A total of 780 base pairs of cytb gene were sequenced to characterize mitochondrial DNA (mtDNA) diversity. The mtDNA marker revealed high haplotype diversity, low nucleotide diversity, and a lack of population structure across both populations of H. mohnikei and H. trimaculatus. A neighbour-joining (NJ) tree of cytb gene sequences showed that H. mohnikei haplotypes formed one cluster. A maximum likelihood (ML) tree of cytb gene sequences showed that H. trimaculatus belonged to one lineage. The star-like pattern median-joining network of cytb and CR markers indicated a previous demographic expansion of H. mohnikei and H. trimaculatus. The cytb and CR data sets exhibited a unimodal mismatch distribution, which may have resulted from population expansion. Mismatch analysis suggested that the expansion was initiated about 276,000 years ago for H. mohnikei and about 230,000 years ago for H. trimaculatus during the middle Pleistocene period. This study indicates a possible signature of genetic variation and population expansion in two seahorses under complex marine environments.Population genetic of seahorses is confidently influenced by their species-specific ecological requirements and life-history traits. In the present study, partial sequences of mitochondrial cytochrome b ( Examining patterns of genetic diversity, population structure, and expansion has become an important part in the management plans of endangered populations, and population size is the major determinant of population well-being and extinction risk For marine species, climatic events can undoubtedly impact their historical biogeography; however, marine patterns are relatively poorly known because of the high geological complexity and biological diversity In marine environments, some species that have long-lived, free-swimming, and feeding (planktotrophic) larval phases probably have relatively high dispersal abilities, and this promotes genetic exchange between populations Hippocampus trimaculatus and Japanese seahorse H. mohnikei are the most abundant and economically important seahorse species along China's coast. H. trimaculatus has a wide distribution range throughout the tropical and sub-tropical regions in Southeast Asia and is about 8–15 cm in body length H. mohnikei is a small body size (about 5–8 cm in body length) and inshore-water species, and is generally found in seagrass areas less than 10 m deep H. mohnikei is limited in the Northeast Asian Sea H. trimaculatus and H. mohnikei along China's coast by sequence analyses of cytochrome b (cytb) and control region (CR) haplotypes. We then compared the characteristic modes of their genetic variations and the evidence for population expansion across past climatic events.The three-spot seahorse H. mohnikei were sampled from Yangmadao and Laizhouwan along North China's coast, which belonged to the Oriental Zone (OZ) H. trimaculatus individuals were sampled from nine localities along Southeast China's coast, and all samples were pooled into three groups from three zoogeographical zones, i.e., Warm-Temperate Zone (WTZ), sub-Tropical Zone (sTZ), and Tropical Zone (TZ) H. mohnikei and H. trimaculatus were collected by researchers on board trawl boats, and a few were obtained with the help of local fishermen and buyers; all seahorses were immediately preserved until DNA isolation. Seahorse project is a key study in the South China Sea Institute of Oceanology, and also a key research focus in the Chinese Academy of Sciences. Seahorses used in this experiment have been absolutely approved for the use of research work, and sampling areas in our study are public, and there is no special policy to protect the seahorses. Some research work in our laboratory aims to obtain detailed information about wild seahorses and then provide data that may lead to the protection of seahorses in some areas. All seahorse samples utilized in this study have received animal ethics approval for experimentation by the Chinese Academy of Sciences. We have provided a scanned certificate for our investigation on seahorses.A total of 50 cytb gene (895 base pairs) was amplified employing the seahorse specific primers: forward shf 5′-CTACCTGCACCATCAAATATTTC-3′ and reverse shr2 5′-CGGAAGGTGAGTCCTCGTTG-3′2O. The thermocycling sequence was conducted as follows: an initial step of 94°C (3 min); a second step of 35 cycles of 94°C (30 s), 50°C (30 s), and 72°C (75 s); and a final step of 72°C (10 min). The primers and amplification conditions used for CR were as described in Teske et al. Cytb genes and CR were commercially sequenced using PCR purified products from both forward and reverse primers . Sequences were assembled and edited using Bioedit 7.0.9.0 cytb for H. trimaculatus KC519325-KC519363 and H. mohnikei KC527556-KC527584; CR for H. trimaculatus KJ158359-KJ158392 and H. mohnikei K J158393-KJ158419).A small amount of tissue from the tail of each seahorse was removed and macerated using phosphate buffered saline (PBS) buffer for extraction. The macerating tissue and muscle from the fresh seahorses were frozen in liquid nitrogen and then ground into powder. Genomic DNA was extracted using the AxyPrep Multisource Genomic DNA Miniprep Kit  following the manufacturer's protocol with minor modifications: the tissue homogenate was incubated at 56°C for 2 hours during cell lysis with Proteinase K. All DNA samples were stored at -80°C until polymerase chain reaction (PCR) amplification. A part of the mitochondrial n), number of variable sites (ns), number of haplotypes (np), haplotype diversity (h), nucleotide diversity (π), and average number of nucleotide differences (k) for each species' population were estimated using the software DnaSP 5.10.00 Genetic diversity indices (based on composition and transition/transversion bias) were calculated using MEGA5 DFsFs is the most powerful test for population growth. Expectations of Fu's Fs and Tajima's D are significantly negative values (P<0.05) in a sudden expansion population −9 per site per year for the seahorse cytb gene was assumed based on a generation time of approximately 1 year AMOVA) was utilized in the Arlequin 3.1 software. We also used the Bayesian skyline plot (BSP) BEASTcytb alignment. Divergence time was estimated to be 2% per million years (My) based on the entire mtDNA molecule, which was widely used for bony fish Pairwise mismatch distributions, sum of square deviations (SSD), and raggedness index (R) were performed using Arlequin 3.1 cytb sequences were subjected to the phylogenetic analysis based on a neighbor-joining (NJ) tree and the maximum likelihood (ML) method in PAUP 4.0b10 H. kuda (JX217831.1) was used as an outgroup for H. mohnikei. H. trimaculatus sequences  were obtained from GenBank as additional reference sequences for phylogeography analysis. Polymorphic sites were determined using DnaSP 5.10.00, and these sites were utilized to construct an unrooted median-joining haplotype network using software package Network 4.6.1.0 The cytb fragment were unambiguously sequenced in 50 specimens of H. mohnikei and 92 specimens of H. trimaculatus. The mean number of nucleotide composition in H. mohnikei was T = 27.1%, C = 15.5%, A = 34.0%, and G = 23.4%; in H. trimaculatus, it was T = 28.1%, C = 15.4%, A = 30.4%, and G = 26.1%. The transition and transversion rates were 1.77 and 8.82, respectively. Of the 780 bp nucleotide sequence of H. mohnikei, there were 25 variable sites, accounting for 3.2% of the length of the sequence. Of these variable sites, there were 12 parsimony informative sites, accounting for 48.0% of the length of the sequence. In H. trimaculatus, the number of variable sites was 45, accounting for 5.7% of the length of the sequence, and the number of parsimony informative sites was 10, accounting for 22.2% of the variable sites.A total of 780 bp of the H. mohnikei cytb sequenced, there were 29 unique haplotypes (Hj1-Hj29), which were distributed across the two sampled populations as follows: 22 haplotypes from Yangmadao and 11 from Laizhouwan. There were four haplotypes shared by the two populations. The overall haplotype (h) and nucleotide diversity (π) in H. mohnikei was 0.946 and 0.00353, respectively , which distributed across the three sampled population pools as follows: seven haplotypes from WTZ, 17 from sTZ, and 24 haplotypes from TZ. The overall haplotype (h) and nucleotide diversity (π) in H. trimaculatus was 0.902 and 0.00281, respectively  and 34 haplotypes (TC1-TC34), respectively.Of the 50 ectively . Of the ectively . Of the cytb was determined using a neighbor-joining (NJ) algorithm for H. mohnikei  indicating no genetic structure. AMOVA analyses also showed that there was not a strong geographic subdivision between the three populations of H. trimaculatus sampled .AMOVA analyses identified that there was not a strong geographic subdivision between the two populations of H. mohnikei and H. trimaculatus populations using neutrality tests and mismatch distributions. Tajima's D and Fu's Fs values in H. mohnikei were significantly negative in all populations. For H. trimaculatus, Tajima's D and Fu's Fs values were significantly negative (P<0.05) in all populations, except that Tajima's D was not significant in the WTZ group , we speculate that the populations may have experienced population expansion in the past. The cytb and CR data sets exhibited a unimodal mismatch distribution  The population of D and Fu's Fs values for all seahorse populations combined in the present study , we speculate that the populations may have undergone population expansion in the past. Furthermore, mismatch distributions were calculated for H. mohnikei and H. trimaculatus to investigate the hypothesis of a population expansion. Previous studies have revealed that population bottlenecks and population expansions have a significant effect on the pattern of genetic polymorphism among haplotypes in the population H. mohnikei and H. trimaculatus populations were unimodal and fully consistent with a population expansion. The expansion was initiated 276,000 years ago for H. mohnikei and 230,000 years ago for H. trimaculatus during the middle Pleistocene period. The Pleistocene, which spans from about 1.6 Myr to 10,000 years before the present, was punctuated by a series of large glacial-interglacial changes Beryx decadactylusHoplostethus atlanticusGlyptocephalus stelleriDue to significantly negative Tajima's H. mohnikei and H. trimaculatus, which are small feeble swimming fish and confidently influenced by their species-specific ecological requirements and life-history traits. Both seahorses have experienced population expansions since the mid-Pleistocene, and the population span of the expansion of H. mohnikei is larger and occurred earlier than that of H. trimaculatus. The observed lack of population differentiation can be explained by this past population expansion and present-day juvenile or sub-adult dispersal. Our study detected the absence of significant genetic divergence across the South China Sea in H. trimaculatus, suggesting that broad-scale conservation management strategies may be appropriate for this species. As a connective study on seahorses, future work will aim to assess the stability of the genetic variation and population expansion in the near future with the possible impact from heavy exploitation of seahorses and environmental change along China's coast.This study demonstrates the genetic variation and population expansion for two seahorses,


