to dispersal along the China coast. It has been suggested that the China Coastal Current in autumn and winter can transport floating marine organisms, such as fragments of Sargassum, along the China coast from the Yellow and Bohai Sea to the East China Sea S. horneri mature twice a year (in spring and autumn), the southward China Coast Current in winter can transport their propagules from the Yellow sea to East China Sea, and then to the South China Sea, and will not be affected by the barrier created by the summer Yangtze discharge. In contrast to S. horneri, the pelagic larval dispersal stage of C. toreuma is limited to only 4–18 days. Most importantly, the reproductive season of C. toreuma occurs only during summer, when the southward China Coast Current is interrupted by freshwater discharge from the Yangtze River which decreases salinity significantly, and therefore acts as a temporary barrier to the demographic expansion for northern C. toreuma populations. The phylogeographic patterns of intertidal species along the China coast are, therefore, closely related to species-specific life cycles and related ecological characteristics.The phylogeographic pattern of Cellana toreuma. Along the coast of China the interplay between these factors is strongly influenced by the timing of intense discharge from the Yangtze River. The coincidence of increased Yangtze River discharge and the resulting weakening of the China Coast Current in summer, when larvae of C. toreuma will be in the water column, act as a physical barrier, inhibiting the southern dispersal of limpet larvae and playing an important role in the phylogeographic patterns of this limpet along the China coast.In general, therefore, both historic events and contemporary oceanographic conditions play important roles in the demographic expansion of the intertidal limpet, Appendix S1Cellana toreuma populations in China coast.The haplotypes frequencies of COI in different (DOC)Click here for additional data file.Appendix S2Cellana limpets as outgroups.Neighbour-joining tree based on COI dataset with bootstrap values of 1,000 above each branch of interest with other (PDF)Click here for additional data file.Appendix S3Cellana toreuma populations.Genetic pairwise distances of the COI mitochondrial gene between 15 (DOC)Click here for additional data file.


CYP3A4 in three South African population groups comprising of 29 Khoisan, 65 Xhosa and 65 Mixed Ancestry (MA) individuals. To identify known and novel CYP3A4 variants, 15 individuals were randomly selected from each of the population groups for bi-directional Sanger sequencing of ~600 bp of the 5′-upstream region and all thirteen exons including flanking intronic regions. Genetic variants detected were genotyped in the rest of the cohort. In total, 24 SNPs were detected, including *12CYP3A4, *15CYP3A4, and the reportedly functional *1BCYP3A4 promoter polymorphism, as well as two novel non-synonymous variants. These putatively functional variants, p.R162W and p.Q200H, were present in two of the three populations and all three populations, respectively, and in silico analysis predicted that the former would damage the protein product. Furthermore, the three populations were shown to exhibit distinct genetic profiles. These results confirm that South African populations show unique patterns of variation in the genes encoding xenobiotic metabolizing enzymes. This research suggests that population-specific genetic profiles for CYP3A4 and other drug metabolizing genes would be essential to make full use of pharmacogenetics in Southern Africa. Further investigation is needed to determine if the identified genetic variants influence CYP3A4 metabolism phenotype in these populations.The CYP3A4 enzyme is the most abundant human cytochrome P450 (CYP) and is regarded as the most important enzyme involved in drug metabolism. Inter-individual and inter-population variability in gene expression and enzyme activity are thought to be influenced, in part, by genetic variation. Although Southern African individuals have been shown to exhibit the highest levels of genetic diversity, they have been under-represented in pharmacogenetic research to date. Therefore, the aim of this study was to identify genetic variation within  CYP3A4 can be inhibited by drugs  and is often involved in unfavorable drug-drug interactions, due to its ability to accommodate two or more similar or dissimilar molecules in its active site  subfamily in facilitating the elimination of drugs, other xenobiotic compounds and endogenous molecules from the body  the most ancient population group: the Khoisan, (2) the most globally-admixed population group: the South African Mixed Ancestry (MA) population, and (3) the largest language family in South Africa: the Bantu-speaking population group, represented by the Xhosa population. The ancient Khoisan population used in this study consisted of individuals from the !Kung and Khwe linguistic groups  and informed consent was acquired from all participants. Genomic DNA (gDNA) was available for 29 Khoisan, 65 Xhosa, and 65 MA healthy individuals. The Khoisan samples used in this study were collected from !Kung and Khwe speaking individuals from the Schmidtsdrift region of the Kalahari desert in the Northern Cape Province of South Africa   and bi-directionally sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit , after which capillary electrophoresis was performed by the Central Analytical Facility of Stellenbosch University on a 3130xl Genetic Analyser (Applied Biosystems). The obtained sequences were subsequently compared to the reference sequence  to detect the presence of variants. The generated sequence data also served to ensure that the reaction conditions used did not amplify any of the associated CYP3A4 isoforms or pseudogenes. The effect of the detected variants was determined using the software programs Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping (PolyPhen) and the Alternative Splice Site Predictor (ASSP)  analysis and additional sequence analyses. In cases where no restriction sites were created or destroyed by the SNPs of interest, mutagenic primers were designed to introduce artificial restriction enzyme recognition sequences , any individuals testing positive for this assay underwent bi-directional sequencing to determine which one, or both, of the SNPs were in fact present. For genotyping specifications, including a list of the specific restriction enzymes used, refer to Table To determine the frequencies of the genetic variants detected in the 5′-flanking region and the coding regions of the CYP3A4 genetic variants detected in the three population groups were compared using MedCalc Version 12.3.0 . Furthermore, we compared the frequencies of the allele defining SNPs detected in the three South African populations to the frequencies reported by the 1000 Genomes Browser (http://browser.1000genomes.org/) and HapMap Phase I + II Project data (http://hapmap.ncbi.nlm.nih.gov/). The 1000 Genomes Browser contains allele frequency information for the African (AFR), American Admixed (AMR), East Asian (ASN) and European (EUR) populations; while the HapMap project contains frequency data for European (CEU), Chinese (CHB), Japanese (JPT) and Nigerian (YRI) populations. Deviations from Hardy-Weinberg equilibrium (HWE) were determined using either a Pearson chi-squared analysis or an analogue to Fisher's exact test, depending on observed genotype distribution, in Tools for Population Genetic Analysis (TFPGA) Software v1.3 (http://www.marksgeneticsoftware.net/tfpga.htm). P-values of <0.05 were considered statistically significant.Allele frequencies of the CYP3A4 DNA sequencing. Three of the intronic SNPs and one SNP in the 5′-flanking region are novel. Genotyping of rs12721624 in intron 8 and rs147972695 in intron 12 could not be performed in the entire cohort, due to technical difficulties. Genotyping of the remaining 22 SNPs was successful, and all SNPs were in HWE  was predicted by both the SIFT and PolyPhen algorithms to affect the function of the protein product. None of the variants were predicted to alter any splice-sites.This study detected 24 SNPs in 45 individuals from three South African populations using P < 0.05) . Overall this study revealed the presence of the previously described *1BCYP3A4, *1GCYP3A4, *12CYP3A4, and *15CYP3A4 alleles, in addition to two novel alleles, *23CYP3A4 and *24CYP3A4. The number of novel alleles reported here is in accordance with the number of novel alleles that we have detected previously through the re-sequencing of other CYP genes in South African populations  in the EUR and AMR populations and is thus unlikely to occur at pharmacogenetically relevant frequencies in the South African populations . The reported functional significance of this intronic *22CYP3A4 variant does, however, highlight the importance of non-coding regions. The significance of these areas, including regions that are not in close proximity to the gene has been further emphasized by the recent release of the ENCODE data  and is thus likely to be affected by the consequences of misalignment or co-amplification of other genes during the use of high-throughput technologies.The detection of novel variants in this study highlights the fact that although large re-sequencing studies such as the 1000 Genomes Project have played an integral role in characterizing the human variome , novel variation still exists, underlining the importance of re-sequencing studies such as this one. These re-sequencing studies are specifically required in African populations, as these populations have been under-represented in genomic studies to date  affects the protein product. p.L373 has been identified as one of the key residues affecting substrate binding, cooperativity and regioselection of metabolism , while in the ASN and EUR population groups the opposite allele occurs more often. Interestingly, both the MA and AMR admixed populations show allele frequencies that are intermediate between the African and EUR/ASN populations. Furthermore, the MA is more similar to the African populations, while the AMR is more similar to the non-African populations. These results are in accordance with the ancestral history of these population groups. The MA have ancestry contributions from the Xhosa, Khoisan, European, and Asian populations , is more similar to the EUR/ASN population groups due to the larger ancestry contribution that these populations have made to the AMR, when compared to the ancestry contribution of Africans (Galanter et al., The differences in allele frequencies observed for the The allele frequency differences observed in admixed populations, as previously reported in admixed Brazilian populations (Suarez-Kurtz, CYP3A4, the relevance of these SNPs in the clinical setting remains unknown. The SNP markers detected in the current study should therefore be included in genotyping panels in future pharmacogenetic association studies involving CYP3A4 substrate medications. Nonetheless, this study provides an excellent example of how re-sequencing studies are required in African populations in order to identify variation that remains novel. These differences in allele frequencies were not only seen when comparing the South African populations to other populations, but also when comparing them to each other. These results demonstrate that a one-size-fits-all approach is not ideal when implementing therapeutic treatment regimes, also within the South African context.Although this study identified both novel and known SNPs of functional significance in all three population groups, due to the current lack of validated evidence regarding the pharmacogenetic application of 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.


