The Qiangic languages in western Sichuan (WSC) are believed to be the oldest branch of the Sino-Tibetan linguistic family, and therefore, all Sino-Tibetan populations might have originated in WSC. However, very few genetic investigations have been done on Qiangic populations and no genetic evidences for the origin of Sino-Tibetan populations have been provided. By using the informative Y chromosome and mitochondrial DNA (mtDNA) markers, we analyzed the genetic structure of Qiangic populations. Our results revealed a predominantly Northern Asian-specific component in Qiangic populations, especially in maternal lineages. The Qiangic populations are an admixture of the northward migrations of East Asian initial settlers with Y chromosome haplogroup D (D1-M15 and the later originated D3a-P47) in the late Paleolithic age, and the southward Di-Qiang people with dominant haplogroup O3a2c1*-M134 and O3a2c1a-M117 in the Neolithic Age. The Sino-Tibetan languages are a family of some 460 languages, including two subfamilies, namely Chinese and Tibeto-Burman. They are spoken by over a billion people all over East Asia and Southeast Asia, and second only to the Indo-European languages in terms of the population size of native speakers Despite intense linguistic and archaeological researches, little has been known about how the Sino-Tibetan people dispersed from western China? During the past two decades, the characterization of genetic diversity has shed light on the history of Sino-Tibetan populations, especially the diversity defined by the maternal mtDNA and the paternal Y chromosome. In the maternal side, mtDNA evidence suggested a northern Asian origin of Tibetans, due to the high frequencies of northern Asian specific haplogroup A, D, G, and M8 Given that a correlation is emerging that suggests language change in an already-populated region may require a minimum proportion of immigrant males, while mtDNA types represent more ancient settlement Y chromosome evidence indicates that Qiang people might be the origin source for the Sino-Tibetan populations We collected blood samples of 407 healthy and unrelated individuals from four Qiaingic populations in western Sichuan province . Our stuThe samples were typed through seven panels of 100 SNPs as listed in the latest Y chromosome phylogenetic tree Haplogroup O panel: M175, M119, P203, M110, M268, P31, M95, M176, M122, M324, M121, P201, M7, M134, M117, 002611, P164, L127 (rs17269396), and KL1 (rs17276338).Corset Panel: M130, P256, M1, M231, M168, M174, M45, M89, M272, M258, M242, M207, M9, M96, P125, M304, M201 and M306.Haplogroup C panel: P54, M105, M48, M208, M407, P33, M93, P39, P92, P53.1, M217, M38, M210, M356, P55, and M347.Haplogroup D panel: P47, N1, P99, M15, M125, M55, M64.2, M116.1, M151, N2, and 022457.Haplogroup N panel: M214, LLY22g, M128, M46/Tat, P63, P119, P105, P43, and M178.Haplogroup R panel: M306, M173, M124, M420, SRY10831.2, M17, M64.1, M198, M343, V88, M458, M73, M434, P312, M269, and U106/M405.Haplogroup Q panel: P36.2, M3, M120, MEH2, M378, N14/M265, M25, M143, M346, L53, and M323.Those binary markers were hierarchically genotyped by SNaPshot (ABI SNaPshot Multiplex Kit) and fluorescent allele-specific PCR. PCR products were electrophoresed on a 3730xl Genetic Analyzer .Seventeen Y chromosomal STRs  were amplified using the AmpFlSTR Yfiler PCR Amplification kit . Amplified products were separated and detected using the ABI 3730xl Genetic Analyzer  according to the manufacturer’s recommended protocol. The data were analyzed using GeneMapper ID v3.2 . For use in the analyses, DYS389II was calculated by subtracting the DYS389I allele size.http://www.phylotree.org/). The mtDNA sequences have been deposited in Genbank with accession numbers KJ783504-KJ783899.The hypervariable segment I (HVS-I) of the control region was amplified by primers L15974 and H16488 4 samples of the program’s output representing 106 MCMC cycles were taken after discarding the first 3×103 samples as burn-in. The Time to the Most Recent Common Ancestor (TMRCA) is calculated using the product of the estimated population size N and the height of the tree T  Principal component analysis (PCA) was performed using SPSS 18.0 software . Networks of Y chromosomal STR data and the mtDNA HVS-I motifs were constructed by reduced median-joining method According to the nomenclature of Y Chromosome Consortium (YCC) The paternal genetic relationships among Qiangic, Tibeto-Burman, and other East Asian populations were discerned with the aid of additional published Y chromosome datasets. We used a PCA based on the distribution of Y chromosome haplogroup frequencies of 51 populations to show the overall clustering pattern . ResultsTo discern the detail relationship between the D3a-P47, O3a2c1a-M117, D1-M15, and O3a2c1*-M134 haplogroups in Tibeto-Burman and other related populations, a median-joining network was constructed based on Y-STR haplotypes of those haplogroups . A clearWe then estimated the coalescence and expansion time of Y chromosome lineages in Qiangic populations . The ageMtDNA HVS-I sequences of 396 individuals from the four studied Qiangic populations have been successfully typed. A total of 214 different haplotypes were defined by 134 polymorphic sites in the HVS-I dataset. The haplotype diversity of those Qiangic groups ranged from 0.978 to 0.994, with the lowest haplotype diversity observed in Horpa-Daofu (0.978) and the highest in Horpa-Danba (0.994). The mean number of pairwise differences (MNPD) and nucleotide diversity (ND) show a similar pattern with the haplotype diversity, as the highest diversity was observed in Horpa-Danba and the lowest in Horpa-Daofu. However, Tibetan-Yajiang has a higher diversity in haplotype but lower diversity in MNPD and ND than Tibetan-Xinlong. Measures of population growth  all gave the negative values for each population, but Tajima’s D, Fu & Li’s D* and F* were not statistically significant in Horpa-Daofu . The not397 samples were successfully assigned to mtDNA haplogroups using a combination of HVS-I sequence motifs and single nucleotide polymorphisms (SNPs) distributed around the coding region of the mtDNA genome. A total of 79 haplogroups or paragroups (unclassified lineages within a clade marked with an asterisk [*]) were identified , all witWe performed a PCA using the mtDNA haplogroup frequencies of Qiangic groups in this study and other 68 populations to see the detailed genetic patterns of those populations . The firMacrohaplogroup M and its subhaplogroups represent the majority of the Qiangic maternal lineages, with frequencies ranging from 65.22% in Horpa-Danba to 57.98% in Tibetan-Xinlong. Haplogroup D4 and G are the most frequent sub-clades of macrohaplogroup M in Qiangic populations, each comprising 13.60%. Haplogroup D4, which is prevalent throughout Central Asia Haplogroup G is found at high frequencies in northeastern Siberia but it is also common among populations of Japanese Archipelago and Korean Peninsula. This haplogroup also comprises an average of 20% of the maternal gene pool of the Tharus from Nepal Haplogroup M8 has two sublineages, haplogroup C and Z. Haplogroup C is a common lineage, which is widespread in East Asia and Siberia and is one of the founder lineages among Native Americans M9a’b is widely distributed in mainland East Asia Haplogroup M13a has been found at its greatest frequency and diversity in Tibet, but it has also been detected at very low frequencies in Siberian Buryat, Yakut, Altaian Kazakh, and Ewenki Qiangic populations also exhibit some basal Eurasian mtDNA lineages. Haplogroup M62, for example, was first reported in Northeast India Haplogroup R and its subhaplogroups (B and F) represent the majority of the lineages branching from the basal N trunk, accounting for 26.09%, 22.50%, 28.57%, and 23.44% of the maternal diversity in Horpa-Danba, Horpa-Daofu, Tibetan-Xinlong, and Tibetan-Yajiang, respectively. Subhaplogroup B4* is the most frequent lineage of haplogroup B in Qiangic, comprising 4.53% of all the samples. In the network of B4*, the root clade composed almost exclusively of non-Tibetan-Burman samples, however, the Tibetan-Burman samples only formed some small clusters or shared the terminal types, suggesting that B4* had already differentiated before its arrival in Tibet. Subhaplogroup F1* is the most frequent lineage of haplogroup F in Qiangic, accounting for 5.54% of all the samples, and even comprising as high as 12.5% of Horpa-Daofu. Age estimate generated for F1* in Qiangic was around 5 kya . The excHaplogroup N* is almost exclusively represented by haplogroup A in our samples. Haplogroup A is widely distributed in northern and eastern Asia, occurring at frequencies of 5%–10% in different populations The Sino-Tibetan linguistic family comprises some 460 languages distributed in East Asia, Southeast Asia, and parts of South Asia, including the Chinese and Tibeto-Burman subfamilies From the paternal Y chromosome perspective, haplogroup D1-M15 originated from D*-M174 during its migration into mainland East Asia Around 20–40 kya, a population with dominant haplogroup O3-M122 Y chromosomes  finally reached the upper and middle Yellow River basin and formed the Di-Qiang populations. During the Neolithic period, the Di-Qiang people experienced relatively huge population expansion. A subgroup of the Di-Qiang people with dominant haplogroup O3a2c1*-M134 and O3a2c1a-M117, now called the Proto-Tibeto-Burman people left their Yellow River homeland, probably also moved along the Tibeto-Burman corridor, embarking on large-scale westward migrations to nowadays Qinghai province and then southward to the Himalayas, or southward migration directly via the WSC corridor to Yunnan and Guangxi, where they mixed with D-M174 linages and developed into Tibeto-Burman populations. However, haplogroup O3a2c1*-M134 might have already reached Tibet predated the above southward migration together with O3a2c1a-M117, judging from the high diversity in the network of O3a2c1*-M134 . In addiThe role of haplogroup O3-M122 lineages played in the origin of Tibeto-Burman populations has suggested extensive genetic input from northern Asians. This suggestion has been supported by previous studies employing autosomal STR Table S1Y chromosome and mtDNA haplogroup frequencies of Qiangic populations.(XLS)Click here for additional data file.Table S2Y chromosome SNP and STR data, mtDNA haplogroups and HVS-I motif of Qiangicpopulations.(XLS)Click here for additional data file.Table S3Y chromosome and mtDNA haplogroup frequencies used in PCA plot and haplogroupcontributions to each PC.(XLS)Click here for additional data file.Table S4Shared Y-STR haplotypes between Qiangic populations and other East Asian populations.(XLS)Click here for additional data file.Doc S1Geographic distribution of Y chromosome haplogroup D1 and D3a, Y-STR neighbor-joining tree based on genetic distance.(DOC)Click here for additional data file.Doc S2Three representative complete mtDNA haplotypes compared to rCRS.(DOC)Click here for additional data file.


