Alternanthera philoxeroides in response to different water availability . This species relies predominantly on clonal propagation in introduced ranges. We therefore expected genetic diversity to be restricted in the two sampled introduced ranges (the USA and China) when compared to the native range (Argentina), but that phenotypic plasticity may allow the species' full niche range to nonetheless be exploited. We found clones from China had very low genetic diversity in terms of both marker diversity and quantitative variation when compared with those from the USA and Argentina, probably reflecting different introduction histories. In contrast, similar patterns of phenotypic plasticity were found for clones from all three regions. Furthermore, despite the different levels of genetic diversity, bioclimatic modeling suggested that the full potential bioclimatic distribution had been invaded in both China and USA. Phenotypic plasticity, not genetic diversity, was therefore critical in allowing A. philoxeroides to invade diverse habitats across broad geographic areas.Phenotypic plasticity has been proposed as an important adaptive strategy for clonal plants in heterogeneous habitats. Increased phenotypic plasticity can be especially beneficial for invasive clonal plants, allowing them to colonize new environments even when genetic diversity is low. However, the relative importance of genetic diversity and phenotypic plasticity for invasion success remains largely unknown. Here, we performed molecular marker analyses and a common garden experiment to investigate the genetic diversity and phenotypic plasticity of the globally important weed  Since Charles Elton published his classic book on biological invasions in 1958, ecologists have been seeking to determine the factors that make a species an aggressive invader  to terrestrial , and shows prominent phenotypic variation  and two introduced ranges (the USA and China). Our major aim was to examine the relative importance of phenotypic plasticity and genetic diversity in determining invasion extent of A. philoxeroides in the USA and China. Molecular marker analyses and a common garden experiment were performed to compare the genetic diversity and phenotypic plasticity of A. philoxeroides among the three regions. We expected that the genetic diversity in the introduced ranges was lower, and phenotypic plasticity was higher, than in native range. In addition, we used a bioclimatic model fitted against native range distribution data to examine whether the full potential distribution of the species in the introduced ranges were invaded. If genetic diversity had played an important role in determining the niche range of A. philoxeroides, we expected that the lower levels of genetic diversity would limit its potential distribution in the introduced ranges.In this study, we conducted a series of intercontinental comparisons using Alternanthera philoxeroides (Mart.) Griseb., alligator weed, is a perennial stoloniferous herb. It can thrive in both terrestrial and aquatic habitats , the USA (9 sites), and China (9 sites)  Table . SpecimeA. philoxeroides  were selected to genotype A. philoxeroides. For each sample, at least two PCR amplifications were performed to evaluate the reproducibility of the bands obtained. Each reaction was carried out in a total volume of 20 μl mixture consisting of 20 ng of template total DNA, 10 mM Tris-HCl (PH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.7 mM primer, 1.5 unit of Taq polymerase and double distilled water. PCR was performed with an Eppendorf Mastercycler programmed for 5 min at 94°C followed by 40 cycles of 45 s at 94°C, 45 s at the appropriate annealing temperature (48–52°C), and 2 min at 72°C. The last cycle was 7 min at 72°C, followed by a 4°C soak. Amplification products were resolved by electrophoresis on 1.5% agarose gels buffered with 1 × TAE.To compare the genetic diversity measured by neutral molecular makers, all field-collected samples were analyzed using Inter Simple Sequence Repeat (ISSR) markers, which have proven effective in discriminating different clones of A. philoxeroides from all sampled ranges in response to different water treatments . Each habitat consisted of four rectangular plots (15 × 2 m). The aquatic habitat was simulated using 1 m deep ponds while the terrestrial habitat was simulated with raised garden beds. The aquatic and terrestrial plots were spatially alternated with each other, with adjacent pairs considered as replicates (blocks).A common garden experiment  was conducted to compare phenotypic plasticity of Regional-level phenotypic plasticity was compared by randomly selecting one clone from each sampling site . As no plants produced seeds in the greenhouse, thick root fragments were used to produce asexual plants as experimental replications. For each of the 25 clones, eight asexual plants with two pairs of leaves grown in pots  were allocated randomly to eight plots  to give a total of 200 pots. Aquatic plants were monitored daily to ensure that the water level remained nearly 2 cm above the pots in ponds. In terrestrial plots, plants received natural precipitation (1200 mm/year) plus supplementary irrigation in continuous sunny days (1L/pot when surface soil in >50% pots are dry).Plants were harvested after 2 months of growth, which was before any flowers appeared. First, six morphological and physiological traits were measured following the protocol reported in Geng et al. : (1) leaGenetic diversity was assessed both by neutral molecular markers (then referred to as marker diversity) and quantitative traits under common garden conditions (then referred to as quantitative variation).P), the Nei's genic diversity index (He), and the Shannon diversity index (I). We performed a re-sampling procedure to control the confounding effect of uneven sample size . Specifically, we randomly sampled 21 individuals from the USA and China datasets respectively, from which we calculated the regional genetic parameters. This re-sampling procedure was then repeated 30 times, and the average value for each genetic variable based on the sub-dataset was reported along with average based on the whole dataset. Second, PAUP 4.0  or absent (0) for each sample. Bands of the same molecular weight were considered to represent the same allele at a given locus. This dataset was analyzed in two ways. First, we used Popgene 1.32 .A. philoxeroides in terrestrial vs. aquatic growth conditions while the regional or clonal effect suggests differentiation of A. philoxeroides in phenotypic traits among different regions or clones. A significant treatment by region or treatment by clone interaction indicates that the level of phenotypic plasticity is different among regions or clones. We performed F tests by testing region effect over clone term; by testing both treatment and treatment-by-region effects over the treatment-by-clone interaction term; and testing treatment-by-clone effects over the error term. We first used Log  as a covariate to examine whether the covariate explained a significant amount of variation. When the covariate was not significant, we performed an ANOVA instead of ANCOVA, and examined the assumptions of homoscedasticity and performed data transformation where necessary.First, we examined the reaction norms at regional level by plotting the mean values of all clones from the same continent against two habitat treatments. We performed two-way nested ANOVAs to examine the effects of treatment, region, clone, and treatment-by-region interaction on each univariate trait, in which clone was nested in region as a random factor. The statistical model included the following terms: treatment, region, clone, treatment-by-region, treatment-by-clone, and error term. A significant effect of treatment suggests significant phenotypic plasticity of post-hoc comparisons based on Bonferroni test to examine whether or not the phenotypic plasticity of A. philoxeroides in China and USA was significantly different from those from Argentina. As all Chinese clones in common garden experiments proved to be the same multi-locus genotype , the plasticity index for Chinese clones might not be independent to each other. To examine the potential effects of pseudoreplication, we also performed a nested ANOVA on plasticity indices, in which region was the main factor and clone was nested in region as a random factor. The overall mean plasticity for Chinese clones was used in this nested ANOVA. In addition we conducted multiple comparisons via t-tests. Specifically, the overall mean plasticity index for Chinese clones was used as a fixed value in two single-sample t-tests , separately. One two-sample t-test was performed when comparing the USA and Argentina. The results of one-way ANOVA, nested-ANOVA, and multiple t-tests were similar (see Supplementary Files). For simplification, we reported the result of one-way ANOVA in main text.Second, quantitative analyses of phenotypic plasticity were also performed based on the plasticity index . The critical probability levels for the correlation coefficients were Bonferroni corrected for multiple comparisons to α/36 = 0.0013 .A. philoxeroides from different regions, using principal component analysis (PCA) conducted on the clone mean value of each trait . Trait data were standardized prior to PCA. We also performed constrained ordination analysis , in which two factors variables  were used as explanatory variables. The result of RDA was highly similar to that of PCA (see Supplementary Files). For simplification, we reported the result of PCA (biplot) in main text. All analyses were carried out with R  for each genotype across terrestrial and aquatic habitats were also examined using the same method. The distance calculations and Mantel tests were done using the ecodist package  was fully invaded. The model was developed using CLIMEX Version 4.0 , supplemented by China distribution , and the USA distribution . A previously published CLIMEX model for alligator weed , the USA (32), and China (126) were analyzed using ISSR markers. The eight ISSR primers generated a total of 60 bands and 61 unique ISSR multi-locus genotypes. For samples from Argentina and the USA, each plant was characterized by a unique multi-locus genotype. In contrast, 94% (119) of the Chinese samples were identical  also showed much higher genetic diversity for clones from Argentina and the USA than those from China, even after the confounding effects of different sample size were controlled  and by quantitative traits (B), of .Genetic diversity as estimated using quantitative traits measure during the common garden experiment showed patterns consistent with those revealed by neutral molecular markers. For both habitats, quantitative variation was much lower among Chinese clones than among clones from Argentina and the USA for most quantitative traits examined Table .P < 0.01), despite