S. haematobium is one of the most widespread causing urogenital schistosomiasis, a major human health problem across Africa, however in terms of research this human pathogen has been severely neglected.Schistosomiasis in one of the most prevalent parasitic diseases, affecting millions of people and animals in developing countries. Amongst the human-infective species Schistosoma haematobium, a DNA ‘barcoding’ study was performed on parasite material collected from 41 localities representing 18 countries across Africa and the Indian Ocean Islands. Surprisingly low sequence variation was found within the mitochondrial cytochrome oxidase subunit I (cox1) and the NADH-dehydrogenase subunit 1 snad1). The 61 haplotypes found within 1978 individual samples split into two distinct groups; one (Group 1) that is predominately made up of parasites from the African mainland and the other (Group 2) that is made up of samples exclusively from the Indian Ocean Islands and the neighbouring African coastal regions. Within Group 1 there was a dominance of one particular haplotype (H1) representing 1574 (80%) of the samples analyzed. Population genetic diversity increased in samples collected from the East African coastal regions and the data suggest that there has been movement of parasites between these areas and the Indian Ocean Islands.To elucidate the genetic diversity of S. haematobium in Africa the population may have passed through a genetic ‘bottleneck’ followed by a population expansion. This study provides novel and extremely interesting insights into the population genetics of S. haematobium on a large geographic scale, which may have consequence for control and monitoring of urogenital schistosomiasis.The high occurrence of the haplotype (H1) suggests that at some point in the recent evolutionary history of  Schistosoma. Species that infect humans are prevalent in developing countries, having a major impact on public health and well-being as well as an impediment to socioeconomic development. More people are infected with Schistosoma haematobium than with all the other schistosome species combined, however mainly due to the inability to maintain S. haematobium in the laboratory system empirical studies on this parasite are minimal. The genetic variation of this Schistosoma species on a wide geographical scale has never been investigated. In this study, we have used a DNA ?barcoding? approach to document the genetic variation and population structure of S. haematobium sampled from 18 countries across Africa and the Indian ocean Islands. The study revealed a distinct genetic separation of S. haematobium from the Indian Ocean Islands and the closely neighbouring coastal regions from S. haematobium found throughout the African mainland, the latter of which exhibited extremely low levels of mitochondrial diversity within and between populations of parasites sampled. The data from this study provides a novel insight into the population genetics of S. haematobium and will have an impact on future research strategies.Schistosomiasis is a disease caused by parasitic blood flukes of the genus  Schistosoma haematobium is one of the most widespread species of Schistosoma and causes urogenital schistosomiasis in humans. More people are infected with S. haematobium than with all the other schistosome species combined. Of the >110 million cases of S. haematobium infection in sub-Saharan Africa, 70 million are associated with hematuria, 18 million with bladder wall pathology, and 10 million with hydronephrosis leading to severe kidney disease S. haematobium and the pathogenesis of the parasite's infection, empirical studies on S. haematobium are minimal, compared to those of S. mansoni and S. japonicum, due, at least in part, to the inherent logistical difficulties of maintaining S. haematobium within the laboratory system S. haematobium has recently been published Schistosomiasis remains one of the world's greatest neglected tropical diseases (NTD). S. haematobium has a large geographical distribution being found throughout Africa, parts of the Middle East, Madagascar and the Indian Ocean Islands and is transmitted by various intermediate snail hosts within the genus BulinusS. haematobium has been the subject of very few molecular studies S. haematobium showed some regional variation and suggested mixing of parasite strains due to human population movements Praziquantel (PZQ) remains the drug of choice for treatment of schistosomiasis and for the control of morbidity. It has a good safety and therapeutic record and is easy to administer , generally improving the health and well-being of schistosome-infected people. National control programmes in several sub-Saharan countries aim to alleviate the burden of schistosomiasis in highly endemic areas through large-scale administration of PZQ cox1) gene is commonly compared between sampled specimens to identify evolutionary differences, as well as, similarities. Such studies have benefited from knowledge of the complete mitochondrial genome of S. haematobium, as well as other schistosomes S. mansoni to date has revealed extensive population diversity with geographical structuring existing between populations S. haematobium worms from Zanzibar also revealed substantial genetic diversity with the worms splitting into two distinct phylogenetic groups S. haematobium from several areas geographically spread across Africa using historical collections of laboratory isolates but also mainly large collections of individual schistosome miracidia and cercariae sampled directly from their natural hosts, thereby avoiding the ethical and biological biases inherent within analyses of laboratory-passaged adult worms S. haematobium on the African mainland and the Indian Ocean Islands could be further elucidated.In recent years, developments in molecular tools, and in particular advances in DNA sequencing, have allowed greater exploration and recording of the genetic diversity of schistosome species and their hosts thereof e.g. . SequencEthical approval was obtained from Imperial College Research Ethics Committee (ICREC), Imperial College London in the UK, in combination with the ongoing Schistosomiasis Control Initiative (SCI) activities. In Senegal, ethical approval was obtained from the ethical committees of the Ministry of Health Dakar, Senegal. In Niger, ethical clearance was obtained from the Niger National Ethical Committee. In Cameroon, ethical approval was obtained from the Commité National d'Ethiqué, Cameroon. In Kenya, ethical approval was obtained from the Ethical Review Board of National Museums of Kenya/Kenya Medical Research Institute. In Tanzania, ethical approval was obtained from the Ethical Review Board of National Institute of Medical Research (NIMR). In Zambia, ethical clearance was obtained from the University of Zambia ethics committee.Before conducting the study, the MoH-approved plan of action had been presented and adopted by regional and local administrative and health authorities. Meetings were held in each village to inform the village leader, heads of the families, local health authority, teachers, parents and children about the study, its purpose and to invite them to voluntarily participate. According to common practice and with approval from the Imperial College Research Ethics Committee (ICREC), due to low levels of literacy all village leaders, teachers, parents and study participants gave oral consent for the studies to take place. Informed consent for the urine examinations was obtained from each study participant and their parents or guardians. Oral consent for each participant was documented by inscription at school committees comprising of parents, teachers and community leaders. All the data were analyzed anonymously and all schistosomiasis positive participants were treated with PZQ (40 mg/kg). In schools or classes where the percentage of infections were more than 50%, mass treatment of all children was carried out at the end of the study.The historical material stored in SCAN has been derived from UK laboratory passage of original specimens collected in collaboration with local authorities abiding by the ethical standards and collection requirements of the day and all specimens were maintained and analyzed anonymously.Laboratory animal use was within a designated facility at the NHM regulated under the terms of the UK Animals (Scientific Procedures) Act, 1986, complying with all requirements therein, including an internal ethical review process at the NHM and regular independent Home Office inspection. Work was carried out under the Home Office project license numbers 70/4687 (-2003), 70/5935 (2003–2008), 70/6834 (2008-).As part of a European Commission Specific Research Project (CONTRAST) ‘A multidisciplinary alliance to optimize schistosomiasis control and transmission surveillance in sub-Saharan Africa’, parasitological surveys were conducted at 13 localities in a total of seven countries across Africa between 2007–2010 . Eggs weIndividual cercariae from naturally infected snails were also sampled from a few localities . Snails Laboratory passaged adult worms from nine additional localities held in the Natural History Museum, London (NHM) liquid nitrogen schistosome repository, Schistosome Collections at the Natural History Museum (SCAN), were also utilised for molecular analysis . A featuFor genomic DNA (gDNA) extraction, a 2.0 mm disc containing the sample was removed from the card using a Harris-Micro-Punch  and incubated for 5 mins in 200 µl of FTA purification reagent . The FTA purification reagent was removed and the disc was incubated for a further 5 mins in 200 µl of fresh FTA purification reagent. This process was repeated for a total of 3 washes and was followed by 2×5 mins incubations in 200 µl of TE buffer. Samples were air dried at 56°C for 10–30 mins and the disc was visually checked to make sure it was dry before being used directly in PCR reactions.Adult worms were thawed on ice and total gDNA was extracted from individual males and females cox1 gene was amplified in separate 25 µl PCR reactions using illustraTM puReTaq Ready-To-Go PCR Beads  and 10 pmol of each primer  (756 bp) was amplified and sequenced as above using DNA available from several of the laboratory passaged samples  datasets were assembled and manually edited using Sequencher V4.6 (http://genecodes.com) to remove any ambiguities between forward and reverse strands. For each sample consensus sequences were aligned in Sequencher and polymorphic positions observed between individuals were checked and confirmed by visualisation of the original sequence chromatograms. The identity (species and gene) of the sequence was also confirmed using the Basic Local Alignment Search Tool (NCBI-Blast). Within each individual locality  to identify individual samples with identical sequences. Each group of identical sequences and also any unique sequences became unique haplotypes for that site and consensus sequences were created and given a unique haplotype identifier code, consisting of the site code and a letter representing the different haplotypes within each locality. The numbers of individuals that presented each haplotype in each locality were also recorded.All sequences for the different (S. haematobium cox1 and nad1 data available on the NCBI database (http://www.ncbi.nlm.nih.gov), which was downloaded and incorporated into the datasets from our collections  to identify any identical haplotypes from different localities.The complete datasets  were aligned in MacClade 4.05, and then collapsed (Collapse V1.2 (cox1 or nad1) haplotype (H1) was used in the analyses to represent all the individuals that it corresponded to, unless stated otherwise. Haplotype sequences were submitted to EMBL/Genbank .To estimate genealogical relationships between haplotypes, the individual haplotype sequences were aligned in MacClade V4.05 and then a minimum spanning network was created in the programme TCS  analysis with 500 replicates was also instigated in Mega V5 using the best fit ML model (HKY+G) which was calculated in Mega V5 and also by using the Akaike criterion in jModeltest V0.1.1 All the aD) between the two main groups found in this analysis was calculated with the Juke-Cantor correction model in DnaSP V5.The net nucleotide divergence  and nucleotide diversity (Π), the latter of which was calculated with Juke-Cantor corrections, which was the most complex substitution model available. Overall