We examined whether the level of synchrony of ILI epidemics in Israel's 12 largest cities is high enough to view Israel as a single epidemiological unit. Two methods were developed to assess the synchrony: (1) City-specific attack rates were fitted to a simple model in order to estimate the temporal differences in attack rates and spatial differences in reporting rates of ILI. The model showed good fit to the data (R2  =  0.76) and revealed considerable differences in reporting rates of ILI in different cities (up to a factor of 2.2). (2) A statistical test was developed to examine the null hypothesis (H0) that ILI incidence curves in two cities are essentially identical, and was tested using ILI data. Upon examining all possible pairs of incidence curves, 77.4% of pairs were found not to be different (H0 was not rejected). It was concluded that all cities generally have the same attack rate and follow the same epidemic curve each season, although the attack rate changes from season to season, providing strong support for the “Israel is one city” hypothesis. The cities which were the most out of synchronization were Bnei Brak, Beersheba and Haifa, the latter two being geographically remote from all other cities in the dataset and the former geographically very close to several other cities but socially separate due to being populated almost exclusively by ultra-orthodox Jews. Further evidence of assortative mixing of the ultra-orthodox population can be found in the 2001–2002 season, when ultra-orthodox cities and neighborhoods showed distinctly different incidence curves compared to the general population.We analysed an 11-year dataset (1998–2009) of Influenza-Like Illness (ILI) that was based on surveillance of  We propose to study the synchrony of influenza in a range of Israeli cities based on a high quality long-term database that has become available from Israel's Maccabi Health Service providers st 1998 and May 31st 2009; a total of 11 influenza seasons. The doctors all made use of ILI ICD9 diagnosis code 487.1 for influenza, as well as several MHS internal codes for influenza and influenza-like disease. In an analysis that appears elsewhere, we have confirmed the relationship between this ILI data and true influenza cases as based on serological data , Israel's second-largest health maintenance organization which serves close to a quarter of Israel's population. (During the period of the research Maccabi's market share increased from 20.8% in 1998 to 24.8% in 2009.) The dataset includes all ILI cases diagnosed in Israel by Maccabi's doctors every day between January 1Most influenza seasons in this period were dominated by the influenza A H3N2 virus, with the exceptions of 2000-1 and 2007-8, which were dominated by a H1N1 strain, 2002-3 and 2005-6 when influenza B was dominant, and 2008-9 which had no dominant strain see . Influen∽17 weeks) in each winter containing the highest number of ILI cases, most of which are influenza cases, was used to estimate the attack rate of influenza in each season. Other definitions for an ILI season length, ranging from 90 days to 180 days, were examined as well and gave similar results.A large fraction of registered ILI cases are in fact not influenza but other diseases with similar symptoms ∽80 km and ∽25 km, respectively) and Ashdod being ∽33 km south of Tel Aviv. The remaining two cities are Jerusalem, ∽50 km south-east of Tel Aviv and Beersheba, ∽90 km south of Tel Aviv.Twelve cities were studied in this work, each having over 30,000 MHS members in 1998. Smaller cities were found unreliable because it was found that cities with few doctors could have large classification biases that introduced major surveillance errors. In the first test, rather than study the daily time series of each city over the 11 year period, we restrict our study to the observed seasonal attack rates. The attack rate of a single epidemic is defined here as the proportion of people diagnosed with ILI during a 120 day season out of the total number of Maccabi members in that city in that season.The reporting rate-attack rate (RR/AR) model rests on two assumptions:s) has the same attack rate as (s  =  1…11) for all cities. The justification behind this assumption relates to each influenza season (c) has its particular reporting rate rc (c  =  1…12) which is constant throughout the 11-season period of the dataset.that each city  and the ratios of reporting rates give indication of each city's reporting properties (e.g. the tendency of doctors to diagnose diseases as ILI).Note that an infinite number of sets of parameters as) and reporting rates (rc) respectively as found through fitting the model to the observed data ic,s for the twelve cities in Israel over the years 1998–2009. The attack rates are all relative to the first season (in 1998–1999) having taken a1  =  1 and the reporting rates are relative to Tel Aviv where rtel aviv  =  1.as and rc fit the data well, and corroborating the validity of the model. ic,s and the expected city-specific attack rate (asrc) in each city for each season. Note that the expected attack rate of each season is identical in all cities up to a scaling factor, which is the reporting rate typical for each city, and the differences in reporting rates of different cities are very pronounced , the table of estimated relative reporting rates  as the probability that any person (in any city) becomes infected on day t. The null hypothesis (H0) that we wish to test is therefore that both cities share the same probabilities of infection tp.In the Methods section, a statistical test is presented for identifying whether the epidemic curves associated with two cities are identical up to a scaling factor (reporting rate), with differences between cities only due to random sampling effects. More formally, by ‘identical epidemic' we mean that individuals in both cities have the same chance of getting infected for any specific day k is Nk, then the expected value for the number of new ILI cases on day t in city k isThe method calculates the expected number of ILI cases in each day of the epidemic in each city, based on the total number of ILI cases that day in both cities. If the population numbers of city kr for city k (1 ≤ k ≤ n) is the probability that a person who becomes infected goes to the doctor, and is diagnosed with influenza.Here the reporting rate T parameters tp and the two reporting rates r1 and r2 from the ILI data for each of two cities. Once the Ek,t are estimated it then becomes possible to simulate epidemics under the null hypothesis that both cities share the same probabilities of infection pt. The simulation of the epidemic is driven by a Poisson modelBased on a maximum likelihood approach, the method is able to estimate the ik,t is the number of newly infected individuals on day t in city k. The null hypothesis is tested by comparing the likelihood of the simulated epidemics to the likelihood of the observed data.where The procedure for testing the null hypothesis is outlined in the following steps for a given pair of cities, say cities 1 and 2:tp and kr are estimated from the ILI data of the epidemic curves of the two cities (see Methods) for k  =  1,2 and t  =  1,2,…,T.The parameters ik,t for both cities are generated  from One thousand simulated epidemics The histogram of the log-likelihoods  of city One-sided 95% confidence limits  that the epidemic curves of Jerusalem and Holon are identical cannot be rejected since the observed log-likelihood falls inside the 95% confidence limits. In contrast the observed log-likelihood of the cities of Beersheba and Bat Yam (2008-2009) falls outside the 95% confidence limits and the null hypothesis must be rejected.The log-likelihood histograms in 0) could not be rejected in 77.4% of the cases. The fraction of city pairs with different incidence curves (i.e. pairs for which H0 was rejected) in each season varied between 30 different pairs in the 2008-2009 season  and 5 different pairs in the 2002-2003 season (7.6%).It is possible to analyse all city pairs in this fashion. For the 12 Israeli cities, there are Considerable variance is also seen in the fraction of different combinations which include specific cities . NetanyaSince this method estimates the reporting rate of ILI in each of the two cities in a given season, we averaged all 121 estimated reporting rates of each city and compared them to the average reporting rate of Tel Aviv, in order to confirm the results of the RR/AR method. A correlation matrix of daily incidence rates was calculated as an additional index of synchrony between all pairs of cities . The aveAn interesting phenomenon of a distinctly different influenza epidemic in a sub-population of Israel, as compared to that experienced by the rest of the country, can be seen in the 2001–2002 season. The epidemics of most cities in Israel were well-synchronized in this season and peaked around January 20th, except Jerusalem and Bnei Brak. The latter cities peaked earlier in January 11th and January 6th respectively and had very similar incidence curves throughout the season. Even more unusually, Ashdod had a double-peak incidence curve in that season, with the first peak occurring on January 8th and the second peak on January 22nd . We find∽62% of the Jewish schoolchildren in Jerusalem attend ultra-orthodox schools Jerusalem and Bnei Brak have very dominant ultra-orthodox communities  combined with a high degree of population mixing potentially lead to a high degree of synchrony, possibly to an extent to which it can be regarded as a “single city”. Additionally, the fact that ∽40% of its population is concentrated in the Dan Metropolitan Area, a small and dense region in central Israel , further decreases the “effective area” of Israel which should intensify the population mixing and affect the epidemic synchronization between the cities in this area, which contains 7 out of the 12 cities analysed in this work. Unlike childhood infectious diseases (e.g. measles) in which transmission is localized due to the contact patterns of children usually limited to their schools, neighborhoods and close families It was previously shown that some degree of synchrony of influenza epidemics exists in all spatial scales up to the scale of a hemisphere On the other hand, some geographical, environmental and social properties of Israel contribute to the de-synchronization. Despite its small size, climate conditions in different areas of Israel are quite different, in both temperature and humidity, both of which are known to affect influenza transmission In order to obtain a measure of the differences in the