Diplodus sargus sargus, as a model species. A multidisciplinary approach was used combining 1) spatial distribution patterns of individuals (post-settlers and adults) assessed through visual census within Torre Guaceto MPA and in northern and southern unprotected areas, 2) Lagrangian simulations of dispersal based on an oceanographic model of the region and data on early life-history traits of the species  and 3) a preliminary genetic study using microsatellite loci. Results show that the MPA hosts higher densities of larger-sized spawners than outside areas, potentially guaranteeing higher propagule production. Model simulations and field observation suggest that larval retention within and long-distance dispersal across MPA boundaries allow the replenishment of the MPA and of exploited populations up to 100 km down-current (southward) from the MPA. This pattern partially agrees with the high genetic homogeneity found in the entire study area (no differences in genetic composition and diversity indices), suggesting a high gene flow. By contributing to a better understanding of propagule dispersal patterns, these findings provide crucial information for the design of MPAs and MPA networks effective to replenish fish stocks and enhance fisheries in unprotected areas.Successfully enforced marine protected areas (MPAs) have been widely demonstrated to allow, within their boundaries, the recovery of exploited species and beyond their boundaries, the spillover of juvenile and adult fish. Little evidence is available about the so-called ‘recruitment subsidy’, the augmented production of propagules (i.e. eggs and larvae) due to the increased abundance of large-sized spawners hosted within effective MPAs. Once emitted, propagules can be locally retained and/or exported elsewhere. Patterns of propagule retention and/or export from MPAs have been little investigated, especially in the Mediterranean. This study investigated the potential for propagule production and retention/export from a Mediterranean MPA  using the white sea bream,  Many studies have highlighted the positive effects of successfully enforced marine protected areas (hereinafter MPAs) on populations of exploited coastal fishes in both tropical and temperate areas Due to a number of processes , MPAs may also provide benefits to outer areas through spillover of juvenile and adult fish  have the potential to increase the occurrence of spawning aggregations (i.e. high-density groupings of conspecific fish gathered together for the purpose of reproducing) and, more in general, to generate a greater propagule production compared to fished areas While there is an abundant literature reporting cases of fish population recovery inside MPAs Empirical estimates of propagule retention/export are rare, due to the difficulties associated with tracking small-sized fish propagules from their natal origins through the pelagic environment to their possible settlement locations Most Lagrangian model applications are based on PLD values and spawning dates from single estimates (in space and in time) that have been extrapolated over larger scales. This is understandable due to the effort (time and resource consumption) and specific competences  required to get reliable information on PLD and spawning or birth dates. There is increasing evidence, however, that these biological traits significantly change in time and space Information on patterns of spatial connectivity of fish populations can be obtained using a number of approaches  was selected as a model species due to its ecological and economic importance The aim of the present work is to investigate the potential propagule production and retention/export of a coastal fish from a Mediterranean MPA . The white sea bream  and in surrounding unprotected areas up to about 100 km away from TGMPA borders. TGMPA is located in southeastern Italy, along the Apulian Adriatic coast , usually inhabits the littoral zone in shallow waters down to about 50 m The white sea bream, Density, size distribution and biomass of adults  for a total of 336 replicates. Due to the high number of replicates, the sampling for settlers was carried out by two operators equally partitioning the number of replicates within each site.Density and size of adults were estimated in July 2010 by scuba underwater visual census using strip transects of 25×5 m D. sargus sargusDensity of settlers was evaluated during the settlement peak in June 2009 by means of snorkeling visual census along strip transects of 25×2 m Sampling sites were randomly selected out of a pool of possible sites having similar features in terms of habitat types’ coverage and exposition.D. sargus sargus were collected for a preliminary genetic analysis. Settlers were collected in June 2009 at three separate sites within the study area: (i) one site (Punta Penna Grossa) within the TGMPA, (ii) one site (Torre Pozzella) located ∼20 km north of the MPA, and (iii) one site (Punta Penne) located ∼15 km south of the MPA. Genetic analyses were conducted on a sub-sample of 96 individuals, including 48 from the MPA, 24 from Torre Pozzella and 24 from Punta Penne. After collection both post-settlers and recruits were immersed in an ice slurry (<5°C) to minimize suffering and then stored in 95% ethanol. The experimental fishing activity was performed in strict accordance with the authorization protocol provided by the Italian Ministry of Agriculture, Foods and Forestry Politics (Permit Number: 0011267–2010).Concomitantly with density estimates, settlers of http://earth.google.com).To test for potential differences in fish densities along the study area, two univariate PERMANOVA analyses were carried out separately for adults and settlers. An asymmetrical sampling design was adopted that included the following factors: Geographic Area  and Site . Linear distance (Di) in km from TGMPA boundary (set to 0 for the two sites within TGMPA) was used as a covariate. Distances were measured using georeferenced satellite imagery from Google Earth .http://gnoo.bo.ingv.it/afs).The study area is covered by the Adriatic Forecasting System (AFS) which provides daily oceanographic model outputs consisting of simulation (for the past time) and forecast for the next 10 days in situ observations.The model used is the POM (Princeton Ocean Model). It is a three-dimensional finite difference, free surface, primitive equation numerical model, based on the Boussinesq and the hydrostatic approximation and a split mode time step. The AFS model domain encompasses the whole Adriatic basin and extends south of the Otranto channel into the northern Ionian Sea, where the only open boundary is located. The AREG  grid has a horizontal resolution of about 1/45° (about 2.2 km), on 31 σ-layers. The surface fluxes are interactively computed using sea surface temperature predicted by the model and realistic atmospheric data provided by the European Centre for Medium Range Weather Forecast (ECMWF) with a frequency of six hours and a resolution of 0.25°. AFS is nested into the Mediterranean Forecasting System, MFS x,y,z) at each time step (t). The depth at which the particle is released at the beginning of the simulation (t = 0) was kept constant along the entire simulation as quantitative data is not available to support alternative assumption on larval behavior and vertical movements for the studied species.The Lagrangian simulations used in this work were performed by using the User Visualization Tool (UVT), a software developed by INGV that ingests the AFS model products allowing the user to derive particle trajectories by simulation. Each Particle (P), representing a single propagule, is described in terms of its spatial position  for each particle location  at time t  using the information provided at each grid point by AFS products. UVT then calculates the movement of the particle ×dt) where dt corresponds to the time step of integration. The new position of the particle at t+dt is then calculated. The horizontal resolution of the AFS model grid does not allow a precise simulation of hydrodynamic processes very close to the coast, and this should be taken into account when interpreting simulated dispersal patterns.UVT calculates current velocity components  were performed. High consistency among repeated simulations  was observed during preliminary analyses.The starting positions of the particles were defined over an equally spaced grid within a circle of 5 km around the actual sampling position. Simulations were started in coincidence of 4 spawning dates that were back-calculated from otolith analysis Minute sections of the caudal fin were digested in a lysis buffer containing 100 µl TE Buffer, 7 µl 1 M DTT (dithiothreitol) solution pH 5.2 (diluted in 0.08 M NaAC) and 2 µl proteinase K solution (20 mg/ml) for at least 8 hours at 56°C. After incubation at 96°C for 10 min, samples were centrifuged at 13,000 rpm for 11 min, and the supernatant was stored at −20°C.Sparus aurataD. sargus sargus. Microsatellites were grouped into two separate multiplexes in order to reduce PCR and genotyping costs  and expected (He) heterozygosities per locus using GENETIX version 4.05 Within sample genetic diversity was assessed by observed  using an Ne value of 1,000. Differences in allele and genotype frequencies among samples were assessed using Fisher's exact test as implemented in GENEPOP. Significance levels for multiple simultaneous comparisons were adjusted using Bonferroni as described above. Population structure was explored by nonhierarchical AMOVA and by calculating pairwise FST between samples in ARLEQUIN 3.5.1.2 4 iterations followed by 106 additional Markov Chain Monte Carlo (MCMC) iterations were performed. The most likely K was determined using the criterion of Prior to the population structure analysis, the statistical power of the markers employed was assessed with POWSIM D. sargus sargus was significantly affected by distance from TGMPA, i.e. it decreased with distance from TGMPA borders. Within TGMPA density was significantly higher than that observed at both northern and southern GAs  and it was significantly higher at TGMPA than at northern and southern areas , followed by that observed at the southern GA (9808.33±1438.75 ind./ha), while the lowest values were found at the northern GA (2202.08±624.65 ind./ha). Mean density of settlers at both TGMPA and southern GA were significantly greater than the mean values in northern GA  generated with the UVT showed that the particles released from TGMPA stayed in part within TGMPA and in part flowed southward, with travel distances ranging between few kilometers and about 180 kilometers , 6, 7.Simulations carried out at three different depths  were in agreement in terms of overall southwards direction for all release dates considered, but differed in terms of dispersal distance. Particles released at the shallowest depth 1 m,  travelleHo = 0.617–0.692) and expected (He = 0.677–0.721) heterozygosity and allelic richness (AR = 7.78–9.77). All comparisons across samples using one-way ANOVA were statistically not significant (p>0.5). Only four out of 34 tests departed from HWE, but none were significant after Bonferroni correction. The software MICROCHECKER showed no evidence for scoring errors due to stuttering or large allele dropout. No linkage disequilibrium was observed between any pair of loci after Bonferroni correction. Simulations using our empirical microsatellite data in POWSIM, taking into account the sample size of each population and a wide range of predefined FST values, showed that our markers have enough statistical power to detect FST values ranging from 0.001 to 0.1 (p = 1.000).Values of genetic diversity in all settler samples are detailed in ST = 0.001), with no significant differences within (FIS = 0.071) or among sites (FIT = 0.072). All pairwise comparisons were not significant (p>0.05). The software STRUCTURE inferred one single population as most likely  in comparison with K = 2 (ln likelihood = −3990.1) or K = 3 (ln likelihood = −4098.6).Comparison of allele frequencies among samples showed no significant differences in settlers at any locus or across all loci (p = 0.221). AMOVA analysis suggested no genetic sub-structuring  of the white sea bream Diplodus vulgaris) that was demonstrated to have a potential egg production approx. 