diversity was measured together with the diversity within different geographic regions and also each locality , Mali (Z21716) and Zanzibar (GU257398). Sequences were aligned and any nucleotide differences recorded.To compare variation found within the mtDNA with nuclear DNA, the complete ITS (1+2) rDNA (927 bp) was amplified from a single male and a single female worm from all localities that had representative adult worms available . This maS. haematobium.There were no differences between the ITS1+2 sequences from any of the samples. This nuclear marker therefore proved uninformative as a population genetic marker for cox1 sequences were analyzed: 46 from cercariae, 241 from laboratory passaged adult worms  analyzed contained 70 variable sites (53 parsimony informative), and the diversity found within the S. haematobium populations sampled was low with the sequences resolving into just 61 unique haplotypes. The percentage occurrence of each haplotype varied but there was a main common haplotype (H1) that was found across most of the localities and appeared frequently on mainland Africa, representing 1574 (80%) out of the 1978 overall sequences analyzed.In total 1978 The minimum spanning TCS network  clearly As with the TCS analysis the same general splitting of the haplotypes was found with all phylogenetic methods separating the 61 haplotypes into two distinct well-supported groups  with H1 S. haematobium groups compared to that of the much larger divergence from their sister taxa S. bovis The net divergence between the groups (0.02145±0.00102) shows a relatively short time between the genetic separation of these two p values were all > than 0.05).Measures of overall haplotype and nucleotide diversity, together with within region and locality diversity, are presented in nad1 haplotypes sequenced from several of the samples . Ninety seven percent of the data generated came from larval stages sampled directly from their human hosts from across 20 localities, with the remainder of the data coming from historical collections based on laboratory-passaged worms. The study has revealed that the genetic diversity of S. haematobium across Africa is unexpectedly low. There were only 61 unique haplotypes found in the 1978 samples collected from 41 locations and 18 countries. The haplotypes split into two distinct groups; one that contains haplotypes predominately from mainland Africa with a few haplotypes from Zanzibar (Group 1) and the other that is made up of samples exclusively from the Indian Ocean islands and the neighbouring African coastal regions (Group 2). The net divergence between the two groups was considerable and was strongly supported by both the cox1 and the nad1 data. This is equivalent to the net divergence seen between some S. mansoni groups spread across Africa, separated by thousands of miles This study is the first time that DNA S. haematobium samples can clearly be seen in the TCS network and phylogenetic analyses with a single haplotype (H1 from 1574 samples) being dominant across Africa with greater diversity found within the samples from the Indian Ocean Islands and the neighbouring African coastal regions. The nuclear ITS data showed no diversity from any sample proving that this can not be used as a population genetic marker for this parasite The lack of the diversity found within and between the S. haematobium populations from coastal Kenya and Zanzibar with complicated networks linking these haplotypes and several nodes not being represented by a haplotype. This suggests that haplotypes may have become extinct or that they have not been sampled indicating there may be more diversity still to be discovered in these areas. However, the basic network of single links around H1 suggests that further sampling in the other areas, reported on in this study, is unlikely to reveal further discrete groupings. With exceptions of haplotypes from the Indian Ocean Islands, Coastal Kenya and Zambia, the network clearly shows a lack of geographic structuring with the same cox1 haplotypes being found in Far West, Central West, East, South of Africa and also in Sudan, Egypt and Zanzibar.The longer branches stemming from the main H1 haplotype in Group 1 on the TCS analysis show that the populations from coastal Kenya, Zambia and Mafia are quite separated from the main haplotype group. The highest diversity was found in the S. haematobium genotype there has also been transfer of Group 2 schistosomes from the Indian Ocean Islands and the neighbouring coastal regions probably associated with the movements of people S. haematobium from Zanzibar. This is not unexpected due to infected children from Mafia having a travel history to the Zanzibar mainland and that urogenital schistosomiasis is suggested to be imported and not endemic on Mafia.The distribution of the haplotypes must reflect in part past movements of people. Group I and 2 parasites have been isolated from the same geographical regions and from the same host. For example, the haplotypes from Mwanza, Tanzania (TA1a + b), resolve into both network groups, one haplotype (TA1a) being identical to the H1 haplotype (Group 1) and one (TA1b) being identical to a haplotype found on Mafia Island and sitting at the end of a long branch within Group 2. This suggests that in this geographic region as well as the main dominant S. haematobium populations from endemic adjacent regions such as Madagascar and the Arabian Peninsula. There is a history of prolific trade links between the Arabian Peninsula, India, the Indian Ocean Islands and the East coast of Africa aided by the Monsoon trade winds, which probably would have facilitated the movements of people and their parasites between these areas.The higher diversity found in the coastal Kenyan populations (15 haplotypes), the close clustering with the Zanzibar haplotypes and the separation of these populations into the two groups also suggests a close association between parasites from coastal Kenya and Zanzibar. There is probably mixing and movements of the populations between coastal Kenya and Zanzibar and vice versa with the movements of people between these areas due to the trade routes between the Indian Ocean islands and the neighbouring East African coastal regions S. haematobium populations separated by 1000's of miles across continental Africa compared to the high diversity found within the populations from the more isolated Indian Ocean Islands and their closely neighbouring African coastal regions is unexpected. It is particularly striking that the dominant haplotype H1 occurs across Africa with 1574 samples analyzed not showing a single nucleotide mutation in the mtDNA analyzed. The success of this haplotype might be attributed to a founder effect following a population ‘bottleneck’ with only a few individual parasites surviving and participating in a later population expansion. The lack of diversity found across Africa and the restriction of the Group 2 haplotypes to coastal regions of East Africa and the Indian Ocean Islands suggests that this may have happened relatively recently in terms of the evolutionary history of these parasites. Given the close phylogenetic relationship of the Indian/Asian and African Schistosoma species S. haematobium populations across mainland Africa is attributed to a re-invasion by a small number of individuals of S. haematobium into Africa from a larger population in Asia across the Arabian peninsula, with a subsequent rapid spread and population expansion across Africa from East to West. The new small re-established population in Africa would be more sensitive to genetic drift and increased inbreeding resulting in low genetic variation. Due to the lack of fossil records it is extremely difficult to accurately define the evolutionary history and phylogeography of the Schistosoma genus In consideration of general population genetic theories, the extremely low levels of genetic diversity between the S. haematobium is very specific and varies in different geographical locations S. haematobium in East Africa and the Indian Ocean Islands is mainly transmitted by Bulinus africanus and B. forskalii group species while elsewhere the same species groups can be involved but snails of the B. truncatus tropicus complex may also play an important role in transmission. However, the study by B. globosus, separates into distinct West and East African clades on a molecular phylogenetic tree, possibly suggesting that the distribution of East African B. globosus could be a limiting factor in the spread of Group 2 type parasites. It is clear that more studies are needed to investigate the role of the different snail species and geographical populations of Bulinus in the transmission of Group 1 and Group 2 type parasites. As well as intermediate snail host compatibility it will be important to determine whether the different groups give rise to infections which result in different pathologies or which respond differently to treatments as genetic diversity has been noted to possibly have an effect on such characteristics One factor that could have influenced the divergence of our populations into the two groups relates to compatibility with intermediate snails hosts. Intermediate host use of S. haematobium miracidial populations from Mali conflicting the data presented here, however only laboratory maintained material from Mali was analysed in the present study and so a direct comparison cannot be made. Due to the difficulties in directly sampling natural schistosome populations there exists a strong sampling bias within this study with the majority of the data obtained from large larval schistosome populations collected from 6 countries. The other countries are represented by far fewer or laboratory maintained samples and whilst providing useful genetic data it cannot be concluded that they are representative of the true genetic diversity in these countries. More samples need to be analyzed from more areas and with more genetic markers to further elucidate the genetic diversity of S. haematobium populations from all it's endemic areas. It would also be beneficial to analyze both nuclear and mt DNA simultaneously from the same individual sample in future population genetic studies and the recent publication of whole-genome sequence of an Egyptian isolate of S. haematobiumThough the small regions of nuclear DNA analyzed in this study proved highly conserved, it is likely that there could be more diversity found in other regions of the nuclear genome, which may or may not correlate to that found in the mt DNA. A recent study S. mansoni and S. haematobium is an area of high interest with respect to the development of drug resistance S. haematobium across most of mainland Africa, as defined by the current genetic markers, may indicate that these parasites may be less likely to change under drug pressure, however the high genetic diversity found on Zanzibar and the neighbouring African coastal region could offer a genetic base for the development of PZQ resistance and hence changes in parasite diversity in relation to chemotherapy needs to be monitored in these highly diverse areas.The genetic diversity of schistosome populations can be influenced by a variety of factors such as; host water-contact patterns, host immunity and susceptibility and moreover, mass chemotherapy has a great potential to promote selection. S. haematobium mtDNA population genetics. It is clear that further sampling in many areas will not dramatically increase the mtDNA diversity found but in areas such as Zambia, the East African coastal regions and the Indian Ocean islands where more diverse populations of S. haematobium have been found, further sampling would add to our understanding of the parasite population movements and diversity. It is also important that the population genetics of the S. haematobium is monitored further to link diversity with morbidity and to provide information on the response of parasite populations to drug treatment pressures. The mtDNA diversity described here, together with other molecular markers, will be of value to monitor the impact of control interventions on different S. haematobium genotypes and may assist in understanding the introduction or re-introduction of parasites associated with human population movements.This study has reported on some very unusual findings in relation to Table S1Sample and haplotype information (Supporting Table).(DOC)Click here for additional data file.