Tabanus nigrovittatus Macquart, is frequently found in coastal marshes of the Eastern United States. The greenhead horse fly larvae are top predators in the marsh and thus vulnerable to changes in the environment, and the adults potentially are attracted to polarized surfaces like oil. Therefore, horse fly populations could serve as bioindicators of marsh health and toxic effects of oil intrusion. In this study, we describe the impact of the April 2010 Deep Water Horizon oil spill in the Gulf of Mexico on tabanid population abundance and genetics as well as mating structure. Horse fly populations were sampled biweekly from oiled and unaffected locations immediately after the oil spill in June 2010 until October 2011. Horse fly abundance estimates showed severe crashes of tabanid populations in oiled areas. Microsatellite genotyping of six pristine and seven oiled populations at ten polymorphic loci detected genetic bottlenecks in six of the oiled populations in association with fewer breeding parents, reduced effective population size, lower number of family clusters and fewer migrants among populations. This is the first study assessing the impact of oil contamination at the level of a top arthropod predator of the invertebrate community in salt marshes.The greenhead horse fly,  Coastal salt marshes are among the most valuable, productive and diverse ecosystems on earthTo date, the largest accidental man-made marine oil spill was the catastrophic explosion of the Deep Water Horizon drilling platform connected to the blowout of British Petroleum’s Macondo well, which occurred on April 20, 2010 and resulted in 4.9 million barrels of oil discharged into the Gulf of Mexico over a period of almost four months3Acute oiling in the Gulf of Mexico and the coastal habitats subsequent to the Deepwater Horizon platform collapse created visible impact on highly prominent vertebrates, particularly birds, marine mammals and turtles along the coast6Contamination is known to alter soil properties and impact the food web from microbes to plants and invertebrate populations and vertebrates that use the marsh for feeding and nursery areasThe salt marsh insect community is the trophic support for both terrestrial and marine vertebrates11Tabanus nigrovittatus Macquart (Diptera: Tabanidae) as a bioindicator of the impact of the intrusion of oil into coastal marshes because this horse fly is one of very few insects that are native to and tightly bound to specific coastal marsh habitats that range from the Texas coast to Nova ScotiaWe evaluated populations of the greenhead horse fly, T. nigrovittatusOur overall hypothesis was that the oil residue impacts the invertebrate ecosystems in tidal marshes, and that tabanid residents of the marsh community would serve as bioindicators for wetland health and the oil intrusion in particular, as they require both aquatic and terrestrial habitat for survival. To test this hypothesis, we sampled adult horse fly populations biweekly immediately after the oil spill (June 2010-October 2011) via canopy traps from oiled areas and those unaffected by oil . We alsoOur trap data with only a range of 1.3–4.8 flies captured per hour in 41 trap days indicated that the adult tabanids had been affected immediately after the oil reached Elmer’s Isle, Grand Isle (Jefferson Parrish) and Grand Bayou  in 3 of the 13 sediment samples from the areas unaffected by oil.F-statistics confirmed population genetic differentiation among samples  and revealed a considerable amount of inbreeding in the total population , and within populations . The local inbreeding coefficient FIS was positive in all 13 populations, with values significantly or marginally greater than zero in 7 populations . Thus, the 13 samples were treated as genetically separated populations. Summary statistics for each locus and population  are listed in ulations , but notTESS analysis assigned the individuals from the 13 sample populations to six major genetic clusters . The ploFST) among populations . Since variances were unequal , additional t-test were performed not assuming equal variances. T-tests confirmed marginally lower genetic distance between populations not reached by oil  compared to oiled populations  and no difference between collection years.Condition  had marginally significant effects on genetic distance (SD = 0.014) was marginally higher than that of oiled populations , while the import rate was not different (P > 0.20). The largest donor among unaffected populations was CP-2010 with gene flow into SC-2010 (0.28) and RWR-2010 (0.11). The largest donor among oiled populations was GIW-2011 with export to GIP-2011 (0.11) and GB3-2011 CP (0.26). Migration rates from unaffected to oiled areas and vice versa were overall low and no directional differences were found . The low migration rate was supported by the fact that genetic distances between unaffected and oiled populations  were significantly larger than among either unaffected  or oiled  populations likely due to the geographic separation (P < 0.001). The only migration between unaffected and oiled areas with a rate exceeding 5% was recorded from SC-2011 to GB2-2011  migration rates among populations ranged from 0.1 to 28% with proportions of 68–99% being derived from the source populations each generation . The emi11 0.07, .Z = 47.02, R2 = 0.13, P = 0.001). No isolation by distance was detected among populations within 150 km  was found only over a large scale up to 360 km between the Western unaffected populations and the Eastern oiled populations . Instead of detecting heterozygote excess as a signature of a recent bottleneck, there was significant heterozygote deficiency detected by at least one mutation model in all but one (RWR-2010) of the unaffected populations, which might indicate a recent population expansion in unaffected areas.P < 0.05, In contrast, we detected significant recent genetic bottlenecks in 2010 and/or 2011 in five out of seven populations from areas that were hit by the oil spill . The average number of offspring that each parent contributed in each population was 2.57 (SD = 1.56) with a maximum of nine. This number is a minimum estimate since it is based on a limited sample size of offspring. It is meant to be a relative estimate to compare between populations from unaffected and oiled areas, not an absolute measure of fecundity. Sexes could not be distinguished from the inferred parental genotypes and thus male and female specific reproductive contributions are unknown. A considerable proportion of full-sisters (avg. 2.09 ± 2.36%) and half-sisters (16.50 ±5.26%) were frequently caught in the same trap. Detailed results for each population are presented in Polygamy was the predominant mating strategy based on a comparison of log likelihood values among different possible mating strategies . Twelve P = 0.048, df = 1, F = 5.21), the effective population size  and the number of offspring per parent . Condition had also a marginal effect on the number of family clusters . The factor ‘year’ (2010 vs. 2011 samples) and the combination of ‘year’ * ‘condition’ had no significant influence on any of the dependent variables . An additional t-test performed for the percentage of fullsib pairs accounting for the unequal variances confirmed the non-significant results from the GLM  as well as the significant results for the number of parents  and effective population size  and the marginal significances for number of offspring per parent . Number of partners each fly mated with and the percentage of full and halfsibs were not significantly different between unaffected and oiled populations . Overall, the effective population size was smaller in oiled populations with fewer parents contributing offspring but with more offspring per mating partner leading to marginally fewer family clusters.General linear model analysis showed that condition (unaffected versus oiled populations) significantly influenced the number of parents contributing offspring to the population . As members of an autogenous species, females do not even need a blood-meal for laying the first clutch of eggsThe differences in adult tabanid population estimates between areas unaffected and impacted by oil sampled immediately after the oil spill were dramatic. The apparently immediate decline in adult populations was surprising, since the summer 2010 generation of flies had developed up to 9 months as larvae in the marsh and had emerged prior to the oil spill. Adults of et al.et al.The most likely reasons for the immediate population crash in oiled areas were the tabanids’ need for fresh water and attraction to oil sheen. Horvath and ZeilLess surprising than the decline of adult populations in oil impacted areas, but equally dramatic was the difference in larval population estimates for the unaffected vs. oiled marshes. WilsonLarvae develop as predators and cannibals for 3–9 months in the marsh soilet al.Subsurface water in Louisiana marshes was shown to have significant levels of polycyclic aromatic hydrocarbons (PAH) and acute toxicity to killifish immediately after the oil spill6We suggest that the initial crashes in adult tabanid populations were associated with acute adult mortality, but that the sustained tabanid population suppression was due to toxic effects directly to larval tabanids and/or important elements of their food web. Therefore, this top predator species with sediment-dwelling