We investigated the spatial genetic structure of the tiger meta-population in the Satpura–Maikal landscape of central India using population- and individual-based genetic clustering methods on multilocus genotypic data from 273 individuals. The Satpura–Maikal landscape is classified as a global-priority Tiger Conservation Landscape (TCL) due to its potential for providing sufficient habitat that will allow the long-term persistence of tigers. We found that the tiger meta-population in the Satpura–Maikal landscape has high genetic variation and very low genetic subdivision. Individual-based Bayesian clustering algorithms reveal two highly admixed genetic populations. We attribute this to forest connectivity and high gene flow in this landscape. However, deforestation, road widening, and mining may sever this connectivity, impede gene exchange, and further exacerbate the genetic division of tigers in central India. Panthera tigris) is the largest extant cat species and has become an iconic conservation emblem for Asian forest ecosystems . This anthropogenic transformation of land may have posed a barrier to dispersal and gene flow among tiger populations and led to genetic subdivision. Therefore, we tested for genetic structure that might have been created by potential impediments to tiger dispersal and risks posed by the features in the existing corridors.FST and genetic distances between tiger populations, (2) by testing the pattern of isolation by distance (IBD) in this meta-population, and (3) using two different Bayesian clustering methods that utilize individual-based information to decipher underlying genetic subdivision patterns. In the last century, the tiger population in India has experienced a dramatic demographic decline that may have eroded the genetic variation and left a genetic signature of a bottleneck in this population. We used two different approaches to test for evidence of a genetic bottleneck in this meta-population.Our tests for genetic subdivision in this tiger meta-population were based on (1) assessing the genetic variation and estimating the population-level genetic difference by calculating and comparing 2 (21.15–22.8°N and 76.5–81.05°E). The Satpura range is one of the oldest mountain ranges in the world and, together with the Vindhyachal range in the north and the Maikal range in the east, forms the catchments of the Narmada and the Tapti rivers and their tributaries . We used DNeasy Blood and Tissue Kit (Qiagen Inc.) for DNA extraction from hair and claw samples. Negative controls were included in every batch of DNA extractions and downstream PCR procedures. Sample processing and genomic DNA extractions were conducted in an area exclusively dedicated to extraction and separate from DNA storage and PCR and post-PCR processing areas. Sterilized conditions were maintained during DNA extraction to prevent cross-contamination of samples.Panthera pardus), whose feces can sometimes be confused with those of tigers, we screened each sample twice using tiger-specific mitochondrial DNA primer that amplifies a 164-bp fragment of the NADH5 region , and extension (72°C for 45 sec); and a final extension (72°C for 30 min). We optimized the seven microsatellite loci to be conducted in two duplex (F42-F43 and FCA672-E7) and three separate  PCR reactions.PCR reactions were performed in a 5-μL volume containing 2 μL QIAGEN Multiplex PCR buffer mix (Qiagen Inc.), 0.2 μmol/L labeled forward primer , 0.2 μmol/L unlabeled reverse primer, 10× Bovine Serum Albumin , and 1- to 1.5-μL DNA template. The PCR conditions for microsatellites loci included an initial denaturation (94°C for 15 min); 45 cycles of denaturation (94°C for 30 sec), annealing  are necessary for obtaining more precise estimates of population structure  and to match identical genotypes. Only those samples that amplified at least three times of four PCRs during multi-tube replication were used to create consensus genotype. A criterion of five of seven matching loci was used to compare each multilocus genotype and identify individuals, because the PID value was low enough (unbiased PID = 1.237e−07 and PID (SIB) = 5.584e−03) even when using the five most polymorphic loci.A set of highly polymorphic loci with a low PGenotyping errors are an inherent problem associated with studies that use non-invasively collected samples such as feces and hair  for every locus typed to address the potential problem of creating false genotypes that may arise as a result of genotyping hairs from multiple individuals. We did not detect any consistent pattern of multiple alleles for any of the loci from any of the hair clumps.Ho), and expected heterozygosity (He) using FSTAT 2.9.3.2 , allelic richness (AR), observed heterozygosity  were estimated using ARLEQUIN 3.11  and the geographical distance matrices. Because the Euclidian distance between study sites is not a true representation of their connectivity, we used the distance measured through forest connectivity between all pairs of study sites on Google Earth (Ver.6.0.3.2197) as the geographical distance.Frantz et al.  suggest P(X|K), the posterior probability of the multilocus genotypic data from individual samples to fit into a number of predefined clusters (K), so as to minimize the deviations from HWE. We conducted 10 independent runs for K ranging from 1 to 10 using admixture model and correlated allele frequencies, using a burn-in for 100,000 steps, followed by 100,000 steps for MCMC runs for data collection. The optimal value of K was selected using the posterior probability of the data for a given K (lnP(K)) and the second-order rate of change of log probability of the data between successive values of K .We used two different Bayesian clustering methods to detect any pattern of genetic structure in this landscape. The first method as implemented in the program STRUCTURE 2.3.2  among populations . We used both admixture models (BYM and CAR) for this analysis , given the sample size (n) for each population sample and for each locus under the assumption of mutation-drift equilibrium (Cornuet and Luikart k) with the allelic size range (r). In a bottlenecked population as rare alleles are lost, k is reduced faster than r, and therefore a low M-ratio relative to a critical value (0.68) indicates population bottleneck  = 7.411e−04). Genotyping error  and scoring error were detected, calculated, and removed using the EB test and the DCH test in the program DROPOUT  and 0.81 . Four loci  were found to be out of HWE in the global test, after Bonferroni corrections, but not consistently when samples were analyzed by study sites as separate populations. Two loci in Melghat and Pench, one in Satpura, three in Corridor, and no loci in Kanha were out of HWE . Similarly, mean DEST values and mean genetic distance were (0.035 ± 0.018 SD) and (0.25 ± 0.003 SD), respectively (R2 = 0.96) for the tree  between geographical distance and the genetic distance matrices using a Mantel's test in ISOLDE sub-programs implemented in GENEPOP. This shows an absence of IBD in this meta-population.There was no significant correlation (P(K) and ΔK method (Q) were variable for different study sites , which was higher than the threshold value of 0.68 found in a meta-analysis . The within-population genetic variation in all of our five study sites was similarly high (He∼0.80), showing no loss of genetic diversity at the landscape scale. Although measures of genetic diversity of tiger populations are available from Western Ghats , and the tiger reserve pairs located most distant from each other and lacking a forest corridor  have somewhat higher FST values . The DEST values showed similar results  and was known to be occupied by tigers Forsyth . By the FST values, and a weak genetic structure. If the anthropogenic forest fragmentation is <100–200 years old in this landscape, its effect on pairwise FST values of this tiger meta-population will not be detected for yet some time. The reason for this is that the FST statistic has a lag time of about 200 generations after the effect on gene flow can be detected due to the formation of a new barrier (Landguth et al. The tiger meta-population in the Satpura–Maikal landscape has high levels of genetic variation, very low Our study establishes the fact that genetic populations exceed the confinement of source populations and tigers require large landscapes consisting of breeding populations interconnected with forest corridors, for their long-term persistence. Tiger conservation efforts should be targeted at a landscape-scale level (Wikramanayake et al. We found that the tiger meta-population in the Satpura–Maikal landscape has low levels of genetic structure. Genetic subdivision was low between tiger reserves that were connected with forest corridors, thus lending support toward the functionality of this connectivity. The future of this tiger meta-population relies on implementing an effective policy that includes further protecting the tiger populations and their habitat in the tiger reserves and forested corridors that connect them.


Plasmodium vivax is observed in the lowlands of Papua New Guinea (PNG), the only country in the Western Pacific Region that experienced an increase in malaria cases over the last decade. Contrastingly, prevalence is lower in the Solomon Islands, a country aiming to eliminate malaria, where the number of confirmed cases decreased by approximately 50% from 2000 to 2010.The highest prevalence of Population structure can inform interventions against malaria. Genetic diversity, gene flow and linkage disequlibrium (LD) between loci are thought to influence the emergence and spread of drug resistance and may affect efficiency of future vaccines. In endemic areas where transmission has been reduced to low levels, genotyping could play an important role in tracking outbreaks and to identify the origin of imported malaria cases.P. vivax endemicity lower than in PNG considerable genetic differentiation between populations was found, suggesting limited gene flow. To understand the population genetic structure of P. vivax in the South Pacific, we have used 14 molecular markers to genotype 295 P. vivax samples from four sites in PNG and from the Solomon Islands. Diversity was very high, with expected heterozygosity values ranging from 0.62 to 0.98 for the different markers. As a result, the effective population size was also found to be high. Among the four PNG sites, a near absence of population structure was observed (Fsτ < 0.015). When comparing PNG and the Solomon Islands, population structure was found to be weak (Fsτ = 0.03 to 0.044).In areas of a P. vivax populations in the South Pacific are much less structured than populations from areas of lower endemicity in Latin America and Asia. In PNG, the presence of a large P. vivax reservoir seem to overcome the geographical barriers to transmission. Intensified, sustained control programs, which are coordinated among endemic countries and also target asymptomatic carriers, seem to be required to eliminate P. vivax in the South Pacific.


