Lessonia spicata. We then evaluated whether chimeric wild-type holdfasts show higher genetic diversity than erect axes (stipe and lamina) and explored the impact of this on the traditional estimation of genetic diversity at the population level. We found a high frequency of IGH (60–100%) and chimerism (33.3–86.7%), varying according to the studied population. We evidenced that chimerism occurs mostly in holdfasts, exhibiting heterogeneous tissues, whereas stipes and lamina were more homogeneous, generating a vertical gradient of allele and genotype abundance as well as divergence, constituting the first time “within- plant” genetic patterns have been reported in kelps. This is very different from the chimeric patterns described in land plants and animals. Finally, we evidenced that IGH affected genetic differentiation among populations, showed lower levels of FST index when we compared holdfast than lamina samples. In the light of this, future studies should evaluate the significance of chimeric holdfasts in their ability to increase kelps resilience, improve restoration and ecosystem service.Chimerism occurs when two genetically distinct conspecific individuals fuse together generating a single entity. Coalescence and chimerism in red seaweeds has been positively related to an increase in body size, and the consequent reduction in susceptibility to mortality factors, thus increasing survival, reproductive potential and tolerance to stress in contrast to genetically homogeneous organisms. In addition, they showed that a particular pattern of post-fusion growth maintains higher genetic diversity and chimerism in the holdfast but homogenous axes. In Chilean kelps (brown seaweeds), intraorganismal genetic heterogeneity (IGH) and holdfast coalescence has been described in previous research, but the extent of chimerism in wild populations and the patterns of distribution of the genetically heterogeneous thallus zone have scarcely been studied. Since kelps are under continuous harvesting, with enormous social, ecological and economic importance, natural chimerism can be considered a priceless in-situ reservoir of natural genetic resources and variability. In this study, we therefore examined the frequency of IGH and chimerism in three harvested populations of  Genetic diversity is important for population viability and survival , 2, 3, 4Lessonia spicata. Chimerism occurs when two genetically distinct conspecific individuals fuse together or coalesce, generating a single entity [Intraorganismal genetic heterogeneity (hereinafter IGH) involves the existence of different genomes within a single body , changine entity . It occue entity , graftine entity , coloniae entity , 18, 19)e entity  and macre entity .Gracilaria and Mazzaella, showed a positive correlation of chimerism with survival [Gracilaria and Mazzaella have shown however a variation of the above pattern, since somatic fusion of genetically different conspecific individuals may produce a chimeric holdfast. It follows that upright axes of the fused individuals emerge by proliferation and vertical growth from a single cell lineage [in-situ reservoir of natural genetic resources and variability.Chimerism provides both benefits and costs for organisms . In red survival , 24, grosurvival , 25, repsurvival , and envsurvival . Therefosurvival  while inIn-situ holdfast fusion in Lessonia berteroana has been associated with positive ecological consequences, protecting thalli from benthic herbivore pressures and wave-induced mortality [Lessonia spicata (Suhr) Santelices, L. berteroana Montagne, L. trabeculata Villouta & Santelices, Macrocystis pyrifera (Linnaeus) C. Agardh and Durvillaea antarctica (Chamisso) Hariot, follows a general pattern of cellular changes [L. berteroana  and. Simila evidencIn Chile, kelp is a major raw material for the alginate and biofuels industry, as well as for invertebrate cultures , 33. TheL. spicata. We then measured the location of chimeric tissues in different thallus zones by evaluating whether wild-type holdfast showed mixed tissues with higher genetic diversity than erect axes (stipe and lamina). Finally, we explored the impact of differential genetic diversity in different thallus zones on the traditional estimation of genetic diversity at the population level. This work provides a descriptive framework for understanding genetic diversity at the intraorganismal level in brown macroalgae, representing a new approach to understanding chimerism in natural populations, and increasing genetic knowledge to improve management and conservation programs.In this study, we therefore examined the frequency of IGH and chimerism in three harvested populations of Lessonia spicata  were collected from three populations—assigned as Management and Exploitation Areas for Benthic Resources (MEABR)—used as harvesting areas by small-scale fishermen, with annual harvested weight of up to 7100 dry tons [L. spicata density . In order to evaluate intraorganismal genetic heterogeneity, we subsampled tissues from the holdfasts (H), basal (B) and medial (M) portions of the stipes, and the apical part of the lamina (L); this was repeated five times for each individual   was firsFrom a total of 45 plants, with 20 samples from different thallus zones, we first estimated different numbers of genotypes by plant, considering a difference of at least one allele in their multilocus genotypes to indicate a different genotype. Secondly, we estimated different numbers of genetic clusters by plants obtained from the Bayesian clustering analysis in STRUCTURE . To thisG-test [We estimated the frequency of plants with IGH in natural populations by quantifying the number of plants showing more than one genotype. As we had 20 samples per plant, we considered a plant to be homogenous when all the thallus zones exhibited the same genotype. In contrast, chimerism was estimated by using two different approaches: i) plants that showed different genotypes that diverged in 2 or more different loci. ii) plants that showed more than one genetic cluster , obtaineG-test .E) using the GenAlEx 6.5 software [ST index among populations by using independent samples from different thallus zones . Population FST values were estimated for each locus and across all loci according to [ST) was calculated among all populations using GENETIX v. 4.0 [In order to describe genetic variability at different levels, standard indices of genetic diversity were estimated. Firstly, at the “within-plant” level, we estimated at different thallus zone the total number of different genotypes, the number of genotypes that diverged in 2 or more different loci, and genetic diversity . Stsoftware  and GLM software . Secondlsoftware  analysissoftware  considerrding to ; pairwisX v. 4.0  and signL. spicata without missing data and registered only a maximum of two alleles per locus, suggesting that all samples was genetically uniform. However, we eliminated the data from microsatellite loci LESS2D1 because the MICRO-CHECKER evidenced null alleles, and from loci LESS2D6 because alleles 146 and 148 were never observed as homozygous, suggesting possible electrophoresis artifacts.All microsatellites were successfully used for genotyping the kelp 2,42 = 10.1; P<0.001), with La Puntilla having the lowest number of genotypes per plant (mean 1.9 ± 0.8) compared to Pichicuy (mean 3.6 ± 1.4) and Maitencillo (mean 6.4 ± 4.8). In the same way, significant differences among populations  were found in terms of the number of genotypes that differed in more than 2 loci , but La Puntilla having the lowest number of genetic cluster (mean 1.0 ± 0.2) compared to Pichicuy (mean 1.3 ± 0.4) and Maitencillo (mean 1.5 ± 0.5). We found that two genetic cluster occurred only in chimeric plants that showed genotypes with 2 to 5 different loci, which in most of cases was a higher threshold than the other approach used in this study.In terms of the numbers of genetic clusters per plant obtained from the Bayesian clustering analysis, we found only two genetic clusters (K = 2) in each studied populations . The numP<0.001, 2,42 = 6.48; P<0.001, The relatedness analysis evidenced significant differences between the three levels , all sampled plants (100%) exhibiting more than one genotype per plant in Pichicuy, 93.3% in Maitencillo, but only 60% (9/15 plants) were found in the La Puntilla population.Considering the number of different genotypes by plant, populations of P <0.001). The La Puntilla population had the lowest chimerism frequency (33.3%), followed by Maitencillo (66.7%) and Pichicuy ; however, the frequencies of chimerism were lower suggesting a conservative method to estimate chimerism compared to the first approach used in our study. Then La Puntilla population had lowest chimeric frequency (6.7%) followed by Pichicuy (26.7) and Maitencillo , we also found significant differences between the three studied populations . A similar tendency of vertical genetic variation “within plant”, though insignificant, was observed in terms of the total number of genotypes  compared to the “among-plant-within-population”  and “among-population”  levels.On the other hand, the three-level AMOVA analysis  suggestsST ranged from 0.14–0.28, independent of each thallus zone analyzed  were consistent with the pattern described above. The first component showed a clear separation of the population in the La Puntilla population, whereas the second component underscored the difference between the Pichicuy and Maitencillo populations , Fig 5. L. spicata, show a high frequency of chimerism and therefore of individuals with high IGH. The frequency of chimerism was different in each of the two approaches used in this study. Using the first approach (genotypes differences in 2 or more loci per plant), chimerism ranged from 33.3 to 86.7% of the sampled plants, varying according to the studied population. However, considering more than 2 different genetic clusters per plant, chimeric estimation was more conservative, ranging from 6.7 to 53.3% of the sampled plants. In addition, we detected a vertical gradient (from holdfast to lamina) of genetic diversity in chimeric plants, with higher numbers of genotypes, allele divergence and unrelated samples in the holdfasts, which decreased towards the apical portions of the axes (stipes and lamina). Finally, we evidenced that IGH affected genetic differentiation among populations, showed lower levels of FST index when we compared holdfast than lamina samples.Results found in the three populations of Lessonia spicata  and La Puntilla (6.7–33%). Most of these frequency values fall within the range of values found in the only other study on chimerism frequency for natural populations of this species in Chile . Howeve. This hypothetical role adds to the known ecological role of holdfasts in larval settlement and in providing an area of refuge for invertebrates [Data from different thallus zones evidenced that chimerism occurs mostly in holdfasts, showing genetically heterogeneous tissues at this thallus levels, whereas stipes and lamina were more genetically homogeneous, generating a vertical gradient of allele, genotype abundance and divergence. These findings constitute the first report of vertical genetic “within- plant” variation in brown macroalgae and a chimeric pattern similar to the patterns recently described in red algae, but very different from the chimerism described for