Plasmodium falciparum populations can be used to assess parasite transmission, which is information vital to evaluating malaria elimination efforts. Here we characterize the genetic diversity of P. falciparum samples collected from patients at seven sites in Haiti using 12 microsatellite markers previously employed in population genetic analyses of global P. falciparum populations. We measured multiplicity of infections, level of genetic diversity, degree of population geographic substructure, and linkage disequilibrium . For low transmission populations like Haiti, we expect to see few multiple infections, low levels of genetic diversity, high degree of population structure, and high linkage disequilibrium. In Haiti, we found low levels of multiple infections (12.9%), moderate to high levels of genetic diversity , low levels of population structure , and moderate linkage disequilibrium . In addition, population bottleneck analysis revealed no evidence for a reduction in the P. falciparum population size in Haiti. We conclude that the high level of genetic diversity and lack of evidence for a population bottleneck may suggest that Haiti’s P. falciparum population has been stable and discuss the implications of our results for understanding the impact of malaria control interventions. We also discuss the relevance of parasite population history and other host and vector factors when assessing transmission intensity from genetic diversity data.Hispaniola, comprising Haiti and the Dominican Republic, has been identified as a candidate for malaria elimination. However, incomplete surveillance data in Haiti hamper efforts to assess the impact of ongoing malaria control interventions. Characteristics of the genetic diversity of  Plasmodium falciparum) [Malaria remains a leading global health threat, accounting for 627,000 deaths in 2012. Reduced morbidity and mortality in the last decade as a result of increased financial support have renewed interest in pursuing global malaria eradication –5 becausciparum) , 3) littciparum) –18 despiP. falciparum-carrying mosquitos. More cases of malaria infection were reported after the 2010 earthquake than before the earthquake, though it is unclear whether this increase was the result of increased exposure to P. falciparum or the result of increased surveillance by visiting aid groups in Haiti [Given that the majority of malaria cases are reported in Haiti, elimination planning in Hispaniola focuses interventions in Haiti. Although Haiti is a strong candidate for malaria elimination, the 2014 World Malaria Report lists Haiti’s malaria program phase as “control” with the note “insufficiently consistent data to assess trends” [in Haiti .P. falciparum population can be a useful tool to characterize parasite populations. The level of genetic diversity and the distribution of that diversity, or population structure, provide insight into trends in parasite transmission and parasite population history, information that is vital to malaria elimination programs seeking to assess the impact of elimination interventions. While advances in sequencing technology allow for whole genome-wide estimates of genetic diversity in P. falciparum populations [P. falciparum populations for epidemiological purposes. Microsatellites markers have been employed extensively to study P. falciparum population structure within countries and across continents [P. falciparum parasite which increases the frequency of recombination and subsequently results in a highly diverse population with low linkage disequilibrium [P. falciparum genetic diversity and population structure.The genetic diversity of the ulations , the morntinents . In genentinents ) have lentinents –23. The ilibrium , 20. In ilibrium –26. Therilibrium . More stP. falciparum microsatellite studies have focused on large geographic scales and it is unclear if the reported trends in population structure and genetic diversity are applicable on a local scale or in small, geographically isolated regions like Haiti. Our study examined microsatellite loci in the P. falciparum population in Haiti with the goal of understating the characteristics of genetic diversity of the malaria parasite population in Haiti. We discuss the results in light of what has been observed in other countries of either high or low transmission. Eighty-five dried blood spot samples collected between 2010 and 2013 from Terre Noire, Leogane, Jacmel, Chabin, Nippes, North Cap Haitian, and Hinche were assayed for 12 neutral microsatellite loci previously used to investigate P. falciparum genetic diversity [Most Dried blood spot samples from patients suspected or confirmed to have malaria infections (by microscopy or rapid diagnostic test) were collected at health centers in seven locations throughout Haiti between May 2010 and March 2013 . The samVery little information is known about differences in transmission among the regions included in this study. While malaria transmission in Haiti is low overall, there have been reports of transmission hotspots in the Southeast Department (includes Jacmel and Chabin), particularly near coastal areas . In thisdhfr, dhps, pfmdr, or pfcrt genes indicating the presence of P. falciparum.Sample collection between 2010 and 2012 has been previously described . Recent All malaria-infected patients were treated with chloroquine or another antimalarial drug as was the standard policy at the clinics. The study was approved by the Haiti Ethical Review Board, UF-IRB, and the Office of Research Protections, US Army Medical Research and Materiel Command (USAMRMC). Written consent was obtained by all participants as approved by the ethics committees listed above.P. falciparum populations worldwide: chromosome 4: Polya; chromosome 5: TA81 and TA42; chromosome 6: TA1, TA87, and TA109; chromosome 10: TA40 and 2490; chromosome 11: ARA2; chromosome 12: Pfg377 and PFPK2; and chromosome 13: TA60 [2, 0.2 mM each nucleotide, 0.25 μM for each primer in the primary PCR (0.4 μM in the semi-nested PCR), and 0.625 U of Go Taq Hot Start Polymerase . The same primers were used as detailed in Anderson et al.[GAAATTGGCACCACCACA, TA40 For AAGGGATTGCTGCAAGGT, TA40 Rev-2 CATCAATAAAATCACTACTA). Fragment analysis of PCR products were conducted at the University of Florida’s Interdisciplinary Center for Biotechnology Research DNA Genotyping Core Laboratory, using BigDyeTM chemistry on an Applied Biosystems 3730 Genetic Analyzer. Raw electropherograms were analysed using GeneMarker . All peaks greater than 1,000 units were called.DNA was extracted from dried blood spots using a Qiagen QIAmp Investigator Kit . Between six to eight punches were taken for each blood spot and final DNA samples were eluted in 60μL elution buffer. Samples were assayed for 12 putatively neutral microsatellites loci previously used to characterize the genetic diversity 13: TA60 , 29, 30.P. falciparum genome is haploid while in human hosts) Minor peaks were not counted if they were less than 1/3 the height of the major peak. We determined the number of multiple infections in the overall sample set, by study sites, and by collection year. For genetic diversity analyses, only the major allele at each locus in multiple infected samples was included.Multiplicity of infection was defined as having more than one peak at one or more microsatellite loci (e). We determined He and number of alleles for each locus and the mean values by study site  and by year of collection (2011 and 2012 only). Other study sites and collections years were not included due to small sample sizes. We also determined the number of haplotypes, defined as having 12 identical microsatellite alleles. To do this, we only included samples with no more than two alleles per locus and only the major alleles/tallest peaks were used for haplotype determination.Genetic diversity was evaluated by the number of alleles at each locus and heterozygosity  and a stepwise mutation model (SMM), since both patterns of variation have been reported for our microsatellite assay , 31. EstSA), was calculated and can be used for across-study comparisons. For our study, we completed 10,000 resamplings.To assess whether alleles from different loci were associated with each other, we tested for linkage disequilibrium (LD) using LIAN . A measust between Terre Noire, Leogane, and Jacmel. Pairwise Fst and accompanying p-values were calculated in Arlequin v3.5x [® 9.2 software .To investigate population substructure, we determined the number of private alleles, i.e. alleles found in only one study site or collection year, and then examined geographic substructure by calculating pairwise Fin v3.5x . We alsoP. falciparum dhfr, dhps, pfmdr, or pfcrt genes. In total, 85 samples were successfully genotyped for least 10 of the 12 microsatellite loci and were included in the final sample for analysis , with a range of 0.28 (TA42) to 0.73 (Polyα and PFPK2). The mean heterozygosity (standard deviation in parenthesis) within study sites was 0.57 (0.17) in Terre Noire, 0.59 (0.14), in Leogane, and 0.57 (0.19) in Jacmel. e) for Haiti overall and for high and low transmission countries previously investigated [P. falciparum population to populations from low and high transmission countries reported in Anderson et al.[The mean number of alleles observed was 4.92 (± 1.68), with a range of 2 (2490) to 7 (TA81) alleles ..e was 0.We determined the number of private alleles . Four study sites carried private alleles at five loci; of those five loci, four contained only a single unique allele (data not shown). TA60 had two private alleles only observed in Terre Noire. Most private alleles were detected only in a single individual with the exception of the TA40 199 allele, which was observed in four individuals from Jacmel.We examined the number of microsatellite haplotypes in our study, excluding samples with any missing genotypes or greater than two alleles at any locus. Of 76 eligible samples, 62 haplotypes were observed (data not shown). Seven haplotypes were observed in more than one sample, while the remaining 55 haplotypes were only observed once. One haplotype was detected in six samples across two study sites  and across three collections years . The other multiple sample haplotypes were found in 2–3 samples each, which limited the number of comparisons that could be done across study sites or collection year.e reported above. Although both He and number of alleles decline after a population bottleneck, He generally declines at a slower rate so excess He is suggestive of a recent population bottleneck. In our study, we found no significant deviation of He based on the observed number of alleles under the stepwise mutation model , suggesting no recent history of population bottlenecks.Overall, we calculated an effective population size of 2084 to 3891 individuals across all samples, based on the infinite alleles (IAM) and single stepwise mutation (SMM) models, respectively. Effective population size by study site and year are reported in P. falciparum samples from Haiti using a Monte Carlo simulation method to calculate the standard index of association. We found significant levels of LD in the total data set . LD was also detected within the three largest study sites: ISA = 0.086 for Terre Noire (p <0.0001), ISA = 0.095 for Leogane (p <0.0001), and ISA = 0.041 for Jacmel (p <0.001).We examined evidence of linkage disequilibrium (LD) between microsatellite markers for the st across the three largest study sites , combined with high levels of linkage disequilibrium and