significant differences among clones (Table P < 0.01).Generally, plants in aquatic plots had longer leaves, longer internodes, thicker stems and larger stem pith cavity, larger specific leaf area, lower root/shoot ratio, lower relative chlorophyll content, and lower storage root/fine root ratio, than those in the terrestrial plots Figure . The twot-tests also found no significant differences among regions for most examined traits when comparing plasticity indices  were consistent across all three regions Figure . The ones Figure . Consists Figure . Althougs Figure . The clos Figure . Indeed,s Figure .Phenotypic correlation analyses showed that some traits were significantly correlated with each other in both habitats Figure . For exar = 0.27, p = 0.04) and aquatic habitat , restricted largely by cold stress to the south and west, and heat stress in areas to the north of Argentina  was high even in areas where A. philoxeroides has not been recorded in Argentina. These could not be excluded from the model by further restricting parameters without losing known locations, therefore suggesting non-climatic factors are also important.Bioclimatic modeling suggested that the full potential distribution of the species in the introduced ranges were invaded in China and the USA. a Figure . Soil moA. philoxeroides has not yet been reported.The potential distribution in China and the USA was largely restricted by cold stress to the north and heat stress within the range and to the west. In both cases most distribution records occurred within the potential range, although records did extend into areas where cold stress was expected to be too high. There were no extensive areas where EI was moderate to high (above 10) in which A. philoxeroides in the native range (Argentina) and one introduced range (USA), but not in another introduced range (China). Specifically, genetic diversity in the USA was similar to that in Argentina. In contrast, the levels of genetic diversity in China were extremely low and many individuals collected from geographically distant sites shared the same multi-locus genotype. Contrary to our expectations that clones in introduced ranges had higher phenotypic plasticity than those in native range, the phenotypic plasticity in response to different water availability  was similar across all three regions. Despite the different levels of genetic diversity, bioclimatic modeling suggests that the full potential bioclimatic distribution had been invaded in both China and USA. Taken together, our results suggest that the ability of A. philoxeroides to successfully invade heterogeneous habitats and broad geographic distributions is the consequence of phenotypic plasticity rather than genetic diversity.Phenotypic plasticity and genetic diversity have long been proposed to contribute to the invasion success of alien plants, especially clonal invaders, but few studies have tested their relative importance Barrett, . In thisA. philoxeroides. Mantel test suggested that the correlation of the two measures is significant but weak , which is similar with previous results (r = 0.217) based on meta-analysis  or the USA (high diversity) as assessed by a bioclimatic model fitted against the native-range distribution. Especially, the genetic uniformity of Chinese clones did not appear to restrict the geographic and ecological distribution of A. philoxeroides. Similar results have also been reported in a few other invasive alien plants in their introduced ranges, e.g., Pennisetum setaceum  acclimation to these habitats.Phenotypic plasticity is frequently envisaged as one of the characteristics that contribute to the adaptability and invasiveness of alien species  in terrestrial habitats, which often kill all the above-ground parts  varied greatly, especially in the clones from Argentina (native region). Indeed, phenotypic plasticity levels of Chinese and USA clones fell within the variation ranges of Argentine clones. In other words, some native clones were even more plastic than the introduced clones. Previous studies on the comparison of phenotypic plasticity between native and introduced populations/species have produced mixed results. For example, Bossdorf et al.  provides valuable information akin to experimental replications. In cases where repeated invasions indeed share common features, comparisons among these replications may help identify the relative importance of different factors in determining invasion success. In this study, we compared the genetic diversity and phenotypic plasticity of A. philoxeroides across three regions. Our results revealed that the pattern of “lower genetic diversity” in one introduced range  was not found in another introduced range , reflecting the heterogeneous nature of biological invasions even for the same invader. In contrast, high levels of phenotypic plasticity were found across all three regions, highlighting the importance of phenotypic plasticity as a common feature underlying successful invasions of A. philoxeroides. Accordingly, this multi-region comparative approach, including two or more biogeographical replicates, may be especially indicative for understanding the relative importance of different factors underlying successful invasion.A biogeographical approach is often proposed to compare the introduced populations with their native counterparts , and the International Foundation for Science (Grant A/4424-1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lucifuga cavefish species have very narrow geographic distribution in Cuba. However, one species, L. dentata, has a wide but disjointed distribution over 300 km in the west of the island. In order to estimate the relative role of vicariance and dispersal in the unexpected L. dentata distribution, we obtained partial sequences of the mitochondrial DNA (mtDNA) cytochrome b (cytb) gene and control region (CR), and then applied Approximate Bayesian Computation (ABC), based on the identification of five genetic and geographic congruent groups of populations. The process that best explains the distribution of genetic diversity in this species is sequential range expansion from east Matanzas to the western Pinar del Río provinces, followed by isolation of groups of populations. We found relative high haplotype diversity and low nucleotide diversity in all but the Havana group, which has high values for both diversity parameters, suggesting that this group has been demographically stable over time. For two groups of populations (Cayuco and Bolondrón), the mismatch distribution analyses suggests past demographic expansion. In the case of the Cayuco region, the star like relationships of haplotypes in the network suggests a recent founding event, congruent with other evidence indicating that this is the most recently colonized region. Over all, the results suggest that a combination of habitat availability, temporal interconnections, and possibly the biological properties of this species, may have enabled its dispersal and range expansion compared to other species of the genus, which are more geographically restricted.Consistent with the limited dispersal capacity of most troglobitic animals, almost all  Some general patterns have emerged from studies on genetic polymorphism in obligate subterranean aquatic species (stygobionts). These patterns are: relatively low genetic diversity, reduced if any gene flow between geographicaly close populations, and few dispersal events over long distances , 9, 10. Lucifuga dentata is a stygobiont (Poey 1858) [Lucifuga species revealed several cryptic lineages restricted to narrow karst patches, with the sole exception of L. dentata sensu stricto, which has a fragmented distribution over 300 km in Cuban south-western karst areas [cytb gene revealed three to four highly isolated geographic groups of populations and showed a significant association between haplotype clades and their geographical distribution. A Nested Cladistic Phylogeographic Analysis (NCPA) suggested long-distance colonization and fragmentation as mechanisms shaping these patterns. Other evidence , together with the highly fragmented nature of underground cave systems, strongly supports dispersal followed by allopatric divergence as the major mechanism promoting speciation in Lucifuga. Here we test these two hypotheses, dispersal versus fragmentation at different scales, in L. dentata by Approximate Bayesian Computation (ABC). We increased the sample size for some regions and added the mtDNA control region sequence to the cytb data set previously used [The blind cave dwelling fish ey 1858) . The mosst areas . A phylosly used .Lucifuga dentata sensu stricto individuals  were digested with proteinase K (100 μg/mL) in 200 μL lysis buffer  at 55°C with slow constant shaking, followed by a phenol: chloroform extraction using Phase Lock GelTM (Eppendorf) tubes. The polymerase chain reaction (PCR) was used to amplify partial sequences of two regions of the mtDNA: 810 bp of the cytb gene  with the primers Glufish (5' CCAATGACTTGAAGAACCACCGTTG) [2. PCR products were purified using the QIAquick® PCR purification kit (QIAGEN) and cycle-sequenced in both directions using the Big Dye terminator sequencing kit (Applied Biosystems). The fragments were resolved on an ABI 3100 automated sequencer (Applied Biosystems). The cytb sequences were deposited in the EMBL database with accession numbers FR716716-FR716717 and FR716721 for cytb sequences and accession numbers FR873577 to FR873609 for the CR sequences. The cytb sequences of 76 individuals previously analyzed [Small pieces of fin or muscle tissue (2–3 mmeinase K 0 μg/mL iACCGTTG) . Five toeinase K 0 μg/mL icytb and CR segments were concatenated and aligned using MEGA6 [D statistics [Sequences were edited using Bioedit Sequence Alignment Editor v. 5.0.9  and manung MEGA6 . To testatistics  was calcatistics . StatistFCT statistic. The level of the haplotype diversity (h), nucleotide diversity (π), and genetic differentiation (FST) were estimated for the inferred groups (populations) using the program ARLEQUIN v. 3.5 [We first explored the spatial structure of genetic variation using a Spatial Analysis of Molecular Variance (SAMOVA) . SAMOVA N v. 3.5 .We constructed a haplotype network using the program TCS v 1.21 , to visuL. dentata, we used the Approximate Bayesian Computation (ABC) method as implemented in the program DIYABC v. 1.0.4.46 [To evaluate the probability of different scenarios of dispersion and/or fragmentation accounting for the observed geographic pattern of genetic variation in 1.0.4.46 . The choi.e. long distance dispersal) to the west (i.e. from Havana to Pinar del Río). This is the inferred NCPA scenario from a previous analysis [In the first scenario an ancestral population, inhabiting the continuous southern karst of Matanzas and Havana regions  split, aanalysis . In the i.e. numbers of haplotypes, number of segregating sites, mean