larvae did not show the quick recovery seen from other insects, such as the community that is built around Spartina herbivoresThe apparently severe population crash and reduced effective population size of tabanid adults and larvae in oiled areas led to genetic bottlenecks in all but one of the oiled populations. The bottleneck test based on faster reduction of allele numbers relative to reduction in heterozygosity is actually considered having little power and requires that extreme bottlenecks have occurred in recent times to show significance, especially in the first generations experiencing the bottleneckWhether these findings translate into reduced evolutionary potential of populations depends on remediation success of oiled marsh sites and recovery of a sustaining food web. Theory37While substantial loss of genetic diversity, increased linkage disequilibrium, reduced heterozygosity and correlative effects on fitness via inbreeding depression are frequently discussed as observed or inferred consequences of genetic bottlenecksGene flow and migration rates among unaffected populations were slightly higher than among oiled populations. This was not due to geographical distance since the distance between oiled populations was actually smaller and no isolation by distance effects were detected within 150 km, i.e., within the range of distance among unaffected (max. 144 km) and among oiled populations (max. 45 km). Interestingly, there was a directional bias in migrations rates with emigration rates being marginally lower from oiled populations, but immigration into oiled populations were equal to those into unaffected populations. This suggests at least a temporary negative effect of oil contamination on dispersal. Other studies showed that sublethal exposure to crude oil causes changes in heart functionThe Deepwater Horizon spill affected over 700 km of marsh shoreline in Louisiana alone with the peak amount of oiling in July 2010; but almost 200 km still displayed some degree of oiling after two years and moreContinued observation of the fate of the tabanids populations in the oiled areas will shed light on population viability. Genetic studies and larval surveys need to continue to discern whether census increase is based mostly on immigrants or if the marsh sediment has detoxified sufficiently to allow local development of larvae. The latter would be a valuable indicator sign of ecosystem recovery.http://www.nytimes.com/interactive/2010/05/01/us/20100501-oil-spill-tracker.html, date of access: 11/05/2015].Collection permits for horse fly larvae and adults were obtained from the Louisiana Department of Wildlife and Fisheries (permit numbers LNHP-10-074 and LNHP-11-092). Adult female flies were collected every other week from June through October in 2010 and 2011, at four locations. Each location had 4–5 trap sites separated by >1km . Since w2 and thus the samples contained exclusively females. The exact daily trapping period (ranging from 2–8 hours) was recorded for each site at each location, and the highest three trap counts were used for analysis. The captured flies were transferred on dry ice and stored at −80 °C. Specimens were identified as T. nigrovittatus using the methods of Sofield et al.Tabanids were captured with canopy traps baited with dry iceA subset of 13 samples of all that were collected for population abundance analyses were used for population genetic studies. For microsatellite genotyping, we used horse flies that were collected in June 2010 and again in June 2011 from single traps representing three different trap sites in the unaffected marshland along the coast of Western Louisiana , Cypremort Point at sites near ACS 1 and 2, Grand Isle near all ACS, and Grand Bayou near all ACS  using thet al.Total DNA was extracted from thoraces of horse flies using the DNeasy Tissue Kit . Up to 30 horse flies per sample were genotyped at ten polymorphic loci. A detailed description of the loci selection process during which unreliable loci  were identified and omitted from population genetic analyses, polymerase chain reaction and genotype scoring procedures can be found in Husseneder Exploratory analyses indicated that data were not normally distributed. The average numbers in each trap collection were transformed to y = log(x+1) to normalize prior to statistical analysis. First, a two way ANOVA  was used to compare log transformed tabanid catch data across years and locations. Model main effects were Year  and Region . Second, a three way ANOVA was performed to add Month  to the previous main model effects. All possible interactions among the main effects in the model, and a Tukey Kramer adjustment (α = 0.05) were used to separate the means.G-Statistics with standard Bonferroni corrections at a 0.001 nominal level for multiple comparisons  were obtained by jackknifing over loci. Tests for deviation from Hardy-Weinberg-Equilibrium were performed with FSTAT for each locus and population based on randomizing alleles within populations and calculating FIS. Statistics such as Kolmogorov-Smirnov tests for normality, parametric (t-tests) and nonparametric tests  and well as General Linear Models were performed with SPSS . P-values ≤ 0.05 were considered significant and P-values up to 0.10 were considered marginal.Samples from different traps were tested for significant genotypic differentiation using pairwise log-likelihood ons FSTAT. GeneticTo obtain ad hoc approximations of the number of genetic clusters in the total population and the genetic relationships among populations, individual flies were probabilistically assigned to genetic clusters based on their multilocus genotypes using TESS 3.1FST into FST/(1 − FST) and performing a regression against the natural logarithm of geographical distance. Matrix correlations were tested for significance using Mantel tests , because these techniques allow for deviation from migration-drift balance and Hardy-Weinberg equilibriumIn addition, we employed Bayesian statistics using Markov Chain Monte Carlo methods (BayesASS v. 1.3) to estimate recent gene flow , as implemented in BOTTLENECK v. 1.2.02Pedigree structure in each population was inferred using the full pedigree likelihood method implemented in COLONY v.2.0.3.1To infer the best configuration of relationship structure, populations of offspring (trap samples) were subjected to full maximum likelihood calculations56To identify the predominant mating strategy in each population independent runs were performed specifying the three possible mating strategies  allowing the occurrence of inbreeding. Log likelihood values for the best pedigree configuration based on the observed genotypes were compared among the hypothesized mating strategies. Only results for the mating strategy model with the best fit (polygamy) were presented in the study.Based on the Best Maximum Likelihood Configuration output of the program, we determined the number of inferred parents contributing offspring  and the number of family clusters in each population. Obtained values were corrected for uneven sample sizes by calculating each for a sample size of 30 individuals genotyped per population. We also estimated the number of partners that individuals of both sex mated with and the number of offspring produced per parent. The percentages of fullsib and halfsib pairs in each population were inferred from sibship assignment plots in COLONY.Ne) was inferred with the sibship assignment method (i.e. via frequencies of a pair of randomly sampled offspring sharing one or both parents) implemented in COLONYNe since it is dependent on the variance in family sizeEffective population size  and with marginal significance when all factors and combinations were considered . Log transformation removed the issue (P > 0.20). Unequal variances were also detected in the percentage of fullsib pairs  when all factors and combinations were considered, but not for ‘condition’ . Transformations failed to remove unequal error variance in this dependent variable. Thus, an additional independent samples t-test not assuming equal variances was performed.Two-Factor Analysis of Variance was performed using General Linear Model Multivariate tests (SPSS) to investigate whether the factors “year” (2010 versus 2011) and/or “condition” (unaffected versus oiled populations) influenced the dependent variables measured to determine population and mating structure. Significant deviations from equal error variances were detected in the number of offpring per parent for the factor ‘condition’ (Levene’s Data availability: Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC). Detailed sample information including GPS location, dates, trap times and horse fly counts and can be found at https://data.gulfresearchinitiative.org/data/R3.x169.000:0001/ in the dataset by LF: Adult tabanid population data and available voucher specimens (doi:10.7266/N75718ZM). Raw data of microsatellite genotypes are available at https://data.gulfresearchinitiative.org/data/R3.x169.000:0002/ in the dataset by CH: Tabanus nigrovittatus microsatellite data for the assessment of population genetics of 13 populations sampled from coastal Louisiana, 2010-2011 (doi:10.7266/N71J97N2).How to cite this article: Husseneder, C. et al. Impact of the 2010 Deepwater Horizon oil spill on population size and genetic structure of horse flies in Louisiana marshes. Sci. Rep.6, 18968; doi: 10.1038/srep18968 (2016).