severity of influenza epidemics in different cities we examined the variation in ILI reporting rates in different cities using a simple linear model (the RR/AR method), which explained 75.5% of the variance in the data. The RR/AR model examines the assumption that attack rates in all cities in a given season are the same. The model has a built-in parallel assumption of constant, typical reporting rate in each city that may differ between cities. Had these two assumptions been absolutely correct, the model would explain 100% of the variance in the data. Although the result of 75.5% is remarkably high, it also indicates that these assumptions are not fully realized in practice and additional causes for differences in attack rates, which are not incorporated in this model, might also exist.2  =  0.38). This finding is in agreement with those of Charland et al (2011) who examined the relationship between rates of influenza and level of material and social deprivation The assumption of common attack rate for each season might be justified on the basis that with the exception of one season, each influenza season is characterized by a single dominant influenza strain Influenza diagnosis affects the variance in reporting rates since different doctors have different tendencies to diagnose a disease as ILI. Influenza shares most of its symptoms with other diseases, such as common cold The “shapes” of ILI incidence curves of all pairs of Israel's 12 largest cities in each season were compared using a likelihood-based test. The test was applied on 726 combinations of two cities and a season, and found that in 77.4% of the combinations the incidence curves are not distinctly different, again indicating a high degree of spatial and temporal synchrony between the cities. Additionally, this method shows that while some cities are well-synced with other cities, a few typical cities show relatively poor synchrony. Each city appeared in 121 pairs out of the total of 726 pairs, and some cities had more pairs that were significantly different in terms of their incidence curves than other cities . The cities with the largest fraction of “out of sync” curves are Beersheba, the furthest large city from the Gush Dan area located in the Negev desert , and Bnei Brak, a city in the heart of Gush Dan populated almost exclusively by ultra-orthodox Jews see . This ouSignificantly different incidence curves between pairs of cities might be the result of random effects. For example, an influx of a few infectives in the early stages of the epidemic might be enough to change the shape of the incidence curve to a degree that makes it significantly different to the other city's curve. However, these cases seem to be uncommon. Excluding Bnei Brak and Beersheba, the fraction of significantly different combinations drops from 22.6% to 14.5%. When Haifa, the third-least synchronized city in the dataset (and the northernmost city in it) is excluded, the fraction drops to 10.6% of the combinations.Although ILI incidence data for the 2009–2010 “swine flu” pandemic was available for this analysis, it was not included in it since this work focuses on seasonal influenza epidemics. However since a 120-day period is used here for both calculating attack rates and comparing incidence curves, the inclusion of the 2009–2010 season gives similar results. Israel had three waves of influenza during the 2009–2010 pandemic. The first two waves, which occurred in the summer and autumn, showed relatively low synchrony ∽25% of the data in both the attack rates and incidence curves. These differences appear to arise for both geographical and social reasons.Overall, it seems that as a first approximation,Israel's ILI epidemics are similar enough in both shape and severity to be considered as a “One City”. Subtle differences can be found in the incidence data, but these deviations from the “One City” hypothesis account for only Ic,s  for n cities over m seasons, we show how to estimate the parameters as (s  =  1…n) and rc, (c  =  1…m) based on the model: Given the observed attack rate data Since there is no single best-fitting set of parameters to this model (see below), we set the sum of all attack rates to unity, that is The typical attack rate of each season is found using where T days, with 1 ≤ t ≤ T, then our null hypothesis is that there is a vector pt (1 ≤ t ≤ T) which gives the probability that any person, in any city, is diagnosed with ILI on day t. The constant reporting rate rk for city k  is the probability that an individual who becomes infected develops symptoms, goes to the doctor, is diagnosed with ILI, and that the case is reported.We present a bootstrap-based approach for testing the null hypothesis that incidence curves of two cities are identical, after taking into account that the cities may have different reporting rates. Given influenza incidence data for two cities over k as Nk. By the above, the expected value for the number of cases on day t in city k isDenote the population numbers of city k becomes infected and is reported is ptrk, so that the number ik,t of reported new cases is binomially distributedThe probability that an individual in city Nk is large, this distribution can be approximated by a Poisson distributionSince tp and rk can be estimated from the data and the fit of the model to the data can be tested.As shown shortly, the model parameters pt by a constant c and divide all the values rk by the same constant, the model is left unchanged. So there is no possibility to identify the parameters uniquely. To overcome this indeterminacy, we set arbitrarilyAs in the attack rate model above, if we multiply all values ik,t, assuming model (2)), is given by:The likelihood function  and (9) we have our maximum likelihood estimate for ptPlugging (16) into (14) we get our maximum likelihood estimate for k (fraction of population who got infected during that season). t, divided by the total number of reported cases in the season. Because of the arbitrary normalization (4), these two parameters do not have their original meaning of reporting rate and probability of being infected, but as we noted above, this fact is of little importance, since we are only interested in the product k on day t is infected and reported.Note that k on day t isFrom (16) and (17) we get that the expected number of cases in city k,t is ‘close’ to Ek,t, for both cities k = 1,2, for all t. This was tested by generating 1,000 simulated epidemics for each one of the cities using the computed Ek,t values by generating If our model is consistent with the data then we should have that iThen, the log-likelihood of each of the simulated epidemics was calculated:Ek,t are computed using the real data, while th smallest value computed out of the 1,000 simulations, and comparing this value to the log-likelihood value computed for the real incidence data:(note that the values 0 cannot be rejected if the log-likelihood of the real incidence data is not significantly smaller than the log-likelihood of the simulated epidemics. H0 is rejected if the log-likelihood of the real data is smaller than that of the 50th smallest likelihood value of the simulated epidemics.In other words, our Hth of the total weekly incidence.Since the raw data has a strong weekly cycle, with very few reported cases during the weekends, the data was summed over weeks and the incidence for each day was set to be 1/7


Phylogeographic studies indicate that many marine invertebrates lacking autonomous dispersal ability are able to achieve trans-oceanic colonization by rafting on buoyant macroalgae. However, less is known about the impact of rafting on on-going population-genetic connectivity of intertidal species associated with buoyant macroalgae. We hypothesize that such species will have higher levels of population-genetic connectivity than those exploiting nonbuoyant substrates such as rock. We tested this hypothesis by comparing nuclear multilocus population-genetic structuring in two sister topshell species, which both have a planktonic larval phase but are fairly well segregated by their habitat preference of low-tidal bull-kelp holdfasts versus mid-to-low tidal bare rock. We analyzed population samples from four sympatric sites spanning 372 km of the east coast of southern New Zealand. The sampled region encompasses a 180 km wide habitat discontinuity and is influenced by a stable, northward coastal current. The level of connectivity was high in both species, and neither of them showed significant correlation between genetic and geographic distances. However, a significant negative partial correlation between genetic distance and habitat discontinuity was found in the rock-associated species, and estimates of migrant movement between sites were somewhat different between the two species, with the kelp-associated species more often yielding higher estimates across the habitat discontinuity, whereas the rock-associated species more often exhibited higher estimates between sites interspersed by rock habitats. We conclude that for species with substantial means of autonomous dispersal, the most conspicuous consequence of kelp dwelling may be enhanced long-distance dispersal across habitat discontinuities rather than a general increase of gene flow. Population connectivity or exchange of genetic material between geographical populations is a key topic in marine ecology, with a wealth of studies having addressed the role of planktonic larvae in facilitating gene flow in otherwise nondispersive species . While a recent genetic study compared large-scale genetic connectivity in two other Diloma species, one of which is specialized on feeding on decaying fragments of bull-kelp in high intertidal and one of which inhabits mudflats  and D. durvillaea  or, occasionally, crawling on rock surfaces nearby . Given that bull-kelp beds, the primary habitat of D. durvillaea, may undergo intermittent local extinction and regeneration cycles .More specifically, if effective dispersal by rafting takes place in n cycles  and alsoThe large-scale oceanography of our study area in the south-eastern coast of New Zealand is characterized by a major north-east flowing Southland Current that is generated by the Southland Front, at the interface between subtropical and subantarctic surface waters . A 180-kDiloma from eight and five sites, respectively (Diloma from both habitats from four sites (nos. 1–4) that spanned a total 372 km of coastline: these samples provided the basis for between-species comparisons. We also collected rock-dwelling Diloma from four additional sites, to provide insight into geographic structuring of D. arida