population structure (estimated with Fst and PCA), are evidence of low transmission populations, while the reverse is observed in high transmission populations. We found that the frequency of multiple infections and presence of linkage disequilibrium (LD) in our study resembled countries with low malaria transmission, but other diversity-related estimates, such as heterozygosity and lack of population structure, resembled higher transmission countries. Specifically, the small proportion of multiple infections in our sample (12%) is similar to the prevalence of multiple infections in other low transmission countries , falling at the lower end of the range observed in other low transmission populations . It is worth noting that pairwise Fst values can vary within a region of low transmission, as seen in Brazil, with pairwise Fst values ranging from 0.04 to 0.30. Given that the greatest physical distance between sites was 100km, with our three largest sites being within 40km of each other, high levels of substructure would not be expected [P. falciparum genetic diversity characteristics do not fall cleanly into what would be expected for a high or low transmission population.Our study employed the use of a commonly used multi-locus microsatellite assay to characterize d Brazil ) and cond Brazil . The mead Brazil , 23, 25)d Brazil , 37). Weondorous , 23). Thexpected . FinallyP. falciparum population than expected for a low transmission country. The level of He across loci (0.61) is suggestive of high malaria transmission countries, as evidenced by previous studies in high transmission populations . Severaimbabwe, , 26, 39,ventions  which maP. falciparum population has not been impacted by interventions such as mosquito bed nets, insecticides, and antimalarial drug use. As mentioned above, widespread antimalarial resistance is not reported in Haiti, further supporting the idea that interventions like antimalarial drugs have not been used to the level that would have a significant impact on the overall P. falciparum population size in Haiti. These results highlight the importance of considering the extent to which long-term regional malaria control interventions have, or have not, impacted parasite evolution and population size when making inferences about transmission intensity from P. falciparum population genetic diversity data.Given the lack of consistent resources for controlling malaria in Haiti, our results may support the notion that the P. falciparum diversity. If multiple vector species exist, multiple parasite strains could evolve to adapt to each vector species. Only one malaria vector species has been confirmed in Haiti, but it possible that more parasite vector species exist. Future studies should determine if additional vector species exist in Haiti and determine what impact, if any, this has on the diversity of the parasite populations in Haiti.In addition, we must consider the role that host genetic diversity has on the parasite population, specifically the role that inherited protective red blood cell disorders, such as sickle cell hemoglobin and glucose-6-phosphate dehydrogenase deficiency, may play in shaping each parasite population. It is worth noting that the initial correlation between genetic diversity measures and transmission intensity across continents was also a comparison across host populations with different genetic backgrounds . In HaitP. falciparum gene flow may be relatively unimpeded. Further studies with larger samples collected across multiple sites in Haiti are needed to confirm our observations. Our study in Haiti highlights some of the challenges that population genetic and epidemiological studies face when working with small endemic populations, particularly those with low malaria transmission and very few number of cases detected and reported.Sample size was a limitation of our study. Most of our sample was collected from the southern portion of Haiti, which may limit our ability to extrapolate our results to the rest of the country. Our across-site comparisons with principal component analysis, however, showed that the smaller sample sets from Hinche, Cap Haitien, Nippes, and Chabin clustered with the larger sample sets, suggesting that geographic population substructure in Haiti may be minimal and P. falciparum population do not fall neatly within expectations for either a low or high transmission population. While the proportion of multiple of infections is suggestive of a low transmission population, we see high allelic diversity similar to that seen in high transmission regions. Our investigation into population bottlenecks in Haiti showed no decline in effective population size, suggestive that the parasite population may be relatively stable. It is possible that other potential for factors other than transmission intensity may shape the diversity of the parasite population, such as the history and extent of malaria control interventions and the high frequency of protective red blood cell disorders in the host populations. The results of this study injunction with continued surveillance of the genetic diversity of P. falciparum population in Haiti may provide further insight as to whether recently adopted malaria control policies are successfully reducing parasite population in Haiti.In conclusion, we found that the characteristics of Haiti’s S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Aedes aegypti is the primary vector of the four serotypes of dengue virus (DENV1-4), Chikungunya and yellow fever virus to humans. Previous population genetic studies have revealed a particular genetic structure among the vector populations in the Americas that suggests differences in the ability to transmit DENV. In Colombia, despite its high epidemiologic importance, the genetic population structure and the phylogeographic depiction of Ae. aegypti, as well as its relationship with the epidemiologic landscapes in cities with heterogeneous incidence levels, remains unknown. We conducted a spatiotemporal analysis with the aim of determining the genetic structure and phylogeography of Colombian populations of Ae. aegypti among cities with different eco-epidemiologic characteristics with regard to DENV.Ae. aegypti across time and space in these cities.Mitochondrial cytochrome oxidase C subunit 1 (COI) - NADH dehydrogenase subunit 4 (ND4) genes were sequenced and analyzed from 341 adult mosquitoes collected during 2012 and 2013 in the Colombian cities of Bello, Riohacha and Villavicencio, which exhibit low, medium and high levels of incidence of DENV, respectively. The results demonstrated a low genetic differentiation over time and a high genetic structure between the cities due to changes in the frequency of two highly supported genetic groups. The phylogeographic analyses indicated that one group (associated with West African populations) was found in all the cities throughout the sampling while the second group (associated with East African populations) was found in all the samples from Bello and in only one sampling from Riohacha. Environmental factors such as the use of chemical insecticides showed a significant correlation with decreasing genetic diversity, indicating that environmental factors affect the population structure of Ae. aegypti lineages are present in Colombia; one that is widespread and related to a West African conspecific, and a second that may have been recently introduced and is related to an East African conspecific. The first lineage can be found in cities showing a high incidence of dengue fever and the use of chemical insecticides, whereas the second is present in cities showing a low incidence of dengue fever where the use of chemical insecticides is not constant. This study helps to improve our knowledge of the population structure of Ae. aegypti involved in the diversity of dengue fever epidemiology in Colombia.Our results suggest that two  Aedes aegypti, the main vector of the dengue virus (DENV), is essential to improving dengue fever prevention strategies in endemic countries. In Colombia, despite the epidemiological relevance of dengue fever, the genetic population structure and phylogeography of the vector Ae. aegypti is little known. In this study, we evaluated the spatio-temporal structure and phylogeography of Colombian Ae. aegypti populations from cities showing different eco-epidemiologic attributes related to dengue fever. Our results indicated that Colombian Ae. aegypti populations harbor two mitochondrial lineages related to West and East African ancestors. The lineage related to West African populations is the most frequent and widely distributed in Colombia, and it was found in cities with a high incidence of the dengue fever. A second lineage related to East African populations, which may have been recently introduced in some regions, was found in cities showing a low incidence of dengue. These findings suggest complex population dynamic is involved in dengue fever epidemiology in Colombia, and indicate further studies about biological attributes of the Ae. aegypti lineages should be performed.Knowledge on the population structure of  Aedes aegypti is the main vector of the four serotypes of dengue flaviviruses (DENV1-4) and yellow fever virus (YFV), and is a known vector of Chikungunya virus [Dengue is one of the major public health problems in the tropics and is the second-most deadly vector-borne disease in the world after malaria . The mosya virus . Around ya virus . Thus faAe. aegypti sensu lato (s.l) have been described according to several molecular and ecological studies [Ae. aegypti formosus (Aaf), a sylvan mosquito restricted to the sub-Saharan region of Africa and the Ae. aegypti aegypti (Aaa) form referred to as Ae. aegypti sensu stricto (s.s), which is widespread across most of the tropical and subtropical regions of the world in association with humans and is considered the main epidemiologically relevant subspecies [Aaa outside of Africa consist of mosquitoes derived from one of two ancestral clades. One clade is basal and is primarily associated with the mosquito population from Western Africa while the second arises from the first and contains primarily mosquitoes from Eastern Africa [Two recognized subspecies of  studies ,5. The pbspecies ,7. Recenn Africa . This din Africa –11.Aaa clades has been extensively reported in several countries of Central and South America, including Mexico [Aaa may also explain this occurrence, thereby generating misconceptions about the true absence of a lineage in a particular location; however, clear evidence for this has not emerged thus far. Therefore, the genetic characterization of natural populations over time may help to further elucidate the behavior of these two lineages over time and may explain any possible effects on the epidemiology of dengue.The sympatric distribution of both g Mexico , Brazil g Mexico ,14, Perug Mexico , Venezueg Mexico  and Bolig Mexico  where thg Mexico . In thisAaa in several cities of Colombia, only two studies were conducted locally in populations in the north and the south of the country, suggesting that the Colombian populations are genetically diverse and are affected by the continued use of chemical insecticides [Aaa individuals from three Colombian cities showing distinct dengue incidence rates that were collected during three samplings during 2012 and 2013. The results indicate two maternal lineages of Aaa in Colombia that are differentially distributed across time and space in cities with different eco-epidemiologic characteristics, suggesting