Sicyopus zosterophorum demonstrated strong isolation across the Torres Strait, which was a geologically intermittent land barrier linking Australia to Papua New Guinea. There was a clear genetic break between the northwestern and the southwestern clusters in Si. zosterophorum (φST = 0.67502 for COI) and coalescent analyses revealed that the two populations split at 306 Kyr BP (95% HPD 79–625 Kyr BP), which is consistent with a Pleistocene separation caused by the Torres Strait barrier. However, this geographical barrier did not seem to affect Sm. fehlmanni. Historical and demographic hypotheses are raised to explain the different patterns of population structure and distribution between these species. Strategies aiming to conserve amphidromous fish should consider the presence of cryptic evolutionary lineages to prevent stock depletion.Both present-day and past processes can shape connectivity of populations. Pleistocene vicariant events and dispersal have shaped the present distribution and connectivity patterns of aquatic species in the Indo-Pacific region. In particular, the processes that have shaped distribution of amphidromous goby species still remain unknown. Previous studies show that phylogeographic breaks are observed between populations in the Indian and Pacific Oceans where the shallow Sunda shelf constituted a geographical barrier to dispersal, or that the large spans of open ocean that isolate the Hawaiian or Polynesian Islands are also barriers for amphidromous species even though they have great dispersal capacity. Here we assess past and present genetic structure of populations of two amphidromous fish (gobies of the Sicydiinae) that are widely distributed in the Central West Pacific and which have similar pelagic larval durations. We analysed sections of mitochondrial COI, Cytb and nuclear Rhodospine genes in individuals sampled from different locations across their entire known range. Similar to other Sicydiinae fish, intraspecific mtDNA genetic diversity was high for all species (haplotype diversity between 0.9–0.96). Spatial analyses of genetic variation in  An increasing number of intraspecific phylogeographic studies on marine species have found that earth history events have influenced connectivity between populations e.g. Isthmus of Panama) that are a consequence of past geologic activity and which physically limit dispersal. Secondly, eustatic changes in sea level occur over evolutionary time scales, for instance during the Pleistocene period Connectivity among populations of amphidromous biota is a consequence of present-day  endemic to the Central West Pacific bioregion. They have similar PLD; about 55 days Smilosicyopus Watson, 1999 and Sicyopus Bleeker, 1857 are mainly found within the middle and upper reaches of rivers and prefer swift, clear and high gradient streams with rocky substrate Little is known about the genetic structure of amphidromous species within the Central West Pacific region Si. zosterophorum and Sm. fehlmanni) were expected to show genetic breaks within the Central West Pacific bioregion. But, if breaks were present, they would be expected to be congruent because both species have similar PLD, biology and spatial ranges. If the two species present a congruent genetic structure, then it indicates the existence of a phylogeographical barrier within the Central West Pacific bioregion. To test hypotheses of genetic connectivity, we employed mitochondrial and nuclear DNA sequences to assay Si. zosterophorum and Sm. fehlmanni sampled from seven and five locations, respectively.The marine dispersal phase of amphidromous species allows migration between specific habitat patches on remote islands and because dispersal occurs only in the larval stage, this kind of life cycle highlights subtle barriers to dispersal that may not be visible if the adult form was movable from a stream to another. Because of their extended PLD (about 55 days), populations of neither species  is a widely distributed species across the Central West Pacific bioregion, covering a longitudinal distance of more than 15,000 km extending from southern Japan and Palau to southern Papua, Vanuatu, New Caledonia and Fiji  has nearly the same distribution as Si. zosterophorumSmilosicyopus chloe  is endemic to New Caledonia and Vanuatu Sm. chloe was used to test whether the two mitochondrial groups of Si. zosterophorum were cryptic species or not by comparing the genetic distance within the two congenerics Sm. chloe and Sm. fehlmanni and the two genetic groups of Si. zosterophorum. Also, the two congenerics Sm. chloe and Sm. fehlmanni co-occur in the rivers of the Vanuatu and New Caledonia islands, showing similar habitat preferences. Their demographic patterns are compared to put forward hypothesis explaining the differences in their ranges. Pelagic larval durations (PLDs) of Sm. chloe and Si. zosterophorum are 53.6±5.7 and 54.6±5.6 days, respectively Sm. fehlmanni is around 54 days Si. zosterophorum were collected from two regions within the Central West Pacific bioregion: the northwest  and the southwest . These regions were chosen because they represent the furthest extent of the species’ distribution on either side of a narrow passage that bisects the species ranges. The passage occurs to the south of the Papua New Guinea mainland and northern Australia and is referred to as the Torres Strait. Twenty-one individuals of Sm. fehlmanni were caught in the southwest region  and the northwest region . Forty-four individuals of Sm. chloe were collected from the southwest Pacific   . Geo-refAll specimens were collected by electro-fishing  or snorkelling. Either a piece of fin was clipped and the fish was released, or the fish was killed with an overdose of clove oil (10%), and a tissue sample taken. Tissues and fish were stored in 95% ethanol. Specimens and tissue samples were collected under permits of sampling and euthanasia No 60912-2320-2010/JJC, New Caledonia; Marine Research permit  No RE-11-06, Palau; Vanuatu Environment Unit No ENV326/001/1/07/DK and ENV326/001/1/08/DK; LIPI (Indonesian Institute of Science) permit No 43/SI/M26/X/2010; General Fisheries Permit 89212, Environment Protection Agency Permit WITK06337909 and Griffith Animal Ethics Committee approval ENV114/09/AEC and ENV10/09/AEC, Australia.To analyse population structure across the species’ ranges, several gene regions were amplified and sequenced. Total genomic DNA was extracted from pectoral fin tissue with the semi-automated ABI PRISM™ 6100 Nucleic Acid Prep Station following the manufacturer’s instructions. To investigate genetic diversity and patterns of genetic connectivity, a 670 bp fragment of the mtDNA cytochrome oxidase (COI) gene was amplified, for each of the three species, using specific fish primers TelF1-5′TCGACTAATCAYAAAGAYATYGGCAC3′ and TelR1-5′ACTTCTGGGTGNCCAAARAATCARAA3′ Si. zosterophorum, and to date vicariant events, a 840 bp fragment of mtDNA cytochrome b (Cytb) gene was amplified using specific fish primers CytbF216-5′TCCGAAAYATACATGCYAATGG3′ and CytbR15537-5′CGTTCTGRGCTGAGCTAC3′ Sm. fehlmanni and Sm. chloe were selected and sequenced as well for this region.In order to test the population structure observed from the mitochondrial COI data in http://www.genoscope.cns.fr/) using the same primers. All gene fragments were sequenced in both directions. Chromatograms were edited manually using Sequencher v4.8 (Gene Codes Corporation).Polymerase chain reactions (PCR) were performed in 25 µL final volume, containing 2.5 µl of the corresponding buffer, 5% of DMSO, 5 µg of bovine serum albumin, 300 µM of each dNTP, 1.7 pM of each of the two primers, 0.3 µM of Taq Polymerase (Qbiogen) and approximately 3 ng of template DNA. Amplification products were generated by an initial denaturation step of 2 min at 94°C followed by 50 cycles of denaturation at 94°C for 20 s, annealing at 52°C for COI, Cytb and Rh for 30 s and extension at 72°C for 60 s with a terminal elongation at 72°C for 3 min. PCR products were purified using Exonuclease I and Phosphatase and sequenced using BigDye Terminator v3.1 kit (Applied Biosystems) and the ABI 3730XL sequencer at Genoscope  of Paris (France) and the California Academy of Sciences (CAS).Si. zosterophorum and Sm. fehlmanni, and for each population represented by more than five individuals. First, genetic diversity indices were estimated by computing the number of haplotypes d, Once the species identities of samples were validated, genetic diversity of the COI gene was examined within Si. zosterophorum and Sm. fehlmanni. The relationships between haplotypes, for both COI and Cytb