15 times higher within TGMPA than in outer fished areas This hypothesis matches the findings related to a congeneric species  suggests a high degree of connectivity between the MPA and adjacent areas at the spatial scale investigated, indicative of transport of propagules well beyond the MPA boundaries. All simulations showed a general southward export, while dispersal distances varied depending on the depth of simulation. Similarly, using dispersal models in large estuarine systems of North America, other authors found a significant variability in spatial patterns of dispersed particles related to depth Lagrangian simulations and settler density observed in the field  agreed in suggesting both propagule retention and export mechanisms directed southwards. Density of settlers, moreover, did not vary significantly with distance from TGMPA, suggesting a fairly homogenous propagule dispersal in open waters over a scale of ∼100 km. On the other hand, density of settlers varied among sites, which could reflect a different suitability for settlement of coastal sites as a function of habitat type.A significant export of larvae beyond TGMPA boundaries, as predicted by model simulations, is in agreement with the observed homogeneity found in our preliminary genetic study. Comparison of samples obtained in TGMPA and neighboring areas showed no differences in genetic variability or genetic composition, which indicates that the TGMPA is not isolated and that there is genetic connectivity among sites at the scale of approximately 50 km. The pattern of dispersion suggested by the oceanographic model could explain the genetic similarity between the TGMPA and southern sites, with down-current sites effectively replenished by TGMPA. However, the genetic homogeneity between TGMPA and northern sites cannot be explained by larval dispersal from TGMPA under the simulated oceanographic regime, as all particles originating from TGMPA either stayed within the protected area or moved southward. As a consequence, the observed genetic homogeneity could be due to gene flow from northern sites towards TGMPA and even more southern sites. In addition, a gene flow between TGMPA and adjacent northern areas could be ascribed also to the exchange of juvenile and adult individuals between TGMPA and northern sites. Evidence of connectivity among TGMPA and the adjacent northern areas has been documented only for juveniles a priori any directionality in dispersal , or differences in hydrodynamic conditions (e.g. current regimes) and topography (e.g. continuous coastline vs islands) among the different study areas.Pelc et al. D. sargus sargus around TGMPA in SW Apulia. These findings support the idea that TGMPA could produce benefits that go well beyond its boundaries.Fishes are generally characterized by relatively high larval dispersal The lack of knowledge on whether and how MPAs can benefit fisheries through propagule export remains a major roadblock to successfully designing and implementing MPAs worldwide D. sargus sargus in the SW Adriatic, a network of effective and self-sustaining MPAs (as it seems to be the case for TGMPA) placed at a distance of 100–200 kilometers could provide benefits for both conservation and fishery output thanks to the replenishment of fished areas through larval export. Carrying on similar studies on a number of representative species and in other areas, and possibly also taking into account propagule inflow in MPAs (e.g. using Lagrangian back-tracking techniques), will make it possible to draw more general conclusions that may help design effective regional networks of MPAs.In conclusion, the present study provides some elements suggesting that, in the case of


Microcystis aeruginosa. Being widely distributed on a global scale but patchily on a regional scale, this prokaryote is an ideal model organism to study microbial dispersal and biogeography.Free-living microorganisms have long been assumed to have ubiquitous distributions with little biogeographic signature because they typically exhibit high dispersal potential and large population sizes. However, molecular data provide contrasting results and it is far from clear to what extent dispersal limitation determines geographic structuring of microbial populations. We aimed to determine biogeographical patterns of the bloom-forming freshwater cyanobacterium M. aeruginosa was studied based on a dataset of 311 rDNA internal transcribed spacer (ITS) sequences sampled from six continents. Richness of ITS sequences was high (239 ITS types were detected). Genetic divergence among ITS types averaged 4% (maximum pairwise divergence was 13%). Preliminary analyses revealed nearly completely unresolved phylogenetic relationships and a lack of genetic structure among all sequences due to extensive homoplasy at multiple hypervariable sites. After correcting for this, still no clear phylogeographic structure was detected, and no pattern of isolation by distance was found on a global scale. Concomitantly, genetic differentiation among continents was marginal, whereas variation within continents was high and was mostly shared with all other continents. Similarly, no genetic structure across climate zones was detected.The phylogeography of M. aeruginosa ITS types is not rare, and that this species might have a truly cosmopolitan distribution.The high overall diversity and wide global distribution of common ITS types in combination with the lack of phylogeographic structure suggest that intercontinental dispersal of  Dispersal, geographic isolation, past range restrictions and expansions, drift processes, founder events and selection all leave their signature in the lineage composition of contemporary populations Prokaryotes are generally smaller and have faster reproduction cycles than the eukaryotic microorganisms that were the subject of these biogeographic studies Microcystis abounds in eutrophic and hypertrophic freshwater bodies worldwide Microcystis often displays mass developments at the surface of lakes, it can easily be detected and sampled, and is therefore a good model organism to study global biogeographical patterns in free-living bacteria. Microcystis forms dense blooms that may be toxic, causing economical and ecological problems worldwide Microcystis is therefore also relevant in the light of control of toxic cyanobacterial blooms.The cyanobacterium Microcystis has a complex taxonomic history. The genus includes, next to the type species M. aeruginosa, a number of other species that have been delimited on the basis of colony morphology. These morphospecies, however, are not supported by molecular data forming a clade of nearly identical 16S rDNA sequences et al.Microcystis into our study, but these all refer to the name M. aeruginosa following Otsuka et al.Microcystis genotypic composition, yet phylogeographic structuring seemed absent Microcystis has been suggested depending on the sampled area or markers used. A global study of biogeographic patterns in relation to climatic conditions is still lacking.On regional scales, significant spatial differences have been observed in Microcystis, considerable ITS variation among Microcystis strains has been shown Microcystis by ITS sequencing as shown for the cyanobacterium ProchlorococcusMicrocystiset al.Microcystis ITS population structure in Tigray (Ethiopia). The Microcystis genome contains two identical rRNA operons, although point mutations may occur occasionally The fast-evolving 16S–23S rDNA ITS region has proven to be a useful marker for phylogeographic studies of various (cyano)bacteria M. aeruginosa on six continents based on sequence variation.This study assesses global biogeographical patterns and dispersal of Microcystis sampled from six continents: Europe (199), Africa (40), Asia (45), North America (7), South America (12) and Oceania (8). All sequences from Belgium (52), South America (12) and Ethiopia (29) were newly obtained in addition to some sequences from Denmark (5), The Netherlands (7) and Spain (2) (Microcystis strains (14). These were completed with 204 sequences from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/), which were also obtained from cultivated isolates (135), sequenced DGGE bands (28) or cloned PCR fragments (41). An overview of all sequences used in this study is shown in Our dataset consists of 311 ITS sequences of pain (2) . New seqMicrocystis colonies from samples from Belgium and Ethiopia were picked out using sterile glass Pasteur pipettes under a stereo microscope. The strains were grown in WC medium −2 s−1 and a 12∶12 h light∶dark cycle. In total, ten ITS sequences from strains isolated from Belgium and two from Ethiopia were included in the molecular analyses.Water samples from lakes and ponds in Europe, South America and Ethiopia were filtered through a 25 mm 0.2 µm GSWP filter (Millipore) and immediately frozen at −20°C. Individual et al.Microcystis strains were amplified using the protocol described by Janse et al.et al.Microcystis ITS sequences. In a first PCR, a specific 16S rDNA primer for Microcystis (CH) described by Rudi et al.et al.DNA from the water samples and isolated strains was extracted using bead beating, phenol extraction and ethanol precipitation as described by Zwart et al.DGGE was essentially performed as described by Muyzer Microcystis-specific ITS sequences obtained from samples from Belgium and Ethiopia were ligated into pGEM®-T Easy Vectors (Promega), and transformed into competent Escherichia coli JM109 cells. The transformed cells were plated on Luria-Bertani (LB) plates containing 20 µg l−1 ampicillin, 20 µg l−1 X-Gal  and 5 µg l−1 IPTG  and incubated overnight at 37°C. White recombinants were picked out and grown overnight in ampicillin-supplemented liquid medium (Luria-Bertani-Broth). The clones were screened for inserts using primers CSIF and ULR (see before). 