Channa striata in Sundaland was carried out using data from mtDNA ND5 gene target to elucidate genetic patterning. Templates obtained from a total of 280 individuals representing 24 sampling sites revealed 27 putative haplotypes. Three distinct genetic lineages were apparent; 1)northwest Peninsular Malaysia, 2)southern Peninsular, east Peninsular, Sumatra and SW (western Sarawak) and 3) central west Peninsular and Malaysian Borneo (except SW). Genetic structuring between lineages showed a significant signature of natural geographical barriers that have been acting as effective dividers between these populations. However, genetic propinquity between the SW and southern Peninsular and east Peninsular Malaysia populations was taken as evidence of ancient river connectivity between these regions during the Pleistocene epoch. Alternatively, close genetic relationship between central west Peninsular Malaysia and Malaysian Borneo populations implied anthropogenic activities. Further, haplotype sharing between the east Peninsular Malaysia and Sumatra populations revealed extraordinary migration ability of C. striata (>500 km) through ancient connectivity. These results provide interesting insights into the historical and contemporary landscape arrangement in shaping genetic patterns of freshwater species in Sundaland.A phylogeographic study of an economically important freshwater fish, the striped snakehead,  Genetic patterning of ichthyofauna is greatly influenced by ecological processes, anthropogenic factors C. striata (Channidae) is native to, and found naturally throughout freshwater sources across many of Southeast Asian countries and is the most widely distributed species among the snakehead members C. striata is often known as haruan or ruan . This species is now commonly found in many freshwater habitats. Its wide occurrence in natural waters coupled by its well-known nutraceutical and pharmaceutical properties, has made it one of the most popular protein food sources, especially among the rural communities The striped snakehead, C. striata to colonize natural and artificial reservoirs C. striata in Malaysia using RAPD markers The ability of Matrilineal markers are potent tools for assessing genetic relationships among individuals within species due to their rapid mutation rates and consequent high genetic variability The present study was focused on the Malaysian populations from both Peninsular Malaysia and Malaysian Borneo. Two populations from Sumatra, Indonesia were also included. These areas represent a major part of the Sundaland biogeographical region. The NADH dehydrogenase subunit 5 (ND5) gene was utilized as an analytical target. The specific objectives of this study were to characterize genetic diversity at each sampling locality and phylogeographic structuring across the region with respect to natural physical barriers. The initial hypothesis was that adjacent populations share common haplotypes, but even close populations may be significantly structured according to prominent geographical barriers.Live specimens were collected from local fishermen and wet markets. Sample locations were determined by interview and confirmed to have originated from a single source prior to collection. Clips were taken from the dorsal or caudal fin rays (approximately 0.2 cm x 2 cm) and preserved in 95% ethanol and stored at room temperature (∼25°C) until use. The fish were then returned to the dealers or brought back to the Aquatic Research Centre at Universiti Sains Malaysia (USM), Penang for further research. This study has been approved by the USM Ethics Committee. All practical steps to ameliorate suffering by specimens were taken throughout this study.C. striata populations were collected throughout its distribution in Peninsular Malaysia and Malaysian Borneo (Sarawak and Sabah) between 2007 and 2010. Populations were provisionally divided into five regions, northwest Peninsular, central west Peninsular, east Peninsular, southern Peninsular and Malaysian Borneo comprising of Sabah and Sarawak  and H13396-ND5 (5′- CCTATTTTKCGGATGTCYTG-3′) 2and 1.67 U of Taq polymerase . The PCR was conducted in 30 µl total reaction volume in an MJ PTC-200 Thermal Cycler . Amplification conditions were: initial incubation at 94°C (2 min); 35 cycles at 94°C (20 sec), 55°C (20 sec), 72°C (1 min 10 sec), final extension at 72°C (5 min) and a final hold at 10°C. The PCR products were visualized on a 1.7% agarose gel and stained with ethidium bromide to confirm successful amplification. The PCR product was purified using QIAGEN purification kits  according to the manufacturer’s instruction. At the final step, 30 µl product was eluted and 5 µl of the total elution was used to assess the quality of purified product on a 1.7% agarose gel. All purified products were sent for DNA sequencing  and reading from both DNA strands. Multiple sequences were aligned and all unambiguous operational taxonomic units (OTUs) were compiled and edited using ClustalW implemented in MEGA 4.0 d) and nucleotide diversity (π) were calculated to describe DNA polymorphism at each sampling site using Arlequin 3.1 DNA templates were isolated using AquaGenomic™ DNA isolation kits  following the manufacturer’s protocol. Aliquots of purified DNA isolates were used as templates for PCR amplification of the complete ND5 gene. The primer pair L12321-Leu (C. micropeltes (HQ 438584) and C. argus (10251173: Gene trees were constructed using Neighbor-Joining (NJ) k) was determined based on the highest value of variance among groups (FCT), incorporating information on haplotype divergence and geographical proximity. Subsequently, a hierarchical analysis of molecular variance (AMOVA) was conducted to infer the relative contribution to variance among groups (FCT), among populations within groups (FSC) and within populations. Based on the SAMOVA population structure estimate (k), the population pairwise comparison statistic, FST that calculates relative genetic differentiation between populations was determined in Arlequin 3.1 to evaluate the significance or otherwise of differences among populations and spatial population structuring. The analysis used Kimura 2-Parameter data and statistically significant pairwise comparisons were tested with 10,000 permutations. Significant probability values were adjusted by performing the False Discovery Rate Procedure (FDR) at α = 0.05 which controls the family wise error rate (FWER), a conservative type I error rate that originates from multiplicity ST) and sequence-based statistics (NSTST*) were also employed using 1000 permutations A spatial analysis of molecular variance was conducted using SAMOVA v.1.0 tics NST and KST*A total of 280 individuals from 24 populations were successfully PCR amplified for mtDNA sequence variation in the ND 5 gene. A final truncated target length of 1017 bp was obtained after alignment and editing of ambiguous sequences. Sample size varied from five to 19 individuals per population with an average of 12. The final alignment of sequences revealed a total of 53 segregating sites (54 mutation sites and 22 parsimony informative sites) defining 27 putative haplotypes with 14 (51.9%) of them being private haplotypes . The aveC. striata from all other regions  shared other common haplotypes – Hap03, Hap04, Hap07, Hap11, Hap12, Hap13, Hap14 and Hap15. However, nucleotide substitution at several sites  further differentiated the central west Peninsular and Malaysian Borneo (except SW) from other populations.Closer observation revealed that these amino acid substitutions were mainly regional specific, if not population specific. Thus, Hap01, Hap05, Hap19, Hap21 and Hap23 were specific to the six northwest Peninsular populations   & S2. InNevertheless, several unexpected findings were made, for example the central west Peninsular Malaysia populations had haplotypes in common with the Malaysian Borneo populations. The dominant haplotype of Malaysian Borneo populations  was Hap02 and this was also found in 28.6% of individuals in one of the central west Peninsular Malaysia population, KJ and in single individuals from KR and KK. Moreover, a single individual from KS population  was also found to share Hap09 with another two central west Peninsular Malaysia populations, TR and KJ.d  =  0.00 to 84.62% and π  =  0.00 to 0.65%). In common with findings made using other variability measures, nucleotide diversity was highest in SG (0.65%), followed by KJ (0.58%), TR (0.57%), JN and KK (0.39%).Overall haplotype diversity was 0.9±0.006. In general, genetic diversity was the highest in central west Peninsular. Here, TR was most polymorphic with 20 variable sites, three being singletons . This waGene trees inferred by NJ and Bayesian tree clustering methods recovered the same topology. All 27 haplotypes could be assigned to three major lineages marked by more or less robust bootstrap values (>70%) as shown in the NJ tree . HoweverThe haplotype network diagram  illustraInterestingly, there were some slight discrepancies as observed in the assignation of Hap11 and 15 using gene tree and network diagram. In the gene tree, they were grouped closely to Clade III while they seemed to be closer to Clade II in the haplotype network diagram.k clusters from 2<k<20 directly increased the values of variance among groups (FCT) suggesting that the populations were highly structured. Therefore, k cluster grouping based on the highest FCT was not very useful to assess population relationships. Thus, based on the phylogenetic NJ analysis, k clusters value was set at 3 (CT = 64.59%), followed by within populations (19.95%) and finally between populations within groups (FSC = 15.45%). The grouping