genes, and their geographic distribution were visualised from a median-joining network (MJN) built using the software Network 4.1.1.2 Intraspecific genealogical relationships were explored to test the null hypothesis of genetic homogeneity among populations within amova, i.e. northwest and southwest). We also assessed genetic differentiation between each pair of populations by calculating pairwise φST values. Significance of F-statistics was calculated from 10,000 replicate analyses based on samples drawn randomly with an alpha value of 0.05.Hierarchical analyses of molecular variance amova,  were perSi. Zosterophorum. To test whether these clades were separate species, the divergence between them was compared to the closely related species Sm. fehlmanni and Sm. chloe. A neighbour-joining phylogenetic tree was constructed using Kimura two-parameter distances (K2P) Lentipes concolor (Gobiidae) were used as an outgroup.There was strong evidence of two intraspecific clades (see results for further details) for Si. zosterophorum and Sm. fehlmanni. First, we used mismatch distributions implemented in Arlequin 0 and θ1, which are values that, respectively, represent effective female population size at the time of the last common ancestor and the current effective female population size. These parameters were implemented in DNAsp to estimate the frequencies of the nucleotide differences expected under exponential growth and constant population size. The proportion of raggedness values that were as small or smaller than observed values for a given growth rate can be considered as the significance level by which the hypothesis of that level of growth is rejected SFFs statistics were performed on genetically homogeneous populations. We performed Fu’s Fs statistics on the two genetically homogeneous clusters  of Si. zosterophorum and on Sm. fehlmanni. Significantly negative Fu’s Fs values are indicative of an expanding population.We used two analytical techniques to test hypotheses on divergence dates of populations and ages of effective biogeographical barriers among populations of Gnatholepis)  approach as implemented in BEAST 1.7.0 We used gene divergence time to test hypotheses of vicariance, which predicts that the divergence time between taxa on either side of a barrier should coincide or precede with the timing of the origin of that barrier Si. zosterophorum studied, all were sequenced for the COI gene (Sm. fehlmanni (Genbank accession number KC407573 - KC407593) and 44 samples of Si. chloe (Genbank accession number KC407529 - KC407572) were sequenced for the COI gene. Three samples of Sm. fehlmanni, two samples of Si. chloe and two samples of Lentipes concolor (Genbank accession number KF016034 - KF016040) were sequenced for the Rh gene. After sequence alignment, contigs of 670, 840 and 760 bp in length were obtained for each species for regions of the COI, Cytb and Rh genes, respectively. For each of the three coding genes, no evidence for pseudo-gene amplification was found . All informative substitutions were synonymous in the Rh gene. There were no instances of misidentification of samples used in this study.Of the 164 samples of COI gene , 113 forSi. zosterophorum, overall haplotype (Hd) and nucleotide diversity (π) was high . Hd and π were similar for the northwest  and for the southwest  regions  and nucleotide diversity (π) was high . However, haplotype diversity for Papua population (Hd = 0.7) was lower than for the other populations   and nucleotide diversity (π) was also high  (For  regions . For Sm.Hd = 1) . For Sm.= 0.046) .There was clear evidence of a genetic break within the widespread distribution of this species in the Central West Pacific. From COI, the distribution of pairwise genetic distances was bimodal and exhibited two peaks at around 0.6 and 2.0% of pairwise differences . The firNetwork analysis confirmed the presence of two genetic groups. The COI median-joining network (MJN) revealed two genetic clusters of respectively 31 and 43 haplotypes . These tST = 0.55737, p-value <10−5, ST within each cluster showed no significant values , exhibited no significant genetic differentiation . On the contrary, pairwise φST values between the two groups were highly significant   showed highly significant genetic differentiation  .Si. zosterophorum, there was no evidence of a genetic break in this species. The distribution of pairwise COI genetic distances was unimodal , ST calculated between the populations   showed no genetic differentiation  .Si. zosterophorum were cryptic species. The data set consisted of 27 sequences of the nuclear Rh gene that were obtained from individuals of the two mitochondrial groups of Si. zosterophorum (13 for the northwest group and six for the southwest group) plus three individuals of Sm. fehlmanni, two of Sm. chloe and two of the outgroup Lentipes concolor. The phylogram showed three clades , which were highly supported by bootstrap values. Clade A included the two congeneric species (Sm. fehlmanni and Sm. chloe). Clade B included all specimens of Si. zosterophorum  than mismatch distributions simulated from the expansion population size model. This was not the case for the southwest cluster , which means that the hypothesis of expansion cannot be rejected. Fu’s Fs value was strongly and slightly significantly negative for the southwest cluster  whereas no significant Fu’s Fs was found for the northwest cluster   .Si. zosterophorum diverged. Parameter estimates for a coalescent model of constant population size were equal or very similar for all three replicates. All effective sample size values were greater than 200 and plots of parameter trends indicated sufficient mixing among chains. The heuristic estimate for t, the time of population splitting, was 306,000 years (306 Kyr) ago (95% HPD 79–625 Kyr BP).We also used coalescence time to predict the time when the two clades of Sm. fehlmanni, the observed raggedness value of 0.226 was not significantly smoother than mismatch distributions simulated from expansion model populations  (For = 0.504) , Table 7Sm. chloe, the observed raggedness value of 0.065 was not significantly smoother than mismatch distributions simulated from expansion model populations   (For = 0.430) , Table 6 0.0045) . The twoSi. zosterophorum and Sm. fehlmanni) were expected to show genetic breaks within the Central West Pacific bioregion. Surprisingly, the analysis of population genetics over the sampled region revealed contrasting patterns of genetic structure between the two species studied. Despite low number of samples, genetic connectivity among Sm. fehlmanni populations was high as expected, suggesting that for this species, neither the fragmentation of freshwater habitat nor the oceanic currents surrounding the Central West Pacific Ocean or the presence of the Torres Strait barrier have created significant barriers to gene exchange. Likewise, the broadly distributed amphidromous goby Sicyopterus lagocephalus, displays low genetic structure among the islands of the West Pacific Sm. fehlmanni will be necessary to confirm these results in future studies.Because of their extended PLD (about 55 days) populations of neither goby species  between the two geographic areas, but not sufficient to ensure mtDNA homogenization among regions. A similar pattern has been found for the diadromous species Si. zosterophorum appears to correspond with the Torres Strait barrier (TSB). This narrow passage connects the northerly and southerly parts of the distribution of Si. zosterophorum and Sm. fehlmanni in the Central West Pacific. The species are not known to occur in streams on the northern coastline of Papua New Guinea Si. zosterophorum clades across the TSB has been documented in other clades of taxa such as reef fish and other marine organisms like crustaceans, molluscs, echinoderms and sharks Si. zosterophorum  is concordant with the most recent closure of the intermittent TSB during the Pleistocene, which is further evidence that the TSB is implicated in the genetic break in this species. Earlier closures of Torres Strait are known, which may have also led to divergence between the clades. Sea level has been at least 10 m below present level during 91% of the time for the last 250,000 years Si. zosterophorum, although other factors such as life history traits, oceanic barriers or habitat availability may have played important roles and cannot be ruled out.The location of the genetic break in Sm. fehlmanni. Despite sea level fluctuations, Sm. fehlmanni shows no phylogeographical structure and numerous haplotypes are shared between geographically