Saccorhizapolyschides, a large brownseaweed that is the main structural species of kelp forests in SW Europe.Signatures of recent bottleneck events were also evaluated to determine whetherthe recently recorded distributional shifts had a negative influence oneffective population size. Our findings show decreasing population density andincreasing spatial fragmentation and local extinctions towards the southernedge. Genetic data revealed two well supported groups with a central contactzone. As predicted, higher differentiation and signs of bottlenecks were foundat the southern edge region. However, a decrease in genetic diversity associatedwith this pattern was not verified. Surprisingly, genetic diversity increasedtowards the edge despite bottlenecks and much lower densities, suggesting thatextinctions and recolonizations have not strongly reduced diversity or thatdiversity might have been even higher there in the past, a process of shiftinggenetic baselines.The genetic consequences of living on the edge of distributional ranges have beenthe subject of a largely unresolved debate. Populations occurring alongpersistent low latitude ranges (rear-edge) are expected to retain high andunique genetic diversity. In contrast, currently less favourable environmentalconditions limiting population size at such range-edges may have caused geneticerosion that prevails over past historical effects, with potential consequenceson reducing future adaptive capacity. The present study provides an empiricaltest of whether population declines towards a peripheral range might bereflected on decreasing diversity and increasing population isolation anddifferentiation. We compare population genetic differentiation and diversitywith trends in abundance along a latitudinal gradient towards the peripheraldistribution range of  Understanding the processes shaping genetic diversity of range-edge populations is animportant current challenge, particularly where rich former glacial refugiapopulations with high conservation value have become isolated in decreasing suitablehabitat islands . AccordiThe genetic diversity of a population reflects both current and past events. Theprediction of lower genetic diversity as a response to reductions in effectivepopulation size and gene flow towards edges ,8 assumeThe relationship between the geographic distributions of abundance and geneticdiversity now appear more complex and interesting than previously assumed. Thiscomplexity strongly alters simplistic biogeographic predictions about populationdynamics , geneticSaccorhizapolyschides, which is the main canopyspecies forming kelp forests in this region. This species sharply declines frombeing a highly abundant dominant species in the north to being rare near itsdistributional limit in the south. Such a spatially unidimensional model haspreviously proven effective in testing phylogeographic hypotheses in marine species[An interesting model to study the genetic implications of distributional ranges froman abundant to a peripheral region is the Portuguese coast along western Iberia.This is a region with a biogeographical interface where a wide range of marinespecies show latitudinal clines in abundance, along a narrow strip of shorelinehabitat essentially in one dimension, from North to South ,26. One  species,27.Spolyschides.. This was achieved byquantifying the latitudinal gradient in population density towards the southern edgeof distribution, and assessing whether it was related to decreasing geneticdiversity and increasing differentiation. We used this information to test whetherpopulations closer to the range boundary show no change in (1) relative densities,(2) fragmentation (3), genetic differentiation (4), genetic diversity and (5)signatures of recent bottleneck events (i.e. population turnover)This study addresses the genetic consequences of a sharp decline in abundance at thedistributional margin of No specific permits were required for the sampling as the sites were notprivately-owned or protected in any way, and the field studies did not involveendangered or protected species.Spolyschides. is an importantecosystem-building species in European waters [The annual kelp n waters . This shn waters .Spolyschides. covered the entirewest coast of Portugal, including searches in areas beyond the current southernlimit of the species in mainland. This region, which happens to coincide withthe boundaries of a political country, is an excellent model coastline to studythe genetic implications of distributional ranges from an abundant to aperipheral region for 3 main reasons: 1) Gradual abundance gradient: Itcoincides perfectly with a sharp linear gradient from abundant continuouspopulations in the North, to small patchy fragmented populations in thecenter-southwest, to complete absence of the species along the southern coast.2) Availability of long-term historical records of the species occurrence showing recent range shifts along thiscoast. 3) Coastal southern limit: The southwest of Portugal is the southernrange edge of the coastal distribution of S. polyschides. Beyond this regionthere are only 2 areas that support S. polyschides. Both are separated byhundreds to thousands of km in opposite directions , are not part of the coastal distribution and thus are not useful toaddress the question of this paper. These two isolated areas are a) the strongupwelling points of Alboran (East) and Morocco (South), and b) the very deepoffshore banks (e.g. Gorringe and Messina) where oceanic waters are sotransparent that they allow the species to occur at depth ranges of about 40-80m, much beyond the coastal depth ranges. Species distributions are not alwayslinear with latitude and pockets or islands can occur beyond the limits of thelinear latitudinal distribution, due to particular unique combinations ofhabitat conditions [Sampling sites for nditions ,30.Along this sampled area populations recruit in spring and reach their highestabundance during summer. Adult individuals (sporophytes) die in the autumn andare absent during the winter, starting to recruit again in spring .° N to 37,0° N, and for better resolution inNorth–South comparisons, the sampling effort was intensified at the 3northernmost and 3 southernmost cells by dividing the 25 km cells into 5sub-cells of 5 km  were placed along three 20 m long transects haphazardly laid inan extant kelp forest, at 5, 10, 15 and 20 m . In eachquadrat all Spolyschides. individuals werecounted. For genetic analyses, 30 individuals were sampled along transects byremoving a piece of the blade above the meristem. These were preserved in silicadrying crystals until DNA extraction.All sampling was conducted at comparable depths, shallower than 8 m, in order toavoid confounding latitude with the effects of depth, since the abundance ofth depth. SpeciesSpolyschides., the presence andabsence records were plotted for both sampling years, together with an extensivelist of historical geo-referenced occurrences gathered from literature (datasetand references can be obtained from the authors upon request). Furthermore, meandensity of Spolyschides. per site was calculated with the quadratcounts for 2010 samples. To infer the fragmentation level of kelp per site, thecoefficient of variation among all quadrat counts was determined as a measure ofdispersion that represents within site landscape spatial heterogeneity [Spolyschides. was less dense ormore heterogeneous at sites towards the distributional edge, linear regressionmodels were fitted between latitude and density, and between latitude and thecoefficient of variation of density. Predictors were transformed if needed(log), homogeneity of variances and normality of models were assessed bygraphical inspection of the residuals versus fitted values [0: theresiduals were normally distributed.To evaluate inter-annual variability in the distribution and abundance ofogeneity  independd values  and by pd values  with H0:2, 3.0 µl of5x PCR Buffer and 0.4 U of GoTaq Polymerase . Cyclingconditions consisted of an initial denaturing step of 5 min at 95°C,followed by 35 cycles of 30 s at 95°C, 30 s at annealing temperature,45 s at 72°C, and a final elongation step at 72°C for 20minutes. All PCR reactions were performed on a GeneAmp 9700 thermocycler . Fragment length was analyzedon an ABI PRISM 3130xl DNA analyzer (Applied Biosystems) using the GeneScan 500LIZ standard.Genomic DNA was isolated from 5 to 10 mg of dried tissue using a CTAB method andFilter Plates  as described in . A totalRaw allele sizes were scored using the software STRand  and binnE), were determined per locus and per site for allloci, using FSTAT. To test whether genetic diversity decreased towards the edge,a linear regression model was fitted between latitude and genetic diversity persite (A and HE). Homogeneity and normality of both models wasassessed. Allelic richness was also computed for each genetic cluster (seebelow), standardised to the number of individuals and coastal distance range ofthe smallest cluster, using StandArich [Genetic diversity, as allelic richness (A) and Nei’s gene diversity [5 bootstrap resamplings.The number of distinct genetic clusters (K) present in the studied region wasinferred by running software Structure  with a b Pr(X/K) for each Pr(X/K). For the Pr(X/K). The pat Pr(X/K). Moreove Pr(X/K) genetic  Pr(X/K) with 1x1FST estimator computed over loci, and withingenetic groups using both FST and Jost’s D [Levels of differentiation between sites were inferred using theJost’s D . HierarcJost’s D , based oJost’s D . VariancFST ⁄ (1-FST)) between sites, against pairwise minimummarine distances. Marine distances were computed with package gdistance for R[5 permutationsas implemented in Genodive.Isolation by distance (IBD) was evaluated within groups, using pairwise estimatesof mean genetic distance  heterozygosity excess  and 2)  2) M-ratPopulations that have experienced a recent bottleneck are predicted totemporarily lose allelic diversity at a significantly faster rate thanheterozygosity . This exNeµ, three parameters known to influence the Mc results[Spolyschides.  sampled sites, and tominimise type I errors, the Mc value for each site was calculated with the meansize of non-stepwise mutations = 3.5 and a highly conservative Theta = 10 (whichassumes larger Ne and lower µ). The proportion of mutations wasset to 0.9 as recommended by Garza & Williamson [The M-ratio test was performed with the software M_P_VAL . This me results. Since tlliamson . ObserveSpolyschides.  