pairwise differences and variance of the pairwise differences) were chosen according to their accuracy and relevance for the parameters of interest for model estimation. We performed six million simulations in order to choose the set of summary statistics that best captured relevant aspects of the data [FST statistics and mean pairwise genetic differences within and between groups were also calculated [The reference table for the scenarios was obtained after six million simulations of the data. This procedure was repeated three times to test for consistency of the results. The posterior probabilities of the competing scenarios were estimated using the logistic regression method  from 10%the data , 32, 33.lculated .i.e. population expansion) a mismatch distribution analysis of pairwise nucleotide differences between haplotypes was performed using ARLEQUIN v. 3.5 [Fs [R2 [To test for demographic changes  comprised of one indel, 65 transitions (TS), 11 tranversions (TV) and 5 sites with both TS and TV (TS/TV = 4.75). A total of 49 haplotypes were identified , as expected under neutral sequence evolution.The total length of the concatenated FCT statistic steadly increased as the number of groups of populations increased, from 0.514 for two groups, up to 0.745 for 12 groups (data not shown). Populations included individuals of single caves (seven instances), two caves , three caves (Chicharrones + Chivo + Pichi) and a group of five caves (Felipe + Pozo Azul + Patrón + El Jagüey + La Raja) in each case, relatively close geographically  that represent the major geographic cores: Guanahacabibes (extreme West) and Cayuco (West), in Pinar del Río; Havana (middle); Bolondrón (East) and Agramonte/Jagüey Grande (extreme East), in Matanzas (see i.e. in Agramonte/Jagüey Grande region), but because of the small sample sizes per locality  and nucleotide diversity (π = 0.01 ± 0.005) were high. The group from Havana has the highest haplotype diversity (h = 0.924) and nucleotide diversity . The other four populations also had relatively high h values, with the exception of Guanahacabibes , but had significant lower values of nucleotide diversity as shown above (FST) were also very high (FST ranging from 0.46 to 0.91) and highly significant for all population comparisons . In contrast, the Bolondrón, Havana and Pinar del Río (excluding El Judio) regions have haplotypes that are shared between intraregional caves separated in several cases by larger karst gaps, suggesting that migrations occur more frequently between these caves. At the scale of the entire L. dentata distribution, the five major regions do not share haplotypes, indicating no exchange of individuals between these populations in recent times. However, the occurrence of haplotypes in Havana genealogically derived from the Matanzas region, and relatively divergent from their most closely related haplotype, is the signature of a more ancient dispersal event (e.g. raise of underground water levels) can be overcome, facilitating transient migration between distant regions [The approach (ABC) used to infer the processes that shaped the current distribution of the genetic variability in al event . These cres 3 to  km. In c regions , 44.L. dentata in southern Matanzas karst and its inferred westward dispersal is further supported by the identification of sister species in the north of this region [Fs and R2 statistics  were inferred in Matanzas between Bolondrón and Agramonte/Jagüey Grande, and in Pinar del Río between Cayuco and Guanahacabibes. In both cases dispersal events appear to have shaped current distributions of genetic variation. Although neither fragmentation nor dispersion between Cayuco and Guanahacabibes were estimated as being more probable in the ABC analyses (see i.e. Cayuco and surrounding areas).At a smaller geographic scale, two other recent simultaneous events  as well as a large geographic distribution. Direct field observations suggest that this species’ census size is larger than that of others Lucifuga species inhabiting the same caves. The relative numbers of L. dentata and L. subterranea in Havana caves and the relative numbers of L. dentata and Lucifuga sp. 3 in Pozo Azul (Cayuco region) are about ten to one or more. The geographic distribution of genetic variation in L. dentata appears most compatible with a historical scenario of a one way stepping stone dispersal from East to West followed by strong isolation, in sharp contrast with the very narrow distribution of the other Cuban Lucifuga species. Overall, this study highlights the importance of genetic data as indirect evidence for underground connectivity patterns, in particular when direct data are unavailable.Compared to other Cuban L. dentata and another Lucifuga species.The ecological characteristics of a freshwater fish species can be used to predict its dispersal capabilities . TherefoL. dentata had lead to this species being considered as not threatened in the Red List of Cuban Vertebrates [Clarias gariepinus) in some aquifers are the main factors of concern.Finally, the high genetic diversity and relatively large distribution of tebrates . NonetheS1 FigDiamonds connected by dash represent observed distributions; continuous and broken lines indicate expected distributions and confidence intervals respectively.(DOCX)Click here for additional data file.S1 TableSee (DOC)Click here for additional data file.S2 Tablecytb+NCR haplotypes of Lucifuga dentata. B. Variable sites characterizing the 49 cytb+NCR haplotypes of Lucifuga dentata.A. Number and geographic distribution of the (DOC)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Anguilla anguilla) underwent a drastic and well-reported decline in abundance in the late 20th century and still displays low recruitment. Here we compared genetic diversity indices estimated from neutral  and adaptive markers (MHC) between two distinct generations of European eels. Our results revealed a clear discrepancy between signatures obtained for each class of markers. Although mtDNA and microsatellites showed no changes in diversity between the older and the younger generations, MHC diversity revealed a contemporary drop followed by a recent increase. Our results suggest ongoing gain of MHC genetic diversity resulting from the interplay between drift and selection and ultimately increasing the adaptive potential of the species.The recent increased integration of evolutionary theory into conservation programs has greatly improved our ability to protect endangered species. A common application of such theory links population dynamics and indices of genetic diversity, usually estimated from neutrally evolving markers. However, some studies have suggested that highly polymorphic adaptive genes, such as the immune genes of the Major Histocompatibility Complex (MHC), might be more sensitive to fluctuations in population dynamics. As such, the combination of neutrally- and adaptively-evolving genes may be informative in populations where reductions in abundance have been documented. The European eel ( Preserving natural biodiversity while allowing species to maintain their adaptive potential is a major challenge in modern conservation biology . AnthropThe genes of the Major Histocompatibility Complex (MHC) have repeatedly been shown to be suitable candidates to evaluate the immune adaptive potential of endangered populations . This hiThe European eel is a highly migratory, semelparous fish whose spawning grounds are located in the Sargasso Sea and whose foraging grounds cover coastal, mixohaline and freshwater habitats  across mAnguillicola crassus  screening of both neutral and adaptive markers and (2) a temporal approach directly comparing two distinct generations of eels. A temporal approach is regarded as a key requirement when investigating the signature of demographic events on genetic diversity in a wild population . SpecifiA total of 683 eels were analyzed in this study, 202 of which corresponded to mature silver eels caught in freshwater while undertaking their spawning migration. The other 481 individuals were glass eels collected from 2009 to 2012 immediately upon their arrival in coastal waters . Number silver eels” age group and the younger “glass eels” group. The first group included individuals born soon after the drop in recruitment (late 1990s and early 2000s) while the second group consisted of very recently recruited individuals (2009 onwards) (silver eels”) or tail clips (“glass eels”) with Qiagen DNeasy Kit© Blood and Tissue kit  following the manufacturer’s protocol.Amongst the 683 individuals, 327 were sequenced at the exon 2 of the MHC class II B gene. This number was achieved as a compromise between costs of 454 sequencing and the need to provide sample sizes sufficient to provide a robust screen for spatial and temporal patterns of local adaptation. Locations and respective sample sizes of fish screened for the MHC gene are highlighted in onwards) . DNA wasND5) exactly replicating (Hd) and nucleotide diversity (π) were calculated for each sampling location in DnaSP v5 .All samples were genotyped for twenty-two microsatellite loci optimized from previous studies . AmplifiHe), observed heterozygosity (Ho) and FIS were calculated for each sampling location in GENETIX  was calculated for each sampling location in HP-RARE v1.0  (Genetic signatures of a bottleneck were tested for each location using the tests available in BOTTLENECK . These mNe) . A two-pNe) . Allele glass eels” n = 481 and “silver eels” n = 202), we performed 10 rounds of re-sampling of the data without replacement using PopTools , observed heterozygosity (Ho), allelic richness (Ar) and FIS were calculated and compared between groups of replicates, i.e., “glass eels” and “silver eels,” with two-sided t-tests in FSTAT   amongst groups of replicates. Demographic history was inferred using two approaches. First, we evaluated the possible genetic signature of the recent population decline using BOTTLENECK  for each age group as previously described. Second, we estimated the effective population size (Ne) of each replicate of “silver eels” and “glass eels” using the linkage-disequilibrium method implemented in NeEstimator V2.01  is the Ne estimate of the ith replicate (Deviations from Hardy-Weinberg equilibrium (HWE) were calculated for each replicate in Arlequin v3.5 . Nei’s unbiased heterozygosity (tations) . The disor V2.01 . We utilimate Ne . All estimate Ne . The unweplicate .