distant populations, which indicates high connectivity. One hypothesis for the lack of phylogeographical structure across the TSB for Sm. fehlmanni is that one of the two putative lineages formed by a TSB vicariant event may have been lost due to local extinction on one side of the Torres Strait, followed by recent re-colonization through the Torres Strait within the last 7,000 years via northern Papua New Guinea even when the TSB was closed. However, the coastal upwelling along the north coast of Papua New Guinea induces cooling of the sea surface temperatures associated with the Pacific warm pool  Sm. fehlmanni exhibits weak differentiation across the TSB, but our small sample sizes limited our ability to detect genetic structure. Additional analyses of this species are needed to test these alternative hypotheses.It appears that similar processes have not resulted in genetic divergence in Si. zosterophorum revealed contrasting patterns. The southwest group had strongly negative and significant Fs values indicating non-equilibrium population dynamics. There was evidence of an excess of low frequency haplotypes as expected from a recent population expansion or secondary contact between previously allopatric populations Fs values only slightly significant) and the sampling bias between the two clusters  affects the robustness of these statistical analyses. Additional data, including a larger number of individuals and the use of additional polymorphic nuclear markers, are required to strengthen our interpretations.The demographic patterns inferred here provide greater understanding of the evolution and biogeography of the Sicydiinae. Clades of Sm. chloe and Sm. fehlmanni cannot be rejected. These two species co-occur in the rivers of the Vanuatu and New Caledonia islands, showing similar habitat preferences . Sm. chloe would be in an early stage  and Sm. fehlmanni would be in an older stage . Other factors, however, must be taken into account else both species would exhibit similar levels of genetic differentiation across the Torres Strait. These may include subtle differences in dispersal and colonization ability, and habitat requirements, which may have direct or indirect effects on genetic connectivity now and in the past. The rarity and widespread nature of the studied species on remote islands makes further research in these areas a difficult task. Additional genetic data for the two widespread species from other regions of their range, especially closer to either side of the Torres Strait , would nonetheless help clarify the extent to which Pleistocene events have influenced the phylogeography of amphidromous species. Our finding suggest that amphidromous species that are widely distributed may harbor cryptic evolutionary lineages. Indeed, the two widespread gobies with similar distributions have differing levels of population structure, implying that one type of conservation action will not fit all. Because Sicydiinae species are rare and susceptible to anthropogenic and environmental perturbations, strategies aiming to conserve them should consider the presence of possible cryptic evolutionary lineages and cannot rely on a widespread population distribution with high-supposed dispersal abilities to prevent stock depletion.In the Central West Pacific, we have found that two widespread amphidromous Sicydiinae species with similar PLD and biology display contrasting patterns of genetic structure. Our data, in addition to similar studies in this region, suggests that a Pleistocene barrier to marine dispersal (the Torres Strait) may have shaped the genetic pattern of one of the species (


Lutzomyia umbratilis (a probable species complex) is the main vector of Leishmania guyanensis in the northern region of Brazil. Lutzomyia anduzei has been implicated as a secondary vector of this parasite. These species are closely related and exhibit high morphological similarity in the adult stage; therefore, they have been wrongly identified, both in the past and in the present. This shows the need for employing integrated taxonomy., 118 sequences of 663 base pairs  were generated from 72 L. umbratilis and 46 L. anduzei individuals captured, respectively, in six and five localities of the Brazilian Amazon. The efficiency of the barcode region to differentiate the L. umbratilis lineages I and II was also evaluated. The data were analyzed using the pairwise genetic distances matrix and the Neighbor-Joining (NJ) tree, both based on the Kimura Two Parameter (K2P) evolutionary model.With the aim of gathering information on the molecular taxonomy and evolutionary relationships of these two vectorsL. umbratilis and 35 for L. anduzei. The mean intra-specific genetic distance was 0.008 , whereas the mean interspecific genetic distance was 0.044 (0.041 to 0.046), supporting the barcoding gap. Between the L. umbratilis lineages I and II, it was 0.009 to 0.010. The NJ tree analysis strongly supported monophyletic clades for both L. umbratilis and L. anduzei, whereas the L. umbratilis lineages I and II formed two poorly supported monophyletic subclades.The analyses resulted in 67 haplotypes: 32 for Leishmania in endemic leishmaniasis areas. However, the barcode region had not enough power to separate the two lineages of L. umbratilis, likely reflecting incipient species that have not yet reached the status of distinct species.The barcode region clearly separated the two species and may therefore constitute a valuable tool in the identification of the sand fly vectors of  Leishmania. Leishmania infection is characterized by a species-specific pathology, varying from cutaneous lesions to the potentially fatal visceral form   is thinner than the terminal ring. In L. umbratilis, the common and individual ducts show sharper transverse striations  at the 5′ end of the mitochondrial DNA (mtDNA) cytochrome oxidase I -  species , althoug species . This re species -30, inclAnopheles,32 and oAnopheles. HoweverAnopheles and not L. umbratilis and L. anduzei using the DNA barcode region to help in their accurate identification and, thereby better determine the role of each one in the leishmaniasis foci. Additionally, this study assessed the efficiency of this fragment in differentiating the two lineages or incipient L. umbratilis species, previously described by Scarpassa and Alencar [In the Brazilian Amazon region, although there are areas of high transmission of leishmaniasis  and of s Alencar .Lumbratilis umbratilis adults were collected in six localities of the Brazilian Amazon region , and one in the State of Roraima (Amajari)  fragment (DNA barcode) was generated from 72 on Table . Adults  females , using aTotal genomic DNA was extracted individually from whole sand flies using the phenol and chloroform method , resuspehttp://www.ncbi.nlm.nih.gov/BLAST/. The haplotype numbers for each species, number of polymorphic sites and other measures of genetic diversity were estimated using the DNASP v. 5.10 [Anopheles triannulatus sensu lato (Diptera: Culicidae: Anophelinae)  as outgroup.All sequences were automatically aligned with the Clustal W and then compiled and edited in BIOEDIT v. 7.0.8.0 . Followi v. 5.10  and TCS  v. 5.10  software v. 5.10 . The phyL. umbratilis analyzed by Scarpassa and Alencar [COI gene. Haplotypes sequences are deposited in GenBank under the accession numbers KF467531 to KF467597.The 663 bp barcode region analyzed in this study overlapped between sites 283 and 663 with those of  Alencar  who studL. umbratilis and L. anduzei (n=118), yielded 75 (11.31%) variable sites and 53 (8%) of these were parsimoniously informative. Transitions were more common than transversions . The amino acid reading frame starts at the first base of the primer-edited sequences. All sequences, including those from ns Table . The aveL. umbratilis specimens, six  were common to the analyzed localities. The sample of Cachoeira Porteira, situated at the north of the Amazonas River (lineage I), however, did not share any haplotype with other localities, likely due to the geographic distance. The samples from the three other locations , situated on the left bank of the Negro River , shared the haplotypes H12, H13, H15 and H19, suggesting gene flow. The samples from Manacapuru and Novo Airão, situated on the right bank of the Negro River (lineage II), shared the haplotypes H26 and H29. H13 was the most frequent in lineage I, whereas the H26 was the most frequent in lineage II. No haplotypes were shared between samples from opposite river banks (lineages I and II), as previously detected by Scarpassa and Alencar [One hundred and eighteen sequences