Chrysodeixis includens , an important defoliating pest species of soybean crops in Brazil. Population genetic variability and the genetic structure of C. includens populations were evaluated by using ISSR markers with samples from the major soybean producing regions in Brazil in the growing seasons 2011/2012. Seven different primers were applied for population characterization of the molecular variability and genetic structure of 8 soybean looper populations from 8 states of Brazil. The seven ISSR loci generated 247 bands in 246 individuals of C. includens sampled. The expected heterozygosity (HE) in the populations varied between 0.093 and 0.106, while the overall HE was 0.099, indicating low genetic diversity. The analysis of molecular variance indicated that 98% of the variability was expressed among individuals within populations . The low level of polymorphism over all populations, the high levels of gene flow, and the low genetic structure are indicatives of the exchange of genetic information between the different sampled regions. Population structuring suggests the presence of two major groups which do not correlate with their geographic sampling location in Brazil. These results may indicate recent recolonization of C. includens in Brazil or migration patterns following source-sink dynamics. Furthermore, the presence of two groups within C. includens suggests that a study on development of resistance or any other genetic-based trait needs to be evaluated on both groups, and pest management in soybean fields should be aware that differences may come to the control strategies they use.This study provides the first genetic characterization of the soybean looper,  Chrysodeixis includens, is restricted to the Western Hemisphere, from the USA to southern South America, and Australia [C. includens is known to migrate, with annual emigrations of moths to the northern states. Florida, Central and South America and the Caribbean islands are reservoirs for overwintering populations [C. includens was even found feeding on radish during the winter (own observations). The duration of the egg stage of C. includens range from 3 to 5 days. The species go through 5 to 6 instars in a period of 23.3 days. As the whole life cycle is 43 days at 27°C [The distribution of the soybean looper, ustralia . The oriulations , 3. The ulations . In Brazulations . In the Chrysodeixis includens has caused significant damage to soybean, especially in the producing regions of Brazil [Anticarsia gemmatalis, in the soybean vegetative phase [f Brazil –8. Untilf Brazil . Indeed,ve phase , 9. The ve phase . Before Bacillus thuringensis, with currently no resistance observed [The control of the soybean looper in Brazil is currently in a transition period, between the use of insecticides and the use of genetic modified soybean that expresses the insecticidal protein Cry1Ac of observed . Howeverobserved , 13. Theobserved , 15.In general, DNA methodologies are contributing to our knowledge of genetic diversity and population structure . MoleculC. includens with the use of Inter Simple Sequence Repeat (ISSR) markers. ISSR markers were widely used to characterize plants [Here in this study, we performed the first molecular characterization of e plants , and aree plants –28, Colee plants , Dipterae plants , 31, Hyme plants , and Neue plants . ISSR maC. includens, and to get information about the population structure and gene flow of this pest species. This information could become essential in designing strategies for controlling this pest species in soybean, and provide more insights useful in the decision-making for resistance management.The aim of the present study was to detect patterns of genetic variation in populations of C. includens were identified in LabMIP by morphologic characterization [Caterpillars were collected from several soybean production regions in Brazil   at 80 V.The caterpillars were dissected, and the integument was used for DNA extraction. The cell lysis based protocol of Sarkosyl  was firsMolecular analysis was performed using seven ISSR primers . The priet al. (2006), with 1 μl genomic DNA, and 0.5 μM of primer. In the amplification reaction samples were initially denatured at 94°C for 2 min, followed by 37 cycles at 94°C for 30 s, annealing at their respective temperature for 45 s  according to Liu for 45 s , and extISSR products were visualized by electrophoresis using 1.5% agarose gel at 100V for 40 min in 0.5X TAE buffer (Tris/Acetate/EDTA). After staining with ethidium bromide buffer, the banding patterns were visualized under U.V.-light and photographed. The Mass Ruler DNA Ladder Mix molecular weight marker  was ran as a standard with each primer. To ensure PCR quality, we repeated the PCR for all primers on some samples. Each primer was capable of producing a reproducible banding pattern.The ISSR band profiles from each DNA sample were analyzed using the Bionumerics software , and scored for the presence (1) or absence (0) of bands for each primer, excluding weak and/or smeared bands. Thus, a binary matrix (1/0) was generated, with 247 multiple bands from 246 individuals.Ne), the mean expected heterozygosity (HE), and the mean Shannon’s Information Index (I), assuming Hardy-Weinberg Equilibrium by using the GenAlEx 6.5 software [Genetic diversity for each population was estimated through the percentage of polymorphic loci (P), the mean effective number of alleles  was calculated as Nm = (1-FST)/2FST [Analysis of Molecular Variance (AMOVA) was conducted to calculate the variance among and within populations . Geneticsoftware , 40. TheT)/2FST .C. includens was determined by performing a Mantel test, Principal Coordinate Analysis (PCA) and Bayesian approach. The Mantel test, which assumes a linear correlation between geographic distance and genetic distance matrices, was done by using the GenAlEx 6.5 software with 9,999 permutations [The population structure of utations , 40. Theutations . The latutations  was perfutations . The grautations .C. includens sampled from eight populations formed from sample locations grouping. All loci were 100% polymorphic. The ISSR primer which amplified the highest number of bands was CI04 with 42 bands, and the lowest number of bands was, with 29, produced by primer CI05 (mean number of bands = 35.28)  .HE) and Shannon’s information index (I), both parameters of genetic diversity, were 0.099 and 0.184, respectively. The population PR showed the highest genetic diversity with HE = 0.106 and I = 0.200. While MT showed the lowest genetic diversity with HE = 0.093 and I = 0.173. The mean effective number of alleles (Ne) within the populations was 1.124 (The population with the highest percentage of polymorphism (P) was PR (86.20%), while the lowest percentage occurred in TO and MT (68.80%) with a mean within population polymorphism rate of 77.48%. The overall populations mean expected heterozygosity (as 1.124 .C. includens populations (AMOVA) indicated a low but significant genetic differentiation among populations , with 98% of the overall genetic variability occurring within populations and only 2% of the variation occurring among populations (p = 0.210). The estimated amount of gene flow between the populations, indirectly calculated from FST, was high (Nm = 23.197), indicating high migration among sampling localities.The analysis of the genetic structure of the ulations . FurtherThe PCA of all individuals from the eight populations revealed two groups. The first Principal Component  explained with 24.21% most part of the variance, while the second Principal Component  explained 17.17% of the total variance. No clear geographical pattern between both groups was observed .The cluster analysis suggested two different genetic groups. Indeed, the Evanno method identified ΔK = 2, this is the best value of K (or number of populations) that fitted our data . Each spC. includens with the use of ISSR markers. To determine the genetic characterization of the soybean looper in Brazil, we conducted an analysis of the genetic diversity and population structure across eight populations, utilizing seven ISSR molecular markers. We have chosen in this project to work with ISSR markers because they allowed us to detect a high level of polymorphic bands (100%). This is comparable with the polymorphism levels detected with ISSR markers in other species. For example, in Lymantria dispar, Ceratitis capitata and Plutella xylostela, the polymorphism levels were 100%, 85% and 100%, respectively [Here in this study, we performed the first molecular characterization of ectively , 31, 47.ectively . In geneFST value, was high (23.197). The Mantel test detected a low but not significant correlation between genetic and geographic distances. Despite this low genetic differentiation between the C. includens populations, the PCA and Bayesian approach test divided all individuals in two major groups which were not correlated with the geographical origin of the specimens. The high number of migrants among the populations of C. includens could be responsible for the low differentiation observed here. Although there is currently no information available on the direction and the migration rate of C. includens in Brazil, the soybean planting pattern together with the polyphagous character of species agrees with facultative migratory habits [C. includens is known to migrate [In our study, the gene flow over all populations, estimated from the was high 3.197. Thy habits . FurtherC. includens in Brazil also suggests a movement of the species between host plants along the cultivated areas in Brazil. Soybean plantations in Brazil start in the Mato Grosso state and progress to other states in the Southeast, Midwest and Northeast following the onset of the rain season, and the increase of the temperature in the Southern regions. These temporal climatic changes could encourage the migration of moths to new areas with