Myristica malabarica Lam., an economically important tree occurring in the Western Ghats, India. We located populations of the trees naturally occurring in different habitat suitability regimes (from highly suitable to poorly suitable) and evaluated them for their regeneration ability and genetic diversity. We also evaluated them for two plant functional traits, fluctuating asymmetry – an index of genetic homeostasis, and specific leaf weight – an index of primary productivity, often assumed to be good surrogates of fitness. We show a significant positive correlation between the predicted habitat quality and plant functional traits, regeneration index and genetic diversity of populations. Populations at sites predicted to be highly suitable had a higher regeneration and gene diversity compared to populations in sites predicted to be poor or unsuitable. Further, individuals in the highly suitable sites exhibited significantly less fluctuating asymmetry and significantly higher specific leaf weight compared to individuals in the poorly suitable habitats. These results for the first time provide an explicit test of the ENM with respect to the plant functional traits, regeneration ability and genetic diversity of populations along a habitat suitability gradient. We discuss the implication of these resultsfor designing viable species conservation and restoration programs.Ecological niche models (ENM) have become a popular tool to define and predict the “ecological niche” of a species. An implicit assumption of the ENMs is that the predicted ecological niche of a species actually reflects the adaptive landscape of the species. Thus in sites predicted to be highly suitable, species would have maximum fitness compared to in sites predicted to be poorly suitable. As yet there are very few attempts to address this assumption. Here we evaluate this assumption. We used Bioclim (DIVA GIS version 7.3) and Maxent (version 3.3.2) to predict the habitat suitability of  The concept of an ecological niche, proposed as early as 1900’s refers to a set of ecological conditions within which a species is able to maintain its population . GrinnelIn the last two decades, a number of efforts have been made to formally treat the concept of ecological niche of a species using several modeling tools e.g. Bioclim,GARP 7]7], MaxenA number of studies have validated the predictions of the ecological niche models in identifying the distribution of the species. Most of these validations have been made using the presence/absence data of the species in those predicted habitats ,19. HoweMyristica malabarica Lam., in the Western Ghats, India. First, using the existing natural distribution data of the species, we generate ecological niche model predictions on the habitat suitability of M. malabarica in the Western Ghats. Second, we evaluate the fitness of the species in areas predicted to be highly suitable and poorly suitable using several direct and indirect measures of fitness [In this paper, we argue that the habitat suitability of a species as predicted by the ecological niche model may also reflect the adaptive landscape of the species. Thus population of species in sites predicted to be highly suitable would be expected to have a higher fitness than con-specific populations in sites predicted to be poorly suitable ,5,20,21. fitness ,22-25namMyristica malabarica is an economically important plant, endemic to India. Field work was carried out in the central Western Ghats regions of Karnataka, with due permission from the Karnataka Forest Department. Tissue sampling was carried out under the supervision of local foresters and used solely for scientific research. The sampling was non-invasive and does not in any way affect the natural growth of M. malabarica.The study was conducted in the Western Ghats, a mountain chain running parallel to the West coast of India and one of the 34 biodiversity hotspots of the world . OccupyiMyristica malabarica Lam. commonly known as “Bombay mace, Malabar Nutmeg or Jaikai” belongs to the family Myristicaceae[M. malabarica has also been fragmented due to human activities leading to decline in the natural populations of M. malabarica. Owing to these pressures, the species has been designated as ‘vulnerable’ [M. malabarica, in recent years there is a growing interest in domesticating the species and in prioritizing areas for its conservation. sticaceae. It is asticaceae-30. The nerable’ . ConsideM. malabarica using a global positioning system (GPS-Garmin12). We used this primary presence only data for modeling the species distribution [Based on a number of field surveys undertaken over a two year period (2008 to 2010) in the Western Ghats, we recorded the latitude and longitude of 56 sites of occurrence of ribution . to develop the potential distribution and generating the suitability surfaces for the species, namely, a) Bioclim  - this is based on presence only data and was run using the envelope method using DIVA-GIS software, b) GARP (Genetic Algorithm for Rule Set Prediction) - this uses pseudo-absences to fit logistic regression to the data using Open Modelers Best subset implementation protocol, c) Maxent, (Maximum entropy) - this uses background points and minimizes the entropy. The softwares used were MaxEnt (version 3.3.2), DIVA-GIS  and GARP (version 1.1.3). We used 19 bioclimatic variables generated by Hijmans et al [We used three algorithmsns et al ; these wns et al . Also ecns et al  and our ns et al , discussWe used Least Training Presence Thresholding method for converting the prediction into habitat suitable indices . In thisWe used these calibrated models to evaluate the model performance using the set of occurrence points that was not used in building the model. As the classic area under curve (AUC)-Receiver operating curve (ROC) gives equal weight to commission and omission error , it is nOf the three models, GARP did not sufficiently discriminate sites based on their habitat suitability values, and hence we did not consider the model prediction further. On the other hand, Bioclim and Maxent discriminated the sites based on their habitat suitability; furthermore the discrimination was comparable between the models  plant functional traits - fluctuating asymmetry (FA) and specific leaf weight (SLW), b) recruitment or regeneration index and c) population genetic variability. Accordingly, we make the following specific predictions:Specifically we ask if populations of the species in sites predicted to be highly suitable (with higher habitat suitability index) have a higher fitness than con-specific populations in sites predicted to be poorly suitable (with low habitat suitability index). Traditionally, the unambiguous measure of fitness is fecundity or reproductive output -41. HoweIndividuals in areas predicted to be highly suitable  would be expected to have a low FA (and hence higher fitness) in contrast to sites predicted to be poorly suitable. Individuals in areas predicted to be highly suitable  would be expected to have a higher SLW (and hence contribute to higher fitness) in contrast to sites predicted to be poorly suitable. Populations in sites predicted to be highly suitable  would be expected to have a higher regeneration index compared to those sites predicted to be poorly suitable.Populations in sites predicted to be highly suitable  would be expected to have a higher genetic diversity compared to those sites predicted to be poorly suitable.M. malabarica and 14 sampling locations is given in At each of the 14 selected sites of varying habitat suitability, the data on the following were collected. The geographic range of Fluctuating asymmetry (FA) is a widely used measure of developmental instability ,43. An iMatured leaves from 10 trees (branches were randomly chosen for sampling) were collected from each of the 14 sites. For each leaf we measured width of the right and left halves from the midrib to the leaf margin at the mid-point , perpendicular to the midrib. Leaf fluctuating asymmetry was then calculated as: FA = (L – R)/size, where L =width of the left side, R = width of the right side, and size = (L + R)/2 .Specific leaf weight (SLW) is the ratio of leaf mass to leaf area. Physiologically, SLW refers to the density of packing of chloroplast; a higher SLW would indicate a higher photosynthetic efficiency and by extension can be expected to be positively related to fitness of the individual. 2). Ten matured leaves from each of 10 trees were harvested from each of the 14 populations. Leaf discs (1 cm diameter) were punched and oven dried at 70 °C for 72 h before being weighed. SLW was calculated as followsThe specific leaf weight was measured as a ratio of leaf dry mass (mg) to its area  to the total number of adults per quadrat.Regeneration index is a direct reflection of the life history strategy of a plant. It integrates the sum total of events from reproduction to the successful establishment of progeny and thus can be regarded as a measure of “realizable” fitness ,48. At ePopulation genetic variability at neutral markers such as simple sequence repeats (SSR) indicates the sum of mating events in a population and hence integrates a wide variety of parameters including population density, gene flow, recruitment success etc. This measure has been used in the past as a measure of population fitness. For example, several earlier studies have reported a direct association between the population genetic variability and fecundity in plants and animals -52. Population genetic diversity estimates were determined for 12 of the 14 sites . Fresh leaf samples were collected from 10-25 individuals per site. Since the species is a canopy tree, we resorted the services of tree climbers to access the tree crown and to fetch the desired leaf sample. The leaves were dried in silica gel and stored at -40 °C until DNA extraction. Total genomic DNA was extracted from the leaves following a standard CTAB procedure . DNA quaM. malabarica [Microsatellite marker analysis of the populations  representing the 12 sites was