resulted in a total of 67 haplotypes Table , reflect Alencar .L. anduzei specimens yielded 35 haplotypes (H33-H67)  and three almost fixed mutations . Table L. umbratilis and L. anduzei, which were higher in the latter.The analysis of 46 7) Table . Of thesL. umbratilis and from 0.008 to 0.014 in L. anduzei. The mean interspecific genetic distance between L. umbratilis and L. anduzei was ~six-fold higher , supporting the barcoding gap. Within species L. umbratilis, the genetic distance between lineages I and II varied from 0.009 to 0.010, corroborating the previous observations of Scarpassa and Alencar [Table  Alencar .L. umbratilis and L. anduzei, with bootstrap values of 99% and 98%, respectively, indicating that these species can be recognized by their barcode region. Clade I, comprising L. umbratilis lineages I and II, formed two monophyletic subclades, but had low bootstrap support. Lineage I haplotypes (H1 to H24) were clustered in several subdivisions, whereas all haplotypes of lineage II (H25 to H32) were grouped in another subclade, with bootstrap support of 62%. Clade II, comprising L. anduzei haplotypes (H33 to H67), consisted of smaller subdivisions, regardless of the geographic origin of the haplotypes  and similar to the values found between members of species complexes in mosquitoes [Lutzomyia, with the probable existence of cryptic species in two of them [L. umbratilis and L. anduzei[L. umbratilis and L. anduzei. These differences may be explained by the fact that the COI fragment at the 3′ end analyzed by these authors was more variable, resulting in higher differentiation. Additionally, in this study the most of the transitions and transversions observed between L. umbratilis and L. anduzei occurred in the third codon position, except for one transition, located in the first codon position , identical to the one found previously by Scarpassa and Alencar [L. umbratilis are incipient species [L. umbratilis requires further inquiry. Moreover, this differentiation between two L. umbratilis lineages, although low, could probably affect the genes that control vector competence and consequently lead to distinct patterns of Le. guyanensis transmission in the central region of the Brazilian Amazon, a hypothesis initially raised by Arias and Freitas [Le. guyanensis, whereas the populations belonging to lineage II (right bank of the Negro River and south of the Amazonas River) are likely not to be involved in the transmission (not vectors) or could be less susceptible to the development of Le. guyanensis. However, further studies, including entomological parameters and transmission dynamics  and experimental infections of these populations are needed to either support or refute this hypothesis.In the present study, the value of the genetic distance between  Alencar . This di Alencar , but the Alencar . However Alencar  may indi Alencar ,48. Take species . Whether Freitas . AltogetL. anduzei has a greater genetic diversity than its closely related species, L. umbratilis, at least with regard to the barcode region. Out of the 35 haplotypes observed in L. anduzei, only two were shared among the samples analyzed, suggesting restricted gene flow between them. Curiously, the genetic distance observed in L. anduzei were slightly larger than those detected between lineages I and II of L. umbratilis, probably indicating that the L. anduzei populations consist of a significant genetic structure or display a large effective population size (Ne) [The findings of this study Tables  and 3 inize (Ne)  or highlize (Ne) . In factL. umbratilis in the present and a previous study [L. anduzei samples from opposite banks of the Negro and Amazonas Rivers. This raises the hypothesis that the large Rivers of the Amazon region may act as possible barriers to the sand fly species, as already discussed by Scarpassa and Alencar [L. anduzei across its range.Similar to what was found for us study , no hapl Alencar . All theLeishmania vectors, L. umbratilis and L. anduzei, from the Brazilian Amazon region. We consider the COI barcode region to be a robust marker for differentiating sand fly species, even those closely related, and it may constitute a valuable tool for epidemiologic studies and for leishmaniasis control programs throughout South America. The barcode region, however, had not enough power to separate the two lineages of L. umbratilis, which may indicate that they are incipient species that have not yet reached the status of distinct species [This is the first study of molecular taxonomy in two  species .The authors declare that they have no competing interests.VMS: Designed the experiment, generated the results, analyzed the data, and wrote the manuscript. RBA: Captured the sand flies in the field, identified the samples, generated and processed morphological analyses and revised the manuscript. All authors read and approved the final version of the manuscript.Variable sites of the 67 haplotypes observed for Lutzomyia umbratilis and Lutzomyia anduzei.Click here for file


Disturbance and habitat destruction due to human activities is a pervasive problem in near-shore marine ecosystems, and restoration is often used to mitigate losses. A common metric used to evaluate the success of restoration is the return of ecosystem services. Previous research has shown that biodiversity, including genetic diversity, is positively associated with the provision of ecosystem services. We conducted a restoration experiment using sources, techniques, and sites similar to actual large-scale seagrass restoration projects and demonstrated that a small increase in genetic diversity enhanced ecosystem services . In our experiment, plots with elevated genetic diversity had plants that survived longer, increased in density more quickly, and provided more ecosystem services . We used the number of alleles per locus as a measure of genetic diversity, which, unlike clonal diversity used in earlier research, can be applied to any organism. Additionally, unlike previous studies where positive impacts of diversity occurred only after a large disturbance, this study assessed the importance of diversity in response to potential environmental stresses  along a water–depth gradient. We found a positive impact of diversity along the entire depth gradient. Taken together, these results suggest that ecosystem restoration will significantly benefit from obtaining sources (transplants or seeds) with high genetic diversity and from restoration techniques that can maintain that genetic diversity. Ecological restoration is the process of augmenting the recovery of a degraded, damaged, or destroyed ecosystem The positive impacts of genetic diversity have been documented in a variety of systems. For example, planting genetically diverse varieties of crops tends to produce greater yields as well as resistance to herbivory and disease Zostera marina (eelgrass) meadows are ideal model systems for studying the relationship between genetic diversity and ecosystem functioning. Eelgrass is a broadly distributed species in the Northern Hemisphere with coverage on both the east and west coasts of both the Atlantic and Pacific oceans Seagrass genetic variability, as for all clonal plants, can be measured as either genotypic or genetic diversity. Positive effects of genotypic diversity (number of clones) can occur when one or a few individuals are particularly well adapted to local conditions. Theoretically where genotypic diversity is high, it is more likely that one or more individuals will be well adapted. While genotypic or clonal diversity measures the number of unique genetically-defined individuals or clones per area, it does not capture the degree of genetic variation among individuals like genetic diversity does. Heterozygosity  is the most often used measure of genetic diversity and is a measure independent of the number of alleles per population. Allelic richness  captures the total diversity present in populations, but is independent of the combinations of alleles. A community with a high genetic diversity and an abundance of genotypes (and thus phenotypes) is likely to have individuals that are occupying various niches. Complementarity is a mechanism by which genetic diversity increase population fitness, and occurs when individuals have a variety of phenotypes, which allows the population access to different pools of resources, and thus limits competition. Increasing the pool of genetic diversity among clonal genotypes also improves evolutionary potential and adaptive capacity under changing environmental conditions.As genetic diversity is more difficult