Arthrobotrys oligospora using 12 single nucleotide polymorphic markers located on eight random DNA fragments. The strains were from different ecological niches and geographical regions from China. Our analyses identified that ecological niche separations contributed significantly, whereas geographic separation contributed relatively little to the overall genetic variation in our samples of A. oligospora. Interestingly, populations from stressful environments seemed to be more variable and showed more evidence for recombination than those from benign environments at the same geographic areas. We discussed the implications of our results to the conservation and biocontrol application of A. oligospora in agriculture and forestry.Nematophagous fungi can trap and capture nematodes and other small invertebrates. This unique ability has made them ideal organisms from which to develop biological control agents against plant- and animal-parasitic nematodes. However, effective application of biocontrol agents in the field requires a comprehensive understanding about the ecology and population genetics of the nematophagous fungi in natural environments. Here, we genotyped 228 strains of the nematode-trapping fungus  Arthrobotrys and Dactylella can lure, then trap, snare, or grip nematodes and other small invertebrate animals in soils and in wood , and symbionts . These strategies enable fungi to colonize a broad range of habitats on plants and animals (including humans), in agricultural and forest soils, and in aquatic environments. They can also be found in sites contaminated by heavy metals and grow on hypersaline microbial mats  contain the largest group of predaceous fungi. These fungi are predominantly soil-living organisms that have the ability to form infection structures, like adhesive hyphae, traps, and knobs, nonconstricting rings and constricting rings to capture nematodes. The taxonomy and diversity of nematode-trapping fungi have been primarily studied using traditional morphological methods and rRNA sequences. These studies identified that the type of nematode-trapping devices was a highly reliable indicator about the evolutionary relationships among these species  and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA gene (ITS) region have found genetic differentiation among strains within species in the genera A. oligospora. SNPs refer to polymorphisms generated by nucleotide substitutions or the insertion and deletion of single nucleotides between homologous DNA sequences. SNPs have become popular molecular markers for studying a variety of biological issues. So far, most such studies have focused on model organisms, and relatively little is known about the SNPs in nonmodel organisms including many fungi  Genetic structure and modes of reproduction in populations of this species. We discuss the implications of SNPs and polymerase chain reaction PCR-RFLP markers in the inference of the population structure and ecological patterns of A. oligospora, and the conservation and biocontrol application of this species in nature.In this study, we used molecular markers based on single nucleotide polymorphisms (SNPs) to analyze the ecology and population biology of A. oligospora occupying different ecological niches, we screened four types of environments in southwestern China: (1) a polluted aquatic environment , (2) a pristine aquatic environment , (3) a high salt containing environment , and (4) a heavy metal polluted environment (lead-zinc mine in Gejiu city in Yunnan province). Soil samples from forests adjacent to these four special environments were also collected simultaneously, in order to detect the relative contributions of ecological niche and geographic distance to the genetic diversity of A. oligospora. Samples from other two geographic sites from Xinjiang province, Kanas Nature Reserve and Turfan, were also obtained for analyses. In short, while two geographic populations in Xinjiang province contained one ecological population each (both were from soil samples), the other four geographic populations from Sichuan province and Yunnan province contained two ecological populations each, with one from natural forest soils and the other from aquatic environments or mines. The detailed information of sampling sites and habitats are given in To compare the level and partitioning of genetic variation within the Chinese A. oligospora were obtained using the standard “sprinkle plate” method in which a small quantity of substrate was placed on agar and entomopathogenic “bait” nematodes were added to selectively enhance the growth of nematode-trapping fungi  and WCC3 . The obtained sequences from the two strains were compared with identify SNPs.HindIII at fragment #2 and #47, NaeI, EcoRI and PvuII at fragment #4, EcoRI at #15, DraI, and XhoI at fragment #26, ApalI at fragment #55, ApaI at fragment #57, Bpu1102I, and NruI at fragment #76. For strain genotyping, the DNA fragments that contained SNPs detectable by restriction enzyme digests were first amplified using PCR. After confirmation of the PCR products using agarose gel electrophoresis, the fragments were digested using the specific restriction enzymes. PCR products and restriction digests were electrophoresed in 1.0% agarose in 1× TBE, the genotypic data were scored by the electrophoresis bands viewed under ultraviolet transillummination  occurring in the sample. A total of 30 replicates were performed of each simulation for K = 1–11, with a burnin of 10,000, and Markov Chain Monte Carlo (MCMC) of 100,000 iterations for the best fixed value of K. Finally, based on results indicated in the above mentioned analyses, the contributions of different levels of separation  to the overall genotypic diversity were assessed through the analysis of molecular variance (AMOVA). In this analysis, each regional population represented a geographic population, whereas the two ecological samples from each of the four regions represented subpopulations within each geographic population  to produce a two-dimensional visual summary of the observed genetic variation  Orbilia auricolor (Pfister A) and rBarD. The null hypothesis for IA is that there is random association  among alleles at different loci. A P-value of <0.05 would indicate that the null hypothesis should be rejected, and statistical significance was derived by comparing the observed dataset to 1000 simulated datasets generated assuming random recombination. The pairwise linkage disequilibria were also measured to assess the role of recombination to the genetic diversity in each population. We standardized the IA value by the number of loci with the rBarD algorithm adjusting for the numbers of loci, because the value of the traditional IA can be influenced by the number of loci analyzed . The adjustment helps facilitate better comparison between populations and studies.The anamorphic   Pfister . HoweverA. oligospora), a phylogenetic incompatibility occurs between two loci with two alleles each when all four possible genotypes are found in the population. The incompatibility ratio (IR), where IR = (number of incompatible pairs of sites in the test dataset)/(number of incompatible pairs of sites in a randomly shuffled dataset), can be used as a test for inferences of statistical significance  was calculated. Phylogenetic incompatibility is an indicator of recombination at the population level. In contrast, the lack of phylogenetic incompatibility implies asexual reproduction. In the simplest case, in a haploid species , commonly used phylogenetic methods developed to test for signals of phylogenetic tree congruence Archie , was adoA. oligospora strains were isolated. These strains were grouped into six geographic populations containing 10 subpopulations total  showed that strains WCC3, K44, and X1 were highly similar to each other. whereas strain DC2 was different from those three in several fragments a. These A. oligospora across geographic regions and ecological niches in western China, 12 polymorphic restriction sites distributed on eight of the 21 sequenced fragments that could be easily amplified in all populations were used to genotype all 228 A. oligospora strains. Our analyses identified a total of 143 genotypes. The clone-corrected dataset which was used for the following analyses included 143 individuals.To analyze the genetic diversity of The overall genotypic diversity for the whole sample was 0.265, and there was a range of variation in genotypic diversity among the 10 subpopulations, from a low of 0.074 in Turfan population to a high of 0.447 in Dianchi population. The genotypic diversity and number of repetition of these genotypes were presented for each sub-population in P = 0.18). No correlation was found between altitudinal differences and population genetic distances (P = 0.52).DNA sequence divergence among populations was estimated using pairwise Nei's genetic distance. The lowest value of 0.011 was found between Kanas and Turfan populations from Xinjiang Province. The highest distance was 0.666, between forest soil populations from Heijing and Turfan populations from Xinjiang Province. The Mantel test indicated a positive correlation between genetic and geographical distances, with a correlation coefficient of 0.276. However, the correlation was statistically insignificant (A. oligospora. PC1 accounts for approximately twice the amount of variation as PC2 (41.89% vs. 23.09%).Using PCA, populations from Dianchi lake (polluted aquatic environment), Gejiu City (lead-zinc mine), and Heijing county  grouped together and were distinct from populations from forests adjacent to these three special environments. However, the two sub-population from Jiuzhaigou Nature Reserve (pristine aquatic environment and forest soil) were grouped together . The dirA. oligospora strains . The most evident differences in cluster pattern were found between high salt containing population and its counterpart from forest soil (HJ vs. HJ_F). However, membership assignments to the inferred genetic clusters revealed similar patterns between populations from pristine aquatic environment and its corresponding forest soils (JZG vs. JZG_F), and between populations from Kanas Nature Reserve and Turfan (KN vs. TF), even though they are geographically separated by over 300 km.P < 0.001). The analysis of molecular variance (AMOVA) revealed that 24% of the total genetic variation was due to ecological niche separations, and geographic distributions showed no contribution to the total genetic variation. The majority of the genetic variance (76%) was found within individual subpopulations  the total sample, (2) samples from each geographic and ecological populations . Due to Candida albicans  collected from terrestrial and aquatic environments, respectively. Sequence alignments with two additional isolates (K44 and X1) from forest soils of Kanas Nature Reserve and Turfan, identified more, although somewhat limited number of additional SNPs. The comparatively large difference between strain DC2 (from a polluted lake) and the other three strains from pristine forest soils suggest that pollution and environmental stress might have contributed to ecological divergence for populations in such environments.Here, we reported the SNP distributions and frequencies for the first time for a nematode-trapping fungal species. We identified 268 SNPs distributed among 21 cloned fragments, with an average SNP frequency of 2.76% per nucleotide site. The frequency is comparable to those observed in several other fungal species. For example, there is a SNP frequency of 2.05% per nucleotide in the model yeast A. oligospora was approximately 5, which is quite similar to that of the recently reported mushroom species T. matsutake   are facultative parasites. Nematodes in these environments could activate the parasitic behavior from the saprophagous habitats (Pathak et al. A. oligospora genotypes. Evidences of genetic