AGXT, PLRP2, MTRR, NAT2 and CYP3A5.One of the most important dietary shifts underwent by human populations began to occur in the Neolithic, during which new modes of subsistence emerged and new nutrients were introduced in diets. This change might have worked as a selective pressure over the metabolic pathways involved in the breakdown of substances extracted from food. Here we applied a candidate gene approach to investigate whether in populations with different modes of subsistence, diet-related genetic adaptations could be identified in the genes CYP3A5, strong signatures of positive selection were detected, though not connected to any dietary variable, but instead to an environmental factor associated with the Tropic of Cancer. Suggestive signals of adaptions that could indeed be connected with differences in dietary habits of populations were only found for PLRP2 and NAT2. Contrarily, the demographic history of human populations seemed enough to explain patterns of diversity at AGXT and MTRR, once both conformed the evolutionary expectations under selective neutrality.At CYP3A5 has been under adaptive evolution during the history of human populations. PLRP2 and NAT2 also appear to have been modelled by some selective constrains, although clear support for that did not resist to a genome wide perspective. It is still necessary to clarify which were the biological mechanisms and the environmental factors involved as well as their interactions, to understand the nature and strength of the selective pressures that contributed to shape current patterns of genetic diversity at those loci.Accumulated evidence indicates that The online version of this article (doi:10.1186/s12863-015-0212-1) contains supplementary material, which is available to authorized users. The most remarkable dietary change over the recent history of human populations was that associated with the change from food collection to food production , which oAGXT, coding for alanine:glyoxylate aminotransferase, the enzyme responsible for the transamination of glyoxylate into glycine [PLRP2, coding for pancreatic lipase-related protein 2, involved in galactolipids hydrolysis, [MTRR, encoding for methionine synthase reductase, an enzyme acting in the complex folate pathway [NAT2 coding for N-acetyltransferase 2, a phase-II enzyme involved in the detoxification of a wide number of xenobiotics [CYP3A5, coding for cytochrome P-450 3A5, a member of the CYP3A enzymes that are involved in the oxidative metabolism of many endogenous substrates and xenobiotics, which is implied in sodium homeostasis [Other metabolic-related genes have been hypothesized to constitute dietary adaptations, among which are included:  glycine -13; PLRProlysis, -17; MTRR pathway , 18; NATobiotics , 19-23; eostasis -27.AGXT was tentatively linked with meat content in diets, PLRP2 with richness in cereals [MTRR and NAT2 with availability of folate in foods and CYP3A5 with health conditions that are influenced by dietary salt intake [AGXT), or not yet replicated (e.g. MTRR and PLRP2), or not clear enough to ascertain whether they can indeed represent genetic adaptations to any dietary variable. This prompted us to address the issue applying of genetic adaptation within those genes.Genetic variation in  cereals , both MTt intake , 27. HowThus, assuming that current modes of subsistence are still good surrogates of main diets in which populations have traditionally relied, the aim of this study was to gain further insights into the relationship between diet-related variables in populations and patterns of diversity at variations in above mentioned five genes.AGXT, PLRP2, MTRR, NAT2 and CYP3A5 were examined in six sub-Saharan populations with distinct modes of subsistence and also in one European population that was also screened to generate a non-African reference group. Results were then combined with previously published information for other African and Eurasian populations to evaluate the contribution of geography and mode of subsistence or other diet-related variables to explain the patterns of genetic diversity observed for the five genes.Functional variants within loci in the seven studied populations are shown in Table The observed genotypic distributions , nor the trend in the frequency distribution sustained the hypothesis that the allele could be positively selected in meat-rich diet populations.To integrate our results in a more comprehensive distribution, data for c.32C > T was retrieved from the literature on populations for which information on the relative predominance of meat in their diets was available , whereas in the Kazaks from Western Uzbekistan, who are traditionally nomadic herders whose diet mainly consists of meat, milk and dairy products, the allele only occurred marginally (1.7 %).AGXT and lifestyle of populations.From these analyses, no connection emerged between the frequency distribution of c.32C > T in In this gene we focused on c.1074G > A, a variant that causes a premature truncation of the pancreatic lipase-related protein 2 resulting in a more active version of the enzyme. In a very recent genome-wide scan for selection in human populations, Hancock et al.  identifiAs long as we know, the association was not further investigated except in the present study, where among the screened African groups, the derived allele was detected to be quite common in the three farmers’ groups (23.3 % - 32.6 %) as well as in the herders from Uganda (39.5 %). Comparatively, the two hunter-gatherers groups showed lower frequencies, specially the Ju/hoansi (2.42 %). The sample from Portugal showed the highest frequency in this study with, 51.1 %  or in Eurasia + Africa . Comparing herders and hunter-gatherers, both integrated in the group of cereal less rich populations . Considering Africa and Eurasia together, the trend that can be extracted from the whole data points to a decreasing frequency gradient of the derived allele at c.1074G > A from populations more specialized on cereals towards those less relying on them, as was also captured by the MDS plot shown in Fig. As a whole, our results do not conflict with the hypothesis that the distribution of c.1074G > A might be related to the weight of cereals in diets, in the sense that at least within Africa, farmers populations tended to have higher frequencies of the derived allele compared to hunters-gatherers who rely less in cereals. These results were then put in a wide-ranging context, recruiting information on c.1074G > A for African and Eurasian populations from several sources, and maintaining the classification in populations that specialize and that do not specialize on cereals when originally presented  [NAT2 locus. Evidence for the diet-related hypothesis provided by Luca et al. [NAT2 worldwide genetic diversity, that were also compatible with a model holding that the slow acetylator phenotypes were selectively favored in populations relying in dietary regimens with reduced folate supply, whereas the fast acetylators were neutral or even advantageous in the presence of folate-rich diets, as those thought to be fulfilled by hunter-gatherers. To extent the population coverage of previous works, frequencies of NAT2 haplotypes and acetylator phenotypes were also estimated in this study . In the Portuguese the slow acetylator phenotype accounted for the high proportion of 52.2 %, which falls within the range typical from other European populations [The dietary availability in folates had also been previously hypothesized to be a modulator of genetic diversity at the gene that encodes for erase 2) . Individa et al.  was reina et al. , based oulations .NAT2*6, because it was the variation with more information accumulated for populations representatives of the three modes of subsistence.Next, we contrasted our data with other results before published for Eurasian and African populations . Between pastoralists and agriculturalists, no significant differences were detected, which means that the clustering of c.590G > A frequencies only showed correspondence with populations that are food producers or food collectors, an observation that otherwise fully meets that reported by Sabbagh et al. [From Fig. h et al. , and theh et al. .Fig. 2AlNAT2 has evolved under a selective factor influenced by human diet.In brief, our analyses reinforce previous indications that CYP3A5, we screened the intronic variation c.219-237G > A, commonly referred to CYP3A5*1/*3 polymorphism, in which the derived allele A results in a premature stop codon that reduces protein expression. It has been firmly demonstrated that the variation possesses a very unusual worldwide distribution whereby the frequency of CYP3A5*3 is significantly correlated with latitude [With regard to latitude .CYP3A5*1/*3 likely influences salt and water retention and risk for salt-sensitive hypertension [CYP3A5*1/*3 could be related with diet of populations.rtension , exertinrtension ,31. Sincrtension , we askeCYP3A5*3 varies quiet narrowly being near-fixation in most populations [P < 0.0001). When the relationship was assessed separately in each of the three continents, no significant rank correlation was observed in Africa  or in Europe , but in Asia the correlation coefficient was again statistically significant . Interestingly, in Asia, where the average allele frequency was 0.793, the significant correlation can be explained since the lowest values are consistently present in populations from the South of the continent, located very near or already inside the intertropical zone. In Africa, the frequency of the allele drastically declines to an average value of 0.286 when inferred from a panel of populations’ majority located inside the tropical zone. In Europe, which is fully situated in a temperate climatic region, the average frequency reaches 0.903. Therefore, being or not located in the tropical zone seems to be a factor that strongly influences the distribution of CYP3A5*1/*3 alleles , disfavoring thus any link between lifestyle and differences in allele frequency across populations. In the Portuguese, the allele reached the very elevated value of 90.2 %, which it is usual in populations from Europe where see Fig. .Fig. 3DiCYP3A5 was the target of a selective factor determined by the geographic location of human populations.These analyses led to conclude that AGXT, PLRP2, MTRR, NAT2 (only for that defining NAT2*6) and CYP3A5, hereinafter referred for simplicity as uniquely by their gene symbols  latitude and ii) the location North and South the Tropic of Cancer, leading to realize that for CYP3A5 the highest value of FCT (which measures the proportion of variance among groups) was achieved when populations North of the Tropic of Cancer were grouped against the southern ones, attaining then 44.9 % of total diversity.Geography was found to significantly account to explain the total genetic variance across Africa and Eurasia at PLRP2, explaining 8.8 % of the total diversity at the locus, while also accounting to residual proportions of diversity at NAT2 (1.6 %) and AGXT (1.5 %). When the criterion to group populations was the content in diets of cereals (for PLPR2), meat (for AGXT), folates (for MTRR and NAT2) or salt (for CYP3A5), significant FCT values were only observed at PLRP2, in which the more or less reliance in cereals contributed to 6.5 % of the total variance, and at NAT2, where differences in the dietary richness in folates explained 3 % of the locus diversity.Concerning mode of subsistence, it was