to manipulate than clonal diversity in natural systems, most studies that have contributed to our understanding of the relationship between genetics and ecosystem function have primarily used clonal diversity as their measure of genetic diversity. Manipulative experiments have shown that as a result of disturbance, clonal diversity–measured as number of unique genotypes–improved habitat quality Previous results demonstrating that clonal diversity enhances ecosystem resistance to disturbance have been used to direct seagrass restoration We conducted a realistic field-based restoration experiment using techniques, sources, and restoration sites currently being employed by ongoing large-scale restoration to demonstrate that a small increase in genetic diversity to a system with high baseline diversity can improve restoration success when measured by the provision of ecosystem services . This benefit of genetic diversity is evident both with and without specific stresses and disturbances. Because our experimental system has high levels of heterozygosity with little variability, we use allelic diversity as our measure of genetic diversity. The outcomes of this study will be broadly applicable to understanding ecosystems and their restoration, and it is novel in that few studies have demonstrated the effects of allelic diversity on ecosystem functioning experimentally in the field.Plants in plots that were seeded from different sources differed in the average number of alleles per locus . Other experiments in this region have shown that regional genetic diversity of seagrass is high 2 = 0.1, p = 0.5).Measured genetic diversity was positively correlated with density and areal productivity during peak growing season (June) during the 3 years monitored . The numST value of 0.01. During the months of high growth (June and July), plants in the high-diversity treatment were more dense   . Maximum= 0.007) . Shoot-s= 0.007) . Nitroge= 0.007) .2 = 0.5, p = 0.03) , maximum daily temperature was greater, often above 30°C,  in shallower water  . This positive relationship decreased during the second year as plants died and eventually the density of all of the deep plots became 0   , and whier water . Plant der water . In both<0.0001) . The plap = 0.2) . BecauseHigh-diversity treatments were more dense across the environmental gradient; however, the magnitude of difference between high and low diversity treatment varied temporally. There was a consistent pattern where in June the difference between the high- and low-diversity plots was greatest when the plots were shallow and heat stressed, while in July the difference between the high- and low-diversity plots was greatest when plots were at a moderate depth and apparently unstressed . DespiteGenetic diversity, measured as allelic diversity, was positively associated with seagrass density, which cascaded upward into positive impacts on invertebrate density, nitrogen retention, and areal productivity. This is in agreement with previous studies that have demonstrated a positive effect of clonal diversity on ecological parameters Zostera marinaPrevious work has shown that genotypic diversity, measured as clonal richness, Allelic diversity was associated with increases in both seagrass density and ecosystem services, including habitat provision, productivity, and nitrogen retention. The mechanism for this enhancement is the increase in density, as there was not a shoot-specific increase in ecosystem services. In this study, as well as another in the same system, density was an appropriate measure of restoration success and a suitable variable for understanding the impact of genetic diversity on seagrass ecosystems This study found a positive impact of allelic diversity on density under different environmental stress regimes: chronic light stress that killed the plants, temperature stress that decreased density, and low stress levels with no apparent effect on the plant. While the importance of diversity in terrestrial systems has been shown in the absence of stress This increased stability is similar to past results using clonal diversity. Manipulative experiments have shown that experimental plots with a larger number of individual clones (range 1–8) have better resisted large disturbances due to geese grazing ST values suggest that there may not be many unique alleles in these more diverse populations. Instead there must be more combinations of similar alleles, which is more likely to lead to complementarity as opposed to dominance.Both complementarity and dominance of a few genotypes have been described as mechanisms for genotypic diversity enhancement of disturbance resistance in other studies One of the strengths of our experiment is that it replicates realistic conditions for restoration. Seeds were collected from sites that are used regularly for restoration projects and planted in the same manner and in close proximity to a site that is being used for large-scale restoration In May of 2007, flowering shoots were collected from three sites: (1) Mobjack Bay (UTM 18S 384784 E 4127127 N); and (2) the York River in Chesapeake Bay (UTM 18S 374285 E 4125059 N), and (3) South Bay (UTM 18S 428005 E 4124724 N), which is part of the Virginia coastal bay system. Seeds from the flowers were then used in a restoration experiment at Hog Island Bay (UTM 18S 435429 E 4140648 N), also part of the Virginia coastal bay system. Hog Island Bay and South Bay are part of the Virginia Coast Reserve Long Term Ecological Research (LTER) site. All necessary permits were obtained for the described field studies. The restoration site is part an area set aside for seagrass research and restoration by the Virginia Marine Resources Commission and access and collection were permitted through collaboration with the Virginia Institute of Marine Sciences.−2 using an approach that has been successfully applied to restoration in this region In Hog Island Bay 32, 2 m×2 m plots were seeded at a density of 100 seeds mTwice during the experiment, differences in light and temperature conditions along the depth gradient were analyzed. Temperature was monitored for one month using HOBO temperature loggers that read every 15 min. Light profiles were taken at the center of each block using a LiCor spherical 4 Pi sensor (n = 3 for each block).The genetic diversity of the plants in each plot was measured once during the experiment, 20 months after seeding. Six mature shoots from each plot were collected. At the time of collection, this was more than 10% of total shoots. DNA was extracted from each shoot using Qiagen DNeasy plant extraction kits, DNA was amplified at 8 microsatellite loci  using standard PCR techniques 2 quadrats, and the maximum height of five haphazardly chosen shoots were measured. Once during the growing season, invertebrate density and productivity were estimated. Three shoots from each plot were carefully extracted and preserved in isopropyl alcohol until invertebrates could be sieved, counted, and identified in the laboratory. Plant productivity was estimated by marking all shoots in a 0.01 m2 quadrat Shoot density, invertebrate density, seagrass productivity, and leaf nitrogen content were measured in each of the plots for three growing seasons (2008–2010). Monthly density (May–August) was counted in four haphazardly placed 0.25 mST  and relatively low (York River and Mobjack Bay both in Chesapeake Bay). To determine overall differences in density and the provision of ecosystem services, summer data were pooled by block and by the diversity categories. Differences in pooled data were analyzed with a t-test. In addition, the direct relationship of measured parameters to genetic diversity was explored with a regression.Differences in light and temperature conditions with depth were analyzed using a standard regression. The potential impact of differences in temperature and light to the plants was considered by comparing the mean density of all plots at each depth.Data were also blocked into three groups by dividing the depth gradient into three equal parts. Environmental stresses varied among those depth groups: higher temperatures in shallow depths, which often rose above 30°C (<0.9 m), lower light in deeper water that led to eventual mortality as predicted from models For those plots that did not survive the light stress, the length of time that each of those deeper plots survived was plotted against depth. A blocked ANOVA  was used to determine if the plants in the high-diversity treatment were more resistant to the light stress of deep water and thus survived for a longer amount of time.