β genes  using Primer v6 (FST matrices (mtDNA and microsatellites) and pairwise Bray-Curtis similarity matrices (MHC).Individual MHC allele numbers detected from the different paralogs, nucleotide diversity, and individual average nucleotide p-distance  were calrimer v6  followinrimer v6  1,000 peRm) and estimates of recombination rate (R) were calculated in DnaSP v5 (R∕θ) contribution of recombination (R) and point mutations (θ) in the generation of genetic diversity  implemented in DnaSP v5.Minimum number of recombination events  betweenA. anguilla, overall positive selection was estimated with a Z-test implemented in MEGA v5 (ω) among sites by applying different models of codon evolution. Three likelihood-ratio tests of positive selection were performed comparing the models M1a  vs M2a (positive selection), M7 (ß) vs M8 (ß + ω), and M8a (ß + ω = 1) vs M8 (ω varies between sites (fixed effect), and between branches at a site (random effect)  that assume constant selective pressures within a branch (In order to test for the mode of evolution of the MHC in  MEGA v5 . We test MEGA v5  and the  MEGA v5  implemen MEGA v5 . The max1) vs M8 . In the 1) vs M8 . We furt effect) . The nula branch .silver eels” and “glass eels” under the assumptions of a constant population size and sudden expansion using DnaSP v5. The historical profile of MHC genetic diversity was investigated with Bayesian skyline plots (BSP) . Theoretically, sites interacting with parasite-derived antigens are expected to be under positive selection while other sites, within the same exon, may evolve differently but still maintain the integrity of the MHC molecules. Therefore, we also concatenated the remaining sites (nPSS) and performed identical analyses as for the PSS. Nucleotide mismatch pairwise distributions were calculated for PSS and nPSS of both “ts (BSP)  in BEASTts (BSP) . Althougts (BSP) , and of ts (BSP) .8. The historical profile of MHC diversity was reconstructed for PSS and nPSS of both “silver eels” and “glass eels.” Piecewise constant skyline model allowing for five skyline groups were used in three independent MCMC runs to verify consistence in parameter space. We then compared the marginal probability distributions of several parameters amongst the runs. Lastly, we constructed lineage-through-time plots. These plots reflect accumulation of lineages through time translated for a given dated phylogeny . All reD values were negative amongst almost all sampled locations, suggestive of population expansion or of population subdivision  and 0.758 (PT), Ho between 0.557 (G_TITA) and 0.667 (DK) and the average number of alleles per locus varied between 3.546 (G_VFRA) and 15.773 (G_AD2012). Allelic frequencies are reported in Data S2. FIS varied between 0.0380 (G_NIRL) and 0.254 (Q)  . Even th testing . Since s testing .None of the sampled locations showed either heterozygote excess or a mode shift in allele frequencies, genetic signatures characteristic of population bottlenecks .silver eels” replicates. No significant differences between generations were apparent for He , Ar  and FIS. Ho, however, was significantly higher in the “silver eel” group . The AMOVA between “silver eel” and “glass eel” groups of replicates revealed a pattern of isolation by time , supporting our a-priori assumption that those groups represent clear age structured cohorts.Twenty-one loci were used to analyze “silver eels” and “glass eels” as locus AjTr-45 consistently deviated from Hardy-Weinberg equilibrium in all “silver eels” nor “glass eels” deviated from an expected L-shape distribution  amongst replicates ranged between 0–625.2 for “silver eels” and 0–2708.9 for “glass eels”. The harmonic mean of effective population size estimates amongst replicates, Estimates of effective population size . However, post-hoc pairwise comparisons showed no significant differences between pairs of populations after correction for multiple testing . The mean nucleotide divergence (p-distance) ranged between 0.078 (BL) and 0.141 (FI) . Post-hoc pairwise comparisons revealed two significant comparisons after correction for multiple testing , suggesting a reduced MHC diversity the stocked freshwater system of the Schwentine river, in Germany.We sequenced a 247 bp fragment of the exon 2 of the MHC class II region  in 327 individuals using 454 sequencing technology. We detected 229 different amino acid coding variants. Among those, 226 (98%) were found to be unique in the dataset but present in both independent replicated reactions and therefore kept as true variants . A total141 (FI)  and was R = 0.001, p = 0.98). Overall, no correlation was found between Bray-Curtis similarity matrices on MHC and pairwise FST for both mtDNA  and microsatellites .The ANOSIM showed no significant differences in MHC allele pools amongst populations . Interestingly, “glass eels” had a significantly higher individual mean number of alleles  and a significantly higher individual mean nucleotide p-distance  (π) and the number of minimum recombination events (Rm) detected between “silver eels” and “glass eels” were similar  while the R∕θ ratio was slightly higher in “silver eels” .Between generations, no difference in MHC allele pools were observed (= 0.032) . Both thglass eels” group but not in the “silver eel” group. The average ψ of the whole segment was found to be 0.0002  (silver eels” and “glass eels” (occurred  and 5. Toccurred  and nPSSes = 10) . Those pes = 10)  and recees = 10) . It was ss eels”  and 5.t = 0 and common to both generations  and heterozygosity estimates (He = 0.687–0.758) were very similar, and neither mode shifts nor heterozygote excesses were observed .Due to high levels of polymorphism, neutrally evolving microsatellites are thought to be sensitive enough to detect subtle shifts in population dynamics . Here asobserved , Fig. S1observed , which wobserved . Note, avs Finland, Germany vs France 2011). Whether it relates to the fact that those samples correspond to a freshwater system, the Schwentine in Germany, where all eels are stocked remains to be investigated. Stocking however might be an issue in other sampling areas as well and therefore understanding why this population demonstrates a reduction in diversity should be the focus of further studies.Regarding the MHC, we found differences in the mean number of alleles amongst locations although none of the post-hoc pairwise comparisons revealed to be significant after correction for multiple testing. MHC allele pool composition did not vary amongst sampled locations. We found that the mean nucleotide distances vary amongst locations, but only comparisons between the German populations and two other sampled locations revealed to be significant after correction for multiple testing  no signature of bottleneck in the frequency distribution of pairwise mismatches. The later rather points towards an historical population expansion  no evidence for a genetic bottleneck, (ii) no differences in haplotype and nucleotide diversities between “xpansion . Given txpansion .silver eels” and “glass eels” replicates. This pattern of genetic variance distribution is in line with previous reports that also attributed higher genetic variance amongst temporal, rather than spatial, partitions of A. anguilla along the European coasts  compared to “silver eels” (Ne = 2941.7). These contemporary estimates are near the lower confidence intervals of historic effective population sizes previously reported . Notewohe 1960s . It is pAnguillicola crassus, for which the MHC was found to respond to in the paratenic host, the three-spined stickleback  genetic diversity, which might have been affected by the recruitment collapse and subsequent population reduction and (2) allele frequency shifts between generations which would be a signature consistent with directional parasite-mediated selection.Extending the evaluation of genetic diversity to the evolutionary analysis of the adaptive immune genes of the MHC was motivated by two main reasons. The first relates to studies suggesting MHC diversity to be more sensitive than neutrally evolving markers in the detection of demographic shifts . The secckleback . Using tN = 102), the large diversity at the MHC displayed in this species may not be surprising.Using next-generation sequencing, we identified a total of 229 MHC alleles amongst 327 individuals. This indicates that the diversity within this species is not low and directly compares to observations made in wild populations of other fishes that are not qualified as endangered, such as for instances, the half-smooth tongue sole  . Of note“silver eels” to exhibit lower mean number of alleles and lower mean nucleotide distance than “glass eels,” suggesting that the “silver eel” generation was under a selective pressure that reduced its pool of MHC alleles to fewer and more similar alleles. This observation suggests that either the selective pressure was widespread amongst continental locations or that it acted when all eels experienced similar conditions; as for instance, during the fastening spawning migration. A hypothetical selective pressure imposed by A. crassus meets both criteria. Not only is this parasite ubiquitous in European freshwater systems  are depicted with *, or ** after correction for multiple comparison after  for each of the replicates, with respective confidence intervals.Estimates of effective population size ; distnt, average nucleotide distance per individual with respective standard error (se).- nHap, number of haplotypes; Click here for additional data file.10.7717/peerj.1868/supp-8Table S4ω = (dN∕dS), the ratio of non-synonymous (dN) per synonymous (dS) substitutions; LRT, likelihood ratio statistic for β + = α (null or no selection) vs β + unrestricted .Click here for additional data file.10.7717/peerj.1868/supp-9Figure S1n) collected for each site and used for mtDNA and microsatellite analyses. Blue dots represent locations where “silver eels” where collected, yellow dots represent locations where “glass eels” where collected and the red dot represents location where both were collected. The G_p prefix stands for “glass eels”. Three numbers for the location of G_AD (Adour) depicts the n for three sampling events: G_AD2010, G_AD2011 and G_AD2012 respectively. The remaining acronyms have the following meaning: LC (Larne Lagoon), BT (Bann Toome), Q (Quoile), BU (Burrishole), BL (BannLower), SLC (LoughComber), DK (Denmark), LL (LarneLagoon), SLB (Boretree), GL (Glynn Lagoon), FI (Finland), PT , Ger (Germany), G_BU (Burrishole), G_BNIRL (Northern Ireland), G_VSWE (Sweden), G_TENG (England), G_EGER (Germany), G_OSPA (Spain), G_VFRA (France), G_WENG (England), G_TITA , G_NIRL (Northern Ireland).Sampling sites and respective number of individuals , with the frequency of the respective class. For purposes of visualization, all values were transformed to their square roots.Above is shown the average distributions of allele’s frequency classes for “silver eels” (grey bars) and “glass eels” (open bars). Error bars represents the maximum and minimum number of alleles observed amongst replicates. Values on the Click here for additional data file.10.7717/peerj.1868/supp-13Figure S5Both the skyline (A), posterior (B) and likelihood (C) overlap, conferring statistical support for the shape of the Bayesian plots produced with MHC data (Click here for additional data file.