40–70 clones per sample were screened by DGGE analysis (see before) to identify groups of clones containing (presumably) the same inserts. One or more representatives of each group were then chosen for sequencing. In total, 34 ITS sequences from Belgium and four from Ethiopia were obtained in this way. Sequencing was performed with the ABI-Prism sequencing kit and the resulting sequencing reaction products were analysed on an automatic sequencer (ABI-Prism 3100).ss statistic, a measure of substitution saturation in molecular phylogenetic data sets, was calculated with DAMBE The 311 ITS sequences were aligned using MUSCLE Statistical parsimony networks The production of PCR artefacts (e.g. chimeras and heteroduplexes) is a potential risk when mixed templates of related sequences are amplified by PCR Microcystis . RNA sequences were folded using Mfold Prediction of the RNA secondary structure of the ITS region was based on the complete rrn operon sequence extracted from the complete genome of The sequence dataset was divided into 32 pre-defined populations, each population consisting of sequences from a single country . For eacst)−1)/4. Geographical distances between the different populations were measured using geographical coordinates of the lake from which the sequences were obtained or if several lakes were sampled per population, the centroid between these lakes was taken. We calculated the great-circle distances as well as the shortest distance over land since Microcystis might disperse easier over land using stepping stones in between sampled locations  than across oceans. The latter distances were calculated using Google Earth (http://earth.google.com/). Mantel tests were performed using both types of geographical distances.To test whether genetic distance was correlated to geographical distance (Isolation By Distance) the program IBDWS Patterns of genetic structuring based on the stripped ITS sequence alignment among geographical localities (continents) and climates were estimated by analysis of molecular variance (AMOVA) using Arlequin 3.1 DFsThe historical demographic structure of the genetic variation at the ITS locus was investigated using Tajima's ss statistic did not reveal significant saturation of the full alignment, the measure of skewness equalled the empirical threshold values in Hillis & Huelsenbeck Specifications of the full and stripped ITS sequence alignments are given in Statistical parsimony analysis of the full ITS alignment resulted in a single, highly interconnected and largely unresolved network with maximum connection limit of 9 steps . SimilarThe parsimony network derived from the stripped ITS sequence alignment was to a certain extent still unresolved, but with a much lower degree of connection uncertainties . One parZ = 28*1011, r = 0.127, one-sided p = 0.958) or the stripped dataset . Similarly, a Mantel test for matrix correlation between genetic similarity (M) and geographic distance using the shortest distance over land showed no significant negative correlation for the full dataset  or the stripped dataset .A Mantel test for matrix correlation between genetic similarity (M) and geographic distance using great circle distances showed no significant negative correlation for the full dataset , but the dominant component of genetic variation was again found within (93.55%) instead of between these super continents (6.45%). AMOVA on the dataset containing all populations showed similar results (not shown). No significant genetic structuring was found among all twelve climate groups, among the six climates represented by more than ten ITS types, or between cold-temperate and (sub)tropical climates  and Fu's test  are indicative of a recent global expansion of M. aeruginosa. Note that the term ‘recent’ only has a qualitative meaning, relative to the rate of mutation and genetic drift at the ITS locus.Both Tajima's test  from true recombination et al.et al.Microcystis aeruginosa is very plastic and displays a high transposon activity. The hypervariable regions found in the rDNA ITS region are too short to be transposons, however, and the exact nature of these regions remains uncertain. Notwithstanding these uncertainties, all analyses on the geographic genetic structure yielded concordant results.Several regions in the ITS alignment were highly variable with only weak correlation between the various alleles of each locus, even among those that were separated by just a few dozen nucleotides. This lack of linkage and high level of homoplasy is very remarkable. A possible but scarcely conclusive explanation for this singularity is that it is the result of recombination within the genome itself. Several studies have revealed exchange of genetic information through horizontal gene transfer and recombination in cyanobacteria Microcystis, along with the lack of geographical genetic structuring and the prevalence of a single, widespread ITS type (excluding the hypervariable regions) may be explained by two radically different scenarios: 1) Microcystis represents an ancient lineage, which acquired its worldwide distribution gradually on evolutionary time scales, or 2) Microcystis represents a young clade that spread globally recently, experiencing ongoing passive dispersal. Several clues favour the second hypothesis. Firstly, 16S rDNA data clearly indicates that Microcystis forms a distinct clade of nearly identical sequences, indicating a relative recent origin D, Fu's Fs) support the hypothesis of a recent worldwide population expansion following a bottleneck or a selective sweep. Fourthly, the isolation-by-distance analysis showed no correlation between genetic and geographical distance, and analysis of molecular variance shows a lack of overall genetic differentiation between continents reinforcing the notion of frequent or occasional ongoing global dispersal of Microcystis. We only found a subtle geographic structure between Afro-Eurasia and the Americas, and ITS types from the Americas were absent in some of the subclusters in the stripped ITS network. However, this could be due to relatively low sampling in the Americas. It is difficult to compare the CTF values from the AMOVA directly with studies on other organisms as other molecular markers are often used. However, the finding that the dominant component of genetic variation was found within, instead of between continents, supports the ubiquitous nature of M. aeruginosa.The global distribution of Microcystis, the role of random drift versus selection and the mutation rate of the ITS sequence. Answers to these questions would require genetic markers with higher resolution as well as a spatially explicit experimental design to study colonization and succession dynamics. So far, the question remains whether the lack of geographic structure in the data is due to frequent global dispersal and gene flow among distant populations, or whether this is due to occasional transoceanic colonisations of new habitat patches combined with persistent founder effects. Population-genetic theory strongly argues against the first scenario, as a result of extremely large population sizes of bacterial populations that would necessitate large amounts of gene flow for immigrant strains to be detected in established populations and contribute to genetic differentiation Microcystis populations is driven by founder effects that arise whenever new habitat patches are created The exact temporal scale of dispersal remains uncertain due to the absence of an evolutionary timeframe of cyanobacteria, as well as limits of the design of the study and the temporal resolution that is inherent to the mutation rate of the studied ITS fragment. We can only state that the elapsed time since the most recent global dispersal events is probably not longer than the average time needed for new mutants to arise in isolated populations and increase above the detection threshold. This, in turn, depends on the population biology of M. aeruginosa contrasts with that of another bloom-forming freshwater cyanobacterium, Cylindrospermopsis raciborskii, in which phylogenetic analyses based on four genes, including ITS, showed a clear clustering of strains according to continent Arthrospira sp., based on the appearance of two discrete ITS clusters corresponding to Africa-Asia and America Microcystis, Cylindrospermopsis and Arthrospira have a narrower ecological amplitude, mainly occurring in tropical to warm-temperate regions The absence of phylogeographic patterns found in this study is remarkable given the fact that freshwater bodies are small and often highly isolated habitat patches in a vast terrestrial matrix, which in itself is separated by extended oceanic regions at a global scale. The pattern in MicrocystisMicrocystis cells have thick cell walls and are grouped into colonies surrounded by a protective mucilage layer, which may protect them from harsh environmental conditions during dispersal. They have been observed in vegetative condition in the sediment Microcystis to tolerate the transport conditions Microcystis to disperse might be by migrating birds Microcystis blooms, colonies may easily be transferred by birds either through internal or external transport. If migrating birds were the dominant dispersal vectors, however, much stronger phylogeographic patterns could be expected, reflecting the north-south migration routes of water birds. The absence of such pattern suggests that transport may also be driven by other factors, such as wind dispersal. Transcontinental wind and dust events have been proposed to form atmospheric bridges over land and sea, facilitating long-distance dispersal of micro-organisms Microcystis strains might also be facilitated by human-mediated dispersal, mainly by the recreational use of water bodies and transport of fish and macrophytes, as already suggested for several other small aquatic organisms Little information is available on the dispersal ability and transport mechanisms of Microcystis, which contrasts with some other studies on cyanobacteria Microcystis ITS types were detected in a wide range of climates, indicating a broad tolerance or capacity for rapid local adaptation to different climatic conditions. We did not find indications that distinct ecotypes of Microcystis can be differentiated by ITS as has been suggested for ProchlorococcusMicrocystis populations are likely phenotypically and/or ecologically differentiated by different suites of functional genes. Indeed, a high functional diversity  in sympatric and allopatric Microcystis strains has been shown by laboratory experiments M. aeruginosade novo mutations might be involved in rapid adaptation to novel environments given the large population sizes and short generation time of M. aeruginosa.No genetic structuring according to climate conditions was found in Dataset S1rDNA ITS dataset with indication of ITS, tRNA and hypervariable regions (x).(TXT)Click here for additional data file.Table S1Overview of the sequences used in this study.(DOC)Click here for additional data file.