land plants and animals . On the tebrates .Traditionally, studies of genetic diversity have considered the individual, population and regional levels. However, our data showed that the intraorganismal level is another key factor that should be considered. In this way, since kelps are ecosystem bioengineers, and their exploitation along the Chilean coastline has an enormous ecological and socio-economic cost , 58, 59,S1 TableL. spicata. H: Holdfast, B: Basal stipe, M: Medial stipe. L: Lamina.Data of allelic and genotype composition of different samples from the three studied populations of (DOCX)Click here for additional data file.S2 TableL. spicata. PIC: Pichicuy, MT: Maitencillo, LP: La Puntilla, QGM = Queller and Goodnight (1989) estimator mean.Pairwise relatedness summary of different samples at Holdfast, Basal stipe, Medial stipe and Lamina from the three studied populations of (XLSX)Click here for additional data file.S1 DatasetL. spicata. N: Sample size, Na: Number of alleles, Ne: Number of effective alleles, I: Information Index, Ho: Observed heterozygosity, He: Expected heterozygosity, uHe: Unbiased expected heterozygosity, and F: Fixation index.S3 Table. Raw data formatted to estimate genetic diversity at different thallus zone. PIC: Pichicuy, MT: Maitencillo, LP: La Puntilla. S4 Table Heterozygosity and F-statistics by thallus zone of different samples from the three studied populations of (XLSX)Click here for additional data file.S2 DatasetST values among populations using holdfast data. S7 Table. Genetic matrix using holdfast data. S1 Text. Principal Coordinates Analysis (PCoA) using holdfast data. Percentage of variation explained by the first 3 axes. S1 Fig. Principal Coordinates (PCoA) using holdfast data from Pichicuy (Pop1), Maitencillo (Pop2), La Puntilla (Pop3). S8 Table. Eigen values by axis and sample eigen vectors using holdfast data. S9 Table. Raw data formatted to estimate genetic distances using basal samples. S10 Table. Pairwise FST values among populations using basal data. S11 Table. Genetic matrix using basal data. S2 Text. Principal Coordinates Analysis (PCoA) using basal data. Percentage of variation explained by the first 3 axes. S2 Fig. Principal Coordinates (PCoA) using basal data from Pichicuy (Pop1), Maitencillo (Pop2), La Puntilla (Pop3). S12 Table. Eigen values by axis and sample eigen vectors using basal data. S13 Table. Raw data formatted to estimate genetic distances using medial samples. S14 Table. Pairwise FST values among populations using medial data. S15 Table. Genetic matrix using medial data. S3 Text. Principal Coordinates Analysis (PCoA) using medial data. Percentage of variation explained by the first 3 axes. S13 Fig. Principal Coordinates (PCoA) using medial data from Pichicuy (Pop1), Maitencillo (Pop2), La Puntilla (Pop3). S16 Table. Eigen values by axis and sample eigen vectors using medial data. S17 Table. Raw data formatted to estimate genetic distances using lamina samples. S18 Table. Pairwise FST values among populations using lamina data. S19 Table. Genetic matrix using lamina data. S4 Text. Principal Coordinates Analysis (PCoA) using lamina data. Percentage of variation explained by the first 3 axes. S4 Fig. Principal Coordinates (PCoA) using lamina data from Pichicuy (Pop1), Maitencillo (Pop2), La Puntilla (Pop3). S20 Table. Eigen values by axis and sample eigen vectors using lamina data.S5 Table. Raw data formatted to estimate genetic distances using holdfast samples. S6 Table. Pairwise F(XLSX)Click here for additional data file.


Schistosoma japonicum, S. haematobium and S. mansoni as determined through microsatellites could pave the way for a better understanding of the transmission epidemiology of the parasite. Thus, the present study aims to apply microsatellite markers in analyzing the populations of S. japonicum from different endemic areas in the Philippines for possible strain differentiation.Microsatellites have been found to be useful in determining genetic diversities of various medically-important parasites which can be used as basis for an effective disease management and control program. In Asia and Africa, the identification of different geographical strains of S. japonicum collected from infected Oncomelania hupensis quadrasi snails in seven endemic municipalities. Adult worms were harvested from infected mice after 45 days of infection and their DNA analyzed against ten previously characterized microsatellite loci. High genetic diversity was observed in areas with high endemicity. The degree of genetic differentiation of the parasite population between endemic areas varies. Geographical separation was considered as one of the factors accounting for the observed difference between populations. Two subgroups have been observed in one of the study sites, suggesting that co-infection with several genotypes of the parasite might be present in the population. Clustering analysis showed no particular spatial structuring between parasite populations from different endemic areas. This result could possibly suggest varying degrees of effects of the ongoing control programs and the existing gene flow in the populations, which might be attributed to migration and active movement of infected hosts from one endemic area to another.Experimental mice were infected using the cercariae of Based on the results of the study, it is reasonable to conclude that genetic diversity could be one possible criterion to assess the infection status in highly endemic areas. Genetic surveillance using microsatellites is therefore important to predict the ongoing gene flow and degree of genetic diversity, which indirectly reflects the success of the control program in schistosomiasis-endemic areas. S. japonicum is the causative agent of the disease. The prevalence of the disease varies in endemic areas, suggesting that the parasite populations might differ in their genetic composition and infectivity to the human host. In this study, DNA samples of adult worms from seven endemic municipalities were analyzed. Characterization of S. japonicum samples in different endemic sites with varying prevalence provides information on the genetic diversity of the parasite. Results of this study showed that samples in high prevalence endemic areas like Irosin, Catarman and Socorro were genetically diverse as compared to other areas. Information on parasite genetic diversity is therefore important in planning disease control strategies. The results suggest ongoing parasite transmission across geographic endemic areas which should be monitored and used as reference for genetic diversity of the schistosomes attributed to geographic areas, thus a safeguarding precaution should be implemented to ensure localized elimination of the disease.Schistosomiasis is one of the important neglected tropical diseases endemic in 78 countries throughout the world. The disease is caused by the parasitic worms known as schistosomes. In the Philippines,  Schistosoma haematobium, S. mansoni, S. mekongi, S. intercalatum, and S. japonicum. Among these species, S. japonicum is considered the most virulent because of the larger number of eggs it can produce as compared to other species, causing severe disease pathology. In addition, the zoonotic nature of S. japonicum contributes to increased disease transmission, making schistosomiasis control more difficult [Schistosomiasis is one of the most important neglected tropical diseases affecting almost 240 million people throughout the world with more than 700 million considered to be at risk of infection . Five spifficult .S. japonicum genetic diversity and in estimating the levels of gene flow in the population. Previous studies have recommended the use of microsatellite markers to determine schistosome genetic diversity because of their codominant expression and their ability to serve as neutral markers [S. japonicum populations provides an opportunity to link some genotypes associated with disease prevalence, which can then be used in formulating effective control measures. Previous studies using microsatellite markers suggested that the prevalence of S. haematobium and S. mansoni infections could be closely related with the parasite’s genetic variation. High prevalence of infection was observed in those areas with high genetic diversity and low prevalence in those areas with low genetic diversity [Microsatellite markers have recently been used in determining  markers , 5. The  markers . Studyiniversity , 8. ThisS. japonicum in China [S. japonicum in the Philippines that can be used as basis to evaluate and modify the widely used current control strategies for human schistosomiasis. For example, cognizant of the genetic types of the parasites, interventions can be modified so as to address differences in disease prevalence.In addition, microsatellite markers had been used in the identification of different geographical strains of in China . Previouin China . In the in China . We hypoS. japonicum.Animal experiments done in this study were conducted according to ethical guidelines for the use of animal samples permitted by the University of the Philippines Manila Institutional Animal Care and Use Committee (2011–009), as well as by Obihiro University of Agriculture and Veterinary Medicine (Permit No. 28–30). Infected mice were anesthesized using isoflurane before they were sacrificed. Perfusion method with normal saline solution was done to collect the adult O. h. quadrasi, were collected from seven municipalities (in seven provinces) in 2013 to 2015 where the disease is endemic, namely Catarman (in Northern Samar), Gonzaga (in Cagayan Province), New Corella (in Davao del Norte), Irosin (in Sorsogon), Talibon (in Bohol), Alang-Alang (in Leyte) and Socorro   . The snaGenomic DNA was extracted from individual male and female adult worms using the DNeasy Blood & Tissue Kit  following the manufacturers’ protocol. 