across distances up to 516 km. In spite of searching, we did not find holdfast cavity-dwelling Diloma in these four localities, but did collect individuals that were grazing on the outer surface of holdfasts and fronds. We collected the kelp-associated Diloma (presumed D. durvillaea) from holdfast cavities of D. antarctica by detaching the holdfasts from their rocky substrate using an axe and/or a wrench bar. We collected rock-associated Diloma (presumed D. arida) by hand from rock surfaces and shallow rock pools in the vicinity of bull-kelp beds. We immersed the animals in 96% ethanol upon collection, with samples from each habitat stored and labeled separately. We replaced the ethanol after 1–2 days, and stored the samples at 4°C in the laboratory prior to analyses.We collected samples of rock-dwelling and bull-kelp-dwelling ectively ; Table 1ectively . SpecifiD. arida having never been encountered inside D. antarctica holdfasts . Although our observations during field collections were generally consistent with this reported ecological segregation, we did find some instances of ecological overlap: specimens with morphological features characteristic of D. arida were occasionally found on the outside of holdfasts and on fronds of D. antarctica. In addition, despite the subtle qualitative differences in adult shell features, we note that the two species are not known to have readily quantifiable, diagnostic morphological characteristics, and that subadult individuals can be especially difficult to distinguish (D. arida from bull-kelp holdfasts/D. durvillaea from rock surfaces), and to avoid erroneous inclusion of D. durvillaea specimens in the D. arida dataset and vice versa, we initially performed a genetic assignment analysis on the whole two-species dataset, assuming that two noninterbreeding clusters are present in the data, and then used the cluster membership estimate of each specimen to assign its species identity .Literature suggests that the habitats of these two species are well segregated, with rock-associated oldfasts . The holD. durvillaea for AG, TG, AAC, AAG, ACT, and ATC repeats following The microsatellite markers for this study were developed by constructing an enriched genomic library of We extracted DNA from the topshells using either Chelex  or an alTaq DNA polymerase , 1×Taq buffer (Bioline), 0.8 µM dNTP, 2.0 mM MgCl2 and 1 µg BSA . We resolved the amplified DNA fragments on 8–10%, vertical acrylamide gels using the Hoefer™ SE 600 Chroma equipment , in 10–11 mA current. We ran 10-base pair DNA ladder (Invitrogen) on the edge and middle lanes of each gel to size the microsatellite fragments. We stained electrophoresed gels in SYBR Green (Invitrogen) solution to visualize the fragments, photographed them with GelDoc 2000 , and later scored the alleles from digital and printed photographs without information about the population or habitat origin of the individual being scored. We controlled the consistency of our allele scoring by rerunning some previously scored, heterozygous samples alongside newly genotyped samples. Amplification and scoring success varied between individuals and loci; we restricted the final sample set and analyses to individuals that were successfully genotyped at four or more loci. Amplification of the DNA samples at more than half of the loci and concurrent amplification failure at some loci despite several PCR trials suggests that the missing genotypes were largely a result of null alleles . Therefore, we adjusted our analyses for presence of null alleles whenever possible. The genotype data used in the analyses, complete with habitat- and museum specimen voucher information, were deposited in the Dryad data repository (http://datadryad.org/).We amplified DNA of the topshells at seven microsatellite loci in separate 10-µl volume PCR reactions. PCR mixture contained 1–4 µl of DNA extraction solution, 0.5 µM of each primer, 0.75 U Mango Q1, Q2) of each individual in two hypothetical populations (K= 2) under the model of nonadmixture and independent allele frequencies. These analyses ignored our prior expectations about species identity based on ecology and/or morphology. We started the simulations with a burn-in period of 50,000 iterations, followed by 200,000 MCMC iterations. We ensured the convergence of the MCMC chains by running five independent iterations of each simulation that yielded virtually identical results. We deemed individuals receiving a >80% membership estimate  for either of the two hypothetical populations to qualify for further analyses as representing D. arida/D. durvillaea and discarded individuals with Q-values between 0.2 and 0.8 from all subsequent analyses.Initially, we analyzed the total 7-locus dataset, consisting of 413 individuals of the two morphologically cryptic sister species, with the individual-based MCMC-clustering analysis in the STRUCTURE software  in orderHS) within sample populations, and total heterozygosity (HT) that took into account the estimated frequencies of null alleles in sample populations. We estimated HS and HT across the total four-population sample of each species and from all population pairs within species.We obtained basic descriptive statistics of the datasets, such as total allele numbers and allele size ranges per locus, using GenAlEx v. 6.4.1 . We usedDest, FST, and G″ST—that complement each other regarding different viewpoints to population structuring, that is population differentiation versus gene flow or migration between populations  unaffected by within-population heterozygosity as per FST and G″ST  and expected heterozygosity within sample populations (HS) were average values over the seven loci, obtained with DISPAN as explained above; k is the number of sampled populations . The estimate of the number of migrants per generation (Nm) under the equilibrium island model assumption was then calculated following mN= (1 –G″ST)/4FST.We measured the global genetic structuring of the species with three types of statistics—ulations . We quanns  corrected for the presence of null alleles. We then analyzed the correlations and partial correlations of population-pairwise DC distances, geographic distances, and major habitat discontinuity (180-km habitat gap in the Canterbury Bight) by conducting Mantel tests in Isolation By Distance Web Service  that looks at mean distance between co-occurrences of an allele in geographically spread data . We expeD. durvillaea in/on bull-kelp versus D. arida on rock) for 76% of the genotyped individuals (315 out of 413). However, 13% (or 48) of the individuals were strongly assigned to the genetic cluster that was unexpected based on their collection habitat  those collected from inside bull-kelp holdfasts that were preliminarily identified as D. durvillaea based on shell characteristics. In contrast, only two individuals collected from inside bull-kelp holdfasts (at sites nos. 1 and 3) were assigned strongly (Q2 > 0.92) to the “D. arida” genetic cluster, members of which were predominantly collected from rock surfaces and identified morphologically as D. arida. In addition, 20 individuals collected from top of bull-kelp holdfasts or fronds at sites nos. 5 and 7 grouped genetically  with the predominantly rock-dwelling cluster and were subsequently regarded as D. arida. Five individuals collected from holdfast outer surfaces associated strongly (Q1 > 0.98) with the “D. durvillaea” cluster. Eighteen of the “D. durvillaea” cluster genotypes were not included in further analyses because they came from localities where too few “D. durvillaea” had been found to warrant meaningful analyses  and were omitted from further analyses due to the apparent uncertainty of their species identity. Most likely their intermediate Q-values were due to data missing at the loci that best diagnose D. arida and D. durvillaea: 39 of the “intermediate” individuals were missing genotypes on at least one of the top three between-species differentiated loci (Ddu5-Ddu7) and in general had a higher proportion of missing data (23%) than the individuals that received Q-values above 0.8 (14%). An alternative explanation is that these individuals are interspecific hybrids, although the present dataset is not sufficient to resolve this issue. Based on the morphological examination of the genetically intermediate specimens by B. A. Marshall at The Museum of New Zealand Te Papa Tongarewa, hybrid status is not out of the question.The remaining 12% of the samples—37 individuals collected from rock surfaces, three from the blades of bull-kelp, and 10 collected from inside bull-kelp holdfasts—were found to be genetically “intermediate” (0.20 < D. durvillaea (91% of single-locus genotypes scored) than in D. arida (83%). Amplification of the locus Ddu7 was particularly unpredictable in D. arida, failing in 56% of the individuals that otherwise amplified at four or more loci, while its failure rate in D. durvillaea was only 16%. Ddu7 was nevertheless included in the analyses, because it showed a consistent, albeit low, scoring rate across the D. arida localities.Among the total 363 study individuals with clear species assignation  indicate this difference is nonsignificant. Also, SAShA suggests that both species are panmictic over the area of overlapping sampling, even though D. durvillaea has approximately 5 km wider mean distance between co-occurrence of shared alleles than D. arida. Summed over all population pairs, the DC distances were slightly, but not significantly, higher in the kelp-associated D. durvillaea  than in the rock-associated D. arida . However, the population-pairwise DC distances of the species were strongly negatively correlated; the statistical significance level of the negative correlation was 6% . By contrast, we inferred the rock-associated species to have exchanged more migrants than the kelp-associated species in two of three comparisons between the sites south of the gap, in an area where rocky intertidal habitat is never uninterrupted by sandy or gravel beaches more than 8 km long, and also between sites nos. 3 and 4, the closest site pair separated by the habitat gap .In the extended set of eight t either . SAShA oD. antarctica) and grazing gastropods , which differ in their reliance on and occupation of D. antarctica beds.In order to obtain a better understanding of the mechanisms and spatial scales of population-genetic connectivity in the marine environment, D. arida versus kelp-associated D. durvillaea (Hypothesis 1). Both species apparently maintain high levels of gene flow across the study area. Nevertheless, in keeping with the predictions made at the outset of this study, we