Corridors are frequently proposed to connect patches of habitat that have become isolated due to human-mediated alterations to the landscape. While it is understood that corridors can facilitate dispersal between patches, it remains unknown whether corridors can mitigate the negative genetic effects for entire communities modified by habitat fragmentation. These negative genetic effects, which include reduced genetic diversity, limit the potential for populations to respond to selective agents such as disease epidemics and global climate change. We provide clear evidence from a forward-time, agent-based model (ABM) that corridors can facilitate genetic resilience in fragmented habitats across a broad range of species dispersal abilities and population sizes. Our results demonstrate that even modest increases in corridor width decreased the genetic differentiation between patches and increased the genetic diversity and effective population size within patches. Furthermore, we document a trade-off between corridor quality and corridor design whereby populations connected by high-quality habitat  are more resilient to suboptimal corridor design . The ABM also revealed that species interactions can play a greater role than corridor design in shaping the genetic responses of populations to corridors. These results demonstrate how corridors can provide long-term conservation benefits that extend beyond targeted taxa and scale up to entire communities irrespective of species dispersal abilities or population sizes. Agriculture and urbanization currently account for 43% of the earth's land area . We constructed habitat patches connected by corridors where we could vary the size of the patches and the length and width of the corridors Fig.A. The ren = 500) and large (n = 1000) and represented the total carrying capacity across all four patches. We chose these population sizes because they reflected a balance between being small enough to remain important from a conservation perspective, but large enough to avoid immediate extinction. We further varied population sizes beyond these two values with little change to qualitative patterns (see Model sensitivity). Carrying capacity in the corridors was proportional to the total corridor area, and corridor population density was set equal to patch density. Each model run was initiated with the placement of habitat patches and corridors in a Cartesian plane. Equal numbers of individuals were distributed randomly throughout each patch, and each individual was characterized by 50 bi-allelic loci with a minor allele frequency of 0.2. Diploid genotypes were created for each individual in accordance with Hardy–Weinberg equilibrium. At the beginning of each trial, allele frequencies were equal between patches  to simulate the fragmentation of a single population. Reproduction was asexual, with each individual producing 20 propagules. Sexual reproduction was not modeled because the mechanism depends upon behavior   was undetectable. From a management perspective, the short time frame means that the sampling of pre-existing variation, not the introduction of new mutations, is the relevant dynamic for measuring the consequences of corridors on genetic diversity and differentiation.Dispersal was modeled as a multivariate normal distribution with mean displacement equals to zero. Thus, dispersal was passive and unbiased with respect to direction. Species dispersed both ‘far’ and ‘short’ distances, where ‘short’ was modeled with a covariance matrix that resulted in 70% less displacement. Increasing or decreasing the variance associated with the two-dimensional dispersal function resulted in greater or lesser amounts of gene flow, respectively. Dispersal distance and direction was determined by sampling from the multivariate normal distribution. Individuals were determined to have dispersed to an area within a patch or a habitat corridor using a modified ray-casting algorithm . Genetic differentiation was calculated using an unbiased estimator for FST . To model positive species interactions, we increased population size by 20%, increased the absolute value of the mean of the dispersal kernel by 20%, and reduced corridor mortality by 20% for the focal species , and we varied population sizes  and dispersal distances  beyond the four ‘species groups’ to establish that the results remained qualitatively identical. We also examined the effect of running the model for 50, 75, and 100 generations. Modifying all of these parameters  resulted in quantitative but not qualitative changes to the presented analyses.We examined the effect of corridor configuration by arranging the patches in a linear fashion such that the two middle patches were connected by two corridors and the two end patches were only connected by one corridor. In this scenario, the population density of each patch had to be independently regulated to prevent extinction in the two end patches. In the preceding scenarios, where each patch was connected by two corridors, the patches could be regulated at the patch or meta-patch  level and no differences in extinction–recolonization rates or genetic response variables were observed. We also measured the effect of fecundity by varying the number of propagules produced by each individual . The mostly linear relationship illustrates that corridor length and width contribute proportionately to genetic diversity within patches , but dispersal distance did not , for the given geographic scales examined. The effect of population size on genetic diversity was greater at large corridor areas than at small corridor areas .For all four species groups, genetic diversity of populations occupying a habitat patch increased as the corridor area connecting patches increased  resulted in lower levels of genetic differentiation, while negative species interactions resulted in higher levels of genetic differentiation between individuals sampled in the patches Fig.A. Both pThe results presented here demonstrate the mitigating effects of corridors on the negative genetic effects associated with habitat fragmentation are achieved through two mechanisms: the exchange of individuals between patches and the reduction in genetic drift. As a consequence, increasing the width of corridors even by a small percentage can substantially reduce the amount of genetic differentiation between patches while increasing the genetic diversity within patches. These benefits partly occur because increasing corridor width creates higher absolute population sizes in corridor areas surrounding the patches and facilitates the exchange of larger numbers of individuals between habitat patches. However, the benefits associated with increasing corridor width were also documented in (i) models where the carrying capacity of both the patches and the corridors were held constant regardless of the total area and (ii) in species that could not disperse very far distances. Thus, an additional benefit of increasing corridor width is to simply facilitate the survival of offspring from a larger number of individuals, which serves to greatly decrease the inflated variance in reproductive success caused by habitat fragmentation and thus decrease the amount of genetic drift that would otherwise occur. The additional benefits of decreased genetic drift within habitat patches provide additional support for the idea that habitat corridors can be an effective conservation and management tool. Previous theoretical work has suggested that increased gene flow provided by corridors is beneficial Harrison , but thiThe benefits associated with increased corridor width are maximized when corridor habitat quality is high. If corridor quality is low , then poorly dispersing species are not be able to traverse between habitat patches to maximize gene flow or persist in the corridors long enough to reduce the effects of genetic drift in the patches. Thus, we recommend that managers connect habitat patches with corridors that are as wide as possible, but caution that if the quality of the corridors differs substantially from the habitat patches, then some of the potential mitigating effects of corridors may be lost. Nevertheless, even long and narrow corridors with low-quality habitat provided populations with greater genetic resilience than habitat patches that lacked corridors entirely Fig. . FurtherSpecies interactions can also play a substantial role in shaping the genetic responses of populations to habitat corridors. Here, we show that positive species interactions can further mitigate the negative genetic effects of habitat fragmentation when corridors are present, while negative species interactions can increase genetic differentiation and reduce genetic diversity. These results highlight the importance of designing corridors for entire communities and not focusing efforts on single species because key ecological principles  can translate into substantial evolutionary consequences. From a conservation and management perspective, it will be most useful to quantify the magnitude of species interactions that influence species dispersal, persistence, and reproduction in habitat corridors (Berlow et al. Animal behavior and matrix quality are two system-specific characteristics worth further consideration. Because dispersal behavior can be multifaceted and difficult to predict, we did not include it in this genetic approach. For some species, it is possible that behavior could facilitate dispersal between patches, including compensatory movement through low-quality habitat (Haddad and Tewksbury Local adaptation and the evolutionary implications of genetic differentiation also warrant further consideration. If there is strong selection specific to each habitat patch that promotes local adaptation, then immigrants from other patches will experience decreased survival and reduced reproductive success (Peterson et al. This is the first study to demonstrate that corridors can reduce the negative genetic effects associated with habitat fragmentation for a wide range of species and taxa with intrinsically different population sizes and dispersal abilities. Our agent-based model is the first to demonstrate that corridors can both reduce genetic drift within and increase gene flow between subdivided populations. We further show that understanding species interactions is vital to predicting evolutionary responses to habitat fragmentation. Minimizing the negative genetic effects of habitat fragmentation will better serve populations of any species to respond to changing environmental conditions. From a management perspective, the benefits provided by increased genetic resilience will have to be carefully weighed against any potential negative ecological effects of corridors (Haddad et al.