2, 0.2 μl of 2.5 mM dNTP, 0.2 μl each of 10 pmol/μl primer, 0.1 μl of 5 U/μl Taq DNA polymerase , and 1 μl of template DNA. The conditions for thermal cycling were as follows: 5 minutes at 94°C, followed by 30 cycles of 1 minute at 94°C, 1 minute at locus-specific temperature, 1 minute at 72°C, with a final extension at 72°C for 10 minutes [PCR amplifications were performed using Veriti 96-well Thermal Cycler . Amplifications were performed in 10 μl reactions containing 1 μl of 10X PCR buffer, 0.4 μl of 1.5 mM MgCl minutes .S. japonicum worm was genotyped using the previously characterized microsatellite loci RRPS, M5A, TS2, MPA, 2AAA, J5, SJP1, SJP5, SJP6, and SJP9 [S. japonicum sample from Gonzaga, Cagayan, which has good DNA volume and concentration, served as the reference genotype for which the microsatellite sizes for the 10 loci had been determined by sequencing. S. japonicum Yamanashi strain (Japanese isolate) was also genotyped as a control group to confirm that the microsatellite markers could differentiate between samples from different origin. A total of 201 DNA samples were tested; however, only 186 were successfully genotyped due to poor DNA quality.The DNA of each individual and SJP9 , 12. In IS), which measures the extent of nonrandom mating, was computed in the study. Nonrandom mating occurs when there is inbreeding.For each population, the genetic diversity was examined by calculating the number of alleles using rarefaction analysis. Expected heterozygosity (gene diversity) (He) and observed heterozygosity (Ho) were determined using the GenAlEx 6.5 software . RarefacS. japonicum population based on their genetic distance using GenAlEx 6.5. Cercariae derived from a snail infected with only a single miracidium is assumed to be genetically identical. Hence, duplicate multi locus genotypes in a population are a consequence of clonal replication within snails [S. japonicum population [Principal Coordinate Analysis (PCoA) was done to determine the clustering pattern of n snails , 15. Thepulation .ST genetic distance using 100 bootstrap replications in POPTREE2 [ST is one of the well-known parameters used in measuring genetic differentiation between populations using microsatellite data [S. japonicum Yamanashi strain was used as an outgroup.To visualize relationships among populations, a Neighbor-joining tree was constructed based on FPOPTREE2 . The FSTite data . The S. Sequences of microsatellite loci reported here have been deposited in GenBank with the following accession numbers, RRPS (U22167), M5A (AF244896), TS2 (AF244896), MPA (U11895), 2AAA (M32280), J5 (M26212), SJP1 (EU262604), SJP5 (EU262608), SJP6 (EU262609) and SJP9 (EU262612).S. japonicum worms collected from seven endemic municipalities were analyzed. Highest gene diversity indices (He) was observed in Catarman (0.727) followed by Irosin (0.694), Socorro (0.677), and Gonzaga (0.605) while the lowest was in Alang-Alang (0.495). Those from New Corella (0.587) and Talibon (0.566) were comparable. Similarly, allelic richness after sample size correction was highest in Irosin (4.630) followed by Catarman (4.500) and Socorro (4.280) and lowest in Alang-Alang (2.570) followed by Talibon (2.920)  .IS) was observed in Irosin (0.239) while the lowest was in Catarman (0.012)  . The lowntiation . Great gntiation .S. japonicum populations  in the Philippines using S. japonicum larval stages, found high levels of parasite gene flow between humans and dogs suggesting strongly the frequent transmission of S. japonicum infection across host species and between villages [S. japonicum from cattle showed high genetic diversity in the marshland areas, whereas parasites from humans and dogs were more diverse in the hilly region [S. japonicum infecting animals should be seriously considered in the increased disease transmission [S. japonicum cercariae shed from infected snails collected from endemic areas will also be analyzed in our future studies. Because there are several studies on parasite’s population genetics by using cercariae-derived samples [Among the seven endemic municipalities analyzed, the Catarman samples showed the highest gene diversity indices . Catarmavillages . Furthery region , 25. Thesmission , 27, 28.smission , 30. The samples , 24, 25,The level of genetic differentiation differs between endemic areas. Great genetic differentiations were observed in the New Corella samples than those from other endemic sites . The larS. japonicum, and suggested that examination of naturally infected snails may exhibit co-evolutionary relationships with their parasites [Oncomelania populations are based on mitochondrial markers. Hence, future studies using microsatellites on the snail populations from each endemic area is essential to obtain results which can be analyzed together with those of S. japonicum population.Furthermore, the possibility of the snail hosts influencing the genetic variation of the parasite population should also be taken into consideration. Presently, there is no study using microsatellites on the genetic variation of the snail population in the sampling areas. However, previous studies on mitochondrial DNA had provided some insights into the genetic variation of the snail population of arasites . Thus, aarasites , 34. NevS. japonicum population (91.95%) than the variation among the populations (8.05%) (S. japonicum population in endemic provinces in China [The genetic variation observed using AMOVA was greater within each  8.05%) . This mi% . This pulation 1.95% thaS. japonicum samples obtained from different endemic areas did not form a particular spatial structuring. The lack of geographical structuring suggests that there is still an ongoing gene flow among the S. japonicum populations in all the study areas despite execution of control measures [S. japonicum across geographic areas, and therefore reflect the inadequate effect of MDA implementation. The current national control strategy for schistosomiasis in the Philippines is annual MDA using 40 mg/kg of praziquantel in all schistosomiasis-endemic villages including the sampling areas. However, the compliance rate was reported to be <50% [measures . These f be <50% , 36.S. mansoni [The ongoing gene flow in the populations might be attributed to migration and movement of infected hosts as also suggested otherwise by previous studies done on  mansoni . The inf mansoni , 22.Two subgroups were observed in the Catarman samples using the PCoA analysis , indicatst century [Some parasite populations in Gonzaga, New Corella and Irosin were identified as migrants using Gene Class 2.0 software . Gonzaga century , and it  century . InteresS. japonicum inside the host should be identified and need to be further studied.Genetic diversity found in this study among the parasite population in each endemic site in the Philippines is vital in the parasites’ ability to survive the effect of selective pressures such as those brought by drug treatment. At the same time, selection pressures increase the frequency of favorable alleles across all populations . In thisS. japonicum population from different endemic areas, indicating the active movement of infected humans and animals from one endemic area to another. Aside from the control programs being implemented in each endemic area, an effective surveillance to monitoring these movements in humans and animals in each endemic site should be in place. Thus, a better cooperation between the medical and veterinary sectors would be highly recommended to ensure a strengthened control program for schistosomiasis. In addition, the diversity will indirectly explain the varying degree of the effects of the ongoing control programs done in these endemic areas. A regular MDA should be implemented and monitored regularly for its efficacy in endemic areas. Considering that only 10 microsatellite markers were analyzed in this study for determining genetic diversity and gene flow of the parasite, we therefore recommend the use of additional highly polymorphic microsatellite markers not only for S. japonicum, but for S. mansoni and S. haematobium to be used in future studies for more precise analysis.In conclusion, the use of microsatellites in this study has shown that there is an ongoing gene flow among the S1 Table(DOC)Click here for additional data file.S1 FigST genetic distance of 0.05.Values on nodes are percentage bootstrap supports based on 100 bootstrap samples. Scale bar represents F(TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Saccharina latissima, a northern hemisphere kelp inhabiting temperate to arctic rocky shores. To protect and document its natural genetic variation at the onset of this novel aquaculture, as well as increase knowledge on its taxonomy and phylogeography, we collected new genetic data, both nuclear and mitochondrial, and combined it with previous knowledge to estimate genetic connectivity and infer colonization history. Isolation‐with‐migration coalescent analyses demonstrate that gene flow among the sampled locations is virtually nonexistent. An updated scenario for the origin and colonization history of S. latissima is developed as follows: We propose that the species (or species complex) originated in the northwest Pacific, crossed to the northeast Pacific in the Miocene, and then crossed the Bering Strait after its opening ~5.5 Ma into the Arctic and northeast Atlantic. It subsequently crossed the Atlantic from east to west. During the Pleistocene, it was compressed in the south with evidence for northern refugia in Europe. Postglacial recolonization led to secondary contact in the Canadian Arctic. Saccharina cichorioides is shown to probably belong to the S. latissima species complex and to derive from ancestral populations in the Asian North Pacific. Our novel approach of comparing inferred gene flow based on coalescent analysis versus Wright's island model suggests that equilibrium levels of differentiation have not yet been reached in Europe and, hence, that genetic differentiation is expected to increase further if populations are left undisturbed.Kelp aquaculture is globally developing steadily as human food source, along with other applications. One of the newer crop species is  This species has been introduced for farming throughout the world, has escaped to the wild in numerous places, and interferes with local species  C.E. Lane, C. Mayes, L.D. Druehl & G.W. Saunders  and passive dispersal by meiospores. Pneumatocysts are gas‐filled bladders that assist in keeping sporophyte blades near the sea surface for photosynthesis, while other species float well without pneumatocysts. Drifting may result in the capacity for long‐distance dispersal Thomas, , but stuFST in terms of dispersal in the hypothetical case of “islands” of equal population sizes and equal migration rates for all possible island pairs. However, it is not the unrealistic landscape model which is the main problem for inferring genetic connectivity in many species, but rather the assumption that equilibrium FST has been reached. The latter assumption is particularly unrealistic for many marine taxa which are often characterized by (extremely) large populations   and genotyped novel EST (expressed sequence tag)‐derived microsatellites. For both phylogeographic inference and genetic connectivity estimation, we took a coalescent approach to simultaneously estimate migration and timing of differentiation, and to allow for the possibility that equilibrium between isolation and migration may not yet have been reached  were obtained from eight locations along northwestern European coasts (Table Samples for 2.2c‐oxidase I (COI)  based on an alignment of COI for kelp species sourced from Genbank. The PCR mixture consisting of 1 μl of DNA template (1:10 diluted), 0.2 mmol/L dNTP, 1× Taq buffer, 0.5 μmol/L of each primer, 1 μl Bovine Serum Albumine , and 1.25 unit Taq polymerase  in a total volume of 50 μl was run on a BioRad T100 thermocycler  with initial denaturation at 94°C for 4 min, 35 cycles at 94°C for 30 s, 40°C annealing for 30 s, extension at 72°C for 40 s, and final extension at 72°C for 10 min. A 623 basepair portion of COI was successfully amplified for a total of 191 individual kelp samples and sequenced at Baseclear B.V. . Sequence data were edited and aligned using BioEdit v7.2.5  were carried out in Arlequin 3.5.1.2 , nor the Arctic data from Churchill, Manitoba, because that area is a secondary contact zone for S. latissima between NEP and NWA : among the Faroe Islands , Brittany , and northwest Atlantic . The noHaslea ostrearia (Genbank accession number HE995416) and Skeletonema ardeus (KM202114), which share a common ancestor less than 65–87 million years ago , we find a p‐distance of 0.04633 and a divergence rate of 0.9572% per million years. This amounts to a maximum substitution rate of 2.98 × 10−6 per year per 623 bp locus. We used the average between this minimum and maximum molecular