detected somewhat differing geographical patterns of connectivity  between the two species (Hypothesis 2), suggesting that habitat associations can have some evolutionary consequences for intertidal marine taxa at the studied spatial scale. Specifically, populations of D. durvillaea were overall more connected across the major Canterbury Bight habitat discontinuity than were populations of D. arida. By necessity, D. arida must rely on larval dispersal alone to traverse this 180 km wide habitat gap, whereas D. durvillaea has the added potential to use rafting as its dispersal mechanism.Contrary to predictions, we did not find differences in the overall level of genetic structuring or in the magnitude of genetic distances between populations between the rock-associated D. durvillaea than of D. arida. The high migration rate estimates of D. durvillaea across the habitat discontinuity are consistent with recent studies of beach-cast bull-kelp in this region. In particular, a genetic survey showed that oceanographic transport of detached bull-kelp drifting across the Canterbury Bight  occurs on a regular basis . Rafting dispersal may be particularly important for local recolonization and population recovery in D. durvillaea, considering the susceptibility of D. antarctica to local population extinctions  higher numbers of migrants across local scales in southern South Island  where no sandy or gravel beaches longer than 8 km separate rocky intertidal habitats. As D. arida is abundant across this rocky region , there are enhanced opportunities for local gene flow. By contrast, local gene flow is presumably weaker in D. durvillaea because of the more limited bull-kelp habitat whose presence is more strictly dictated by the slope and wave exposure of the shore. The very large effective population size of D. arida  flow of the Southland Current , there iveligers .Diloma species depends on the comparability of the microsatellite data between the species. Given that we inferred null alleles to be abundant in the data, it is important to consider whether they could have biased our between-species comparisons of patterns and strengths of population-genetic structuring, on one hand by disproportionately inflating all population-pairwise FST and DC estimates  had affected the between-species comparisons and found out that it had not: for example, population-pairwise DC distances from analyses that omitted Ddu7 correlated closely with those from the seven-locus data in both species (Pearson r > 0.98), and the absolute differences in the distances were small .The validity of the inferences we have made on the patterns and strength of population-genetic structuring in the two stimates  of one sD. durvillaea has a very fragmented and sparse distribution compared to D. arida, it is remarkable that the two species exhibited comparable levels of genetic connectivity. Either the less common D. durvillaea populations must produce more gametes and larvae than D. arida, or their gene flow is enhanced by some other mechanism such as rafting of reproductively active adults. Genetic studies of species pairs with more balanced population distributions and population sizes are still needed to more robustly test the evolutionary significance of rafting dispersal for epifauna associated with buoyant macroalgae. To this end, we are preparing a comparison of population-genetic structuring in rock-associated versus kelp-associated chitons of the genus Sypharochiton along the eastern coast of New Zealand, using AFLP-markers. Importantly, this additional study will provide much needed phylogenetic replication for testing the dispersal-by-rafting hypothesis.Our study suggests that for intertidal snails with a planktonic larval dispersal stage, ecological association with a buoyant macroalga does not confer dramatic consequences to population-genetic structuring, at least on the geographic scale and genetic resolution achieved by our study. However, given that


Channa striata, as the species has been overfished due to its high market demand. Using maternal markers ), we discovered that evolutionary forces that drove population divergence did not show any match between the genetic and morphological divergence pattern. However, there is evidence of incomplete divergence patterns between the Borneo population and the populations from Peninsular Malaysia. This supports the claim of historical coalescence of C. striata during Pleistocene glaciations. Ecological heterogeneity caused high phenotypic variance and was not correlated with genetic variance among the populations. Spatial conservation assessments are required to manage different stock units. Results on DNA barcoding show no evidence of cryptic species in C. striata in Malaysia. The newly obtained sequences add to the database of freshwater fish DNA barcodes and in future will provide information relevant to identification of species.Conservation is imperative for the Asian snakeheads  Channa striata , from the family Channidae, has a wide range of habitats ranging from rivers, swamps, ponds, canals, lakes, and land of rice fields. Their natural populations are extensively distributed across southern Asia, southern China, Indochina, and Sunda Islands  , 49 C. sst value  from theC. striata, this species did not show the highest level of intraspecific divergence, which was instead observed among the two Rasbora paviana collected from sites A and B (0.90%). The maximum divergence among C. striata individuals was 0.60%, less than the mean divergence between C. striata and the congeneric C. lucius (1.70%), indicating that the divergence between these species was comparable to the divergence observed at the same taxonomic level among other congeneric groups. On average, there was almost 38-fold more variation among congeneric species (mean divergence of 6.03%) than among con-specific individuals (mean divergence of 0.16%), with the minimum congeneric divergence (1.70%) more than twice the maximum intraspecific divergence (0.90%). This pattern of divergence is reflected in the phylogenetic tree  and West Malaysia (Kedah and Pulau Pinang) are genetically alike with the evidence of sharing of the same haplotype (hap2) and nonsignificant C. striata, validation of COI barcode in discriminating freshwater fish species in Malaysia is required. Among all 27 freshwater fish species surveyed for DNA bar coding, divergence among these 27 species surveyed here was relatively high, suggesting that the COI marker was able to differentiate between the Malaysian freshwater fish species examined in this study. In every case where multiple haplotypes were sampled from a single species, all haplotypes were closely related, with no haplotypes common among more than one species and strong statistical support that each individual species sampled was monophyletic. Previously bar coding studies have reported the same incidences of shared haplotypes among species . Although when considered independently from divergences among the entire dataset the pattern of divergence within the Channa does not conform to the 10X threshold for species delineation proposed by Hebert et al. [Channa species is likely to increase estimates of average congeneric divergence among this group. Furthermore, this study reveals that although highly divergent populations of C. striata have been found in other parts of its natural geographical distribution [C. striata in Negeri Sembilan through the molecular dispersal patterns of C. striata using rapidly evolving microsatellite marker [In our study, in order to make any judgment of unidentified cryptic taxa in ies (see ). These ies . This could be explained by the possibility of migration of fish across adjacent drainage systems due to flooding [Acrossocheilus paradoxus found in the model in Taiwan [In the population study, gene flow is predicted to occur in adjacent populations such as between Kedah and Pulau Pinang and between Negeri Sembilan and Johor which recorded a nonsignificant flooding . This thflooding  that alln Taiwan .C. striata between West and East of Malaysia. Although physical barriers could eventually separate the allopatric populations in central region in Peninsular Malaysia, the historical event of Pleistocene as a whole will explain the historical genetic divergence pattern of C. striata in Malaysia. Our finding with overlapping haplotype distribution between the West and the East Malaysia indicates that the fluctuating sea-water level during Pleistocene played a predominant role in permitting genetic exchange between the mainland of Peninsular Malaysia and the island of Borneo through the formation of Sunda River which allow migration of C. striata and thus permitting gene flow.Physical separation of an inhabited geographical range allows a particular organism to acquire different genetic structures. It appears that freshwater species is a casualty of physical barriers such as mountains and dam construction effectively disconnecting them from the drainage basin. An interesting finding in this study was the sharing of hap2 between West Malaysia (Kedah and Pulau Pinang) and East Malaysia (Sarawak) and the nonsignificant population pairwise difference among these two isolated locations, indicating the presence of gene flow and migration of fish among the mainland of Peninsular Malaysia and the island of Borneo during the historical event of Pleistocene Epoch. This epoch encountered with the multiple scenarios of glaciations and deglaciations with the resulted rising and lowering sea-water level and hence periodically changes the exposed land mass. The historical event during Pleistocene with the accompanied fluctuation in sea level could explain the incomplete divergence of C. striata, until the end of the Pleistocene era when this great river system was submerged due to rising in sea-water level as a result of the deglaciations period [C. striata. It is suspected that hap1, hap4, and hap5 were formed after separation of Peninsular Malaysia from the island of Borneo due to these haplotypes not being any haplotype leading towards formation of those Sarawak's haplotypes (hap2 and hap7). Hence, we predict that complete population divergence will occur between West and East Malaysia after a long period of separation by South China Sea. Indeed, in our study, it is believed that there is no colonization of C. striata on the island of Borneo and it is expected that the population in Sarawak is the migrated population from the mainland of Peninsular Malaysia. This is supported by previous study of Adamson et al. [Channa species including C. striata happened in South East