Sigaus australis) widespread and the other (Sigaus childi) a narrow endemic.Gene flow is traditionally considered a limitation to speciation because selection is required to counter the homogenising effect of allele exchange. Here we report on two sympatric short-horned grasshoppers species in the South Island of New Zealand; one  all but one marker we examined showed extensive allele sharing, and similar or identical allele frequencies in the two species where they co-occur. We found no genetic evidence of deviation from random mating in the region of sympatry. However, analysis of morphological and geometric traits revealed no evidence of morphological introgression.Based on phenotype the two species are clearly distinct, but their genotypes thus far reveal no divergence. The best explanation for this is that some loci associated with the distinguishing morphological characters are under strong selection, but exchange of neutral loci is occurring freely between the two species. Although it is easier to define species as requiring a barrier between them, a dynamic model that accommodates gene flow is a biologically more reasonable explanation for these grasshoppers.The online version of this article (doi:10.1186/s12862-014-0216-x) contains supplementary material, which is available to authorized users. Although taxonomy implies abrupt disjunctions between biological entities, we know that speciation usually involves non-instantaneous change ,2. The ea priori assumption that species are always reproductively isolated is readily demonstrated [Genetic introgression occurs when two genetically distinct populations come into contact enabling individuals from each to interbreed. When this occurs through secondary contact the process has usually been regarded as hybridisation ,8, howevnstrated ,11. Starnstrated ,13.Since the 1960’s, direct measures of variable genetic loci have provided strong evidence that genomes are not unitary and exchange of loci between populations may be uneven ,9,14,15.Gene flow may therefore result in species with mosaic genomes, comprised of alleles from different ancestral populations, which has been described as a potentially important evolutionary mechanism for the formation of many animal species ,13,15. IBrachaspis robustus, Sigaus minutus and Sigaus childi [Historically, one of the most informative animal groups in this field of study have been Orthoptera and in particular grasshoppers ,31-35. Hs childi -42).Sigaus australis appears, on the basis of mtDNA sequence data, to encompass several narrow endemics and one widespread species [Sigaus australis are relatively large  and abundant in South Island subalpine grasslands between 1000 and 1800 m asl. Sympatric with this widespread species is the microendemic Sigaus childi, which is restricted to a low-lying, semi-arid region of about 100 km2 around the town of Alexandra   that grows on rocks in some areas of Central Otago. Although, inferences of camouflage are subjective they support the conjecture that these grasshoppers are under selection by visual predators. Sigaus australis are more boldly patterned, often with longitudinal stripes, and tend to be colour-pattern variable within locations.The species  species ,43. TypiSigaus childi, although morphologically distinct, could not be distinguished from S. australis using mtDNA data [S. childi evolved recently and has retained ancestral mtDNA haplotypes (Incomplete Lineage Sorting), or has exchanged genetic information since diverging [. In order to understand the evolution of this system we applied six types of data; morphology, mtDNA sequencing, microsatellite genotyping, multi-copy nuclear sequencing, single nucleotide polymorphisms (SNP) and spatial position. We used these putatively independent data to contrast species integrity as characterised by morphology  and neutral characters that allowed us to test the stability of species delimitation, assess the extent and evenness of gene flow and thus gain an understanding of where these grasshopper populations are in the speciation continuum.DNA data . Perhapsiverging . Or perhSigaus childi and S. australis  and S. australis in the area of sympatry (P < 0.001 T-square: 935.5225).Geometric analysis of the grasshopper pronotum gave a similar result to that of traditional morphology. PCA analysis of the 14 landmark measurements showed two major groupings  but there was no overlap between the two forms. Thus we found no evidence of morphological intermediates in adults or juveniles.The PCA of pronotum shape from those grasshoppers sampled within the area of sympatry was scrutinised for evidence of hybrids Figure a. HoweveS. australis and 33 S. childi). Six haplotype clusters were identified that were each geographically restricted within the range of Sigaus australis . Although the mtDNA diversity within clades also did not fit a model of isolation by distance there was a non-significant positive relationship (p = 0.1410) and the power of this test was limited by smaller within-clade sample sizes. There was no statistical support for population genetic differentiation between S. childi and S. australis from either the central group FCT = 0.09175 (P = 0.09677), or from the area of sympatry FCT = 0 (P = 1)  supported a model of isolation by distance for Sigaus australis . Analysis of S. australis microsatellite data (excluding S. childi samples) using STRUCTURE [The three microsatellite loci surveyed each had between 16 and 18 alleles. No evidence of linkage disequilibrium was detected and Hardy-Weinberg expectations were met in the majority of population samples. A positive relationship between geographic distance and genetic differentiation , or the area of sympatry FCT = −0.07424 (P = 0.65494), although these samples were not all taken from the same generation. This result was consistent with the inference from STRUCTURE.As with the mtDNA data we sought evidence of genetic structure concordant with taxonomy and morphology using analysis of the correlation of genotypes between species by grouping the samples according to the morpho-species S. childi and 10, S. australis) collected near the township of Alexandra, 16  had more than one sequence which differed by the presence of an INDEL approximately 100 bp from the ITS1 forward primer. Sequences of ITS2 from these individuals were unambiguous except at single nucleotide polymorphic sites (SNPs), confirming that these grasshoppers carried more than one ITS sequence per genome. There were 16 SNPs in the set of unambiguous sequences. However the presence of an INDEL near the start of ITS1 meant grasshoppers with more than one sequence had only 13 observable SNPs. Only one of the 15 grasshoppers from outside the Alexandra area (a specimen from Raggedy Range) appeared to have more than one ITS sequence per genome that involved the large INDEL. However, many individuals (mostly collected from the northern part of the species’ range) had an independent 8 bp insertion that occurred in all their copies of ITS. When more than one ITS sequence was detected in a grasshopper DNA, we found that ambiguity could parsimoniously be explained by combinations of unambiguous (single copy) sequences that we separately identified in other grasshoppers. We detected only one copy of ITS in a genome-wide survey of Sigaus australis, suggesting that there was only one family of ITS in these grasshoppers; sequence variation within this family occurs where individuals have recently exchanged genetic material.We amplified and sequenced the ITS region  from 40 grasshoppers. Some of the grasshoppers had unambiguous single ITS sequence but many had more than one ITS sequence, consistent with these grasshoppers being heterozygotes of mixed ancestry. Of twenty-five grasshoppers  were removed due to low coverage. From these data sets we identified 8,958 loci that occurred in >2 individuals and these were subjected to further selection based upon coverage per population. The Lindis population was removed from subsequent analysis as several individuals failed to produce data of sufficient quality  and most of the putative-loci resolved were not represented in the other samples. Of 8,958 loci, 74 were retained as they occurred in ≥ 50% of the two populations . The relatively low proportion of loci that were represented across both population samples was due to insufficient representation of their very large genome despite our protocol involving quanitification and compensation for this. Nevertheless we obtained ample data for our purpose.The Illumina sequencing provided 9,789,323 forward reads of 100 bp for the 30 grasshoppers, of which 8,934,377 were retained after quality checks in process_radtags.pl (part of the Stacks package). These comprised between 3,743 and 978,246 reads per individual with a total of 30,439 loci. Three individuals  FST values for each of the 74 putative-loci revealed the high frequency of low scores expected in the absence of significant structure , with a confidence interval that effectively included zero , providing little evidence that these samples represent more than one population, with random mating. Population differentiation estimated with STRUCTURE suggested extensive sharing of genetic material among populations, with no species structure detected  gene flow. The SNP data show no population structure and extensive gene flow between the two species in sympatry, with one marker showing some sign of diversifying selection. The presence of more than one ITS1-ITS2 sequence within the genomes of single grasshoppers is also consistent with recent gene flow near the township of Alexandra. Concerted evolution normally results in homogenisation of variation in the rRNA cassette containing ITS [We found morphological support for two distinct entities consistent with their existing taxonomic treatment as species: ning ITS .S. childi is a phenotypic variant of S. australis generated by different local environmental conditions. Extreme plasticity is known to occur in other grasshoppers such as the locust (Locusta migratoria) which has two distinct life history strategies that are partitioned in time [S. childi is restricted to a limited lowland habitat in contrast with the subalpine environment experienced by most S. australis. The lowland conditions of Central Otago have been described as semiarid [S. australis and S. childi are sympatric in the lowland semiarid environment of Central Otago. They occur at the same places at the same times with, for example, specimens of both morphotypes used in our analysis collected as adults within metres of each other in Little Valley, Alexandra on the same day. These circumstances are inconsistent with an interpretation of phenotypic differences being driven by environmental induced plasticity. Although we cannot exclude this possibility, a novel type of