differentiation based on habitats have also been reported for several other microorganisms. For example, the genomic divergence of Bacillus simplex strains was found to significantly correlate with ecological contrasts, but not with geographic distance (Sikorski and Nevo Grosmannia clavigera, from the same host species in different geographic areas were genetically closer than those collected from different host species occurring in the same geographic region (Alamouti et al. Metarhizium anisopliae (Bidochka et al. Claviceps purpurea (Douhan et al. Venturia inaequalis (Gladieux et al. Colletotrichum gloeosporioides agg. (Gazis et al. Our analyses identified that ecological niches exerted more effect on genetic diversity and differentiation than geographic separation among subpopulations of A. oligospora suggested a general pattern of continuous range expansion coupled with recent local adaptation within individual lineages and sublineages (Zhang et al. A. oligospora.DNA sequence variation among strains of Stressful conditions are known to increase genetic polymorphism, recombination, mutation, gene conversion, and sex in biological organisms (Korol et al. In our study, as expected, a predominantly asexual reproductive mode was found for the whole samples. However, we also found unambiguous evidence for recombination in populations from polluted aquatic, high salt containing, and heavy-metal polluted environments. As different genotypes may display different fitness in different environments, mutation or recombination could have slowed the loss of genetic variation due to genetic drift and local selection in these populations. The high genotype diversity in more stressful conditions likely reflected the adaptive significance of high genetic diversities in stressful environments. The high genotype diversity was partly due to the increased rate of recombination in stressful environments, as demonstrated in our tests. This result is consistent with previous observations that recombination frequencies tend to increase under stressful conditions (Hoffmann and Parsons A. oligospora populations may have different reproductive strategies, and the levels of genotypic diversity were at least partly associated with the levels of recombination. How this variation in reproductive mode across A. oligospora populations is controlled and whether these distinct populations use either sexual and/or asexual reproductive modes to regulate gene flow, to select more rapidly adapted genotypes, and to respond to heterogeneous environments remain open questions. Extensive environmental sampling and laboratory mating assays would allow full understanding the mating behavior and differentiating the relative contribution of clonal versus sexual reproductive modes in this fungus.It is known that both asexual and sexual reproductive modes can confer significant advantages as well as disadvantages to fungal populations (Sun and Heitman A. oligospora seemed to have a strong ability to increase the rates of mutation and recombination under environmental stress, it might be better to select wild strains from stressed soils as biocontrol strains. Such strains could generate recombinant genotypes by crossing with native strains and enhance their environmental adaptability and parasitizing ability. Additional field investigations are needed to evaluate the effectiveness of this strategy.Given the close relationship between ecological divergence and reproductive isolation and consequently the presumed role of ecological adaptation in speciation, an evaluation of the studied organism's ecology could also help species delimitation in nematode-trapping fungi. A general decrease in genome-wide within-population genetic variation, and an increase in between-population genetic variation should be tested using adaptive genes that are directly involved in responding to environmental changes. These genetic differences could be compared with phenotypic variation among genotypes in their physiological ability to cope with variable microenvironments. Understanding the ecological adaption of this fungus could help design sustainable and effective biocontrol application strategies. For example, because


Dicamptodon tenebrosus) is classified as threatened at the northern periphery of its range in British Columbia (BC), Canada, primarily due to forestry practices and habitat fragmentation. Characterising dispersal behaviour and population connectivity is therefore a priority for this region, while genetic differentiation in core versus peripheral locations remains unstudied in this wide-ranging species. We present seven new polymorphic microsatellite markers for use in population genetic analyses of D. tenebrosus. We examine locus characteristics and genetic variation in 12 streams at the species' northern range limit in BC, and within two regions representing sub-peripheral (North Cascades) and core localities (South Cascades) in Washington State, United States. In BC, the number of alleles per locus ranged from 2–5 and observed heterozygosity ranged from 0.044–0.825. Genetic differentiation was highest between BC and the South Cascades, and intermediate between BC and the North Cascades. Across loci, mean allelic richness was similar across regions, while private allelic richness was highest in the core locality (corrected for sample size). These new microsatellite loci will be a valuable addition to existing markers for detailed landscape and population genetic analyses of D. tenebrosus across its range.The Coastal Giant Salamander ( Dicamptodon tenebrosus) is endemic to the Pacific North-west coast of North America, from northern California up to an approximately 100 km2 area in the Chilliwack Valley of southern British Columbia (BC), Canada. In Canada, D. tenebrosus is designated as Threatened and is on the provincial Red List in BC (COSEWIC 2000: http://www.sararegistry.gc.ca/species/speciesDetails), due to high susceptibility to decline from habitat degradation owing to forestry practices D. tenebrosus, complementing existing microsatellite loci developed for D. tenebrosusD. copeiD. tenebrosus at their northern range limit to evaluate genetic variation at these new microsatellite markers. We also provide locus characteristics for two regions in Washington State (WA) to examine for differences in allelic diversity, heterozygosity, and genetic differentiation between peripheral populations in BC, with sub-peripheral (North Cascades) and core (South Cascades) regions of the species' range. These new loci have potential use in other Dicamptodon species and combined with existing loci, will enable range-wide genetic analyses using high-resolution microsatellite data.Species at the periphery of their range may show reduced genetic diversity that can limit microsatellite variation and the potential for detailed genetic analyses that are relevant for conservation This research was conducted with approval of the Animal Care Committee of the University of British Columbia (permit A08-0241) in accordance with the Canadian Council on Animal Care.D. tenebrosus throughout the Chilliwack Valley of British Columbia in Canada (∼70 km2 in area), and from two regions in Washington State, United States (each ∼30 km2 in area). All individuals were anaesthetised in a 0.05% solution of MS-222 (0.5 g/L), before a 2–4 mm2 sample of tail tissue was cut from the tail tip and preserved in 90% ethanol. Individuals were immediately recovered in a stream water bath for 10–20 minutes before being returned to their capture location. Genomic DNA was extracted from British Columbian samples using a standard phenol-chloroform extraction method 13, (GT)13, (GTAT)7 and (GATA)7 oligonucleotide repeats D. tenebrosus in British Columbia . We then compare BC locus characteristics with those from individuals collected within the North Cascades (NC) , and the South Cascades (SC)  of Washington State, which are located approximately 60 km and 350 km south of the Chilliwack Valley respectively. NC may therefore be regarded as sub-peripheral, and SC as core within the entire range of D. tenebrosus. Individuals with missing genetic data were excluded from the dataset. Genotypes from each region  were screened for genetic relatedness in the program COLONY 2.0 D. tenebrosus in the Cascade Mountains Samples were collected from Microsatellite loci were amplified for all individuals by polymerase chain reaction (PCR) using a QIAGEN Multiplex PCR Kit. All loci were labelled with fluorescent dye (forward primer) and multiplexed in a 20 µl reaction on a PTC-100 Thermal Cycler (MJ Research). PCR reagents were: 2x QIAGEN Multiplex PCR Master Mix , 0.2 µM primer mix (forward and reverse), 5x Q-Solution  and 10-20 ng DNA. Amplification conditions followed the QIAGEN protocol for Multiplex PCR using Q-Solution: 15 min at 95°C , followed by 30 cycles consisting of 94°C for 30 s, 57°C annealing for 1.5 min, 72°C for 1.5 min, and a final extension step at 72°C for 10 min. Electrophoresis of multiplexed PCR products was performed on an ABI3730 automated sequencer (Applied Biosystems) with a LIZ500 size standard run with each sample. Genotypes were manually scored using Genemapper v.3.7 (Applied Biosystems). The presence and frequency of null alleles (Oosterhout method) was examined using MICROCHECKER He) across loci increased with sample size of each region, with mean He (± s.e.) being highest in BC , intermediate in SC  and lowest in NC (0.266±0.09)  showed an expected increase with sample size in each region . However, when corrected for sample size (n = 9 samples with 18 genes based on NC), allelic richness in BC was more similar to NC and SC , indicating that estimates of allelic richness in NC and SC may be underestimated due to sample size. Despite a higher number of private alleles in BC (as expected for a greater sample size), their mean frequency was very low (mean 0.04±0.02) compared to NC (0.295±0.0) and SC (0.22±0.15) . FurtherPairwise Fst was calculated excluding Dicten 27 and Dicten 11 in all regions. Genetic differentiation (Fst) was significant (P<0.05) for all pairwise regional comparisons, and followed expectations of isolation by distance, with the lowest differentiation between the northern peripheral (BC) and sub-peripheral (NC) regions (Fst = 0.176) The highest genetic differentiation was between the most spatially distant BC and SC regions (Fst = 0.330) and was intermediate between NC and SC (Fst = 0.192). Genetic differentiaton within BC populations was low to moderate and significant for ∼31% of all pairwise comparisons, while all 12 populations differed significantly from NC and SC , furtherD. tenebrosus, and show variation in allelic richness and increasing genetic differentiation between peripheral (BC), sub-peripheral (NC) and core (SC) regions of the species' range. Although our sample sizes restrict conclusions regarding differences in genetic variation between regions, our data indicate that allelic richness is comparable between regions, with rare alleles being more common in the range core. Previous studies at the species' northern periphery have found monomorphism or low genetic diversity in loci for D. tenebrosus (range of He for three loci  = 0.0-0.24) He for nine loci  = 0.18-0.85) D. tenebrosus. However, our data provide the first indication of high genetic differentiation between British Columbian and Washington State regions, lending it to broader questions relating to ecological adaptation in this species. Not only will the new loci provide increased potential for high data resolution in genetic studies of this species, but may also be useful in studies of three other Dicamptodon species occurring in the Pacific Northwest . We suggest these new loci will complement existing loci We present seven new polymorphic microsatellite loci for Table S1Locus characteristics by population, British Columbia(0.15 MB DOC)Click here for additional data file.