Asia, specifically, the north part of Indochina. The biogeographically structure between India and South East Asia coupled with historical climatic changes caused fish flow through southeastern route from India. Geographically, the population on the east coast of Peninsular Malaysia which is closer to the island of Borneo has a greater tendency to migrate than the west coast due to the draining of water from the east of mainland towards Sunda River. However, the population group of Kedah, in the northwest of Peninsular Malaysia, may have migrated closer to the northern region of Peninsular Malaysia and then eventually flowed through the river streams draining out towards Sunda River during Pleistocene glaciations. This hypothesis is supported by previous study of Esa et al. [Tor tambroides which shows a possibility of a migration route between northern Peninsular Malaysia and Borneo due to the same haplotype between populations from Batang Ai, Sarawak, and Perak, northern Peninsular Malaysia. In short, historical Pleistocene glaciations and deglaciations accompanied with changing in land mass configuration will describe the historical coalescence of a freshwater species such as in previous studies on Hampala macrolepidota [Tor tambroides [Barbonymus schwanenfeldii [The Pleistocene glaciations  period hs period , 13. Recn et al.  which hya et al.  on Tor tlepidota , Tor tammbroides , and Barenfeldii .C. striata was not correlated to genotypic divergence pattern due to the morphology of certain terrestrial and marine animal having an additive effect of genetic components and environmental factors. A simple explanation would be that there is no connection between variation at the single gene investigated and variation in body size and shape.The morphology divergence pattern of C. striata is more appropriate to be significantly affected by environmental heterogeneity that influence the development of certain morphology characters and such development changes might be a response to adaptive survival in certain harsh environmental conditions. These developmental changes need not be correlated with the underlying genetic changes due to morphology, as it is the additive effect of the genetic component and environmental conditions that induce changes in certain observable character for survival fitness. Although C. striata is able to survive in harsh environments, conservation planning on this species is required due to its highly commercialize value which has led overfishing to further reduce its effective population size. In this study, we provided information on spatial pattern of conservation planning on this species. Although our study did not reveal any high diversity population in one of the surveyed locations, however, conservation of this species at its natural variation level is required as C. striata forms a diverse group of taxa across Malaysia with seven haplotypes distributed across Malaysia and the majority of populations show significant differences among each other. Our study also revealed that there are two groups with populations within each group which are genetically alike and hence exchangeable for stock management purpose; there are group of Negeri Sembilan and Johor and another group of Kedah, Pulau Pinang, and Sarawak. The current data compiled did not reveal any commercial characters such as larger body size, which will produce more flesh, but the morphological divergence pattern of C. striata indicates that environmental factors had a significant impact on the evolution of certain traits. Our molecular divergence pattern is not congruent to the pattern of morphology variation due to environmental conditions playing a significant role in population morphology divergence or simply the genetic variation across the whole genome would have to be investigated if we wanted to explore the connections between genotype and phenotype. The divergence patterns of both genotypic and phenotypic data reveals that spatial conservation assessment on C. striata is needed to conserve this species. Spatial conservative management could be applied to maintain the genetic diversity and to improve aquaculture quality.In summary, the current dataset did not include sample's sex details due to the evidence of the absence of cytological distinguishable sex chromosome . The mor


Drosophila melanogaster to test the effects of crossing different bottlenecked populations as a method of ‘genetic rescue’ for endangered species lacking outbred donor populations. Two strains differing in the origin of the founders were maintained as isolated populations in a laboratory environment. After two generations of controlled full-sib matings, the resulting inbred fruit flies had significantly reduced breeding success and survival rates. However, crosses between the two bottlenecked strains reversed the effects of inbreeding and led to increases in breeding success and survival that persisted into the second generation of hybrid offspring. In contrast, crosses within each strain (but between different replicate lines) resulted in only slight improvements in some fitness components, and this positive trend was reversed in the second generation. This experiment highlights the potential value of translocations between different inbred populations of endangered species as a tool to mitigate the negative effects of inbreeding, but this benefit may depend upon the origin of the populations. Our results also confirm the importance of maintaining adequate levels of genetic variation within populations and that severely bottlenecked populations should not be discounted as possible donors in genetic rescue programs for endangered species.We produced replicated experimental lines of inbred fruit flies  Tympanuchus cupido, Vipera berus, Anthropogenic influences such as habitat loss and fragmentation, the introduction of exotic predators, excessive hunting, and pollution have forced many species through severe population bottlenecks. Decreased effective population size during a bottleneck can lead to increased inbreeding and the loss of genetic diversity, which both adversely affect population viability Drosophila melanogaster) and houseflies (Musca domestica) support the prediction that immigration of individuals into inbred lines can lead to rapid improvements in fitness traits such as viability, productivity and survival Canis lupus baileyi) to both captive and reintroduced populations of this species. As only three captive lineages of Mexican wolves survived from a total founding population of 7 animals, no outbred individuals were available as donors. Despite low levels of genetic variation and fixed deleterious alleles within each lineage, crosses between lines experienced increases in the proportion of live births, litter size, and survival of offspring The use of genetic rescue as a management tool depends on the availability of suitable outbred donor populations. However, for many endangered species there are no outbred populations left to act as a donor. Instead, many endangered species survive only as a series of small, fragmented populations, with each likely subject to some loss of genetic variation and increased levels of inbreeding. Theoretical models suggest that by crossing individuals from one inbred population with those of a second inbred population, the severity of inbreeding depression should decrease in the hybrid offspring Drosophila melanogaster to test changes in the viability of the hybridised population.Despite the apparent success of the genetic rescue technique using inbred donors in lab and field studies, the general effectiveness of using inbred individuals as donors is not clear, nor whether the suitability of inbred donors varies with their source. In some species, prospective donor populations may share a recent common ancestry with a recipient population , and may not be differentiated enough to introduce new genetic variation. Alternatively, a donor population may be so differentiated  that it may lead to a deterioration of fitness traits, in a process termed outbreeding depression Drosophila melanogaster originating from different parts of the world  were used to create inbred lineages. Both source populations were maintained in cages supporting >500 individuals with overlapping generations. Despite the maintenance of large populations, stocks of fruit flies are known to lose genetic variation, with the degree of loss increasing with greater periods of time in captivity Two strains of All cultures were maintained on standard commercial medium  with a supplement of live yeast. The stocks were kept in an incubator at 25±1.0°C, and a 12∶12 hour light:dark photoperiodic cycle. The position of vials within the incubator was re-randomised on a regular basis.Four inbred replica of the Slg14–15 strain  and 3 inbred replica of the Wild type Oregon-R strain  survived for 2 generations. The flies from these 7 lines  were used for replicate crossing ( =  “genetic rescue”) experiments. To test the effects of hybridising individuals from inbred lines on the fitness of offspring, we paired inbred flies within each strain, but between different replica . The resulting offspring are referred to as F1 within-strain hybrids. This tested whether inbred populations founded from the same population could still act as genetic rescue donors. We also paired inbred flies from one strain with inbred flies from the other strain , to test the effects of hybridising inbred individuals from differing strains on the fitness of their offspring. These are termed F1 between-strain hybrids  was bred within each F1 within-strain and between-strain hybrids. Within each of the three groups , F1 hybrids were either paired with other F1 hybrids of the same group , or with inbred flies of the same strain , or of both strains in the case of the between-strain crosses (Slg-Wt: 30 pairs), resulting in six groups in the F2 hybrid generation  to account for the end of the observation period.Upon eclosion, flies were counted and sexed under COAs measures of breeding success did not differ significantly between the Slg and Wt strains , the two strains were pooled to facilitate comparison with the between-strain hybrids. This resulted in two groups in the first generation (hybrid F1 within-strain (Slg and Wt) vs. hybrid F1 between-strain (Slg-Wt)). As mean trait values of reproductive success of hybrid-hybrid and hybrid-inbred pairs were not significantly different in both F2 within-strain and F2 between-strain crosses , we pooled this data, resulting in two groups of F2 hybrids (hybrid F2 within-strain and hybrid F2 between-strain). We therefore compared a total