soybean, considering that moths of C. includens have preference to oviposit on soybean leaves [The high level of gene flow of n leaves . Indeed,n leaves , and youn leaves . Howevern leaves . In thisn leaves .C. includens in Brazil. Indeed, the soybean looper is able to migrate long distances as (i) is seen in the USA where C. includens is recognized as a migration species, (ii) by the absence of overwintering and recolonization of C. includens in some states [C. includens in Florida where it can survive at average temperatures of 16°C [C. includens [C. includens in Brazil may be the Caribbean islands or Central America. However, to investigate the origin of Brazilian C. includens populations, a new analysis including samples from different Central and North American countries, and the Caribbean islands would be necessary.The results of this study may also be indicative of a recent recolonization of e states , and . Values greater than nine migrants per generation were sufficient to homogenize populations . AlthougC. includens. Our results showed high levels of gene flow and low genetic differentiation between the different populations, which may indicate recent recolonization of C. includens in Brazil and/or migration patterns following source-sink dynamics. Further research is needed to identify the migration patterns of C. includens in Brazil. Furthermore, the presence of two groups within C. includens suggests that any management tactic dependent on insect genetics such as the effect of insecticides and the development of resistance, needs to be evaluated on both groups. Taking this into consideration, pest management in soybean fields should be aware that differences may come to the control strategies they use.In conclusion, we present here the first molecular study on the populations of the soybean looper,


Beta vulgaris subsp. maritima and Beta macrocarpa. Using nuclear and mitochondrial markers, we surveyed 93 populations from Brittany (France) to Morocco – the southern limit of their species' range distribution. Whereas B. macrocarpa showed a genotypic structure and a high level of genetic differentiation indicative of selfing, the population genetic structure of B. vulgaris subsp. maritima was consistent with an outcrossing mating system. We further showed (1) a strong geographic clustering in coastal B. vulgaris subsp. maritima populations that highlighted the influence of marine currents in shaping different lineages and (2) a peculiar genetic structure of inland B. vulgaris subsp. maritima populations that could indicate the admixture of distinct evolutionary lineages and recent expansions associated with anthropogenic disturbances. Spatial patterns of nuclear diversity and differentiation also supported a stepwise recolonization of Europe from Atlantic-Mediterranean refugia after the last glacial period, with leading-edge expansions. However, cytoplasmic diversity was not impacted by postglacial recolonization: stochastic long-distance seed dispersal mediated by major oceanic currents may mitigate the common patterns of reduced cytoplasmic diversity observed for edge populations. Overall, the patterns we documented here challenge the general view of reduced genetic diversity at the edge of a species' range distribution and provide clues for understanding how life-history and major geographic features interact to shape the distribution of genetic diversity.Understanding the factors that contribute to population genetic divergence across a species' range is a long-standing goal in evolutionary biology and ecological genetics. We examined the relative importance of historical and ecological features in shaping the present-day spatial patterns of genetic structure in two related plant species,  Plant populations are often composed of distinct breeding units, and patterns of genetic variation within and among populations are determined primarily by mating system and the neutral microevolutionary process of gene flow , leading to sharp genetic discontinuities or nonintuitive genetic affinities among populations  and its close relative, Beta macrocarpa. Sea beet is a typical coastal taxon, mainly outcrossing, with a wide distribution from the Canary Islands in the west, northward along Europe's Atlantic coast and Baltic Seas  the sea beet , including fodder, leaf, or sugar beets; (3) Beta macrocarpa, which is an annual self-compatible plant thought to reproduce predominantly by autogamy; (4) Beta patula, which is endemic to an islet of the Madeira Archipelago; and (5) Beta vulgaris subsp. adanensis, a self-compatible species that is only found in Eastern Mediterranean areas, Greece and Turkey  gynodioecious species, has an outcrossing mating system – enforced by a gametophytic self-incompatibility system with up to four gametophytic S loci – and relies on wind pollination  and tetraploid (2n = 36) cytotypes occur in B. macrocarpa .Bvm3  the two species may possibly hybridize  from 30 runs along a range of K varying from 1 to 50 with 2.106 Markov chain Monte Carlo replications following a burn-in period of 100,000 iterations without any prior information on the putative population affiliation of individuals. Among the 30 replicates, 15 with the highest likelihood were retained for subsequent analyses. The ad hoc statistic ΔK was then calculated to determine the most accurate number of K clusters  MCMC iterations from K = 1 to K = 20  using the BYM admixture model described in Durand et al.  were retained. We set the Dirichlet parameter of the allele frequency model, the trend degree to 1.0, and the admixture and spatial interaction parameters to default values. To reveal which K may provide the best fit to the genetic data, average DIC values were plotted against K.Additional analyses were conducted using spatially explicit Bayesian clustering, that is, incorporating spatial trends and autocorrelation in the prior distribution on individual admixture coefficients, as described in Chen et al.  and Durad et al.  and the Similarity coefficients between runs and the average matrices of individual membership proportions were estimated using CLUMPP version 1.1.2 , allelic richness (Ar), and level of gene diversity (HE), all using FSTAT version 2.9.3.2 (Goudet ArP) were computed following a rarefaction procedure (n = 8) using ADZE software . Statistical significance of FIS values was subsequently assessed using the randomization procedure provided by FSTAT. Sequential Bonferroni corrections for multiple comparisons were applied following Rice  were compared between the genetically distinct groups depicted through Bayesian clustering and multivariate analyses described later. Statistical significance of differences was tested using a one-way analysis of variance followed by Tukey's multiple-comparisons test. To assess whether a northward-declining diversity gradient occurred in B. vulgaris subsp. maritima, linear regressions were applied on multilocus genetic diversity estimates and spatial attributes of populations: latitude and geographic distance measured along the coastline from the most southern sampling site. Linear regressions were performed on separate population data sets to take into account the presence of genetic discontinuities. A potential bias could arise because levels of allelic diversity often varied among the loci. Therefore, we also performed linear mixed models in analyzing genetic diversity as a function of geographic locations, with loci as random intercepts.Levels of genetic variation . Furthermore, we compared the mean pairwise genetic differentiation between genetic clusters following the statistical procedure accounting for nonindependent data described in Coulon et al.  described in Jombart et al. . The advdata see . To evaldata see . Main redata see . All thedata see .DCE chord distance . All analyses were performed using PASSAGE version 1.1.2.3 . Among the two groups of sea beets, a significant hierarchical differentiation supported this clustering: Genetic differentiation among populations within these two clusters accounted for most of the genetic variability , and the differentiation between the two clusters, although weaker, was highly significant . The K versus ΔK distribution was multimodal with another mode at K = 6 suggesting further hierarchical structure within the sampled area  after Bonferroni correction (119 expected from type I error at α = 0.05). LD was only found in 13 of the 85 B. vulgaris subsp. maritima populations and involved different pairs of loci. This suggests spurious LD due to genetic drift and/or population substructuring effects  had a highly negative mean FIS value due to fixed heterozygosity at multilocus genotypes  to 56 (Bvm3), with an average of 32.25 alleles and a mean gene diversity (HE) of 0.679 (Table TR2) to 15 (TR1). Based on a single haplotype determined by combining the alleles at four loci, we found a total of 45 haplotypes, of which four represented 77.31% of the total number of haplotypes. In sharp contrast, B. macrocarpa displayed much lower levels of genetic diversity, with an average of 7.750 alleles for nuclear variation and a total number of 5 haplotypes for cytoplasmic polymorphism  varied greatly among B. vulgaris subsp. maritima populations . In contrast, no significant trends were observed for Moroccan B. vulgaris subsp. maritima populations at lower latitudes  and inland ruderal (R) populations. Inland ruderal populations exhibited higher levels of gene diversity , allelic richness , and mean number of alleles  for nuclear data. Inland ruderal populations also showed a higher level of allelic richness for cytoplasmic data . However, no significant differences were observed for private allelic richness . Regarding genotypic structure, mean multilocus FIS values marginally differed between inland ruderal populations and coastal populations , and no difference was observed for nuclear multilocus FST .We then focused on Morocco, a region considered as a hot spot of genetic diversity, to compare coastal  with a mean multilocus FST of 0.148  and from 0 to 0.973 (44.14% at P < 0.05) for nuclear and cytoplasmic data, respectively  or local (P = 0.184). When performing sPCA on nuclear data, a significant global structure was observed (P < 0.001), indicating that neighboring entities are genetically more similar than would be expected at random. The three first axes in the sPCA unambiguously captured all global spatial patterns (see K = 6 (see P < 10−2). Overall, the general trend was that populations located in Morocco and along the Strait of Gibraltar displayed the highest levels of cytoplasmic and nuclear genetic diversity. Figure FST values observed along the Portuguese coastline  previous