clock rate as well as the range in the IMa2 simulations. For the microsatellites, we used a mutation rate of 0.001564, which is the average across all multicellular taxa for which multiple loci were studied as reported in Brohede . These haplotypes (a–g) were closely related, as can be seen in the minimum spanning network Figure . Despitend Ayala . After cnd Ayala . Variabind Ayala .3.2FST = 0.268 (p < .001)). This can also be seen visually from principal coordinates analysis of the microsatellite data , as well as from Brittany to the Wadden Sea (2Nem = 1.214).At the smaller intra‐European scale, and based on microsatellite data, estimated population subdivision times were much smaller and in the order of decades to a few hundred years Table B. The de3.5r = .5129; p = .017424) between connectivity estimates assuming equilibrium and those not assuming equilibrium ; the other does not make this assumption . A significant correlation was found  to reach half its equilibrium value theoretically equals ln2/(2m + 1/(2Ne)), where m equals migration rate and Ne effective population size. In other words, the more migration, the faster is the approach to equilibrium, and the larger the population is, the slower that approach should be. Faurby and Barber  colonized areas.The pairwise migration estimates not assuming equilibrium differentiation are all well below the line of unity in Figure d Barber  demonstrS. latissima has not yet reached its maximum in Europe is further emphasized by the fact that COI is not differentiated in northern Europe, while this is the case for the microsatellite DNA markers studied. This may seem counter‐intuitive, because mitochondrial haplotypes are theoretically expected to coalesce faster than nuclear alleles  and ITS  sequences. They suggest that an initial circumpolar distribution of the species was compressed to the south during early glaciation episodes leading to isolation. From this state of isolation, recolonization of the northeast Pacific and the northwest Atlantic would have occurred postglacially from a European source. Finally, contact is thought to have reestablished and to have resulted in hybridization in the Canadian Arctic and perhaps also the northwest Atlantic  are found to be genetically closest to those S. latissima that are at present found in the northeast Pacific  and congruent with knowledge on postglacial recolonization of European Atlantic coasts  and in the additional files (microsatellite markers tested and microsatellite data). Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Swiftia simplex collected from Eastern Pacific waters along the west coast of the United States. We utilized high-throughput restriction-site associated DNA (RAD)-tag sequencing to develop the first molecular genetic resource for the deep-sea octocoral, Swiftia simplex. Using this technique we discovered thousands of putative genome-wide SNPs in this species, and after quality control, successfully genotyped 1,145 SNPs across individuals sampled from California to Washington. These SNPs were used to assess putative population structure across the region. A STRUCTURE analysis as well as a principal coordinates analysis both failed to detect any population differentiation across all geographic areas in these collections. Additionally, after assigning individuals to putative population groups geographically, no significant FST values could be detected (FST for the full data set 0.0056), and no significant isolation by distance could be detected (p = 0.999). Taken together, these results indicate a high degree of connectivity and potential panmixia in S. simplex along this portion of the continental shelf.Deep-sea corals are a critical component of habitat in the deep-sea, existing as regional hotspots for biodiversity, and are associated with increased assemblages of fish, including commercially important species. Because sampling these species is so difficult, little is known about the connectivity and life history of deep-sea octocoral populations. This study evaluates the genetic connectivity among 23 individuals of the deep-sea octocoral  Deep-sea coral communities are increasingly acknowledged as critical components of deep-sea ecosystems. They are recognized as regional hotspots for biodiversity, and appear to be associated with increased assemblages of fish, including commercially important species . These cLife history variation can have a large effect on the distribution of corals, and their ability to recover from both natural and anthropogenic disturbances –13. CoraSwiftia simplex, is found in the deep-sea , and initially assigned to the genus Psammogorgia based on colony morphology. While this nomenclature is still found in some literature and databases  [Psammogorgia as currently defined, and recent publications place the group in the genus Swiftia [S. simplex is gonochoric, and recent evidence suggests it is a broadcast spawner, as no brooding or larvae have been observed in any of the samples examined [S. simplex females had the highest fecundity, calculated as the count of previtellogenic and vitellogenic oocytes within the imaged polyps, of any of the eight octocoral species analyzed.The plexaurid gorgonian, ow 100 m ) across ow 100 m –20. S. s (WORMS) ), the sc Swiftia , 22. S. examined . Additioexamined  found S.Given the complexities involved in tracking planktonic larvae, indirect methods for measuring connectivity, including both hydrodynamic modeling and genetics, currently provide the best predictions of dispersal and connectivity . To dateSwiftia simplex through RAD-tag sequencing, and to apply the SNPs developed in this process to define the population structure and connectivity of this species across a portion of the United States West Coast.The advent of high-throughput genotyping-by-sequencing techniques, including restriction-site associated DNA (RAD)-tag sequencing, has greatly expanded the speed and availability of genome-wide marker development in non-model organisms . RAD-tagS. simplex individuals included in this study came from three sources: the NOAA West Coast Upper Continental Slope Groundfish Trawl Survey (RACE), the NOAA West Coast Groundfish Bottom Trawl Survey (WCGBTS), and seamount surveys carried out by the Monterey Bay Aquarium Research Institute (MBARI). The specimens provided by the trawl surveys were collected as a regular part of NOAA’s annual WGCBTS program (RACE was a previous iteration of this survey). The permits for these collections were issued by the California Department of Fish and Game, the National Marine Sanctuaries, NOAA, Oregon State, and U.S. Fish and Wildlife. Detailed descriptions of the two trawl survey methods and goals can be found in Keller et al. [n = 29) individuals spanning the United States West Coast .Swiftia simplex and to examine regional variation in mitochondrial haplotype, the mitochondrial sequences for the regions MutS and igr+COI were Sanger sequenced [MutS+igr+COI) whenever possible. Amplicons that were successfully sequenced were compared to a collection of sequences from voucher specimens identified by morphological taxonomists using phylogenetic analyses to confirm species identification  were ligated to each individual  were identified and removed from further analysis. The remaining twenty-three individuals were run through the remaining Stacks pipeline: ustacks, cstacks, sstacks and populations. To choose optimal parameters, ustacks and cstacks were run multiple times, varying the values of m, the minimum number of reads to create a stack, from 2 to 6, and n, the number of mismatches allowed between stacks when creating the catalog, from 0 to 3. Results of these runs were compared and final parameters were selected. In all ustacks runs the–r and–D options were used to limit highly repetitive stacks. Ultimately, ustacks was run with m = 5 and a catalog was constructed from this output in cstacks with n = 2. As a limited number of individuals were included in this study, all individuals were included in the catalog. Next, ustacks was run again, with m = 3, in order to improve genotyping accuracy in low coverage individuals. This output was compared to the catalog using sstacks. Finally, genotypes were called using populations with m, the minimum stack depth to include a locus equal to 7, and grouped individuals into four putative populations based on sample location . RAD-tag library preparation was subsequently carried out on the remaining twenty-six individuals, following the methods described in Baird et al. . Brieflylocation , Table 1All filtering of SNP loci was carried out using vcftools v. 1.12b. PGDSpider (v. 2.0.5.2)  was usedST values and significance tests between all putative populations were carried out in Arlequin (v. 3.5) with 10,000 permutations and a significance level of p<0.05. An AMOVA was also carried out in Arlequin to examine the variance within and between populations, and significance was calculated via a 10,000 permutation test, p<0.05. GenAlEx (v. 6.205) [ST/ (1-FST)) using the Mantel test carried out in adegenet with 1000 randomizations.Initial analysis of putative population structure was carried out in STRUCTURE (v. 2.3.4). K values were varied from 1–6, using a burn in of 500,000 replicates followed by an additional 500,000 MCMC repeats. Each K value was tested 10 times. An admixture model was selected, using the putative population groupings based on region as a prior. STRUCTURE output was examined to determine optimal K values using Structure Harvester , and whe. 6.205)  was usedMutS+igr+COI mitochondrial sequence was obtained for thirteen of the twenty-nine individuals. Ongoing phylogenetic work carried out by our laboratory has indicated that species assignment in S. simplex is successful with MutS alone (data not shown), thus in the remaining sixteen individuals only the octocoral specific MutS gene was amplified. Successful sequence data was deposited in GenBank, see igr+COI were identified. Pairwise genetic distances among the MutS haplotypes were less than 1% and, following the precedent described in McFadden et al. [Mitochondrial amplicon sequence was successfully obtained for all twenty-nine individuals to check species identification. The full concatenated n et al. , we consTwenty-six individuals yielded sufficient DNA to be included in the RAD-tag library construction. RAD-tag sequences were deposited in NCBI’s Short Read Archive (SRA) (Accession number SRR4296452). After quality filtering and demultiplexing the resulting RAD-tag sequences, three individuals had extremely low sequence counts  and were excluded from all further analysis. An average of 355,393 reads was obtained for each of the remaining twenty-three individuals, though there was considerable variation in reads per individual , likely caused by the range of DNA qualities observed on the agarose gel. Lower quality DNA can result in a loss of raw reads due to low sequence quality scores  S2 Tabl. After rAt this point two subsets of data were created see  and eachInitial tests for population structure were carried out in STRUCTURE. To reduce linkage in the data, only the first locus in any tag containing more than one SNP was included, so the initial tests were run on 786 of the 1,132 (n = 23) and 1,145 of the 1,726 (n = 20) SNPs genotyped. In the data subset containing all twenty-three individuals the STRUCTURE results indicated the highest