of six groups of varying inbreeding status .iy as a function of an additive genetic component ia. This model can accommodate the pedigree of the individuals through the use of a relatedness matrix. In addition to the genetic component, the model included a group effect with the six levels of inbreeding status. The number of eggs laid was analysed using a Gaussian distribution with an identity link function, while the proportion of eggs that pupated (pupae/eggs) and the proportion of pupae that eclosed (adults/pupae) were analysed using a binomial distribution with a logit link function. Inference for the animal model relied on a Bayesian framework, using Integrated Nested Laplace Approximations (INLA) to calculate the marginal posteriors for all parameters Data from the breeding experiments were fitted using an ‘animal model’ survreg function within the “survival” package in the R statistical environment , the two strains were pooled to facilitate comparison with the between-strain hybrids. Data for each fruit fly in the survival trial consisted of the time (in days) until exit from the study (either by death or by censoring), a censoring indicator specifying whether an individual survived until the end of the experiment or not , and the group  that the individual belonged to as factor. We analysed the survival data using the Inbreeding within each line lead to a significant reduction in the absolute numbers of eggs laid . The effThe proportion of eggs that developed into pupae in inbred flies decreased by 18% compared to outbred flies . In contIn between-strain hybrids, pupating success in the F1 generation was virtually identical to that found in inbred flies; however, there was a significant increase in pupating success in the F2 generation and levels were not significantly different to those recorded in outbred flies  and 3. PIn F1 within-strain hybrids, the proportion of eggs that pupated tended to increase compared to inbred flies, but this increase was not significant . In the F2 generation of within-strain hybrids, the proportion of eggs that pupated decreased to levels observed in inbred flies. Similarly, eclosing success of pupae increased in the F1 generation of within-strain hybrids and was virtually identical to eclosing levels in outbred flies, but decreased in the F2 generation to levels that were intermediate between inbred and outbred flies  and 3.Daily mortality probabilities and results of the survival analyses  are summarised in As expected, the severe bottlenecks we induced in the two strains of fruit flies and the subsequent forced inbreeding led to a decline in individual breeding success and survival. Even after only two generations of inbreeding, half of our lines went extinct through reproductive failure. However, subsequent crossings within each strain of inbred flies (but between different replicate lines) as well as crossings between the two strains resulted in significant increases in survival and some measures of reproductive success. The positive effects of the crossing experiments were more pronounced in the between-strain hybrids, which increased even further in the F2 generation. Most importantly, between-strain hybrids exhibited a significant increase in both the absolute (number of eggs produced), and the relative reproductive output . This was coupled with a marked increase in survival probability, which even exceeded the survival probabilities of outbred individuals. Our results thus support the potential value of genetic rescue as a management tool for endangered species that survive only as a series of fragmented and bottlenecked populations.Mustela nigripes; For within-strain crossings, the results were of mixed nature. In terms of absolute reproductive output (number of eggs laid), the first generation of within-strain hybrids showed no increase compared to inbred flies. Significant positive effects were, however, observed in the second generation of within-strain crossings. Relative reproductive output  tended to increase in F1 within-strain hybrids, but in F2 individuals this positive trend was reversed to levels measured in inbred flies. Similarly, survival probabilities increased significantly in F1 within-strain hybrids compared to inbred flies, but this positive effect did not persist into the second generation. Nonetheless, the increase in number of eggs laid observed in the second generation of within-strain hybrids constitutes a fitness improvement compared to inbred flies. Whether the improvement in fitness is sufficient to warrant the implementation of genetic rescue between populations of endangered species stemming from the same source population is not clear and would first require determining whether such crosses would actually introduce any new genetic variation into the recipient populations. Given that a number of endangered species currently managed as discrete populations originated from the same single source population and yet show some genetic differentiation  were expected to be genetically similar. Nevertheless, even replica of the same strain were unlikely to be genetically identical, and thus deleterious alleles could still be masked by crossing flies from different replica within each strain. The subsequent exposure of deleterious alleles in the second generation of within-strain hybrids could then cause the reversal of the positive effects seen in the F1 generation . As the biggest improvements in reproductive success and survival were observed in between-strain hybrids , and these improvements persisted into the second generation, our observations are consistent with a concomitant increase in levels of genetic diversity in the hybrid offspring that persisted for at least two generations. However, an analysis of genotypes would be necessary to determine the exact mechanism for this fitness effect.Outbreeding depression  typically becomes apparent in the F2 generation of crosses, when the original parental gene combinations are split up by recombination processes such as chromosomal crossover and segregation, which can cause the disruption of extrinsic interactions between genes and the environment  or inherent interactions between genes As previously found in other studies e.g. , fitnessPetroica traversi: 1 pair Falco punctatus: 1 pair The bottleneck that the two original populations used in this study were forced through (two generations of full-sib matings) was particularly severe. Although bottlenecks of this severity are unlikely to occur to the same extent in most natural populations, at least a few species have passed through bottlenecks that approach this level Click here for additional data file.Script S2Survival analysis script used in R.(R)Click here for additional data file.Data S1Breeding data used in INLA analysis script.(CSV)Click here for additional data file.Data S2Pedigree data used in INLA analysis script.(CSV)Click here for additional data file.Data S3Survival data used in survival script.(TXT)Click here for additional data file.


Proceratophrys moratoi) inhabits Brazilian moist savannas and is critically endangered due to its very limited geographic distribution, reduced number of isolated populations, and evidence of population decline and local extinctions. With the objective of providing tools for the genetic study of the species, 22 polymorphic microsatellite loci were isolated and screened using DNA extracted from samples of oral mucosa cells obtained from 113 individuals representing five remnant P. moratoi populations in the Brazilian state of São Paulo. These markers presented 2–18 alleles per locus, polymorphism information content (PIC) of 0.02–0.87, observed heterozygosity of 0.02–0.96 and expected heterozygosity of 0.02–0.87. Three of the loci deviated significantly from Hardy–Weinberg equilibrium in one of the populations, possibly due to the presence of null alleles. Significant linkage disequilibrium was also detected between three pairs of loci. The molecular markers developed in this study were able to discriminate each of the individuals sampled . This means that they will be extremely useful for future genetic studies applied to the conservation of P. moratoi, providing a baseline for estimating the levels of genetic diversity, pedigrees, inbreeding, and population structure, which will be essential for the development of effective genetic management programs.The Morato’s digger toad ( The class Amphibia has experienced a major global decline in recent decades, becoming more endangered than birds and mammals, due to a combination of factors . HabitatProceratophrys moratoi is a digger toad of small size, typically with a snout-vent length of no more than 35 mm, which is endemic to the Cerrado savanna of the Brazilian state of São Paulo [campo sujo habitats , invariably near the gallery forests associated with the headwaters of streams [P. moratoi. Due to its very restricted geographic distribution and the evidence of population decline and local extinctions [ão Paulo . The spe streams ,7. In Sã streams . Current streams , which hinctions , the speinctions , and is inctions  and São inctions .P. moratoi include molecular analyses of mitochondrial and nuclear genes [P. moratoi.The available genetic studies of ar genes  and cytoar genes . HoweverN/GTN repeats (130 motifs identified) were the most numerous, followed by CTN/GAN (48 motifs). This predominance of CAN/GTN repeats is typical of the eukaryote genome [N/TAN (36 motifs), the trinucleotides CATN/GTAN (18 motifs), CTTN/GAAN (17), AATN/TTAN and CTCN/GAGN (6 motifs each), and the tetranucleotides CTATN/GATAN (13 motifs) and CATTN/GTAAN (6 motifs). These data provide a baseline that will support the development of additional probes for the isolation of new microsatellites in P. moratoi.A total of 384 clones were isolated and sequenced bidirectionally. The Codoncode Aligner 3.7.1 software  revealed a redundancy of 17% in the library. Of the unique clones selected for analysis in Microsatellite Repeats Finder , 176  and was excluded. Six loci—Pmoratoiμ2, Pmoratoiμ3, Pmoratoiμ4, Pmoratoiμ9, Pmoratoiμ20, and Pmoratoiμ22—were monomorphic. With the exception of Pmoratoiμ22, all these monomorphic loci represent interrupted or interrupted compound microsatellites characterized by a small number of repetitions, with predictably low polymorphism [Pmoratoiμ7, Pmoratoiμ8, Pmoratoiμ10, Pmoratoiμ11, Pmoratoiμ14, Pmoratoiμ17, Pmoratoiμ18, and Pmoratoiμ21). The Pmoratoiμ5 locus was not amplified in some populations, possibly due to local mutations in the primer annealing site. The identity analysis calculated using Cervus 3.0.3 [A total of 29 pairs of primers