carried out using 5 polymorphic primer pairs out of 11 published SSR primer pairs developed for labarica . The prolabarica . The 5’ end of forward SSR primers were labelled with fluorescent dye  and samples for genotyping were prepared by mixing 10 μL of deionized formamide, 0.1 μL of 35-500 bp internal size standard  and 1 μL of PCR product. The mixture was denatured at 95 °C for 2 min and immediately placed on ice for a minimum of 5 minutes and loaded onto an ABI 310 Genetic Analyzer  for capillary electrophoresis and fluorescent scanning detection. The electropherograms of genotypes were analyzed using GeneScan 3.7 and Genotyper 3.7 (Applied Biosystems).The data was subjected to population genetic analysis to measure the overall genetic variability of populations sampled across the different habitat suitability regimes predicted by ENM. The following measures were calculated using different population genetic software: the mean number of alleles per locus (averaged across 5 loci) and to avoid bias caused by uneven sampling, a standardized estimate of allelic richness and private alleles  independent of sample size  was calcWe used general linear model (GLM) to relate the three fitness measures  with habitat suitability index. The three fitness measures were defined as the response variables and the habitat suitability index as a fixed explanatory variable. The relationship of the three fitness measures, namely, functional traits (FA and SLW), regeneration index and genetic diversity parameters with habitat suitability index was also evaluated using simple linear regression models. Besides, we also analysed for differences in the frequency distribution of FA and SLW across highly and poorly suitable habitats using a non-parametric Kolmogorov-Smirnov (KS) two sample test. For theAs a possible alternative explanation, we also analyzed if the population genetic parameters were correlated with the latitude from where the samples were collected. The relationship was statistically evaluated using univariate general linear models . M. malabarica yielded satisfactory results . Amongst the 19 input Bioclim variables only 12 contributed to model variation  . Leaves Individuals occurring in highly suitable habitats had significantly higher SLW compared to those occurring in the poorly suitable habitats. The SLW was positively correlated with habitat suitability index . The freThere was a significant positive relationship between the number of regenerants per adult (regeneration index) and habitat suitability index based on both, Bioclim and Maxent models . HabitatOver the 5 SSR loci, a total of 102 alleles were recovered from 194 individuals sampled from 12 populations. The mean allele number over all the loci for all the population was 15.4. Observed allele number per locus was significantly correlated with habitat quality (p=0.005 for Bioclim and p=0.008 for Maxent) . The, geAnalysis of univariate ANOVA under general linear model (GLM)  also indIn recent years a number of ecological niche modeling (ENM) tools have been used in predicting the habitat suitability of species ,11,13. AIn this study we considered three parameters of species performance, namely, plant functional traits (FA and SLW), regeneration index and genetic diversity. All measures are inter-dependent and reflect the cumulative effects of the life-history processes of a population, and have been used in the past to indicate the health of species’ population. For example, seed production or fecundity of plants (as a direct measure of fitness) has been found to be significantly higher in genetically diverse compared to inbred populations –66; hencFluctuating asymmetry is a widely used measure of developmental instability. The instability could be genetically driven  or environmental induced (as under stressful environments). Our study showed that individuals occurring in highly suitable habitats were significantly less asymmetric in their leaf morphology compared to those in poorly suitable habitat. In other words, it appears that individuals and hence population in highly suitable habitats might be more “fit” than those from the poorly suitable habitat. The difference in FA between the habitats might reflect the underlying genetic adaptations of respective populations; individuals in the highly suitable habitat being better adapted to their habitats than are individuals in the poorly suitable habitats. In the latter scenario, individuals may also been seen to be “stressed” in the poorly suitable habitats. Indeed, several studies have reported a positive association between leaf FA and environmental stress –69. For Another functional trait, the specific leaf weight (SLW), is an indicator of leaf thickness and the degree of mesophyll development within a leaf blade. The extent of mesophyll development largely determines the photosynthetic capacity of a leaf, although intracellular effect or other factors such as nutrient supply may also influence photosynthetic capacity . ClearlyThe number of regenerants per adult was significantly higher for populations in highly suitable sites than in poorly suitable sites indicating that as a direct measure of fitness, it pays to be in habitats that are highly suitable. The higher number of regenerants per adult (seedlings and saplings) might arise due to a number of features in the populations in the highly suitable sites. These might range from a higher mating probability to a better survivability of the seeds in such habitats. It would be interesting to tease out these different variables in order to understand the critical role they play in shaping the lifetime fitness of plants across the gradient of habitat suitability. However, we did not find any significant differences in the density of adults across the gradient of habitat suitability. In other words, the increased regeneration in high habitat suitability sites does not seem to be translated in to increased density of adults. Lack of realization of recruits in to adults could be influenced by number of contemporary variables such as human disturbances, grazing pressures, man-made fire etc., all of which were observed at one time or the other during the field visits . These variables are extraneous and do not reflect the intrinsic habitat quality. Thus the differences in recruitment between the highly and poorly suitable habitats may actually reflect the real population differences due to differences in the habitat suitability.A) and gene diversity per locus were significantly affected by habitat suitability. Also the highly suitable habitats had higher number of private alleles compared to poorly suitable habitats. These differences across the habitat suitability could arise due to evolutionary ecological processes. The highly suitable habitat may in fact indicate the geographical foci of the species origin and diversification. Accordingly these sites would be expected to represent the allelic diversity of the species over the evolutionary time frame. On the other hand, the poor habitat might represent the limits of the species distribution and therefore could be expected to have shed a number of alleles due to genetic drift [Our study also demonstrated that observed mean number of alleles per locus  increased and genetic parameters  decreased with increasing distance from the “abundant center” of species distribution. Assuming that the abundant center of the species may actually represent the niche centroid as proposed by Meyer et al. [Recently, Dixon et al. evaluatedr et al.  it is clBut why should predictions about habitat suitability reflect variation in plant fitness? Albert and Thuiller  argued tThe findings of the study provide a powerful handle for, and direction to, the conservation of rare, endangered and otherwise threatened species. Over the last few years, ecological niche models have become very popular in rationalizing choice of habitat for species re-introduction and ex-situ conservation –79. ThesM. malabarica itself, from our results it is evident that not all regions in the Western Ghats are uniformly suitable for the species. In fact within the Western Ghats, certain areas are highly suitable and others are unsuitable. Areas that have highest “habitat suitability index” would be ideal locations for domestication/cultivation of the species. The Karnataka Forest Department, for example, is keen to enrich the population status of M. malabarica in the Western Ghats. The outputs of this study, specifically the prediction of the ENM provide a robust platform for identifying sites for establishing such enrichment plantations.Finally with specific reference to Figure S1M. malabarica inthe Western Ghats.Distribution records and sampling sites of (TIF)Click here for additional data file.Figure S2M. malabarica at the sampling sites in the Western Ghats.Density of adults of  Note: Regions represented in black indicate areas of high genetic diversity and allelic richness.(TIF)Click here for additional data file.Figure S3Myristica malabarica at sampling locations in the Western Ghats.Map showing genetic diversity parameters of  A) Allelic richness B) Genetic diversity. Note: Regions represented in black indicate areas of high genetic diversity and allelic richness.(TIF)Click here for additional data file.File S1Supplementary tables. S1, Geo-coordinates and habitat suitability index of the fourteen populations selected for the study. Table S2, Skewnessand Kurtosis tests for frequency distribution of fluctuating asymmetry across highly suitable and poorly suitable habitats.Table S3, Kolmogorov-Smirnov (KS) two sample tests for frequency distribution of fluctuating asymmetry and specific leaf weight across highly suitable and poorly suitable habitats. Table S4, Skewnessand Kurtosis tests for frequency distribution of specific leaf weight across highly suitable and poorly suitable habitat. Table S5, The univariate ANOVA under general linear model (GLM) for different genetic diversity parameters across latitudinal gradient.Table (DOC)Click here for additional data file.


Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics).  Clostridium perfringens is a Gram-positive, spore-forming anaerobe residing in soil, water as well as the gastrointestinal tracts of various mammals, including humans. This ubiquitous bacillus is one of the most “toxic” of all known bacteria, collectively producing more than 15 different protein toxins/enzymes with diverse modes of action [Clostridium species synthesize some of the most potent toxins that include tetanus and botulinum neurotoxins, respectively produced by Clostridium tetani and Clostridium botulinum. These bacteria are found in similar environments  as long-lasting, quiescent spores that await a mammalian host and infection/intoxication opportunity. f action . PathogeC. perfringens was first isolated by William Welch and George Nuttall (1892) at Johns Hopkins Hospital  following autopsy of a cancer/tuberculosis patient, eight hours post-death. In particular, they noted flammable gas bubbles perfused throughout the cadaver and especially within blood vessels. Gas (carbon dioxide plus hydrogen) and organic acids  are common byproducts of anaerobic metabolism by C. perfringens. C. perfringens has also been successively known in the literature as Bacillus aerogenes capsulatus, Bacillus welchii or Clostridium welchii [C. perfringens are linked to protein toxins, and the next section succinctly describes the “major” and “minor” toxins produced by this bacterium. One of those toxins, epsilon, is this review’s focus as this protein impacts in many ways the veterinary and biodefense fields throughout the world.  Nuttall 892 at Jo welchii . VariousC. perfringens and have been a research focus of various laboratories around the world. For bacterial pathogens, toxins possessing diverse modes of action often play critical roles during disease, including food gathering and suppressing the host’s immune system. The four major toxins produced by C. perfringens either affect cell membranes directly by increasing permeability and causing ion imbalances , or destroy the actin cytoskeleton (iota toxin) [Clostridium and Bacillus species that enable survival in soil, protein toxins can play a pivotal role in bacteria surviving and subsequently thriving in an animal or human host. Protein toxins are important virulence factors of a toxin) ,2. IntoxC. perfringens based upon the production of one or more major protein toxins [There are five toxin types  of n toxins ,2  is another possible therapy for mitigating alpha-toxin induced myonecrosis [Type A strains of d humans ,13,14,15d wounds ,12. Deep and leg ,14,15. Pnecrosis ,14,16. Rnecrosis ,18. C. perfringens alpha was the first bacterial toxin ascribed enzymatic activity [C. botulinum neurotoxin [Alpha toxin is a zinc-containing phospholipase C (43 kDa), composed of two structural domains, that destroys eukaryotic cell membranes ,20. In 1activity . Like beurotoxin . InteracStaphylococcus aureus [Ascaris lubricoides) that release trypsin inhibitor into the intestinal lumen. Unusually high concentrations of protein in the intestinal tract facilitate C. perfringens types B  or C (human) overgrowth, leading to lethal levels of beta toxin, which forms cation-selective channels in lipid membranes [1 antagonists, capsaicin and an omega conotoxin (Conus magus MVIIA) that specifically blocks N-type calcium-channels.Originally purified in 1977, beta toxin is a 35 kDa protein that shares sequence similarity with the alpha and gamma hemolysins of s aureus ,24. The s aureus . For somembranes . Tachykiembranes . This paC. perfringens by consisting of two non-linked proteins that form a multimeric complex on susceptible cells, akin to the edema and lethal binary toxins from Bacillus anthracis [C. perfringens, B. anthracis is a Gram-positive sporulating bacillus found in soil that uniquely causes various forms of anthrax  in animals as well as humans. For iota toxin, the cell-binding component (Ib or iota b) is an 81 kDa monomer that forms a heptamer following proteolytic activation of the 94 kDa protoxin. The complementary enzymatic component is an ADP-ribosyl transferase (Ia or iota a) of 45 kDa, which in complex with the Ib heptamer, travels from the cell surface via lipid rafts into endosomes. The Ia component translocates from an acidified endosome into the cytosol, through a transmembrane pore formed by the Ib heptamers. In the cytosol, Ia covalently transfers an ADP-ribose moiety from nicotinamide adenine dinucleotide (NAD) to Arg177 on globular (G) actin, which then prevents filamentous (F) actin formation. Destruction of the cytoskeleton ensues, ultimately killing an intoxicated cell unable to maintain intracellular trafficking necessary for homeostasis.Iota toxin, discovered in 1943 by Bosworth , is uniqnthracis . Like C.C. perfringens enterotoxin that causes a major form of food poisoning linked to meat consumption [C. perfringens proteins designated as minor toxins that can play prominent roles in pathogenesis. A list of these latter toxins/enzymes with putative activities is presented in In addition to the major toxins listed above, there is a sporulation-linked sumption . FinallyC. perfringens types B and D is involved in animal  enterotoxemias that can be rapidly fatal and economically devastating [C. perfringens toxins as determined by a 50% lethal dose , subsequently activated by extracellular serine-type proteases (trypsin/chymotrypsin) that remove 10–13 amino- and 22 or 29 carboxy-terminal residues, depending on the protease [protease . The proAeromonas hydrophila aerolysin and C. perfringens enterotoxin  composed of three domains sharing conformation with other bacterial pore-forming toxins (PFTs) of the aerolysin-like family, such as erotoxin  [30,34,3Enterolobium contortisiliquum), mushroom (Laetiporus sulphureus) and freshwater hydra (Chlorohydra viridis) [Furthermore, this protein family is produced by other diverse life forms that include a Brazilian tree (viridis) . AlthougTwo groups of PFTs exist, based upon using either amphipathic alpha-helix  or beta-hairpin (beta-PFT) structures for membrane insertion. Etx is a beta-PFT. Putative roles for Etx domains include: receptor binding (domain I–amino terminus); membrane insertion plus channel formation ; and proteolysis activation plus monomer-monomer interaction sites (domain III–carboxy terminus) ,35. Follvs. 8.0), perhaps favoring more productive receptor interactions [C. perfringens lambda toxin, a 35 kDa thermolysin-like metalloenzyme) and the host are abundant [C. perfringens until stationary/death phase and is then released into the environment after autolysis [Proteolysis is a common process that activates many bacterial toxins. For epsilon, this process induces conformational changes that facilitate homo-oligomerization of activated toxin on the external surface of a targeted, eukaryotic cell. This “protein priming” enables Etx to quickly act after binding to diverse target cells that include those of neuronal, renal and endothelial origins (described in detail below). Loss of the amino- and carboxy-termini from the epsilon protoxin generates a more acidic protein  becomes internalized from the cell surface, promoting vacuole formation in the late endosomes and lysosomes [Etx induces pore formation in eukaryotic cell membranes via detergent-resistant, cholesterol-rich membrane domains (lipid rafts) that promote aggregation of toxin monomers into homo-heptamers ,43. The ysosomes ,47. Epsiysosomes . Pretreaysosomes . C. perfringens enhance binding of the bacterium and Etx to cultured cells [C. perfringens. Increased toxin binding following sialidase activity has been described previously for another enteric pathogen, Vibrio cholerae, perhaps suggesting a conserved pathogenic mechanism between diverse, enterotoxin-producing bacteria [Sialidases (neuraminidases) produced by ed cells . Interesbacteria . When applied to cells, Etx forms transmembrane pores (≥2 nm diameter) that rapidly facilitate: (1) free passage of 1 kDa-sized molecules; (2) decreased intracellular potassium levels; and (3) increased intracellular levels of chloride and sodium ,50. In aC. perfringens Etx, cattle less so [C. perfringens type D strains are more common than type B for epsilon-induced disease, although both toxin types produce Etx. Various factors can play a role in disease, including: (1) consumption of feed rich in fermentable carbohydrates; (2) poor nutritional status; (3) parasite infestation; (4) pregnancy toxemia; (5) tranquilizer (phenothiazine) use; and (6) overdose of a broad spectrum anthelmintic (netobimin) [Sheep and goats are the most frequent, natural hosts for  less so ,55. C. ptobimin) ,55. Althcluding:  consumptydrates;  poor nuttobimin) ,56,57,58tobimin) .Post-mortem findings in kidneys from either lambs or mice given Etx show similar results, including congestion, interstitial hemorrhage and degenerated epithelium in the distal tubules. Toxin accumulating in the kidney may be a host defense attempting to prevent lethal toxin concentrations in the brain . During C. perfringens Etx rapidly disrupts the blood-brain barrier, binds neuronal cells and causes lethality [n = 13 members in mammals) of 30 kDa-sized proteins that form tetrameric, membrane-channel complexes regulating water flow in various cell types, perhaps representing an exploitable therapeutic target against Etx. There is already a keen medical interest in aquaporins as: (1) a diagnostic indicator for certain autoimmune diseases ; and (2) a therapy for glaucoma, meningitis, stroke, epilepsy, cancer, pain as well as weight regulation [ethality ,52,56,60ethality . There cethality . Aquaporgulation . Cerebra50/kg body weight dose of Etx, clinical signs are evident in all within 95 minutes [50/kg body weight dose of Etx show clinical signs within 170 minutes. These animals experience a myriad of symptoms involving labored breathing , excited/exaggerated movements, intermittent convulsions, loss of consciousness and death. Additional signs of epsilon intoxication include elevated blood pressure, fluid in the lungs and brain congestion with edema [Clinical signs linked to Etx given intravenously to calves, lambs and kid goats are dose dependent and occur within minutes for calves and up to three hours in lambs ,65. For  minutes . In thisth edema ,65. TherC. perfringens type D leads to: (1) diarrhea ; (2) respiratory distress (lung edema and froth in trachea/bronchi); (3) glycosuria ; and (4) central nervous system dysfunction  [More natural than an intravenous injection of purified toxin, duodenal inoculation of Angora goats (12–14 kg kids) with either whole culture, culture supernatant or washed cells of