Plasmodium vivax in South Korea since 1993 represents a serious public health concern. Despite the importance in understanding genetic diversity for control strategies, however, studies remain inconclusive with the general premise that due to low rate of malaria transmission, there is generally low genetic diversity with very few strains involved. In this study, the genetic diversity and population structure of P. vivax in South Korea were explored by analysing microsatellite polymorphism.The reemergence of P. vivax were obtained from 58 South Korean isolates collected during two sampling periods, namely 1997-2000 and 2007. The sequences were used for the analysis of expected heterozygosity and multilocus genotype diversity. Population structure was evaluated using STRUCTURE version 2.3.2. Linkage disequilibrium was also analysed to investigate the extent of outbreeding in the P. vivax population.Sequences for 13 microsatellite loci distributed across the twelve chromosomes of P < 0.05). The number of multilocus genotypes was 7 and 27; and genotype diversity was statistically significant (P < 0.01) at 0.661 and 0.995 in 1997-2000 and 2007, respectively. Analysis by STRUCTURE showed a more complex population structure in 2007 than in 1997-2000. Linkage disequilibrium between 13 microsatellites, although significant in both time points, was notably lower in 2007.Mean expected heterozygosity significantly increased from 0.382 in 1997-2000 to 0.545 in 2007 (The present microsatellite analysis clearly showed recent increase of genetic diversity and recent relaxation of the strong population structure observed in 1997-2000. These results suggest that multiple genotypes not present previously recently migrated into South Korea, accompanied by substantial outbreeding between different genotypes. Plasmodium vivax is the most prevalent in Asia, Melanesia, the Middle East, South and Central America, accounting for 70-80 million cases annually , where a = 2(n-2){∑pi3-(∑pi2)2} and b = ∑pi2-(∑pi2)2  for each K value. Also, in order to determine the most appropriate number of K, ΔK was calculated as described by Evanno et al [Population structure was evaluated using the STRUCTURE version 2.3.2 software . This Bano et al . Illustrno et al .n = 58 isolates in total). There were two cases in the 1997-2000 group where three sequences could not be obtained. All microsatellite loci were polymorphic, exhibiting three to six alleles per locus (Table hE) for each locus ranged from 0.069 to 0.548 for 1997-2000, and from 0.133 to 0.778 for 2007  was 0.382 ± 0.061 and 0.545 ± 0.063 in 1997-2000 and 2007, respectively (Table P = 0.023), indicating a recent increase in the genetic diversity of the P. vivax population in South Korea.Microsatellite sequences from 29 isolates in 1997-2000 (Group 1) and another 29 in 2007 (Group 2) were obtained in this study (gd) was 0.661 ± 0.005 for 1997-2000, and 0.995 ± 0.000 for 2007 (P = 0.00004). Hence, the diversity of multilocus genotypes appears to have significantly increased between 2000 and 2007.A total of 33 multilocus genotypes, which were defined as the unique association of alleles in the 13 microsatellite loci, were detected: seven in 1997-2000 and 27 in 2007 with one genotype (no. 25) found in both groups  for 1997-2000, while it was significant in 33 of all possible pairs (42%) for 2007  suggests that isolate no. 6 in 1997-2000 was a clearly distinct from others. Isolates from 2007 were mainly derived from three clusters at K = 3 and four clusters at K = 5. Of note, one cluster at K = 3 (shown in orange) and two clusters at K = 5 (shown by red and orange colours) predominated in 2007, but were rare in 1997-2000. Clusters that predominated in 1997-2000 were minor in 2007. Some isolates in 2007  were assigned to multiple clusters , diversity is still low as compared to levels obtained from other geographic areas with the exception of Peru: thus, HE = 0.71-0.86 in Brazil, Colombia, India, Laos, Thailand, Sri Lanka, Vietnam, Ethiopia, and Myanmar [vs HE = 0.44 - 0.69 in Peru  as compared with other geographical areas  of P. vivax microsatellite is unknown, accumulation of mutation cannot be completely excluded. However, the present study does reveal the appearance of very many novel alleles at almost all the microsatellite loci examined. Previous studies have also reported rapid increase in genetic diversity in antigen-encoding genes [P. vivax populations collected in 1999 and one in 2002 [P. vivax in neighbouring geographic areas can elucidate and verify the population mechanism.Two mechanisms can be proposed to account for the observed increase in the genetic diversity of ng genes ,11. It i in 2002 . FurtherP. vivax populations. In this study, the extent of inbreeding inferred from linkage disequilibrium was reduced in 2007, compared with that in 1997-2000; although overall linkage disequilibrium was significant in both sampling groups. These results indicate very strong linkage disequilibrium, leading to a high inbreeding rate in the parasite population in 1997-2000, whereas relatively weak linkage disequilibrium, leading to elevation of outbreeding levels in 2007. Analysis by STRUCTURE (Figure P. vivax population resulted partly from an increase in the level of outbreeding among different parasite genotypes.The rate of inbreeding also contributes to the extent of genetic diversity in E Figure  and 2D sP. vivax is more virulent than previously thought in tropical endemic areas [P. vivax has been reported in South Korea [P. vivax population in South Korea, monitoring of the parasite genetic diversity in this country as well as in neighbouring endemic areas will provide valuable information for developing malaria control strategies in South Korea.Moving forward, molecular tools and techniques can elucidate parasite genetic diversity and population structure that can help malaria control efforts and understand mechanisms of pathogenicity and drug resistance. It is generally believed in a parasite population with a high genetic diversity, protective immunity against malaria is slow to develop. There is accumulating evidence, which suggest that ic areas , where gth Korea , which eP. vivax population in South Korea. This expansion is suggested to be due to recent migrations of different genotypes from other geographic areas into South Korea. Drastic change of P. vivax population structure during the period was also detected, in which a substantial increase in outbreeding partly contributes to the increased genetic diversity. Compared with genetic diversity in P. vivax populations from other geographic areas, the genetic diversity in South Korea is still low, although significant increase has been noted in recent years. This suggests that control of vivax malaria in this country could be achieved in line with WHO's agenda for malaria eradication.The present microsatellite analysis showed a recent expansion in the genome-wide genetic diversity of the The authors declare that they have no competing interests.HH performed experiments, data analyses and paper writing. JYK and WL coordinated the sampling. NMQP, TM, JYK and TH critically reviewed the manuscript. TM, JYK, WL and KT participated in acquisition of funding. KT made substantial contributions to conceive the study design, paper writing and reviewing. All authors read and approved the final manuscript.P. vivaxPrimers for PCR and sequencing of 13 microsatellite loci of . Primer sequences for PCR amplification and direct sequencing of microsatellite loci are detailed. Forward and reverse primers are designated with "F" or "R", respectively.Click here for fileP. vivax populations in South KoreaAllele frequency in 13 microsatellite loci of . The alleles for each locus and their frequencies in two sample groups are presented. An allele for each locus corresponds to the number of repeat-motifs.Click here for file