Thus our results do not support the prediction of generally high mfr and suggest that intersexual constraints on the evolution of colouration traits may be weaker than expected. We hypothesise that observed patterns of genetic correlations result from sex-specific selective pressures acting on nestling plumage colouration.In some bird species, both adult and juvenile individuals are often brightly coloured. It has been commonly assumed that identical plumage colouration present in both sexes results from strong intersexual genetic correlations in colour-related traits. Here, we aimed at testing this hypothesis in juvenile individuals and looked at genetic parameters describing carotenoid-based colouration of blue tit nestlings in a wild population. To separate genetic and environmental sources of phenotypic variation we performed a cross-fostering experiment. Our analyses confirmed the existence of sexual dichromatism in blue tit nestlings and revealed a significant, although low, genetic component of carotenoid-based colouration. However, genetic effects are expressed differently across sexes as indicated by low cross-sex genetic correlations ( In this species, both adults and juveniles express yellow colouration of breast feathers Our aim was to explore genetic patterns in sexually dimorphic traits in blue tit   were recorded. Nestlings were uniquely marked by nail clipping on day 2 followed by ringing on day 11. Two days post-hatching nestlings were blood-sampled for molecular sex identification (see th day post-hatching) and placed between two glass slides together with colour standards helping to calibrate subsequent colour sampling. The arrangement of feathers resembled that on the living bird in such a way that they formed two superimposed layers. Samples were photographed  in a black-box using an unidirectional halogen light source. Photographs were analysed blindly with respect to the origin of the sample using Colour Analysis Tool software (http://wwwfxc.btinternet.co.uk). We averaged RGB colour values from ten 2×2 mm squares per individual. Measurements were highly repeatable . We made all efforts to minimize the time required to handle the nestlings and thus to decrease the stress associated with all procedures.Colour data were analysed with using linear mixed models implemented using the Markov Chain Monte Carlo method in R 2.9.2 The models fitted experimental treatment (brood-size manipulation) and year as fixed effects. Additionally, sex was introduced in the univariate models. All models contained random effects of nest of origin, nest of rearing and dyad. Inclusion of each of these effects was decided upon the deviance information criterion (DIC) (Table S2 in 2H). Since we defined sex-specific variances for nest-of-origin effect we were able to estimate also related covariances. Dividing genetic covariance by geometrical mean of the respective variances yielded genetic correlations Using such kind of full-sib analyses assumes that there is no relatedness between the analysed families. In such a case, nest-of-origin approximates half of additive genetic variance plus quarter of dominance variance and maternal effects (ME) if present V = 1, covariance COV = 0, ν = 1.002). However, all models were robust to prior modification – changing priors by doubling variances had no effect on qualitative conclusions drawn from our analyses.All analyses were checked for possible problems resulting from autocorrelations in MCMC time series by visual inspection of the time-series plots and by the calculation of autocorrelations between successive samples. No autocorrelation-related issues were detected in any of the models. All models were fitted with relatively uninformative, inverse-Wishart distributed priors  of about 8% . Thus, our estimates of heritability and genetic correlations might be downwardly biased. It is hard to quantify the strength of this bias as, to our knowledge, the influence of misassigned paternities on the estimates of heritability and genetic correlations has rarely been rigorously assessed 2 for colour related traits reaches 8% ), and does not differ significantly between sexes (see respective CIs in mfr) in all three colour traits, with mfr for brightness being even weakly negative  and yellower  selection acting on adults would require demonstrating that juvenile and adult plumage traits are strongly correlated genetically. Compelling evidence for such correlations in blue tits does not exist. Only one study estimated phenotypic juvenile-adult correlation  in carotenoid-based plumage traits in a closely related species – the great tit - Firstly, juvenile plumage may be ontogenetically correlated with that of adult birds. In such a case juvenile plumage would be a mere dysfunctional side-product of selection acting on adult individuals – and all genetic patterns observed in offspring plumage would in fact reflect underlying genetics of adult traits. Usually, strong genetic correlations between analogous traits expressed throughout the ontogeny are expected mfr – remains to be studied.Secondly, carotenoid-based plumage colouration in juvenile blue tit might be adaptive at this stage of life independently of its function in the adulthood. It is unlikely that plumage colouration plays any role in the competition inside a nest or in parental favouritism as tits breed in cavities with limited light availability To conclude, our results provide first evidence that genetic constraints on the independent evolution of male and female ornamentation observed among nestlings may be weak. Lack of strong intersexual genetic correlation is contrary to a common assumption of shared genetic background of the sexes. However, the potential sex-specific selection forces responsible for diminishing the expected genetic correlation between sex remains to be identified. Further studies, focusing on sex-specific function of carotenoid-based plumage colouration in juveniles, should explain evolutionary mechanisms that produce low cross-sex genetic correlations among nestlings, and will certainly open new and exciting research perspectives.File S1Tables S1–3 providing repeatabilites of analysed traits, fixed-effects estimates and DIC-based model selection.(DOC)Click here for additional data file.


Peromyscus leucopus), and test the hypotheses that park size and time since isolation are associated with genetic variability using nonlinear regression and information-theoretic model selection. White-footed mice have previously been documented to exhibit male-biased dispersal, which may create disparities in genetic variation between males and females in urban parks. We use genotypes of 18 neutral microsatellite data and four different statistical tests to assess this prediction. Given that sex-biased dispersal may create disparities between population genetic patterns inferred from bi- vs. uni-parentally inherited markers, we also sequenced a 324 bp segment of the mitochondrial D-loop for independent inferences of historical demography in urban P. leucopus. We report that isolation in urban parks does not necessarily result in genetic bottlenecks; only three out of 14 populations in NYC parks exhibited a signature of a recent bottleneck at 18 neutral microsatellite loci. Mouse populations in larger urban parks, or parks that have been isolated for shorter periods of time, also do not generally contain greater genetic variation than populations in smaller parks. These results suggest that even small networks of green spaces may be sufficient to maintain the evolutionary potential of native species with certain characteristics. We also found that isolation in urban parks results in weak to nonexistent sex-biased dispersal in a species known to exhibit male-biased dispersal in less fragmented environments. In contrast to nuclear loci, mitochondrial D-loop haplotypes exhibited a mutational pattern of demographic expansion after a recent bottleneck or selective sweep. Estimates of the timing of this expansion suggest that it occurred concurrent with urbanization of NYC over the last few dozens to hundreds of years. Given the general non-neutrality of mtDNA in many systems and evidence of selection on related coding sequences in urban P. leucopus, we argue that the P. leucopus mitochondrial genome experienced recent negative selection against haplotypes not favored in isolated urban parks. In general, rapid adaptive evolution driven by urbanization, global climate change, and other human-caused factors is underappreciated by evolutionary biologists, but many more cases will likely be documented in the near future.Severe fragmentation is a typical fate of native remnant habitats in cities, and urban wildlife with limited dispersal ability are predicted to lose genetic variation in isolated urban patches. However, little information exists on the characteristics of urban green spaces required to conserve genetic variation. In this study, we examine whether isolation in New York City (NYC) parks results in genetic bottlenecks in white-footed mice ( Populations in fragmented habitats are predicted to lose genetic variation due to drift and local adaptation through natural selection , althougSevere fragmentation is a typical fate of native remnant habitats in cities , and urbPeromyscus leucopus). We then use nonlinear regression and information-theoretic model selection to test the hypotheses that park size and time since isolation are associated with genetic variability. Our previous research on this system found substantial genetic structure among urban white-footed mice, and indicators of genetic variation at neutral microsatellites were moderately high but not uniform across parks  parks results in genetic bottlenecks in white-footed mice .Mice were trapped over 2–3 nights at each site using Sherman Live Traps (9″ × 9″ × 3″) baited with birdseed. For genetic analysis, we snipped the terminal 1 cm or less of each mouse’s tail before releasing them alive at the site of capture. Tail snips were stored in 80–95% ethanol until DNA extraction. Next, we genotyped all mice at 18 unlinked microsatellite loci, and calculated for each population across all loci the mean number of alleles, effective number of alleles , number of private alleles, and observed heterozygosity in GenAlex 6.2 . All aniWe tested for genetic bottlenecks in each park using the program BOTTLENECK 1.2  and the cAI), average relatedness, and STF calculated separately for males and females. The assignment index calculates the probability that an individual’s genotype occurred by chance in a population, and thus individuals of the dispersing sex should exhibit lower mean cAI values , effective number of alleles (EN), number of private alleles (PN), and observed heterozygosity (OH) reported in EμN where μ = mutation rate) estimated using MIGRATE-n  indicate that most parks were surrounded by urban development by 1924, and thus isolated concurrent with or before their founding. Many maps exist from throughout NYC’s history, but unfortunately these maps