micro-environmental control of grasshopper development would need to be invoked.A possible explanation for this lack of genetic differentiation is that  in time . Indeed,semiarid ,48, but Alternatively, the observed morphological divergence but lack of genetic structure is consistent with strong character specific selection in the presence of high levels of gene flow or incipient speciation Figure b. Under Sigaus childi, is a small, highly cryptic species of grasshopper suggesting it has been or is under selection from visual predators. In contrast, Sigaus australis, is a larger grasshopper, usually with more striking colour markings that are more easily observed against the substrate, suggesting a different mode of predator avoidance. Although the two species appear to share the same neutral alleles; they remain morphologically distinct with a single genetic marker showing some evidence of diversifying selection between the two species. All individuals examined fell into one of two morphological groups and no specimens could be classed as morphologically intermediate within the area of sympatry even when juveniles were examined. The apparent lack of morphological intermediates (hybrids) suggests that selection, even in today’s highly modified environment, is intense. It should be noted, however, that F1 phenotypes are often not intermediate between parentals and this might also explain our observations [S. childi limited our sample sizes and precluded any observation of mating behaviour and juvenile colouration and survival where the species are sympatric. Further examination might reveal morphological intermediates.Different evolutionary responses to selection could explain the observed morphological distinction. rvations . The conSigaus grasshoppers. The South Island of New Zealand was settled by Polynesian colonists starting about 800 years ago, and this was accompanied by episodes of scrub and forest fire [S. childi and S. australis was further modified by European introduction of plants and grazing animals, and mining practices in the last 150 years. These changes could have facilitated increased gene flow, but were analogous to the effects of Pleistocene climate cycling. Disentangling their respective influence on the grasshoppers is not simple [Situations where speciation and selection are most likely to be observable in nature are those with high environmental heterogeneity, temporal instability and/or novel environments ,58. Not est fire ,62. Expat simple .S. childi is not genetically isolated from S. australis it is morphologically well differentiated, and in our relatively small SNP dataset we were able to find one marker possibly under selection. Models of speciation with gene-flow predict a continuum from partially isolated populations to reproductive isolation [Sigaus system divergence seems to be at an early stage; Sigaus australis and Sigaus childi do not appear to be losing morphological distinction, but our neutral genetic data does show extensive gene flow. The absence of any morphological hybrids suggests selection is intense, removing relatively conspicuous intermediates and holding these two species apart. This may provide them the opportunity to diverge at other loci.The taxonomic status of these species is problematic, as traditional methods cannot resolve the conflicting information from morphology and genetics resulting from the process of evolution. Although solation ,23. In tThe findings of our study endorse Charles Darwin’s original dynamic view of speciation , but areS. australis complex  from their full geographic range  areas in some locations. Sigaus childi occurs only at a single low elevation site in Central Otago where it is sympatric with S. australis. Due to the legal protection given to the endangered S. childi, sample sizes were limited and sampling spanned more than one overlapping generation . Sampling from different generations, which are already overlapping is, however, not likely to increase the similarity of population allele frequencies, and therefore we do not consider this will have hindered any of the analyses. Sigaus obelisci and S. homerensis are each recorded from single subalpine locations within the range of S. australis. The identity of S. obelisci and S. homerensis specimens were confirmed by Simon Morris (pers comm. to SAT). Individuals were preserved by freezing or in 95% ethanol and identified following Bigelow [We collected grasshoppers by hand when they were active during the New Zealand summer season . Sampling included all recognised members of the  Bigelow , Morris  Bigelow , and Jam Bigelow .Sigaus australis complex grasshoppers in two ways. The first used the traditional species diagnostic characteristics, although we note that much of the information used to distinguish some of these taxa has been geographic location and altitude [Sigaus childi, for which the sinuous caudal margin of the pronotum and tegminal size are diagnostic [S. homerensis and S. obelisci, are each described as being near identical to S. australis and S. “remarkables”, which is a synonym of S. australis [S. australis, but uninformative for species delimitation [S. homerensis and S. obelisci are incorporated into S. australis here.Morphological data were collected for all adult altitude ,66. The agnostic . Male geustralis -66. Thusmitation ,40,64. Bmitation  S. homerS. childi) coded 0, not beyond first abdomen segment coded as 1, and so forth to a maximum of 4 (no tegmina ever reached beyond the posterior margin of the 5th tergite).The traditional species diagnostic characters for the grasshoppers were examined and measured with the aid of a dissecting microscope. Four metrics were recorded for each grasshopper using callipers accurate to 0.01 mm; maximum pronotum width, mid-line pronotum length, femur length and body length. In addition, six characters with discrete states were examined; sex, length of tegmina (see below), shape of pronotum posterior margin (sinuous or concave), cuticle rugosity (rugose or smooth), shading on pronotum posterior margin (pigmented or not), and shape of pronotum lateral margins (irregular or smooth). For most characters, the alternative and intermediate states were coded as 2, 0 or 1 respectively. The length of tegmina was classified by reference to the number of abdominal tergites across which they extended; not beyond the pronotum  approaches implemented in MINITAB 15 . The disSigaus genus relies on the pronotum shape, but descriptions are often vague, based on discrete states and inferred from few individuals making species identification difficult [S. australis, 34 S. childi) that were obtained with the aid of a dissecting microscope we tested whether shape variation could be detected from metric data. Using IMAGEJ [S. childi and 113 S. australis). These analyses were also separately applied to the S. childi and S. australis individuals collected within the area of sympatry   mtDNA Cytochrome Oxidase Subunit I (COI) data for the  complex . Tissue  complex ,71. DNA  complex  were use complex ,74.GENEIOUS was used to estimate a neighbour-joining tree for all the lineages. NETWORK version 4.5.1.6.  was usedCT) using ARLEQUIN version 3.5.1.2 [S. childi against all populations in the central group , this is unlikely to influence the detection of genetic differentiation [S. australis population samples were linearly regressed against their pairwise FST estimates. Nineteen populations had sufficient sampling for this analysis  and central group S. australis  using ARLEQUIN version 3.5.1.2 [S. childi against all populations in the central group and the second only those S. childi and S. australis individuals from the area of sympatry . Alignment and comparison of unambiguous with ambiguous sequences allowed us to identify the most likely combinations of sequences that gave the observed heterozygotes (S3). Where sequence variants differed by single nucleotide substitutions we could identify and resolve the polymorphism. Where sequence variation involved INDELs the resulting length polymorphism was evident by abrupt onset of sustained nucleotide ambiguity at the INDEL position, but sequencing in both directions allowed identification of the combination of sequences involved. To examine the number of families of ITS per grasshopper genome we interrogated a DNA dataset generated by high throughput sequencing. Genomic DNA from a single Sigaus australis individual was sequenced on an Illumina Hi-Seq 2000 (Beijing Genomics Institute) resulting in >1GB of sequence. The sequence was de-novo assembled via VELVET [Nuclear sequences representing the internal transcribed spacers (ITS1 and ITS2) of the rRNA cluster and the intervening rRNA 5.8S gene were obtained using the primers ITS4 and ITS5 . PCR cona VELVET  with mapa VELVET  and the a VELVET . The resSigaus genome were approximately 11.9 pg (consistent with estimates of other short-horned grasshopper species (http://www.genomesize.com)). In light of this information we used the restriction enzymes PstI and BamHI to digest the whole genomic DNA extracted from 30 grasshoppers . The DNA fragments were tagged with DNA sequences that identified each individual before size selecting at 300-400 bp and pooling as per [Single nucleotide polymorphic (SNP) anonymous nuclear markers were generated using high throughput sequencing, with individual DNA fragments coded so we could identify individual grasshopper genotypes. The double digest Rad-Seq protocol  was applg as per .ST as calculated for each putative-locus across all loci in STACKS, and an AMOVA was run in GENODIVE version 2.0b24 [ST across populations. STRUCTURE version 2.3.4 [S. australis and S. childi populations, and occured in ≥ 50% of the individuals.Data were generated using an Illumina Hi-Seq , and sorted using the STACKS version 0.99992 pipeline . Settingn 2.0b24  to deteron 2.3.4  was usedon 2.3.4 . Charts on 2.3.4 ,83. STRUST with subsequent runs using the resulting θ and M values. The uniform prior distributions were used for both parameters with slice sampling; one long chain was run recording every 5 steps after a burnin of 50,000 with a static heating scheme with five chains. Four runs were conducted on half the data before three final runs on all loci were undertaken using the starting values for θ and M of the previous run. BAYESCAN version 2.01 [Gene flow between the two populations in sympatry was estimated using MIGRATE-N version 3.5.1 ,93, althion 2.01 ,94,95 waion 2.01  and markion 2.01 .http://www.ncbi.nlm.nih.gov/bioproject/261083) are available on genbank. ITS sequence data from de novo assembly is available on genbank (KM576254). ITS data indicating SNP and indel variation is available in Additional file All sanger sequenced data (KM576255-KM576292) and raw Illumina reads (bioproject-261083 This study did not require ethical approval.