Wuchereria bancrofti (Wb) is the primary causative agent of lymphatic filariasis (LF). Our studies of LF in Papua New Guinea (PNG) have shown that it is possible to reduce the prevalence of Wb in humans and mosquitoes through mass drug administration . While MDAs in the Dreikikir region through 1998 significantly reduced prevalence of Wb infection, parasites continue to be transmitted in the area.We sequenced the Wb mitochondrial Cytochrome Oxidase 1 (CO1) gene from 16 people infected with Wb. Patients were selected from 7 villages encompassing both high and moderate annual transmission potentials (ATP). We collected genetic data with the objectives to (i) document contemporary levels of genetic diversity and (ii) distinguish between populations of parasites and hosts across the study area.We discovered 109 unique haplotypes currently segregating in the Wb parasite population, with one common haplotype present in 15 out of 16 infections. We found that parasite diversity was similar among people residing within the same village and clustered within transmission zones. For example, in the high transmission area, diversity tended to be more similar between neighboring villages, while in the moderate transmission area, diversity tended to be less similar.In the Dreikikir region of PNG there are currently high levels of genetic diversity in populations of Wb. High levels of genetic diversity may complicate future MDAs in this region and the presence of dominant haplotypes will require adjustments to current elimination strategies. Wuchereria bancrofti (Wb). The primary approach to LF elimination has been through mass drug administration (MDA), which serves to interrupt transmission by killing the microfilaria required to continue the parasite life cycle through mosquito transmission. Despite success of MDA, evidence indicates that transmission can rebound if drug administration is discontinued. In the void of well-characterized genetic markers, it is difficult to understand how a Wb population will be impacted by or recover from MDA. Here we use recently described mitochondrial DNA polymorphisms to evaluate the diversity of a Wb population that has been previously exposed to MDA in Papua New Guinea. Our data analyses reveal significant genetic diversity and evidence that MDA has not significantly reduced the genetic complexity of the Wb population. This study describes a population genetic approach for assessing the impact of MDA and other transmission control strategies.The Global Program to Eliminate Lymphatic Filariasis (LF), initiated by the World Health Organization (WHO), aims to eliminate LF from endemic regions, where 1.34 billion people live at risk of this disease. The causative agent responsible for 90% of LF is the nematode parasite species  Lymphatic-dwelling nematodes that cause damage to the lymphatic system (lymphatic filariasis—LF) contribute to significant permanent and long-term disability in the world, second only to mental illness W. bancrofti (Wb), Brugia malayi, and Brugia timoriAs some countries approach completion of MDA programs, priorities have changed to focus on monitoring elimination success through development of post-MDA surveillance tools cytochrome oxidase I gene polymorphisms. Our results provide the first description of genetic diversity in a Wb parasite population by: i) constructing a haplotype network of the infecting strains, ii) quantifying the diversity of Wb at both the infrapopulation and host village level, and iii) determining if the population is genetically structured. Finally, we demonstrate how genetic diversity data can be used to aid in LF elimination efforts by reconstructing the parasite population history in light of the recent MDA efforts in this region.Through genotyping populations of Wb, we can acquire additional information that is likely to contribute to elimination success. This allows us to move beyond mere detection and track individual Wb strain prevalence through time. Until recently, the availability of genetic markers for differentiating between strains of Wb has been limited, so it has not been possible to evaluate changes in parasite populations in the context of LF elimination programs. Through our recent sequencing of the Wb mitochondrial genome (mtGenome) we have identified numerous genetic polymorphisms that can be used to evaluate population structure and to characterize infection complexity In 1993, 14 communities in Dreikikir District , East Sepik Province of Papua New Guinea, underwent a randomized field trial of MDA [Diethylcarmabazine (DEC) with/without ivermectin] to reduce the prevalence of Wb in human and mosquito infections Institutional review boards (IRBs) of University Hospitals of Cleveland, the PNG Institute of Medical Research, and the PNG Medical Research Advisory Committee approved the study. All study participants provided informed consent; parents/guardians provided consent on behalf of all child participants. Consent for all study participants was written.(CO1)et al. 2012 Genomic DNA (gDNA) was extracted from whole blood using a QIAamp 96 DNA Blood Kit . PCR Primers were designed to amplify 690 base pairs of the cytochrome oxidase 1 gene Sequence processing). Corrections included removal of polymorphisms that occurred less than twice in individual alignments (treated as Taq DNA polymerase errors) and were modified to match the consensus sequence (see Sequence processing). After correcting polymerase errors, UCHIME, a chimera detection program, was used to detect PCR-based recombinant molecules www.ncbi.nlm.nih.gov/genbank) as of January 2013 as popset (KC558603–KC559091).All sequences were edited and assembled using CodonCode Aligner 3.5 ; hereafter a sequence refers to a sequence collected from a single clone. Primer sequences were deleted and remaining sites with PHRED scores <30 were visually inspected and recorded as ambiguities. Sequences with less than a minimum of 300 high quality bases (PHRED score >30) were removed from further analyses. Edited sequences were then imported into Geneious 5.3 and aligned against the complete Wb mitochondrial genome sequence (GenBank Accession No. JF 557722) to verify that all the sequences were in identical translation frames and contained no stop codons. Afterward, a correction for Taq DNA polymerase errors was applied to aligned sequences within each infrapopulation (Wb population within a single human host) (see θ) i) among parasite infrapopulations (ΦST-H), ii) among parasite infrapopulations within a host village (ΦST-HV), and iii) among host villages (ΦST-V). AMOVA was performed using Arlequin 3.5 Sequence alignments for each parasite infrapopulation were generated using Geneious 5.3 . Pairwise nucleotide diversity (π) A multiple linear regression was used to build a predictive model of the number of Wb strains within a host. Here we define strain as a unique haplotype isolated from a single infrapopulation; each unique CO1 haplotype is taken to represent a maternal strain. Individual host factors were compiled into a data frame and analyzed using a generalized linear model, with strains as the dependent factor, in R statistical software. The best-fit model was determined based on the lowest Akaike's Information Criteria (AIC) value as given by step-wise addition and removal. A test for multi-collinearity was performed to exclude any factors inflating variance. Model fit was also visually examined using the CAR package in R. Further tests for violations to linear model assumptions were performed using the GVLMA package in R ST-HJ-H) J-H = 0; corrected p-value, α = 0.0004).Pairwise measures of genetic differentiations used both a comparison of sequence difference among populations as well as the difference in allele frequency among populations. Pairwise genetic differentiation, ΦST-V, and pairwise statistics were used as a measure of panmixia and permitted the grouping of infrapopulations into host villages  (see Grouping Infrapopulations). The host village dataset was assessed for genetic diversity and genetic differentiation following the methods indicated in the infrapopulations section above. The only deviation in analysis was the Bonferroni's adjusted p-value, where α = 0.002 for both the ΦST-V and DJ-V statistics. Genetic differentiation results for host villages were visualized in multi-dimensional scaling plots using XLSTAT Addinsoft software Power Tests of Genetic Differentiation).Non-significant values of AMOVA analysis, pairwise ΦHaplotype networks were created using the program NETWORK 4.6.1.0 (fluxus-engineering.com) using only unique sequences from each infrapopulation. Pre-processing was performed via a Star Contraction to collapse nodes <2 mutational steps apart. Networks were then constructed using median joining with maximum parsimony S) to the average number of pairwise mutations (π) θSTajima's D statistic We collected a total of 487 sequences from 16 infected individuals residing in 7 study villages. These individuals were selected based on age (<40 years of age), migration history (same location in 1998 and 2008), and parasitemia (greater than 50 MF/ml of blood) to represent each host village location. From the total of 487 sequences generated, 109 were identified as unique haplotypes, thereby representing individual strains. A summary of individuals included in the study is found in Overall the level of genetic diversity was highly variable among parasite infrapopulations with mean nucleotide diversity (π) varying as much as 9-fold between infections (e.g. T0097PN: π = 0.222% and T0346A2: π = 2.073%) . We alsoθ were highly variable among the infrapopulations . The remaining variance was divided among infrapopulations within a host village  and among host villages . Fixation indices were only significant in the case of ΦST-H (p = 0.019), indicating a significant difference among the ungrouped infrapopulations.Consistent with the high levels of diversity observed within infections, the analysis of molecular variance (AMOVA) attributed 90.31% of the genetic variance in the overall parasite population to be within infrapopulations  or two different clusters (ΦST-H). The remaining two clusters contained