were performed on 48visited cells . Atotal of 96 alleles were obtained from 676 genotyped individuals, ranging from 7to 20 alleles per locus , and on a single site from 23to 41 alleles . SignificantFIS values were obtained, particularly insouthern sites . No linkage disequilibrium wasdetected between all pairs of loci . Below the dense northern kelp forests, two sharp declines in densitywere observed along the coast, the first below latitude 41° N  followed by an even sparser region inthe south , below latitude38° N. The among site variation in kelp density, quantified bythe coefficient of variation of the densities, was lowest in the north,increasing significantly towards the south .The density of kelp per sampling site varied between ca. 2 and 26individuals·mThe Structure analyses, based on both the Evanno  and the The genetic differentiation illustrated by the FCA and by the NJ network alsorevealed differentiation of 3 well supported clusters correspoFST and Jost’s D levels ofdifferentiation were higher between the southern sites than between the centralor northern sites . A considerable number of marine species also exhibitlatitudinal abundance declines along this coast towards their distributionallimits . SoutherSpolyschides. tends to be lessdense and more isolated towards the South, sharply increasing genetic distancesand consequently IBD in this marginal zone. Such low densities may also increasethe variation in mating success, which in turn explains the higher inbreedingvalues of the southern sites. Low sporophyte densities might be reflected invariable and patchy gametophyte densities, decreasing effective population sizeas only the spores that happen to settle in close proximity to others  will form gametophytes close enough to achieve reproductivesuccess [Significant and strong isolation by distance (IBD) was only present at thesouthern edge region, a likely consequence of habitat fragmentation as seen inother studies ,63,64. Msuccess ,67.Along the studied range of western Iberia, our results reveal two major geneticgroups, North and South, with an admixture region in the Centre. Their highgenetic diversity and high number of unique alleles indicate that both regionsrepresent populations that have been large, stable and persistent for longenough to accumulate unique mutations and maintain allelic diversity. They mightthus represent genetic groups that were separated at distinct glacial refugia, arole that is also supported by their degree of differentiation from otherpopulations from central Europe , similarly toother marine species for which the Iberian Peninsula was a glacial refugium,5,6,68. Macrocystispyriferain southern California [Laminaria digitata in the English Channel [Spolyschides. , with high dispersalpotential in areas with strong unidirectional currents [The geographical areas between the northern, central and southern forests weresampled and the absence of kelp forests reflected the paucity of suitable rockyhabitat. Rocky reefs occur throughout the sampling region, but extensive sandyareas separate these kelp groups  able topersist over unfavourable periods. These could maintain genetic diversity incryptic stages despite apparent temporary local extinctions and bottlenecks.Experiments on other kelp species, demonstrated that microscopic gametophytescan be maintained in culture for over 7 years  and thatdigitata . FurtherSpolyschides.  exist on underwatermounts (at ca. 40-80 m depths), these are located a few hundred km offshore ofthe southern distributional edge , andLocal bottlenecks could also be rescued by connectivity from just the fewneighbouring remaining patches in the area. Yet, once more, the higher levels ofdifferentiation found between these patches do not support the idea thatmigration from local remaining sites would be a frequent process. Still, thishypothesis cannot be ruled out since it’s difficult to survey the bottom of theocean fully and extant populations might occur in areas that we are not awareof. In such a scenario, founder effects could lead to rapid differentiation ofpatches putatively recolonized by very few occasional migrants from otherpatches. Yet, this would have been associated with a strong reduction indiversity in such recolonized patches, which is not supported by the presence ofmany alleles and of alleles that are absent in their neighbours .A last, but not the least likely hypothesis, is that of shifting geneticbaselines, whereby information about the past is lost with increasingextinctions, a problem already reported for other species along this coastline,6. The hExtinction of genetic variants is likely to happen frequently without it havingbeen recorded to have ever existed before. This problem calls for studies of thepotentially rich and unique genetic diversity that might still exist at pocketrange edges. Rear edges below postglacial expansion zones are likely frequentalong northern Atlantic shores, and in cases of expansion from introgressedgenomes at contact zones, the rear edges may even represent the only survivingpopulations with the native genomes for the species, as has been reported forother brown algae ,94. MargSpolyschides.  in particular, in acontext of future climate change where bottleneck events may prevail as a resultof increasing environmental pressures [Spolyschides. [Spolyschides.  negatively affects thediversity and abundance of many associated species.Our results have clear implications for the conservation of ressures . In addiressures  and presschides ,97, the Figure S1(A) Mean log-likelihood of the data per K, i e. standard output fromStructure. (B) Mean absolute difference of the second order rate of changewith respect to K .(PDF)Click here for additional data file.Figure S2Spolyschides.  per site for the2008 and 2010 surveys.Allele sizes are indicated on the x axis and sites on the y axis. Presence(+) and absence (-) of (PDF)Click here for additional data file.Figure S3Numbers above the branches are Bayesian posterior probabilities (> 0.50).Inferred groups are divided by dotted lines; and by (B) a FactorialCorrespondence Analysis of population multiscores.(PDF)Click here for additional data file.Table S1Spolyschides.  for sampling years2008 and 2010. Mean density records for the 2010 survey.Records of presence and absence of (XLS)Click here for additional data file.Table S2FIS) per site and perloci and deviations from Hardy–Weinberg equilibrium and for linkagedisequilibrium between pairs of loci.Inbreeding coefficients ((XLS)Click here for additional data file.Table S3Hierarchical analysis of molecular variance (AMOVA) under 999 permutationswith 3 hierarchical levels.(XLS)Click here for additional data file.Table S4Site number, number of samples per site (n), Wilcoxon test probability , Critical MC value, M-ratio (M) and the probability of a smaller MRatio under equilibrium (Mp).(XLS)Click here for additional data file.


However, they are currently excluded from control programmes. Molecular phylogenetic studies have provided insights into the evolutionary origins and transmission dynamics of S. mansoni, but there has been no research into schistosome molecular epidemiology in pre-school children. Here, we investigated the genetic diversity and population structure of S. mansoni in pre-school children and mothers living in lakeshore communities in Uganda and monitored for changes over time after praziquantel treatment. Parasites were sampled from children (<6 years) and mothers enrolled in the longitudinal Schistosomiasis Mothers and Infants Study at baseline and at 6-, 12- and 18-month follow-up surveys. 1347 parasites from 35 mothers and 45 children were genotyped by direct sequencing of the cytochrome c oxidase (cox1) gene. The cox1 region was highly diverse with over 230 unique sequences identified. Parasite populations were genetically differentiated between lakes and non-synonymous mutations were more diverse at Lake Victoria than Lake Albert. Surprisingly, parasite populations sampled from children showed a similar genetic diversity to those sampled from mothers, pointing towards a non-linear relationship between duration of exposure and accumulation of parasite diversity. The genetic diversity six months after praziquantel treatment was similar to pre-treatment diversity. Our results confirm the substantial genetic diversity of S. mansoni in East Africa and provide significant insights into transmission dynamics within young children and mothers, important information for schistosomiasis control programmes.Significant numbers of pre-school children are infected with S Schistosoma mansoni, which causes intestinal schistosomiasis. However, there has been no work published on the molecular epidemiology of Schistosoma in children under six or the role that these children play in parasite transmission. We analysed the genetic structure of parasite populations collected from mothers and young children living on the shores of Lakes Albert and Victoria in Uganda. Parasite populations were different at the two lakes indicating that there is little flow of parasite genes between the lakes. We were surprised to discover a large amount of genetic variation in parasites sampled from both children and mothers, suggesting that genetic variation is not directly related to duration of exposure to infested water. In addition, we found some evidence that young children are involved in S. mansoni transmission. The substantial genetic variation of S. mansoni in young children suggests that these parasites could be operating as a source of a variety of genetic traits, including drug susceptibility. Overall our findings offer significant insights into population genetics of S. mansoni in pre-school children and their mothers and provide important information for effective control of intestinal schistosomiasis.Many pre-school children in sub-Saharan Africa are infected with the parasite  SchistosomaSchistosoma mansoni is responsible for intestinal schistosomiasis and is associated with pathologies ranging from diarrhoea and anaemia to hepatosplenomegaly and portal hypertension S. mansoni infects tens of millions of individuals across SSA, its distribution is heterogeneous as its life-cycle depends on water contact, poor sanitation and the presence of suitable habitats for the intermediate host, freshwater snails of the genus BiomphalariaThe neglected tropical disease schistosomiasis, remains a significant public health problem, particularly in sub-Saharan Africa (SSA), and