obtained was precisely the same as that defined by the previous phylogenetic analysis.In the SAMOVA analysis, increasing the number of set at 3 , Table 3ST values, 89.5% of the comparisons showed significant population differentiation (p<0.05) after FDR adjustment (table not shown). Out of 276 possible comparisons, three of the pairwise estimates were negative. Adjacent populations in the same group  population was closely related to the Malaysian Borneo population, SS, while the KP population (Sumatra) was not differentiated from SG (east Peninsular) and nor from two populations in southern Peninsular, LG and MS. These observations were in agreement with the gene trees which divided the populations we investigated into the three clades.Based on pairwise FST) and nucleotide sequence-based statistics (NST and KST*) were high (0.67 to 0.72) and significant (p<0.001), consistent with the FST statistic results. Gene flow estimates among populations were low .Genetic differentiation estimates for all populations based on haplotype-based statistics (HST between groups showed the highest differentiation between groups 1 and 3 (74.35%) followed by between groups 2 and 3 (58.86%) and the lowest between groups 1 and 2 (57.63%). This pattern was further supported by the divergence analyses. All pairwise comparisons were significantly structured in FST at 95% confidence level.Mean genetic distances between populations computed based on Kimura 2-parameter values ranged from 0.00%–1.20% (table not shown) with an overall mean distance of 0.62%. Comparisons of pairwise FC. striata populations within the regions studied. Overall, relatively high haplotype diversities were observed in most C. striata populations (C. striata populations mainly from the Mekong and Chao Phraya rivers found them to exhibit relatively high population haplotype diversity (0.97) inferred from mtDNA cytochrome b gene sequence data Acrossocheilus paradoxus (Hd = 1.00) inferred from multiple mtDNA segments Lentipes concolor, Stenogobius hawaiiensis, Sicyopterus stimpsoni and Awaous guamensis (0.47 to 0.98) inferred from both coding and non-coding regions of mtDNA The SG, TR, KJ and LG samples were shown to have come from among the most highly variable populations  i.e. thoulations , a phenoC. striata populations showed a total absence of genetic variation, which was not simply related to sample size. Several of the smaller populations  harbored higher genetic variation than some of the larger ones  which showed monomorphism or lower haplotypic variability nHemibagrus nemurus within the Southeast Asian region has shown a genetically and morphologically characteristic form of H. nemurus is found in the Kapuas River (west Borneo) as distinct from other Sarawak populations (Sadong River at Serian and Rajang River at Kapit and Sibu) C. striata population with the southern Peninsular and Sumatra populations suggest ancient drainage connectivity, possibly via the North Sunda River system. This ancient drainage was believed to have connected many rivers of Sumatra and Borneo, and thus having a major influence on freshwater fish dispersal between these two regions H. macrolepidota populations from southern Peninsular Malaysia with southern and western Sarawak populations has been taken to reflect recent geographic isolation between the two regions Tor tambroides between Sarawak and Perak  populations, inferred from partial COI gene was hypothesized to be a consequence of a historical drainage connection during the last Pleistocene glaciation period Barbonymus schwanenfeldii from Peninsular Malaysia and Sarawak inferred from mitochondrial cytochrome b gene sequence data was in concordance to the separation of the Borneo Island from mainland Peninsular during the late Pleistocene k, was set at a value of 3, the SW population (west Sarawak) showed closest relationship to the populations belonging to east Peninsular, southern Peninsular and Sumatra though they are presently separated by a great expanses of sea namely the South China Sea and Straits of Malacca respectively.Our study provides yet further evidence of historical events associated with the palaeo North Sunda River system during the Pleistocene era Population isolation over a prolonged period of time, most prominently due to geographical barriers may have permitted the development of characteristic mtDNA variants in individual populations in this species. Evolutionary processes occurred independently in the various isolated regions and have now resulted in the clustering of three distinct monophyletic molecular groups. The first group consisted of six Peninsular Malaysia populations situated in northwest Peninsular  where they were physically isolated from populations to the south of them by the Bintang Mountain Range. This mountain acted as an effective barrier to gene flow, which would have constrained short-range migrations between populations separated by this divide.Labiobarbus leptocheilusOxyeleotris marmoratusBatagur baskaAnabas testudineusThe other significant structuring was observed between populations from the central west Peninsular and east Peninsular Malaysia (Clade III). The main mountain range (Titiwangsa Mountains) which was formed during the late Triassic (200mya) had acted as a natural divider to terrestrial as well as riverine biota since its formation. A similar pattern of separation has been reported by several researchers investigating freshwater fishes within this region; the freshwater cyprinid, C. striata as reported by the United State Geological Survey  shows that this species is not indigenous to most of the eastern part of Sarawak and the state of Sabah. Thus, in this case data for populations SS, SB and KS strongly suggest human mediated translocation as the reason for the presence of these populations in these regions. However, there has been one report documenting C. striata as native to Sabah C. striata from Borneo to Singapore took place back in the 1950s as well as by unintentional translocation due to human activities in the past Lack of genetic differentiation between two adjacent populations is not unexpected but when two distantly located populations are found to be homogenous, then historical ecology and demographic explanations may be important factors to consider when trying to understand the lack of genetic patterning. As discussed above, based on similar investigations of other freshwater species in this area several of the unexpected findings of this study may be attributed to ancient connectivity. The native range of C. striata in this study as revealed by haplotype-based (HST) and sequence-based (NST and KST*) statistics. The population pairwise FST values are also consistent with the low degree of gene flow estimates between populations (Nm = 0.25 and 0.20 respectively for haplotype-based and sequence-based). High genetic structuring particularly of non-migratory freshwater fishes have been well documented C. striata is well-known to be capable of short distance migration over land A high level of genetic structuring was apparent among the populations of Another interesting finding in this study was the private unique haplotype, Hap27, from the KP population, Sumatra. Despite geographically distant separation, KP was grouped with the SG population (east Peninsular) and LG and MS (southern Peninsular); see Anabas testudineus from Aceh (Sumatra) and Terengganu  In support of this account, another population from Sumatra, CS was also found to be clustered in the same group, further illustrating the close genetic relationship between populations from these two regions. Similar close genetic relationships were also observed between populations of the climbing perch, C. striata population leading to the pattern of genetic diversification which still persist until today. Though the populations from east Peninsular harboured higher genetic variation, the lower genetic variation detected in the KP population might be due to demographic bottleneck. Further investigation on samples from Sumatra should be undertaken to elucidate the phylogenetic relationships of this group.During unfavourable periods of marine invasion, local adaption or migration for survival even beyond atypical migratory range  was the only means of escape for this C. striata in the Sundaland was highly influenced by the interplay of several factors. The most prominent finding of this study was the segregation of this obligate freshwater species into three highly structured and significant phylogenetic groups, limited by effective geographical barriers and thus low gene flow between each population and phylogenetic region. Anthropogenic activity may also have played a major part via the probable translocation of this species from central west Peninsular Malaysia to as far away as Malaysian Borneo as detected here using a maternal lineage marker. Furthermore, ancient dispersal through the palaeo river system running across two presently isolated regions was also apparent indicating the typical Pleistocene glacial signature of the genetic structure of freshwater fishes in the Sundaland. This study has indirectly revealed the dispersal power of C. striata when dispersal limitation was broken down and its high mobility and rapid adaptability into a newly colonized area.Genetic patterning among populations of the striped snakehead, Table S1Haplotype frequency and nucleotide polymorphic sites encoded by 27 haplotypes. Non-synonymous amino acid substitution is indicated by *(DOCX)Click here for additional data file.Table S2Haplotype distribution across 24 populations. Abbreviation of population name is as listed in (DOCX)Click here for additional data file.