were designed and optimized successfully for the PCR amplification of microsatellite loci . Of the morphism . Twenty-us 3.0.3  detectedNA) varied between 2 and 18 (HO) ranged from 0.02 to 0.96, expected heterozygosity (HE) from 0.02 to 0.87, and polymorphism information content (PIC) from 0.02 to 0.87. As might be expected from the relatively large number of markers developed for this study, evidence of linkage disequilibrium was found in three pairs of loci  following Bonferroni correction (p < 0.002). Significant deviations from Hardy–Weinberg Equilibrium (HWE) were found in Pmoratoiμ24 and Pmoratoiμ27 from the São Carlos population and in Pmoratoiμ29 from Brotas, due to a deficit of heterozygotes. These deviations can be attributed to the presence of null alleles in these populations. The estimated null allele frequency for the Pmoratoiμ24 locus from São Carlos was 0.23, and that for Pmoratoiμ27 from this same population was 0.18. The estimated frequency for Pmoratoiμ29 from Brotas was 0.11.The total number of alleles per locus (2 and 18 . ObservePmoratoiμ13 (0.10), Pmoratoiμ23 (0.19), and Pmoratoiμ24 (0.10) loci from Bauru, and in Pmoratoiμ27 from Brotas (0.07). In all these cases, the evidence of the presence of null alleles was relatively weak and thus insufficient to confirm a significant departure from HWE following the Bonferroni correction. Micro-Checker 2.2.3 [Pmoratoiμ13 from Bauru. The identity analysis indicated that the combination of all the loci would permit the individual identification of each of the specimens.In addition to the loci with deviations from HWE, null alleles were detected in the er 2.2.3  did not P. moratoi using the procedure of Sambrook et al. [Afa I (Invitrogen) and the fragments were then ligated to Rsa I linkers (Rsa21: 5′-CTCTTGCTTACGCGTGGACTA-3′/Rsa25: 5′-TAGTCCACGCGTAAGCAAGAGCACA-3′) using 2 units of T4 DNA ligase (Promega). The fragments were then amplified by polymerase chain reaction (PCR) with a reduced number of cycles (20 cycles) using the primer Rsa21. The PCR products were purified, denatured and hybridized with biotinylated microsatellite probes (GT8 and CT8) at room temperature for 20 min. The hybrid mixtures containing microsatellites were then collected by streptavidin-coated magnetic beads (Promega). The selected fragments were amplified via PCR and the products were ligated into a pGEM-T easy cloning vector (Promega). Escherichia coli XL1-Blue cells (Stratagene) were transformed with recombinant plasmids by electroporation and grown overnight in solid Luria-Bertani agar medium containing ampicillin, IPTG and X-Gal. The positive colonies were selected and grown in liquid medium with 2YT HMFM containing ampicillin. After growing for 16 h, they were stored at −80 °C.We constructed an enriched partial microsatellite genomic library using an approach based on the selective hybridization method of Kijas . The libk et al.  with modOf the total of 596 clones obtained, 384 were sequenced bidirectionally in an ABI Prism 3100 automatic sequencer . The DNA sequences were exported into Codoncode Aligner 3.7.1 (CodonCode Corporation) which assembled the contigs and verified the redundancy of the library. The Bioedit program was used to check the quality of the sequences by chromatogram and to align them to form a consensus sequence. The repetitive elements were located using the Microsatellite Repeats Finder program . After ret al. [P. moratoi populations in the Brazilian state of São Paulo: 41 samples were collected in the municipality of São Carlos , 41 in Brotas , 27 in Bauru , 3 in Avaré , and 1 in Lençóis Paulista . The PCRs were prepared in a final volume of 15 μL containing 10 ng of the DNA template, 1× reaction buffer, 0.3 mM dNTP, 0.6–4.0 mM MgCl2  following a modified version of the procedure described by Pidancier et al. . SamplesmM MgCl2 , 0.6 μM mM MgCl2 , and 1 mNA), observed (HO) and expected (HE) heterozygosity calculated by Popgene 1.32 [The levels of polymorphism of the microsatellites were evaluated as the number of alleles per locus (ene 1.32 . The polene 1.32 , which wene 1.32  was usedene 1.32 , with 10ene 1.32 . Micro-Cene 1.32  was usedene 1.32 .Proceratophrys. These markers constitute a powerful tool for the study of P. moratoi populations, allowing the identification of untagged individuals and providing a database for the development of kinship studies for future ex situ conservation programs. It will also be possible to analyze inbreeding, genetic diversity and structure, and gene flow in natural populations, which will be vital for the development of effective in situ conservation measures.These are the first microsatellite markers developed for Morato’s digger toad, and in fact, the first for any member of the genus


Population-level studies of parasites have the potential to elucidate patterns of host movement and cross-species interactions that are not evident from host genealogy alone. Bat flies are obligate and generally host-specific blood-feeding parasites of bats. Old-World flies in the family Nycteribiidae are entirely wingless and depend on their hosts for long-distance dispersal; their population genetics has been unstudied to date.Pteropus species  from eight localities in Malaysia, Cambodia, and Vietnam. We identified specimens morphologically and then sequenced three mitochondrial DNA gene fragments  from a subset of 45 bat flies. We measured genetic diversity, molecular variance, and population genetic subdivision (FST), and used phylogenetic and haplotype network analyses to quantify parasite genetic structure across host species and localities.We collected a total of 125 bat flies from three Cyclopodia horsfieldi with the exception of two individuals of Eucampsipoda sundaica. Low levels of population genetic structure were detected between populations of Cyclopodia horsfieldi from across a wide geographic range (~1000 km), and tests for isolation by distance were rejected. AMOVA results support a lack of geographic and host-specific population structure, with molecular variance primarily partitioned within populations. Pairwise FST values from flies collected from island populations of Pteropus hypomelanus in East and West Peninsular Malaysia supported predictions based on previous studies of host genetic structure.All flies were identified as Cyclopodia horsfieldi is most likely due to frequent contact between flying fox species and subsequent high levels of parasite gene flow. Specifically, we suggest that Pteropus vampyrus may facilitate movement of bat flies between the three Pteropus species in the region. We demonstrate the utility of parasite genetics as an additional layer of information to measure host movement and interspecific host contact. These approaches may have wide implications for understanding zoonotic, epizootic, and enzootic disease dynamics. Bat flies may play a role as vectors of disease in bats, and their competence as vectors of bacterial and/or viral pathogens is in need of further investigation.The lack of population genetic structure and morphological variation observed in  Intraspecific evolutionary studies of parasites can give insight into vector ecology, and uncover patterns of connectivity in host species populations not inferred by host genealogy alone ,2. In soCyclopodia parasitize only bat species in the family Pteropodidae, and global distributions of the two groups closely coincide , and 2 μl of template DNA. PCR conditions for cytB were identical to those in Dittmar and Whiting (2003), and for the CoII PCR conditions were an initial denaturation period of 3 min at 94°C, followed by 35 cycles of 94°C for 1min, 47°C for 1min, and 74 C for 1min, with a final extension at 74°C for 7min. PCR mix and cycling conditions for CoI followed Hebert et al.[et al.[Bat flies were sequenced at three mtDNA genes, cytochrome B  AmPure magnetic beads, cycle sequenced with Big Dye v.3.0 terminator mix , and final DNA was precipitated using Agencourt CleanSeq magnetic beads. All sequencing was performed on an ABI 3730xl capillary sequencer (Applied Biosystems) at the Sackler Institute for Comparative Genomics at the American Museum of Natural History, New York. Sequences were edited in Sequencher v.4.6 and manually corrected for ambiguous base calls. Alignment of sequences was done using MAFFT v.6 using default parameters . Alignedhttp://www.phylo.org/sub_sections/portal/. Bayesian analysis was conducted using MrBayes version 3.2 [Eucampsipoda sundaica (#KB5) were used as an outgroup to Cyclopodia horsfieldi in all phylogenetic analyses because of its basal position within the Cyclopodiinae [Phylogenetic relationships and nucleotide/haplotype diversity were initially determined for each mtDNA gene fragment independently. As there was a paucity of variable and parsimony-informative sites for each marker alone, sequence data from the three mtDNA genes were concatenated for a total of 1744 bp for each specimen. Phylogenetic analyses on the pooled dataset were conducted using PAUP* for maximum parsimony (MP) analysis , RAxML vsion 3.2 , with dasion 3.2 . Sequencpodiinae .http://users-birc.au.dk/biopv/php/fabox/). Fifty-one bat flies were sequenced, but outgroup and individual flies that did not have complete sequences for all three genes were excluded in subsequent analyses , leaving 45 individuals. Statistical parsimony networks, useful for inferring relationships among sequences that have recently diverged, were created using TCS v.1.21 [ST values were calculated in Arlequin using a method based on pairwise differences in sequence data, and statistical significance was assessed with 1000 permutations [C. horsfieldi populations from Malaysia only, using a Mantel test [Aligned FASTA files were collapsed into variable sites and haplotypes for parsimony network reconstruction with the online tool FaBox 1.31 , was used to test hypotheses regarding the partitioning of genetic variation for calities . AMOVA wcalities  using thCyclopodia horsfieldi  from Kuala Berang, Malaysia  and Mann–Whitney test . We also inadvertently collected Neolaelaps spinosa (Acari: Mesostigmata) mites as we removed individual flies with forceps. These mites appear to be phoretic with bat flies [All bat fly specimens examined were all identified morphologically as i Figure  with the sundaica collectes #070904 from Kual Figure ; and somat flies , a relatat flies -63.C. horsfieldi mtDNA dataset  was 0.002 ± .0004 , CoII , and cytB . There was an overall high amount of haplotype diversity across all markers and specimens (0.903 ± .026). Although sample sizes were limited, overall nucleotide and haplotype diversity were both higher for