generally do not contain information on the quality or extent of vegetation  the years since each park was officially founded, and (2) years since major infrastructure projects (primarily multi-lane parkways and expressways) were erected around park perimeters. Our rationale for the former date was that many parks in NYC were likely the last large green spaces in the general area at the time of their establishment, and thus their founding dates should reflect the order in which they became isolated. We used a time since isolation based on infrastructure projects because many NYC parks are at least partially circled by major roadways that likely present major obstacles to wildlife. Networks of parkways were constructed during the Robert Moses era of park management in NYC , and thegetation . Generatgetation .c) corrected for finite sample sizes  variables were examined with eighteen candidate models, each of which consisted of various combinations of the three geographic covariates and time since founding (F): an intercept-only model, the four univariate models , the eight combinations of two covariates,the four combinations of three covariates, and a global model with all four covariates (TA + HA + PH + F). Intercept-only models were included in the candidate model sets of each genetic variable to serve as a baseline for detecting a covariate effect: if a model performed substantially better than the intercept-only model, we interpreted this result as evidence for an effect of that model’s covariates upon the respective diversity index. We calculated maximum likelihood estimates of model parameters for each model, and then models were ranked using Akaike’s Information Criterion  is applied to non-normal data to facilitate the use of regression; however, this approach may be inferior to using regression techniques that directly match the distributions of the variables in question . All modP. leucopus D-loop sequences available on GenBank in September 2009 (accession numbers available from authors upon request). We created the consensus sequence from a ClustalW alignment conducted in BioEdit 7 (KF986735–KF986771), and a Nexus haplotype file used for the analyses below is available on the Figshare digital repository (DOI 10.6084/m9.figshare.881830).We sequenced a 324 bp region of the mitochondrial D-loop for a subset of 110 individuals from above to examine historical demography and genetic variation using a maternally inherited marker. We designed D-loop PCR primers from a consensus sequence created using all ioEdit 7 , and choioEdit 7 . We condD and Fu’s Fs, and assessed their significance using 10,000 coalescent simulations. We also calculated mismatch distributions  under a model of population expansion to examine demographic changes, and assessed significance of the observed distributions using 10,000 coalescent simulations of the raggedness statistic, r  and performed well in a number of other analyses, and thus we feel it is unlikely that we are failing to detect true bottlenecks in these populations. Changes to the assumed frequencies of single- vs. multi-step mutations in our Bottleneck runs did not significantly alter the results.Tests for genetic bottlenecks did not detect significant heterozygosity excess in most NYC parks , indicatsiliency . Howeversiliency , and drisiliency . This tyc), but this sex difference was not robustly supported. Males in our sample had less likely genotypes than females given the overall genetic characteristics of our sample, as has been argued previously to support male-biased dispersal in this species  can avoid genetic bottlenecks if they maintain high population densities in small urban parks. Common forest dwellers in eastern North America that are known to thrive in urban parks include red-backed salamanders, cinereus , and norevicauda . The foriversity . We predhabitats , but thitersheds .AN, EN, and PN were right skewed continuous and thus were modeled using gamma regression . Θ was highly right-skewed and thus was best modeled using a Tweedie distribution with an inverse Gaussian dispersion parameter . The inverse Gaussian parameterization of the generalized Tweedie distribution is useful in modeling variables that are right-skewed and continuous ; however, models with these combinations were universally poor.The values for gression , while Hntinuous . In all ntinuous , indicatntinuous . Θ increntinuous ; Fig. 1SSciurus carolinensis), although the relative proportions of tree and building cover also influence these densities , and white-footed mice in northeastern forest fragments is weak to nonexistent, except in certain sites with idiosyncratic characteristics  had one of the largest habitat areas but the lowest measures of genetic diversity . This siPark isolation time was generally not successful at explaining genetic variation. Similar to park size, there may not be enough variation in the time since isolation of NYC parks to observe a general trend in genetic variation. Alternatively, our use of the years since a park was founded or the years since construction of major infrastructure as proxies for how long parks have been ecologically isolated may not have been accurate. Given that much of NYC was heavily urbanized concurrent with the founding of these parks (particularly outside Manhattan), we feel that our choices were justified. However, future historical reconstructions of the NYC landscape  may fruiWe identified 37 haplotypes among 324 bp mitochondrial D-loop sequences obtained for 110 individuals . HaplotyD and Fu’s Fs values for the total NYC sample and Queens indicate that urban P. leucopus underwent a recent population expansion after a bottleneck or selective sweep. Alternatively, the D-loop may have experienced genetic hitchhiking  due to negative selection on linked mitochondrial genes. The observed mismatch distributions for NYC and Queens closely fit the expected unimodal distribution for a recent population expansion  in humans  and the entire NYC sample . These results suggest that demographic expansion after a bottleneck or selective sweep was concurrent with urbanization of NYC , assuming a conservative generation time in urban P. leucopus between 0.5 and 1.0 years. The expansion also occurred more recently in Queens than NYC overall, potentially because Queens was not heavily urbanized until after the construction of bridges, tunnels, and commuter rail connecting Queens to Manhattan in the early twentieth century. However, these estimated times should be interpreted cautiously given that we used D-loop mutation rates estimated from human pedigrees. These human estimates were calculated over sequences that were a few hundred bp longer than those analyzed in this study. If mutation rates are heterogeneous along the D-loop, then these human estimates may over- or under-estimate the mutation rate for P. leucopus. Additionally, mitochondrial mutation rates vary widely across mammals with variation in body size, temperature, metabolic rates  since the bottleneck or selective sweep reflected in the mismatch distributions  using ther 2 µt;  and fourn humans . To our ic rates , age at ic rates . All of In the absence of independent evidence, it is difficult to distinguish between bottlenecks and selection as explanations for the mismatch distributions observed in this study. Only a few NYC populations exhibited evidence of recent bottlenecks at nuclear microsatellite loci, thus undermining the bottleneck argument for the mtDNA data. Alternatively, the mtDNA results may reflect a bottleneck that occurred further back in time than could be detected by the microsatellite data, or the mtDNA reflects a bottleneck specific to this matrilineal marker. These alternatives are unlikely given that the D-loop is also a hypervariable marker appropriate for detecting recent demographic events, and the events creating the mismatch distribution were estimated to occur in the last few dozens to hundreds of generations. A matrilineal bottleneck signature is also unlikely given that males and females did not differ at multiple population genetic parameters estimated using nuclear data .The mismatch distributions may be better explained by negative selection against unfavorable mtDNA haplotypes once these populations became isolated in urban habitat patches with novel selection pressures. In this scenario, the D-loop would have hitchhiked along with the surviving haplotypes containing favored alleles in mtDNA protein-coding regions. The mitochondrial genome is now widely acknowledged to have experienced selective sweeps in many if not most taxa, and thus some authors have called into question its utility in demographic estimation . TheoretP. leucopus from the Chicago area indicated that mtDNA haplotypes changed rapidly over a timeframe corresponding to human development of natural areas , suggesting that mitonuclear pathways may be active targets of selection in urban populations (Mitochondrial DNA sequencing of contemporary and museum specimens of al areas . Mismatcal areas  closely NYC area . Two of ulations . Full miP. leucopus populations in different NYC parks. In contrast to nuclear loci, mitochondrial D-loop haplotypes exhibited a mutational pattern of demographic expansion after a recent bottleneck or selective sweep. Estimates of the timing of this expansion indicate that it occurred concurrent with urbanization of NYC over the last few dozens to hundreds of years. Given the general non-neutrality of mtDNA in many systems and evidence of selection on coding sequences in urban P. leucopus, we argue that the P. leucopus mitochondrial genome experienced recent negative selection against haplotypes not favored in isolated urban parks. In general, rapid adaptive evolution driven by urbanization, global climate change, and other human-caused factors is underappreciated by evolutionary biologists, but many more cases will likely be documented in the near future.We report here that isolation in urban parks does not necessarily result in genetic bottlenecks or substantial loss of genetic variation in urban wildlife populations. White-footed mouse populations in larger urban parks, or parks that have been isolated for fewer years, do not generally contain greater genetic variation than smaller or older parks, although we could not address site-specific variability between parks that may exert a greater influence on genetic variation than size alone. These results should be encouraging to conservation biologists working in human-dominated landscapes, as even small networks of green spaces may be sufficient to maintain self-sustaining populations and evolutionary potential of some native species . We also