distinct colonization events and microclimate variables that modulate the frequency and distribution of each lineage.Colombia is a hyperendemic country for DENV, and the number of cases of DHF and deaths due to dengue have increased dramatically during the last few years, mainly in the Central and Eastern regions ,19. For cticides ,21. HoweA total of 343 adult mosquitoes were collected in three cities in Colombia between August 2012 and September 2013 with the assistance of the staff of the Vector-Borne Diseases program of the Instituto Nacional de Salud (INS de Colombia). Three samplings were performed in each city, including at least one during the rainy and dry seasons . To miniAe. aegypti, the city manifests low incidence rates of between 25.9 to 52.3 cases  and a reduced use of chemical insecticides [The cities sampled are localized in three eco-geographic regions with distinct climates and dengue incidence . Bello  with 50 μl reaction mixes/total volume containing 4 μl of 1/16 template DNA dilution, 5 μl of 10X reaction buffer (Fermentas), 0.2 mM dNTP, 20 pmol of each primer and 1 UI of Taq polymerase (Fermentas). For the ND4 gene, reaction conditions were conducted at 94°C for 5 min, 35 cycles at 94°C for 30 s, 50°C for 30 s and 72°C for 30 s with a final extension at 72°C for 5 min. For the COI gene, 40 cycles were conducted at 94°C for 30 s, 48°C for 30 s and 72°C for 1 min followed by 10 min at 72°C. PCR products were detected by agarose gel electrophoresis in Tris-borate-EDTA buffer (TBE), stained with GelRed 10,000X diluted 1:10,000 in agarose gel, and visualized under UV light. All positive PCR products were purified and sequenced using the Sanger methodology at Macrogen sequencing service, Seoul, South Korea.D [Sequences were aligned using CLUSTAL W  as impleD  tests waD  using DnD .Mismatch distribution analysis was performed to identify the pairwise differences spectrum between haplotypes in the different isolates of time and space using DnaSP v.5.0 . A MediaAe. aegypti was assessed by ΦST pairwise comparison, and the genetic structure of isolates in time and space was also assessed using a hierarchical analysis of molecular variance (AMOVA) in GenAlEx v.6.4 [Genetic differentiation in Colombian Ex v.6.4 . The stain situ temperature and number of chemicals interventions with adulticides and larvicides performed in each city during the study using non-parametric Spearman`s correlation coefficients (ρ) with post-hoc Holm’s method for multiple comparisons in Hmisc and Rcmdr packages in R software.Nucleotide (π) and haplotype diversities (Hd) values were compared with the quartiles of relative humidity (HR-Q), Ae. aegypti groups, an analysis of Inverse Distance Weighting (IDW) interpolation between the number of positive and negative houses, and the geographic area of each neighborhood studied was performed. The search classes in the IDW interpolation analysis ranked between 0 (absence) to 1 (presence) individuals for each group, with a maximum search area of 2.5° without anisotropy  using ArcGis software  [The spatiotemporal distribution of genetic groups was first evaluated through the frequency of individuals in the positive houses. Also, due to any particular spatial arrangement was observed in geographical distribution of nds, CA) . This annds, CA) .Phylogeographic analysis was performed on the combined COI-ND4 dataset because congruent polymorphism and diversity proportions were observed for separated COI and ND4 genes . The phyAll nucleotide sequences are available with GenBank accession codes for COI: KM203140—KM203248 and ND4: KM203249—KM203336.For the combined COI-ND4 dataset (1178 bp), we identified 160 haplotypes harboring 147 variable sites (12.47%) of which 79 (53.74%) were parsimony informative and 68 (46.25%) were singleton sites . The oveThe first two coordinates of the PCoA, harboring 87.2% of the genetic variability in the dataset, roughly indicated that the two groups of haplotypes are inferred , as suppThe haplotype network inferred for combined CO1-ND4  in all samplings from VI, and in the second sampling in RI, indicating that an excess of rare haplotypes  is remained across the studied time in VI and eventually in RI . Moreover, similar significant values of genetic differentiation was observed among cities and among samplings , but higher variance component was attributed to among cities (29%) (ST = 0.79), and between groups within cities (FST = 0.81)  . Because = 0.81) . These rpost-hoc Holm`s test, significant (P < 0.05) negative Spearman correlation coefficients (ρ) was observed only for nucleotide diversity (π) and number of chemical interventions with adulticides (ρ = -0.73), indicating possible selective pressure is drown in the studied sample  showed two groups of haplotypes that were associated with the haplotypes of America, Asia and Africa belonging to previously suggested mitochondrial lineages I and II –17,44 S. Further Aegypti . In the  Aegypti .Ae. aegypti supported by a Bootstrap of 100% , showed two main monophyletic clades of  of 100% . One cla of 100% . The oveAe. aegypti populations of Colombia. Our results reveal a significant level of genetic variation between two previously reported mitochondrial lineages circulating in distinct frequencies across cities with different eco-epidemiologic characteristics during 2012 and 2013. These frequencies, which have fluctuated over the last few years in cities showing distinct dengue incidence rates, indicate that a complex vector-population composition might potentially drive dengue epidemiology in Colombia. However, this specific issue is outside the scope of this work; this hypothesis must be further explored in studies of the association between endogenous factors such as vector competence and insecticide resistance with the distribution of genetic groups in Colombia and dengue fever incidence.This is the first study of the genetic variation and the molecular phylogeography of In this study we observed that values of nucleotide diversity (π) in Colombia are similar to those detected across several Brazilian localities ,14, VeneAe. aegypti in the same locality may significantly increase the genetic variability of the vector populations [Ae. aegypti are the main control measures against dengue [Since the introduction of different genetic groups of ulations . We suggt dengue , whereast dengue . These fAe. aegypti studied. This attribute, as well as the star-shape of the haplotype network exhibited by group 1 suggests that this group is most likely more ancient in Colombia because it presents as central  and presents the most widespread haplotypes in the country. Additionally, for this group negative and significant values of neutrality testing were observed in the three cities, indicating that it has been subjected to selection pressures across the country. Thus, for example in VI (where only group 1 was found), recent studies demonstrated that mosquito populations have high levels of physiologic resistance to DDT, lambda cyhalothrin and deltamethrin [Similar to observations in Brazilian populations , higher amethrin , as wellamethrin ,49. Thesamethrin . FurtherAe. aegypti groups, in BE the spatial analyses indicate that both groups are widespread across complete areas in the neighborhoods studied, but group 2 might have higher geographic dispersion  compared with group 1 in this city, where the use of chemical insecticides was not reported for sampling periods. These results support the conclusions of some studies, which have shown that mosquito populations with lower levels of resistance to chemical insecticides  have high fitness compared to those with a higher degree of resistance  without this selection pressure [As relates to the local distribution of pressure . HoweverAedes aegypti is a poikilothermic species; therefore, changes in response to environmental factors such as humidity, temperature and rainfall can affect the genetic structure of its populations [Ae. aegypti populations [ulations ,53. In tulations ,54,55. WAe. aegypti was considered eradicated between 1952 and 1960 as a result of the eradication program initiated in the 1940s by the Pan American Health Organization (PAHO) [In Colombia, n (PAHO) . Unfortun (PAHO) .Ae. aegypti, which is in accordance with mitochondrial lineages previously suggested in the Americas [Based on the phylogeographic analysis, our data indicate the presence of two genetic groups of Americas –17,44. TAe. aegypti s.l. worldwide, it has been suggested that the populations that invaded North America belong to the ancestral clade associated with West African populations, whereas those that invaded South America and the Caribbean region derive from a clade belonging to East African populations [Since the work conducted by Tabachnick and Powell  in analyulations . Accordiulations . A diffeulations ,46. ThesAe. aegypti in the northern regions South America, we suggest that group 1 might have been introduced into Colombia through the land border with Venezuela or by the increase in imports from the USA [According to the colonization routes of  the USA , since H the USA . However the USA . These hAe aegypti populations is thought influencing their ability to transmit arbovirus as YFV, Chikungunya and dengue in endemic areas [Ae. aegypti from West Africa historically are involved in the transmission of YFV, while East Africa population has not been implicated in outbreaks [Flaviviride), DENV vectorial capacity of the American mitochondrial genetic groups should be still addressed.Genetic structure of ic areas . Accordiic areas , the vecic areas . Althougutbreaks . Based oAe. aegypti is due to a genetically linked faulty production during Numts amplification [Finally, whereas some works suggest that the presence of two mitochondrial lineages of fication ,25, our fication . Second,fication . FinallyAe. aegypti in Colombia, inferring that a distinct ancient origin is involved. Cluster analysis clearly showed two genetic groups, each of which share haplotypes with populations of West Africa and East Africa, respectively. Our results suggest that the group 1 specimens related to West Africa might be present in cities where the use of insecticides is constant, whereas the group 2 specimens related to East Africa are associated with cities exhibiting lower incidence rates, without any selection pressure due to insecticides. The presence of genetically distinct groups in Colombia might imply differences in vector competence for transmitting dengue and urban yellow fever viruses, as well as a differential response to vector control strategies [The results demonstrated in this study indicate multiple introductions of rategies ,11,60, aS1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S1 FigThe size of the nodes corresponds to the frequency of the haplotypes. The black bar represents the number of mutational steps between the nodes of the suggested groups (see text). Color indicates the collection origin: green BE, blue RI and red VI.(TIF)Click here for additional data file.S2 FigAe. aegypti (gray solid) were included in the haplotype network. The haplotypes with (*) are subsets of the haplotypes represented in the Vectors of each Colombian city studied here are represented as fill (BE) green; (RI) blue and (VI) red. South America haplotypes of Bolivia (fill yellow); Brazil ; Venezuela ; North American haplotypes ; Africa ; Asia ; Caribbean region (Martinique = green phosphorescent solid) and the Liverpool strain of (TIF)Click here for additional data file.