average likelihood with the lowest variance was found for K = 1 across all 23 individuals. In this situation Evanno’s test for detecting the number of clusters of individuals is not applicable . To testth-7th highest missing marker counts in the full data set . Pairwise FST values ranged from -0.0016 to 0.0123, and none were statistically significant (p = 0.1862 to p = 0.6828) (ST and geographic distance (p = 0.099).As removing the three lowest individuals did not have any apparent effect on possible population structure, the population statistics were calculated on the full twenty-three individual dataset. The overall F 0.6828) . The res 0.6828) . Observe 0.6828) . The resSwiftia simplex. We successfully identified and genotyped more than 1,000 SNP loci in twenty-three S. simplex individuals. Within the limited number of samples examined, we found no genetic structure amongst geographic groups spanning the entire United States West Coast region, indicating the possibility of high connectivity and possible panmixia among all regions.This study represents the first genomic sequence resource and the first population level study in S. simplex, and applied 786 of these to our population analyses. We tested the possible trade-offs between limiting the number of missing genotypes in the sample and maximizing the numbers of individuals analyzed, including between 228  and 1,145  SNPs to obtain our results. In all cases we detected no discernable population structure, indicating the likelihood of high gene flow along the U.S. West Coast; however, we cannot rule out the possibility that including samples from a larger geographic area such as S. simplex individuals from Alaska, would reveal genetic structure over a wider geographic area.The application of large numbers of SNP markers has enhanced the ability to detect previously cryptic fine-scale population structure in a number of species –33. Our Carassius carassius, the authors demonstrated that RAD-tag markers were able to detect finer structure among populations than microsatellites , despite including only 17.6% of the individuals genotyped with microsatellites  [ST values can be obtained with sample sizes as low as 4–6 individuals when numbers of sufficient bi-allelic markers are included. Subsequently, the use of large numbers of SNP markers has been successfully applied to detecting population structure in biological data obtained from small samples sizes. For example, a study in seahorses successfully detected population structure among twenty-three individuals from across the United States East Coast [Given the difficulty of sampling deep-sea corals, the need for large numbers of individuals can be a barrier to population genetics analyses. The current study included relatively few individuals (n = 23) compared to many traditional population genetics studies; however, recent studies have shown that larger sets of markers can overcome low sample sizes in population studies. In Cructh SNPs) . The usest Coast . The abist Coast .Paragorgia arborea from various museum and laboratory collections in their study of global haplotype diversity. Our study included bycatch samples inadvertently caught during the course of regular annual trawl surveys [The cost and difficulty of obtaining deep-sea coral samples can be a barrier to large scale population studies in these organisms. In addition to using high-throughput genotyping-by-sequencing to compensate for limited sample sizes, our study successfully utilized an existing collection, obtained largely as fisheries bycatch . The use surveys , in addi surveys . While tS. simplex, can impact their ability to recover from anthropogenic disturbances, including fisheries activities, and thus have implications for their protection and management [The life history traits  , and thenagement , 27, 55.nagement . Speciesnagement . Thus knnagement , 55, 57.Corralium rubrum identified significant population structure at all spatial scales tested [Callogorgia delta from the Gulf of Mexico. A study by Miller et al. [Desmophyllum dianthus had significant genetic subdivisions among populations and regions, but four additional scleractinians, including one occurring in the same family as D. dianthus, showed no evidence that groups from across Australia/New Zealand are distinct, with haplotypes shared across all included regions. All the species included in the study are believed to be seasonal gonochoric broadcast spawners, suggesting that the larval dispersal potential in D. dianthus is lower than for the other species, even within family. In L. pertusa, an isolation by distance pattern is supported across the entire geographic area (Gulf of Mexico to the Eastern North Atlantic) [Paragorgia arborea, Herrera et al. [P. arborea individuals from the North Eastern Pacific. This diversity in the degree of genetic connectivity among corals highlights the need for additional study and the development of additional molecular tools that can be applied towards understanding the connectivity amongst deep-sea coral communities.Within deep-sea corals, population structure and degree of connectivity can vary over both geographic and depth scales and can be influenced by factors such as larval duration and oceanographic circulation. A handful of studies have examined a number of deep sea corals with differing dispersal capabilities, and found a range of results regarding population structure: A study examining microsatellite and mitochondrial markers in mesophotic populations of s tested . This sts tested  found der et al.  demonstrtlantic) . Examinia et al.  found thS. simplex, from the United States West Coast. Our study demonstrates the apparent lack of genetic structure along the United States West Coast and suggests a high level of genetic connectivity in S. simplex. This is consistent with the expectations for a highly fecund broadcast spawner [S. simplex may have the capability to recover from or recruit following disturbance, at least on a regional scale, given sufficient time for immigration of new settlers.We have successfully developed the first large scale molecular sequence resource for  spawner , and sugS1 FigSTRUCTURE plots for K = 2-K = 6 generated using the full twenty-three individual dataset. No pattern was observed in for any K value, consistent with a panmictic, K = 1 population.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Fraxinus excelsior L.) is affected by several human activities in Central Europe, including habitat fragmentation, agroforestry expansion, controlled and uncontrolled transfer of reproductive material, and a recently introduced emerging infectious disease, ash dieback, caused by Hymenoscyphus fraxineus. Habitat fragmentation may alter genetic connectivity and effective population size, leading to loss of genetic diversity and increased inbreeding in ash populations. Gene flow from cultivated trees in landscapes close to their native counterparts may also influence the adaptability of future generations. The devastating effects of ash dieback have already been observed in both natural and managed populations in continental Europe. However, potential long-term effects of genetic bottlenecks depend on gene flow across fragmented landscapes. For this reason, we studied the genetic connectivity of ash trees in an isolated forest patch of a fragmented landscape in Rösenbeck, Germany. We applied two approaches to parentage analysis to estimate gene flow patterns at the study site. We specifically investigated the presence of background pollination at the landscape level and the degree of genetic isolation between native and cultivated trees. Local meteorological data was utilized to understand the effect of wind on the pollen and seed dispersal patterns. Gender information of the adult trees was considered for calculating the dispersal distances. We found that the majority of the studied seeds (55–64%) and seedlings (75–98%) in the forest patch were fathered and mothered by the trees within the same patch. However, we determined a considerable amount of pollen flow (26–45%) from outside of the study site, representing background pollination at the landscape level. Limited pollen flow was observed from neighbouring cultivated trees (2%). Both pollen and seeds were dispersed in all directions in accordance with the local wind directions. Whereas there was no positive correlation between pollen dispersal distance and wind speed, the correlation between seed dispersal distance and wind speed was significant , indicating that strong wind favours long-distance dispersal of ash seeds. Finally, we discussed the implications of establishing gene conservation stands and the use of enrichment planting in the face of ash dieback.Gene flow dynamics of common ash ( Pollen and seed dispersal patterns are important to determine the consequences of ecological and biological threats on plant populations, such as landscape changes, climate change and newly emerging pathogens. Habitat fragmentation can reduce genetic connectivity of plant populations  by reducNatural forests are often replaced with managed large-scale monocultures, in order to meet the demand for wood and other forest products . The gloEmerging infectious diseases (EIDs) are another potential cause behind loss of genetic diversity and local genetic patterns. Increasing in number worldwide , EIDs arFraxinus excelsior, hereinafter ash), is a keystone species in natural plant communities in European forests [Hymenoscyphus fraxineus , which causes dieback [Common ash  and maternal (ovule-seed) contributions are identified. With seeds sampled on specific mother trees, pollination distances and directions can easily be calculated. However, in the case of seedlings, additional information regarding the gender of the both candidate parents is required. A prior assumption of the closest parent being the maternal parent is potentially misleading and can underestimate the seed dispersal distances . UnipareThe process of propagule dispersal is complex, and is influenced by both biotic and abiotic factors. For wind-mediated dispersal, wind conditions are obviously expected to play a substantial role in determining the dispersal directions and distances from parent trees. For example, dry, windy weather conditions promote long-distance dispersal (LDD) of seeds –43. LikeThe aim of the present study is to investigate genetic connectivity among ash trees in a fragmented landscape in Rösenbeck, Germany. A combination of categorical parentage and the neighbourhood model approaches was applied to estimate mating parameters and to describe dispersal patterns in our study site. Specifically, we studied the degree of reproductive isolation of a 2 ha natural population from cultivated trees planted within a few hundred meters. Gender information was used to calculate pollen and seed dispersal distances and directions. Actual wind and precipitation data were utilized to assess the effect of wind on the dispersal patterns. The outcomes are discussed in relation to the genetic management of ash, including its conservation and reproductive material use, especially under the current epidemic ash decline.F. excelsior. Large numbers of female flowers are produced and pollinated in spring, before leaf flush. The pollination season in Northern and Central Europe lasts from April to May [Both pollen and seed dispersal are mediated by wind in l to