is caused by infection with parasites of the genus National Control Programmes for schistosomiasis have been established in a number of African countries Schistosoma species of medical and veterinary importance have been studied in some detail using mitochondrial and nuclear markers Schistosoma species. DNA barcoding approaches cox1) have been particularly informative S. mansoni samples from across the globe revealed that S. mansoni separates geographically into five major lineages S. mansoni was recently introduced into the New World, perhaps with slaves infected with intestinal schistosomiasis from West Africa The molecular evolution and phylogenetics of S. mansoni populations S. mansoni collected from school-aged children and Biomphalaria snails on the shores of Lake Albert (LA) in Uganda and Lake Victoria (LV) in Kenya, Tanzania and Uganda revealed extensive population diversity with genetic differentiation between LA and LV Particularly high levels of genetic diversity have been observed in East African Schistosoma populations in pre-school children. It could be hypothesised that because the cumulative exposure and infection window has been shorter for younger children than school-aged children or adults, parasite diversity in pre-school children may be lower than in older individuals. However, heterogeneities in local transmission may also be more manifest in younger children S. mansoni parasites in a cohort of pre-school children and their mothers living in Ugandan lakeshore communities before and after praziquantel treatment, in so doing we hoped to address the role of young children in local parasite transmission.To date there have been no studies investigating the genetic structuring of The London School of Hygiene and Tropical Medicine (LSHTM 5538.09) and the National Council of Science and Technology, Kampala, Uganda, granted ethical approval for the Schistosomiasis in Mothers and Infants (SIMI) study. Before selection, all families received an information leaflet describing the study objectives and procedures, which were explained in detail by the local Vector Control Division district officer. Informed consent documented by signature or fingerprint (in cases of illiteracy) was obtained from each mother on her own behalf and on behalf of her child or children who were participating in the study. Fingerprint consent procedures were specifically approved by the research ethics committees of the London School of Hygiene and Tropical Medicine and the Ugandan National Council of Science and Technology.S. mansoni was carried out Schistosome parasites were collected during the SIMI longitudinal study carried out in six communities on the shores of LA and LV in Uganda S. mansoni infection. Particular effort was made to obtain eggs from family groups. During subsequent surveys, eggs were collected from the same individuals sampled at baseline or members of the same family (if S. mansoni positive). In addition, for the 6 month survey at Bugoigo a selection of samples for egg isolation was chosen at random from a list of schistosome-positive individuals. To obtain eggs, stool samples were diluted in bottled water and an adaptation of the Pitchford-Visser funnel method was used At baseline schistosome eggs were isolated from the stool of participants who were egg-patent or CCA-positive for 2.0 mm punches were taken from the centre of each parasite spot on FTA® cards and processed as described cox1 sequences from previous studies Cox1 sequences representing the five lineages of S. mansoni across Africa S. rodhaini cox1 sequence was used to root trees. Bootstrapping (1000 replicates) was carried out to test branch reliability.DNA sequences were manually edited using CLC Workbench v6 based on inspection of sequence chromatograms and truncated to the 396 bp ASMIT region for comparison with ST estimator together with permutation tests (10000 random permutations) of genetic differentiation. The net mean genetic distance Sequences from all parasite samples were imported into DNASP v5 For baseline data, statistical associations were investigated (using non-parametric tests) between haplotype and nucleotide diversity in individual hosts (from whom ≥6 parasites were sequenced) and lake, host type (mother or child), host age, infection intensity (number of eggs per gram of stool) and a history of praziquantel treatment (mothers only). To examine associations between morbidity and schistosome diversity, study participants were classified as anaemic if they had a haemoglobin level <11.0 g/dL and FOB positives were categorised as described S. mansoni cox1 haplotypesV7: FJ750538; V3: FJ750534; A7: FJ750530; H1: GQ415163; H2: GQ415167; H4: GQ415169; H8: GQ415171; H10: GQ415173; H14: GQ415176; H16: GQ415179; H17: GQ415182; H18: GQ415185; H20–H23: GQ415189–92; H29: GQ415200; H31: GQ415202; H35: GQ415208; H36: GQ415211; H38: GQ415215; H41: GQ415218; H42: GQ415220; H46: GQ415227; H47: GQ415228; H50: GQ415231; H53: GQ415234; H54: GQ415235; H60: GQ415242; H63: GQ415245; H65: GQ415247; H67: GQ415249; H75: GQ415258; H77–H79: GQ415261–3; H84: GQ415268; H87: GQ415271; H89: GQ415273; H92: GQ415276; H99: GQ415283; H100: GQ415284; H103: GQ415288; H106: GQ415291; H107: GQ415292; H116: GQ415301; H117: GQ415302; H123: GQ415308; H124: GQ415309; H126: GQ415311; H128: GQ415313; H131: GQ415316; H136: JF290433; H137: HM055378; H140: HQ839768; H146: JF274042; H150: JF274046; H153: JF440336; H155: JF508492; H158: JF508495; H176–H359: KC964660–848 (release date 1 Aug 2013); CA1b: JQ289589; CA1f: JQ289593; CA1h: JQ289595; CK1e: JQ289600; CK1g: JQ289602; CK1h: JQ289603; CK1j: JQ289605; CK1l–CK1n: JQ289607–9; CK1u: JQ289616; EG1: JQ289620; KE1: JQ289618; NI1a: JQ289624; NI1g: JQ289630; NI1h: JQ289631; NI1k: JQ289634; NI1m: JQ289636; NI1p: JQ289639; SA1b: JQ289653; SE2a: JQ289655; SE2c: JQ289657; SE2j: JQ289668; SE3e: JQ289682; SE4c: JQ289690; TA1c: JQ289693; TA1g: JQ289697; TA1h: JQ289698; TA1n: JQ289704; TA1p: JQ289706; UG3a: JQ289716; UG3b: JQ289717; UG4a: JQ289721; UG4e: JQ289725; ZA1b–ZA1j: JQ289728–36.S. rodhaini cox1: JQ314100Schistosoma mansoni cox1 sequences were obtained from 1347 parasites collected from 35 mothers and 45 children. The cox1 region was highly diverse with 246 different haplotypes identified, of which 184 were novel. cox1 haplotype diversity at either lake, as the curves did not approach the asymptote after 36 infrapopulations for LA or 28 infrapopulations for LV , lake, history of praziquantel treatment, infection intensity and morbidity were investigated at baseline. There was no evidence for an association between host type, lake and praziquantel treatment history and haplotype or nucleotide diversity (data not shown). Interestingly, nucleotide diversity at non-synonymous sites was associated with lake . There was no correlation between infection intensity and haplotype diversity , or nucleotide diversity  . In addition, there was some evidence for a positive correlation between nucleotide diversity at non-synonymous sites and age in children . Finally, no association was observed between any individual morbidity marker and genetic diversity (data not shown), nor was there evidence of an association between overall morbidity and haplotype diversity  or nucleotide diversity .The associations between schistosome  N = 50) , howeverAMOVA analysis, with groups defined based on location or host type, revealed that variation in parasite populations within individual hosts accounted for most of genetic variation observed  and S3. S. mansoni in pre-school children. We found similar high levels of genetic diversity in pre-school children and their mothers, both at baseline and after praziquantel treatment. Overall diversity was comparable between LA and LV and between individual villages. However, nucleotide diversity at non-synonymous sites was significantly higher in LV than LA. There was also evidence of genetic differentiation between parasite populations at the two lakes.This is the first study to investigate the population genetics of S. mansoni is highly diverse in East Africa cox1 diversity at either lake  rather than more miracidia per host leads to more robust estimates of parasite population diversity S. mansoni diversity in East Africa.Our results are consistent with a number of studies demonstrating that her lake . AlthougSchistosoma infrapopulations could be anticipated to depend on the degree of host exposure to genetically diverse parasites in the environment and on parasite interaction with the host immune system. Our findings contradict the hypothesis, based on a trickle model of infection S. mansoni genetic diversity than mothers due to a shorter cumulative exposure window. Using small GPS dataloggers, we have demonstrated that young children come into contact with water at the margins of Lake Albert for around 30 minutes each day S. mansoni from six months of age cox1 diversity at non-synonymous sites in children, potentially because of the more developed immune system in older children. Diversifying selection by host immune systems is an important explanation for pathogen antigenic variation cox1 encodes an intracellular protein, it is not exposed to the immune system in intact schistosomes, but it is conceivable that particular cox1 haplotypes are associated with specific polymorphisms in antigens under selection pressure. As a mitochondrial gene, cox1 variation is only related to the diversity of female worms, but it is possible that host immunity also drives diversity in female schistosomes. Inspection of genetic variation within genes whose products are under immune-surveillance would be informative.Our observation that genetic diversity is similar in mothers and young children is somewhat unexpected. Diversity in S. mansoni populations at LA and LV cox1 diversity at non-synonymous sites was higher at LV than LA, which may reflect differences in diversifying selection caused by host immunity at the two lakes. The snail intermediate host may also play a role, since the composition of Biomphalaria snail populations is different at the two lakes, with B. stanleyi found only at LA, B. choanomphala only at LV and B. sudanica and B. pfeifferi at both lakes S. mansoni-associated morbidity have been observed between LA and LV which may be due, at least in part, to genetic differentiation between schistosome populations, leading to somewhat different host pathologies Consistent with previous work, we found evidence for genetic differentiation between S. mansoni