infrapopulations from high transmission villages with the occasional exclusion of T0097PN from Peneng (only DJ-H). T0557Y2 from Yautong2 also clustered with infrapopulations from moderate transmission villages in the case of ΦST-H statistic.Pairwise comparisons among all parasite infrapopulations produced 4 clusters for both DST-HV; p = 0.134), pairwise ΦST-HV, and pairwise DJ-HV. Non-significant values suggest that infrapopulations within host villages may act as a single population. Following this assumption, infrapopulations occupying the same host villages were concatenated to facilitate a larger spatial scale analysis of genetic differentiation. In the unique case of Yautong2, where DJ-HV was significant, all analyses were conducted first with the infrapopulations separated and then concatenated.Infrapopulations within a host village were not significantly differentiated as given by AMOVA , therefore on the MDS plot they are tightly clustered. Compare this to Peneng and Moilenge, which are geographically distant (∼9.5 kilometers), genetically different  and are not clustered. Pairwise analysis using DJ-V was comparable to ΦST-V, except in the placement of Yautong1 relative to Peneng.Pairwise comparisons of both DS (data not shown) We found Tajima's D values non-significant for all infrapopulations . When weAs we did not find values of Tajima's D concordant with a recent population contraction, we determined which parameter combinations would lead to a positive value of Tajima's D. If we assume that MDA reduces Wb effective population size by a given percentage each time the drugs are applied (once per year for 5 years), then we can calculate the amount of reduction needed per year to give a positive value of Tajima's D statistic. We constructed a simple model of Wb populations that assumed a single closed population with an effective population size of 300,000 individual Wb worms and 5 generations per year. We then subjected this population to 5 successive contractions, corresponding to the time period of the MDA in Wb generations (e.g. first treatment was 75 generations in the past), after which the population recovered to its current size. We found that only when we contracted the population by >90% each year for 5 consecutive years did we find a positive Tajima's D value.We included 16 individuals from seven villages in the Dreikikir District, East Sepik Province of Papua New Guinea that all received five years of MDA i) the effectiveness of drug regimens, ii) the optimal time-course of drug administration, iii) the potential for drug resistance, iv) vector characteristics and v) human host migration dynamics To increase success of LF elimination we need to understand several parameters including T = 0.15; .20–.34) e = 0.20,0.24,and 0.27) Our goal was to characterize the amount of genetic diversity in the overall parasite population of Wb in the in the Dreikikir District, East Sepik Province of PNG. As this is the first study of Wb population genetics in PNG we do not know the pre-MDA levels of genetic diversity and therefore we cannot objectively define if the diversity was affected by past treatments. Previous studies using DNA fingerprinting (RAPD) have shown that Wb populations were highly heterogeneous across India and Southeast Asia .Within PNG, we may interpret the amount of genetic diversity by comparing it to a simplified expectation. First, we calculated an upper boundary on the expected amount of genetic diversity by assuming that Wb first colonized PNG 50,000–75,000 years ago and has a mutation rate equal to that of other nematodes see . Then asAt smaller spatial scales, we observed that diversity varied widely among infections with both high and low diversity found in the same village . We examThe haplotype network provides additional perspective on the overall relationship between Wb strains in the study area. While it is interesting that strains 1 and 2 appear in every infection, in this study it was not possible to determine whether these strains are highly fecund, or may be resistant to drugs included in the MDA strategy. Despite this current limitation, we do know that the next MDA will have to reduce the prevalence of these strains if it hopes to be successful in eliminating Wb in the Dreikikir region. A haplotype network constructed during an ongoing MDA would essentially resemble a tree with few long branches (pruned), where rare strains are continually lost until only the most common strains (trunk) remain. The length of time needed for singleton strains to emerge in a treated population is not known. Therefore, it may be helpful to sample and genotype the Wb population at multiple time points throughout the course of MDA treatment and compare it to the pre-treatment population.Wb parasites disperse across the landscape via movement of infected individuals, vector dispersal, or a combination of these factors. If infected individual people had recently moved to a new village we would expect their infrapopulation of parasites to be more closely related to their previous village. This pattern was not clearly evident in our data, as in most cases individuals from the same host village clustered together. There were exceptions in our dataset such as individuals T0145A2 from Albulum2 and T0557Y2 from Yautong2 that clustered more closely with infrapopulations from Moilenge .In other ecological systems the pattern of genetic diversity observed in Dreikikir is typically produced by short distance dispersal taking place over multiple generations The drugs currently used in MDA are effective against the parasite transmission stage (MF), but not as effective at killing adults As genetic markers have not previously been available for Wb it follows that genetic diversity of the parasite has not been measured resulting in the inability to quantify Wb breeding population size or its potential for variation. With newly identified genetic markers it is now possible to use population genetics to assess potential for emergence of strains that are not responding to MDA strategies, and thereby monitor progress toward LF elimination by more than simple prevalence assessments. Here we have used population genetic data to infer that the parasite population is composed of many independent strains with overall high genetic diversity. We have also determined that the Wb population in Dreikikir is genetically structured. How vector dispersal, human migration and intervention have influenced this population structure remains to be determined. Beyond using these genetic markers to characterize basic population genetic characteristics of the Wb population in Dreikikier, we have shown how the frequency and distribution of polymorphisms can be used to evaluate the effects of a past MDA on genetic diversity. These results provide new capacity for evaluating and optimizing strategies for Wb elimination.Figure S1Relationship between the number of strains (haplotypes) and parasitemia (MF/ml).(TIF)Click here for additional data file.Figure S2Relationship between the number of strains (haplotypes) and the number of collected sequences from each individual samples.(TIF)Click here for additional data file.Figure S3J-H) measuring genetic differentiation among parasite infrapopulations. An independent test using K-means algorithm in Genodive J-H supported a significant difference among all infrapopulations .Multidimensional Scaling plot of Jost's D statistic (D(TIF)Click here for additional data file.Figure S4ST-H statistic measuring genetic differentiation among parasite infrapopulations. Overall ΦST-H supported a significant difference among all infrapopulations .Multidimensional Scaling plot of Φ(TIF)Click here for additional data file.Table S1Frequencies of all haplotypes observed in infrapopulation samples. Each infected individual is represented as a column heading with haplotype names as rows. Corresponding entries refer to the haplotype frequency found within each infrapopulation. Corresponding information on sample sizes and number of unique haplotypes can be found in (PDF)Click here for additional data file.Table S2Cumulative density of the number of strains in each infrapopulation given a value of diversity, θ, and the current number of sequences. Projections to capture 95% of the total probability are also provided. Information on number of sequences and diversity are given in (PDF)Click here for additional data file.Table S3Cumulative density of the number of strains in each village level sample population given a value of diversity, θ, and number of sequences. Projections to capture 95% of the total probability are also provided.(PDF)Click here for additional data file.Text S1Supplemental text document containing additional details on methods used throughout the manuscript. Initial sections highlight data generation techniques such as sequencing and cloning while later sections detail data analysis. All methods parallel the main manuscript merely providing additional detail when needed.(DOCX)Click here for additional data file.