May . Seeds ml to May . A fractl to May . The disl to May .3) can remain in the air between 10–20 days [The species presents a polygamous reproductive system where the individuals are phenotypically classified in a continuum from purely male to purely female, with a wide range of hermaphroditic intermediates ,54, but –20 days .www.tim-online.nrw.de/tim-online/initParams.do) revealed the historical fragmentation of the region, indicating that the landscape fragmentation must be at least 200 years old. In a radius of 5 km around the study site, ash mainly exists as single trees in different stands, mostly mixed with beech, and only exists seldom in more or less small pure tands. Thus the estimated abundance of ash in the area is less than 5% .The location of the study site and the sampling scheme are shown in The cultivated alley trees stretch for 1 km along both sides of the highway . Apical th -17th of April in 2014 and 21st -22nd of April in 2015 because flowering started earlier in 2014 relative to 2015. The trees were recorded as male, female or hermaphrodite according to their flower types. In case of the absence of flowering, any additional observations informative for gender identification, such as presence of dry seeds and seed stalks from the previous year and flower galls [The geographical position of each adult tree was recorded during the sampling period using Garmin GPSmap® 62s. The position of each seedling on the transect line relative to the start point was recorded, and the heights of the seedlings were measured. Visual observation of the adult tree flowers was conducted in two subsequent years in 2014 and 2015 when both cultivated and native trees were flowering at the same time. The flowers were observed between 11er galls , were reLeaves and buds were stored at -20°C until they were used for DNA extraction. Seeds were collected and stored at 4°C in labelled bags. Prior to embryo extraction, randomly picked seeds were embedded in water for 1–2 days after pericarp tissues were removed. After the rehydration, seeds were sliced into two halves with a razor blade and embryos were carefully picked up to avoid any contamination with maternal tissues. Total DNA was extracted from leaf, bud and embryo tissues using DNeasy® 96 Plant Kit (Cat No. 69181) according to the manufacturer’s protocol .F. excelsior [F. excelsior or other Fraxinus species. The primers were labelled with two different fluorescent dyes , and were combined in 4 Multiplexes (M) . Amplifications were performed in 14 μl reactions. Each reaction contained 1.5 μl of 1× Reaction Buffer: 0.8 M Tris-HCl (pH 9.0), 0.2 M (NH4)2SO4, 0.2% w/v Tween-20 , 2.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 μl 1 U of Taq DNA polymerase , and 0.3 μM forward and reverse primer, and ~ 10 ng genomic DNA. A combination of hot start and touchdown procedure was applied for PCR conditions. Thermal cycling conditions were as follows: 95°C for 15 min followed by 10 cycles of 94°C for 1 min, annealing temperature step downs each cycle of 1°C (from 60°C to 50°C) for 1 min, 72°C for 1 min. The annealing temperature for the final 25 cycles was 50°C for 1 min with the denaturation and extension steps as above. The final extension step was at 72°C for 20 min. PCR products were analyzed using Applied Biosystems 3130xl Genetic Analyzer . Allele size calling was carried out with GeneMapper 4.1®  using the software developer’s default settings for the analysis parameters, except for the parameters for allele peak quality. Signal levels for minimum homozygous and heterozygous peak height were set to 200 RFU and 100 RFU, respectively. Minimum peak height ratio was set to 0.5 for heterozygote balance. Each individual genotype was checked for errors, and the analyses were repeated for missing or uncertain peaks. Mother-offspring mismatches in all loci were checked and repeated when necessary to avoid scoring PCR artefacts because of null alleles. Manual allele binning was conducted based on the microsatellite repeat units and the cumulative allele size distributions were plotted in Excel for each locus. Manually binned alleles were compared to the ones generated by TANDEM, an automated allele binning software [Microsatellite analyses were performed using 13 primers. FEMSATL8, FEMSATL11 and FEMSATL19 are frequently used primers developed for xcelsior . ASH2429xcelsior , FRESTSSxcelsior  and Fp12xcelsior  are EST-e), observed heterozygosity (Ho), Hardy-Weinberg expected heterozygosity (He) of cultivated and native adults and offspring were calculated using GENALEX 6.5 [R) was computed using rarefaction method in HP-RARE [IS). The software jointly estimated inbreeding coefficients and null allele rates according to the Bayesian approach at the parameter settings of 200 000, 2000 and 20 000 for the number of cycles, thinning and burnin, respectively. Pairwise genetic differentiations (FST) and their significance were determined based on 9999 permutations using GENALEX 6.5 [Genetic diversity parameters including number of alleles (A), effective number of alleles  while 15 of them presented another (Genotype 2) . Only onParentage assignments were performed using CERVUS 3.0.7 . Parent After the parentage analyses, each confident assignment was subjected to further elimination using the gender information of each adult tree acquired from field observations in 2014 and 2015, in order to improve the reliability of the assignments. The assignments were excluded from further calculations when there was a discrepancy between the genders of the two assigned parents or no information about the genders of the assigned parents. This was only the case for 37 (out of 332) paternity, and 11 (out of 121) parent pair assignments. The confidently assigned single parent was only accepted as a seed parent when it was female and having no mismatching loci with the seedling. This was the case for 23 (out of 67) of the assigned single parents.x, y) of the seedlings relative to their respective transect lines were calculated using Euclidean distances. These coordinates were later transformed into the same geospatial coordinates with the adult trees to calculate the pollen and seed dispersal distances. To identify the directions of the dispersal events, azimuths  were calculated using QGIS 2.16 [Point coordinates  , located 26 km north of the study site. The data were further arranged by including only the time period between 10 am-6 pm for each day. Because long-term historical data was not available for Haaren Station, the next closest wind station (40 km southeast), Arolsen-Volkhardinghausen (ID No. 197), was chosen to extract the wind data for the seed dispersal periods between October 2008 and February 2012. The precipitation data from Brilon-Thülen station (ID No. 6264) (4 km to the study site) was used to remove the effect of high relative humidity by subtracting the hours with precipitation from the corresponding wind data. The dispersal and wind data were analyzed in 18 angle classes each with 20° (18×20° = 360°). The patterns of pollen and seed dispersal direction and wind direction were assessed using ORIANA 4.02  by constructing circular histograms, calculating mean directions, and computing circular correlations between dispersal directions and wind directions. Since the wind directions were also recorded as azimuth (0° North), there was no need for further conversion to make pairwise comparisons. To determine the effect of the frequency of strong winds on LDD, linear relationships between dispersal distances and wind speeds in the same angle classes were assessed using paired t-test for sample means. Pearson’s correlation coefficients and their p-values were calculated using (1) the means of the dispersal distances and wind speeds, and (2) the cumulative dispersal distances and wind speeds in each angle class.To assess the effects of wind direction and speed on the realized dispersal events, publicly available meteorological data was acquired from Climate Data Centre of German National Meteorological Service (hourly/) . Based oR was higher in the native adult trees (6.61) than in the cultivated adults (4.69), and compared to the native adults, it was slightly lower in the seedlings (6.47) and in the seeds (6.19) (IS level of the cultivated adults (0.012) was slightly higher than that of the native adults (0.006) (IS (0.021) while the seeds had lower (0.002) , and showed that the offspring were differentiated from the cultivated roadside trees  . The FIS (0.006) . Compare (0.002) . All paiTotal exclusion probabilities for both parentage analyses were high (0.999) in CERVUS. Thirteen loci provided the mean heterozygosity of 0.49, and the mean numbers of alleles per locus were 10.3 for paternity and 9.5 for parent pair analyses. Using Δ, unique candidate fathers were confidently assigned to 332 (66.4%) seeds, and among them 200 (62.1%) were strictly confident. No mismatching loci were detected in 228 (70.8%) of the assignments. Twenty paternal assignments were not statistically confident enough even though the candidate fathers had no mismatching loci with the mother and seed. In the case of the seedlings, the number of confidently assigned unique parent pairs was 121 (49%). The level of confidence was strict for 41 (17%) assignments. The overall number of trio loci with no mismatch was 110 in the parent pair analysis. Sixty-seven (27%) single parents were confidently assigned and among them 28 were strictly confident.The paternity and parent pair analyses revealed 64.4% and 74.9% of gene flow from the native trees while 2% and 1.2% from the cultivated trees, respectively . Althougbp = 0.269, b < 1) while seed dispersal kernel rather follows a normal distribution   is the best model for the seed and pollen dispersal in the study site . The est, b = 2) . The est, b = 2) .Based on the best-fitting model, the parental profiles of the progenies were assessed according to the posterior probabilities of the two most-likely parentages inferred for each progeny . AccordiThe frequency distribution graphs of pollen and seed dispersal distances were constructed based on the dispersal events identified using two approaches to parentage analysis . A totalds, bs, dp and bp, and the approximate 95% confidence interval around them were given in ds) and pollen (dp) were 39 m and 571 m, respectively  and their significances were calculated for linear associations between dispersal distances and wind speeds in the same angle classes, in the corresponding dispersal period (p < 0.01).Pearson’s correlation coefficients . The estimated prevailing direction of pollen dispersal (ap) with the fitted model was 27.36° . However, when only direction was taken into account, the mean seed dispersal direction was towards the northeast (59.84°), which was similar to the neighbourhood model estimations for the mean prevailing seed dispersal direction  into the angles  . The seee across the 3 genomic SSR loci was very high: 0.89 for the native adults and 0.87 for the seedlings and seeds, although lower, 0.72, for the cultivated adults. 