are substantially different at LA and LV: overall infection prevalence at baseline was higher at LA than LV and individuals were more rapidly re-infected at LA Intriguingly, whereas there was little genetic structuring between parasite populations in individual hosts at LA, there was genetic differentiation between infrapopulations at LV. This could reflect the fact that the lake shore of LV is more convoluted than that of LA, providing micro-environments for transmission. Such micro-environments could mean that even individuals living in the same village are exposed to different parasite populations, depending on where and when they come into contact with infested water. In all villages there was generally little evidence of genetic differentiation between parasite infrapopulations from hosts belonging to the same family. The transmission dynamics of S. mansoni genetic diversity were observed at the lake, village and host level in follow-up surveys after praziquantel treatment, suggesting that six months is sufficient for acquisition of diverse infections and that therapy did not impact on the host immune system to reduce diversity. There was generally little genetic differentiation between parasite populations pre- and post-treatment at LA. At LV schistosome populations did show different genetic structures at baseline and six months, although this could be because parasites were successfully sampled from only three children at six months compared with nine at baseline. When parasite populations from the same hosts were compared between surveys, there was generally little evidence of genetic differentiation between surveys with infrapopulations clustering by lake rather than timepoint. Overall these results suggest that parasite population structure in the lake-shore communities was relatively stable over the study period. Although we did not directly test the clearance of parasites after praziquantel treatment in the present study, we have recently published data from the same villages demonstrating that cure rates were low in very young children and those with a history of previous praziquantel treatment S. mansoni sampled from Tanzanian school children was observed after one round of treatment and genetic differentiation between pre- and post-treatment populations Similar levels of S. mansoni at an early age S. mansoni transmission is still somewhat unclear. A comparison of cox1 haplotypes identified only in parasites from pre-school children with haplotypes discovered in cercariae shed by BiomphalariaS. mansoni transmission but further work is required to confirm this. If true, the high genetic diversity of S. mansoni in young children suggests that they could be currently operating as local refugia of meta-populations, providing pools of susceptible genes to dilute genes conferring praziquantel tolerance selected in treated populations (i.e. school children) It is now evident that pre-school children can become infected with S. mansoni in young children and mothers in Uganda, important information for effective ongoing control of intestinal schistosomiasis.To conclude, this study provides novel insights into the epidemiology, genetic diversity and population dynamics of Table S1Cox1 diversity stratified by individual host.(DOC)Click here for additional data file.Table S2Cox1 diversity in infrapopulations summarised by host type, lake system and survey timepoint.(DOC)Click here for additional data file.Table S3AMOVA results showing little evidence of genetic differentiation between villages at Lake Albert or Lake Victoria at baseline.(DOC)Click here for additional data file.


Callitropsis guadalupensis  is endemic to Guadalupe Island, Mexico, where it is the dominant species of the only forest. The species has suffered declining numbers following the introduction of goats to the island over 150 years ago. Callitropsis guadalupensis is closely related to Callitropsis forbesii (Tecate cypress), distributed in small isolated populations in mainland Baja California and southern California. The objective of the present study was to compare the genetic diversity of the island endemic to the continental species.Callitropsis guadalupensis (n = 54) from Guadalupe Island and in Callitropsis forbesii (n = 100) from five populations in mainland Baja California. The plastid DNA trnS-trnG spacer and the trnL-trnF region were chosen for characterization. Thirty-four haplotypes were observed, of which six were shared between both species. One of these haplotypes was also shared with three other species, Callitropsis lusitanica, Callitropsis montana, and Callitropsis stephensonii. Haplotype diversity (h) and nucleotide diversity (π) were significantly higher for Callitropsis guadalupensis  than for Callitropsis forbesii .We measured genetic diversity in Callitropsis guadalupensis shows no evidence of a founder effect or of a genetic bottleneck, and can be added to a growing list of insular species with higher genetic diversity than their mainland relatives. The importance of genetic diversity within and between populations lies in the possibility that the variation confers a capacity to respond to environmental changes, including climate change, pollution, introduced species, disease, pathogens, or parasites Callitropsis guadalupensis (S. Watson) D. P. Little  is endemic to Guadalupe Island, where it forms four forest patches of 8.8, 46.9, 4.3, and 87.7 ha at the north end of the island at an elevation of 950–1300 m  that were introduced to the island over 150 ago and have decimated the native flora C. guadalupensis, killing mature trees by eating their bark, and impeding new recruitment by browsing seedlings C. guadalupensis is currently classified as endangered by the Mexican government 0–1300 m . The preCallitropsis forbesii (Jeps.) D. P. Little (Tecate cypress) is closely related to Callitropsis guadalupensisC. forbesii, but their sizes are probably smaller than those of C. guadalupensis. Callitropsis forbesii is considered in some treatments as a variety of C. guadalupensisCallitropsis forbesii is classified as “subject to special protection” by the Mexican government Cupressus, but phylogenetic studies have shown that the genus is polyphyletic, prompting the reclassification of New World taxa (together with Xanthocyparis and one species of Chamaecyparis) under the name CallitropsisHesperocyparis has also been proposed for the New World taxa Callitropsis. In this paper we adopt the name Callitropsis.The generic name of these taxa also is in dispute; until recently they were included in the circumscription of the genus Callitropsis guadalupensis provoked by grazing may have reduced its genetic diversity and negatively affected its ability to respond to future changes. An exploration of the state of its genetic diversity is a necessary condition for predicting the future prospects of the species. The main objective of this study was to characterize the genetic diversity and population structure of C. guadalupensis on Guadalupe Island with plastid DNA markers, which are paternally inherited in Cupressaceae C. guadalupensis with five populations of C. forbesii, we observed that in contrast to general trends, the insular species  maintains higher levels of genetic diversity than the continental species (C. forbesii). Furthermore, both species share presumably ancestral haplotypes with each other and with other New World species of Callitropsis. We compare the levels of DNA variation in this group with levels in other tree species, and discuss other cases of shared polymorphism among closely related species of conifers.The reduction in size of the only population of Callitropsis guadalupensis, five linear 200 m transects were randomly arranged in the larger of two northern forest patches and seven transects in the larger of two southern patches on Guadalupe Island. Five collections were made from trees separated by 50 m along each transect in the northern patch and one to five collections were made along the seven transects in the southern patch for a total of 54 samples. For C. forbesii, 20 samples were collected randomly from each of five populations from the Baja California mainland . Most of the primers evaluated were originally designed for Pinus (Pinaceae) and did not recover variation in Callitropsis. Only 11 primer pairs resulted in successful amplifications (trnS-trnG spacer and the trnL-trnF region (including the trnL intron and the trnL-trnF spacer), both of which have been used in previous intraspecific studies of Cupressaceae Total DNA was isolated from individual plants by grinding foliar tissue with liquid nitrogen and then using a DNeasy Plant Mini Kit (Qiagen). We evaluated 34 DNA primer pairs in a total of four individuals  80°C for 5 min, (2) 30 cycles of 95°C for 1 min, and 55°C for 4 min (no separate extension step), and (3) 66°C for 10 min trnL-trnF region were: (1) 94°C for 3 min, (2) 30 cycles of 94°C for 1 min, 50°C for 50 s, and 72°C for 1.2 min, and (3) 72°C for 5 min.The During early stages of the study, PCR products were purified with a Geneclean III kit (Qbiogene), diluted in double distilled water, and mailed to Macrogen, Inc.  for Sanger sequencing. For the remainder of the study, unpurified PCR products were mailed to the University of Washington High Throughput Genomic Unit  for purification and sequencing. Forward and reverse sequence reads were edited and manually aligned in BioEdit Sequence Alignment Editor trnS-trnG spacer and trnL-trnF region sequence alignment by comparing the number of haplotypes, adjusting the sample size to 50 individuals for each species with a rarefaction analysis h) and nucleotide diversity (π)Genetic diversity was estimated for the concatenated trnS-trnG spacer and trnL-trnF region alignment was used to estimate a genealogical network with statistical parsimony The concatenated trnS-trnG spacer and trnL-trnF region were obtained from single individuals representing three additional species from Mexico, C. stephensonii (C. B. Wolf) D. P. Little, C. montana (Wiggins) D. P. Little, and C. lusitanica (Mill.) D. P. Little. These were identical to one another and with a haplotype recovered from C. forbesii and C. guadalupensis (see below). Therefore, to include an outgroup for the phylogenetic analysis in PAUP*, sequences from the more distantly related Juniperus monticola Martínez were added and the matrix was realigned. The alignment was deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S11036). A parsimony