found to be a considerable modulator of diversity at FST-based approach that assumes that genetic differentiation among populations is expectedly higher or lower for loci under directional or balanced selection, respectively, expected under neutrality.To dissect better whether from the levels of genetic differentiation across Africa and Eurasia signs of selection could be captured, we used a conventional FST employing two different models, the finite Island Model (IM), which assumes the classical island model at migration-drift equilibrium [loci for selection [Viewing that, we have firstly generated null sampling distribution of the empirical ilibrium ; and theilibrium . Besideselection .FST values plotted against scaled heterozygosity estimated for the SNPs at AGXT, PLRP2, MTRR, CYP3A5 and NAT2 [The simulated null-distributions are presented in Fig. and NAT2 .Fig. 4JoFSTs for MTRR and AGXT did not differed significantly from the null expectations , noteworthy in Africa the differentiations at PLRP and CYP3A5 were significantly higher than expected under the neutral expectations derived from the two demographical models  surrounding each of the three genes, viewing which a genomic window was considered that encompassed the adjacent genes. In Table D’ and r2 values with our target SNPs, identified in African populations, which were the unique with genome data available. The correspondent LD plots for each gene across different African populations are present in supplementary material , whose functional consequences are unknown, are in strong LD with our target SNP at PLRP2, which in addition was at high LD with rs475199, a non-synonymous substitution of unknown functional effect, also located in PLRP2.In order to assess whether the examined genetic variants were in fact those responsible for the selective signals detected The analysis of patterns of human genetic diversity at wide geographical scales can disclose remarkable features difficultly explained by demographic events or pure neutral processes, that rather might represent the first symptoms of environmental adaptations.AGXT, PLRP2, MTRR, NAT2 and CYP3A5, five genes assumedly involved in the metabolism of substances (including xenobiotics) that gain entry into the organism through dietary food stuffs, for which it has been previously posited that they could represent instances of gene-culture coevolution in humans [In this study, we draw attention to variations in n humans , 15, 20., PLRP2, NAT2 and CYP3A5 were found to present signs in their distribution patterns evoking the action of environmental selective pressures, though of diverse nature and strength.Out of those genesCYP3A5 that displayed a level of inter-population differentiation dramatically surmounting even the most conservative neutral expectations. Contrarily to our starting hypothesis, however, the amount of salt presumed to be ingested across main dietary habits did not accounted for the distribution of CYP3A5, which instead was highly determined by the geographical location of populations in the North or in the South of the Tropic of Cancer. So, the analyses here undertaken fully support previous findings indicating that CYP3A5*3 evolved under a selective pressure determined by an environmental factor correlated with latitude [CYP3A5 has been intensively explored in the context of the genetic factors contributing to hypertension susceptibility, known to vary widely across different human populations. Nearly 40 years ago Gleibermann [CYP3A5 is being often quoted to address the evolutionary perspective of hypertension susceptibility, due to the demonstrated role of CYP3A5 enzymes in sodium homeostasis, even though the many studies that analyzed the relationship between CYP3A5 genotypes and blood pressure/hypertension have provided quite inconsistent results . So, totonicity .PLRP2, the explorations here undertaken led in essence to corroborate the findings of Hancock et al. [PLRP2 to the content in cereals in diets of populations, on the grounds of the important role of the protein encoded by PLRP2 in plant-based diets once, unlike other pancreatic lipases, this enzyme hydrolyzes galactolipids, which are the main triglyceride component in plants [PLRP2, suggesting for instance a likely major influence in fat digestion in newborns [PLRP2 substrate, or amount of substrate, differing in hunter-gatherers, herders and farmers is the factor that exerted the selective pressure contributing to shape the current pattern of PLRP2 diversity in human populations.Concerning k et al. , indicatk et al.  have assn plants . Howevern plants , which snewborns . This rePLRP2 and PLRP1. The two genes encode lipases that show high sequence homology and that assumedly participate in dietary fat metabolism, although not being yet clarified their differences in substrate specificity. Consequently, for the moment is not possible to discriminate between which variations at PLRP2 or PLRP1 are the best candidates to be causative of the selective signals detected.Before, however, it is necessary to overcome the uncertainty raised by the presence of significant LD between NAT2, in agreement with earlier studies [NAT2*6 allele, revealed an unusual low level of geographical structure across Africa and Eurasia, indicating that it was subjected to drifting constrains that likely could arise under the action of a mode of selection resulting in such a homogeneous allele distribution. In the tests for selection involving NAT2, significant departure from the neutral expectation was captured when the simple IM demographic model was assumed, which can raise uncertainty on whether signs of more subtle selective pressures might mistakenly escape the stringency of the HIM. The adaptive evolution of NAT2 has been supported by a number of different studies [NAT2 sequence data whose patterns of diversity made it plausible that slow-acetylating variants have been subject to weak selective pressures [NAT2 acetylator phenotypes and historical dietary habitudes in India with the slow acetylator prevalence being higher in regions where is higher the proportion of vegetarians populations [NAT2 human enzymes in folate metabolism [NAT2 are not known, although being well established that NAT2 catalyzes the acetylation of many xenobiotics [NAT2 deserves further attention.With respect to  studies , 22 we a studies , 41, 42,ressures . Howeverulations . A majortabolism . Endogenobiotics . Since tPLRP2 and NAT2 harbor signatures of genetic adaptations that might have been triggered by the diversification of modes of subsistence and diets that human populations began to experience after the rise of the Neolithic. Before that, modern humans had started to leave their original homeland in Africa, traveling out of the continent to colonize all regions in the globe. They progressively reached a wide range of new environments, facing new selective pressures that may have contributed to shape human genetic diversity. In CYP3A5, the compelling correlation with regions North and South the Tropic of Cancer, makes it likely that it belongs to the yet not fully understood catalog of genetic adaptations triggered by environmental stresses. Furthermore, the genetic signatures that CYP3A5 harbors seem strength enough to have been driven by very long-lasting selection.In summary, we provided added evidence that diversity at AGXT and MTRR could also be diet-selected genes, since their diversity patterns could be well reconciled with demographic history at least of African and Eurasian populations.Finally, we were unable to confirm the hypotheses at stake that PLRP2, NAT2 and CYP3A5, three genes assumedly involved in the metabolism of substances (including xenobiotics) that gain entry into the organism through dietary food stuffs, can represent instances of gene-culture coevolution in humans. Concerning PLRP2, it is still needed to clarify whether the signal detected is not a hitchhiking effect of its neighbor PLRP1. In addition, it is also necessary to demonstrate which were the biological mechanisms, and the environmental factors involved as well as their interactions, to understand the nature of the selective pressures that contributed to shape current patterns of genetic diversity at those loci. Furthermore, functional studies are needed to demonstrate the putative biological impact of the variations assessed, which ultimately would also exclude that the detected signs of selection could be due to other variations in linkage disequilibrium with those that were here examined.In this study, we found signs that http://jama.jamanetwork.com).The current study was approved by the Institute of Molecular Pathology and Immunology of the University of Porto institutional review board. All samples involved were anonymised DNA extracts previously obtained from healthy unrelated individuals. The samples were collected under written informed consent. The current study complies with the ethical principles of the 2000 Helsinki Declaration of the 206 World Medical Association (http://lucy.ukc.ac.uk/cgi-bin/uncgi/Ethnoatlas/atlas.vopts), Encyclopedia of World Cultures, Africa: An Encyclopedia for Students [A total amount of 361 individuals from six sub-Saharan African populations with different modes of subsistence were analyzed, i) 144 were farmers/agriculturalists from three populations: 32 from Angola (ANG), 82 from Equatorial Guinea (EQG) and 30 from Mozambique (MOZ); ii) 116 were herders/pastoralists from the Karamoja region in Northern Uganda (UGN); and iii) 101 belonged to two hunter-gatherer/forager populations: 39 Baka Pygmies from Gabon (BPY) and 62 Ju/hoansi from Tsumkwe, a small settlement in North Eastern Namibia (KNA). Forty-eight individuals from the Portuguese population (PTG) were additionally studied. Each population was assigned to a specific mode of subsistence and main diet component according to Murdock Ethnographic Atlas (Students .From blood samples stored in FTA™ cards (Whatman), total DNA was extracted using a standard phenol-chlorophorm protocol  in the sAGXT gene), c.1074G > A , c.1130A > G , c.219-237G > A  and the following 4 variants in NAT2 gene (NM_000015.2): c.191G > A (rs1801279), c.341 T > C (rs1801280), c.590G > A (rs1799930) and c.857G > A (rs1799931). The last 4 SNPS, defining the alleles NAT2*14, NAT2*5, NAT2*6 and NAT2*7, respectively, were selected in this study because reportedly they allow inferring the acetylator phenotypes with high accuracy [The screened SNPs were c.32C > T .Chromosomal locations and genomic segments from the 5 genes were obtained using the latest version of the human genome assembly GRCh37 (FST). Regarding the NAT2 gene, to account for linkage disequilibrium (LD) between SNPs, from the unphased multilocus genotypic data, haplotypic frequencies defined by the 4 SNPs were computationally estimated also with Arlequin software ver. 3.5.1. [The Arlequin software ver. 3.5.1  was used. 3.5.1.  and the . 