Giardia duodenalis is a protozoan parasite that can cause significant diarrhoeal diseases. Knowledge of population genetics is a prerequisite for ascertaining the invasion patterns of this parasite. In order to infer evolutionary patterns that could not be uncovered based on the morphological features, a population genetic study with the incorporation of molecular marker was carried out to access the genetic structure of G. duodenalis isolated from the Malaysian population and the global populations.Giardia, collected from different Malaysian communities, were subjected to DNA amplification and sequencing targeting three genetic loci . The tpi sequences together with sequences from the global data obtained from the NCBI GenBank were used for genetic diversity analyses including identification of haplotypes, haplotype diversity, nucleotide diversity, Tajima’s D and Fu and Li’s D, gene flow and genetic differentiation tests.A total of 154 samples positive for Analysis of the Malaysian and global data showed that assemblages A, B, and E , have different levels of genetic diversity. Assemblage B had the highest level of both haplotype diversity and nucleotide diversity, followed by assemblage E. The analysis also revealed population expansion and high gene flow in all assemblages. No clear genetic structure was observed across five continents . However, median joining network of assemblage B formed a cluster that was exclusively isolated from Asia while other haplotypes were well dispersed across the continents.Giardia assemblages in different geographical regions. The significant result shown by gene flow and genetic differentiation analyses as well as test of neutrality among the populations should have brought a clearer picture to the dynamics and distribution of Giardia infection.This study provides new insight into the genetic diversity of  Giardia is a gastrointestinal flagellate with a worldwide distribution affecting both developed and developing countries. It is also well known as a ubiquitous parasite of a wide range of vertebrates from mammals to birds and amphibians. Giardia trophozoites produce energy by bacterial anaerobic pathways, lack of subcellular organelles such as mitochondria, peroxisomes and have only rudimentary Golgi apparatus [Giardia represents the transitional form between prokaryotes and eukaryotes which developed eukaryotic features before mitochondrial symbiosis [Giardia had been described in literature as ‘early branching eukaryotes’ and was commonly used as a model system to study the development of basic cellular processes and evolution of eukaryotes [Giardia near the base of ribosomal RNA tree was attributed to long-branch attraction caused by rapid evolution of the organisms [Giardia. Thus it is suggested that, the microbial organisms are probably more advanced eukaryotes that underwent loss or modification of complex structures as a consequence of their adaptation to parasitic life style [pparatus , 2. The ymbiosis . Alongsirganisms , 5. Emerfe style , 6.Giardia which used to have over 50 described species [G. duodenalis being isolated from humans and other mammalian hosts. It contributes to approximately 5 % of gastroenteritis incidences in developed countries and as high as 15–55 % in developing countries [Despite its ubiquitous presence, the genus of  species  now compountries . This spountries .G. duodenalis isolated from Malaysia as well as to understand the transcontinental distribution of the parasite genotypes by comparing with sequences recovered from other countries based on triosephosphate isomerase (tpi) gene.DNA-based genotyping approaches have been widely applied in epidemiology and epizootiology of parasites. From the perspective of population ecology, molecular approaches provide a means for any parasitologist to understand processes such as transmission of parasites, patterns of speciation, evolution of host specificity and spread of drug resistance . The evoG. duodenalis isolates collected from different places in Malaysia were subjected to DNA extraction and amplification with subsequent sequencing targeting the tpi, glutamate dehydrogenase (gdh), and beta-giardin (bg) genes as indicated previously [G. duodenalis in Malaysia. Ethical approval: The protocol that involved the sample collection was approved by the Medical Ethics Committee of the University of Malaya Medical Centre (Ref. no: 788.74 and 878.19). Informed consent was obtained from the participants or their parents/guardians before the sample collection was done.A total of 154 eviously . The Maltpi gene was chosen as the candidate as it produced the highest number of amplifications compared to the other two genes and had the relatively longer length of nucleotide sites for sufficient analysis. In addition, molecular characterization analysis using tpi was well established [tpi sequences as possible from NCBI GenBank database. Of the 1465 tpi sequences available on NCBI GenBank, only sequences with known sources were selected. A total of 932 sequences  were enrolled in the analyses. The sequences were trimmed and sorted into assemblages by visual inspection according to the positions of the single nucleotide polymorphisms (SNPs) using multiple alignments implemented by Clustal W [For the population genetic analysis for the worldwide dataset, ablished  and the lustal W . In ordetpi sequences obtained from previous study [G. duodenalis genotypes across different regions. Sequences of Malaysia generated from tpi gene comprised isolates of assemblages A and B obtained from different states in Malaysia  were analysed within its own subset to access the genetic variation and the pattern of genotypes distribution.A Median Joining Network analysis were performed with all individual us study  and GenBus study  accordinD (D) and Fu and Li’s D (D*), gene flow and genetic differentiation tests were performed using DnaSP v.5.10.01 [Genetic diversity analysis including identification of haplotypes, haplotype diversity (Hd), nucleotide diversity (Pi), Tajima’s .5.10.01 . The lev.5.10.01 . Huge di.5.10.01 .tpi, gdh and bg showed that all the three markers produced consistent results, with sequences of assemblage B having the higher level of both haplotype diversity (Hd) and nucleotide diversity (Pi) compared to assemblage A  were included in the Median Joining Network analysis. Haplotype network of assemblage A formed two distinct networks. The first and the largest network comprised all the haplotypes except three haplotypes that were included in the second network and were isolated from HIV patients  and unimodal mismatch distributions  and with American  populations. Genetic differentiation of negligible to moderate differentiation was estimated among the populations for assemblage B except that populations between America-Africa recorded level of differentiation of 0.25445 (Nm = 0.73). The level of genetic differentiation for assemblage E was above 0.15 and significant for populations between Europe-Africa (Nm = 0.69) and between Asia-Europe (Nm = 0.91) whereas the rest of the population pairs had negligible to moderate differentiation. The overall results showed moderate divergence among the populations with 25/30 of the population pairs had FST less than 0.25 and half of them were statistically significant. These results reflected the spread of the same population of Giardia isolates across the geographical regions. In general, gene flow levels were high (i.e. Nm > 1). The highest gene flow was observed in assemblage B (2.41) followed by assemblage A (1.50) and assemblage E (1.48).For assemblage A, huge differentiation levels were recorded between Asian population with Australian  was obtained in the Malaysian population Fig. , but sucGiardia is closely tied with its host as it is only in the intestine of the host where this flagellate can proliferate by binary fission. Moreover, the dispersal ability of this food and waterborne parasites is dependent on the host in which the cysts are spread and continue the lifecycle at other places. From the distribution pattern of haplotypes in the network analysis, corroborated with observation of moderate gene differentiation and high gene flow across continents, it was shown that G. duodenalis with similar genotype were well dispersed over the globe. In light of the rapid pace of globalization in sectors such as intercontinental travelling, migration, trading of livestock and agricultural activities, host dispersal can be a major determinant of parasite gene flow. Blouin et al. [Giardia that is the formation of resistant cysts, enables the spread of the parasite genome over long distances and large areas. This genome dispersal is a kind of gene flow known as genotype flow which occurs in asexual reproduction pathogen (with little or no recombination) where entire genotypes can be transmitted from one population to another [As a kind of parasite, n et al.  in the pn et al. . The sur another .D and Fu and Li’s D signifies an excess of low frequency of polymorphism compared to the expectations under neutral processes such as mutation, genetic drift and population size equilibrium [bg gene was possibly influenced by ongoing purifying selection [gdh gene was under neutral selection [Giardia undergoes reproduction asexually as it is generally assumed, the population would be made up of independently evolving lineages and mutations are expected to confine to the lineage in which they began [The negative sign shown in the tests of neutrality, Tajima’s ilibrium . In addiilibrium –39. Howeelection  while thelection . On the ey began , therebyAs the population analyses were performed based on single gene, stronger inferences could be made if molecular data from multiple genes were included. In addition, it is well known that assemblages A and B can infect both humans and animals while assemblage E predominantly cause infection in livestock with a few rare cases reported in humans , 40. DesD and Fu and Li’s D can be the consequence of population expansion after a bottleneck event or the presence of purifying selection. These findings provide fundamental evolutionary information of G. duodenalis and enhance our understanding of the dynamics and distribution of Giardia infection.In general, high gene flow was observed in all of the assemblages and this could pose a threat in the transmission of the parasites especially the spread of the virulent alleles. The different genetic diversity observed in assemblages A, B and E could be due to their different evolutionary patterns especially between lineages of assemblages A and E with assemblage B. The departure from neutral expectations from the test results of Tajima’s