Carcinusmaenas and the Mediterranean green crab C.aestuarii, has raised questions regarding the possibility ofhybridization between these sister species. Here I present analysis based onboth nuclear microsatellites and the mitochondrial cytochrome C oxidase subunitI (COI) gene which unambiguously argues for a hybrid origin of JapaneseCarcinus. Despite the presence of mitochondrial lineagesderived from both C. maenas and C. aestuarii,the Japanese population is panmictic at nuclear loci and has achievedcytonuclear equilibrium throughout the sampled range in Japan. Furthermore,analysis of admixture at nuclear loci indicates dramatic introgression of theC. maenas mitochondrial genome into a predominantlyC. aestuarii nuclear background. These patterns, along withinferences drawn from the observational record, argue for a hybridization eventpre-dating the arrival of Carcinus in Japan. The clarificationof both invasion history and evolutionary history afforded by genetic analysisprovides information that may be critically important to future studies aimed atassessing risks posed by invasive Carcinus populations to Japanand the surrounding region.Interspecific hybridization plays an important role in facilitating adaptiveevolutionary change. More specifically, recent studies have demonstrated thathybridization may dramatically influence the establishment, spread, and impactof invasive populations. In Japan, previous genetic evidence for the presence oftwo non-native congeners, the European green crab  The anthropogenic introduction of populations into novel ecological contexts allowsexploration of a wide range of phenomena that may influence evolutionarydiversification The rising frequency with which anthropogenic dispersal brings together previouslyallopatric lineages provides numerous opportunities to examine genetic exchangeassociated with recent hybridization events. To date the bulk of such research hasfocused on interspecific hybridization between native and invasive species. The vastmajority of this literature has addressed plant taxa Carcinus provide a promising opportunity toexamine interspecific genetic exchange between invasive animal species. While theEuropean green crab Carcinus maenas Linnaeus has achieved acosmopolitan distribution through anthropogenic dispersal, with establishedpopulations on all non-polar continents, its sole congener Carcinusaestuarii Nardo has been introduced to a more limited geographic rangeC. maenas found along the Atlantic coast of Europe and Africafrom northern Scandinavia to Mauritania and C. aestuarii limited tothe Mediterranean, although there has been some speculation as to the possibleexistence of a transition zone in southwestern Iberia Shore crabs of the genus C. aestuarii were first reported inTokyo Bay in 1984 and had spread as far southwest as Dokai and Sagami Bays by the1990s C. maenas in Japan was suggested only later, whengenetic analysis revealed the presence of mitochondrial haplotypes from both sisterspecies C. maenas has still generally not been recognizedas a distinct presence in Japanese populations. Morphological analysis of JapaneseCarcinus has largely supported the view that these crabs belongto C. aestuariiC.aestuariiC. aestuarii butpossessing carapace width to length ratios characteristic of C.maenas provides the only morphological indication that some crabs mayderive from mixed parentage Carcinus. Based onmitochondrial DNA haplotypes, Geller et al.C. aestuarii and C. maenas hadindependently invaded Japan. In contrast, Bagley and Geller C.maenas and C. aestuarii mitochondrial haplotypes. Thisargument was supported primarily by the observation of low microsatellite diversityin Japan and the inference that multiple introductions from both Atlantic Europe andthe Mediterranean would likely have conferred a much more diverse foundingpopulation. Both studies noted the possibility of hybrid origins for Japanesepopulations, but the absence of direct comparisons of nuclear and mitochondrialdatasets precluded direct tests of that hypothesis. More recently, Darlinget al.Carcinus inrecent assessments of invasion risk in that region In Japan, crabs described as Carcinus population in Japan. Bothmitochondrial COI sequence data and multilocus genotypes based on nine nuclearmicrosatellite loci were generated for 159 crabs collected from Tokyo and DokaiBays. Analysis of these data confirms hybrid origin for the JapaneseCarcinus population, and suggests that hybridization hasresulted in massive introgression of a C. maenas mitochondrialhaplotype into a predominantly C. aestuarii nuclear background. Inlight of the observed patterns of genetic variation in Japan, it seems most likelythat the Japanese invasion derives from a single introduction from a hybridpopulation in the native range. This conclusion, along with genetic characterizationof this previously undescribed hybrid lineage, may have important ramifications forunderstanding the invasion risks posed by Japanese Carcinuspopulations.This study extends on previous work by comprehensively addressing the hypothesis of ahybrid origin for invasive C.maenas and C. aestuarii, consistent with previousstudies  and the distance between invasive haplotypes H1 and H65 was10.1%. H1 previously has been recognized as the single most commonC. maenas haplotype in both the native and invasive rangesof that species; H65, in contrast, has been reported only from Japan . However, H65 does belong to a strongly supported clade (100%posterior probability) comprising haplotypes derived entirely from the easternMediterranean  and not observed in a more western population.Bayesian inference of phylogenetic relationships based on the mitochondrial COIgene clearly indicates the divergence between the two sister species studies .MonophyP<0.0001, Fisher's exact test). In contrast, AMOVAbased on nuclear microsatellite loci revealed no overall genetic differentiationbetween populations in the two Bays  are never distinguished in the analysis from those with the C.aestuarii haplotype (H65), even at values of Khigher than 3. For example, at K = 4population structure is observed within native C. maenas while no structure is observed within Japan despite thepresence of both C. meanas and C. aestuariiCOI haplotypes . Again, coancestry was completely independent of mitochondrial haplotype;mean C. aestuarii coancestry for Japanese individuals withhaplotype H1 was 0.9881, compared to 0.9924 for individuals with haplotype H65.When STRUCTURE analysis was conducted with native ancestry . For allC. maenas and C. aestuarii. Despiteestimates of divergence times between the two species on the order of 5 to 8 millionyears ago based on mitochondrial sequence data C. maenas andC. aestuariiC. maenas have been observed among C.aestuarii populations off the coast of Tunisia in the westernMediterranean basin . These results lend credence to thehypothesis that natural hybrid zones may exist near the mouth of the MediterraneanSea Carcinus population in the native range has yet revealedgenetic evidence of hybridization; even crabs collected from the Palmones estuarywere found in a study separate from that noted above to be unambiguosly C.maenas by genetic criteria A number of studies have previously suggested the possibility of hybridizationbetween C. maenas and C.aestuarii, and several studies have recognized the possibility ofhybrid origin for this population Carcinus population. Phylogeneticreconstructions indicate that invasive Japanese COI haplotypes H1 and H65 derivefrom C. maenas and C. aestuarii, respectively. This would explain both the observedpattern of introgression  as well as the genetic uniformity of Japanesesamples.Alternatively, it may be that the interspecific hybridization event leading tointrogression of Carcinus to Dokai Bay in the 1990's.This secondary invasion event is supported both by historical records It is important to note that while some departure from that genetic uniformity hasbeen observed, these are still consistent with this most likely invasion scenario.The only significant deviation from genetic equilibrium across the Japanesepopulation is seen in mitochondrial haplotype frequency differences between theTokyo and Dokai samples. These may be the result of founder effects associated withthe secondary spread of C. aestuarii origin of the nuclear genomes ofJapanese green crabs, that population remains well differentiated from samplednative sources  and Banyul-sur-Mer, France(C. aestuarii) in the northern Mediterranean, so the mostlikely source regions for the Japanese invasion have not been explored.Additionally, any introgression of the C. maenas nuclear genomeinto the C. aestuarii background could also have drivendifferentiation from the parental types. Unfortunately, without knowledge of thetrue source populations for C. aestuarii and C.maenas parental types, it is very difficult to distinguish betweenthese two hypotheses; in fact, it is likely that some combination of the two has ledto the observed differentiation from native sources. Alternatively, it is alsopossible that the differentiation of Japanese Carcinus from nativesamples has resulted from substantial genetic drift imposed by populationbottlenecks associated with initial introduction and subsequent absence of gene flowwith native sources. This phenomenon has been observed for other invasiveCarcinus populations, but only in cases where time sinceintroduction was greater than 100 years It should also be noted that while genetic analysis is consistent with apredominantly ces e.g. .It is pC. aestuarii COIhaplotype H65 observed in Japan belongs to a well-supported subclade of C.aestuarii  is expected to have a native range extending wellinto the western Mediterranean.Generally speaking, a single introduction from a hybrid source appears to be the mostparsimonious explanation for the Japanese invasion, and is broadly consistent withboth the observed genetic pattens and known invasion history. Only one observationapparently contradicts this scenario. The estuarii , and memCarcinus in Japan and surrounding locales.For instance, a recently developed model of green crab range expansion in the regionsuggests that primary introduction from the native range is likely a rare eventCarcinus, it appears that such admixture may in factpredate the invasion. This raises important questions regarding the possibility ofecologically relevant distinctions between C. maenas, C.aestuarii, and their hybrids. The ecology of C. maenashas been particularly well studied, and organismal and ecological traits likely toaffect range expansion and invasiveness have been incorporated into various riskassessments C.aestuarii, and what is known derives largely from study of Japanesepopulations Carcinus will reflect the ecological characteristics ofeither parent species. For instance, introgression of C. maenasmitochondrial genomes throughout the Japanese population suggests the possibilitythat those populations possess capacity for thermal adaptation significantlydifferent from native C. aestuarii. Invasion of Hokkaido Island,with minimum seawater temperatures apparently more suited to C.maenas than C. aestuarii, therefore may be more likelythan assumed by current risk assessments The results of genetic analyses presented here may prove relevant to assessments offuture risks posed by C. maenas and C. aestuarii, havebeen described previously Live crabs were collected from