10 EST-SSR loci showed much lower mean He, which was 0.37 for the cultivated and native adults and for the seedlings, and 0.35 for the seeds. Lower polymorphisms are well known in EST-SSRs because they are located within expressed genes and therefore are more conserved compared to genomic SSRs [e calculated for all 13 SSRs was higher in the native adults (0.49) and in the natural regeneration (0.48) compared to the cultivated trees (0.45) (F. excelsior adults (He = 0.767–0.818) and progenies (He = 0.777–0.805) have previously been reported in two sites in Ireland [ST = 0.013 in Clonee and FST = 0.014 in Kildalkey). Our pairwise FST values showed higher genetic differentiation between cultivated and native trees  . Using 6s He = 0.7–0.805 hs 0.053; . This reF. excelsior populations, reflecting a likely mix of Wahlund effect, null alleles, inbreeding and large allele drop-out [IS (0.067) in ash populations in Northern Ireland has been reported [IS in two native ash populations (FIS = 0.053 and 0.057), but higher mean FIS in their progenies (FIS = 0.131 and 0.122). In our study, the mean inbreeding coefficients for the adults and the offspring were much lower. The FIS of the native adults was 0.006, lower than that of the seedlings (FIS = 0.021) but higher than that of the seeds (FIS = 0.002)  . The higd et al.  also repCategorical parentage approaches are powerful in detecting gene flow when microsatellite markers are highly polymorphic and sampling is efficient . HoweverF. excelsior in Scotland. When including the reproductive success parameters such as plant size and flowering intensity to the neighbourhood model, Goto et al. [F. mandschurica in a 10.5 ha plot in Japan. However, since the estimated immigration rates are sensitive to the defined neighbourhood size and the selection gradients influencing male mating success, model comparisons still remain as a challenge [The overall successes of the both parentage analyses using CERVUS were high in our study. Not only because using 13 loci with no missing allelic information provided us high exclusion probabilities (0.999), but also because the exhaustive sampling scheme of the adult trees played a role in increasing the chances of sampling the true parents. We also allowed mismatches (1%) in the parentage analyses to increase the overall success rate . We confo et al.  estimatehallenge . The resF. excelsior based on a prior assumption of the nearest, or the single parent identified being the maternal parent. However, this assumption may not always be true and therefore may cause an erroneous conclusion such that seed dispersal distances are always shorter than pollen dispersal distances [Molecular markers provide enough information to assign parentage to offspring. However, reliable identification of pollen and seed dispersal distances requires additional information regarding the gender of the parental trees. This is relatively easy for the pollen dispersals as long as prior information about the genotypes of mother trees from which the seeds were collected is available. It presents a greater challenge to determine pollen and seed parents of established seedlings. Seed trapping can provide information about the genotype of maternal tissue ,90 that istances . Additioistances  because istances . Two-yeaF. excelsior is one of the tree species silviculturally managed in the last 30–40 years in Europe, mostly due to its high quality wood [th and 17th of April. The German Weather Service also recorded first ash pollen release in the region on the 15th of April in 2014 (Station ID: 9247) . The small stand size of the cultivated trees compared to the native stand, their relative distance from the native mothers and the local wind direction may have affected the pollen clouds in our study site. On the other hand, Steinitz et al. [Pinus halepensis plantations to two conspecific native populations; the more isolated population had a lower gene flow level (22%) than the less isolated one (49%). The differences in seed and pollen dispersal results among different sites are highly dependent on phenological synchrony and relative fecundity of trees [Plant improvement is based on the breeding of plants with desirable traits; however, gene flow from domesticated species cannot be regarded as universally beneficial . In someity wood . It is aity wood . Plantinity wood . Accordiity wood . Accordiity wood . In the z et al.  estimateof trees ,56, spatof trees , size ofof trees , topograof trees , wind diof trees  as well of trees , intrinsof trees  and on tof trees .bp = 0.269, b < 1; bs = 2.085, b = 2; F. excelsior in Scotland. In the same population, however, the predicted seed dispersal distribution was also fat-tailed, suggesting that the seed dispersal may also spread over tens of kilometres, and the authors concluded that deforested landscapes favors LDD of seed due to the increased likelihood of winged-seed to be uplifted [F. excelsior populations has been previously reported for both pollen [in situ gene conservation practices of the species.Short distance dispersal (SDD) mainly shapes the local distribution of genotypes . LDD of uplifted . Based ouplifted . On the h pollen  and seedh pollen  using pap < 0.01; term) during the fall. According to their results, turbulence is positively correlated to LDD for the species with Vterm ≥ 0.8 m/s and the effect of LDD changes from positive (Vterm = 3.2 m/s) to negative (Vterm ≤ 0.4 m/s). This is consistent with our results when considering the medium Vterm of F. excelsior (1.58 m/s). However, pollen dispersal distances were negatively correlated with the wind speeds , on the other hand, has been predicted to cross the western border of Russia soon [F. excelsior is promising for the species’ existence in the European forest ecosystems and for its adaptive potential. Recent reports on the presence of genetic variation in dieback tolerance among ash trees [cosystem ,110. Thesia soon . The funsia soon . High stsh trees ,113 suggsh trees . HoweverF. excelsior, and the relative distributions of individuals, sampling design, the method of choice and local wind conditions seem to influence the estimated seed and pollen dispersal patterns in the study site. A combination of different analytical approaches to parentage analysis can provide a better resolution to gene flow studies. Available wind data from weather stations in proximity can be utilized for planning purposes.Our results indicated an effective landscape process of gene flow in ash including the LDD of pollen and seed and the effect of the wind conditions on the dispersal patterns. In our study, the cultivated roadside trees in the landscape only provided limited gene flow into the old growth forest. However, our results still suggest that gene conservation stands should be planned carefully in ash, considering the LDD of ash propagule. A maximum of 100 m distance among trees may be recommendable for effective enrichment plantings in order to facilitate gene flow from healthy to unhealthy individuals. However, our results are based on a single small stand of S1 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.


For geometric morphometric analysis, specimens were collected in the Northeast Atlantic Ocean—Azores, Madeira and mainland Portugal, and for genetic analysis, these samples were complemented with samples collected in the Mediterranean Sea—Spain, Italy and Croatia, in order to cover the entire distribution area of the species. Body shape of the forkbeard from the Northeast Atlantic was found to be highly variable. This variation was probably associated with different environmental factors between the study areas. Despite morphological variation, a low genetic differentiation between samples from different areas was found, most likely due to gene flow that occurred in the past or with the demographic history of the species. Moreover, the presence of unique haplotypes in the Northeast Atlantic and in the Mediterranean suggests that recent gene flow between populations from these areas should be limited. Altogether, a high haplotype diversity, a low nucleotide diversity, a “star-like” network and the results of the mismatch distribution, indicate a possible signature of recent population expansions, which probably started during the end of the Last Glacial Maximum and led to the colonization of the Northeast Atlantic and the Mediterranean.The knowledge of population structure of a species is essential to effectively assess and manage fisheries. In the present study, genetics, by mitochondrial DNA cytochrome  In marine environments, population genetic structure is influenced by life-history traits and species-specific ecological supplies and, therefore, genetic differentiation in marine organisms is highly influenced by their dispersal capability . The theMorphological variation of populations can be caused by additive genetic variation or processes of phenotypic plasticity in response to different selective pressures or environmental constraints , 8. For A recommended procedure for population identification is the use of a holistic approach where, at least, one phenotypic-based method and one genetic technique should be used . The comPhycis phycis . This species is a gadiform benthopelagic fish with a wide distribution in the Northeast Atlantic (from the Bay of Biscay to Morocco and Macaronesia islands) and in the Mediterranean Sea [In the present study, a comparison between variation and adaptation at the morphological and genetic levels was performed, using a fish species: the forkbeard, nean Sea . The fornean Sea , taking nean Sea –17. In tnean Sea –20, withnean Sea  and abounean Sea . In the nean Sea . The fornean Sea –20. Despnean Sea .b gene (cyt b) and of the first intron of the nuclear S7 ribosomal protein gene (S7). These genes have been widely used in both genetic structure of fish populations  cytochrome ns e.g. –25) and  and b gens (e.g. ), provinns (e.g. . Variatins e.g. –36). Thr. Thrb geSampling for the present study focused on the forkbeard from the Northeast Atlantic Ocean and the Mediterranean Sea. Ethical or government approval, specific permissions or licenses for sample collection were not required for this study, as all specimens were collected as part of routine fishing procedures by fishermen of commercial fleet. As the forkbeard is a deep-water species, fish are killed during the hauling of fishing gear due to differences in atmospheric pressure. No animals were killed specifically for this study. Dead specimens were acquired in the commercial ports of Horta , Funchal , Peniche , Fuengirola (Spain), Fiumicino  and Split (Croatia). Sampling was performed in the laboratory. The forkbeard is not an endangered or protected species.For geometric morphometric analysis, forkbeard specimens were collected from commercial landings of fishing vessels operating off Azores and Madeira archipelagos, and mainland Portugal—Northeast Atlantic , betweenFor genetic analysis, a piece of fin of sampled specimens from each population from the Northeast Atlantic—Azores (n = 29), Madeira (n = 26) and mainland Portugal (n = 27) was collected and preserved in 96% ethanol. In order to cover the entire distribution area of the forkbeard, fins of specimens from 3 populations from the Mediterranean Sea—Spain (n = 17), Italy (n = 2) and Croatia (n = 26) were also collected , preservTo quantify body shape variation of the forkbeard from Northeast Atlantic, a total of 13 anatomical landmarks were defined , which cAll sampled specimens from the Northeast Atlantic were analysed by geometric morphometrics (n = 268)  was performed to detect morphometric differences in the body shape of forkbeard from the three study areas and to investigate if body shape could