C. horsfieldi individuals from Pteropus vampyrus  compared to P. hypomelanus  , which corresponded with the smallest host population census size (n=200 bats). Total A-T content was uniformly high for all sequences (77.3%), as is common in other insect mitochondrial genomes [Average nucleotide diversity for the combined 5), CoII 8bp, 11/2 genomes . SummaryC. horsfieldi sampled across sites in Southeast Asia. No significant population structure that corresponded to either geography or host species was detected in any of the analyses  vs. eastern  island populations (Table ST between P. vampyrus and the western P. hypomelanus population (PPH) was significant and high (FST =0.443)   but non-significant in a randomized mantel test (p=0.34).Despite an overall lack of geographic structure observed in phylogenetic and haplotype network analyses, pairwise Fus hosts . Signifins Table , suggestFST =0.44 , but had much higher levels of gene flow between the east coast islands (FST =0.0 to 0.4). Here we observe a similar pattern for the parasite, Cyclopodia horsfieldi, with significant pairwise FST values between flies from Pulau Pangkor off the west coast and Pulau Tioman (FST =0.560) and Pulau Perhentian (FST =0.435) off the east coast of Malaysia. In contrast, FST values among flies from the two east coast islands were low and not significant (FST =0.031). We also observed low and non-significant FST values between mainland populations of Pteropus vampyrus and island populations of P. hypomelanus, with the exception of Pulau Pangkor (PPH). These data suggest ongoing or recent gene flow among Cyclopodia horsfieldi parasites between mainland Pteropus vampyrus and eastern P. hypomelanus; and less contact between P. vampyrus and the small western island of Pulau Pangkor.Overall, we found that populations of ox hosts . Olival ox hosts  found thPteropus vampyrus was found to be essentially panmictic across a very large geographic range of thousands of kilometers in Southeast Asia at mtDNA markers [2) across Malaysia, Thailand, and Indonesia [Cyclopodia horsfieldi populations have recently diverged or are subject to ongoing gene flow. Frequent contact between flying fox host species and subsequent ectoparasite gene flow may best explain the lack of parasite population structure observed. In particular, we suggest that the highly volant Pteropus vampyrus may be acting as a “vector” spreading bat flies to other conspecific populations and Pteropus species in the region during long-distance dispersal events. Satellite telemetry studies have shown that P. vampyrus uses small islands as stopover sites when migrating to and from Peninsular Malaysia and Sumatra [P. vampyrus relative to those from P. hypomelanus. This suggests that populations of Cyclopodia horsfieldi from Pteropus vampyrus may have larger effective population sizes and may be acting as a source for founding island populations of parasites [Pteropus vampyrus is actively dispersing parasites to the outlying island populations of P. hypomelanus and making contact, and not vice versa.In contrast,  markers , and thendonesia . The sha Sumatra . We alsoarasites ,75. In sCyclopodia horsfieldi, may explain our results. First, the observed lack of geographic or host species population genetic structure seen in C. horsfieldi could simply be due to invariability or insufficient variance of the molecular markers examined. We suggest this is not the case as a number of previous studies of winged, free-living dipterans using the same markers at similar geographic scales have found significant geographic population structure [Wolbachia or Arsenophonus, may have influenced the observed lack of population structure and genetic variation via mitochondrial sweeps, as seen in other dipteran species [Wolbachia infection and immunity can create striking differences in mtDNA diversity and lead to speciation, e.g. in fig wasps [Wolbachia infection in insects, high levels of migration may still be a more prominent factor reducing genetic differentiation in species with potential to disperse long distances [Cyclopodia horsfieldi[Cyclopodia spp. and their flying fox hosts is likely not recent [Three alternative explanations, beyond host-mediated gene flow of tructure -39,76. A species ,39,77,78ig wasps . A numbeig wasps -82, and ig wasps . While wistances . Third, orsfieldi. This sct recent , and we Pteropus vampyrus, P. lylei, and P. hypomelanus are considered three of the most important natural reservoir hosts for this virus [Cyclopodia horsfieldi, suggest high levels of contact among Pteropus species in Southeast Asia – a pattern not apparent from host genealogy or prior ecological studies alone.Bats are important reservoir hosts for a large number of emerging zoonotic viruses , includiis virus -94. The Pteropus species in Southeast Asia, and also potentially with other fruit bat species not sampled here. Two individuals of the fly Eucampsipoda sundaica were collected from Pteropus vampyrus in Kuala Berang, Malaysia. This is an ectoparasite species most commonly associated with Eonycteris spelaea, a cave-dwelling, nectivorous bat species in Malaysia [Cynopterus and Rousettus[Pteropus spp. and other Pteropodid species with different roosting ecologies may occur on occasion, potentially during interactions over shared food resources [Pteropus giganteus, Rousettus leschenaultii, and Cynopterus brachyiotis was observed with infrared cameras at shared date palm sap feeding sites [Eonycteris spelaea[Cynopterus brachyotis individuals were also positive for NiV antibodies in these surveys [Pteropus spp., and fit with our observations of occasional ectoparasite sharing between these species.The data we present here on host specificity also contributes to a better understanding of interspecific contact between Malaysia , but alsRousettus,73. Thisesources . For exang sites . This sus spelaea. Similar surveys . These rCyclopodia horsefeldi, C. ferrarii. More specimens of bat flies, especially females, collected from Pteropus vampyrus should be examined in the future to see if this truly represents Cyclopodia ferrarii and another case of parasite host switching between bat species – this time between Pteropus vampyrus and P. giganteus. Horizontal transfer of bat flies between sympatric fruit bat species deserves further research attention, as results are relevant for understanding the mechanism of cross-species viral spillover and maintenance of diseases amongst reservoir host species. NiV has a short infectious period and long-term immunity in bats, suggesting that very large populations, or metapopulation dynamics, are necessary to sustain the virus in bat reservoirs [Pteropus hypomelanus[Pteropus spp in the region [We also observed one genetically divergent fly, RC24, that could represent a morphologically indistinguishable male of the sister species to servoirs -100. Hospomelanus, and whye region -94,102.Hepatocystis sp.) were identified using morphological and molecular methods from the same population of Pteropus hypomelanus sampled here [Hepatocystis infection in bat flies [Culicoides sp. midges have been demonstrated to be competent vectors for these parasites [Plasmodium murinus, were found in 7 of 26 dissected Nycteribia kolenatii from Myotis daubentoni bats [Hepatocystis and other related parasites, and if they move regularly between fruit bat populations and species (as demonstrated here) this could disrupt expected co-evolutionary patterns between chiropteran hosts and their malarial parasites.Other diseases endemic in bat populations, such as apicomplexan parasites, may not cause significant morbidity or mortality to their hosts, but have evolved long-term co-evolutionary relationships with them . Malarialed here . The rolat flies -107, andarasites . More reoni bats . Electrooni bats  which suBartonella spp. in bats globally [Bartonella parasites [gltA genotypes of Bartonella from Cyclopodia greefi flies collected from Eidolon helvum (Family Pteropodidae) in Ghana grouped closely with Bartonella genotypes from related Cyclopodia horsfieldi flies sampled from Pteropus hypomelanus in Malaysia, suggesting an underlying co-phylogenetic pattern for Bartonella-bat-bat fly associations [Bartonella[Bat flies also play an important role in the evolution and transmission of globally . There iarasites . For exaciations . Additioartonella,111.Cyclopodia horsfieldi, and show it to be a useful tool to understand host movement and interspecific contact among bat species. We observed an overall lack of morphological variation and phylogenetic structure across geographic regions and host species for C. horsfieldi. For some bat fly populations, elevated pairwise genetic differentiation (FST) did correspond to a lack of gene flow in host populations, i.e. insular populations of P. hypomelanus in Malaysia. By combining our data with previous studies of bat genetics, telemetry, and parasite host range, we suggest that P. vampyrus may facilitate movement of bat flies through frequent physical contact among the three Pteropus species in the region, and occasionally with other fruit bat species. Our approach and findings have wide implications for understanding zoonotic disease dynamics and cross-species transmission in bats, in particular the transmission and ecology of Nipah virus. Bat flies may also play a critical role in bat disease transmission and evolution (e.g. Bartonella or apicomplexan parasites), and their ecology, dispersal, and competence as vectors of bacterial and/or viral pathogens are in need of further investigation.For the first time, we investigate the population genetic structure of an Old World bat fly species, The authors declare that they have no competing interests.KJO lead and implemented the sample collection, laboratory work, molecular analysis, and writing of the manuscript. CWD examined the morphology for all specimens and helped to draft the manuscript. KD examined specimens and helped to revise the manuscript. JCM, DM, NBS, SLP, PD, and RD assisted in the study design, project coordination, analysis, and manuscript revisions; NBS additionally helped collect specimens on Pulau Perhentian, Malaysia. All authors read and approved the final manuscript.Dr. Olival is currently a Senior Research Scientist at EcoHealth Alliance. His research focuses on understanding the drivers of zoonotic disease emergence and the ecology and evolution of bats, their associated parasites and viruses. This study was part of his dissertation research at Columbia University with the laboratory work completed at the Sackler Institute for Comparative Genomics at the American Museum of Natural History.