results on population memberships and (2) a sufficient number of pairwise comparisons within distance classes to ensure robust statistical support, we only focused on three groups of sed Fig. C. Regardsed Fig. E. FinallB. vulgaris subsp. maritima and B. macrocarpa. One may wonder whether the modal K depicting three genetically distinct clusters clearly discriminated the two species. Nonetheless, we are very confident of the species assignation for the following reasons: (1) Concordant patterns of genetic distinctiveness were obtained using both sPCA and Bayesian clustering, (2) the hierarchical genetic differentiation was the highest between the two clusters of sea beets and the putative cluster composed of Beta macrocarpa individuals, and (3) contrasting patterns of genetic differentiation among populations and genotypic structure within populations were found between the two clusters of sea beets and the cluster composed of Beta macrocarpa individuals, the observed patterns being concordant with what is known about the mating system in these two species  the larger effective population size for nuclear genes as compared to organellar genes, indicating a stronger effect of genetic drift on cytoplasmic DNA, and (2) higher gene flow for nuclear DNA, which occurs via seed and pollen dispersal, than for cytoplasmic DNA, which takes place via seed movements only  accessions from Canary Islands. Along with the recent discovery of diploid and tetraploid forms occurring in sympatry in south Portugal  colonization before barrier formation and/or ii) effective dispersal after barrier formation. During the last glacial maxima, the relative sea level dropped 120 m relative to its present level and the distance between the two continents was thereby reduced to 10 km (Yokoyama et al. B. vulgaris subsp. maritima, genetic continuities on both sides of the strait have been reported for several herbaceous plant species characterized by ruderal life-history traits (e.g., Caujapé-Castells and Jansen The Mediterranean-Atlantic transition has been reported to be an efficient barrier to the expansion of many coastal plants and marine species along the coast (Kadereit et al. B. vulgaris subsp. maritima seeds are dispersed by gravity in the vicinity of the mother plant (De Cauwer et al. B. vulgaris subsp. maritima populations along the Portuguese Atlantic coast (see Martins et al. Seed dispersal is a major determinant of the spatial genetic structure of populations Ennos . In mostB. vulgaris subsp. maritima survived the Würm glaciation: Due to ice and permafrost, it would not have been able to maintain a continuous distribution along large parts of the Atlantic coast of Europe. Today, the northern range limit of B. vulgaris subsp. maritima is situated at approximately 58°N near the 8°C January and 14°C July isotherms. Therefore, populations probably had to retreat to at least northern Portugal, where the 14°C July isotherm would have been located during the last glacial maximum (see Eryngium maritimum, a coastal species that shares the same habitats, temperature requirements, and distribution range as B. vulgaris subsp. maritima, was estimated to have retreated to as far south as North Africa (Clausing et al. B. vulgaris subsp. maritima. Indeed, the Gibraltar region is considered as a buffer zone that was climatically stable during geological times due to its latitude and the influence of the Atlantic Ocean and the Mediterranean Sea (Rodríguez-Sánchez et al. It seems unlikely that imum see . In accoSubsequently, the significant decrease in nuclear gene diversity and allelic richness northward from the Strait of Gibraltar region can be attributed to a series of founder effects during range expansion along the shoreline. In this type of narrow expanding population front, drift effects may be accentuated by growth of genetically uniform populations ahead of the main colonization front that would inhibit subsequent gene flow (Waters et al. In contrast, we did not find any IBD patterns for cytoplasmic variation, nor any evidence for decreasing levels of cytoplasmic diversity or increase in differentiation among newly established northern Atlantic populations following the postglacial recolonization. A migrant-pool model – where colonization occurs from multiple source populations Slatkin  – would B. macrocarpa. Gradients in allelic richness and gene diversity in B. v. subsp. maritima suggested a scenario of postglacial recolonization from the Mediterranean-Atlantic region and supported the status of southern Iberia and Morocco as a long-term refuge as documented for a wide range of organisms. Further, specific life-history traits along with the admixture from genetically differentiated lineages are likely to increase invasiveness for ruderal lineages in anthropogenically disturbed habitats. Our results also indicate that environmental factors, such as marine currents, may play a role in shaping genetic diversity in coastal plant species concordantly of what can be observed in marine organisms releasing larvae in water flows. Range expansions do not always have straightforward consequences: in our case study, long-distance seed dispersal through marine currents may drive to a mixing of cytoplasmic diversity and buffer the northward decrease in nuclear allelic richness observed for edge populations recently established. This challenges the usual view that populations located at the edge of a species' geographic distribution are genetically depauperate.Nearly complete autogamy was verified in


Bacillus, Exiguobacterium, and Pseudomonas) that are abundant in the aquatic systems of the Cuatro Cienegas Basin, Mexico. We tested the hypothesis that a common habitat leaves a signature upon the genetic variation present in bacterial populations, independent of phylogenetic relationships. We used multilocus markers to assess genetic diversity and (1) performed comparative phylogenetic analyses, (2) described the genetic structure of bacterial populations, (3) calculated descriptive parameters of genetic diversity, (4) performed neutrality tests, and (5) conducted coalescent-based historical reconstructions. Our results show a trend of synchronic expansions across most populations independent of both lineage and sampling site. Thus, we provide empirical evidence supporting the analysis of coexisting bacterial lineages in natural environments to advance our understanding of bacterial evolution beyond medical or health-related microbes.Comparative population studies can help elucidate the influence of historical events upon current patterns of biodiversity among taxa that coexist in a given geographic area. In particular, comparative assessments derived from population genetics and coalescent theory have been used to investigate population dynamics of bacterial pathogens in order to understand disease epidemics. In contrast, and despite the ecological relevance of non-host associated and naturally occurring bacteria, there is little understanding of the processes determining their diversity. Here we analyzed the patterns of genetic diversity in coexisting populations of three genera of bacteria ( The present-day distribution of biodiversity is a consequence of the evolutionary dynamics of populations and the history of the regions in which they occur . LineageIt is clear that patterns of biodiversity across phylogenetically distinct taxa may be influenced by shared historical factors . DespiteIn this context, the Cuatro Cienegas Basin (CCB) harbors high levels of bacterial diversity, arguably due to environmental variation  across the aquatic systems within the basin . HoweverBacillus and Exiguobacterium, as well as lineages of Gammaproteobacteria from the genus Pseudomonas. The selected lineages are all common in the natural setting of the CCB aquatic system, and we anticipated that the genetic variation present among coexisting lineages would reveal a signature of common historical dynamics independent of phylogenetic relationships. Multilocus sequence typing (MLST) markers were used to (1) perform comparative phylogenetic analyses, (2) describe the genetic structure of bacterial populations, (3) calculate descriptive parameters of genetic diversity, (4) perform neutrality tests, and (5) conduct historical demography reconstructions.In the present work, we tested the hypothesis that a shared history in the CCB region has left common genetic signatures across phylogenetically diverse microbial populations. Using population genetics and coalescent-based approaches, we assessed the population history of lineages from two closely related genera of Firmicutes, Consistent with our hypothesis, we identified a common trend of expansion in the studied populations over a similar time frame that occurred independently of phylogenetic relationships. These results provide empirical evidence that analyzing coexisting bacterial lineages in natural environments can advance our understanding of bacterial evolution, beyond medical or health-related species.Pseudomonas isolates were collected only from C. All isolates were obtained using previously described methods  glycerol.Water and sediment samples were diluted at 1:10; 1:100; 1:1,000; and 1:10,000 using saline solution (1% NaCl). Subsequently, 200 µl of each dilution was plated using either marine agar (Difco 2216) for Firmicutes or GSP agar  for Pseu16S rRNA gene was amplified and sequenced using 27F and 1492R primers, according to the conditions described by Bacillus, Exiguobacterium or Pseudomonas, we amplified and sequenced a set of housekeeping genes commonly used for MLST (Bacillus citC (KC900996–KC901178); Bacillus gltx (JQ241465–JQ241624); Bacillus hsp70 (KC901179–KC901361); Bacillus recA (JQ247793–JQ247952); Bacillus spo0A (KC901362–KC901540); Exiguobacterium citC ; Exiguobacterium hsp70 (JF952111–JF952292); Exiguobacterium recA (JF952293–JF952475); Exiguobacterium rpoB (JF952476–JF952658); Pseudomonas acnB (KC953704–KC953753); Pseudomonas gyrB (KC920532–KC920576); Pseudomonas recA (KC961435–KC961462); Pseudomonas rpoD (KC920481–KC920531).DNA was extracted from all isolates using a DNAeasy Tissue Kit , following the manufacturer’s instructions. The for MLST . Genus-sfor MLST  were use16S rRNA gene and partial sequences of the MLST genes were edited and aligned using BioEdit  using the “PHI test recombination” function implemented in SplitsTree4  concatenated alignments of MLST sequences. The program jModelTest v.2.1.3 was used to determine the best nucleotide substitution model for each alignment  phylogenies were constructed using (i) lignment . For theML v.3.0 , with thSTF values among sampling sites within lineages. With this approach we defined a single