Saguinus geoffroyi, a small cooperatively breeding Neotropical primate. Mitochondrial sequences and microsatellite genotypes from three sampling localities revealed genetic structure across the Chagres River and the Panama Canal, suggesting that both waterways act as barriers to gene flow. F-statistics and exact tests of population differentiation suggest population structure on either side of both riverine barriers. Genetic differentiation across the Canal, however, was less than observed across the Chagres. Accordingly, Bayesian clustering algorithms detected between two and three populations, with localities across the older Chagres River always assigned as distinct populations. While conclusions represent a preliminary assessment of genetic structure of S. geoffroyi, this study adds to the evidence indicating that riverine barriers create genetic structure across a wide variety of taxa in the Panama Canal watershed and highlights the potential of this study area for discerning modern from historical influences on observed patterns of population genetic structure.The role of physical barriers in promoting population divergence and genetic structuring is well known. While it is well established that animals can show genetic structuring at small spatial scales, less well-resolved is how the timing of the appearance of barriers affects population structure. This study uses the Panama Canal watershed as a test of the effects of old and recent riverine barriers in creating population structure in  The distribution of genetic variability across geography is affected by multiple biotic and abiotic factors , includiZapus trinotatus) readily bound large topographic barriers, with gene flow explained more appropriately by habitat connectivity (Ovis canadensis nelsoni). Similarly, anthropogenic barriers that red grouse could theoretically cross in one flight acted as barriers to gene flow  in the Daraina region of Madagascar. Bodies of water may also be barriers for volant animals, as reported by (Scutiger spp.) in the Hengduan Mountains of China . Second, previous work has shown that riverine barriers are important for structuring primate populations in general  increased toward the narrow headwater streams of the river, as predicted by Alouatta palliata and capuchin monkeys: Cebus capuchinus), tamarins may be most likely to exhibit rapid population differentiation in response to landscape changes. Tamarins exhibit high intragroup relatedness  at mtDNA and microsatellite loci to investigate population genetic structure between sampling localities, and (3) use Bayesian clustering algorithms to determine the number of likely populations.To examine the role of rivers in creating population genetic structure at a small spatial scale, I sampled three populations distributed across two prominent riverine barriers—the Panama Canal and the Chagres River—to test whether the age of a physical barrier to gene flow has an effect on the level population genetic structure. I predict that there will be significant differentiation between populations separated by the Chagres, whereas differentiation across the Panama Canal will be more modest, owing to its novelty as a barrier: whereas the individuals included in this study represent approximately 20–40 generations since the construction of the Canal, the number of generations since the appearance of the Chagres is at least three orders magnitude higher. To test these predictions, I: (1) examine differences in genetic variability among sampling localities at mtDNA and microsatellite loci, (2) use S. geoffroyi  for sampling : (a) Theeoffroyi . I seleceoffroyi . The useI captured tamarins from Gamboa and Soberanía using hand-activated live traps baited with bananas as described by later and frozen at –20°C until extraction. Soberanía animals were represented by hair samples and Gamboa individuals were represented by hair and tissue samples. To verify the reliability of microsatellite genotypes from hair samples, I genotyped all Gamboa individuals using tissue samples to corroborate the results obtained from hair samples.Field collected samples included: (a) Hair samples plucked from the base of the tail and saved in coin envelopes and stored dry and (b) Ear tissue collected from the pinnae using surgical scissors and stored in RNA2 piece of tissue from the edge of the flat skin. I stored tissue samples in an empty microcentrifuge tube until extraction, which occurred within the week. A list of sampled individuals is included in Museum skins were preserved as dry flat skins. I used surgical scissors to extract ca. 1 mmI extracted Genomic DNA using Qiagen DNA Micro kits , according to manufacturer instructions for each sample type. I soaked museum tissue samples in 70% ethanol for 24 h prior to extraction. I quantified DNA yield using a NanoDrop Spectrophotometer . I extracted DNA from hair and museum skin samples in a “clean” room dedicated to low-copy sample extractions. Both sampling and extraction negative controls were used to monitor for possible contamination at every step of the genetic workflow.Saguinus  and geneious 4.8.5 (Biomatters). To ensure sequence accuracy, I obtained, aligned, and manually edited sequences from both strands (derived from each forward and reverse primer) belonging to the same individual. I used the consensus sequence of each individual for further analyses. I deposited consensus sequences in GenBank (JN849580-JN849633).Mitochondrial sequences are extensively used in population genetic studies  owing toSaguinus . To desiSaguinus —to desig. To ensure robustness of genetic data, I typed homozygote genotypes from the museum skin samples and field collected hair samples from at least two independent PCR reactions. I genotyped a subset of samples from the Gamboa population from both hair and tissue samples. Additionally, a subset of samples was genotyped de novo from independent extractions. I deposited microsatellite genotypes in the Dryad data repository (doi:10.5061/dryad.mg87590q).In order to maximize the chances of detecting population structure across the Panama Canal, I genotyped microsatellite loci. Due to their higher mutation rates , microsaicrochecker 2.2  diversity for mitochondrial sequence data. I calculated number of alleles, expected and observed heterozygosities for microsatellite loci using arlequin 3.1 and genalex 6.1  values  over 5 × 105 iterations  values . I conduneclass2  to deterneclass2 , based oerations , using terations . I generS. geoffroyi across the Panama Canal watershed. The number of individuals from which microsatellite  data were collected in each population were: Gamboa: 19 (17), Panama West: 22 (21), and Soberanía: 18 (16).Genetic samples were collected from a total of 59 Microsatellite loci showed no evidence of null alleles or genotyping errors. Loci did not show evidence of departure from Hardy–Weinberg equilibrium or linkage disequilibrium in any of the populations after correction for multiple tests at the 0.05 nominal level. Genotyping of hair samples and ear tissue samples from Gamboa yielded identical genotypes at all loci, suggesting that genotypes from hair samples were reliable.P = 0.8008, unbiased heterozygosity P = 0.5118, allelic richness P = 0.3991, rarefaction-calculated private allelic richness P = 0.3882). Gamboa appeared to have the lowest allelic diversity across measures, but these differences were not statistically significant. The lack of difference in microsatellite diversity included the Panama West locality, despite the differences in the nature of the sampling regime .Analysis of mitochondrial sequences revealed 12 variable sites and 13 haplotypes. As expected for a larger geographic sample, the Panama West population had the largest number of haplotypes, but it did not have the greatest nucleotide diversity, as did Gamboa . MeasureP < 0.0001; 30,000 Markov steps).The haplotype network  revealedfst values yielded significantly different (P < 0.001) values between all pairwise comparisons of sampling localities .AMOVA for mitochondrial data attributed 27.85% of variance to among-group  variation, compared to 11.11% for microsatellite data . The fixtructure detected k = 2 as the most likely number of populations, with the Gamboa locality distinct from the combined Panama West and Soberanía localities. The assignment plot is depicted in eneclass2 analysis correctly assigned 78% of individuals to their sampling localities when three populations were assumed. When two populations were assumed, assignment success increased to 88.1% of individuals as did the quality index (k = 3: 62.06%k = 2: 80.77%), which represents the mean value of individual assignment scores . All analyses except Structure and Geneclass2, indicate that there is detectable population differentiation among sampling localities across the Panama Canal. As expected, the level of differentiation was smaller across the Panama Canal than across the Chagres River.The results suggest that both the Chagres River and the Panama Canal have contributed to population genetic structure in S. geoffroyi.There were possible limitations imposed by the sampling regime for interpreting differentiation of sampling localities across the Panama Canal. In particular, the wider geographic sampling at the Panama West locality may cause additional allelic and haplotypic variation to be sampled. Analyses of microsatellite data suggest that this is not the case. Although the number of haplotypes is larger in the Panama West locality, it was Gamboa that had the greatest nucleotide diversity. However, because of these differences in sampling regime, the results of this study should be interpreted as preliminary evidence. More generally, the limited number of sampling localities also underscores the need for caution absent broader geographic sampling. Previous studies investigating the role of riverine barriers have found discrepant results when sampling at different localities along riverine barriers e.g., . Future The degree of differentiation among sampling localities inferred using mitochondrial data was larger that that calculated with microsatellite genotypes. This was true across both the Chagres River and the Panama Canal. The AMOVA conducted on both genetic datasets indicated that a greater proportion  of among-group variance was explained by mitochondrial sequence differences, as expected for a uniparentally inherited marker.tructure and Geneclass2 suggest two populations in the dataset, Bayesian algorithms have been reported to perform poorly at detecting populations with low differentiation  have shown that multiple bird species have become locally extinct, most likely as a cause of the limited dispersal across the Canal . There ivational  and censvational  suggestsThe lower differentiation across the Panama Canal suggested by this study points to only modest structure. However, in the absence of migrants these populations may diverge in the future, as has happened more clearly across the Chagres. In fact, divergence may be hastened across the Panama Canal, due to decreasing habitat availability west of the Panama Canal. In contrast, the Chagres headwaters are < 25 km from both Soberanía and Gamboa populations and well forested. This may ensure that over time gene flow will persist across, that is around, this barrier, which will likely not be the case for the Panama Canal. This study adds to the growing body of literature on the effects of recent anthropogenic barriers on population structure and genetic diversity . The res