be used to classify samples in terms of area of origin. Pairwise comparisons of mean shapes of forkbeard from different areas were based on Procrustes distances, and a permutation test with 10,000 runs was used to assess the null hypothesis of no difference between samples .The mean shapes of the forkbeard from the three study areas were estimated by warped outline drawings, which were performed using the wireframe function .All geometric morphometric analyses were done in the software MorphoJ version 1.05c . A signiCanonical discriminant analysis (CDA) was also performed to detect morphometric differences in body shape of the forkbeard from the three study areas. The Jackknife cross-validation was used to calculate an unbiased estimation of classification success, being each specimen allocated to the group with the nearest centroid and the proportion of correctly allocated specimens calculated . The disb (cyt b) gene was amplified by polymerase chain reaction (PCR) for all 127 forkbeard specimens. A pair of primers purposely designed by the authors for this study, based on 3 aligned cyt b complete sequences of Phycis phycis obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank)  was used to amplify the region of interest: FORcytbF (forward) 5’-GCCAGCCTTCGAAAAACAC-3’ and FORcytbR (reverse) 5’-GCTTCGTTGTTTTGAGGTGTG-3’. In addition, PCR amplification of a 751 bp fragment of the first intron of the nuclear S7 ribosomal protein gene (S7) was performed with the following pair of primers: S7RPEX1F (forward) 5’-TGGCCTCTTCCTTGGCCGTC-3’ and S7RPEX2R (reverse) 5’-AACTCGTCTGGCTTTTCGCC-3’ [Genomic DNA was extracted from fins preserved in 96% ethanol using the E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek), according to the manufacturer's instructions. An 899 bp fragment of mitochondrial DNA (mtDNA) cytochrome TCGCC-3’ .2, 1.4/1.2 μl BSA (10 ng/μl), 4.0 μl 5x Colorless GoTaq Flexi Buffer, 0.05 U GoTaq DNA Polymerase (Promega) and 2.0 μl of template DNA . PCR conditions used were composed by an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 55°C or 68°C, for cyt b or S7, respectively, for 35 sec and extension at 72°C for 1 min, with a final extension period at 72°C for 10 min. Amplification results were confirmed by 1% TBE agarose gel electrophoresis. All PCR products were purified with the SureClean kit (Bioline) following the manufacturer’s protocol, and sequenced in the forward and the reverse directions (using the same primers) by an ABI PRISM 3700 DNA analyser at Macrogen (http://www.macrogen.com). The existence of haplotypes present in one single specimen was confirmed by performing independent PCR amplifications and sequencing (as previously described). The obtained cyt b and S7 sequences were compared against a database in GenBank using the basic local alignment search tool (Blast) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm that they belong to both genes.PCR amplifications were performed in a 20 μl total reaction volume with 0.6 μM of each primer, 0.175/0.2 mM dNTPs, 0.925/1.125 mM MgClSequencher version 4.0.5 (Gene Codes Corporation) and BioEdit version 7.0.9 [ClustalX version 2.1 [Concatenator version 1.0.1 [Fragment sequences of both genes were verified and edited using the software on 7.0.9 . For eacsion 2.1  and convon 1.0.1 .Data analysis of nuclear S7 was suspended because this gene did not show genetic variation.a priori: Northeast Atlantic Ocean, composed by samples of Azores, Madeira and mainland Portugal, and Mediterranean Sea, constituted by samples of Spain, Italy and Croatia.Two different geographical groups were defined h), and nucleotide diversity (π) were calculated for each sampled population, as well as for both geographic groups and for the total samples, using the software DnaSP version 5.10.01 [Network version 5.0.0.0 (Fluxus Technology Ltd.). Analysis of molecular variance (AMOVA), with 10,000 permutations, was performed using Arlequin version 3.5.1.2 [The number of polymorphic sites and haplotypes, haplotype diversity  was used to detect and estimate the timing of population expansion . Estimatunimodal . To testunimodal . Statistunimodal , 62. Neuunimodal  and Fu’sunimodal  were perilibrium , 66.t), as well as the 95% confidence interval, was calculated from τ = 2ut, where τ is the expansion time parameter generated by Arlequin and u is the mutation rate per nucleotide per year multiplied by sequence length (i.e. number of nucleotides) [Time since population expansion in years (eotides) , 62. A geotides) , and a meotides) , since np < 0.031) of shape variation due to allometric growth was detected . Therefore, data were allometrically corrected to remove the size effect between variables and the residuals were used as “size-free” variables in the statistical analyses.In this study, a similar length range of specimens from the Northeast Atlantic was used, based only in adult mature specimens. However, and despite the fact that juveniles and very large specimens were excluded from analyses to minimize the effect of size on shape, a very small amount, but statistically significant , so sexes were analysed together for each geographical area.No statistically significant differences were found between males and females shape within the three study areas from the Northeast Atlantic . Their score plot  , meaningb gene fragment (GenBank accession numbers: KM252661-72 and KX215149-52) and only one haplotype of the nuclear S7 gene fragment (GenBank accession number KP005452) were found. The results of the latter gene were not shown due to its lack of genetic variation. Pseudogenes were not found in any of the samples sequenced, since two real peaks in the same position during the base calling in the chromatograms were never obtained.Within the 127 samples of forkbeard from the Northeast Atlantic and the Mediterranean, a total of 16 haplotypes of the mitochondrial cyt b showed 17 polymorphic nucleotide positions and 8 of the 16 haplotypes were unique (h) and nucleotide diversity (π) for the entire sample were 0.675 ± 0.038 and 0.00144 ± 0.00013, respectively. The Northeast Atlantic and the Mediterranean groups showed similar haplotype and nucleotide diversities . In the Atlantic group, the highest haplotype richness was found in Mainland Portugal, where, for 27 samples, nine different haplotypes were present, and Azores showed the lowest one (5 different haplotypes were detected in 29 samples). In the Mediterranean Sea, the highest haplotype richness was found in Croatia, with 8 different haplotypes in 26 samples. A summary of the distribution of haplotypes per population and group, as well as a summary of haplotype and nucleotide diversities indices, are presented in The 846 bp amplified fragments of cyt e unique . The AtlThe haplotype network did not show a phylogeographical structure among the sampled geographical region, presenting a “star-like” shape where the most common haplotype (H1) is shared between all sampled geographic areas, and the less frequent and unique haplotypes are connected to the central most common haplotype by only one or two mutation steps .p = 0.721) instead of among groups   and AMOVA was performed. AMOVA also revealed a lack of genetic structure, indicating that the overall source of variation was within populations  . The pai= 0.301)  showed nSSD and PRAG showed that the observed distributions did not differ significantly from those expected under a sudden expansion model (The distribution of pairwise nucleotide differences (mismatch distribution) showed a unimodal shape , suggeston model . Tajima’on model , which dA substantial variation in forkbeard body shape was found, suggesting a clear separation between the three populations from the Northeast Atlantic. However, this morphological differentiation is not in accordance with the genetic population structure evidenced by the molecular marker used.A large component of morphological variation in fishes is induced by exposure to different environmental factors during their development , namely Results of body geometric morphometric analysis obtained in this study suggests its usefulness as a population differentiation tool and indicate the existence of at least three phenotypically distinct local populations in the Northeast Atlantic, as reported by Vieira et al.  using otb suggest that the forkbeard forms a panmictic population along the Northeast Atlantic and the Mediterranean. F-statistics and AMOVA indicated no genetic structure over space, probably due to the high frequency of the most common haplotype (H1) (which minimizes the effect of rarer haplotypes), and also due to similarities between sequences caused by the low nucleotide diversity. These facts suggest that high levels of gene flow between geographically separated areas must have occurred in the past, or that the current distribution is an expanded area of an ancestral refugial and panmitic population, with low levels of diversity but widespread common/ancestral haplotypes. Moreover, and taking into account the existence of unique haplotypes in the Northeast Atlantic and the Mediterranean, recent gene flow between populations from these groups is unlikely to occur or, if present, occurs at low levels. Although larval behaviour of the forkbeard is unknown, other species of the genus Phycis present pelagic larvae for several months [Genetic data from cyt l months , 80 and,l months . In addil months  of this b haplotypes was defined by a “star-like” network topology, in which rare haplotypes are derived from the most common haplotypes , presumably ancestors, frequently by one or two mutation steps. This could indicate that the forkbeard populations have recently expanded in size from one or from a small number of founders following a population bottleneck [The genetic diversity pattern of the forkbeard might be consequence of population expansion after a period of low effective population size due to founder events or bottlenecks . The phyttleneck , suggestttleneck , 84).Chromis limbata [One of the major focuses in studies of phylogeography of the Northeast Atlantic specimens has been the location of refugia, where these organisms survived the successive glacial peaks during the Pleistocene . These r limbata ).At the present, little information exists about the population structure of the forkbeard and from a management point of view the knowledge of this issue is crucial.Genetic data suggest that the forkbeard forms a panmictic population in the Northeast Atlantic and the Mediterranean, and body shape variations indicate, as otolith shape analysis had already shown , a clearThe information presented in this study shows that, along its distribution area, the forkbeard presents, at least, three phenotypic stocks—mainland Portugal, Azores and Madeira, in the southern NE Atlantic, and two genotypic stocks between the NE Atlantic and the Mediterranean. Any depletion in one of these populations might not be compensated by migration from the others, at least at a sufficiently rapid rate to ensure resource sustainability. The most precautionary management approach should be to consider the different populations of the forkbeard from the Northeast Atlantic and the Mediterranean as separate stocks, to ensure sustainability of resources and genetic biodiversity maintenance . A failuFuture studies using a holistic approach, which employ a broad spectrum of complementary techniques  , as well