four sites in Tokyo Bay in 1995 and 1996 and asingle site in Dokai Bay in 1997 . SpecimeC oxidasesubunit I (COI) was conducted as previously described using universal primersLCO1490 (GGTCAACAAATCATAAAGATATTGG) TAAACTTCAGGGTGACCAAAAAATCA) for C. maenas haplotypesand degenerate universal primers COIF-PR115 (TCWACNAAYCAYAARGAYATTGG) andCOIR-PR114 (ACYTCNGGRTGNCCRAARARYCA) for C. aestuarii and HCO2198,Charybdis japonica (#EU586120), and Portunussanguinolentus (#EU284152). Kimura 2-parameter genetic distancesbetween invasive haplotypes and within and between C. maenasand C. aestuarii were calculated in MEGA v. 4 COI sequences were aligned using ClustalX FST) on microsatellitedata with ARLEQUIN v. 3.0 C. maenas vs.C. aestuarii) and tested for partitioning of geneticvariance within and between groups. Significance of difference in thedistribution of the two Carcinus COI haplotypes between Tokyoand Dokai Bays was determined by Fisher's exact test. In addition, geneticrelationships between individual multi-locus genotypes were assessed usingFactorial Correspondence Analysis (FCA) conducted with the software GENETIXv4.05.2 Carcinus populations,pairwise Cavalli Sforza-Edwards chord distances were calculated based onmicrosatellite data using MICROSATELLITE ANALYZER Genotypic data were assessed for departures from Hardy-Weinberg equilibrium (HWE)using Fisher's exact test in GENEPOP v3.4 Carcinus population,Bayesian model-based cluster analysis was implemented using the programSTRUCTURE v.2.2 a priori classification of populations. Known C.maenas and C. aestuarii individuals from thenative range of both species, as well as all Japanese Carcinus,were assigned probabilistically to populations or jointly to multiplepopulations if their genotypes indicated admixture. For this analysis,likelihood of models was assessed with K (the user-definednumber of clusters) ranging between 1 and 5. In addition, the coancestry ofJapanese individuals in parental gene pools defined by native C.maenas and native C. aestuarii was determined byadopting an ancestry model that used prior population information to determineclustering. Specifically, native C. maenas and C.aestuarii individuals were classified as known samples belonging totwo pre-defined parental clusters, and all Japanese individuals were classifiedas of unknown origin. By setting K = 2,this procedure allowed estimation of admixture proportions of known C.maenas and C. aestuarii allelic states in the testpopulation (Japanese Carcinus). For both analyses, fiveindependent runs were conducted, each run consisting of 1,000,000 iterationswith the first 100,000 iterations discarded as burn-in. STRUCTURE results werevisualized using the software DISTRUCT v. 1.1 To assess admixture in the Japanese


Mel-13  and Morchella eohespera , using fragments of 4 DNA sequences, to analyse their genetic structure. Our results indicated significant differentiation among geographic locations in both species, whereas no obvious correlation between genetic and geographic distance was identified in either species. M. eohespera exhibited a predominantly clonal population structure with limited recombination detected in only 1 of the 14 geographic locations. In contrast, relatively frequent recombination was identified in 6 of the 11 geographic locations of Mel-13. Our analysis indicated that the sympatric species Mel-13 and M. eohespera might have divergent evolutionary patterns, with the former showing signatures of recent population expansion and the latter being relatively stable. Interestingly, we found no heterozygosity but strong evidence for genealogical incongruence, indicating a high level of inbreeding and hybridisation among morel species.As highly prized, popular mushrooms, morels are widely distributed in the northern hemisphere, with China as a modern centre of speciation and diversity. Overharvesting of morels has caused concern over how to effectively preserve their biological and genetic diversity. However, little is known about their population biology and life cycle. In this study, we selected two sympatric phylogenetic species,  Morchella rufobrunneaM. importuna3Morels are among the world’s most prized edible fungi, and are collected by both fungal enthusiasts and food specialists due to their unique culinary flavour and rarity. Although cultivation of Morchella might be heterothallic and could outcross in nature11121314Morchella species are still not clearly known, and it is unknown whether inbreeding or clonality exists in the life cycle of morels. Dalgleish and Jacobsonet al.Fungal mating systems play important roles in shaping the genetic structure of fungal populations56789MorchellaMel-13 Morchella sp. proved as the valid phylogenetic speciesM. eohespera) from China using multi-locus sequence data. We aimed to address the following questions. (1) What is the spatial genetic structure, the level of genetic diversity within and among populations and the degree of population differentiation within each of the two sympatric species? (2) Are their evolutionary histories similar to or divergent from each other? (3) What are their potential modes of reproduction? Are their reproductive modes dominated by clonality, recombination or a mixture of both? Our findings will help in understanding the reproductive modes and the distributional patterns of genetic variability in natural populations of morels, which is essential for developing effective strategies for the management and conservation of these two morel species.China is known for its complex geological and ecological diversity. Our recent study identified China as the modern species diversity centre of Mel-13 and 156 samples of M. eohespera). No heterozygosity was observed for any site in the four genetic makers of both species.We successfully obtained all of the sequences from four DNA fragments for 280 samples /total aligned nucleotide sites for B1, F1, F2 and ITS rDNA were 1 (0.16%)/638, 6 (0.85%)/706, 4 (0.59%)/673 and 11 (0.17%)/666, respectively, separating the 124 collections into 2, 8, 4 and 7 sequence types or haplotypes, respectively. For M. eohespera, the observed polymorphic sites (percentage)/total aligned nucleotide sites for B1, F1, F2 and ITS were 5 (0.78%)/638, 8 (1.24%)/644, 10 (1.5%)/670 and 16 (2.3%)/690, respectively, separating the 124 collections into 5, 6, 8 and 15 sequence types or haplotypes, respectively.For Twenty-two variable sites were found among the 2682 aligned nucleotide sites for the combined four-gene dataset, the variable site proportion of which was 0.82%. Of the 29 haplotypes detected in this species , 23 79..d and π, for each population are summarised in d = 0.889) was detected in the XJZS and SXFS populations, with nucleotide diversity (π) of 0.00083 and 0.0015, respectively. The QHZM population had the lowest Hd (0), with only one haplotype (H23).The molecular genetic diversity indices, Hp < 0.001).Nei’s genetic distance between pairwise members of the population ranged from 0.009 (between QHBB and QHZG) to 0.663 (between SCJZ and SCMEK) . The estCT value was highest when K = 9. However, as it made little difference when the 11 populations were defined as 9 groups, we do not define the groups in Mel-13 here.The SAMOVA analysis showed that the Φp = 0.2) and a negative correlation for altitude (p = 0.19), although the correlations were not statistically significant.The AMOVA revealed that 49% of genetic variation was within populations and 51% between populations , and theMel-13 indicated a sudden population expansion, inferred by the near-perfect fit between the observed and the expected mismatch distribution, and supported by non-significant SSD and HRI statistics  were private haplotypes, each found in only one population . H22 wasd and π, for each population are summarised in d = 0.9) and XZRW had the highest number of haplotypes . The XJWLMQ and GSZQ populations had the highest nucleotide diversity  and the lowest Hd (0.417), respectively.The molecular genetic diversity indices, Hp < 0.001).Nei’s genetic distance between pairwise members of the population ranged from 0.001 (between GSDB and GSZQ) to 0.254 (between QHBB and XZRW) . The aveCT value was the highest, corresponding to the XZRW population as one group and the remaining populations as the other. Based on the groups defined by SAMOVA, the AMOVA analysis revealed that 35% of genetic variation was within populations, 7% between populations and 58% between groups (p = 0.25) and altitude (p = 0.52), but they were not statistically significant  were indicative of limited recombination by the PrC values; and the other five  showed no evidence of recombination. Of the fourteen populations in M. eohespera, the PrC value indicated limited recombination only in the XZRW population. Thus, Mel-13 showed more evidence of recombination than M. eohespera.Of the 11 populations within M. importuna, D502 and D546 nested within Mel-13, D518 nested within M. eximioides, D390 and D554 nested within M. eohespera. However, the results based on the F1 gene fragment showed that D129 was nested within M. eximia, D390 and D554 were nested within Mel-13, D165 and D502 were nested within M. eohespera and D518 and D546 were nested within Mel-34  belonged to three species, Mel-13 (n = 10), Mel-14 (n = 7) and M. eohespera (n = 2). Similarly, in the HBES population, 11 samples collected less than 30 m apart were found to represent 3 species, M. eohespera (n = 1), Mel-21 (n = 9) and M. pulchella (n = 1). In the SCSJS population, 9 samples collected less than 50 m apart represented 2 species, Mel-14 (n = 1) and M. eohespera (n = 8). In the SCQC population, 10 samples collected from less than 100 m apart represented two species, Mel-21(n = 8) and M. pulchella (n = 2). Their sympatric distribution affords plenty of opportunities for hybridisation among the lineages.Sympatric distribution was also found to be common in M. eohespera than in Mel-13. Some studies have suggested that increases in genetic polymorphism, mutation and recombination might ensure higher levels of genetic diversity, thus providing greater potential for genetic adaptation in changing environments17181920et al.M. esculenta complex (Esculenta Clade) were haploid due to no heterozygosity found, as similar results were observed in Mel-13 and M. eohespera (Elata Clade). It is presumed that selfing might be very common in these morel species or they were homothallic and their fruiting bodies were developed from haploid mycelia (see the latter discussion).This is the first study conducted on the population biology of two closely related sympatric morels species. Both species showed rich and divergent genetic diversity. The ITS region and the B2 gene fragment, respectively, harboured the most polymorphic sites and the fewest polymorphic sites in both species and all four DNA fragments showed more variation in Mel-13 and M. eohespera. However, no correlation was detected between genetic distance, geographic distance and altitude (232627Mel-13 (79.3%) and M. eohespera (51.5%) indicated potential multiple