hotonos) . Similarhotonos) ,67. In li.e., lesions) while the latter are more affected in the gut  [vs. controls, post-Etx administration. There is also fluid volume change in toxin-treated ileal loops of goats, but minimally so in sheep . Sodium efflux into the lumen may play a role in fluid accumulation within the ileum and colon, post-Etx exposure. Etx differentially affects sheep and goats, as the former have more overt brain effects (testine) . Regarditestine) . Gross ptestine) . Colonicin vivo involves ion imbalance, endothelial disruption and edema. A vicious cycle is established by the toxin within the digestive tract and includes increased intestinal permeability that promotes higher, circulating levels of toxin throughout the body [in vivo that often include small animals such as mice. Furthermore, results from animal models critically advance vaccine and therapeutic developments. The mode of action for Etx the body . To more50) yields seizures within 60 minutes [Various laboratories have published efforts involving different routes of murine intoxication by Etx. One laboratory reveals that an intravenous injection of Etx into mice –platelet aggregation, swelling of astrocyte end-feet corresponding to clear, perivascular spaces. Perhaps hypoxia linked to capillary damage affects more drastically cell types with relatively low levels of oxidative enzymes, such as astrocytes and granule cells. This extensive study by Finnie also points out that varying results from similar studies involving Etx-induced damage to the brain could be linked to fixation techniques: immersion vs. perfusion [In mice injected intraperitoneally with sub-lethal/lethal doses of Etx, electron microscopy of brain reveals that within just 30 minutes the end-feet of astrocytes (cerebellum) begin swelling with evident damage to the capillary endothelium . Cytoplaerfusion . C. perfringens type D culture supernatants , injected intravenously into mice, shows that Etx is required for lethality [in vitro, yet by definition each of these bacteria contain the toxin gene. Such findings of silent Etx genes clearly have implications upon diagnosis and taxonomy. These studies nicely mimic those subsequently done in sheep, goats and mice, revealing that a genetic knockout of Etx derived from a wild-type D strain  does not cause disease following intraduodenal injection [One study with ethality . Trypsinnjection . Complemnjection ,73. et al. shows that orogastric-administered Etx causes fluid accumulation in the murine ileum, with toxin binding preferentially throughout the villus, vs. crypt, surface [C. perfringens Etx. Interestingly, this same study reveals that Ussing chamber experiments of either mouse or rat ileum, incubated with Etx , does not disrupt transepithelial resistance. However, a similar experiment with basolateral application of toxin (30 LD50) shows damage within 40 minutes, likely disrupting tight junction gaps. Entry of toxin into the blood stream could facilitate this latter event. An extensive study by Goldstein  surface . Such a In this same study, horseradish peroxidase (44 kDa) minimally passes from the intestinal lumen into the zonula occludens of murine loops injected with Etx, suggesting minimal intestinal damage from the mucosal surface . Intraveet al. uses oral administration of Etx-producing C. perfringens type D (n = 10 strains given at 8 × 109 colony forming units (CFU)/mouse), leading to strain-dependent degrees of lethality (0%–100%) [in vitro production (protein amounts) of Etx and lethality. Another study by Fernandez-Miyakawa 0%–100%) . SeizureC. perfringens type-specific antisera is used less these days, given non-animal alternatives. Relatively large numbers of C. perfringens type D (104–107 CFU/mL) can be isolated from Etx-affected sheep, but such data for goats are lacking to our knowledge [n = 15), each inoculated intraduodenally with C. perfringens type D culture , reveals respective 92%, 64% and 57% detection rates of Etx (0.075 MLD50/mL detection limit) in the ileum, duodenum and colon [The classic mouse assay involving toxin neutralization with nowledge ,76. As aet al. assesses four techniques for detecting Etx (200–0.0075 MLD50/mL) spiked in intestinal contents, pericardial fluid and aqueous humor [50/mL detection limit) adsorbed to the plate well, is more sensitive than a monoclonal antibody capture ELISA (25 MLD50/mL), counter-immunoelectrophoresis (50 MLD50/mL) or toxin neutralization in mice (6 MLD50/mL). One major caveat with the polyclonal capture ELISA, counter-immunoelectrophoresis and mouse-lethal neutralization assays is that all use polyclonal antibodies against C. perfringens type D that could recognize antigens other than Etx. However, specificity of each assay is confirmed by using intestinal contents from sheep (n = 12) that died of causes other than Etx. Further studies using intestinal contents from goats with experimental or natural epsilon intoxication essentially confirm results from the spiked toxin experiments. Altogether, these results and those from other laboratories reveal that a properly crafted ELISA can be an effective method for detecting various toxins of C. perfringens [An extensive, ovine-based study by Uzal us humor . A captu in mice  MLD50/mLfringens ,9. A novel liquid chromatography–mass spectrometry technique uses immunoaffinity beads to initially concentrate Etx or protoxin from a complex matrix, such as serum or milk . Detectiet al. discovered that a cell culture assay employing MDCK cells, purified Etx diluted in culture medium and specific neutralizing antibodies  against Etx correlates well with the mouse lethal assay [Payne al assay . Of the in vitro studies that support discovery of therapeutics and vaccines for biodefense should use human, vs. canine, kidney cells? In addition to established cell lines, freshly isolated human kidney cells  behave like MDCK cells following Etx, which includes blebbing and large complex formation ,78. PerhC. perfringens type D (in particular Etx) are common reagents used for managing sheep and goat herds around the world [in vivo/in vitro studies suggest that therapy is possible. Perhaps a proteomics-based approach following Etx exposure can reveal even more interesting, and unique, host-based targets for therapeutic intervention? This has recently been done using mice given Etx intravenously, with subsequent analysis of select organs , plasma and urine for differentially expressed proteins [Effective vaccines against Etx (described below) are readily available for animal use, thus obviating the need for a therapeutic in susceptible populations given this prophylaxis. In fact, vaccines that target he world . There iproteins . The stuproteins , unique in vitro for testing novel therapeutics. A murine-based study using brain slices incubated with Etx and Etx-specific antibody shows that the toxin binds preferentially to the cerebellum, particularly oligodendrocytes and granule cells . Currentin vivo by Miyamoto et al. reveal that riluzole, a benzothiazole (234 Da) therapeutic for human amyotrophic lateral sclerosis that prevents presynaptic glutamate release, minimizes murine seizures as well as glutamate release induced by Etx [50) of Etx. However, the drug was evidently not used as a therapeutic . Experiments with rats also show that riluzole (8 mg/kg body weight), as well as glutamate antagonists MK-801 or CNQX , decrease hippocampus damage following intravenous injection of Etx  [vs. the mouse cerebral cortex and granular layer of the cerebellum [Related murine endeavors d by Etx ,82. Thesin rats) ,82. Etx-et al. ultimately found three, structurally-unique inhibitors that afford protection against Etx but do not prevent toxin binding or oligomerization. These inhibitors seemingly affect pore function and/or an unidentified co-factor important in Etx intoxication. Two of these compounds  protect cells using various criteria. One experiment used a constant concentration of inhibitor added concomitantly with increasing concentrations of Etx needed to kill 50 percent of the cells (CT50). A post-exposure experiment shows that addition of either compound (50 μM) up to 10 minutes after toxin (25 nM) exposure is protective in vitro. Such results logically lead to efficacy studies in animals, yet to be done or at least published to our knowledge. Furthermore, these inhibitors are specific for Etx as the related A. hydrophila aerolysin is not inhibited in similar assays [A small-molecule library  and high-throughput screening have also been used with a MDCK cell-based assay (384-well plates) for discovering novel therapeutics against Etx . In thisr assays . Bacillus anthracis, Escherichia coli and Helicobacter pylori [in vitro. Although used only as an Etx inhibitor in vitro (MDCK cells), dose-dependently effective at a 1, 2, 4 or 8 (wild-type toxin):1 (dominant negative) mole mixture, these unique constructs should perhaps be tested in vivo for therapeutic and vaccine potential. Another therapeutic approach against Etx includes dominant-negative inhibitors. These protein-based therapeutics have been used for other oligomer-forming bacterial toxins produced by Gram-positive and Gram-negative pathogens such as r pylori ,85,86. Dr pylori . These pd ~ 4–6 nM) on MDCK cells [Additional studies in mice show that the epsilon protoxin delays time to death when given intravenously before activated toxin. Protection presumably occurs by competitive occupation of cell-surface receptors, namely in the brain, by the protoxin . These dCK cells . Plasma CK cells . To dated values of Etx binding to rat brain homogenates and synaptosomal membrane fractions are 2.5 and 3.3 nM, respectively, thus very similar to Kd values in kidney cells [Bungarus multicinctus) that blocks acetylcholine release dose-dependently decreases binding of Etx, suggesting a common receptor for these protein toxins from diverse organisms [C. botulinum type A had no effect upon binding of Etx. Furthermore, sialidase pre-treatment of kidney cells and synaptosomes suggests different receptors for Etx [Identifying a toxin’s cell-surface receptor and understanding how toxin molecularly interacts with it can be very useful in formulating receptor-based therapies. The latter include toxin-binding antagonists and therapeutic targeting of susceptible cell populations throughout the body. Early receptor-binding studies employing radio-iodinated Etx reveal a heat-labile sialoglycoprotein, as pretreatment of rat synaptosome membranes with heat (70–80 °C for 10 minutes), neuraminidase, lipase or pronase effectively reduces saturable binding of the toxin . The Kd ey cells ,90. Furt for Etx ,90. i.e., homo-oligomer