3.5.1. .http://vassarstats.net).In order to determine whether means of allele frequencies were statistically different between groups of populations defined according to various criteria, One-Way ANOVA tests were performed. The Spearman Rank Correlation Score Corrected (SRCSC) was used to evaluate the correlation between allele frequencies and latitude. Both statistical analyses were performed on the website for statistical computation VassarStats  procedure implemented in StatSoft, Inc. (2007), Statistica version 8.0 (www.statsoft.com).The graphical representation of a FSTs conditional on heterozygozity based on genotypic data from a validated panel of 52 assumedly neutral SNPs for human identification [forID browser, to draw a neutral dispersion cloud 50,000 coalescent simulations of 100 demes were carried on Arlequin software. For variants at AGXT, PLRP2, MTRR, NAT2 and CYP3A5, FST, average heterozygosity within populations (Het) and the scaled heterozygosity Het/(1-FST) were also computed. The null sampling distribution of the empirical FST values was calculated using two distinct models, both implemented in Arlequin software ver. 3.5.1 [FST strongly correlates with heterozygosity [P-value for each locus was calculated within bins of 2,0000 SNPs grouped according to Minimum Allele Frequency (MAF), as the proportion of the bivariate probability distribution which was less probable than the estimated values, in the same way as calculated in the DFdist software package [To investigate possible signals of selection we obtained the neutral distribution of fication , using tr. 3.5.1 , consider. 3.5.1 , for whizygosity , the emp package .CYP3A5, populations groups were also defined according to latitude and location North or South the Tropic of Cancer.Hierarchical Analysis of Molecular Variance (AMOVA) was performed in the Arlequin software ver. 3.5.1. defining population groups according to: i) mode of subsistence, ii) main diet component, and iii) geography in the context of Eurasia and Africa. Additionally for http://pngu.mgh.harvard.edu/purcell/plink/) [D’  and r2 (squared correlation coefficient measure of LD between the two loci) parameters and visualize the LD plots, considering a window of 200, 300 and 500 Kb for PLRP2, CYP3A5 and NAT2 genes, respectively.Finally for LD patterns analyses, the .ped files containing the data sets were first manipulated with gPLINK software (/plink/)  and then/plink/)  to calcuhttp://genapps.uchicago.edu/software.html) from the Di Rienzo laboratory, which includes information from the International HaPMap Project Phase III (http://hapmap.ncbi.nlm.nih.gov/), the Human Genome Diversity Project Panel-Centre d’Etude du Polymorphisme Humain (HGDP-CEPH); and also we included data already published in other works [Viewing comparative analyses, data for other populations were retrieved from the database dbCLINE (er works -59. The


Varanus niloticus) have been identified in Florida, USA, located in Cape Coral, West Palm Beach and Homestead Air Reserve Base. This large, predatory lizard could have negative effects on Florida's native wildlife. Here, we infer the source of the introduced populations using genetic and statistical approaches, as well as estimate the potential non-native distribution of V. niloticus in North America. We collected genetic data from 25 Florida individuals as well as utilized genetic datasets from reference individuals spanning the full native distribution throughout sub-Saharan Africa. Using occurrence data from the inferred source population and the full species range, we built ecological niche models (ENMs) and projected them onto North America to determine regions with suitable climate. Our results indicated that the introduced populations resulted from three separate introduction events, and all originated from the southern coastal region of West Africa. The ENM built from the West African source population predicted only the southernmost portions of North America to be suitable. Conversely, the model derived from the full species’ range predicted suitable climates across a large portion of the United States. This information can be used to focus management and eradication efforts.Three separate breeding populations of the Nile monitor ( WhileVaranus niloticus, was added to Florida's long list of introduced species in 1990, when individuals were first observed in the Cape Coral region [V. niloticus sightings in West Palm Beach and Homestead, respectively, compared with 389 public sightings [V. niloticus is largely dependent on permanent bodies of water throughout its native distribution [V. niloticus represents a severe threat to Florida wildlife, able to prey upon a variety of species and quickly outgrowing potential predators [V. niloticus, including early sexual maturation (around 2 years of age), high fecundity (laying up to 60 eggs in a single clutch) and high motility (occupying home ranges up to 50 km2), are indicative of a high invasive potential [The Nile monitor lizard, l region . Currentl region ,16. The l region , whereasightings ,18. Owinribution ,18, and ribution . This spribution . Becauseredators . Other lotential ,19.FiguV. niloticus populations has not been confirmed; however, the pet trade likely played a dominant role. Approximately 84% of the introduced amphibian and reptile species in Florida have resulted from the pet industry [V. niloticus introduction in Florida. One theory suggests that individual pets escaped or were released by owners when the animals became too difficult to manage [V. niloticus individuals were released by reptile dealers in order to establish a breeding population in which to capture individuals for resale, thus avoiding the costs of husbandry or international trade [The introduction history of the industry . Nile moindustry . A few to manage ,15. The al trade ,15.V. niloticus populations throughout Africa identified three geographically structured lineages [V. niloticus, understanding the origin of the introduced populations is of utmost importance in predicting their spread.A recent genetic analysis of native lineages . This stlineages . The othlineages . Restriclineages –25. Evidlineages –28 and alineages ,29,30. BGenetic methods can be used to understand the source of introduced organisms and to estimate the number of introduction events. These approaches rely on first characterizing the genetic variation across the species’ native range. One method uses DNA sequence data to establish the genealogical relationships among the introduced individuals and reference individuals from known localities throughout the native range . In thisV. niloticus population in Cape Coral is expanding into new regions [V. niloticus populations using both genealogical methods and frequency-based approaches. Furthermore, we estimate the number of independent introduction events that have led to the current V. niloticus populations in Florida. Because the hypothesis of local adaptation among the V. niloticus lineages has not been tested, we predict suitable climates in North America under both assumptions: (i) local adaptation among populations and (ii) constant fitness levels across the full range of the species.Evidence suggests that the  regions , and the2.2.1.V. niloticus populations, consisting of 15 individuals from Cape Coral, 5 from West Palm Beach and 5 from Homestead. Individuals were collected from Cape Coral by trapping [ND1 (457 bp), ND2 (528 bp) and ND4 (816 bp); as well as three nuclear loci, RAG-1 (781 bp), KIAA1217 (595 bp) and KIAA1549 (781 bp), totaling 4251 bp. PCR amplification was carried out in 21 µl reactions and conditions followed those of Dowell et al. [KT894381–KT894551.We obtained a total of 25 tissue samples from the introduced Varanus komodoensis—K7, K10, K11, K15, K22 and K23 [V. salvator—VARSA10 and VARSA07 [V. acanthurus—VA17, VA38 and VA74 [et al. [We amplified 11 microsatellite loci originally developed for  and K23 ,37; V. s VARSA07 ; and V. and VA74 , followi [et al. . We carret al. [V. niloticus originating from throughout their native distribution . These reference samples from known localities across Africa included both contemporary samples and historic museum specimens [JN673347, JN673350–JN673352, JN673363–JN673365 and KT720497–KT721289). The reference microsatellite dataset was composed of all 11 loci for contemporary samples (N = 67) and five loci for museum samples (N = 42), including K7, K11, VARSA10, VA38 and VA74 . This discrepancy in the number of loci was due to a lower amplification success for museum specimens [We additionally used the recent dataset of Dowell et al. , totallipecimens . The DNA2.2.DNA sequences from the introduced populations were analysed together with sequences from reference individuals across the species’ native range in Africa. We aligned all DNA sequences using Clustal-W  implemenV. niloticus individuals, DNA sequence data from reference and Florida individuals were analysed using maximum-likelihood (ML) with RAxML 8.1.15 [Γ distribution and four discrete rate categories [a posteriori bootstrap convergence tests with the frequency-based and majority-rule consensus criteria, as implemented in RAxML [To infer the source lineage(s) of the introduced L 8.1.15 . We analL 8.1.15  with rattegories . We impltegories . To confin RAxML .To examine the level of connectivity among introduced and reference haplotypes, indicative of the source population as well as the number of introduction events, we constructed a minimum-spanning haplotype network in TCS 1.21  using th2.3.V. niloticus populations by conducting assignment tests with the microsatellite data. We assigned introduced and reference individuals to populations with GeneClass 2.0 [et al. [α level of 0.01.We further examined the origin of the introduced lass 2.0  using thlass 2.0  which halass 2.0 . Probabi [et al.  algorithV. niloticus individuals with the continuous assignment method (CAM) implemented in SCAT 2.0.1 [V. niloticus across Africa . The median coordinates across all runs were then plotted in ArcMap 10.1 [We also estimated the geographical origin of the Florida AT 2.0.1 . This prAT 2.0.1 . This isAT 2.0.1 . Ten repMap 10.1 . ConfideMap 10.1 .V. niloticus [We assessed the reliability of the CAM estimates by analysing 30 reference individuals from across the full distribution, 10 from each of the three known genetic lineages within iloticus . Runs weLastly, to visualize the relationships among reference and introduced individuals, we conducted a principal component analysis (PCA) on raw microsatellite genotypes using the gstudio package  in R 3.12.4.V. niloticus species across sub-Saharan Africa. Occurrence points for the source population were limited to reference individuals belonging to the same genetic subclade as the Florida samples. For both ENMs, reference individuals lacking specific locality data were removed from the analysis. In total, nine reference individuals from the African source population and 71 from the full native V. niloticus distribution exhibited unique and reliable GPS coordinates and were used in subsequent ENMs .We generated ecological niche models (ENMs) for the inferred source population as well as for the full V. niloticus source population and full species using the elevation and environmental data from the WorldClim database [We modelled the climatic niche of the database  at 2.5 adatabase . The varWe generated ENMs using the machine-learning maximum entropy software Maxent 3.3.3 . This apThe final models were evaluated using a fivefold cross-validation method, with AUC scores calculated for each fold along with the omission rate of the test databased on binary predictions with the minimum training presence threshold . We calcV. niloticus under future climate projections, we used two global climate models, the Hadley Center for Climate Prediction and Research's model, HadGEM2-ES [2) and the most extreme category (RCP 8.5) to examine a range of potential outcomes.To infer the potentially inhabitable regions for dGEM2-ES , and thedGEM2-ES , projectdGEM2-ES . We sele3.3.1.a; electronic