Populus × jrtyschensis, an assumed natural hybrid between two distantly related species. This hybrid poplar occurs mainly on the floodplains along the river valleys between the overlapping distributions of the two putative parents.The factors that contribute to and maintain hybrid zones between distinct species are highly variable, depending on hybrid origins, frequencies and fitness. In this study, we aimed to examine genetic origins, compositions and possible maintenance of P. × jrtyschensis, P. nigra and P. laurifolia. We genotyped them based on the sequence variations of one maternally inherited chloroplast DNA (cpDNA) fragment and genetic polymorphisms at 20 SSR loci. We further sequenced eight nuclear genes for 168 individuals from 31 populations. Two groups of cpDNA haplotypes characteristic of P. nigra and P. laurifolia respectively were both recovered for P. × jrtyschensis. Genetic structures and coalescent tests of two sets of nuclear population genetic data suggested that P. × jrtyschensis originated from hybridizations between the two assumed parental species. All examined populations of P. × jrtyschensis comprise mainly F1 hybrids from interspecific hybridizations between P. nigra and P. laurifolia. In the habitats of P. × jrtyschensis, there are lower concentrations of soil nitrogen than in the habitats occupied by the other two species.We collected 566 individuals from 45 typical populations of P. × jrtyschensis suggested that it is typical of F1-dominated hybrid zones. This finding plus the low concentration of soil nitrogen in the floodplain soils support the F1-dominated bounded hybrid superiority hypothesis of hybrid zone maintenance for this particular hybrid poplar.Our extensive examination of the genetic composition of The online version of this article (doi:10.1186/s12870-016-0776-6) contains supplementary material, which is available to authorized users. The nuclear genes sequences datasets are available in the Genebank repository Accessions no: KT626975-KT629820 [see Additional file


Distylium lepidotum is a small tree endemic to the Ogasawara Islands located in the northwestern Pacific Ocean. This species is a sole food for an endemic locust, Boninoxya anijimensis. Here, we developed microsatellite markers to investigate genetic diversity and genetic structure and to avoid a genetic disturbance after transplantation to restore the Ogasawara Islands ecosystem.D. lepidotum populations on Chichijima and Hahajima Islands of the Ogasawara Islands. The number of alleles for the markers ranged from three to 23 per locus in the two populations. Expected heterozygosity per locus in each population ranged from 0.156 to 0.940 and 0.368 to 0.845, respectively.Microsatellite markers with perfect dinucleotide repeats were developed using the next-generation sequencing Illumina MiSeq Desktop Sequencer. Thirty-two primer pairs were characterized in two D. lepidotum and provide a basis for conservation management of the Ogasawara Islands.These microsatellite markers will be useful for future population genetics studies of  Microsatellite markers, or simple sequence repeats, are widely applicable as DNA-based markers for population genetics studies. Moreover, their cost-effective development has been increasingly facilitated by applying next-generation sequencing (NGS) technologies .Distylium lepidotum Nakai (Hamamelidaceae) is a small tree endemic to the oceanic Ogasawara Islands in the northwestern Pacific Ocean. The species is the dominant tree in the Distylium–Pouteria dry scrub [Boninoxya anijimensis Ishikawa, a locust recorded as a new genus and species [D. lepidotum as the sole food, i.e., it is monophagous [Anolis carolinensis. Conservation/benign introduction measures of B. anijimensis are needed on the Ogasawara Islands, except Anijima Island, to protect the B. anijimensis populations. As D. lepidotum is an essential food source, it may be possible to transplant the species. Therefore, it is important to reveal the genetic structure of the species to minimize any genetic disturbance due to the transplant. Here, we developed microsatellite markers to investigate the genetic diversity and structure in D. lepidotum.ry scrub , which i species . The locophagous , 9. AlthD. lepidotum using an Illumina MiSeq Desktop Sequencer . Total genomic DNA was extracted from one silica-gel dried D. lepidotum leaf sample collected from Chibusayama (26°39′17.4″N 142°10′03.6″E) on Hahajima Island of the Ogasawara Islands using a DNeasy Plant Mini Kit . A shotgun library was prepared using the Nextera DNA Sample Preparation Kit v2 (Illumina), and the raw de novo sequencing data were obtained using the MiSeq Reagent Kit v2 (500 cycles) (Illumina). The raw reads were divided into each index, extra sequences (adapters and indices) were trimmed, and FASTAQ files were generated using the MiSeq Reporter v.2.5.1 (Illumina). The paired-end reads were merged using PEAR 0.9.6 [Microsatellite markers were developed for AR 0.9.6  with defAR 0.9.6 . A totalThe microsatellites were identified and the primer pairs were designed with QDD2.1 . A totalAmplification and polymorphism were confirmed in 48 selected primer pairs after considering the microsatellites (one single dinucleotide motif with more than ten repetitions), design type (“A” or “B” in QDD2.1), and PCR product size to apply multiplex amplification  with the GeneScan 600 LIZ Size Standard (Life Technologies). The fragments were sized using GeneMapper 4.0 (Life Technologies).NA), observed heterozygosity (HO), expected heterozygosity (HE), and fixation index (FIS) were calculated to characterize each locus using GenAlEx 6.501 [FNull) were estimated with CERVUS 3.07 [FST was calculated using FSTAT 2.9.3.2 [FST from zero was tested based on 1000 randomizations. Genetic structure was also evaluated by a Bayesian clustering method implemented in STRUCTURE 2.3.4 [K value from one to five under an admixture model with correlated allele frequencies. The log-likelihood probability at each run and the rate of change in the log-likelihoods between adjacent K values, ΔK [K values to determine the best fit for the data.We finally tested two populations from Chichijima and Hahajima Islands in the central part of the Ogasawara Islands to evaluate the allelic polymorphisms: 24 individuals from Asahiyama (27°05′40.7″N 142°12′35.6″E) on Chichijima Island and 20 individuals from Omotohama (26°37′28.9″N 142°10′41.7″E) on Hahajima Island. Voucher specimens of the representative individuals were deposited in the Makino Herbarium (MAK) of the Tokyo Metropolitan University, Japan . The number of alleles per locus . The Bayesian clustering analysis represented the highest ΔK value at K = 2 (ΔK = 121.4; Of all the 397 alleles that were detected, the 193 alleles which were detected in the Chichijima population were not found in the Hahajima population. On the other hand, the 53 alleles which were detected in the Hahajima population were not found in the Chichijima population. In addition, the two populations were significantly differentiated (D. lepidotum, since they have enough polymorphisms and they can clearly distinguish the two populations. The genetic data would be useful to investigate the genetic diversity and structure of D. lepidotum which is necessary for a food source of the endangered locust species on the Ogasawara Islands.These 32 novel microsatellite markers will be valuable for elucidating the genetic diversity and structure of


Transitional phase) compared to the population from the initial phase (Tlamimilolpa) as well as the population from the final phase  of the history of Teopancazco. Genetic studies showed no differences in genetic diversity between males and females in the adult population of Teopancazco, this data along with ample archaeological evidence, suggest a neolocal post-marital pattern of residence in Teopancazco. Nevertheless, genetic analyses on the infant population showed that the males are significantly more heterogeneous than the females suggesting a possible differential role in cultural practices by sex in the infant sector. Regarding interpopulation analysis, we found similar indices of genetic diversity between Teopancazco and heterogeneous native groups, which support the multiethnic character of Teopancazco. Finally, our data showed a close genetic relationship between Teopancazco and populations from the “Teotihuacan corridor” and from Oaxaca and the Maya region, in agreement with previous archaeological evidence.Multiethnicity in Teopancazco, Teotihuacan, is supported by foreign individuals found in the neighborhood center as well as by the diversity observed in funerary rituals at the site. Studies of both stable and strontium isotopes as well as paleodietary analysis, suggest that the population of Teopancazco was composed by three population groups: people from Teotihuacan, people from nearby sites , and people from afar, including the coastal plains. In an attempt to understand the genetic dynamics in Teopancazco we conducted an ancient DNA (aDNA) analysis based on mtDNA. Our results show that the level of genetic diversity is consistent with the multiethnicity phenomenon at the neighborhood center. Levels of genetic diversity at different time periods of Teopancazco’s history show that multiethnicity was evident since the beginning and lasted until the collapse of the neighborhood center. However, a PCA and a Neighbor-Joining tree suggested the presence of a genetically differentiated group (buried at the  The ancient city of Teotihuacan, located in the Central Mexican highlands , can be Ciudadela [Tlamimilolpa (AD 200–300) was the initial phase of the Teopancazco’s history; previous stable and strontium 87/86 isotopes studies, and a paleodietary analysis of burials from this phase [Tlamimilolpa (AD 300–350), differential rituals were found which suggested the end of a constructive phase and/or drastic social changes originated by population rearrangements  [Xolalpan , and has been also characterized by previous isotopic studies [The population of Teotihuacan was organized in different social and spatial units; one of them was the multiethnic neighborhood center headed by intermediate elites . Teopanciudadela . Differeis phase –8 have ris phase , having l phase) , 10–12. l phase) –9. The l studies –9 by theWide archaeological evidence supporting the multiethnicity of Teopancazco has been reported: the different burial practices found in this site, the large number of faunal elements associated with the coast, foreign pottery from the Gulf Coast, cotton cloth, and foreign materials processed in Teopancazco , 13. ThiRegarding the population of Teopancazco characterized by sex, previous osteological analyses showed an unusual higher proportion of adult males (48.38%) in relation to adult females (ca. 15%) , 20. SevOur genetic study from Teopancazco is the first attempt to genetically characterize the ancient population of Teopancazco assessing changes in genetic diversity through its history, from the initial period to the final construction phase of the neighborhood center, as well as to assess and compare genetic diversity by sex and by age at death of the Teopancazco individuals. Likewise, our data allowed us to assess the genetic relationships between Teopancazco and other Mesoamerican populations.th field season: C.A. 401-36/0787 July 11, 2003. 12th field season: C.A. 401-36/0824 July 9, 2004. The sampling process was carried out under strict criteria in order to avoid exogenous contamination. The human burial remains from Teopancazco are kept in the Instituto de Investigaciones Antropológicas (UNAM) under the custody of Dr. Linda R. Manzanilla. The samples are part of the Mexican National Heritage.The population under study came from Teopancazco, Teotihuacan, Central Mexico: Latitude North: 19.67464167, Longitude West: 98.84532778. UTM: 2, 14, Q, 516213, 2175485, “TEOPANCAZCO”. Bone and tooth samples were collected in several extensive excavation field seasons (1997–2005) and provided by Dr. Linda R. Manzanilla, head of the project “Teotihuacan. Elite and rulership. Excavations in Xalla and Teopancazco” 19.2013/36/0579 of the National Institute of Anthropology and History). Archaeological permits for Dr. Linda R. Manzanilla, director of the project “Teotihuacan. Elite and rulerhip”, by the Consejo de Arqueología  of the Instituto Nacional de Antropología e Historia  are as follows: First field season: C.A. 401-36/1077 October 16, 1997. 