Pristipomoides filamentosus, with special focus on Hawai'i. We applied mtDNA cytochrome b and 11 microsatellite loci to 26 samples  collected across 17,000 km from Hawai'i to the western Indian Ocean. Results indicate that P. filamentosus is a highly dispersive species with low but significant population structure  due entirely to the isolation of Hawai'i. No population structure was detected across 14,000 km of the Indo-Pacific from Tonga in the Central Pacific to the Seychelles in the western Indian Ocean, a pattern rarely observed in reef species. Despite a long pelagic phase (60–180 days), interisland dispersal as adults, and extensive gene flow across the Indo-Pacific, P. filamentosus is unable to maintain population connectivity with Hawai'i. Coalescent analyses indicate that P. filamentosus may have colonized Hawai'i 26 K–52 K y ago against prevailing currents, with dispersal away from Hawai'i dominating migration estimates. P. filamentosus harbors low genetic diversity in Hawai'i, a common pattern in marine fishes, and our data indicate a single archipelago-wide stock. However, like the Hawaiian Grouper, Hyporthodus quernus, this snapper had several significant pairwise comparisons (FST) clustered around the middle of the archipelago  indicating that this region may be isolated or (more likely) receives input from Johnston Atoll to the south.In the tropical Indo-Pacific, most phylogeographic studies have focused on the shallow-water taxa that inhabit reefs to approximately 30 m depth. Little is known about the large predatory fishes, primarily snappers (subfamily Etelinae) and groupers (subfamily Epinephelinae) that occur at 100–400 m. These long-lived, slow-growing species support fisheries across the Indo-Pacific, yet no comprehensive genetic surveys within this group have been conducted. Here we contribute the first range-wide survey of a deepwater Indo-Pacific snapper,  The effort to understand patterns of genetic connectivity in the Indo-Pacific has largely focused on shallow-water reef associated taxa and pelagic species that support multinational fisheries. The use of molecular tools to identify patterns of population subdivision and the geographic distribution of genetic diversity has provided insights into gene flow and the definition of management units, historical demography, and the impacts of biogeogeographic barriers on dispersal. While there is some incongruence among datasets, large scale patterns have emerged. A growing number of studies indicate a lack of genetic subdivision in reef fishes across nearly 10,000 km from French Polynesia in the Central Pacific to Western Australia and Cocos Keeling in the Indian Ocean Species distributions and a growing number of phylogeographic comparisons of shallow-water species indicate that Pleistocene sea level fluctuations had major impacts on population sizes and connectivity patterns between the Pacific and Indian Oceans. A defining barrier in the Indo-Pacific is the shallow Sunda shelf, surrounding the Malay Peninsula and western islands of Indonesia, and the Sahul shelf off northern Australia and New Guinea. This region, which is exposed during periods of low sea level, separates the Pacific and Indian Oceans and is known as the Indo-Pacific Barrier (IPB) Pristipomoides tend to live longer and spend more time as pelagic larvae than their shallow-water counterparts While our understanding of the impact of biogeographic barriers and historical processes on shallow-water reef organisms is developing, there are no studies that have examined these same processes in deepwater species across the Indo-Pacific. Of particular interest, are the demersal snappers (subfamily Etelinae) found between 100–400 m on the continental shelves and islands throughout the tropical Indo-Pacific and that support important fisheries. While the majority of shallow-water reef species are restricted to a narrow band of habitat in the upper 30 m of tropical oceans it is not unusual for deepwater species to inhabit much wider depth ranges (e.g. 100–300 m). Field studies indicate that shallow-water reef fishes have high site fidelity and move only short distances as adults (<2 km) Pristipomoides filamentosus . This species is better understood than most deepwater fishes due to fishery-oriented research and successful culturing efforts. Adults are found in rocky habitat at depths of 100–360 m from Hawai'i in the Pacific to East Africa and the Red Sea P. filamentosus is a slow growing and long lived species (≥40 years) P. filamentosus can remain planktonic at a large size (37–70 mm TL) with a pelagic duration lasting 60–180 days P. filamentosus to move great distances as adults indicate that this species may be more dispersive than shallow-water reef associated species. However, this is not a foregone conclusion, as the only other molecular appraisal in this genus (Pristipomoides multidens) indicated limited dispersal between Indonesian islands separated by less than 500 km P. filamentosus at nine locations across the Indo-Pacific. Additionally we sampled 17 locations in Hawai'i, a 2,600 km linear array of islands and atolls, to test for fine-scale population connectivity in the archipelago. Specifically we addressed the following questions: 1) Does this deepwater species demonstrate high levels of genetic connectivity across the Indo-Pacific as would be predicted from life history characteristics? 2) Is there gene flow between Hawai'i and Central Pacific populations of P. filamentosus? 3) Does Hawai'i contain one or more populations pertinent to management efforts? 4) Does the IPB limit dispersal in this species as has been demonstrated for shallow-water taxa?Here we present an Indo-Pacific wide survey of a deepwater snapper, the Crimson Jobfish, P. filamentosus were collected from 26 locations across the species range including 17 locations within the Hawaiian Archipelago  were amplified using the primers H15020 b-07L eneious Pro 5.0 . In all cases, alignment was unambiguous with no indels or frameshift mutations. Unique haplotypes were identified using the Hapotype Collapser and Converter option in FaBox v.1.35 (http://birc.au.dk/fabox), and deposited in GenBank (accession numbers: JQ083084–JQ083155).Approximately 560 bp of mitochondrial cytochrome h) and nucleotide diversity (π), were estimated in arlequin 3.5 h and π between populations we conducted Welch's t-tests, which allow for unequal variances, using the t-test calculator of GraphPad Software . Median-joining networks were constructed using the program network 4.6 with default settings arlequin, to determine whether the number of pairwise differences among all DNA sequences reflected expanding or stable populations FSFS indicate an excess of low-frequency haplotypes, a signature characteristic of either selection or a recent demographic expansion Summary statistics for the mtDNA dataset, including haplotype diversity  at the NSF EPSCoR sequencing facility at the Hawai'i Institute of Marine Biology.Fifteen microsatellite loci previously designed for mstools 3.1 micro-checker 2.2 genepop 4.0 arlequin. We maintained α = 0.05 among all pairwise tests by controlling for the false discovery rate as recommended by Benjamini and Yekutieli reeNA with 1,000 bootstrap replicates FST outlier method implemented in lositran 1.0 HO) and expected (HE) heterozygosities, number of alleles, and allelic richness were calculated for each locus per population using fstat 2.9 Most samples were collected by proxy. To avoid using replicate specimens of the same individual we used N = 812). Second, we compared samples from nine locations (plus Hawai'i) across the Indo-Pacific (N = 485). Due to the large sample size from Hawai'i (N = 812) in comparison to other populations across the Indo-Pacific (range N = 7–75) we randomly sub-sampled 75 individuals from the Hawaiian dataset for the range-wide comparison. This sub-sample was used in all Indo-Pacific comparisons . To test for hierarchical population genetic structure in P. filamentosus, an analysis of molecular variance (AMOVA) was performed in arlequin. Two different F-statistics were calculated: Wright's FST based on allele frequencies (microsatellites) and an analogue of FST (ΦST), which incorporates the model of sequence evolution (mtDNA). Significance of pairwise F-statistics were tested using 20,000 permutations. The AIC implemented in jmodeltest 0.1.1 b sequence evolution. Because this model is not implemented in arlequin, we used the most similar model available ST values.Population structure was assessed at two geographic scales. First we examined genetic structure among 17 locations in the Hawaiian Archipelago , N = 812-Pacific , N = 485structure 2.3.3 structureharvester 0.6.1 to determine which K was the best fit to the data http://taylor0.biology.ucla.edu/struct_harvest/). Based on the results we conducted an additional 10 replicates for each value of K ranging from 1 to 3 for each dataset.Population structure was also assessed using the Bayesian clustering analysis implemented in ST (mtDNA) or FST (microsatellites) values and geographic distance using the Isolation-by-Distance Web Service 3.16 FST/ΦST values and again with negative FST/ΦST values converted to zeros.Mantel tests were performed to determine whether significant isolation-by-distance exists among populations by testing for correlation between pairwise Φstructure we divided the dataset into K groups, randomly sampled 50 individuals from each group, and estimated long-term average migration rates between groups with migrate 3.1.6 Neμ) and M (m/μ) were calculated under a Metropolis-Hastings sampling strategy with three replicates of one long MCMC chain run for 1 million