supplementary material, S4) with high bootstrap support. All introduced V. niloticus individuals were assigned to subclade 1a, containing reference individuals from a region of coastal West Africa, located from Liberia to Cameroon (figure 2c).Our ML analysis, constructed with a concatenated mitochondrial and nuclear dataset, produced geographically structured groups across reference individuals (figure 2b). Each Florida population was represented by a different, fixed mitochondrial haplotype. Introduced individuals from the West Palm Beach population directly shared a haplotype with reference individuals originating from Benin (N = 1), Burkina Faso (N = 1), Ghana (N = 2) and Togo (N = 1). There were two mutational differences between the West Palm Beach and Homestead haplotypes, while the Cape Coral and West Palm Beach haplotypes differed from each other by a single base pair.The haplotype network, created with the mitochondrial dataset (2094 bp), also assigned all introduced individuals to the 1a subnetwork, indicating that all Florida individuals originated from the southern coastal region of West Africa b. Each F3.2.The assignment test results were consistent with those obtained from the DNA sequence analyses. All introduced individuals were assigned to the reference population located in southern coastal West Africa, denoted 1a, with probabilities exceeding 0.945 . All othThe CAM analysis implemented in SCAT showed varying levels of accuracy and confidence in assigning reference individuals to their locality of origin. Overall, median locality estimates were most accurate for reference individuals in the western clade (1a and 1b), assigning the geographical origin to within an average of 520 km from the actual origin (range 69–1082 km). Reference individuals from the northern (2a and 2b) and southern (3a and 3b) clades produced lower levels of accuracy, with straight-line distances between estimated and actual localities averaging 845 km (range 119–2318 km) and 984 km (range 236–1974 km), respectively. Similarly, confidence in the estimated origin was generally higher for the western clade, which exhibited tighter geographical clustering of point coordinates . These estimates from reference individuals provide a general idea of the level and accuracy and precision we can expect in identifying the origin of the introduced individuals with the CAM.V. niloticus individuals were located within West Africa  and 0.5 for the remaining fold (p = 0.148). For the full V. niloticus distribution (71 occurrence points), the ENM produced and average test AUC of 0.770 (0.683–0.830), and an average omission error rate of 0.029 (p = 0.007–0.084). Projecting the source population ENM onto the African continent showed that coastal West Africa was predicted to be highly suitable (greater than 0.2 based on the minimum training presence threshold) and produced a more restricted range than the full V. niloticus ENM  produced an average test AUC of 0.924 (0.824–0.97), and an average omission error rate from the binomial test of 0 for all but onefold  showing smaller predicted ranges . For these future projections, areas predicted to be suitable for the West African source population extended farther north than under current climatic conditions; however, for the United States, the potentially inhabitable areas were confined to Florida, as well as small portions of Texas and California . For thed ranges .Figure 4.4.1.V. niloticus populations to be the lineage inhabiting coastal West Africa. Reference individuals within this subclade range from Liberia to Cameroon, with the northernmost occurrence in Burkina Faso. The DNA sequence data produced a greater degree of genetic partitioning among reference populations and were able to assign Florida individuals more precisely than the microsatellite data. Introduced V. niloticus individuals were recovered within the coastal West African subclade, visualized in the ML tree and haplotype network , Ghana  and Niger , these numbers pale in comparison with imports from Togo and Benin [V. niloticus populations. Furthermore, populations of V. niloticus in West Africa are also intensely harvested for the leather industry, with the top exporting country, Mali, reporting an annual average of approximately 47 000 skins (http://trade.cites.org). While V. niloticus is managed as a single taxonomic unit by the Convention on International Trade in Endangered Species  and considered a low conservation concern due to its widespread distribution [et al. [V. niloticus populations [V. niloticus lineage in West Africa could represent a greater conservation concern than previously thought.These results are consistent with import and export data of live annually . Althougnd Benin . This prribution , Dowell  [et al.  identifiulations . TherefoV. niloticus populations inhabiting Cape Coral, West Palm Beach and Homestead resulted from three separate introduction events. This inference was based on differing mitochondrial haplotypes among each of the introduced populations. We found that each Florida population shared a single mitochondrial haplotype, indicative of low genetic diversity; however, independent introductions and subsequent admixture could increase the genetic variation, and possibly the invasive potential, of introduced species [V. niloticus sightings in other parts of Florida [Our results suggest that the introduced  species ,72. Thes Florida , call fo4.2.V. niloticus populations in the United States were confined to southern Florida, even in future climate projections. These potentially inhabitable regions represent the most specific outcome and are based on the assumption that the West African lineage exhibits local adaptation. Under this assumption, individuals belonging to the western lineage, including the Florida individuals, would have a fitness advantage in environmental conditions most similar to those in West Africa, and reduced fitness levels in other environments. Because reduced gene flow among populations in heterogeneous environments can facilitate local adaptation [V. niloticus lineages. In this outcome, all three introduced populations reside in regions predicted to be suitable under current climate conditions; however, the Cape Coral population is near the northernmost extent of the predicted distribution. Although confirmed sightings of V. niloticus have occurred outside of this predicted range [Based on the climatic conditions of the West African source population, the regions predicted to have suitable climates for the introduced aptation ,23, thised range , there ied range , possiblV. niloticus genotypes in varying environmental conditions, it is necessary to examine alternative outcomes. The contrasting scenario shows the potentially inhabitable regions based on climate data throughout the full native V. niloticus range spanning sub-Saharan Africa. This prediction is based on the assumption that members of the western lineage, as well as those in Florida, do not exhibit local adaptation, but instead show similar fitness levels in all environmental conditions throughout the full native range of the species. Under this assumption, introduced V. niloticus individuals would be able to tolerate climatic conditions throughout a large expanse of the United States and Mexico, as well as Cuba and the Caribbean islands. Because some populations of V. niloticus are known to hibernate in dry, cool periods [V. niloticus could potentially lead to additional populations in regions other than Florida. It is important to note, however, that both models were created based solely on climate and elevation data. Therefore, more specific habitat conditions, including permanent bodies of water and appropriate substrate for egg laying [V. niloticus is able to become established in a particular region. Future studies assessing the degree of local adaptation among native V. niloticus lineages could help distinguish between these two contrasting scenarios.Without information regarding the relative fitness of  periods ,73, intrg laying , could aV. niloticus populations can aid in focusing management strategies and eradication efforts. Even under the most conservative scenario, our analyses showed that the southern regions of Florida are highly suitable for V. niloticus. Therefore, the immediate management focus should target the southernmost regions, and wildlife officials should act quickly when new V. niloticus individuals or populations are observed in these areas. Because V. niloticus individuals occupy large home ranges and are capable of dispersing large distances [V. niloticus may have on native wildlife species in these regions, including ground-nesting birds, aquatic turtles and other vulnerable species, such as the American crocodile and the gopher tortoise [V. niloticus imports [V. niloticus populations.Understanding the geographical origin of the introduced istances , sensititortoise . Althoug imports , other s5.V. niloticus across Africa, we determined the source of all three introduced populations to be the cryptic West African evolutionary lineage. Furthermore, by identifying the geographical origin of the introduced V. niloticus populations, we have established a baseline from which future studies can expand upon. With this information, subsequent investigations can make direct comparisons between introduced and native populations, comparing diet, morphology, gene expression, etc. to examine if and how V. niloticus is adapting to the non-native habitat.Our study illustrates the importance of developing range-wide genetic assessments and their utility in conservation. This library of genetic variation across a species' distribution can serve as a database to source unknown individuals, with applications to invasion biology ,71,74 as