Rumex acetosa  L. and Commelina communis  L. are two pseudo-metallophytes thriving in both normal and cupriferous soils along the middle and lower reaches of the Yangtze River in China. Their non-metallicolous and metallicolous populations are often sympatric thus providing an ideal opportunity for comparative estimation of genetic structures and divergence under the selective pressure derived from copper toxicity.Patterns of genetic divergence between populations of facultative metallophytes have been investigated extensively. However, most previous investigations have focused on a single plant species making it unclear if genetic divergence shows common patterns or, conversely, is species-specific. The herbs R. acetosa  and C. communis , including metal tolerance, genetic structure and genetic relationships between populations, were investigated and compared using hydroponic experiments, AFLP, ISSR and chloroplast genetic markers. Our results show a significant reduction in genetic diversity in metallicolous populations of C. communis  but not in R. acetosa . Moreover, genetic differentiation is less in R. acetosa  than in C. communis , the latter species also shows a clustering of its metallicolous populations.In the present study, patterns of genetic divergence of R. acetosa  and C. communis , and the contrasting responses of the two species to copper contamination, might be attributed to the differences in their intrinsic physiological and ecological properties. No simple and generalised conclusions on genetic divergence in pseudo-metallophytes can thus be drawn.We propose that the genetic divergences apparent in  RAPD, RFLP, ISSR, and AFLP) employed in different studies. Moreover, it should also be noted that until now, most previous studies have focused on a single plant species only, which inevitably influences the comparability of results from different studies. Therefore, choosing two or more sympatric metallophytes with similar geographic distributions and population history, and then comparing their characteristics of genetic divergence might shed light on the evolutionary processes of plants under heavy metal pressure.Understanding the genetic basis of how organisms adapt to extreme and novel environments remains as one of the major challenges facing evolutionary biologists. Intraspecific differentiation of metallophytes is one of the most striking adaptive changes that result from natural outcrops of heavy metal-rich rock or mining activities -3. Metalet al.  have sug. lyrata ,19-21. BRumex acetosa  L. (Polygonaceae) and Asiatic dayflower Commelina communis  L. (Commelinaceae ) have consistently been documented as dominant plants on most of these cupriferous habitats and are also present in surrounding non-metalliferous sites [R. acetosa  is well-known metallophyte thriving on metalliferous soils of NW & SW Europe. Many edaphic ecotypes of R. acetosa  from a wide range of soil types have been reported, some of which exhibit high potential for phytoremediation of contaminated soils [The copper (Cu) belts distributing along the middle and lower reaches of the Yangtze River are one of the largest areas of Cu mining in China. In this area, there are considerable spoil heaps and mineral deposits, some of which are currently in practice. A national ruin of an ancient Cu mine built over 1000 years ago (Da-Gong Mountain Mining Ruin) is located in this area, suggesting a long history of mining activities. Extensive Cu mining activities in this area have resulted in many waste heaps with elevated concentrations of Cu in soils, and the plant species growing in this area have experienced long-term selection under Cu toxicity derived from mining activities dating back to at least 1000 years. Although these waste heaps are often far away from each other, they harbor similar assemblages of metallophytes, which are distinctly different from the surrounding communities in non-metalliferous soils -24. Bothus sites ,22. Besied soils ,26.Rumex acetosa  is a perennial herb with an erect stem, about 40 to 120 cm high. Its flowering season ranges from March to May and the fruiting season ranges from April to June. Unisexual and dioecious flowers are arranged in acrogenous panicle inflorescences. Plants are capable of producing large numbers of small achenes encircled with an aliform membrane and therefore can be easily dispersed by wind. The species is commonly found along roadsides, in forests, and in habitats along rice fields in most regions of China [C . communis  is an annual multi-branched herb with erect stems in the upper part and creeping stems in the lower part. It is able to grow clonally by elongation of the creeping stem, which bears a number of erect ascending shoots. It flowers from May to September and fructifies from June to November. Bisexual flowers are arranged in a cyme. Commelina communis  often occupies habitats similar to those of R. acetosa  and is mainly distributed in southeastern China [of China . In contrn China .R. acetosa  and C. communis  have different life histories  and reproductive systems , which result in them being in different functional groups. Therefore, their metallicolous and non-metallicolous populations provide an ideal opportunity to study genetic divergence under selective pressure derived from Cu toxicity between taxa of two distinct functional groups.Despite their similarities, the two pseudo-metallophtes R. acetosa  and C. communis  would differ greatly in population genetic divergence. To test our hypothesis, 12 R. acetosa  populations and 13 C. communis  populations collected from different sites along the Yangtze River of Eastern China were used to investigate comparative genetic differentiation patterns. AFLP (Amplified Fragment Length Polymorphism) and ISSR (Inter-Simple Sequence Repeats) were employed to assess genetic diversity and population structures. Six R. acetosa  and 10 C. communis  populations were selected to determine Cu tolerance of the metallicolous and non-metallicolous populations. In addition, two cpDNA regions (psbJ-petA  and 3′rps16-5′trnK ) were sequenced and analyzed to infer uniparental patterns of colonization.Previous studies have shown that many factors such as life history and reproductive system may significantly influence population genetic divergence through their effects on genetic drift and gene flow ,15-17. WR. acetosa  and 13 C. communis  populations were investigated at areas along the Yangtze River  and non-metallicolous (NM) populations; tolerance indices of M populations were significantly higher than those of NM populations for both species.  Interestingly, tolerance indices of NM populations of both species were also similar. Commelina communis  M populations exhibited higher tolerances than those of R. acetosa  in the 160 μM and 320 μM Cu treatments. Shoot elongation was, with few exceptions, considerably depressed in all NM populations investigated when seedlings were treated with 40 μM Cu or above. Moreover, the treatment with 320 μM Cu which killed all NM plants revealed a higher tolerance in C. communis  M populations than R. acetosa  M populations . In contrast, for C. communis  significant (P  < 0.001) differences in genetic diversity were detected between the M and NM populations, the latter having higher values than the former. Furthermore, significantly negative correlations between genetic diversity and DTPA-extractable Cu concentrations were detected .For R. acetosa  and C. communis  populations was investigated by an Analysis of Molecular Variance (AMOVA). Relatively high genetic differentiation among populations of both C. communis ,  and R. acetosa   was detected from AFLP data. Conversely, for R. acetosa  ISSRs were not able to detect significant differentiation among populations , whilst a low differentiation was detected for C. communis  . Mantel’s tests carried out to investigate the existence of any correlations between geographical and genetic distances .The genetic structure of F ST   were clustered in one main branch in both AFLP and ISSR analyses . Edaphic differentiation was strongly supported for C. communis  , whereas lower levels were found for R. acetosa  (3.68 and 4.04% for ISSR and AFLP data). However, for C. communis,  a geographical structuring of populations was also strongly supported by the ISSR data .Hierarchical partition was then computed at three levels . Groupings were related to both edaphic and geographic differences  of each population investigated were sequenced  were included in one single group, suggesting a common chloroplastic  origin.Two cpDNA non-coding regions . Interestingly, there was no significant difference in Cu tolerance of non-metallicolous populations between the two species, suggesting a possible similar level of selective pressure on the ancestral non-tolerant colonizers of metalliferous sites. Although the concentrations of Cu in the contaminated sites we sampled were extremely high when compared with those of the non-metalliferous sites, distinct differences in Cu concentration were also observed among contaminated sites. Therefore, heterogeneity in the metalliferous sites could partly explain the difference in Cu tolerance among metallicolous populations. However, considering that the Cu concentrations in contaminated sites were generally 100 times greater than those in uncontaminated sites, the significant differences in Cu tolerance should be mostly attributed to the intense and long-term selection due to heavy metal stresses and to the differences in biological traits of the two metallophytes. Because they were sympatric in most sampling sites, and so subjected to the similar edaphic conditions. On the other hand, it is possible that the heterogeneous nature of the metalliferous sites could also partly explain the difference of Cu tolerance between the two species. Because there were some significant differences in Cu concentration among the metalliferous sites . In contrast, the similar levels of genetic diversity in metallicolous and non-metallicolous populations of R. acetosa  might be due to substantial gene flow between populations, as indicated by the low values of pairwise F ST .  