population as all isolates from a lineage that exhibited no significant genetic differentiation as measured by pairwise STF estimates. Pairwise STF estimates were obtained for each monophyletic lineage across all sampling sites using Arlequin 3.5  that are not site-restricted we investigated population structure by genetic differentiation, which is defined as changes in the frequency distribution of haplotype variants among subpopulations . We estiquin 3.5 , with 1,STF approach implicitly incorporates geographic location and genetic criteria in the investigation of population structure. However, it is possible that genetic structure exists for each lineage when looking at individual genetic variants within each population. Thus, we investigated potential substructure within the groups defined by STF by taking a Bayesian approach implemented in BAPS 6 (The n BAPS 6 . The Bayn BAPS 6 . A maximπ) and the mutation parameter Watterson’s θ were estimated, together with neutrality tests  using DNAsp v4.1  analysis as implemented in Beast v.1.7.5 . The EBSion size .Bacillus and Exiguobacterium) was 7 × 10−9 substitutions/per site/generation, obtained from a dated phylogenetic tree . All analyses were run for 50–100 million generations, until adequate mixing was achieved. Ten percent of burn-in was removed and the sampling was done every 1,000 chains. The rest of the parameters were set according to the guidelines of For the EBSP analysis, we used all MLST genes, a strict molecular clock, and the same nucleotide substitution models used for phylogenetic reconstruction to estimate changes in population size for each genetically homogeneous population. The substitution rate used for the two Firmicutes genera  sampled from water bodies in the geographic region of CCB, Mexico.To investigate the influence of a common habitat in the genetic variation of bacterial populations, we analyzed MLST data from a collection of isolates across three bacterial genera  was detected in one population (B1_HPa) that included most B1 isolates. Plots for the other two B1 populations (B1_M and B1_C) showed constant population size across generations , in one of the two E2 populations (E2_CH) and a trend of expansion  in the only E3 population (E3_CM). Finally, the plots for all Pseudomonas populations displayed signals of expansion, although only P2_C was statistically supported. Overall, a trend of expansion was observed for the studied populations independent of phylogenetic relationships or sampling sites. Moreover, the skyline plots revealed a common time frame for the start of expansions  analyses of populations (genetic pools) showed signals of expansion in 9 of the 13 populations, of which 4 have statistical support for at least one change in population size . Within erations . The B2 Diversity patterns in nature are a consequence of the evolutionary dynamics of populations and the history of the regions in which populations occur . A powerSTF statistic include different sampling sites with contrasting environmental conditions  and Pseudomonas to parameterize the coalescent simulations. We used the same substitution rate for Bacillus and Exiguobacterium since little information is available for Exiguobacterium and also because Population expansions in our data were identified to occur during the same time frame . Our estimation of time to the most recent population change is calculated in number of generations. Although generation times may vary considerably among bacterial lineages , laboratDespite the fact that expansions are expected in bacterial populations, the time of such expansions may differ among lineages because they depend on specific traits such as mutation rate, life history, and population structure . The synEscherichia coli see Currently, we do not have the necessary information or samples from the appropriate temporal and spatial scales to determine the environmental changes associated with the historical events that influenced population dynamics of the studied lineages. However, we can propose an approximate timespan in which these demographic events occurred and hypothesize on the environmental causes behind our observations. Most estimates of generation times for bacteria have been determined in laboratory conditions, which may be considerably shorter than generation times in the wild  Bacillus lineages with Geobacillus jurassicus as outgroup, (b) Exiguobacterium lineages with E. auranticum as outgroup, and (c) Pseudomonas lineages with Escherichia coli as outgroup. Bold font denotes representative sequences of the lineages studied. The scale of the bar represents number of substitutions per site.Phylogenies were constructed using the maximum likelihood approach and the complete sequence of the Click here for additional data file.10.7717/peerj.696/supp-3Table S1Primer sequences obtained from previous publications and this study are indicated.Click here for additional data file.10.7717/peerj.696/supp-4Table S2P values, which account for recombination. NC denotes non-computable estimations due to small number of informative sites. E1, E2 and E3 values were previously published in Values were obtained for all the housekeeping loci analyzed for each studied lineage using “PHI test recombination” function implemented in Splits Tree4 . Bold foClick here for additional data file.10.7717/peerj.696/supp-5Table S3Exiguobacterium and Bacillus lineages. Bold font denotes significant STF values (P < 0.05).Values are shown for Click here for additional data file.10.7717/peerj.696/supp-6Table S4π is Nei’s pairwise nucleotide diversity value and θ is Watterson’s theta per site = 2Neµ; neutrality was tested using Tajima’s D, Fu & Li’s D* and F*, and Fu’s SF. Bold fonts denote significant values (P < 0.05) for neutrality tests  are shown in bold.Click here for additional data file.


Malacosoma californicum pluviale, in two periods of peak or pre-peak density separated by 8 years. Populations showed high levels of genetic variation and little genetic differentiation either temporally between peaks or spatially among sites. Mitochondrial haplotypes were also shared between one island population and one mainland population in the two years studied. An isolation-by-distance analysis showed the FST values of the two geographically closest populations to have the highest level of differentiation in both years. We conclude that high levels of dispersal among populations maintain both synchrony of population dynamics and override potential genetic differentiation that might occur during population troughs. As far we are aware, this is the first time that genetic similarity between temporally separated population outbreaks in insects has been investigated. A review of genetic data for both vertebrate and invertebrate species of cyclic animals shows that a lack of spatial genetic differentiation is typical, and may result from high levels of dispersal associated with fluctuating dynamics.Cyclic or fluctuating populations experience regular periods of low population density. Genetic bottlenecks during these periods could give rise to temporal or spatial genetic differentiation of populations. High levels of movement among increasing populations, however, could ameliorate any differences and could also synchronize the dynamics of geographically separated populations. We use microsatellite markers to investigate the genetic differentiation of four island and one mainland population of western tent caterpillars,  The geographic structure and genetic differentiation of populations are determined by demographic processes and dispersal Three mechanisms have been proposed as potential causes of the synchronous dynamics among geographically separated populations of cyclic species: (1) sufficient movement of individuals synchronizes populations, (2) widespread weather patterns influence the populations, and/or (3) widespread movement of natural enemies synchronize population declines The genetic structure of cyclic populations could be related to their dynamics, in several ways. Elton Patterns of genetic variation of species of forest Lepidoptera with cyclic population dynamics have received little attention compared to studies of mammals. Social interactions among mammals are likely to influence the occurrence and timing of dispersal over the population cycle and this is important for maintaining high genetic variation and low differentiation Malacosoma californicum pluviale (Lepidoptera: Lasiocampidae). Western tent caterpillars occur primarily in western North America from British Columbia to California. They have one generation a year and feed on a variety of deciduous trees. Gregarious larvae form silken tents and live in family groups until the last instar. On the Southern Gulf Islands in British Columbia, Canada , on four islands in southern British Columbia (BC) Canada including: Galiano Island , Mandarte Island , Pender Island  and Saturna Island . The fifth population, which we consider to be a mainland population, is on Westham Island that is separated from the mainland by two arms of the Fraser River  . Larvae For the isolation by distance analysis we determined the shortest distance between study sites based on their geographic coordinates (latitude and longitude) using Google Map. Since these were island sites, much of the distance between them is over water.M. c. pluviale were selected for the analysis of population structure 2 from 1.0 mM to 0.75 mM to reduce stutter for primers A106 and B101.DNeasy Blood and Tissue kits  were used to extract DNA from larvae and moths. DNA was extracted from between 11 and 41 individuals from each of the sites in 2003 and 2011 for a total of 248 individuals. Only one individual was used per tent caterpillar family. Eight microsatellites that had been proven to be highly variable when characterized in a previous study of PCR products were denatured, loaded on a 6% polyacrylamide gel, and electrophoresis was performed for 1.5 hours on a LICOR 4300 automated sequencer . Four size standards (50–350 bp or 50–700 bp LICOR Inc.), negative controls (no DNA) and two reference samples were run on each 64 well gel to ensure that assigned scores were consistent among gels. Gels were scored using SAGA GT version 3.3A for microsatellites (LICOR Inc.).To compare haplotypes and genetic similarity between sites we chose two populations for further analysis: Westham Island and Galiano Island. Westham Island lies approximately 30 km to the northeast of Galiano Island across the Strait of Georgia and the two sites are separated by open water. Prevailing westerly winds (data from Environment Canada) could assist moth movement between these locations and thus similar