habitat fragmentation events. Considering that the speciation time of Mel-13 and M. eohespera has been estimated at around 2.855 Mya and 0.443 Mya respectivelyMel-13 and M. eohespera.Significant differentiation was observed among geographic locations within altitude . AlthougMel-13 and M. eohespera have sympatric distributions in southern China, our results indicated that they probably had different evolutionary histories. The mismatch distribution analysis and the median joining network indicated that Mel-13 probably experienced a sudden population bottleneck followed by expansion, while M. eohespera did not. The speciation time for Mel-13 has been estimated at about 2.855 Mya. During its evolutionary history, the climatic oscillations of the Quaternary led to repeated drastic environmental changes (<2.0 Mya)30Mel-13 and stimulated the emergence of very rich private haplotypes (79.3%) and more frequent recombination detected in 54.5% of the Mel-13 population, which may have helped it to survive the extensive environmental changes and habitat fragmentation. Different genotypes may display different fitness in different environments, so the highly localised genetic diversity could have accelerated adaptation to heterogeneous and changing biotic and abiotic environments32Although M. eohespera emerged around 0.443 Mya, during the Pleistocene3435M. eohespera, which partly explains why fewer private haplotypes (51.5%) were found in M. eohespera than in Mel-13, and why limited recombination was identified only in the XZRW population. Our results suggested that the XZRW population was the centre of diversity of M. eohespera in China because this population possessed the most haplotypes (n = 10), including the most broadly distributed haplotype, H22, and the SAMOVA analysis separated it from the other populations.It has been estimated that M. esculenta, M. deliciosa and M. snyderi are heterothallic and could be outcrossed in the laboratory121437M. esculenta and some individuals of M. snyderi were clonal or had selfing as their dominant reproduction mode.The reproduction mode of morels has been debated by various authors. Studies have indicated that collections identified as Mel-13 and the ITS region of M. eohespera, however, no evidence of recombination was found in the other DNA fragments in either species  appeared to be asexual, and five populations  appeared to have limited recombination. Notably, only one population (SXFS) showed evidence of recombination according to both rBarD and PrC values, and this population had the highest haploid diversity and nucleotide diversity. Among the 14 populations in M. eohespera, 13 showed no evidence of recombination, consistent with asexual reproduction. Only the XZRW population showed limited recombination according to the PrC value. Similar to Mel-13, XZRW, the only population where recombination was detected in M. eohespera, also had high haploid and nucleotide diversity. Interestingly, two other populations, XJWLMQ and XZLL, in M. eohespera also had high haploid and nucleotide diversity, but no recombination was identified in them.Our current results identified limited recombination within the four combined loci of the total samples for both species, and within the F1 gene fragment of  species . In Mel-et al.40414243M. eohespera. The results were supported not only by the results of a multilocus linkage disequilibrium analysis, but also by the identification of a few over-represented genotypes (H3 and H22), consistent with clonal expansion. This strategy might facilitate the dispersal of individuals colonising a new habitat, as proposed by Dalgleish and Jacobson4546Mel-13, but recombination events were much more frequent in Mel-13 than in M. eohespera, which probably stimulated the increase in genetic diversity that might have enhanced the adaptation of some populations to hostile environmental and climate changes. The different populations in Mel-13 may have had different reproductive strategies, whereas most populations in M. eohespera possesses clonal reproductive mode, and the levels of genotypic diversity were at least partly associated with the level of recombination.Taylor It is not known how variation in reproductive mode across morel populations is controlled, or whether these distinct populations use sexual and/or asexual reproductive modes to regulate gene flow, select more rapidly adaptive genotypes and respond to heterogeneous environments. Extensive sampling and laboratory mating assays would help to gain a full understanding of the mating behaviour and determine the relative contribution of asexual versus sexual reproductive modes in morels. We cannot reject the possibility that both species are heterothallic, and speculate that the unknown mating system may also facilitate outcrossing considering the high level of genetic variation in the populations of both species.Mel-13 and M. eohespera were heterothallic, the lack of heterozygosity in any of the samples from any of the four DNA fragments would suggest that inbreeding must be highly prevalent. The presence of hybridisation, as suggested by the gene genealogical incongruence, indicates that mating must have occurred in nature. If this were true, the lack of heterozygosity would indicate that inbreeding must have been the predominant mode of sexual reproduction.If the life cycles of Morchella were found to be sympatric in our study, but whether there is reproductive isolation between them and what’s the isolation degree is unknown. Some studies have proposed greater reproductive isolation is involved in sympatric species than allopatric ones48At least five species of Morchella was conducted for the first time in this study. Rich genetic diversity and significant population differentiation were found in Mel-13 and M. eohespera, however, M. eohespera possessed more variation than Mel-13 at the four molecular markers. We conclude that the XZRW population was probably the diversity centre of M. eohespera in China. Prevalent clonality, limited local recombination and potential hybridisation or horizontal gene transfer were detected in the Morchella taxa, but the mating systems of both species remain to be determined. Taken together, our findings suggest that the two sympatric species have a potentially divergent evolutionary history.The population genetics of two sympatric closely related species of Genomic studies have become a research hotspot. Sequencing morel genomes will provide unprecedented insights on fruiting-related genes, the mating system and genes essential for sexual reproduction of morels.M. sexelata, M. eximia, M. exuberans, M. importuna, Mel-13, Mel-14, M. eximioides, M. eohespera, Mel-21, M. pulchella and Mel-34. Based on the results combined with the phylogenetic and biogeographic results in Du et al.Mel-13 and M. eohespera for conducting comparative population genetic studies. Additionally, Mel-13 and M. eohespera were also respectively distributed in India and Europe, we tried to obtain collections from both regions for this study but failed. More effort will be made in the future for a study on the continental disjunction distribution and evolutionary history of morels. Finally, 124 collections of 11 populations representing Mel-13 distributed in 6 provinces  region with high nucleotide variation rates in morels, were chosen to genotype all 280 strains belonging to Mel-13 and M. eohespera from China.The cloned fragments in the range 0.5–1.0 kb were amplified using vector primers and the PCR products were confirmed using agarose gel electrophoresis and then sequenced. We developed PCR primers from 15 sequences chosen from the cloned fragments using the online software Primer3 to directly amplify and sequence the DNA from four isolates: D139 (positive control), D47, D300 and D112 , all of which belonged to 2O. PCRs were conducted in an Applied Biosystems 2720 thermocycler , using the following cycling parameters: 94°C for 3 min, 35 cycles of 94 °C for 1 min, 50 °C (ITS and B2), 49 °C (F1) and 53 °C (F2) for 30 s, 72 °C for 1 min, followed by a final extension of 10 min at 72 °C. Amplicons were electrophoresed in 1.2% agarose in 1 × TAE, stained with GoldView™ , then photographed over an ultraviolet transilluminator. PCR products were purified using a Bioteke DNA Purification Kit . They were used for bidirectional sequencing using the PCR primers with ABI BigDye v3.1  and run on an ABI 3730 DNA analyser. All newly acquired sequences have been deposited as KR073664-KR073911 in GenBank.Amplifications of ITS, B2, F1 and F2 were conducted using ITS4 and ITS5http://www.mbio.ncsu.edu/bioedit/bioedit.html). As no sequence ambiguities or heterozygosity were detected, the genetic profile exhibited by each individual on the basis of four DNA fragments was treated as a haplotype on the assumption that they were haploid fungi.The raw sequence data of each gene fragment was edited in SeqMan  and they were aligned using MUSCLE v3.8.31Mel-13 and M. eohespera. Each variable nucleotide site was treated as a locus and different nucleotides at the same site were viewed as different alleles.To identify the overall population structure and compare potential differences in the structure, in the following analysis we combined the four DNA fragments into one dataset respectively comprising d) were calculated for each population and each species using DNASP v5.10Indices of nucleotide diversity (π) and haplotype diversity (Hhttp://www.fluxus-engineering.com) with the MP criterion applied. The single ambiguity (closed loop) observed in the network was resolved using the frequency criterion and the geographical criterion based on coalescence theory61A median-joining haplotype network was constructed using NETWORK v4.5.1  and rBarD. The null hypothesis for IA was that there were random associations  between alleles at different loci. A p value of < 0.05 indicated that the null hypothesis should be rejected. We standardised the IA value by the number of loci using the rBarD algorithm, because the value of the traditional IA can be influenced by the number of loci analysed. In the second test, the proportion of pairwise loci that were phylogenetically incompatible (PI) was calculated. A lack of phylogenetic incompatibility implies asexual reproduction. The incompatibility ratio (IR), where IR = (number of incompatible pairs of sites in the test dataset)/(number of incompatible pairs of sites in a randomly shuffled dataset), can be used as a test of statistical significanceThe following two tests were conducted to examine evidence of recombination in an individual population and the role of sexual recombination in natural populations. First, we calculated the index of association (IAccession codes: Nucleotide sequences associated with this article are available through GenBank (Accession nos: KR073664-KR073911).How to cite this article: Du, X.-H. et al. High inbreeding, limited recombination and divergent evolutionary patterns between two sympatric morel species in China. Sci. Rep.6, 22434; doi: 10.1038/srep22434 (2016).