11A subset of 50 samples from 46 different individuals buried at Teopancazco was sent by Dr. Linda R. Manzanilla to the aDNA laboratory in LANGEBIO, CINVESTAV, Mexico, in order to be analyzed . Additionally, a subset of eleven samples  was sent to the Universitat Autònoma de Barcelona (UAB), Spain, in order to replicate results.DNA extraction was carried out under strict conditions for the analysis of ancient DNA, in a dedicated clean room, positively pressurized with ultra filtered and UV-irradiated air, with separation of pre- and post-PCR areas. Laboratory equipment was treated with 30% bleach for DNA contamination removal. The use of disposable filter-plugged pipette tips, tubes, protective cloths, hair covers, laboratory gloves, surgical masks, glasses, and shoe protectors to prevent contaminations was mandatory. Solutions and buffers were irradiated with ultraviolet light. Negative extraction controls and negative PCR controls were always employed.Buccal swab samples from the personnel related to samples handling were analyzed in order to discard the possibility of contamination during excavations or laboratory procedures.Bioanalyzer 2100 (Agilent) to quantify and assess the quality of extracted DNA. The result of this assay shows the distribution of DNA fragment sizes in the extract, and helps to determine the DNA fragmentation pattern of each sample. Highly degraded samples show a distribution with a peak of less than 100 bp  [For each individual analyzed, a fragment of bone or a complete teeth, in a good state of preservation (without fissures), was used. Samples were processed in a type II B2 biological security hood located inside the clean room. The outer surface of the bone or teeth was UV-irradiated for 15 min and a dental drill was used to remove 0.1 g of powdered material. Powdered samples were added to 5 ml of extraction buffer  and 50μl of Proteinase K solution (10 mg/ml). Samples were incubated at 37°C for 24 hrs, followed by a phenol/chloroform DNA extraction . Aliquot 100 bp) .Ancient DNA extraction and mtDNA amplification and sequencing in the UAB were performed as previously described .et al. [2, 200 μM each dNTPs, 0.4 μM each primer, and 0.12 U of Bioline TaqDNAPolymerase (Biotaq). Thermal cycling was carried out under the following conditions: 5 minutes at 94°C; followed by 40 cycles of 0.5 minutes at 94°C, 0.5 minutes at 52°C, 0.5 minutes at 72°C; and a final step of 7 minutes at 72°C. The PCR product was then cloned using a TOPO TA Cloning kit and TOP10 competent cells (Invitrogen) following manufacturer’s instructions. A minimum of 50 colonies were selected from each cloned sample for colony PCR using the M13 primers. We then sequenced several clones and obtained between 3 and 11 clones per sample that contained the transformed vector with the fragment of interest. DNA sequencing was performed in both directions at the Genomic Services Unit in LANGEBIO, CINVESTAV. Sequences obtained were aligned to the mtDNA reference sequence (rCRS) [A 149 base pair (bp) portion located between positions 16190 and 16339  . The hape (rCRS) , MitoMape (rCRS)  and Phyle (rCRS) .A D-loop fragment (700 bp) was amplified and sequenced in order to discard possible contamination from personnel related to samples handling.2 1X LightCycler Master-Mix (with dUTP instead of dTTP) and 0.2 μM of each primer. Thermal cycling was carried out under the following conditions: 10 minutes at 95°C; followed by 80 cycles of 30 seconds at 95°C, 30 seconds at 60°C; and a final step of 45 seconds at 72°C. After PCR the HRM analysis was performed as described [We developed an HRM method to determine the mitochondrial haplogroup of the ancient samples. This analysis is based on the melting temperature (Tm) difference of the amplified DNA fragments carrying diagnostic mutations located in the mitochondrial coding region belonging to Native American Haplogroups A, C, D and X. Haplogroup B was identified by the presence of a 9-pb deletion. Primers were designed to amplify short fragments (59–63 bp) and therefore the analysis was efficient even for samples for which amplification of the D-Loop fragment (149bp) was unsuccessful. Primers and conditions used are shown in escribed . The curTlamimilolpa , Transitional , and Xolalpan  [Intrapopulation statistical analyses were carried out on the haplogroup and the haplotype (sequence) level to assess if there were changes in genetic diversity in a temporal scale through the history of the neighborhood center. Teopancazco was analyzed as a whole, and with the samples divided into three different historical phases: 350–550) , 12, 38.Ĥa) and haplotype (Ĥb) data. A one-way ANOVA was performed in order to compare Nei’s genetic diversity indices at the haplogroup level among the three chronological groups analyzed. A two-tailed t-test was performed in order to compare Nei’s gene diversities indices at the haplotype level among the temporal groups analyzed. These analyses were carried out using GraphPad Prism software v6.00 . An homogeneity test of haplogroup frequency distributions was carried out between the analyzed populations using an exact test of population differentiation [Nei’s genetic diversity indices  were calntiation , with 20ntiation .Ĥa) data for both groups. Gene diversities were compared by a two-tailed t-test using GraphPad Prism software v6.00 . A homogeneity test of haplogroup frequency distributions was carried out by an exact test of population differentiation [The sex of the infants was previously determined by real-time PCR amplification of small fragments of the amelogenin gene, followed by High Resolution Melting analysis (HRM) . Nei’s gntiation  as descrĤa) [www.graphpad.com). A homogeneity test of haplogroup frequency distributions was carried out by an exact test of population differentiation [The sex of the adult individuals from Teopancazco was previously determined by osteological methods , 29. NeiĤa)  was calcntiation  as descrĤa) between Teopancazco and, Maya, Otomi, Nahua populations [Interpopulation statistical analyses were carried out on the haplogroup and the haplotype (sequence) level. Mitochondrial haplogroup frequencies from all reference populations are reported in ulations , 49–53 aulations  were carwww.xlstat.com). A Pima population was used as external group (Aridoamerican population) due to its relatively higher genetic distance to Mesoamerican populations [A Principal Component Analysis (PCA) based on frequencies of mitochondrial haplogroups was done with XLSTAT software v2014.1.03 . All these 29 samples amplified for short fragments (59-63pb) of the coding region, however, only 16 of them  also amplified for the 149bp fragment of the HVR-I. These 16 sequences have been deposited in GenBank (Accession numbers KR813318-KR813333). No matches were detected between sequences recovered from skeletal remains and from personnel related to samples handling .We sent eleven samples (bone and teeth) to the Universitat Autònoma de Barcelona (UAB), Spain, in order to replicate results. For two of the samples  no amplification was obtained. Nine samples were amplified; however, the sequences of five of them  showed an unusual number of substitutions constituting haplotypes without phylogenetic sense and therefore were considered artifacts. Reliable sequences were obtained for only four of the eleven samples (36.36%). These sequences showed SNPs in agreement with the SNPs previously determined in the LANGEBIO laboratory for those samples . The percentage of efficiency (sequences) obtained in the UAB was similar to the efficiency obtained in LANGEBIO (34.78%). This result confirms the poor DNA preservation at the site.Tlamimilolpa (N = 10), the Tlamimilolpa-Xolalpan Transition (N = 11), and Xolalpan (N = 8). The Tlamimilolpa and Xolalpan periods showed a similar frequency of haplogroup A (~ 60%) and B (~10%) while the frequencies in Transitional period were 50% and 30% respectively. Regarding haplogroup C, a twofold increase in frequency was observed in the Tlamimilolpa and Transitional periods (20%), in comparison to the Xolalpan phase, and, notably, the presence of haplogroup D was observed only in the Tlamimilolpa and Xolalpan phases   is reported in  period) .p> 0.05). However, the absence of haplogroup D in the Transitional period might be significant in relation to the genetic structure of this population even if not statistically significant, as discussed below. Haplogroup frequencies were used to estimate Nei’s genetic diversity for the three periods of the Teopancazco´s history (Ĥa) are similar in the three periods analyzed: Tlamimilolpa 0.6444 ± 0.1518, Transitional 0.6909 ± 0.0861, and Xolalpan 0.6429 ± 0.1841, and the differences were not significant  = 0.381, p> 0.05). However, the PCA . Haplotype diversity in Xolalpan was not detected.D-loop sequences from 16 individuals buried at Teopancazco  were used to assess haplotype diversity  in the tĤa) than female infants , and in this case the difference between them was significant . However, no difference in haplogroup frequencies distributions was detected between both groups .As we were able to determine sex in the infant population from Teopancazco, we analyzed genetic diversity by sex in the infants . As in tĤa) in males  than in females  nevertheless, no significant difference in genetic diversity was found .We analyzed genetic diversity by sex in a total of 12 adult individuals . An exacF  = 385.6, p< 0.05). Pairwise comparisons indicated that Teopancazco shows no significant differences to one of the Maya population from Yucatán (MAY II) , two Otomi populations  and three Nahua populations . Nevertheless, pairwise comparisons indicated that Teopancazco’s diversity is significantly different (p< 0.00036) from the diversity found in a Lacandon population, which is one of the most isolated Mexican Native group and shows a lower diversity index (0.0426 ± 0.0403).Differences in Nei’s genetic diversity among Teopancazco, Nahua, Otomi, and Maya populations were statistically significant . This result is in agreement with the PCA , Oaxaca and Maya region. No significant differences among populations were found  was composed mainly by local people and by foreigners from sites belonging to the “Teotihuacan corridor to the Gulf Coast” [Xolalpan phase), characterized by the possible expansion of exchange routes between Teotihuacan and Mesoamerica. However, Nei’s genetic diversity at haplogroup (Ĥa) and haplotype level (Ĥb) in this phase , was similar to the Xolalpan phase, suggesting that the population from Teopancazco was heterogeneous since the beginning. At the end of the Tlamimilolpa phase (AD 300–350), differential burial rituals were found, in which some intentionally decapitated individuals, mainly males, were considered foreigners coming from low altitudes and some others coming from the “Teotihuacan corridor to the Gulf Coast” according with previous isotopic analyses [Transitional period showed a different pattern in comparison with the data observed in Tlamimilolpa and Xolalpan times. In the Transitional phase, the diversity at haplogroup (Ĥa) and haplotype (Ĥb) levels  was higher than those in the initial phase in the history of Teopancazco (Tlamimilolpa), although the differences between both periods were not significant (p> 0.05). However, the absence of haplogroup D in the Transitional period might be indicative of a differential genetic structure of this sample, in agreement with the differential burial ritual applied to these individuals. Further evidence for the genetic differentiation of the Transitional group can also be observed in both the PCA plot and the Neighbor-Joining tree  was characterized, according with isotopic studies, by the presence of people from several origin sites, suggesting demographic changes caused by during this phase in Teopancazco [p> 0.05) in genetic diversity (Ĥa) or haplogroup frequencies between this period and the Tlamimilolpa or Transitional periods. These data suggests that the genetic diversity levels were constant from the initial phases of Teopancazco until the last part of the neighborhood history . These data also support the multiethnic character of Teopancazco. Genetic diversity at haplotype level in Xolalpan was not determined because the three haplotypes obtained were identical.Genetic analyses of the Teopancazco population from the initial to the later phases of its history allowed us to assess if there were changes in genetic diversity and composition in accordance to previous isotopic and paleodietary studies as well as archaeological evidence –9. Theseanalyses –9. The iing tree . Future  history , and thaĤa) between infant males and females in Teopancazco. Our results indicated that there is a significantly higher genetic diversity in male infants compared to female infants . This means the female group was more homogeneous than the male group, as observed for the adult population (described above). In order to find out if the differences in genetic diversity between sexes can be explained by cultural practices, such as funerary rituals, we then analyzed when and where the infants were buried. In the Transitional period, six perinatal individuals were placed in a flexed position on top of a large pit  [Previous studies had reported that approximately 29% of the formal burials found in Teopancazco were infants [ll pits)  with aduĤa) was higher in males than in females ; however, this difference between them was not significant (p> 0.05). Likewise, the two groups were not different in haplogroup frequencies (p> 0.05). These data suggest that females were as heterogeneous as males, which is a characteristic of a