analysis was performed both with all gaps treated as missing data and with simple indel coding trnS, trnL and trnF) with and without enforcing a molecular clock and the likelihood ratio test was used to determine whether the data evolved at a homogenous rate Phylogenetic analyses also were performed with PAUP* v.4.b10 trnS-trnG spacer  and the trnL-trnF region  from 54 samples of Callitropsis guadalupensis and 100 samples of C. forbesii. Twenty-eight distinct haplotypes were detected with the trnS-trnG spacer, and an additional six were detected with the trnL-trnF region, giving a total of 34 haplotypes. For the latter marker, all variation occurred in the trnL-trnF spacer; the trnL gene and trnL intron were invariant. Variable sites in the trnS-trnG spacer included a dinucleotide microsatellite (TA) that varied from 6 to 15 repeats occurring between positions 381 and 410 of the alignment , a single bp indel (site 438), a 43 bp indel (site 499), and a nine bp indel (site 544).Intra- and interspecific variation was observed in both the lignment . The micCallitropsis guadalupensis had significantly higher haplotype diversity  than the five populations of C. forbesii  and significantly higher nucleotide diversity (π = 0.00071±0.00009) compared to C. forbesii . Twenty-one of the 34 (62%) haplotypes were detected in C. guadalupensis and 19 (56%) in C. forbesii. The two species shared six haplotypes ; the other three haplotypes occurred at lower frequencies , the number of haplotypes observed in C. guadalupensis (20.3) was statistically greater (at the 95 and 99% confidence levels) than the number for C. forbesii (13.7). The Hudson, Boos and Kaplan permutation test found that genetic distances among C. guadalupensis haplotypes were closer than expected at random . Also, the permutation test within C. forbesii was not significant (Snn = 0.19205) suggesting that the taxon lacks phylogeographic structure.C. forbesii; only San Vicente, the most southerly population, lacked unique haplotypes. The highest haplotype and nucleotide diversity were observed in the northernmost population of C. forbesii, Rancho El Escondido , and the lowest haplotype and nucleotide diversity were observed in the second northernmost population, El Dieciséis . The Spearman rank test found no significant correlation between latitude of C. forbesii populations and h or π (p>0.05).Unique haplotypes were observed in four of the five populations of Callitropsis , occurred at an internal node, consistent with it being ancestral. The most abundant haplotype (H09) was shared between C. guadalupensis and C. forbesii and also occurred at an internal, putatively ancestral node. Haplotypes of C. guadalupensis tended to occur on one side of the network and those of C. forbesii on the other, consistent with slow plastid DNA divergence. Also, a mismatch analysis indicated a recent population expansion for C. forbesii but not for C. guadalupensis (Fu & Li's F* and D* <0.05). This expansion was not statistically significant when Tajima's D was used for either of the species.In the genealogical network , H24, whtrnS-trnG spacer and trnL-trnF region with Juniperus monticola resulted in a concatenated matrix that was 1589 bp in length, with 45 variable but parsimony uninformative sites (singletons) and three parsimony informative sites. The parsimony heuristic search with gaps treated as missing recovered one equally most parsimonious tree (MPT), with a length (L) of 48 steps, a consistency index (CI) of 1.0, a consistency index excluding uninformative characters (CIexc) of 1.0, and a retention index (RI) of 1.0 (not shown). Using simple indel coding added 30 binary characters to the matrix, resulting in 67 variable but parsimony uninformative and 11 parsimony informative characters. The heuristic search of the matrix with simple indel coding recovered >10,000 MPTs . Only a single clade with two C. guadalupensis haplotypes (H12 and H14) was resolved in the strict consensus (not shown). For the maximum likelihood analysis, the best model using the AIC test was TPM1uf, and likelihood settings were specified as determined in JModeltest: A = 0.3435, C = 0.1458, G = 0.1590, T = 0.3517; nst = 6, rmat =  . The heuristic search recovered a single tree with –ln = 2383.33014 . The log-likelihood ratio test strongly rejected the null hypothesis of rate homogeneity for the 1,453 noncoding sites .Realignment of the 83.33014 . In bothPinus radiata D. Don (Pinaceae) has insular populations on both Guadalupe Island and Cedros Island in Baja California with lower levels of genetic diversity, smaller effective population sizes, and higher rates of inbreeding than disjunct continental populations in California Pinus torreyana Parry ex Carrière are genetically depauperate on both the mainland near San Diego and on Santa Rosa Island Callitropsis guadalupensis has appreciable plastid DNA variation and shows no evidence of a founder effect or of a population bottleneck. The level of plastid DNA haplotypic diversity found in C. guadalupensis is similar to that found in Cunninghamia konishii Hayata (Cupressaceae) endemic to Taiwan  π) in C. guadalupensis (π = 0.00112) are within the range of tree species in Taiwan which are hypothesized to have preserved high values of genetic diversity in one or more refugia during the Pleistocene (Castanopsis carlesii (Hemsl.) Hayata, Cunninghamia konishii, Cyclobalanopsis glauca Oerst., and Trochodendron aralioides P. F. Siebold & Zucc.; π = 0.00050 to 0.00200) C. guadalupensis joins a growing list of examples of narrow endemics with relatively high genetic polymorphism Arctocephalus townsendi) Mirounga angustirostris) Delphinium variegatum Torr. & A. Gray (Ranunculaceae) Castilleja grisea Dunkle (Orobanchaceae) Jepsonia malvifolia (Greene) Small (Saxifragaceae) Lithophragma maximum Bacig. (Saxifragaceae) Picea mexicana Martínez Pinus rzedowskii Madrigal & M. Caball. Island endemics make up a disproportionate number of rare and endangered species, and although anthropogenic disturbance is a primary factor in driving species to extinction, relatively low genetic diversity of island populations is also important Cryptomeria japonica D. Don (Cupressaceae) includes a population on Yagu Island, Japan with relatively high isozyme diversity C. guadalupensis was 15,000 adults at the time of sampling C. guadalupensis or C. forbesii. The greater genetic variation found in C. guadalupensis plastid DNA compared to C. forbesii may be due to effectively larger historical population sizes in C. guadalupensis, which would suggest that it represents a stable population with a long evolutionary history C. forbesii, the populations appear smaller than those for the only population of C. guadalupensis. The maximum population size of C. guadalupensis has probably been limited by the size of Guadalupe Island, but this still could have exceeded the population size of C. forbesii on the continent.Rare species with large local populations have shown high levels of genetic variation h and π found in C. forbesii are similar to other arboreal species with a wide distribution in Taiwan and East Asia, Cyclobalanopsis glauca [π = 0.00065], Trochodendron aralioides [π = 0.00052], Castanopsis carlesii . The low values are consistent with the hypothesis that populations of this species recently passed through a prolonged demographic bottleneck or a founder event Calocedrus macrolepis Kurz  were shared between C. forbesii and C. guadalupensis. The result of the phylogenetic analyses of the Callitropsis species compared in this study illustrates the extent of haplotype sharing among species and the resulting lack of reciprocal monophyly (C. forbesii) and the existence of the oceanic geographic barrier. The hypothesis of homoplasy would explain the proportion of shared haplotypes due to a series of parallel and independent changes in the two species resulting in the same haplotypes arising more than once C. forbesii and C. guadalupensis plastid DNA and the discovery of the second-most frequent haplotype (H24) in three other allopatric New World species of Callitropsis. The most frequent haplotype overall (H09) was also shared by both species and occurred at a high frequency in all populations: Guadalupe cypress (15%) and Tecate cypress . The persistence of ancestral haplotypes could be associated with the climatic or ecological conditions of the habitats where it occurs , but it is more likely that they are the result of slow coalescence favored by large effective population sizes, long generation times, and a low mutation rate.Of the 34 haplotypes found for onophyly , 3. This−9 substitutions/site/year in the conifer family Pinaceae −9 estimated in angiosperms Pinus rate is probably closer to the rate for Callitropsis because the two genera have similarly long life spans and large population sizes and because of their relative phylogenetic proximity. Although the substitution rate of the trnS-G and trnL-F spacers deviated significantly from a molecular clock, a single substitution in 1453 bps (excluding the trn genes) would be expected to occur every ∼ 1,600,000 to 3,100,000 years. Callitropsis haplotypes differ by zero to four substitutions. Two clades with haplotypes exclusive to Guadalupe Island  are separated from the remaining haplotypes by a single substitution, suggesting that they originated subsequent to the formation of the island approximately 7 million years ago CallitropsisThe silent substitution rate of plastid DNA is estimated at 0.22–0.42×10PinusAbiesJuniperusPinus lambertiana Douglas Non-monophyly of plastid DNA may be a common pattern in recently diverged conifer species pairs and in species complexes. It has been documented in other species rich conifer genera such as C. guadalupensis and C. forbesii. In the case of C. forbesii, genetic diversity is highest in two of the three most northerly populations . The greatest number of plastid haplotypes would be preserved if conservation efforts focused on northern populations, but studies of more populations using higher resolution multi-locus markers are needed.Seed collection and in situ and ex situ conservation need to be sustained, and should continue to distinguish between seed collected from Table S1Callitropsis.Plastid primer pairs that gave successful PCR reactions in (DOC)Click here for additional data file.Table S2trnS-trnG intergenic spacer and the trnL-trnF region.Variable sites and their position in the alignments of the (DOC)Click here for additional data file.