generations, a burn-in of 25%, and four short heated chains. Initial runs were conducted with default priors with an unrestricted migration model. Some microsatellite loci consisted of complex repeat motifs and could not be coded by repeat number, therefore, migrate runs for the nuclear dataset were conducted using the infinite allele model of evolution. Posterior distributions for θ and M from initial runs were used to inform priors for subsequent runs. Estimates of the number of migrants between groups per generation (Nm) were calculated as θ×M for the mtDNA dataset and as θ×M/4 for the microsatellite dataset, where θ belongs to the recipient population Using the results from ST) we grouped the specimens from the Hawaiian locations of Northampton Seamounts and Laysan Island (Northampton), Pioneer and Lisianski (Pioneer), and Maui and Kaho'olawe Islands (Maui).Pairs of identical genotypes were detected twice, in specimens from Rowley Shoals and Scotts Reef. Only one of each genotype and the corresponding mtDNA sequence were retained for analyses. Due to geographic proximity and a lack of genetic differentiation , number of haplotypes (NH), haplotype diversity (h), and nucleotide diversity (π) for each location are provided in P. filamentosus was π = 0.0010 while the corresponding haplotype diversity was h = 0.38. Across all samples π = 0.0002–0.0024 and h = 0.00–0.81. Hawaiian samples (excluding populations N≤5) demonstrated significantly lower values of π  and h  compared to other locations in the Indo-Pacific.We resolved a 503 bp segment of cytngletons , Fig. 2.P. filamentosus is a star shaped phylogeny with no more than four bp differences between any two haplotypes. The most common haplotype (78.4% of specimens) was found at every location sampled  indicating an excess of low-frequency haplotypes. When the dataset was divided by geographic region (Hawai'i vs. Indo-Pacific) Fu's FS was still significant . The mismatch distribution for all datasets was unimodal . Together these data indicate expanding populations. Under the spatial expansion model we found τ = 0.889, θ0 = 0.00, and θ1 = ∞. Based on an estimated generation time of 7 years 6 years between lineages eNf0 = 0 and current effective population size estimate is eNf1 = ∞. When only the Hawaiian populations  were considered τ = 0.262, θ0 = 0.00, and θ1 = ∞ with a coalescence time of roughly 26 K–52 K y, initial female effective populations size estimate is eNf0 = 0 and current effective population size estimate is eNf1 = ∞. While estimates for the Indo-Pacific population excluding Hawai'i indicated similar effective population sizes, this group had a much older coalescence .The median-joining network for  sampled . Fu's FFST values or the overall patterns of population structure. Therefore, we excluded the loci Pfi1.6B3 and Pfi1.3A from all analyses. Among the remaining eleven markers the following showed evidence of linkage disequilibrium at only one location: Pfi2.9C/Pfi2D , Pfi2.9C /Pfi1.9C , Pfri2.8A/Pfi1.5C . The following samples did not meet HWE expectations at single loci: Scott Reef , Christmas Island , New Caledonia , Tonga , Lana'i . In each case an excess of homozygotes was indicated which could reflect the presence of null alleles. However, FreeNA indicated a low frequency of null alleles across loci (range = 0.00–0.06). Calculating FST values using the corrected allele frequencies we found overlapping 95% confidence intervals for all pairs of corrected and uncorrected values. To ensure that patterns of population structure were not driven by any single locus we calculated F-statistics by removing one locus at a time. In no case did the patterns change substantially. Using an FST outlier method we found no evidence of selection at any loci.After correcting for multiple comparisons, there was evidence of physical linkage among the loci pairs Pfi1.3A /Pfi1.5C and Pfi1.6B2 /Pfi1.6B3 in 16 of 17 and 14 of 17 Hawai'i populations, respectively. Pfi1.3A/Pfi1.5C were also in linkage disequilibrium (LD) in 5 of 9 populations outside of Hawai'i. Removal of one of each loci pair from the dataset did not make substantial difference in N≤5) demonstrated significantly lower values of allelic richness (AR = 1.92–2.02) and HE (0.043–0.060) than the other Indo-Pacific locations . For unknown reasons microsatellite primers failed to amplify fragments in most of the specimens from Gascoyne. Even after a second extraction of DNA from the original tissue we could not get these specimens to amplify. Here we report the data from the Gascoyne specimens (7 out of 44) that amplified for at least 7 of the 9 microsatellites.All microsatellite loci were polymorphic with the number of alleles per locus ranging from 3–32 . HawaiiaP. filamentosus across the range . However, when only the Hawaiian populations were considered overall population structure was not significant (N = 810) . Only 9 of 272 pairwise comparisons within the archipelago were significant (N = 75), we found low but significant structure across the Indo-Pacific . Pairwise comparisons indicate that most of the genetic structure is due to the distinct population in Hawai'i . The only other significant pairwise comparisons were between Seychelles and Tonga  and Seychelles and Scott Reef . When Hawai'i was removed from the analysis overall population structure was no longer significant .We detected low but significant population structure in  Hawai'i . This postructure analyses corroborate the patterns of population differentiation revealed with F-statistics. No population subdivisions were detected in Hawai'i , whereas the analysis separated the Indo-Pacific into two populations (K = 2): Hawai'i and the other nine Indo-Pacific sites  (chelles) , 4. Migrchelles) . MigratiST) and geographic distance across Hawai'i in mtDNA . However, after excluding Salmon Banks, which is at the furthest end of the sampled range in the archipelago, the correlation was no longer significant . There was no evidence of isolation by distance across the Indo-Pacific in either the mtDNA or microsatellite datasets.We found only weak evidence for isolation by distance in the mtDNA: a finding not supported by the microsatellite dataset. Mantel tests showed a correlation between genetic distance  across the Indo-Pacific. However, pairwise population comparisons and the Bayesian clustering analyses indicated that the genetic structure is due to the isolation of Hawai'i. This population is divergent from all other locations in both the mtDNA and nuclear datasets. Overall population structure within Hawai'i was non-significant but we did detect several significant pairwise comparisons that included the adjacent sites of St. Rogatien, Brooks Banks, and Gardner in the middle of the archipelago. Our results indicate that P. filamentosus is a highly dispersive species that displays little to no population structuring across 14,000 km from Tonga in the Central Pacific to the Seychelles in the western Indian Ocean, a pattern documented in only two shallow-water reef associated fishes  so that while the shallow-water fauna underwent widespread extirpations, some deepwater taxa could have persisted and even maintained low levels of gene flow between ocean basins. However, this scenario seems unlikely in P. filamentosus. The mtDNA haplotype network for P. filamentosus is a tight star shaped phylogeny with haplotypes differing by a maximum of only four mutations and offers no signal of past isolation events. If populations had diverged between oceans during the Pleistocene we would expect a network with greater complexity as detected in a variety of shallow-water taxa Lutjanus kasmira and L. fulvusA lack of population structure across the Central Pacific (exclusive of Hawai'i) has been demonstrated in many shallow-water taxa The Hawaiian Archipelago is geologically young with Kure, in the northwest, having emerged approximately 30 million years ago P. filamentosus  A. nigroris, to 185 K–371 K y for the Ornate Butterflyfish, Chaetodon ornatissimusP. filamentosus near the lower end of the range at 26 K–52 K y , Hawaiian populations demonstrate lower genetic diversity compared to populations in the Central Pacific . Only thP. filamentosus constitutes the largest proportion of catch in the commercial bottomfish fishery in Hawai'i P. filamentosus, Shaklee and Samallow P. filamentosus at six locations within Hawai'i using five allozyme markers. They detected no population structure across about two-thirds of the archipelago. Here we increase the geographic coverage to include 17 locations from the Big Island to Salmon Banks at the Northwest end of the Island chain and employ 11 microsatellites. Overall we found no evidence of genetic structure within the Hawaiian Archipelago, as is the case for many shallow-water reef fauna Hyporthodus quernus (previously Epinephelus quernus) P. filamentosus when there appears to be a single genetic stock at the ends of the range (Main Hawaiian Islands and upper NW Hawaiian Islands), but possibly a separate stock in the middle? Seven of the eight significant pairwise comparisons involve St. Rogatien, Brooks Banks, and Gardner Atoll in the middle of the archipelago (RO, BB, and GA in FST values indicate not an isolated stock, but input from the Johnston Atoll, the only island outside the archipelago that is regarded as part of the Hawaiian biogeographic province based on faunal similarities P. filamentosus is unable to overcome the formidable barriers that isolate Hawai'i from the rest of the Indo-Pacific.Given the sea level changes that accompany glacial cycles and disrupt reef habitats <130 m deep, we expected to find that this deepwater snapper had older, more stable, and more diverse populations than shallow-water fishes. This genetic survey reveals the opposite. We find it remarkable that a snapper with broad depth preference and a vast geographic range coalesces to a common ancestor within the last 100 K–200 K y indicating historical population reductions or a selective sweep during Pleistocene sea level fluctuations. We found evidence of recent population expansion and a lack of population structure across a geographic scale rarely seen in coastal marine fishes. If oceanographic currents in the past are similar to contemporary conditions then it is not just geographic distance isolating Hawai'i. Instead prevailing patterns of larval dispersal indicate that it is much more difficult to get to Hawai'i than to leave. Despite a long early pelagic phase, an ability to disperse as adults, and evidence of extensive gene flow across the Indo-Pacific,