High levels of gene flow between metallicolous and non-metallicolous populations of metallophytes have previously been reported in some other comparable studies [R. acetosa  showed higher levels of background tolerance to Cu  compared to those of C. communis.  Considering that metallicolous populations are presumed to evolve from non-metallicolous populations, this evidence may imply that selection for Cu tolerance in C. communis  has been greater than in R. acetosa , although present-day levels of Cu tolerance in metallicolous populations are lower in R. acetosa .In order to determine more thoroughly whether the different phenotypes observed are linked to population histories and are reflected in the genetic diversity of populations, three different molecular techniques  were employed to analyze the genetic structures and genetic relationships among different populations of the two metallophytes. Firstly, we found that levels of genetic diversity in the two co-occurring species were distinctly different. For i et al.  reported halleri , N. caerulescens  and Onoschioides  maintainocations . In the  studies ,17,31. IR. acetosa , metallicolous populations branched in separate positions in the NJ dendrograms obtained by AFLP and ISSR data, supporting the hypothesis of independent origins from nearby normal populations. Furthermore, we also found that there was a direct correlation between genetic differentiation and geographical distances between pairs of R. acetosa  populations  of genetic differentiation.Concerning that the habitats of metallicolous populations of metallophytes are often fragmented and disjunct, it is unlikely that dispersal from a single tolerant ancestral population could have produced the wide ranges of geographic distribution today. Hence, the hypothesis of polyphyletic origin is proposed and has been subsequently corroborated by considerable data ,17-19,31C. communis  and R. acetosa  was also confirmed by the analysis of chloroplast markers. Though this analysis was performed on only one individual per population, results for C. communis  were similar with those obtained with AFLP, suggesting the presence of one main cluster of metallicolous populations. On this basis, it could be reasonable to hypothesize that metallicolous populations of C. communis  may derive from seeds of one single ancestral population. Interestingly, we observed a very low variability of chloroplast sequences of R. acetosa , which seemed to be consistent with the idea of a recent spread and low differentiation between metallicolous and non-metallicolous populations.The contrasting pattern of genetic differentiation between C. communis  were more remarkably influenced by heavy metal stress than those of R. acetosa , although the genetic structures of the two species were influenced by geographic isolation and heavy metal contamination.The contrasting results of the two metallophytes also implied that populations of Anthoxanthum odoratum [Sedum alfredii  than geographic distance [R. acetosa . In contrast, Cu contamination seemed to play a more important role in determining the population structure of C. communis  than geographic distance.Although ecogeographic isolation has long been viewed as the most important reproductive barrier in plants , barrierodoratum . Additiodistance . Based oR. acetosa  and C. communis  populations in the present study almost share the same edaphic conditions (mainly Cu toxicity) and geographic distribution pattern, the different characteristics of genetic divergence might result largely from their different life history and reproductive system. On the one hand, as a perennial, unisexual, and dioecious species, R. acetosa  may have more opportunities to experience gene exchange among individuals and gene flow between populations than the bisexual annual plant C. communis . In addition, the winged seeds of R. acetosa  are presumed to have higher dispersal capacity than those of C. communis , which might enhance the success of colonization by R. acetosa [In previous studies, it has been shown that the genetic divergence and evolutionary processes of plants could be affected by various factors, such as edaphic conditions, stress intensity and duration, geographic isolation, bottleneck and founder effects, life history, reproductive system, and so on ,29. Cons acetosa ,28.e.g.  seed dispersal, background tolerance) and geographical patterns of colonization. Yet, no simplistic conclusions (e.g.  the presence of founder effects etc. ) can be drawn concerning the microevolutionary dynamics of all metallophytes.Contrasting patterns of genetic divergence in two pseudo-metallophytes that experience similar selective pressures provides insight into evolutionary processes under heavy metal stress. Our findings indicate that genetic divergence of metallophytes is the result of the interactions between biological properties of a species  [From 3 to 10 soil samples were collected at each sampling site (0–20 cm depth). Soil samples were air-dried and passed through a 2-mm sieve. For analysis of total Cu, soil samples were digested with 65% HNOO4 (5:1) ; for anaO4 (5:1) . Cu concR. acetosa  populations and 10 C. communis  populations were selected for hydoponic experiments to investigate Cu tolerance in each population . The composition of the nutrient solution was as follows: Ca(NO3)2·4H2O 2.00 mmol L−1, KH2PO4 0.10 mmol L−1,MgSO4·7H2O 0.50 mmol L−1,KCl 0.10 mmol L−1,K2SO4 0.70 mmol L−1,H3BO3 0.01 mmol L−1,MnSO4·H2O 0.50 × 10−3 mmol L−1,ZnSO4·7H2O 0.50 × 10−3 mmol L−1,CuSO4·5H2O 0.20 × 10−3 mmol L−1,(NH4)6Mo7O2·4H2O 0.01 × 10−3 mmol L−1,Fe-EDTA 0.10 mmol L−1. Hydroponic culture vessels were positioned randomly in the growth chamber and rearranged once a week. The nutrient solutions were renewed every 5 days. Before Cu treatment, roots of all seedlings were blackened with activated charcoal and rinsed in deionized water to remove the excess powder. Seedlings were then treated with a range of Cu concentrations , supplied as copper sulphate (CuSO4·5H2O). Here, 0.25 μM CuSO4 was the control. Because Cu is an essential plant micronutrient and our preliminary tests did suggest that 0.25 μM CuSO4 ensured good growth in all populations. Each treatment was repeated 6 times. After 25 days of Cu treatment, 6 seedlings were harvested from each treatment for Cu tolerance measurement. The presence of new roots (uncoated) visible beyond the charcoal-coated roots were recorded. The roots are in direct contact with the nutrient solution  and are therefore the first target for any toxic effect. Hence, the response of root growth is generally considered to be a very appropriate measure of the metal tolerance of plants. The determination of tolerance indices followed the methods described by Baker & Walker [Six n Figure . A bulk & Walker , and werR. acetosa  and C. communis  were selected, respectively  protocol of Lodhi et al.  with minet al. [Eco RI/Mse I and synthetic adaptors were ligated to the restriction fragments using T4 DNA ligase. A pre-amplification PCR was performed using AFLP primers having a single selective nucleotide (EcoRI  + A/MseI  + C). PCR products of the pre-amplification reaction were diluted 30-fold in sterile water then used as template for selective amplification using a combination of AFLP primers. In total, six selective primer pair combinations were used to generate fingerprints for the two species  in duplicate PCR reactions. Totally, 8 and 9 primers which produced highly readable, reproducible and polymorphic band patterns were selected for R. acetosa  and C. communis , respectively  from each species were selected to screen the ISSR polymorphisms by using 93 primers . Primers and PCR protocols are as described in Shaw et al. [psbJ-petA  and 3′rps16-5′trnK  cpDNA regions were sequenced with a 100% overlap.One individual from each sampled population was randomly selected for sequencing of two cpDNA regions  and non-parametric tests were used to assess the statistical significance of data.t -test as available in the SPSS statistical package was used to detect the significant differences in genetic diversity values between populations from cupriferous sites and those from uncontaminated sites. The same software was used to assess the correlation between the values of genetic diversity of each population and the associated Cu concentrations in soils. The partitioning of the genetic variance within and among populations and groups of populations was obtained with an analysis of molecular variance . Geneti  conduct . HaplotMY designed experiments, carried out laboratory and statistical analysis, and drafted the manuscript. BL designed experiments and carried out the molecular marker studies. JTL, MH, WCL performed the analyses, AM constructed sequence alignment and helped to draft the manuscript. WSS designed and coordinated the project and drafted the manuscript. All authors discussed data and drafted the manuscript. All authors read and approved the final manuscript.Rumex acetosa  populations and 10 Commelina communis  populations in the four CuSO4 treatments.This file provides the tolerance indices for the 6 Click here for fileThis file provides some supporting information for the article, including the result of the Mantel’s test, the results of Slatkin’s linearized pairwise FST, the results of AMOVA, GenBank accession numbers of two cpDNA non-coding regions, the lists of primers for AFLP and ISSR analysis.Click here for file