neolocal pattern of post-marital residence, the establishment of a new couple in an independent place of residence away from the relatives of either spouse [Xolalpan phase. In addition, it has been proposed that when an elite controls resources and property, the remaining population follows a pattern of neolocal residence [Teopancazco was a neighborhood center characterized by an unusual higher proportion of adult males (48.38%) in relation to adult females 10–15%) , 20. Befr spouse . Sample r spouse –68, a chesidence , 70, and–15% , 20esidence , 18. Furesidence .Levels of mitochondrial genetic diversity found in Teopancazco, at haplogroup (0.6404 ± 0.0738) and haplotype level (0.9167 ± 0.0643), supported the heterogeneous character of the site, previously proposed based on evidence indicating trade and migration with different areas and populations. Our genetic data shows genetic diversity levels similar to populations that can be considered heterogeneous due to their high genetic fluxes with Mesoamerican populations –52, 71.p> 0.05), represented also in a PCA plot  originally suggested by García-Cook . These rPCA plot  which shPCA plot  and in aPCA plot , which sOur results show that the genetic relationships between Teopancazco and populations from the corridor are closer than the relationships with Maya and Oaxaca regions, in agreement with the idea that most of the emigrants from Teotihuacan did not travel very far . Our resTransitional phase between Tlamimilolpa and Xolalpan has been detected, in agreement with the peculiar way in which they were ritualized. Regarding the post-marital residence pattern in Teopancazco, our genetic data, compared with archaeological evidence, suggest the presence of a neolocal pattern. Genetic analysis of the infant population from Teopancazco allowed us to put forward hypotheses about the termination ritual found in the Transitional period. We found similar indices of genetic diversity between Teopancazco and other heterogeneous civilizations such as Maya, Otomi and Nahua, which support the multiethnic character of Teopancazco. Our data also suggest a close genetic relationship between Teopancazco and populations from the “Teotihuacan corridor to the Gulf Coast” and from Oaxaca, in agreement with previous archaeological evidence.In summary, our results imply that the population of Teopancazco was heterogeneous at mtDNA level since the initial phase of the history of Teopancazco until its abandonment, although the presence of a possible genetically differentiated group, buried during the S1 FigTEO (Teopancazco), TEO-TLAM (Tlamimilolpa period), TEO-XOL , TEO-TRAN , PIMA , ZAP , OTO , OTO I , OTO II , NAH , NAH I , NAH II , NAH III , MAY , MAY II , MAY III , MAY IV , TEPE , HUA  MIXT , MIXT II .(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table* Ancient DNA Studies.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Gene bank managers collect ex situ accessions that represent a comprehensive snap shot of the genetic diversity of in situ populations at a given time and place. Although simple in theory, achieving complementary in situ and ex situ holdings is challenging. Using Trifolium thompsonii as a model insect-pollinated herbaceous perennial species, we used AFLP markers to compare genetic diversity and structure of ex situ accessions collected at two time periods  from four locations, with their corresponding in situ populations sampled in 2009. Our goal was to assess the complementarity of the two approaches. We examined how gene flow, selection and genetic drift contributed to population change. Across locations, we found no difference in diversity between ex situ and in situ samples. One population showed a decline in genetic diversity over the 15 years studied. Population genetic differentiation among the four locations was significant, but weak. Association tests suggested infrequent, long distance gene flow. Selection and drift occurred, but differences due to spatial effects were three times as strong as differences attributed to temporal effects, and suggested recollection efforts could occur at intervals greater than fifteen years. An effective collecting strategy for insect pollinated herbaceous perennial species was to sample >150 plants, equalize maternal contribution, and sample along random transects with sufficient space between plants to minimize intrafamilial sampling. Quantifying genetic change between ex situ and in situ accessions allows genetic resource managers to validate ex situ collecting and maintenance protocols, develop appropriate recollection intervals, and provide an early detection mechanism for identifying problematic conditions that can be addressed to prevent further decline in vulnerable in situ populations.A standard conservation strategy for plant genetic resources integrates  In situ conservation allows the natural trajectory of evolution to continue to mold genetic variation, and ensures sustained access to genetically adapted populations. An important strength of ex situ conservation is that it readily provides the diverse germplasm needed by plant breeders, natural research managers and basic and applied researchers. In contrast to in situ populations, ex situ accessions provide a genetic snapshot, reflecting a wild (or on-farm) population’s adaptation to the biotic and abiotic conditions it was collected in. The static nature of ex situ conservation is frequently cast as a disadvantage; however, there is evidence that these snapshots conserve alleles that can be lost from in situ populations. For example, in a comparison of barley (Hordeum vulgare L.) landraces conserved in situ (on farm) in Morocco, and ex situ (since 1985), for resistance to current strains of powdery mildew (Blumeria graminis f.sp. hordei), ex situ accessions had more qualitative resistance than in situ accessions, for some pathotypes of powdery mildew, due to rare resistance genes being preserved ex situ, but lost in situex situ samples that have disappeared from current in situ populations in situ and ex situ approach Since utilization of plant genetic resource (PGR) conservation is an important aim, it’s long been recognized that effective strategies need to integrate ex situ samples that represent the inherent diversity of in situ populations at the time they are sampled. The literature suggests this might not be a straight forward proposition. In situ and ex situ accessions were compared in maize (Zea mays L.) landraces using morphological Ex situ accessions of American Indian Hopi landraces differed significantly from their in situ counterparts that had been maintained on farm in Arizona and the differences were attributed to original collecting bottlenecks and seed multiplication in a different environment (Iowa) in situ and ex situ samples of Jala, a Mexican maize race, were attributed to good initial sampling and effective regeneration practices Phaseolus vulgare L.) landraces conserved ex situ and on farm were compared using SSR markers ex situ subpopulations. SSR data indicated significant genetic differentiation in ex situ subpopulations as well as loss of alleles, gain of new alleles, and reduction of rare alleles and increase of common alleles ex situ subpopulations could be attributed to regeneration practices. Using ISSR markers similar results were found in Oca , a tuber crop species grown in the Andes Gene bank curators and managers generally aim to have ex situ and in situ methods are also evident. RAPDs were used to examine three remaining natural populations and one ex situ  subpopulation of Vatica guangxiensis (X. L. Mo), a rare endemic of southwestern China. The ex situ subpopulation contained 88.31% of the total genetic variation and was thought to adequately represent extant natural variation Parashorea chinensis H. Wang, a rare endemic timber tree of southwest China and adjacent areas of Laos and Vietnam. The ex situ subpopulation (conserved in a field bank) was found to contain only 77.1% of the total variation found in samples taken from seven in situ sites. Additional sampling was recommended ex situ samples  of Berberidopsis corallina Hook. f., a threatened vine endemic to southern Chile, and found to only represent the northern part of the natural range of the species ex situ accessions represent the genetic diversity of wild species. In contrast, in situ populations of Agropyron cristatum (L.) Gaertn., a widely distributed wind pollinated grass species, were compared with gene bank counterparts collected 28–30 years previously, using SSRs. Although there were individual loci differences for number of alleles and genetic diversity, when averaged across all loci there was no significant difference among recent recollections and ex situ stored accessions Solanum jamesii Torrey (diploid outcrosser) and Solanum fendleri A. Gray (tetraploid inbreeder), crop wild relative (CWR) species of potato (Solanum tuberosum L.). Subpopulations were originally sampled in 1958, and recollected in 1978 and 1992. Significant genetic differences were found between all ex situ and re-collected in situ subpopulations of S. jamesii, and 12 of 16 comparisons of S. fendleri. Relative differences were attributed to mating system, vulnerability of small populations (<100 plants) to genetic change, and difference in original sampling procedures The challenges of maintaining complementary samples of wild species using ex situ samples that reflect an accurate snapshot of in situ populations. One, comprehensive initial sampling is imperative at both population and taxon level; and two, changing selection regimes–from either the ex situ regeneration/field site, or in situ site itself, can contribute to the divergence of in situ and ex situ accessions. Although a large body of literature outlines collecting parameters to ensure adequate sampling of PGR ex situ samples that represent in situ populations. Recollection, rather than regeneration, may be a useful approach. This has been proposed for wild species that are difficult to regenerate ex situ, because natural conditions are too costly to replicate, or the material itself is difficult to propagate ex situ samples. Should ex situ managers also consider how quickly the genetic structure of in situ PGR might be changing? Seven farmer varieties of rice, conserved ex situ and in situ (on farm) over a period of time when production practices changed, were compared using allozymes, agronomic, stress resistance and morphological traits. Although yield and genetic diversity remained the same, traits associated with adaptation to new production practices significantly changed between ex situ and on farm subpopulations in a period of seventeen years in situ populations change over time, and how this may impact ex situ resampling.These studies emphasize two factors that are important to obtain and maintain Trifolium thompsonii Morton (2n = 2x = 16) is a restricted endemic herbaceous species that grows on the east slopes of the Cascade Mountains, in central Washington State, USA. The species is ranked as globally imperiled T. thomsonii, effectively returning the area consumed to an early seral community. We used this species as an insect-pollinated, strongly out-crossing model to study population genetic change, since it is diploid, occurs in diverse environments that are undergoing successional change, occurs in populations greater than 10,000 plants, and is readily accessed and easily sampled. The aims of our study were to examine four distinct populations of T. thompsonii using Amplified Fragment Length Polymorphism (AFLP) markers to i) understand general patterns of population genetic diversity and structure, including the relative influence of gene flow, selection and genetic drift, and ii) examine the extent that diversity and structure changed over time. Specifically, this information would help us understand how adequate our efforts have been to conserve T. thomsonii using in situ and ex situ methods and help us determine if ex situ resampling intervals should be driven by declining seed viability in storage, or the occurrence of significant change in the genetic structure of in situ populations.Collecting permits for populations growing on US Forest Service land were obtained from the Entiat District Ranger of the Okanogan-Wenatchee National Forest. Verbal permission to collect on state land was obtained from the Natural Areas Ecologist of the Washington Department of Natural Resources. Verbal permission from the landowner (Weaver family) was obtained to collect from private land.T. thompsonii is reported to occur at 9–14 sites within 187 km2 on the east slopes of the Cascade Mountains, in southeastern Chelan and adjacent Douglas County in central Washington State, USA ex situ storage (−18°C). In 2009, leaf tissue was sampled from in situ populations growing at the four sites. For the purposes of this study, each site was designated as a population, and samples collected from a specific site during a specific year were considered subpopulations. Seed and leaf tissue samples were collected