Incarvillea sinensis is widely distributed from Southwest China to Northeast China and in the Russian Far East. The distribution of this species was thought to be influenced by the uplift of the Qinghai-Tibet Plateau and Quaternary glaciation. To reveal the imprints of geological events on the spatial genetic structure of Incarvillea sinensis, we examined two cpDNA segments ( trnH- psbA and trnS- trnfM) in 705 individuals from 47 localities.GST =0.843, NST = 0.975, P < 0.05). The survey detected two highly divergent cpDNA lineages connected by a deep gap with allopatric distributions: the southern lineage with higher genetic diversity and differentiation in the eastern Qinghai-Tibet Plateau, and the northern lineage in the region outside the Qinghai-Tibet Plateau. The divergence between these two lineages was estimated at 4.4 MYA. A correlation between the genetic and the geographic distances indicates that genetic drift was more influential than gene flow in the northern clade with lower diversity and divergence. However, a scenario of regional equilibrium between gene flow and drift was shown for the southern clade. The feature of spatial distribution of the genetic diversity of the southern lineage possibly indicated that allopatric fragmentation was dominant in the collections from the eastern Qinghai-Tibet Plateau.A total of 16 haplotypes was identified, and significant genetic differentiation was revealed (The results revealed that the uplift of the Qinghai-Tibet Plateau likely resulted in the significant divergence between the lineage in the eastern Qinghai-Tibet Plateau and the other one outside this area. The diverse niches in the eastern Qinghai-Tibet Plateau created a wide spectrum of habitats to accumulate and accommodate new mutations. The features of genetic diversity of populations outside the eastern Qinghai-Tibet Plateau seemed to reveal the imprints of extinction during the Glacial and the interglacial and postglacial recolonization. Our study is a typical case of the significance of the uplift of the Qinghai-Tibet Plateau and the Quaternary Glacial in spatial genetic structure of eastern Asian plants, and sheds new light on the evolution of biodiversity in the Qinghai-Tibet Plateau at the intraspecies level. Incarvillea sinensis Lam., which is widely distributed from the southwest China through to the northeast China and the Russian Far East  including 16 species, and it also is the type of the subgenus Incarvillea including two species. Compared with other members of the genus centered in the Himalaya-Hengduan Mountains, this species has a broad range and altitudinal distribution  were used to distinguish intraspecific taxa, but the annual populations with red flowers were confusing owing to the disjunct distribution between the northern region and the southernmost Lancangjiang Valley  contain all three morphological types, and appears to be the center of diversity for this species. We suspected that the spatial pattern of morphological characteristics likely dropped a hint to the potential divergence in genetics.After investigating all of the specimens of s Figure , Table 1Incarvillea suggests that its evolution may be related to the uplift of the Qinghai-Tibet Plateau. In addition, the continuous distribution of the subgenus Incarvillea, from the eastern Qinghai-Tibet Plateau to the Russian Far East, could partially be the result of the Quaternary expansion since part of its range was glaciated [Incarvillea sinensis occupies almost the full range of the subgenus except in Mongolia. Considering the morphological variations correlative to geography, we proposed that the uplift of the Qinghai-Tibetan Plateau potentially resulted in the genetic variations between populations within and outside of the Qinghai-Tibetan Plateau. Furthermore, the Pleistocene glaciation possibly induced the extinction of some northern populations and the modern distribution likely resulted from the postglacial and interglacial expansion. In order to test the hypothesis, we examined the genetic diversity of populations within and outside the Qinghai-Tibet Plateau and identified potential genetic lineages within this species.A study on the biogeography of laciated . IncarviIncarvillea sinensis using phylogeographical methods that can indicate how historical events such as range fragmentation, range expansion, and long distance dispersal, as well as current levels of gene flow, have influenced present-day distributions. The goals of this study are to provide new insights into the population genetic structure and responses of I. sinensis to geological events and reveal the imprints of geological events on the spatial pattern of the genetic diversity.Since the colonization of new habitats occurs through seeds, and chloroplast DNA is generally maternally inherited in angiosperms (meaning that the plastid genome is moved only by seeds) , chloroptrnH- psbA (377 sites) and trnS- trnfM  sequences from 705 individuals representing 47 collections, in which 38 polymorphic sites were confirmed by sequencing in both directions. Sequences with polymorphisms between each other were deposited in GenBank (Accession No. JQ858492-JQ858511). Of these polymorphisms, five were indels (insertions and deletions). Two indels were single nucleotides and the other three were six bp, five bp and seven bp long respectively. In subsequent analysis, indels with length more than one base were considered as a single mutation. All five indels were coded as binary characters, 1 s and 0 s, so that the matrix was shortened to 1381 characters in length: 372 sites from trnH- psbA and 1,009 sites from trnS- trnfM. The adjusted matrix included 23 variable sites, five of which were due to the presence of indels, and all of variable sites were informative and involved in all analysis. All of the sequences from the 47 localities contained 16 unique haplotypes when indels were included . The GST and NST at the regional level implicated that significant structures existed in the SEQPT, NEQTP and their combined regions, but not in the northern region. Genetic parameters at different levels are listed in Table The total gene diversity The analysis of molecular variance (AMOVA) of the data from the two cpDNA fragments indicates the presence of a strong differentiation in genetics among the lineages and regions Table . When por = 0.001, P = 0.48, Figure Fsts with pairwise Nei’s genetic distances. The positive correlation between genetic difference and geographic distance (isolation by distance) was encountered in the species . Because the haplotype H15 was detected in the DQ population, the annual DQ population with red flowers phylogenetically clustered together with all of the other perennial populations with red flowers from the southeastern Qinghai-Tibet Plateau, not together with the annual populations with red flowers from the northern region  with red flowers was most closely related to the perennial populations with yellow flowers from the northeastern Qinghai-Tibet Plateau . We note that both the differentiation index (GST) and the diversity index (hT) of the sorthern clade is higher than those of the southern clade. The degree of subdivision of the cpDNA diversity (GST) reflects the dispersal ability of the species and the effect of long-term range fragmentation, while the low GST value is indicative of high levels of gene flow through seeds [Incarvillea sinensis split, the seeds are ejected and the seed wings contribute to seed dispersal by wind. In the southern region accommodating the southern clade, orogenesis resulted in range fragmentation, and populations of I. sinensis were distributed in valleys, except for those populations with haplotypes H7 and H8  are widely distributed in NEQTP, except in Wenxian and Diebu in Gansu Province, and the remaining eight haplotypes are relatively rare and endemic to one valley or locality Figure , Table 1gh seeds . Once caGST > NST). Distantly related haplotypes were more often found in the same populations, as case 3 proposed by Pons & Petit [In contrast, the analogous limiting factors of seed movement did not exist in the northern region, and we could not identify the existence of a significant geographic structure in the northern clade . The long evolutionary history allowed this species to accumulate not only mutations relative to diversity, but also high differentiation between regions.The two major cpDNA lineages were revealed by means of reconstructing the phylogenetic tree and the haplotype network, which can be recognized by a connection of 11 mutations. The deep gap between these two clades indicates the early origin of the corresponding lineages in the evolution of the species, and the long-standing separation thereafter. We dated the divergence between the two lineages at approximately 