genetic patterns could indicate migration of moths between them.Malacosoma species . We sequenced this region plus part of the COI gene for 12 individuals from Galiano Island in 2003, 9 individuals from Galiano Island in 2011 and 9 from Westham Island in both 2003 and 2011. PCR reactions used 10 ng of genomic DNA, 0.2 mM dNTP, (1x) PCR reaction buffer (Invitrogen), 0.5 units of native Taq (Invitrogen), 2 mM MgCl2 , 5 pmol of forward primer (K698: 5′ TAC AAT TTA TCG CCT AAA CTT CAG CC 3′) and reverse primer (K699: 5′ AGG AGG ATA AAC AGT TCA (C/T)CC 3′) , and 16.4 µl H20 for a total reaction volume of 25 µl. PCR conditions were as follows: 94°C for 2 min, cycled 35 times at 94°C for 30 s, 45°C for 1 min, and 72°C for 30 s, with a final extension of 72°C for 5 min. Products were purified using sodium acetate precipitation and sent to Eurofins MGW Operon  for sequencing. Bioedit Sequence Alignment Editor We sequenced an intergenic spacer region that had previously been found to be highly variable in a number of T) was calculated in FSTAT version 2.9.3 for each population in the two sampling years The number of observed alleles (Na), number of effective alleles (Ae), allele frequency, expected heterozygosity (He), and observed heterozygosity (Ho) were estimated using GenoDive version 2.0b22 ST values for all populations sampled and separately for the two sampling years. Pairwise FST values were calculated in Genodive and significance was assessed using 100,000 permutations. Bonferroni correction was applied to correct for multiple comparisons. An isolation-by-distance analysis was carried out using standardized FST values and log distance between sites using GenAIEx 6.5 M. c. pluviale populations. First, simulations were run including all eight microsatellite loci for the 248 individuals surveyed from the five populations in both 2003 and 2011. Following this, separate analyses were performed for each of the survey years. These analyses included 124 individuals in 2003 and 124 individuals in 2011. We followed the recommendations of K values) from 1 to 10 we ran 20 replicate simulations using a burn-in length of 100,000 and 500,000 Markov chain Monte Carlo (MCMC) repetitions. These parameter settings were appropriate given the convergence of all summary statistics . Simulations were run using the admixture model with correlated allele frequencies. To detect the true number of clusters we calculated the ad hoc statistic, ΔKK value and DISTRUCT FSTAT was used to estimate global FM. c. pluviale and therefore eight generations elapsed between sampling periods. In addition, we have compared these data to two other methods which use the temporal approach to estimate genetic drift and Ne: the likelihood method developed by Wang We used TEMPOFs IS values for each locus for the five islands and two years were significant after Bonferroni correction . This is due to significant departures from Hardy-Weinberg equilibrium observed at three of the loci   to 32 (A106) with an average of 13.12 alleles per locus for the eight loci. Seven of the F and B6) . These lAnalysis of the intergenic spacer region and partial CO1 revealed 11 distinct haplotypes for Galiano and Westham populations . AlthougThe number of alleles observed on each island varied from 6.25 to 9.75 over both years and after correction for sample size the effective number of alleles ranged from 4.54 to 6.33 . ExpecteST value for all populations sampled from the five islands in 2003 and 2011 was 0.009, which was significantly different from 0 at P<0.001. This indicated a very low level of structure among populations. When examined separately the pairwise FST values for populations surveyed in 2003 were lower than those in 2011 , although none of the pairwise FST values were significantly different after Bonferroni correction was applied , with the counter intuitive result of FST decreasing (becoming more similar) with increasing distance. No significant pattern occurred in 2011 (P = 0.40)  .ST results, the clustering method implemented in structure identified little population structure when all populations from 2003 and 2011 were analyzed together. Ln Pr(X|K) was highest for the model with K = 1 indicating there is no spatial or temporal structure among M. c. pluviale populations . Based on the Evanno method, ΔK was highest for K = 3, however inspection of the bar plot and individual assignment probabilities (q-hat) indicated a low probability of individual membership into each cluster for K = 3 and lack of structure for this K value. Separate examination of populations surveyed in 2003  and 2011  indicated that ΔK was largest for K = 2. However, the individuals had a low probability of membership into each of the two clusters  and supported the grouping of all individuals into a single cluster in both survey years.In agreement with FST values and the STRUCTURE results that indicated a lack of genetic differentiation among island populations.Estimates of effective population size for each island population based on Jorde & Ryman’s ST values, the low likelihood estimates of Ne, and the low diversity of haplotypes shared between populations. The isolation by distance analysis did indicate a weak relationship in 2003 and curiously this was the opposite to the anticipated pattern; closer populations were more genetically differentiated. There is no obvious explanation for this and, given that the result was not repeated in 2011, further data need to be gathered to assess whether this is a consistent pattern. However, it is interesting that the two closest populations have almost exactly the same FST value in both 2003 and 2011.Estimates of the genetic structure of four island and a mainland western tent caterpillar populations indicate low levels of genetic differentiation among these populations over scales of tens of kilometers. In addition to spatial similarity, population peaks 8 years apart did not differ. This conclusion is based on the genetic similarity of pre-peak populations as indicated by STRUCTURE, low FMalacosoma disstria was observed by Brown It is likely that the lack of genetic differentiation is related to movement of moths among populations including from the islands to the mainland. This suggestion is supported by several lines of evidence. First we know that moth dispersal can reestablish “sink” populations of tent caterpillars when source populations are high In addition to the high levels of genetic similarity among tent caterpillar populations, their population dynamics also show similar temporal patterns. Tent caterpillar populations cycle over four orders of magnitude and can become locally extinct. The resulting population bottlenecks could possibly allow for genetic drift to occur and influence the ability of populations to adapt and evolve. It could also result in genetic differentiation among populations if movements were restricted. However, given the rapid population increase over three to four generations following the trough of population density, genetic diversity should be reestablished Estimates of effective population size were less than 200 with the use of two of the methods and could not be accurately estimated for two of the populations. We found in an earlier study that multiple paternity was likely to have occurred in a high proportion of families from high-density populations M. americanum populations were shown with electrophoretic markers to have high genetic homogeneity among widely separated populations Zeiraphera diniana, in Switzerland using allozymes showed a low FST of 0.002 among larch feeding populations Operophtera brumata, was studied in Belgium using allozymes ST estimates while four species with low dispersal had higher FST estimates. An example of a species with strong geographical structure based on AFLP and mitochondrial markers, and one that does not show cyclic dynamics, is pine processionary moth, Thaumetopoea pityocampaTrichoplusia ni, migrates annually to British Columbia from permanent populations in southern North America. A high genetic connectivity occurred over the migration route, but some genetic differentiation occurred among seasonal, green house populations in British Columbia A lack of genetic differentiation appears to be typical of outbreak and cyclic species of forest Lepidoptera. Eastern tent caterpillars, Locusta migratoria, showed much greater genetic homogeneity than did non-outbreaking populations Chortoicetes terminifera in Australia is characterized by outbreak dynamics and showed no genetic structure across the country Comparisons of the genetic structure of outbreak and non-outbreak populations have also been carried out in another insect order, the Orthoptera. Outbreak populations of the migratory locust, ST (0.015) for snowshoe hares in the northern Boreal Forest of Alaska and Yukon. These values are similar to those we report here for cyclic tent caterpillars. They concluded that hares have large evolutionary effective populations. Berthier et al. Arvicola terrestris, maintain high genetic diversity through migration of increasing populations that wipes out the genetic drift among demes that occurs in low-density populations. Similarly Ehrich and Jorde Lemmus and Dicrostonyx. They suggest that population fluctuations possibly destabilize the spatial organization and favor the success of migrants. Four-fold variation in mean density of root voles (Microtus oeconomus) over six years revealed no change in genetic heterogeneity Myodes glareolus, populations over eight consecutive years of population fluctuation. They found relatively high levels of genetic heterogeneity but a lack of genetic differentiation among populations at the scale of 10 to 40 km during that time. No signature of population bottlenecks was apparent.Other non-insect cyclic species also have been shown to lack genetic differentiation even though populations periodically decline to low density. Burton et al. Arvicola terrestrisST at high densities. Genes associated with the major histocompatibility complex that is related to immune function showed evidence of balancing selection and geographic differentiation among populations at low density. At high density, genetic homogenization was greater than could be explained by drift and migration alone and therefore could have been associated with a general pattern of selection across all populations.Of particular interest with the western tent caterpillars is the potential for strong selection associated with the epizootic of a nucleopolyhedrovirus that occurs at peak densities which could potentially select for different resistant genotypes during sequential outbreaks Melitaea cinxia Click here for additional data file.