by walking multiple haphazard linear transects of 150 m throughout the study site. Two inflorescence or three young and healthy leaves were sampled from a single plant if it occurred at 5 m intervals along thetransect. This distance was considered sufficient to minimize within family sampling based on observations of seed dispersal ex situ conservation of plant genetic resources. Seed was cleaned and stored in a −18°C freezer. For the analysis, seed was scarified and subjected to an 18 hour water rinse that we have found effective in overcoming seed dormancy. Seeds were germinated in petri dishes and fresh and healthy seedlings were transported to the molecular laboratory where seedling tissue was frozen at −80°C and lyophilized in a Vertis Lyo-Centre Freeze Dryer . A third of the 1995 seedlings were inadvertently frozen during transit and had to be discarded. The 2009 leaf samples were placed in envelops, kept at 4°C for 24 hours then placed in a −80°C freezer. Leaf tissue was lyophilized using the same procedure as the seedling tissue.In 1995 and again in 2004, seed was sampled between July 22 and August 10 from the four sites, and placed in Although less informative than codominant markers, AFLP markers have been used widely to investigate population genetic structure in insect-vectored out crossing species i.e. . As withFreeze-dried seedlings or leaf tissues were pulverized in a SPEX SamplePrep 2000 Geno/Grinder, and DNA extraction was automated using the Wizard Magnetic 96 DNA Plant System (Promega). The AFLP analysis was based on individual plants germinated from seed collected at the same locations in 1995 and 2004, and on leaf samples of individual plants collected from the same locations in 2009.AFLP markers were generated using locally developed procedures based on technology by Adapter sequences  were obtained through Eurofins MWG/Operon . Each adapter pair was adjusted to 100 pM/ul (EcoRI) and 200 pM/ul (MseI), mixed in equal amounts, and allowed to anneal for 1 hour at 37°C and cooled to room temperature. The annealed pairs were then diluted to 5 pM/ul (EcoRI) and 50 pM/ul (MseI), aliquoted to 100 ul amounts and frozen for future use.Ligation was done at 20°C for 2 hours in a 20 ul reaction containing 10 ul of the remaining restriction digest, 1X T4 Ligase Buffer (New England BioLabs), 5 pMoles EcoRI adapter, 50 pMoles MseI adapter, 0.5 mM ATP, and 80 cohesive end Units of T4-ligase (New England Biolabs). The completed ligation reaction was diluted 10∶1 and used for the Pre-amplification. Pre-amplification and selective amplification were done on an ABI 9700 thermocycler using cycling programs prescribed by Since we had unequal sample sizes ., a subsFor each year, band patterns were calculated for overall population and subpopulations for the following: total number of bands (NB), number of common bands (CB) (frequency >0.05), number of rare bands (RB), (frequency <0.05), number of not widely shared bands (NWS), (common bands found in 25% or fewer populations) and number of private bands (PB). Allele frequencies were estimated using AFLPsurv 1.0 To further examine population differentiation and partitioning of genetic variance, a euclidean distance metric was estimated and used for an analysis of molecular variance (AMOVA) to calculate Phi statistics , using GENALEX 6.5  is an important parameter used to assess genetic drift eV using a method developed for dominant markers by eVeN reasonably large, The occurrence of genetic drift can also influence genetic diversity, the rate unfavorable alleles are fixed, and the efficiency of selection ex situ samples collected in 1995 and 2004 and the in situ sample from the same populations in 2009. Across all populations, the number of common alleles was the same in all three sampling periods; however, more rare alleles were sampled in 2004 and 2009, compared to 1995. In the individual populations, the proportion of rare alleles was greater in TENAS and KEYSTONE, compared to BADGER and RNA, and this pattern was observed in all three sample years. The occurrence of private alleles was very low; however, there were the occurrence of common bands that were not widely shared among populations. KEYSTONE had the lowest proportion of polymorphic loci, followed by TENAS. BADGER and RNA had the highest percentage of polymorphic loci and were comparable. For all populations, percent polymorphic loci did not change over the fifteen years, with the exception of BADGER, where 2004 (79.8) was greater than 1995 (75.2) and 2009 (76.0).The four primer pairs revealed a total of a 129 readily discernible polymorphic bands. st was 0.05. Pairwise Fst between geographically close sites ranged from.03 in 1995 and 2004 to.04 in 2009. In contrast, Fst between BADGER and KEYSTONE, which were the furthest away, ranged from.07 in 1995, .05 in 2004 and.06 in 2009. Population pairwise PhiPT values showed a similar result  from 1995 to 2009. r result .Based on the AMOVA analysis, 90% of the variation occurred within populations. Seven percent of the variation occurred among populations and 3% occurred among subpopulations collected within each location  . The neiThe STRUCTURE analysis also supported strong spatial structure. The plot of P(X|K) indicated that K = 2 and K = 4 were the most likely groupings . Fig. 3 The assignment tests suggested that limited gene flow did occur between the four populations. With AFLPOP, , individOutlier loci detection suggested that most of the markers we examined were neutral but there was some signature of selection. BayeScan 2.1 identified no significant outliers among the 4 K groups at either prior odds of 10∶1 or 1∶1 for all three years sampled. However, DFDIST showed a high probability (>95%) for divergent selection for 9, 7 and 9 loci in 1995, 2004 and 2009, respectively. In one instance, the same locus experienced selection in all three years, while a second locus experienced selection in 1995 and 2004, and a third, experienced selection in 2004 and 2009.When we estimated effective population size, we found that NeV declined in RNA from 163 to 113. However, it increased in BADGER and KEYSTONE . This coT. thompsonii is a narrowly distributed endemic species, our results showed that it did not have a narrow genetic base. The overall level of genetic diversity (0.26) was similar to comparable AFLP studies on widespread perennial insect-pollinated congeners, T. montanum L., (0.23), T. alpinum L., (0.24), T. repens, (0.1) T. montanum (58%) T. repens (31%) Silene chlorantha (Willd.) (0.20) Abronia alpina Brandegee (.28) Echinacea laevigata (Boynton and Beadle) Blake (0.26) Although Among the four populations, KEYSTONE and TENAS, the northern most populations, had less overall diversity and a lower percent of polymorphic loci compared to BADGER and RNA. They also had about twice as many rare bands. An explanation may be that these populations are relicts of the leading north edge of the species during the Pleistocene glaciations. These populations lie due south of the furthest extent of the Okanogan lobe of the Cordillera ice sheet, and although major valley glaciers extended eastward from the Cascade Range, the closest stopped at the upper Entiat river, approximately 10 km west of KEYSTONE T. repens, also an insect-pollinated outcrossing perennial species BADGER was the only population that showed a significant decline in genetic diversity from 1995 to 2004. It was also the only population where a private band was detected. Considering that this population is located in open shrub steppe habitat, as opposed to the other three populations that are located in the forest transition zone, it may contain unique alleles that help it persist in a drier, hotter environment. Although resampling suggested that uneven sampling sizes did not bias our genetic estimates, we would expect if bias was present, sampling too few plants would have underestimated diversity. Although we sampled fewer plants in 1995, compared to 2004 and 2009, 20 individuals were determined to be adequate to assess genetic variation in Although genetic differentiation between the four populations was significant , as was our test of isolation by distance, there was sufficient evidence to suggest that gene flow did occur among the studied populations.T. thompsonii or contribute to the long distance movement of insect pollinators, since RNA is located northwest of BADGER Trifolium populations has been reported and has been concordant with data generated from morphologic The assignment test suggested infrequent gene flow occurred, despite the distance between populations. The asymmetrical flow of genes was concordant with site specific characteristics, in terms of climate and topography. The occurrence of twice as many migration events from RNA to BADGER, compared to BADGER to RNA supported the hypothesis that prevailing northwesterly winds may disperse fruits of Although there was some evidence that selective forces were responsible for influencing Fst values for some of the markers we examined, it was only for a few. However one marker was identified as being under divergent selection in all three years examined, and may be associated with an adaptive trait. Although succession has been occurring in the area where RNA, TENAS and KEYSTONE are located since the 1988 fire, our marker set did not detect any overall temporal trends in changing selection pressure. Studies similar to in situ reserve of Thompson clover.There was limited evidence for genetic drift although we did detect a decline in effective population size in RNA, which was concordant with consensus data. However, we did not detect a decrease in genetic diversity. Our study suggested that further monitoring is warranted in RNA to ensure population decline does not have a negative impact since the population is the largest and oldest ex situ samples of Thompson clover could be greater than 15–20 years.Altogether, we tracked the genetic structure of 4 different populations of Thompson clover over 15 years. With the exception of BADGER, the populations did not appear to have undergone substantial change during the time period of our study. Changes in genetic structure of the RNA, TENAS and KEYSTONE populations due to succession following the 1988 fire, were not evident. Although gene flow was evident, admixture tended to decline over time, especially in BADGER and KEYSTONE. This suggested that despite the occasional influx of migrants, individual populations tended to return to equilibrium, as unadapted genotypes were selected against. Overall, spatial differences among the 4 study locations accounted for more than twice the variation as temporal differences within study locations. These results suggested that recollecting intervals for thompsonii populations sampled from four diverse locations over a 15 year time period. Although there was some evidence for gene flow, selection and drift, there was no difference in overall diversity between ex situ samples collected in 1995 and 2004 and in situ samples collected in 2009. Only one population showed a decline in genetic diversity over the 15 years studied. Our results echo other reports that have found that perennial out crossing species generally showed less difference in genetic diversity between in situ and ex situ accessions compared to annual, self-pollinated species In this paper, we report the spatial and temporal genetic variation and population substructure revealed by AFLP markers in T. ex situ samples of insect-pollinated herbaceous perennial species provide clear snap shots of in situ populations. Our study suggested that a successful ex situ collecting strategy is to collect from a large number of plants (>150 plants), equalize maternal contribution by sampling the same number of heads per plant, and sample along random transects with sufficient space between plants to minimize intrafamilial sampling. We also learned that long distance dispersal can occur, and appeared to play an important role in limiting the extent that our four populations diverged from one another. Long distance gene flow by insect pollinators not only maintained genetic connectivity, but can also help in maintaining and increasing effective population size We can use the lessons we learned in our study to formulate some general guidelines to help ensure ex situ and in situ conservation of PGR is the value of comparing the genetic structure of ex situ and in situ samples. Quantifying genetic change over time not only allows genetic resource managers to validate conservation protocols, develop appropriate recollection intervals, but provides them with an early detection mechanism for identifying problematic conditions that can be addressed to prevent the local extinction of vulnerable in situ populations.The most important lesson we can apply to the effective Figure S1The plot of P(X|K) indicated that K = 2 and K = 4 were the most likely groupings.(TIF)Click here for additional data file.Table S1Results from resampling to determine unequal sample bias.(DOCX)Click here for additional data file.