Hynobius retardatus) is endemic to Hokkaido Island, Japan, and shows intriguing flexible phenotypic plasticity and regional morphological diversity. However, to date, allozymes and partial mitochondria DNA sequences have provided only an outline of its demographic histories and the pattern of its genetic diversification. To understand the finer details of the population structure of this species and its evolution since speciation, we genotyped five regional populations by using 12 recently developed microsatellite polymorphic markers. We found a clear population structure with low gene flow among the five populations, but a close genetic relationship between the Teshio and Kitami populations. Our demographic analysis suggested that Teshio and Erimo had the largest effective population sizes among the five populations. These findings regarding the population structure and demography of H. retardatus improve our understanding of the faunal phylogeography on Hokkaido Island and also provide fundamental genetic information that will be useful for future studies.The Hokkaido salamander ( One of the major goals of molecular ecology is to understand the current genetic structure and pattern of genetic diversity of organisms. Moreover, these data are useful for conservation management, because genetic diversity is an essential factor in morphological and behavioral phenotypic variation. Salamanders have been used as model species for studies of genetic structure and diversity  because Hynobius is monophyletic in East Asia. Most salamander species belonging to this genus are endemic to the Japanese archipelago, and phylogenetic and phylogeographic analyses of local populations of several Hynobius species, including H. boulengeri )hipelago . The HokH. retardatus is about 18–14 Ma [The estimated divergence time of 18–14 Ma , but the18–14 Ma . There a18–14 Ma . The conWe clarified the population structure of the Hokkaido salamander and gene flow among populations. Populations of this species show distinct genetic differences and generally low gene flow. Thus, each population has different genetic features and evolved independently. These findings are applicable to the conservation management of this species. Because the populations are genetically clearly different, it is important to protect all of the local populations. In the future, a more detailed population structure and the evolutionary history of this species should be examined by using more comprehensive data, such as genome-wide polymorphic markers. The causal relationships between genetic background and the phenotypic differences among populations also need to be resolved.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Aedes japonicus japonicus, a potential vector of several arboviruses, has become one of the most invasive mosquito species in the world. After having established in the USA, it is now spreading in Europe, with new populations emerging. In contrast to the USA, the introduction pathways and modes of dispersal in Europe are largely obscure.Originally native to East Asia, Ae. j. japonicus in The Netherlands and northern Germany go back to new importations or to movements within Europe, the genetic makeup of mosquito specimens from all known European populations was compared. For this purpose, seven microsatellite loci from a representative number of mosquito specimens were genotyped and part of their mitochondrial nad4 gene sequenced.To find out if two recently detected populations of nad4 haplotype found in the newly discovered Dutch population of Ae. j. japonicus suggests that this population is not closely related to the other European populations but has emanated from a further introduction event. With five nad4 haplotypes, the Dutch population also shows a very high genetic diversity indicating that either the founder population was very large or multiple introductions took place. By contrast, the recently detected North German population could be clearly assigned to one of the two previously determined European Ae. j. japonicus microsatellite genotypes and shows nad4 haplotypes that are known from West Germany.A novel Ae. j. japonicus are geographically separated but genetically mixed, their establishment must be attributed to passive transportation. In addition to intercontinental shipment, it can be assumed that human activities are also responsible for medium- and short-distance overland spread. A better understanding of the processes underlying the introduction and spread of this invasive species will help to increase public awareness of the human-mediated displacement of mosquitoes and to find strategies to avoid it.As the European populations of  Aedes (Finlaya) japonicus japonicus   indicated a much greater distribution area at that time. In the same year, a population was found widely distributed on both sides of the Austrian-Slovenian border [Also in 2008, larvae of n border . A monitn border . Anothern border . Severaln border . By 2013Ae. j. japonicus seems to have been spreading continuously across Germany. Populations have been shown to exist in the federal states of North Rhine-Westphalia and Rhineland-Palatinate [Ae. j. japonicus female already trapped in July 2012 during routine monitoring in the municipality of Lelystad (province of Flevoland). Extensive surveillance from April to October 2013 in the surroundings of the first finding identified numerous breeding sites over large parts of the municipality [Since its first detection in Baden-Wurttemberg in 2008, latinate  and, morlatinate . In Janucipality . The firAe. j. japonicus populations detected in Europe, only the Belgian one is known to be due to an importation in used tyres. For the other five populations there is no information on their mode of introduction nor on their origin or relatedness.Of the six Aedes j. japonicus is a potential vector of several arboviruses including West Nile virus (WNV) and Japanese encephalitis virus [is virus ,20. In tis virus . In addiis virus -24 and iis virus . Its sprAe. j. japonicus and, in particular, (ii) to assign the newly discovered populations in Lelystad, The Netherlands, and in Lower Saxony, Germany, to already known genotypes, in order to detect genetic proofs for introduction or migration events. For this purpose we analysed highly polymorphic simple sequence repeats (microsatellites) and maternally inherited, rapidly evolving mitochondrial nad4 sequences which are characterised by variable numbers of repeats or nucleotide sequences, making them appropriate targets for population genetics and the identification of source populations [The objective of the present study was (i) to learn more about the relationships between the various European populations of ulations ,27.Mosquito larvae and eggs were collected between May and October 2013 from flower vases and other small artificial water containers and from ovitraps in cemeteries and gardens .Individuals central to the study were collected from two sites, about 60 km apart, in the North German federal state of Lower Saxony (NG) and from six sites within the municipality of Lelystad, The Netherlands. In addition, specimens from two towns in the South German federal state of Baden-Wurttemberg (SG) and from 18 sites in Slovenia as well as two individuals from two close collection sites in Croatia were examined  according to the manufacturer’s instructions. In the case of the Croatian individuals, DNA was extracted from single legs using the same kit. DNA was eluted in 80 μl EB buffer (Qiagen) and kept frozen until use.PCR amplification was performed in a C1000™ 96 well thermal cycler (BioRad). The thermoprofile consisted of a 3 min denaturation step at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 56°C and 30 s at 72°C, and a final 10 min elongation step at 72°C. For each of the seven targeted microsatellite loci  one pair of primers was used as previously described . Only thBecause frequency-based microsatellite analysis requires a minimum population size that was not given for every sampling site in Slovenia, individuals from there were assigned to five groups according to the geographic regions where they were sampled.nad4) gene of the mitochondrial DNA of the sampled specimens was sequenced using a modification of the protocol of Fonseca et al. [Anopheles gambiae genomic sequence (GenBank accession no. L20934). DNA amplification was preceded by a 10 min denaturation step at 96°C and consisted of 35 cycles of 40 s at 94°C, 40 s at 56°C and 60 s at 72°C. A final extension step of 7 min at 72°C was added. PCR products were checked by electrophoresis on a 1.5% agarose gel run for one hour and visualised by ethidium-bromide staining. DNA bands were excised and recovered with the QIAamp Gel Extraction Kit (Qiagen). Afterwards, they were cycle-sequenced in both directions with the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies). PCR products were cleaned with SigmaSpin Sequencing Reaction Clean-Up Columns (Sigma-Aldrich) before being run on a 3130xl Genetic Analyzer. FASTA files of the obtained sequences were aligned with MultAlin [Additionally, part of the sodium dehydrogenase subunit 4  [Nei’s genetic distance and pairwise population s (PCoA) . Furthers (PCoA) . Shannons (PCoA) ,40. The Ae. j. japonicus specimens from the Dutch, German and Slovenian/Croatian populations were analysed. In some cases, previously obtained data from West German, Swiss and Belgian samples [Microsatellites of a total of 215  samples  were incThe obtained microsatellite signatures enabled the examined specimens to be assigned to one of two genotypes described for Germany Figure . The NG A Hardy-Weinberg equilibrium check showed significant deviations at most microsatellite loci of specimens from the two SG sites, Korntal and Waldshut-Tiengen. A few more departures from the Hardy-Weinberg equilibrium, mostly at loci OJ5, OJ10 and OJ187, were found across all populations. A closer inspection of these did not produce considerable effects of null alleles . Deviatinad4 gene sequence provided analysable results. Nine different nad4 haplotypes were obtained: H1, H3, H5, H9, H12, H21, H33, and two haplotypes that have not previously been described , we suggest naming the newly found haplotypes H44 and H45. Haplotype sequences are available in [nad4 haplotype, and their microsatellite signatures were different too. The Croatian mosquitoes could not be assigned to a defined haplotype as both specimens were characterised by mitochondrial heteroplasmy, the coexistence of multiple mitochondrial haplotypes in a single organism [Ae. j. japonicus.Of the 215 genotyped individuals, 154 examined for their lable in  and GenBorganism  which isFST values  are apparently more closely related to the Swiss samples of the same geographic population than to the Korntal (SG) samples, as only the first two of them show haplotype H33. This haplotype has not been reported from the USA, and in Europe has so far only been found in Swiss specimens . This suAe. j. japonicus in Europe. The Dutch population shows an admixed microsatellite signature as displayed by Bayesian cluster analysis. Its genetic makeup looks like a mixture between Slovenian/SG and WG/NG individuals, which is supported by PCoA, suggesting that at least two introductions of mosquitoes into The Netherlands have taken place. However, the fact that the Dutch population shows a relatively large number of five nad4 haplotypes including one novel haplotype suggests a different conclusion: a large number of individuals with high genetic diversity could have been introduced recently from overseas. The genetic diversity may in this case be a measure of the time that has passed since the introduction. With time passing, founder populations normally experience a loss of genetic diversity, known as the founder effect [Ae. j. japonicus population. Haplotypes H9 and H12, which account for 42% of the Dutch individuals, are the only haplotypes described by Fonseca et al. [To date, Lelystad in The Netherlands is considered to be the northernmost location infested by r effect . If presa et al.  for popunad4 haplotype H9. Additionally, some Slovenian individuals displayed haplotype H5, which is widely distributed in the NG and WG populations, indicating a possible link between these two and the Slovenian population.Of the 18 Slovenian sampling localities examined, seven were located close to the Austrian border, eight close to the Croatian border and three in the centre of the country  of the identified blood sources were human [Ae. j. japonicus has not attracted attention as an important vector in its native distribution area in East Asia, it was susceptible to several arboviruses in the laboratory, including WNV. A new study claims that German Ae. j. japonicus are refractory to WNV [Regarding intercontinental trade and travel as well as the transport of mosquitoes and pathogens between countries, it is to be expected that further populations of re human . Whilst y to WNV . Howevery to WNV ,47 or otAe. j. japonicus from Europe is no longer possible, but efforts should be made to eliminate sources of introduction and to prevent the present populations from spreading further.There is consent that the eradication of