Rosa soulieana Crép. is an important wild rose species that is widely distributed in the Hengduan Mountains. To provide better future utilization of this wild rose, and also to add some possible proof of the effect of geomorphological and ecological characteristics of the Hengduan Mountains on the current spatial distribution and genetic diversity of local species, the genetic diversity and genetic structure of 556 individuals from 37 populations of R. soulieana were studied using fluorescent amplified fragment length polymorphisms (AFLPs). R. soulieana showed a moderately high level of genetic diversity and a high level of genetic differentiation at the species level. The total percentage of polymorphic loci, total heterozygosity (Ht), Shannon index (I), and heterozygosity value within populations (Hs) were 97.8%, 0.253, 0.339, and 0.139, respectively. More than half of the total genetic variation (54.0%) occurred within populations, and the overall gene differentiation coefficient (Gst) was 0.451. The genetic differentiation among populations was positively and significantly correlated with geographic distance. The neighbor-joining cluster and the Bayesian analysis divided all the populations and individuals into 3 groups, and did not support the morphology based intraspecific varieties. The results confirmed that the ancient R. soulieana of the third group survived in northwestern Yunnan and Yalongjiang valley and then moved upnorth along the valley. The spatial distribution of the other two groups was the result of allopatric divergence due to long period of adaptation to the different climatic conditions of its distribution at either side of the Yalongjiang River.The Hengduan Mountains are the core of the Sino-Himalayan Floristic Region.  Rosa DC. (formerly Cinnamomeae DC.), the section Synstylae DC., the section Pimpinellifoliae DC. and the section Microphyllae Crép. in the Hengduan Mountains, 43 of which are endemic , 15Rosa hylogeny , which cGenetic variation at the intraspecific level is critical for the adaptation to environmental changes and for long-term survival of a species . KnowledR. soulieana can be directly used both for the ecosystem revegetation of dry valleys and for landscape utilization. It can also be potentially used for improving modern rose cultivars in rose breeding programs. To provide better future utilization of this wild rose, and also to add some possible proof for the effect of the geomorphological and ecological characteristics of the Hengduan Mountains on the current spatial distribution and genetic diversity of local species, we use AFLPs analysis to study the genetic variation of R. soulieana, the aims of which are as the follows: 1) To determine the extent and structure of the genetic diversity of R. soulieana in the Hengduan Mountains Region. 2) To determine whether the intraspecific varieties are independent evolutionary units or just arbitrary units plastically responded to the geological and ecological environment in the Hengduan Mountains. 3) To assess possible causes of the existing genetic diversity and genetic structure of R. soulieana.As a fragrant wild rose species, which is also highly tolerant to the dry and barren environment, R. soulieana in the Hengduan Mountains and Zayü in the eastern Himalayas, southwestern China . The detailed procedures were as the follows. For the digestion, a 30 μl volume of digestion mix containing 4 μl NEBuffer , 3 U EcoR I, 1.5 U Mse I, and 4 μl 10 mg/ml BSA was added into 10 μl of genomic DNA (approximately 100 ng). The reaction was incubated at 37°C for 1.5 h. Next, 10 μl ligation mix, which included 3 μl 5μM EcoR I adaptor, 3 μl 5 μM Mse I adaptor, 1 μl 10×Ligase Buffer and 5 U T4 ligase, was incubated at 65°C for 10 min and then slowly reduced to room temperature in the machine. Then, the ligation mix was added to the 40 μl restriction digested DNA and kept at 16°C for 3 h. After ligation, the mix was diluted 10-fold with sterile deionized water (sdH2O). A pre-selective polymerase chain reaction (PCR) was done using a Bio-Rad machine with a single selective nucleotide extension. The reaction mix  included 5 μl diluted restriction/ligation temple DNA, 0.5 U Ex-Taq polymerase, 2 μl 10×PCR buffer (Takara) (Mg2+ free), along with 1.2 μl MgCl2, 1 μl 10 μM EcoR I Primer (+1), 1 μl 10 μM Mse I Primer (+1), and 1.6 μl 2.5 mM dNTPs (Takara). After an initial incubation at 65°C for 5 min, 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 1 min were performed with a final extension for 5 min. We electrophoresed 5 μl pre-amplified product on a 1.0% agarose gel to identify the most variable selective primer extensions. The pre-amplified products were then diluted 10-fold with sdH2O and stored at -20°C. Four selective primer pairs: EcoR I (FAM) -AAG/ Mse I -CAT, EcoR I (FAM) -AAG/Mse I- CGT, EcoR I (FAM) -AAG/Mse I- CTC and EcoR I (FAM)-AAG/Mse I–CTT  (Mg2+ free), along with 1.2 μl MgCl2, 1 μl 10 μM EcoR I Primer (+3), 1 μl 10 μM Mse I Primer (+3), and 1.6 μl 2.5 mM dNTPs (Takara). After an initial incubation at 94°C for 2 min, 13 cycles of 94°C for 30 s, 65°C and touch down 0.7°C for each cycle  for 30 s, and 72°C for 1min, 23 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 1min were performed with a final extension for 5 min. Then, 5 μl final selectively amplified product was electrophoresed on a 1.0% agarose gel to determine if the length of the amplified product was approximately 100 bp-500 bp.The total genomic DNA of the collected specimens was extracted with Plant DNA-Easy kit . AFLP reactions were performed according to Vos et al. with somhttp://www.softgenetics.com/GeneMarker). Peak patterns were converted to dominant presence—absence (1–0) matrices. Electropherograms were checked manually to exclude doubtful peaks, and only peaks with sizes between 100 bp-500 bp were included in the analysis.The amplicons were detected using an ABI 3100 capillary sequencer (Applied Biosystems) by MicroRead Gene Technique Company in Beijing. For each individual, 2 μl of the AFLP products were separately combined with 0.5 μL GeneScan ROX 500 (Applied Biosystems) as an internal size standard (Applied Biosystems) and run on the sequencer. In order to estimate the stability of band pattern, 5 samples in each plate were repeated during selective amplification and capillary sequencing. Electropherograms were analyzed using GeneMarker software v2.2.0  using the default parameters recommended as optimal for AFLP markers by the manufacturer (NP), the percentage of polymorphic loci (P), Nei’s [He) assuming Hardy–Weinberg equilibrium, and Shannon’s diversity (I) [Ht), heterozygosity within populations (Hs), and gene differentiation coefficient (Gst) using the same software.Such indices of genetic diversity as the number of polymorphic loci , Nei’s  unbiasedsity (I)  were calsity (I) . At the Nm = (1/Gst- 1)/4 [To assess the hierarchical genetic structure among populations and within populations, the AMOVA analysis was first performed by partitioning genetic variation among and within populations at the species level using ARLEQUIN version 3.0 . The amost- 1)/4 .Fst values between populations and bootstrap values  were calculated using the program AFLP-SURV version 1.0 [Ht), and heterozygosity within populations (Hs) were further calculated for each group using POPGENE Version 1.31[A data matrix of pairwise sion 1.0 . These dsion 1.0 . The neision 1.0 . The popsion 1.0  was alsosion 1.31.To assess the correlation between geographic and Nei’s genetic distances, the Mantel  test wasFinally, we examined the assignment of individuals to groups based on their multilocus genotypes using the Markov Chain Monte Carlo (MCMC) Bayesian clustering method in STRUCTURE version 2.2 . The ‘ad10.5061/dryad.4q53p). The number of scored bands for each primer combination varied from 96 to 109 with a mean of 104.3 bands per primer. The maximum number of polymorphic bands was produced by primer combination EcoR I-AAG/Mse I–CTT (109 bands). The percentage of polymorphisms was from 95.5% to 100% with an average polymorphism percentage of 97.8%; the highest percentage of polymorphism was also produced by primer combination EcoR I-AAG/Mse I–CTT (100%).The four AFLP primer combinations produced a total of 417 fragments in 556 individuals from 37 populations, 408 of which were polymorphic (NP) per population varied from 101 (SPzjg) to 283 (LJwh), with an average of 173.6 loci per population. The percentage of polymorphic bands (P) ranged from 24.2% (SPzjg) to 67.9% (LJwh), with an average of 41.5%. At the population level, the Nei’s gene diversity (He) of population varied from 0.085 (SPzjg) to 0.374 (CYc), with an average of 0.145. Population SPzjg, BY, BSbm, DG, LJxz, LX, HS and BSc had the lowest gene diversity, less than 0.110, while population JD, DQyl, ZD, YJ, DR, LJwh and CYc had the highest gene diversity, greater than 0.160. As far as Shannon’s information index (I) was concerned, it ranged from 0.127 (SPzjg) to 0.329 (LJwh), with an average of 0.209. Similar to Nei’s gene diversity, population SPzjg, BY, BSbm, DG, LX, LJxz, and BSc had the lowest Shannon’s information index, less than 0.165, while population MLmdl, JD, DQyl, ZD, YJ, DR and LJwh had the highest Shannon’s information index, greater than 0.242.As shown in The first and the second axes of the Principal Coordinate analysis of 556 individuals from 37 populations explained 13.0% and 