4.4 MYA, which corresponds to the early Pliocene. The uplift of the Himalaya-Hengduan massifs was a complicated geological event, and the chronology of this process is still under debate -45. It iFor the northern lineage, the long history should be enough for localized gene flow to interact with drift to produce a pattern of isolation by distance across the region , but the correlation estimation of pairwise genetic and geographical distances show that drift was much more influential than gene flow on the distribution of genetic variability Figure E. PopulaOn the contrary, within the southern clade, including all haplotypes from the eastern Qinghai-Tibet Plateau, a significant association between the genetic variability and the geographical distribution was revealed by the Mantel test. The Mantel tests also showed that there was a significant effect of isolation by distance, which means that a regional equilibrium was reached and that more closely situated populations tended to communicate and be more genetically similar to one another . ObviousIncarvillea sinensis, as mentioned by Chen et al. [We detected two lineages connected by a deep gap within the species, and significant difference in genetic diversity between them. The significant genetic divergence concordant with geography and the features of genetic diversity seemed to reveal the imprints of geological events on the plant evolution. We preferred the significance of the uplift of the Qinghai-Tibet Plateau and the Quaternary Glacial in the distribution of the genetic diversity and differentiation of n et al. .Incarvillea sinensis populations, however, offers clues to the mechanisms of the evolutionary of the diversity of plants in the Qinghai-Tibet Plateau. Our present study not only shows that the spatial genetic structure within this species likely is the result of tectonic events and long term adaptation to distinct habits, but also supports an evidence on the higher diversity in the Qinghai-Tibet Plateau influenced by origins.The Qinghai-Tibet Plateau and its adjacent areas have been listed as one of the world’s biodiversity hotspots ,49. Two Incarvillea sinensis from CDBI, E, K, KUN, PE, and SZ. A number of populations, in some locations, became extinct due to habitat destruction. Numerous specimens were collected in Henan, Shandong, Inner Mongolia, Jilin, Liaoning, and Heilongjiang provinces during the 1920s–1950s. Subsequently, fewer collections have been recorded; and no new specimens were recorded since 1985. We were able to obtain only eight samples in these areas, seven of which , 0.5 μL of dNTPs (2.5 mM each), 1.25 μL of each primer (10 μM), 1 U Taq polymerase, 1 μL of template ( c. 50–100 ng double-stranded DNA), and 17.25 μL of sterile water.Two regions of cpDNA, trnH- psbA segment was amplified using the primers trnH [psbA [trnH was used for sequencing. The PCRs consisted of an initial denaturation at 94°C for 3 min, followed by 30 cycles of 30 s at 94°C (denaturation), 30 s at 52°C , and 30 s at 72°C (extension), and concluding with a final extension period of 7 min at 72°C.The ers trnH  and psbAnH [psbA , and trntrnS- trnfM fragment was amplified using the trnS and trnfM primers [The  primers  and bothtrnH- psbA and trnS- trnfM PCR products were sequenced in both directions using the ABI BigDye version 3.1 terminator cycle sequencing chemistry, with sequences read by an ABI 3730 Genetic Analyser (Applied Biosystems).The PCR products were purified with DNA-purification kits . The Incarvillea sinensis using MrBayes V3.1.2 [Incarvillea arguta was used to root the phylogenetic trees, which is similar to I. sinensis on morphology such as the flower, fruit and habit. We ran 500,000 Markov chain Monte Carlo (MCMC) generations for the Bayesian analysis, with a sampling frequency of 1 in 100 generations. The consensus tree was constructed from trees saved after reaching the stationarity . Tree-building methods might not always be the most appropriate way to represent genealogical relationships among haplotypes within a species, owing to the possibly shallow genetic divergence [Sequences were aligned using ClustalX , and ins V3.1.2 . Based overgence ; thus, avergence  with thevergence  and Cranvergence . Ambiguivergence .The genetic structure was evaluated by the analysis of molecular variance  using ARhS), total diversity (hT), and the level of population differentiation (GST and NST) at the species and regional levels. The occurrence of a significant phylogeographical structure was inferred by testing whether GST (the index that only considers haplotype identities) and NST (the index that takes into account a measure of haplotype divergence) were significantly different using 1000 permutations in PERMUT [We calculated within-population diversity  were compared with the matrix of geographical distance by means of a simple Mantel test to detect isolation by distance and to evaluate the relative influences of gene flow and drift on the regional population structure. We used 999 random permutations to test for the Mantel statistic significance.Traditional empleton  proposedGenAlEx6 , in whicDA = diXY - (diX + diY)/2 [dA) using ARLEQUIN, and then calculated the divergence time following the formula T = dA/2 μ [-9 substitutions per site per year [trnH- psbA and trns- trnfM fragments owing to the lack of any fossils for this group to use as calibration points.The net averaged population genetic distance, + diY)/2 , account+ diY)/2 . To date= dA/2 μ . In seedper year , which cper year ,80; etc.Shaotian Chen carried out field work, the molecular genetic studies, the statistical analysis, and drafted the manuscript. Yaowu Xing and Tao Su participated in sample collections and sequencing. Zhekun Zhou designed the study. David L. Dilcher and Douglas E. Soltis polished the manuscript, and David L. Dilcher helped to conceive of the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum in an area of high ITN coverage in western Kenya.Insecticide-treated bed nets (ITNs) reduce malaria transmission and are an important prevention tool. However, there are still information gaps on how the reduction in malaria transmission by ITNs affects parasite genetics population structure. This study examined the relationship between transmission reduction from ITN use and the population genetic diversity of P. falciparum-infected children (< five years) before introduction of ITNs  and five years after intervention .Parasite genetic diversity was assessed by scoring eight single copy neutral multilocus microsatellite (MS) markers in samples collected from A = 94% and He = 0.75) and follow up (%MA = 95% and He = 0.79) years. However, locus specific analysis detected significant differences for some individual loci between the two time points. Pfg377 loci, a gametocyte-specific MS marker showed significant increase in mixed infections and He in the follow up survey (%MA = 53% and He = 0.57) compared to the baseline (%MA = 30% and He = 0.29). An opposite trend was observed in the erythrocyte binding protein (EBP) MS marker. There was moderate genetic differentiation at the Pfg377 and TAA60 loci (FST = 0.117 and 0.137 respectively) between the baseline and post-ITN parasite populations. Further analysis revealed linkage disequilibrium (LD) of the microsatellites in the baseline (14 significant pair-wise tests and SA I= 0.016) that was broken in the follow up parasite population (6 significant pairs and SA I= 0.0003). The locus specific change in He, the moderate population differentiation and break in LD between the baseline and follow up years suggest an underlying change in population sub-structure despite the stability in the overall genetic diversity and multiple infection levels.There were no significant changes in overall high mixed infections and unbiased expected heterozygosity between baseline (%MP. falciparum population maintained an overall stability in genetic diversity after five years of high ITN coverage, there was significant locus specific change associated with gametocytes, marking these for further investigation.The results from this study suggest that although  Plasmodium falciparum malaria transmission by 70-90%. Most importantly, these trials have provided substantial evidence that use of ITNs significantly reduces all-cause mortality and malaria morbidity in children less than five years of age [1-Σni = 1 2i p] where n is the number of isolates sampled and i pis the frequency of the ith allele while sampling variance for He (Vs(He)) was calculated as Vs(He) = 2(n-1)/n3[2(n-2)][Σpi3-(Σpi2)2] [p-values.The infinite allele model which is more appropriate for analysis of the complex patterns observed in microsatellite loci in analysis . Allele analysis . The mea(Σpi2)2] ,22. The The prediction of the ITN-mediated decrease in transmission intensity and consequent reduction in genetic diversity was a possible increase in LD in the post-ITN parasite population. LD measures the degree of association between or among gene loci under the null hypothesis of no association . As suchSAI) using LIAN programme [0: VD = Ve. A significant LD was when the observed variance (VD) was greater than expected (Ve). Index of association was expressed as SA I= (VD/Ve-1)(r-1) where r is the number of loci tested. A 95% confidence limit was determined by Monte Carlo simulation.individual pair-wise LDs for baseline and post-ITN parasite populations were first obtained respectively by the Fisher's exact test adapted for haploid data using ARLEQUINS 3.11 programme . Multilorogramme  for bothST estimator [ST is a comparison of the sum of genetic variability within and between populations based on allele frequency differences in