Drosophila pseudoobscura males respond to rivals by extending mating duration and increasing ejaculate size. Whilst such responses are predicted to be adaptive, the extent to which the magnitude of response is heritable, and hence selectable, is unknown. We investigated this using isofemale lines of the fruit fly D. pseudoobscura, estimating heritability of mating duration in males exposed or not to a rival, and any genetic basis to the change in this trait between these environments (i.e. degree of plasticity). The two populations differed in population sex ratio, and the presence of a sex ratio distorting selfish chromosome. We find that mating duration is heritable, but no evidence of population differences. We find no significant heritability of plasticity in mating duration in one population, but borderline significant heritability of plasticity in the second. This difference between populations might be related to the presence of the sex ratio distorting selfish gene in the latter population, but this will require investigation in additional populations to draw any conclusions. We suggest that there is scope for selection to produce an evolutionary response in the plasticity of mating duration in response to rivals in D. pseudoobscura, at least in some populations.Phenotypic plasticity is a key mechanism by which animals can cope with rapidly changeable environments, but the evolutionary lability of such plasticity remains unclear. The socio-sexual environment can fluctuate very rapidly, affecting both the frequency of mating opportunities and the level of competition males may face. Males of many species show plastic behavioural responses to changes in social environment, in particular the presence of rival males. For example,  Phenotypic plasticity, the ability of a genotype to exhibit a range of phenotypes depending on the environment, is widespread and a fundamental component of fitness sensuMale reproductive success depends on the ability to compete for matings  and, if females mate multiply, also for fertilisations after mating , which can create biased sex ratios in natural populations, and its prevalence can fluctuate over the course of a year D. pseudoobscura. As mentioned above, males of this species show a similar response to rivals as D. melanogaster, increasing copulation duration when exposed to a rival for four days prior to mating SR, as the SR chromosome is itself likely to modify male behaviour. Instead we focus on how non-SR males respond to the risk of sperm competition. We also test the hypothesis that populations with different prevalence of the sex ratio distorter, hence different propensity for variation in sex ratio, will show a difference in their plasticity. Specifically, that a population with low levels of SR will show lower plasticity, or difference in mating duration, between isolines.Here we investigate whether there is heritable variation in male response to rivals in the fruit fly SR in each population was determined by mating 100 wild caught males to stock females. Males which produced broods that were more than 95% female were assigned as SR males SR males in Lewiston, whereas 11% carried SR in Show Low, and previous work confirms that SR is found at less than 1% frequency in the Lewiston population, whereas in Show Low population it is found at 10–20% prevalence SR, as revealed both by genotyping with a PCR marker for SR [see methods in 32] and examination of the sex ratio of broods fathered by males from the isoline. Isolines maintain genetic diversity and prevent evolution and adaptation to the laboratory by reducing heterozygosity Drosophila food We collected flies using standard banana baits 2 anaesthesia at 24 hours old, which does not affect the response to these males by focal males at 4 days old D. pseudoobscura). Isoline males were randomly allocated to “exposed to rivals” (R) or “not exposed to rivals” (NR) treatments. R males were placed in a vial with a marked stock male, whereas NR males were kept singly in a vial. Flies were 5–6 days old at the time of mating, at which age both sexes are fully sexually mature For this experiment we used 13 isolines from Lewiston and 14 from Show Low. We collected males from each isoline within 18 h of eclosion to ensure virginity Genetic variation and genotype by  environment interactions in mating duration were tested by fitting standard linear mixed effects models in ASReml Where µ is the intercept, pop is a fixed effect of population of origin of the lines, pop.line is the random effect of line nested within population and pop.ε is the random error term nested within population. Genetic variation is estimated from the variance in mating duration that is explained by pop.line. This model was run separately on data from each environment  to provide estimates of the total genetic variance in each environment. Broad sense heritability for mating duration in each environment was calculated as the variance due to pop.line expressed as a proportion of the sum of the variance due to pop.line and pop.ε.To test for environment specific genetic variation and differences in the response to environments across populations (G×ssE) we fit a model of the form:2) distributed with the number of degrees of freedom equal to the difference between the models in the number of parameters estimated. However, it has recently been highlighted that this approach is over-conservative and that the actual distribution is a mixture of χ2 distributions with different degrees of freedom Where terms are as above except env represents a fixed effect of environment, and population and environment specific line variances and genetic covariances between lines represented in multiple environments are estimated. To test for genotype by environment interactions, this model was compared to models where genetic correlations between copulation duration in different environments in the two populations were fixed to one and where genetic variances across environments and populations were constrained to be equal. This tests the hypothesis that the genetic correlation between copulation duration in the two environments in the two populations is 1 and that the genetic variances in the two environments in the two populations are equal. Genetic correlations of less than one or unequal genetic variances between environment indicate significant G×E for this trait. Model comparisons were made using LogLikelihood ratio tests. Traditionally, significance has been based on an assumption that twice the difference in LogLikelihoods of the models is chi-squared .20&1df  = 0.04, p = 0.42), showing that the genetic variation in copulation duration is equal in each population. In addition there was no significant effect of population of origin on the mean copulation duration in the absence of rivals  estimate of 0.078±0.054, which was significantly greater than zero (χ20&1df  = 3.32 p = 0.034).Univariate models of copulation duration in the absence of a rival revealed no significant difference between the amount of variation explained by line in the two populations  and again there was no effect of population on mean copulation duration . Total genetic variation and broad sense heritability were therefore again estimated from a model assuming line variation in both populations is drawn from the same distribution. There was significant heritability in this environment .Line variation for copulation duration was also not significantly different between the two populations when expressed in the presence of a rival . To show that these changes in mating duration differed between isolines, and thus had a genetic basis, requires significant genotype by environment interaction . There was some marginal evidence of genotype by environment interactions in this study. The genetic correlation (rg) between mating duration with and without a rival present was marginally non-significantly different from 1 in Show Low  , but not significantly different from 1 in Lewistown , . However, these correlations were not significantly different from each other . This provides some evidence for G×E in Show Low, suggesting that the rank order of genotypes in mating duration changes when exposed or not to a rival, but that this is not the case in Lewistown, although it should be noted that the genetic correlation in Show Low is not significantly different to that in Lewistown. The total genetic variation did not differ between environments in either population  and the genetic variance did not differ between populations . Taken together these results provide marginal evidence of genotype by environment interaction for mating duration in Show Low, but not in Lewistown, with the genetic correlations between mating duration in different environments being marginally non-significantly different from 1 in Show Low. However, this result is technically non-significant, and so is only weak evidence of G×E effects. The data presented are for untransformed copulation duration. However, transforming copulation duration such that in each environment and population, the mean mating duration was 0 and the variance was 1, and thus controlling for any effects of scaling differences between populations and environments, gives qualitatively similar results (data not shown).Males exposed to rivals significantly increased their copulation duration, with presence of a rival increasing the predicted mating duration by 79.81±11.85 seconds . Mating duration showed significant heritable variation in both conditions, with the broad sense estimate being greater for males exposed to rivals, although not significantly so. There was some variation in the degree of response to rivals amongst lines, with borderline significant variation between lines in response in the southern Show Low population, but no evidence for this in the northern Lewistown population, although this difference between populations was not significant. This provides some evidence that the degree of plasticity of mating duration is more variable in the southern Show Low population, suggesting that plasticity in copulation duration may be heritable. However, this conclusion is only supported by a borderline significant level of heritable variation, not a clearly significant result. Further examination of variation in plasticity between isolines is needed to strengthen this conclusion.Males exposed to rivals increased their copulation duration, as in previous experiments D. melanogaster found that heritability of mating duration varied between sexes, with 2h = 0.23–0.46 for father-son comparisons, but 2h = 0 for mother-daughter comparisons 2H or 2h estimates), but no effect of father genotype 2h = 0.58 in Onthophagus taurusScatophagia stercorariaCallosobruchus maculatusA genetic basis and heritable variation are required if a trait is to be evolutionarily labile. In this study, copulation duration was significantly heritable, both with and without the presence of rivals. However, in both cases the heritability was small . A previous investigation of the heritability of mating duration in Jadera haemotoloma) in the USA SR allele, due to seasonal fluctuations in its abundance and the resulting sex ratio SR affecting the overall degree of response to rivals between populations, although with only two populations investigated, this is a very weak test.We found no evidence for overall differences in copulation duration in our two populations. To our knowledge, the only previous study to assess population differences in a response to rivals was of the soapberry bug  Ficedula albicollisLarus canusParus majoret al.The lack of conclusive evidence for the heritability of plasticity in this study is possibly due to the difficulties of detecting such heritability. Phenotypic plasticity is accepted to have a genetic basis, but this is rarely quantified D. pseudoobscura, responses to rivals are beneficial in terms of number of offspring D. melanogasterDrosophila species so far tested, 6/7 respond to the presence of a rival in the same manner Firstly we must consider the power of our design to detect heritable variation. Our number of isolines is lower, and replicates per line higher, than some other GxE studies We found evidence of heritability of mating duration, both in the presence and absence of rival males. We also found evidence suggesting genetic variation in degree of plasticity in mating duration depending on exposure to rivals in one of the two populations, although this evidence was borderline non-significant and hence very weak. However, we did not find overall significant differences in plasticity between populations that are expected to show different variation in sex ratio, suggesting that this is not a strong enough selective pressure to globally diminish or increase plastic responses to mating rivals. Nevertheless, we suggest that the extension of mating duration after exposure to rivals is probably heritable, at least in one population, and so has the potential to respond to selection.