formation). There are eight other proteins, including sphingomyelin synthase 2, that when not expressed lead to varying cell resistance towards Etx [Unique studies involving gene-trap mutagenesis show that hepatitis A virus cellular receptor 1 (HAVCR1) is a receptor, or co-receptor, for Etx in MDCK cells ,92. It iards Etx . These rvs. wild-type toxin. Although recent studies with Etx and HAVCR1 are intriguing, further studies must be done to more clearly understand the intimate interactions between these molecules.The natural role of HAVCR1  involves T-regulatory cells and maintaining immunological balance throughout the body. HAVCR1 is a class I, integral-membrane, O-linked glycoprotein containing multiple isoforms varying within a mucin-like domain containing multiple glycosylation sites. The 100, but not 90, kDa variant of HAVCR1 binds Etx, perhaps due to increased length of the extracellular, mucin-like domain containing approximately 57 glycosylation sites . Domain et al. interestingly suggests that aforementioned tyrosines 29, 30, 36 and 196  do not play a role in binding/cytotoxicity of ACHN cells, in contrast to MDCK cells [vs. an electronegative glutamic acid, is too subtle of a change. Cumulative data from different laboratories indeed make this aspect of understanding epsilon intoxication complex and clearly unresolved to date. A subsequent, and contrasting, study by Bokori-Brown CK cells ,93. SuchC. perfringens type D (Etx), more lambs died from enterotoxemia than all other diseases combined [vs. lambs from ewes vaccinated six weeks before parturition. Ewe-derived antibodies can evidently protect lambs up to twelve weeks of age. Recommendations that lambs be vaccinated twice before six weeks of age, regardless of ewe vaccination status, are evidently not warranted [As stated earlier, Etx is primarily of veterinary concern and vaccines are used in the field ,95,96. Bcombined . Lambs farranted . From a arranted . vs. a heterogeneous population of antibodies in serum or colostrum [C. perfringens Etx, when timely present. Parenteral hyperimmune sera can also generate passive protection for three to four weeks in weaned lambs . Howeverolostrum ,100. AntC. perfringens  and associated toxins, are frequently formaldehyde toxoids of culture filtrates that may also contain whole cells [Human and animal vaccines have been quite effective over time against various diseases. However, many veterinary vaccines, such as those against le cells . Althougle cells . Currentle cells . This lavs. sheep. An attempt to remedy this problem involved a specific study in goats, comparing liposome- vs. aluminum hydroxide- based vaccines  of epsilon toxoid [The vaccination schedule to counter epsilon enterotoxemia varies too, depending upon the animal species . Two dosn toxoid . The latE. coli expressing Etx that is then inactivated by 0.5% formaldehyde at room temperature for 10 days can evidently be used as a cost-effective vaccine [C. perfringens [i.e., formaldehyde) or recombinantly  detoxified versions of purified protein. In contrast to relatively crude (multiple antigen) vaccines for Etx, recombinant technology can be helpful in many ways towards a better vaccine. The gene for Etx was first successfully cloned, sequenced and expressed in 1992, making subsequent work possible . As one  vaccine . This vafringens . The stafringens . Use of 50 of intravenous, wild-type toxin. In regards to recombinant-based attenuation, replacement of just one amino acid (histidine 106) with proline results in a non-toxic form of Etx (H106P) when tested by MDCK cytotoxicity and mouse lethality . In contE. coli heat-labile toxin B subunit) [vs. that found on native protein) and subsequently elicit antibodies that don’t readily bind native protein. This particular study did not determine if this construct elicits protective antibodies in an animal model for epsilon intoxication. In our opinion, replacement of select amino acids involved in a critical step during the intoxication process  typically does not grossly alter native conformation and thus represents a better vaccine strategy offering multiple epitopes to the immune system. Further vaccine refinement, with minimal risk of aforementioned toxicity, involves use of a linear, B-cell epitope (amino acids 40–62 of Etx) linked to a carrier molecule (subunit) . In theoC. perfringens is one of the most “toxic” bacteria known, using different protein toxins in different ways to perpetuate itself. Many of these toxins are commonly linked to various diseases in many mammalian species. In particular, the Etx has been studied by various groups and is primarily a veterinary concern for some large animals. Vaccines of varying quality are available to combat epsilon enterotoxemia, for veterinary use only. A human equivalent for biodefense is not commercially available. As a therapeutic or prophylactic for humans, toxin-specific immunoglobulins might be helpful following nefarious application of Etx. Clearly, there is much more to learn regarding Etx, how it works and how to protect against it. Such knowledge goes a long way towards bettering the lives of animals and humans.


Tuberculatus Mordvilko (Hemiptera: Aphididae) exhibit a variety of interactions with ants, ranging from close associations to non-attendance. A previous study indicated that despite wing possession, ant-attended Tuberculatus species exhibited low dispersal rates compared with non-attended species. This study examined if presence or absence of mutualistic interactions and habitat continuity of host plants affected intraspecific genetic diversity and genetic differentiation in mitochondrial DNA cytochrome oxidase I (COI) sequences. Sympatric ant-attended Tuberculatus quercicola (Matsumura) (Hemiptera: Aphididae) and non-attended Tuberculatus paiki Hille Ris Lambers (Hemiptera: Aphididae) were collected from the daimyo oak Quercus dentata Thunberg  in Japan and examined for haplotype variability. Seventeen haplotypes were identified in 568 T. quercicola individuals representing 23 populations and seven haplotypes in 425 T. paiki representing 19 populations. Haplotype diversity, which indicates the mean number of differences between all pairs of haplotypes in the sample, and nucleotide diversity were higher in T. quercicola than T. paiki. Analysis of molecular variance (AMOVA) showed higher genetic differentiation among populations within groups of T. quercicola (39.8%) than T. paiki (22.6%). The effects of attendant ant species on genetic differentiation in T. quercicola were not distinguishable from geographic factors. Despite low dispersal rates, host plant habitat continuity might facilitate widespread dispersal of a T. quercicola haplotype in Hokkaido. These results suggested that following T. quercicola colonization, gene flow among populations was limited, resulting in genetic drift within populations. However, frequent T. paiki dispersal is clearly evident by low genetic differentiation among populations within groups, resulting in lower haplotype diversity.Aphid species within the genus  Tuberculatus Mordvilko (Hemiptera: Aphididae) feeds primarily on Fagaceae, does not alternate host plants during its life history, and exhibits various interactions with ants, ranging from non-attendance to strong associations. Tuberculatus species exhibit cyclical parthenogenesis; During the summer months, Tuberculatus nymphs develop into winged  viviparous females, regardless of host plant nutritional quality, aphid colony density, or ant attendance. In autumn, alate males and apterous oviparous females appear. After mating, oviparous females move from the leaves to the branches to deposit eggs.The aphid genus Attending ants often have negative impacts on aphids, including decreased body size and/or embryo number due to the costs of increased honeydew production (Stadler and Dixon Tuberculatus quercicola (Matsumura) (Hemiptera: Aphididae)  and Tuberculatus paiki Hille Ris Lambers (Hemiptera: Aphididae)  a and an hididae) b: the toaiki Yao . MoreoveQuercus dentata Thunberg  is a deciduous broad-leaved tree, which is host to T. quercicola and T. paiki. The species is native to Japan, Korea, and China, and its habitat ranges from the seacoast to highland regions. Pollen fossil records indicate that daimyo oak was widely distributed along the Sea of Japan  to serve as a windbreak, or lost due to the development of more desirable landscapes. Such an anthropogenic fragmentation of Q. dentata populations would have significant impact on the population genetic structure and intraspecific phylogenetic divergence of T. quercicola, but not T. paiki.Genetic diversity is influenced by many factors, including animal and seed dispersal capacity, and habitat continuity. The daimyo oak Tuberculatus aphids was examined using mitochondrial DNA cytochrome oxidase I (COI) sequences. The two species are known to exhibit contrasting dispersal patterns, therefore unique haplotypes were expected in T. quercicola populations, and widespread haplotypes in T. paiki populations.Organellar genomes are well accepted as appropriate in estimating population history, and information regarding genealogical relationships between and within samples can typically be obtained from the appropriate genes. Furthermore, between and within taxa comparisons are rapidly and easily performed. In this study, genetic structure of T. quercicola and non-attended T. paiki species collected from an approximately 1800 km length in Japan. Population histories of the two species are discussed in terms of dispersal capacity and habitat continuity of host plants.This study examined whether the presence or absence of mutualistic interactions affected intraspecific genetic diversity in mitochondrial COI sequences, and focused on ant-attended T. quercicola. The seven regions were assigned to three islands divided by the strait: Hokkaido, Honshu, and Kyushu , which appeared from late May to mid September. Colonies of T. quercicola were attended by eight ant species, including Camponotus japonicus Mayer (Hymenoptera: Formicinae), Crematogaster teranishii Santschi (Hymenoptera: Myrmicinae), Formica fukaii Wheeler (Hymenoptera: Formicinae), Formica japonica Motschoulsky (Hymenoptera: Formicinae), Formica yessensis Forel (Hymenoptera: Formicinae), Lasius japonicus Santschi (Hymenoptera: Formicinae), Lasius sakagamii Yamauchi and Hayashida (Hymenoptera: Formicinae), and Pristomyrmex punctatus Smith (Hymenoptera: Myrmicinae)  . MultiplμL volumes, which included 1 μL of 10× PCR buffer , 0.8 μL of dNTP mixture (2.5 mM of each), 0.5 μL of 2 pM of each primer, 10 ng/μL of genomic DNA, and 0.025 units of Ex-Taq DNA polymerase (Takara-Bio). The reaction cycle parameters were as follows: 94°C for 3 min; 30 cycles of 94°C for 30 sec, 45°C for 20 sec, and 65°C for 90 sec. For heteroduplex formation, 2 μL of T. quercicola PCR products was mixed with 2 μL of T. paiki PCR product amplified from a single aphid using the same above conditions, and standard loading buffer. The mixture was heated to 94°C for 5 min, and slowly cooled to room temperature. One microliter of mixture was electrophoresed on 13-cm non-denaturing gels consisting of 10% acrylamide (36:1 acrylamide:bisacrylamide) in 1× TBE buffer at 150V for approximately 5 h. Gels were stained for 20 min in 1× TBE containing 0.5 μg/ml ethidium bromide, and examined using a ultraviolet transilluminator.Total DNA was extracted from the entire aphid following the Chelex procedure , 1.6 μL of dNTP mixture (2.5 mM of each), 1 μL of 2 pM of each primer, 2μL of 10 ng/μL of genomic DNA, and 0.5 units of Ex-Taq DNA polymerase (Takara-Bio). The reaction cycle parameters were as follows: 94°C for 3 min; 30 cycles of 94°C for 30 sec, 45°C for 20 sec, and 65°C for 90 sec. For each fragment, the entire PCR product was purified using the QIAquick PCR purification kit . A 5-μL sequencing reaction volume was used and consisted of 2 μL of Quick Start Mix , 0.5 μL of 10 pM forward or reverse primers, and 2.5 μL of 10 ng/μL template DNA. The reaction cycle parameters were as follows: 33 cycles of 94°C for 30 sec, 50°C for 15 sec, and 65°C for 90 sec. DNA sequencing was performed using CEQ2000XL DNA Analysis System (Bechman Coulter). Totals of 875 and 862 bp were aligned for T. quercicola and T. paiki, respectively. Alignment was conducted manually using MacClade 4.08 (Maddison and Maddison T. quercicola haplotypes 1 to 17 and AB679259 – AB679265 for T. paiki haplotypes 1 to 7.Each different haplotype identified in a population was sequenced. All PCR reactions were performed in 20-π), and mutation rates (θ (S)) were calculated to examine species genetic diversity. Haplotype diversity is defined as the mean number of differences between all pairs of haplotypes. Mutation rate is an estimate of the scaled mutation rate determined from the number of segregating sites (S) in a sample of DNA sequences. A segregating site is any of the total number of nucleotide sites that maintain two or more nucleotides within population (Hamilton D (Tajima Ne (effective population size) over time tend to exhibit a bimodal distribution, where two clusters of values in the mismatch distribution are evident  and the number of segregating sites (S) observed in a sample of DNA sequences to make two estimates of the scaled mutation rate, θ (S) and θ (π). D < 0 (θ (π) < θ (S)) indicates populations that had experienced rapidly growing. D > 0 (θ (π) > θ (S)) indicates populations that had experienced recent bottleneck. Genetic differentiation among populations was assessed using an analysis of molecular variance (AMOVA). Populations analyzed under AMOVA were a priori divided into six and seven of the eight major regional geographic groups for T. quercicola and T. paiki, respectively. An additional AMOVA was performed following a subdivision according to T. quercicola attendant ant species. Population genetics data were analyzed using Arlequin , Kyushu (2.7), and all samples from Japan (0.66), but negative value in Hokkaido populations (−0.36). Mismatch distribution of T. paiki showed left-skewed and bimodal shapes in Honshu and Kyushu populations, respectively  and negative values in Honshu (−0.97) and all samples from Japan (−0.47). Mismatch distribution of all samples from Japan exhibited unimodal and positively skewed distributions in T. quercicola (T. paiki (Seventeen  samples a and c. T. paiki . The folectively b and d. ectively a–c. Tajiectively e and f. ercicola d and T. T. paiki g populatT. quercicola and 54.2% in T. paiki). However, genetic differentiation among populations within groups was higher in T. quercicola (39.8%) than in T. paiki (22.6%). AMOVA for subdivision by attendant ant species exhibited high genetic variance within species, responsible for 61% of the total variation  throughout several groups. One of the primary factors affecting genetic diversity is the level of gene flow among populations. AMOVA results for T. quercicola indicated the variation in the second and third hierarchical levels  accounted for approximately 40% and 3% of the total genetic variation, respectively, indicating limited gene flow within relatively small geographic areas. The low T. quercicola dispersal rates may be associated with physical flight difficulties and fitness benefits from ant attendance. A comparative study on flight muscle in T. quercicola and T. paiki showed that flight muscle development was significantly lower in T. quercicola than T. paiki ), and three times the mean number of pairwise differences and nucleotide diversity in T. quercicola compared with T. paiki, indicating that mutation following colonization by T. quercicola in each newly colonized population might lead to genetic differentiation. Mismatch distribution and positive Tajima'D of all samples from Japan indicated that T. quercicola populations underwent recent bottleneck. Limited dispersal by ant attendance makes aphid colonies small populations, so that bottleneck would have been more effective in such small populations.This study revealed that T. quercicola Kyushu group haplotypes. Definitive phylogenetic relationships were observed among Kyushu group haplotypes in populations 22 and 23. Phylogenetic analysis showed a monophyletic group derived of clade 3 supported by a high bootstrap value. Furthermore, the bimodal pattern in mismatch distribution  accounted for 61% of the total genetic variation. However, ant species habitat and geographic subdivision  were not independent, suggesting T. quercicola, frequent dispersal of T. paiki is responsible for low genetic differentiation among populations within groups, resulting in lower haplotype diversity. Mismatch distribution and negative Tajima's D of all samples of T. paiki from Japan also indicates recent expansion. Haplotype 1 was widely distributed from the Chubu to Tohoku areas and in Hokkaido across the Tsugaru Strait, where the narrowest point between the Tohoku and Hokkaido is approximately 19 km. The mismatch distribution . Tuberculatus annulatus (Hartig) (Hemiptera: Aphididae), a non-attended Tuberculatus aphid, was caught by aerial netting at ∼200 m above land  and highlands surrounded by mountains (populations 18 and 19). The disruptive effects of these geographic boundaries can serve as a barrier to gene flow, and subsequently the observed haplotype distribution.Haplotype 3 detected in the Kinki and Chugoku groups, and haplotypes 4 and 6 in the Kyushu group exhibited discrete geographic distribution patterns in T. paiki haplotypes  exhibited little phylogenetic relationship with other haplotypes in each population. This was further clarified from the haplotype network. Missing haplotypes between H4 and H6, and between H5 and H1 or H2 indicated that phylogenetically distant haplotypes entered a population by chance likely due to widespread dispersal. Considering the phylogenetic relationships between H4 and H6, and the mono-haplotype in population 18, H6 is likely to have migrated from outside Japan.The three T. quercicola and non-attended T. paiki. However, recent interspecific comparative studies have been developed in the framework of phylogenetic relationships (Felsenstein T. quercicola and T. paiki are not sister species (Yao Tuberculatus species would contribute toward elucidating the evolutionary processes in aphid–ant interactions.This study clearly revealed contrasting patterns of genetic diversity between ant-attended cies Yao , it is pcies Yao  used phy


Dispersal and gene flow determine connectivity among populations, and can be studied through population genetics and phylogeography. We here review the results of such a framework for free-living marine nematodes. Although field experiments have illustrated substantial dispersal in nematodes at ecological time scales, analysis of the genetic diversity illustrated the importance of priority effects, founder effects and genetic bottlenecks for population structuring between patches <1 km apart. In contrast, only little genetic structuring was observed within an estuary (<50 km), indicating that these small scale fluctuations in genetic differentiation are stabilized over deeper time scales through extensive gene flow. Interestingly, nematode species with contrasting life histories (extreme colonizers vs persisters) or with different habitat preferences  show similar, low genetic structuring. Finally, historical events have shaped the genetic pattern of marine nematodes and show that gene flow is restricted at large geographical scales. We also discuss the presence of substantial cryptic diversity in marine nematodes, and end with highlighting future important steps to further unravel nematode evolution and diversity. Estim5 to 108[45[8, body si5[8, life hi5[8. Next to5[8, with a 5[8, marine 5[8,14 or pa5[8. These s5[8. DefininGene flow describes the exchange of genetic information between populations through migration, whereas dispersal is defined as the movement of individuals from one genetic population to another . ConsequIn what follows, we aim to provide a state of the art on dispersal and gene flow at ecological (i.e. one to a few generations) and evolutionary time scales (i.e. hundreds of thousands of generations), and the factors that may influence them , for marine nematode populations. We also discuss the current knowledge on cryptic marine nematode diversity and end by identifying key questions for future population genetic studies of marine nematodes.Dispersal is one of the most important life history traits for species evolution and persistence. Dispersal allows organisms to escape unsuitable environmental conditions, avoid competition and increase their distribution range. Dispersal distance is generally correlated with the presence and duration of pelagic larval stages in the water column , but theLitoditis marina typically frequents decaying and standing macroalgae in the intertidal, which form a rather transient habitat with frequent local extinctions when algae are decomposed. In such temporal habitats, the ability to disperse enables them to survive the strong fluctuations in habitat availability. L. marina produces dauer larvae, and due to its high reproductive capacity and short generation time it is an efficient colonizer that can establish populations from one or a few gravid females. To investigate the effect of colonization