populations. Interpretation of the FST values at each locus was based on three categories defined earlier as no differentiation (0), low genetic differentiation (0 > 0.05), moderate differentiation (0.05-0.15) and great differentiation (0.15-0.25) [In order to further assess genetic diversity before and after ITNs intervention, genetic differentiation was tested using the Fstimator  implemenstimator . FST is 15-0.25) .p-value for the comparisons were adjusted using Bonferroni's correction [When multiple tests were conducted, significant levels of rrection .P. falciparum positive samples from baseline and post-ITN surveys, respectively. Locus P195 had an overall amplification rate of 90% for the baseline parasite population. All other loci had amplification rates of 94-100%.Results in this study comprise outcomes from the analysis of the eight microsatellite loci on 69 and 74 The extent of multiple infections was assessed based on the proportion of multiple alleles and mean allele counts Table . Overallp = 0.0076) after Bonferroni correction. There was no significant difference in the mean allele counts for all other microsatellite markers between the two parasite populations. Overall, mean allele count was similar in the baseline parasite population (3.4 ± 0.15) compared to the post-ITN parasite population (3.1 ± 0.12)  in the post-ITN survey than in the baseline parasite population (1.36 ± 0.07) although it was not a statistically significant difference  and post-ITN parasite populations (0.79 ± 0.038). However, He of individual loci showed extensive range in gene diversity .The allele size, number, frequency and richness for each MS shown in Figure p = 0.0018) as shown in Table Pair-wise LD for each individual MS locus for the baseline and post-ITN parasite populations was assessed. Of the 28 possible pair-wise tests LD was highly significant in a total of 14 pairs in the baseline parasite population while only 6 pairs were significant in the post-ITN population (D) of 1.21 and variance expected (Ve) of 1.10 (p = 0.01) compared to non-significant LD in the post-ITN population where VD and Ve were 1.183 and 1.180 respectively (p = 0.57) as shown in Table SAI) was significant in the baseline survey (0.016) compared to the post-ITN survey (0.0003). The multilocus LD results coupled with the significant pair-wise LD observed in individual microsatellites suggest that the existing non-random association between the MS loci in the baseline was broken in the post-ITN parasite population.The multilocus analysis showed significant LD in the baseline population with a variance in difference  respectively while all other remaining six loci had little differentiation. The results suggest that a moderately significant genetic variability at Pfg377 and TAA60 arose from differences between the two parasite populations while the majority of genetic variability remains within each population. Conversely, low genetic differentiation in the other six markers suggested much of the genetic variability resulted from within each parasite population.Results of the overall and locus specific genetic differentiation between the baseline and post-ITN surveys are shown in Table P. falciparum parasites was examined in this study. The overall proportion of mixed infections and heterozygosity were high at over 90% and 0.75 respectively both before and after ITN use with no significant reduction in these two parameters as well as the overall mean allele counts. This indicates an extensive multiplicity of circulating parasites in the area in spite of a dramatic decline in EIR post-ITN intervention. The results from this study are consistent with those of an earlier study conducted in areas with EIRs ranging from 0.4 to 31.8 in western Kenya, which also recorded over 80% mixed infections in both low and high malaria transmission areas [Plasmodium parasites in western Kenya despite reduction in EIRs.The effect of five years of high coverage with ITNs on the genetic diversity of on areas . This super se and natural selection. To minimize variation in host age, seasonality and geographic scale between baseline and post-ITN surveys, the current study sampled children less than five years of age, during similar transmission seasons and matched by villages in the two surveys. In addition, previous studies conducted in the ITN trial area showed that transmission reduction by use of ITNs changed humoral immunity in children and reduced childhood malaria morbidity and infant mortality resulting in overall decreased anti-malarial treatment [The stable overall genetic diversity after dramatic reduction in transmission intensity observed in the current study was unexpected by the initial prediction. The counter-intuitive results suggest that other factors may be involved in offsetting the effect of transmission reduction on parasite genetic diversity and/or stabilization of the overall genetic diversity of malaria parasite. Indeed, several previous studies suggest that genetic diversity of malaria parasite measured by different markers could be shaped directly or indirectly by multiple factors such as seasonality, geographic scale, migration, disease severity, and host age and immunity -55 in adreatment ,56,57. Te) in the EBP marker, but an increase at the Pfg377 locus. EBP MS locus flanks the functionally important erythrocyte binding protein gene expressed in the asexual stage of the life cycle and the gene may be under selection by human immune response [e for EBP MS marker observed in the post-ITN survey could reflect an indirect effect of ITNs on parasite genetic diversity but this will need further investigation. Likewise, PfPK2 MS which showed a decrease in mean allele counts flanks a putative protein kinase gene expressed in young trophozoite although the exact function is still not clear [While the overall stability in the parasite genetic diversity show the transmission reduction by ITNs had insignificant impact on parasite population, locus specific changes suggest there were some differences in the parasite population sub-structure. For example, PfPK2 microsatellite marker showed a decrease in the mean allele counts in the post-ITN survey, while Pfg377 microsatellite locus showed a significant increase in the proportion of infections with more than one allele. There was also a decrease in genetic diversity (Hresponse . It is pot clear . On the ot clear  showed aST was much lower in our study at other remaining loci examined in the same study in western Kenya [ST estimates at other loci observed in this study are expected since the samples were from the same area for baseline and post-ITN surveys. It is possible that the differentiation at TAA60 could represent random temporal effect/drift on the parasite population independent of transmission reduction. However, the differentiation observed at Pfg377 most likely resulted from the decreased transmission intensity by the use of ITNs rather than mere temporal effect since Pfg377 locus showed consistent increases in multiple infection and He after ITN intervention.Consistent with the genetic diversity data described above, overall genetic differentiation between the baseline and post-ITN parasite populations was low, mainly arising from variations in the Pfg377 and TAA60 microsatellite markers. The differentiation observed in this study for Pfg377 and TAA60 were higher between the baseline and post-ITN surveys than that observed between three geographically different areas in western Kenya . Yet FSTrn Kenya . The lowP. falciparum even in areas with intense and perennial transmission. It would be expected that decreasing transmission intensity by use of ITNs increases LD level based on a generalized assertion of higher LD in low transmission areas [The inter-relationship among LD, transmission intensity and genetic diversity of malaria parasites is complex and is still far from conclusive. The stronger LD observed in the baseline survey in our study area is consistent with the trend observed in previous studies conducted in the Democratic Republic of Congo, Zimbabwe and western Kenya lowland areas where malaria transmission is intense ,27,28,44on areas . HoweverP. falciparum parasites contributing to the observed differentiation between the two parasite populations. Of note, the data on Pfg377 locus showed an increase in diversity ecologically associated with reduction in transmission intensity. Further studies are necessary to evaluate the usefulness of this marker and other gametocyte-specific gene markers as molecular tools for monitoring how changes in transmission reflect gametocyte population dynamics [P. falciparum for extended periods, and in different geographic areas and in changing ITN coverage.This study suggests that although the parasite population maintained an overall stability after ITN use, there were locus specific changes in the dynamics . It is aThe authors declare that they have no competing interests.WG carried out genotyping work and genetic data analysis, and wrote the manuscript. SK was responsible for sample processing and laboratory diagnosis for the ITN trial, and participated in the design of this study. WH, FTK, DT, PPH, JG and KL implemented and conducted the Kenya ITN trial including collection of samples and epidemiological data. WH, BN and KL were PIs for the two phase ITN trial. JG, EW, MH and LS participated in the design of this study. SC and JW assisted in sample size calculation, sample selection and data analysis. YPS was the PI and responsible for the design of this study, participated in data analysis, and wrote manuscript. All authors contributed to data interpretation, read and approved the final manuscript.