Aedes albopictus is expanding globally, raising the threat of emerging and re-emerging arbovirus transmission risks including dengue and chikungunya. Its detection in Papua New Guinea's (PNG) southern Fly River coastal region in 1988 and 1992 placed it 150 km from mainland Australia. However, it was not until 12 years later that it appeared on the Torres Strait Islands. We hypothesized that the extant PNG population expanded into the Torres Straits as an indirect effect of drought-proofing the southern Fly River coastal villages in response to El Nino-driven climate variability in the region (via the rollout of rainwater tanks and water storage containers).The range of the Asian tiger mosquito Examination of the mosquito's mitochondrial DNA cytochrome oxidase I (COI) sequences and 13 novel nuclear microsatellites revealed evidence of substantial intermixing between PNG's southern Fly region and Torres Strait Island populations essentially compromising any island eradication attempts due to potential of reintroduction. However, two genetically distinct populations were identified in this region comprising the historically extant PNG populations and the exotic introduced population. Both COI sequence data and microsatellites showed the introduced population to have genetic affinities to populations from Timor Leste and Jakarta in the Indonesian region.Ae. albopictus invasion into the Australian region was not a range expansion out of PNG as suspected, but founded by other, genetically distinct population(s), with strong genetic affinities to populations sampled from the Indonesian region. We now suspect that the introduction of Ae. albopictus into the Australian region was driven by widespread illegal fishing activity originating from the Indonesian region during this period. Human sea traffic is apparently shuttling this mosquito between islands in the Torres Strait and the southern PNG mainland and this extensive movement may well compromise Ae. albopictus eradication attempts in this region.The  Aedes albopictus is expanding globally, raising the threat of emerging and re-emerging arbovirus transmission risks, including chikungunya and dengue. Detection of Ae. albopictus in southern Papua New Guinea (PNG) in 1988 and 1992 placed it 150 km from mainland Australia. In 2004–05 it reached the islands in Australia's Torres Strait that separate the mainland from PNG. Suspecting a range expansion from PNG driven by human adaptation to climate variability, we employed population genetics methodologies to investigate possible origins of the introduced population, as well as population structure of previously existing populations from New Guinea. Mitochondrial cytochrome oxidase I sequences and 13 novel microsatellite markers revealed a clear genetic distinction between regional populations in PNG and the newly introduced population in the Torres Strait and Fly Region. The closest genetic relative to the introduced population was found in the Indonesian region to the west and it is now suspected that this species may have been brought into the Torres Strait by sea vessels involved in extensive illegal fishing activities during this period.The range of the Asian tiger mosquito  Aedes (Stegomyia) albopictus, originally described by Skuse from Calcutta, India, in 1894, is considered native to the Southeast Asian region where the larvae are often found in forest tree holes – a characteristic that assists its current global expansion via rapid adaptation to human-made container habitats Ae. albopictus had spread to several islands in the Indian Ocean, as well as to the Hawaiian Islands in the Pacific The Asian tiger mosquito Aedes (Stegomyia) aegypti, Ae. albopictus has been the epidemic vector of dengue viruses in Hawaii, Macao and China Ae. albopictus is highly susceptible to the CHIKV, with the species not only responsible for these outbreaks but also able to transmit the virus after only two days Alongside this species' global expansion, its status as a vector of human pathogens is also of increasing concern. As a laboratory vector of over 25 arboviruses, its role in arbovirus transmission cycles has mostly been secondary to other incriminated vectors Ae. albopictus was first detected in 1963 in Jayapura on the West Papua Province of Indonesia , dated its arrival to 2004. Subsequent surveys in the Torres Strait revealed its presence on 10 of the 17 inhabited islands Ae. albopictus establishing on mainland Australia, the obvious question was why Ae. albopictus had only expanded into the Torres Strait Islands in 2004–05 when it was known to have been extant on Daru Island (northern Torres Strait) and in Kiunga in 1992 – 12 years earlier? A number of potential sociological and ecological factors may have contributed to the mosquitoes' proliferation and led to its dramatic expansion into the Torres Strait islands in the mid-2000s. For example, the increase in human-made water storage containers and sundry smaller discarded disposable containers may have served as potential larval habitats, leading to a population expansion.In 2005, Ae. albopictus in the Torres Strait in the mid 2000s led to the question of whether recent adaptation to climatic variability had played a role in its expansion – as we had suggested in earlier work on other container inhabiting Aedes species in this region El Nino conditions contributed to the worst drought in PNG for 100 years: traditional groundwater supplies were greatly affected, either drying up or becoming contaminated Ae. albopictus already present in PNG, leading to its rapid population expansion and a subsequent spillover into the Torres Strait Islands. Once Ae. albopictus was established on the islands, the continuous human ocean traffic would have rapidly shuttled mosquitoes through the region. Thus our working hypothesis was that climate variability driven by the 1997–98 El Nino resulted in water storage management changes in PNG's southern Fly River coastal villages and was indirectly responsible for the invasion of a local Ae. albopictus population through the Torres Strait Islands.The discovery of Ae. albopictus into and through the Torres Strait islands, as well as the population structure of this species throughout PNG. The maternally inherited mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) is used as both a population genetics marker and a proxy for female movement between islands and southern PNG villages. The rationale here is that the DNA sequence of each female is (barring mutation) identical to that of her offspring, providing insights into the dynamics and diversity of the females' contribution to each population. This proxy would ultimately be an underestimate of movement as different females of the same sequence cannot be distinguished. Additionally, we developed and ran 13 microsatellites markers that permitted the evaluation of the nuclear background of these mosquitoes.In this study, we use extensive regional mosquito collections and population genetics methodologies to investigate the origins and dynamics of the introduction of Ae. albopictus collected in 1992 from Daru Island (northeastern Torres Straits) and the Kiunga Port area in Western Province were provided by the Australian Defence Force. When samples were collected from private residences, permission was granted prior to entry. In most cases larvae were sampled from different containers at each location and preserved in 70% ethanol, and in some cases adults were collected. In many cases only a few individuals were collected at each location in order to reduce the chance of sampling siblings (as larvae in the same container). Larvae were initially identified as Ae. albopictus using the morphological keys of Aedes (Stegomyia) scutellaris taxonomic group.Container-inhabiting mosquitoes were collected from throughout the Torres Strait and PNG's southern Fly River region villages by Queensland Health between 2004 and 2010 . PopulatAe. albopictus had genomic DNA extracted using a salt extraction method 5′ CAY CCT GGT ATA TTT ATT GG ′3 and reverse primer 5′AAT TAA AAT ATA AAC TTC TGG were modified from 2SO4, 67 mM Tris-HCl pH 8.8 (at 25°C), 0.45% Triton X-100, 0.2 mg/ml gelatin, 1.5 mM MgCl, 0.2 mM of each dNTP, 0.4 µM of each primer. One unit of Taq polymerase (Bioline) and 2–10 ng of purified genomic DNA (1 µl of gDNA) were used per reaction. Cycling  was 94°C for 3 min followed by 30 cycles of 94°C for 1 min, 40°C for 1 min, and 72°C for 1 min using minimum transition times between steps. The PCR products were visualized on a 1% agarose gel containing 0.5 µg/ml ethidium bromide and visualized at 312 nm. PCR product purification was via QIAGEN (QIAquick) PCR purification columns using manufactures recommendations.Specimens identified as FST values were estimated in Arlequin version 3.5 (distance method) FST comparisons were assessed using permutation tests , also implemented in Arlequin. DnaSP 5 All sequences were edited and aligned using the Geneious software Ae. albopictus and performed by Macrogen (Korea). To design primers for microsatellite loci, we ran the resultant data through the program msatcommander taq buffer (Bioline)(containing pre-optimized concentrations of MgCl and dNTPs), 0.4 µM of each primer, 0.5–1.0 unit of MyTaq polymerase (Bioline) and 5.0–10.0 ng of extracted genomic DNA (1 µl of extraction). The cycling involved an initial denaturation of 95°C for 3 min, then 13 cycles of 95°C for 30 s, 56°C for 40 s with a gradient decrease of 0.5°C/cycle, and 72°C for 30 s, followed by 25 cycles of 95°C for 30 s, 50°C for 40 s and 72°C for 30 s, and a final 72°C for 5 min using minimum transition times. M13 labeled products for 13 microsatellite markers  generated from genomic DNA of Alleles for each marker were scored