dynamics of L. marina on neutral genetic variation within and among patches in close proximity (≤ 1 km) to each other, Derycke et al. [L. marina on newly colonized algae was surveyed during one month at two contrasting sites in an intertidal salt marsh: in one site, defaunated algae were incubated amongst permanent algal stands that can act as a source population, while no algal stands were present in the second site [L. marina colonized these distant patches within 10 days, showing that dispersal of this nematode at this scale is substantial. In these distant patches, mtDNA haplotypes that were rare in the source population of the permanent algal stands were abundant suggesting that founder effects and genetic bottlenecks structured these populations . Next to these theoretical aspects, the genetic structure of marine species can be influenced by a variety of biological  and -history ), as wel-history . In whaties only ).Life histories are known for only a limited number of marine nematode species e.g. ,64-66. B. B66. BoLitoditis marina and Halomonhystera disjuncta have very short generation times and a high reproductive output [H. disjuncta if the presence of a dauer stage is taken as a prerequisite for a cp-score of 1 [L. marina species complex (PmI) within the Westerschelde estuary was low but significant , there was no significant differentiation among populations when the four seasons were pooled by locations . Since L. marina populations are highly unstable over time, this result suggests that the genetic differences caused by extinction-colonization dynamics become unimportant or balance each other when several time points are taken into account. Similar patterns of genetic structure have been observed in the dominant species (GD3) of the Halomonhystera disjuncta species complex in the Westerschelde . Again, when the genetic data from the two seasons were pooled within locations, no significant spatial differentiation was observed within the Westerschelde estuary . Although colonization-extinction dynamics can lead to genetic structuring at small geographical scales, such effects are no longer observed at time scales covering several generations.e output ,68, enabe output . Both spe output  or 2 forore of 1 . Both moore of 1 ,71 and pted from , Figure L. marina, changes 0.0001, ). This cL. marina. AlthougThoracostoma trachygaster lives in holdfasts of kelp along the Californian coast [Thoracostoma has a cp value of 5 [T. trachygaster because of its association with algal holdfasts, intuitively its very long generation time (probably only one or two generations per year) and few offspring would render successful establishment in new habitats less likely compared to L. marina and H. disjuncta. To exclude effects of well known biogeographic barriers, such as Point Conception and the Los Angeles Region, we investigated the genetic structure of T. trachygaster using populations that were continuously distributed along the coast with exclusion of these two barriers, and at a geographical scale of less than 100 km, to be comparable with the geographic scale of the sampling in the Westerschelde . When tBathylaimus assimilis is an endobenthic nematode species that in contrast to L. marina, H. disjuncta and T. trachygaster, is not associated with macroalgae. Dispersal through rafting is therefore unlikely for B. assimilis. Moreover, several endobenthic nematode species show vertical migration in the sediment [B. assimilis is expected to be lower than that of L. marina and H. disjuncta. Because B. assimilis is a less efficient colonizer than L. marina and H. disjuncta, and because it has an endobenthic life style, its population genetic subdivision in the Westerschelde is expected to be more pronounced than that of L. marina and H. disjuncta. Yet, a COI sequence analysis of 173 specimens from four locations in the Westerschelde suggested only a weak, but still significant structuring  had an impact on the genetic patterns observed within L. marina (PmI): 11.15% of the variation could be explained by habitat type , but a comparable amount of variation (13.09%) was observed by differences between populations within each habitat type , a genetic break along the British Isles was observed, and two possible postglacial recolonization routes of northern areas were suggested, one around the British Isles, and one through the English Channel [st values were non-significant, despite populations being separated by obvious geographical barriers . This illustrates again the chaotic genetic patchiness and suggests that additional ecological factors are influencing the genetic structure of marine nematodes.All species of the three in ), demons Channel  , with a significant amount of this variation being explained by differences between populations north and south of PC and, within the Southern Californian Bight, between populations north and south of LAR . Assuming that these lineages represent cryptic species, the presence of cryptic species seems therefore not to be correlated with life history traits. Instead, the prevalence of cryptic diversity in species with different life histories and from different areas suggests that it is a common phenomenon for marine nematodes. Nevertheless, we did not find any indications of cryptic species in the endobenthic enoplid Bathylaimus assimilis within the Westerschelde , so caution is needed when making such generalizations based on the limited data available.Nematode morphology is generally thought to be conserved, leading to speculations on the presence of substantial cryptic diversity in this taxon . Howeveris marina, five indisjuncta and threchygaster,104. The seasons  and at a seasons . When loSpecies in species complexes were delimited using the phylogenetic species concept with reciprocal monophyly of nuclear and mitochondrial gene trees. For nematodes, this approach is well-suited . SubsequL. marina species (PmI and PmIII) from the Westerschelde show a faster population development at a salinity of 15 psu than at a salinity of 25 psu [L. marina were combined in a multi species treatment, interspecific interactions reduced the population development of species PmII and even led to the total extinction of species PmIV. These interspecific interactions were also clearly affected by salinity, suggesting that fluctuations in abiotic factors may at least in part drive the coexistence of cryptic nematode species at local scales [Despite the substantial increase of the discovery of cryptic species over the last decade ,108, onlf 25 psu . Furtherl scales .Caenorhabditis elegans[The population genetic data of marine nematodes are exclusively based on COI, the usefulness of which has been well-documented ,111. Alts elegans,119, no L. marina and H. disjuncta, the effects of life history on population structure and nematode evolution can be explored. Similarly, including species with different habitat preferences should highlight habitat related effects on genetic structure. Since both L. marina and H. disjuncta occur on macroalgae, they are likely to have substantial passive dispersal capacity, and their dispersal ability may thus be much larger than that of typically endobenthic nematode species. Although for the time being this is not confirmed by our data on Bathylaimus assimilis, one would expect that if an endobenthic life style restricts dispersal in marine nematodes, then one should find higher Φst values in these species.Life history, morphology, behavior and habitat-associated traits all contribute to dispersal ability, but hitherto their relative importance for the genetic structure in marine nematodes remains largely unknown. Comparing the genetic structure between several species differing in one of these traits can contribute to unravel the relative importance of these traits for micro-evolutionary processes . For insst values are often significant between populations in close proximity, while they are not for populations that are further apart. This pattern may well be caused by the hydrodynamic currents in the study area, as well as by other ecological factors. Coupling hydrodynamic [Next to species-specific ecological characteristics, dispersal in marine nematodes may be driven by environmental parameters. The genetic structuring in marine nematodes so far does not seem to correlate with geographic distance, but instead shows chaotic genetic patchiness: population pairwise Fodynamic ,120 and odynamic  with genodynamic , can helNext to the analysis of neutral genetic variation, understanding the importance of selection on genetic structuring is essential for predicting how populations will respond to changing environments and to understand evolutionary diversification. Neutral loci across the genome will be similarly affected by demography and the evolutionary history of populations, while loci under selection will often behave differently and therefore reveal ‘outlier’ patterns of variation . Next geCaenorhabditis elegans and C. briggsae have revealed extensive intrachromosomal rearrangements, but remarkable conservation of chromosome organization and synteny [The advance in sequencing technology has generated an unprecedented amount of genome and transcriptome data from, mainly parasitic, nematode species ,128. Hor synteny . Compari synteny . Further synteny  and mult synteny . The effExperimental field studies have demonstrated that dispersal of marine nematodes at ecological time scales (i.e. < 10 generations) is substantial in the estuarine environment. The use of genetic data revealed that colonization dynamics strongly affect the genetic composition of local patches, with founder effects and bottlenecks causing strong differentiation among nearby patches. At deeper time scales, these genetic differences seem to disappear and populations become homogeneous. Consequently, gene flow in the marine nematodes analysed so far is substantial at geographical scales of 50 km, but is strongly restricted at larger geographical scales . This scale is tentative, and depends on a variety of environmental factors. Our data suggest that life history (short generation time and high reproductive output vs. long generation time and low reproductive output) and habitat preference  may be less important drivers for dispersal in marine nematodes, but additional analyses of the genetic structure in other nematode species are required to confirm these observations.The authors declare that they have no competing interests.SD conceived and wrote the manuscript, and analysed the data. TB and TM critically reviewed the manuscript and improved its intellectual content. All authors read and approved the final manuscript.