Stipiturus mallee is an endangered passerine whose global distribution is confined to fire-prone, semi-arid mallee shrublands in south-eastern Australia. This species, with poor capacity for dispersal, has undergone a precipitous reduction in distribution and numbers in recent decades. We used genetic analyses of 11 length-variable, nuclear loci to examine population structure and processes within this species, across its global range. Populations of the Mallee Emu-wren exhibited a low to moderate level of genetic diversity, and evidence of bottlenecks and genetic drift. Bayesian clustering methods revealed weak genetic population structure across the species' range. The direct effects of large fires, together with associated changes in the spatial and temporal patterns of suitable habitat, have the potential to cause population bottlenecks, serial local extinctions and subsequent recolonisation, all of which may interact to erode and homogenise genetic diversity in this species. Movement among temporally and spatially shifting habitat, appears to maintain long-term genetic connectivity. A plausible explanation for the observed genetic patterns is that, following extensive fires, recolonisation exceeds in-situ survival as the primary driver of population recovery in this species. These findings suggest that dynamic, fire-dominated landscapes can drive genetic homogenisation of populations of species with low-mobility and specialised habitat that otherwise would be expected to show strongly structured populations. Such effects must be considered when formulating management actions to conserve species in fire-prone systems.Fire is a major disturbance process in many ecosystems world-wide, resulting in spatially and temporally dynamic landscapes. For populations occupying such environments, fire-induced landscape change is likely to influence population processes, and genetic patterns and structure among populations. The Mallee Emu-wren  Fire is a major disturbance process that changes landscape structure in many ecosystems worldwide The effects of fire-induced landscape change on ecological and population processes are complex, and major gaps remain in our knowledge Following disturbance by fire, subsequent population recovery will be influenced by many factors including the number and demographic parameters of survivors Stipiturus mallee. The Mallee Emu-wren is one of the tiniest members (4–6.5 g) of the family Maluridae, endemic to the semi-arid zone of south-east Australia Eucalyptus spp. ‘Mallee’ refers to the growth form of Eucalyptus spp. characterised by a large underground lignotuber from which multiple stems sprout. The Mallee Emu-wren is a resident habitat specialist and has a patchy distribution. Multi-scale studies of the habitat requirements of this species show that its distribution is strongly influenced by the post-fire age of vegetation, as a consequence of seral changes in the structure of ground-storey spinifex grass (Triodia scariosa) on which it depends Triodia-mallee vegetation older than 15 years since last burnt, with a slight preference for vegetation 15–29 years of age This study examines the genetic diversity and structure of a globally endangered bird species, the Mallee Emu-wren In recent decades, the Mallee Emu-wren has undergone a precipitous reduction in numbers and distribution. Large wildfires (>10 000 ha), exacerbated by drought, have caused the extinction of populations in parts of the south-west of its range. Large fires and inappropriate fire regimes that reduce the amount of habitat of suitable age are considered a significant threat to remaining populations. Geographic range contraction has occurred such that the Murray-Sunset and Hattah-Kulkyne National Parks in northern Victoria now support an estimated 92% of the global population. Small, scattered and isolated populations occur in South Australia in Billiatt and Ngarkat Conservation Parks and only one pair was recorded in the Big Desert/Wyperfeld reserve complex in Victoria in 2006 This study examines the genetic diversity and structure of the Mallee Emu-wren across its global range. The primary objective was to examine spatial genetic patterns among populations across the species' range to draw inferences about population structure and processes. Its occurence in a fire-prone environment, coupled with the intrinsic characteristics of the Mallee Emu-wren (e.g. poor flight capability) strongly suggest that it may exhibit a metapopulation structure at the landscape-scale, with relatively strong patterns of genetic divergence (i.e. population genetic structure) expected among geographically-dispersed sampling units The Mallee Emu-wren is inconspicuous and rare, has patchy distribution and occur in low density Long-term ecological and genetic studies on other species of the family Maluridae show males are philopatric, and related males (e.g. brothers) have a tendency to occupy neighbouring territories. Dispersal tends to be female biased, although the pattern and distance varies within populations and among species Mallee Emu-wrens were captured either by trapping in monofilament mist nets, or by throw nets after being lured by playback recordings of calls (©David Stewart/Nature Sound). The species is sexually dichromatic and males are easily distinguished from females by the presence of brilliant sky-blue throat and breast feathers All work was conducted under Deakin University Animal Ethics Committee approval (A35/2005) and research permits issued by the Department of Sustainability and Environment, Victoria (10003389) and the Department of Environment and Heritage, South Australia .Malurus splendensM. cyaneusStipiturus malachurusDNA was extracted from samples using a standard ethanol/chloroform extraction method Taq DNA polyermase, 5× buffer, 0.25 mM dNTPs, 0.5–4.5 mM MgCl2 (Promega/MBI Fementas) and 5–10 pmol of each primer pair in a total volume of 20 µl. Microsatellite products were run on an ABI® Capillary Analyser (Perkin Elmer) or a Li-Cor 4200 and 4300 Global IR2 two-dye DNA sequencer for separation and sizing. Putative homozygotes were amplified and genotyped twice to confirm their status. Positive and negative controls were used in all reactions.PCR reactions for microsatellites and product separation were performed in two different laboratories, with cross-referencing quality control. PCR reactions for each marker were optimised using the following: 20–40 ng of sample DNA, 0.5 Units Go ISF) were calculated in FSTAT 2.9.3.2 Standard measures of genetic diversity for length-variable markers were obtained in various programs, treating the six geographic locations as separate populations. GENALEX V6.0 FST calculated in GENEPOP V4.0 D, adjusted for small sample sizes (estD)FST and estD as measures of genetic differentiation each have different strengths and shortcomings, so we present both to infer population differentiation FST and estD values theoretically range from zero to one, with zero indicating no differentiation  FST is a widely used estimator of population genetic structure, with which many researchers are familiar D may be more appropriate for highly-variable markers (e.g. microsatellites) ST, .Enhanced effects of genetic drift in small, isolated populations are expected to lead to increased genetic differentiation among sites. The extent of genetic differentiation between each pair of sites was estimated using two allele-frequency-based measures: FIS - which is an expression of excess of heterozygotes relative to proportions expected under Hardy-Weinberg equilibrium The heterozygosity excess test in the program BOTTLENECK V1.2.03 To assess the extent of genetic population structure across the species' global range, individual genotype-based Bayesian clustering algorithms were implemented in both STRUCTURE 2.3.3  tructure was run using the admixture model with correlated allele frequencies, and K values 1 to 10. Twenty replicate runs were performed for each K value. Each run was 3×106 Markov Chain Monte Carlo (MCMC) repetitions following a burn-in period of 106 repetitions.SK≈5 populations K values 2 to 9. The Deviance Information Criterion (DIC) was used to select the model that best fit the genetic data K, and the most likely value of K was selected by visually assessing the point at which DIC first reached a plateau and the number of clusters to which individuals were proportionally assigned. The 10 runs with the lowest DIC values for the selected K-value were retained and their admixture estimates were averaged using CLUMPP V 1.1.2 TESS incorporates information on individual geographic coordinates and has been shown to be more powerful than non-spatial algorithms, especially in weakly differentiated populations Analyses of the nuclear loci show overall moderate to low levels of genetic diversity across the global range of the Mallee Emu-wren, with signatures of recent population bottlenecks in two locations, and local effects of genetic drift in others such that only a single sampled population showed neither phenomenon , 3 & 4.FIS values were positive and significant (p = 0.05) for Hattah-Kulkyne National Park, with two loci  showing significant homozygote excess FIS, none were significant. Linkage disequilibria were not detected for any loci pairs.e excess . One locA) was variable among loci, ranging from 2 to 32 alleles . As was expected based on the theoretical and empirical behaviour of the metrics, significant estD values were higher than their respective FST values  heterozygosity excess was detected for the Murray-Sunset National Park (East) and Ngarkat Conservation Park samples under the two-phase mutation model, indicating evidence of recent bottlenecks in these two populations (Significant (ulations .K = 1). Increased power resulting from the incorporation of spatial information in TESS, revealed the presence of two weak spatial genetic clusters across the study region (o. 6) were assigned to one cluster (cluster 2 in o. 2), the remaining populations within the Murray-Sunset and Hattah-Kulkyne reserve complex  were more strongly assigned to the alternative cluster  (consistent with reported demographic declines) and some evidence of genetic drift in other locations, indicate these localities may have experienced recent declines in effective population size. Bottlenecked and strongly inbreeding populations are important to identify for conservation management because of genetic threats to individual fitness and population viability The prevalence of several monomorphic loci and evidence of a bottleneck in the Ngarkat Conservation Park population  & 4 is cFingilla teydea polatzeki, a critically endangered passerine endemic to the island of Gran Canaria. Despite a 50% decline in the global population of this sub-species , temporal sampling found no genetic signature of a bottleneck. Furthermore, the post-fire population retained a high level of genetic diversity Our results contrast with those from a study of the genetic effects of a forest fire on the Blue Chaffinch A second genetic threat to the long-term viability of the Mallee Emu-wren can presumed to be the erosion of quantitative genetic variation necessary for adaptive evolution FST values ; that is, populations have similar allelic frequencies indicating substantial genetic connectivity  from within the fire footprint in-situ survivors is less likely to be accompanied by loss of much original genetic diversity, except in the presence of very strong, sustained or repeated bottlenecks Collectively, the genetic patterns and population structure found in this study can be attributed to the spatial and temporal patterns of fire in mallee ecosystems. Most fires are small (<100 ha in size), but intense landscape-scale wildfires exceeding 10,000 ha occur within the region every 10–20 years in-situ survival as the primary means of population recovery of the Mallee Emu-wren. These large fires  create vast homogenous areas in which the ground layer , shrub and canopy strata are all consumed Amytornis spp. of the arid-zones of Australia Ningaui yvonneaeGiven large, severe wildfires dominate the mallee landscape in-situ survival may occur in environments where fires leave numerous unburnt refuges, as in the case of the Blue Chaffinch of Gran Canaria discussed earlier. Unburnt refuges were prevalent throughout the fire area and were thought to enable a sufficient proportion of individuals to survive and persist, thereby mitigating the loss of genetic variability in the post-bottleneck population In-situ survivorship in unburnt refuges has been attributed to the rapid demographic recovery in birds , and weak genetic structure (STRUCTURE/TESS) is an optimistic message for the conservation of this species. The lack of marked population differentiation across its global range means that for management purposes  this species can be treated as a single genetic unit. Nevertheless, the finding of disrupted fine-scale population processes, as illustrated by the demographic and genetic impoverishment in the Ngarkat Conservation Park population, supports implementation of actions to assist population recovery. Reintroduction programs or the genetic restoration The apparent genetic connectivity of the Mallee Emu-wren inferred from the low population differentiation Click here for additional data file.Text S1Summary of CERVUS parentage analysis.(DOCX)Click here for additional data file.