Coregonus clupeaformis and Prosopium cylindraceum) on Lake Huron, Canada, in the immediate vicinity of thermal effluent from nuclear power generation. Niche metrics using δ13C and δ15N stable isotopes showed high levels of overlap (48.6 to 94.5%) in resource use by adult fish captured in areas affected by thermal effluent compared to nearby reference locations. Isotopic niche size, a metric of resource use diversity, was 1.3- to 2.8-fold higher than reference values in some thermally affected areas, indicative of fish mixing. Microsatellite analyses of genetic population structure  indicated that fish captured at all locations in the vicinity of the power plant were part of a larger population extending beyond the study area. In concert, ecological and genetic markers do not support the presence of an evolutionarily significant unit in the vicinity of the power plant. Thus, future research should focus on the potential impacts of thermal emissions on development and recruitment.Thermal pollution from industrial processes can have negative impacts on the spawning and development of cold-water fish. Point sources of thermal effluent may need to be managed to avoid affecting discrete populations. Correspondingly, we examined fine-scale ecological and genetic population structure of two whitefish species ( Worldwide a variety of energy generating stations use natural lake or river water as an industrial coolant, which may be released as warmed effluent into the environment. Thermal pollution of this nature can result in localized changes in water temperature ranging from 2 to 15°C above ambient values . FreshwThe population structure of fish is often assessed using neutral genetic markers to identify discrete subpopulations that result from reduced gene flow due to a variety of factors, including geographic barriers –16, humaStable isotopes of carbon and nitrogen are ecological markers that provide a quantitative means of comparing resource use among groups of fish. The range and variance of isotopic values present a two-dimensional metric of niche that incorporates information about carbon source, a proxy for habitat in freshwater, and nitrogen source, indicative of trophic position –31. TogeCoregonus clupeaformis) comprise one of the most important commercial fisheries within the Great Lakes [Prosopium cylindraceum) may be an important indicator species for environmental monitoring [Canada currently has four major nuclear power generating stations on the Laurentian Great Lakes, the largest freshwater system in the world. All three stations operate multiple CANDU reactors and use lake water for once-through cooling processes to cool steam condensers –42. The at Lakes –49, and nitoring . Recent nitoring –53. Earlnitoring –56. FromBoth genetic and ecological population structuring occur among lake whitefish spawning aggregations in Lake Huron on the spatial scale of hundreds of kilometers. Previous studies have identified genetic differentiation among populations in different basins or separated by large geographic distances  isotopic niche characteristics; and (2) genetic population structure. The results of this study will inform whitefish management within the study area.et al. [Our study took place in the vicinity of the Bruce Power site in eastern Lake Huron, Ontario, Canada . Stationet al.  has showBoth whitefish species we studied are broadcast spawners that cast their eggs over reefs and rock-cobble substrate in shallow (2-8m depth) near-shore areas in the late fall . Lake whAll animal research was approved by the University of Regina President's Committee on Animal Care, following the guidelines of the Canadian Council on Animal Care. The approved Animal Use Protocol was AUP 11–13 "Population and Conservation Genetics of Freshwater Fish". The authors did not require a permit to sample fish; the permit was issued to Bruce Power and fish specimens were received after collectionAdult lake and round whitefish were collected as part of Bruce Power’s environmental assessment follow-up program (see ) using bδ13CVPDB and δ15NAIR  in units of per mil (‰). Replicates of standards and unknowns generally varied less than 0.2 ‰. We arithmetically lipid-corrected δ13C with the McConnaughey & McRoy [Sub-samples of dorsal muscle from individuals from reference and affected sites were rinsed with distilled water, dehydrated in a drying oven at 50°C for 120–168 hours, ground to a fine powder using a dental amalgamator and weighed (0.5–1.0 mg) into tin capsules . Stable  & McRoy  formula ‰2) and is directly related to resource use diversity [c; [b) to obtain an average SEA with 95% credibility. To compare resource use similarity between fish in the reference and affected sites we quantified isotopic niche overlap by calculating the area shared between standard ellipses of each reference site with each affected site. To compare resource use diversity we calculated the Bayesian probability that SEA of fish in affected sites was larger than fish in reference sites based on SEAb estimations. All niche metrics were calculated with the Stable Isotope Analysis in R (SIAR) package [We created Bayesian standard ellipses using SIBER Metrics to define and compare the isotopic niches for each species at each sample site. Bayesian ellipses encompass 40% of the data, thus this metric enables a robust comparison of niche that is less biased by differences in sample size than traditional metrics ,77. The iversity . We corrsity [c; ). The SE package ,79.et al. , with the exception that we added 28 U of RNase A  and extended proteinase K digestion to 8–12 hours at 56°C. DNA was quantified using a Qubit 2.0 Fluorometer  prior to diluting to a standard concentration (5 ng/μl) for PCR reactions. Lake whitefish were genotyped at 9, and round whitefish at 6 microsatellite loci previously developed for each species . PCR reactions were performed in a total reaction volume of 25 μL containing 1X PCR Master Mix , 2 μM forward and reverse primer, and 10 ng of template DNA. Thermocycling conditions for whitefish primers were: 5 min at 95°C, followed by 30 cycles at 95°C for 30 s, 30 s at the locus-specific annealing temperature see , 60 s atO and HE). Deviations from Hardy-Weinberg Equilibrium (HWE) were examined for each locus using GENEPOP v4.3 [PCR products were size fractionated using a Beckman Coulter GenomeLab GeXP Genetic Analysis System with a 400 bp internal size standard . Alleles were scored using GENEMARKER 2.20 software  under default settings, with the exception of a bin width of 1 nucleotide to reflect the resolution limit of capillary electrophoresis. Automated allele calling was verified manually and confirmed by a second, independent observer. The program MICRO-CHECKER  was usedPOP v4.3 .ST; [ST values were evaluated by comparing reference and affected regions, individual sampling sites, and sampling years for both lake and round whitefish. Second, we used Bayesian clustering in the program STRUCTURE to identify the number of potential populations in our data sets [et al. [ST and Bayesian analyses. Finally, the data were analyzed using Discriminant Analysis of Principle Components (DAPC), a multivariate ordination method in the R package ADEGENET [Population structure among spawning groups was assessed using several different approaches. We calculated fixation indices  in the ST; . FST valata sets . The ana [et al. . Only loADEGENET . DAPC doδ13C and δ15N values were similar for fish of each species collected in reference sites to those in affected sites, but varied between species (C) was generally similar between reference and affected sites for lake whitefish, with the exception of site A4, which was substantially larger than all other sampling locations with high probability . The outcome was similar when comparing among sampling years . Pairwise comparisons among zones also produced very small FST values not different from 0, with an average of -0.0015 for both lake and round whitefish . Co. Coδ13Cs ; or (2)g guilds ). Our daet al. [There was no genetic differentiation between affected and reference zones for either lake or round whitefish, which were both part of single populations across our study area. This finding was not unexpected for lake whitefish, which have significant population structure throughout the Great Lakes system ,106, andet al.  showed net al. –111. Inta priori expectations about population structure. Lake whitefish have been much more intensively studied because of their commercial value –117a prie e.g., ,118). Ro. Roa prie S1 TableN; k is number of alleles observed; Ho and He are observed and expected heterozygosity, respectively; PI is the probability of identity for each locus, and TD refers to the touchdown protocol used for PCR (see text).The size indicates the range of observed alleles in base pairs and includes the length of the CAG tag; number of individuals genotyped is (DOCX)Click here for additional data file.S2 Tablek refers to the number of alleles observed; HO and HE are observed and expected heterozygosities, and PIC is the polymorphic information content.The repeat size indicates the length of the repeat within each locus; N is the number of individuals genotyped; (DOCX)Click here for additional data file.