manually in the program GeneMarker K) in the region sampled, based on the microsatellite data. STRUCTURE was run using the admixture model, and using sampling locations as priors. Including information on sampling locations in STRUCTURE analyses has been shown to be useful for detecting subtle genetic structure, without detecting structure that is not present K = 2 to K = 8, for a total of 1 million generations per iteration with a burn-in of 200 000 iterations. STRUCTUREHARVESTER, a program that implements the Evanno et al. Delta K method K and CLUMPP v. 1.1.2 K = 5 . We used the Greedy algorithm in CLUMPP with 1000 repeats. The output from CLUMPP was run through DISTRUCT FST values between the eight populations defined above.The Bayesian program STRUCTURE, v. 2.3.2 A total of 16 mtDNA COI haplotypes were identified from 377 individuals (haplotype diversity Hd = 0.769) collected throughout the southern Fly River villages, the Torres Strait islands, north and south PNG, Timor Leste and Jakarta – 10 of these haplotypes were present in the Torres Strait Islands and southern Fly River region . The FST value between Torres Strait and Fly Region populations is significant but small (0.043), and most comparisons between PNG populations are non-significant . Indonesian populations are not significantly differentiated from each other but have significant FST values in all other comparisons, with the Torres Strait populations being the most closely related to them, followed by the Fly Region and then by PNG populations.Mitochondrial COI pair-wise ions see . Again tIS) of the majority of loci were positive, and observed heterozygosity was less than expected heterozygosity in most cases, indicating an excess of homozygote genotypes at most loci  inferred by STRUCTURE HARVESTER was K = 5 The most likely number of genetic clusters  than from the extant PNG populations, as had been previously hypothesized. The Jakarta population is relatively isolated on the FCA plot.The factorial correspondence analysis performed in GENETIX  supportsAe. albopictus in southern Papua New Guinea (PNG) in 1988 and 1992 placed it only 150 km across the Torres Strait from mainland Australia. In 2004–05 it appeared on the Torres Strait Islands and we initially suspected a range expansion from PNG potentially driven by human adaptation to climate variability. The AusAID funded drought-proofing expansion of rainwater tanks and 200 L water containers into the southern Fly River region villages immediately adjacent to the Torres Strait was completed in 2002 as a response to climate variability in the region Detection of Initially the mtCOI suggested that two genetically distinct populations were present in this region, providing the first piece of evidence that the invading population may not have originated from the population previously extant in PNG. Shared haplotypes between the southern Fly Region, the Torres Strait Islands and Indonesian populations provided the first clue as to where the invading population may have originated. Despite haplotype diversity being biased by the larval sampling method (which favors the collection of siblings of the same haplotype), the Torres Strait Island populations revealed four more haplotypes  than the southern Fly River . This difference in haplotype diversity may suggest that the initial introduction into the region started in the Torres Strait Islands from the Indonesian region, however more COI sequencing from the southern Fly region may be needed to clarify whether or not this is the case. Interestingly however, genetic diversity appears higher in the Torres Strait and Fly Region than in the Indonesian populations, possibly suggesting multiple introductions of closely related populations from different parts of Indonesia, or that the founding population was more genetically diverse than those sampled from the Indonesian region.Ae. albopictus mtDNA haplotypes  were moving between islands. This suggests that attempts to eradicate the species from individual islands would likely be unsuccessful given the high potential for re-introductions. Indeed, this information has assisted Queensland Health – the regional state health authority – in its decision to move from the island eradication program implemented in 2006 to a cordon sanitaire in 2008, whereby surveillance and control was focused on the inner Torres Strait islands of Waiben, Muralug and Ngurupai  adjacent to mainland Australia. In particular, Muralug and Ngurupai act as the major regional transport hubs and are thus the most likely staging point for the species' introduction onto the Australian mainland. This cordon sanitaire was breached in 2009 and Ae. albopictus now exists on Waiben and Ngurupia, less than 30 km from Australia's Cape York Peninsula. In 2010, Aedes albopictus larvae were collected from New Marpoon on mainland Australia's Cape York although no other individuals have been collected in this area since.The mitochondrial DNA was informative at another level with the discovery that multiple Ae. albopictus being present in Timor Leste dating to the 1920s Ae. albopictus into the Torres Strait and southern Fly River region came from the Indonesian region.The 13 microsatellite loci reaffirmed the findings of the mitochondrial data that the introduced population was genetically distinct to the populations already present in PNG. As microsatellites evolve more rapidly than mitochondrial sequence data, they were more informative, resolving five genetically distinct populations in total with three historically extant populations in PNG that have experienced various levels of admixture and one distinct population encompassing the Torres Strait Islands and the southern Fly River region – the introduced population. Samples from Madang in northern PNG, as well as from Port Moresby on the southern Papuan Peninsula, appear to be admixtures, with individuals from Port Moresby being more similar to samples from Kiunga, and individuals from Madang more similar to the Daru Island population  and 4. AAe. albopictus specimens which then infested the islands of the Torres Strait and/or the southern Fly region. Indonesian/Macassan visitations to the coastline of northern Australia have a long history predating European settlement, and Indonesian fishermen have been known to illegally enter Australian waters more recently to fish for shark fin . This activity is reported to have peaked between 2005–07, during which time several hundred landings occurred where fuel, water, shark fin, fishing nets and lines were often cached. Many of these landings were associated with well-established camps that received multiple visits. Uninhabited islands in the Torres Strait were one focus for these activities and evidence gathered from apprehended vessels indicates that most shark boats  carried large open-topped water drums of which a significant proportion harboured Ae. aegypti and Ae. albopictus larvae. There have been documented collections of various Aedes (Stegomyia) species, including Ae. albopictus, from illegal fishing vessels that were intercepted at the port of Darwin in the Northern Territory that clearly indicate that the mosquitoes' survival in smaller vessels is possible Since it now appears highly unlikely that the introduced population originated from PNG but rather came from the Indonesian region (west of the Torres Strait), it is conceivable that the introduction was driven by foreign fishing vessels that travelled from the Indonesian region, harboring Ae. albopictus in this region, populations on Daru Island (which adjoins the Torres Strait) appear to have been unaffected by this exotic invasion up until 2008 and follow-up collections are now warranted to determine if the introduced population has established there since. Despite Daru Island sitting geographically adjacent to the southern Fly River coastal region, its Ae. albopictus population seems to be clearly isolated from the introduced population. The occurrence of two genetically distinct populations adjacent to each other in the northern Torres Strait/PNG region can be best explained by their different jurisdictions. While the Torres Strait operates as part of Australia, Daru Island is politically part of PNG and its function as the international customs clearance station into Western Province means that it encounters different incoming and outgoing transport movements.With regard to the expansion and movement of Ae. albopictus appears to be extensive in the Australasian region, particularly where the Torres Strait Islands' junction separates New Guinea and mainland Australia. With the maximum reported flight range of Ae. albopictus being 1 km Ae. albopictus between the islands and back and forth from the southern Fly region's villages. This movement is sanctioned under The Torres Strait Treaty (Miscellaneous Amendments) Act 1984 that allows for cross-border movement for trade, fishing, and family gatherings or for seeking medical attention without the need for customs protocols Ae. albopictus.The movement of Ae. albopictus is more than likely to arrive in a town or city on the Australian mainland via human transport. Due to the intrinsic ecological plasticity in both larval habitat  and host feeding patterns Given its current location, its mobility and its phenotypic fondness for containers, Aedes albopictus's particular biology permits its container inhabiting ecological niche to once again facilitate its global expansion. It is important to consider it's potential to rapidly exploit the outcomes of any socio-economic or policy-driven interventions, such as the recent and dramatic expansion of domestic rainwater tanks throughout Australian urban regions as a drought-proofing adaptation to observed and forecasted climate change