7.8% of the genetic similarities among all the individuals, respectively. As shown in For the PCoA analysis of the 37 populations , the firFst) were calculated (10.5061/dryad.4q53p). The neighbor-joining phylogram constructed from the population pairwise Fst value produced 3 groups  revealed that the highest estimation of the posterior log probability given the number of clusters chosen, L(k), occurred at k = 3 . ConsideAccording to Figs R. soulieana  correlation between Nei’s genetic diversity and geographic distance (km) in oulieana . This reHt) of R. soulieana at the species level was 0.253±0.029; the heterozygosity value within populations (Hs) at the species level was 0.139±0.011. The overall estimate of genetic structure, i.e., the gene differentiation coefficient (Gst), is 0.451 (p<0.001). Genetic differentiation based on pairwise Fst comparisons between populations ranged from 0.016 (BSbm and BSc) to 0.384 (LJxz and BY). The gene flow (Nm) per generation is 0.608. AMOVA revealed that 46.0% of the total variation resided among populations while 54.0% of the total variation resided within populations  than in group II  and in group I . When applied to the whole dataset, hierarchical AMOVA  was 0.253, which was comparable to other outbreeding woody perennials having a moderate to high level of AFLP genetic diversity, e.g., Rosa arvensis (0.237) [Junipers oxycedrus subsp. macrocarpa (0.2097) [Cedrela balansae (0.222) [Buddleja crispa (0.3135) [R. soulieana covers almost the whole Hengduan Mountains range, although it is endemic to this area [R. soulieana is an entomophilous plant pollinated by bees, Bombus and butterflies. Although both geitonogamy selfing and xenogamy exist in this species, xenogamy can get much more achenes in each hip [R. soulieana is a long-lived woody perennial, and long-lived woody perennials in general have higher genetic diversity at the population level than short-lived perennials and annuals [The genetic diversity based on AFLPs of  (0.237)  of the s(0.2097)  from the (0.222)  from Nor(0.3135)  from the(0.3135) , 43, beshis area . The larhis area . Second,each hip , indicateach hip . Third,  annuals , 47–48.R. soulieana was within the populations. The gene differentiation coefficient (Gst) was 0.451, which was much higher than that of most outcrossing species (approximately 0.20–0.23) and lower than that of inbreeding species (0.50–0.59) analyzed with dominant markers [R. soulieana are dispersed close to the mother plant by gravity [R. soulieana, because hips of other wild roses are known to be widely dispersed by birds in Europe  was less than unity, indicating that there was restricted gene flow at the species level. Furthermore, the positive and significant correlation between genetic and geographic distances further explained the relatively high level of genetic differentiation among populations due to its wide distribution in the Hengduan Mountains.As far as genetic structure is concerned, more than half (54.0%) of the genetic diversity of  markers , 49. The markers , especia gravity , which wpecies 0.–0.59 anaarvensis ), North  sericea ). This mene flow . The Nm R. soulieana were clustered into 3 groups. The first group included several populations distinguished as R. soulieana var. soulieana and most populations of R. soulieana var. yunnanensis. The second group included most populations of R. soulieana var. soulieana and the only population of R. soulieana var. sungpanensis. The third group included the only population of R. soulieana var. microphylla (BSbm), several populations of R. soulieana var. soulieana  and some populations of R. soulieana var. yunnanensis, even though their relationship was not so close to each other, as shown in the results of PCoA  and west to Baxoi (BS)  . This isngiflora  and Arctica nana . Second,ountains , 66 thanR. soulieana in the east of the Yalongjiang River belongs to the Mountain Warm Zones in the Southeastern Monsoon Climatic Realm, while the second part in the west of Yalongjiang River belongs to the Qinghai-Xizang Plateau Cold Climate Realm . The populations between the first group (Group I) and the second group (Group II) had a very limited gene flow. According to the result of Bayesian analysis, genotypes of both groups concentrated significantly in the populations along the Yalongjiang River belonging to the third group (Group III)Figs , and onl persica . Thus, tR. soulieana based on AFLP genotypes in this study and the cpDNA haplotypes reported by Jian et al. [According to the genetic structure of n et al. , it is pS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


While most populations on this route have been characterized, the genetic background of others remains poorly understood, and little is known about past migration patterns. The scientific expedition “Marco Polo” has recently collected genetic and phenotypic data in six regions  along the Silk Road to study the genetics of a number of phenotypes.The ancient Silk Road has been a trading route between Europe and Central Asia from the 2We characterized the genetic structure of these populations within a worldwide context. We observed a West-East subdivision albeit the existence of a genetic component shared within Central Asia and nearby populations from Europe and Near East. We observed a contribution of up to 50% from Europe and Asia to most of the populations that have been analyzed. The contribution from Asia dates back to ~25 generations and is limited to the Eastern Silk Road. Time and direction of this contribution are consistent with the Mongolian expansion era.We clarified the genetic structure of six populations from Central Asia and suggested a complex pattern of gene flow among them. We provided a map of migration events in time and space and we quantified exchanges among populations. Altogether these novel findings will support the future studies aimed at understanding the genetics of the phenotypes that have been collected during the Marco Polo campaign, they will provide insights into the history of these populations, and they will be useful to reconstruct the developments and events that have shaped modern Eurasians genomes.The online version of this article (doi:10.1186/s12863-014-0131-6) contains supplementary material, which is available to authorized users. The route traverses a geographical region that was central during the human expansion from Africa [The ancient Silk Road has been a trading route for several centuries in the past , theWe compared genetic data at 299,899 single nucleotide variants for 441 individuals from six populations along the Silk Road (abbreviated in SR from now on) with 943 individuals from 53 populations worldwide from the Human Genome Diversity Project (HGDP) panel . To makeversus cities, see First, we explored global patterns of genetic variation in our study populations. In the principal component analysis (PCA) of all 53 populations . Approval for information collection and processing has been obtained by the Ethical committee of the Maternal and Child Health Institute IRCCS-Burlo Garofolo Hospital . All individuals signed an appropriate consent form  after they were instructed about the project, and all samples were completely anonymous.All communities belong to the Terra Madre organization .Subjects in this study have been enrolled as part of the scientific campaign “Marco Polo”, aimed at collecting social, genotype and phenotype information from populations of central Asia and Caucasus along the Silk Road. Populations in this study originate from West to East: Georgia, Armenia, Azerbaijan, Uzbekistan, Kazakhstan, Tajikistan and for the sake of simplicity we will collectively refer to them as the Silk Road populations, even if they are not fully representative of the entire range of countries along the Silk Road , consisting of 186267 SNPs.Shared ancestry between populations was evaluated using ADMIXTURE v 1.22 and the number of cluster that better represent the data was established by cross-validation as described in . Each AD–het implemented in PLINK. Demography and long-term effective population size were estimated by using linkage disequilibrium information as described in [Runs of homozygosity (ROH) were calculated using 299,899 SNPs. A ROH is defined as a chunk of genome ≥ 2000 kb long, containing at least 100 SNPs, with SNP density > = 1 per 50 kb. Within a ROH one heterozygous SNP and five missing calls are allowed. Two consecutive ROHs are considered as a single unit if their distance is < = 1 Mb. Coefficient of inbreeding was estimated using the function ribed in . Since tribed in  implemenribed in , a unrooribed in . The gen-se option was used to calculate standard errors. The tree was rooted with Yoruba when using the dataset with all the populations. Migration edges were added until the model explained 99% of the variance and also the migration edges were significant. Migration rate was estimated from Treemix run for the best tree selected. A Three-population test [Treemix v1.12  was usedion test  was usedion test , consideSignificance of the admixture was assessed after Bonferroni’s correction and consisted in the decay of linkage disequilibrium. In addition the level of admixture was assessed for each population using as reference for the population of the Silk Road only one population at the time. As an additional approach, we applied a machine learning method, SupportMix , to esti