Chthamalus montagui was investigated to understand its genetic structure and quantify levels of population divergence, and to make inference on historical demography in terms of time of divergence and changes in population size. We collected specimens from rocky shores of the north-east Atlantic Ocean (4 locations), Mediterranean Sea (8) and Black Sea (1). The 312 sequences 537 bp) of the mitochondrial cytochrome c oxidase I allowed to detect 130 haplotypes. High within-location genetic variability was recorded, with haplotype diversity ranging between h = 0.750 and 0.967. Parameters of genetic divergence, haplotype network and Bayesian assignment analysis were consistent in rejecting the hypothesis of panmixia. C. montagui is genetically structured in three geographically discrete populations, which corresponded to north-eastern Atlantic Ocean, western-central Mediterranean Sea, and Aegean Sea-Black Sea. These populations are separated by two main effective barriers to gene flow located at the Almeria-Oran Front and in correspondence of the Cyclades Islands. According to the ‘isolation with migration’ model, adjacent population pairs diverged during the early to middle Pleistocene transition, a period in which geological events provoked significant changes in the structure and composition of palaeocommunities. Mismatch distributions, neutrality tests and Bayesian skyline plots showed past population expansions, which started approximately in the Mindel-Riss interglacial, in which ecological conditions were favourable for temperate species and calcium-uptaking marine organisms.The model marine broadcast-spawner barnacle  The understanding of current and historical factors that shape species’ genetic structure in broadcast spawning coastal marine invertebrates is an intriguing topic. Most marine invertebrate species spend a part of their life cycle in the pelagic realm as free-moving gametes, larvae, juveniles or adults, providing moderate to high potential for dispersal and favouring genetic homogenisation even over large distances. As a consequence, the identification of population borders in marine broadcast spawning species is not an easy task. However, the tendency to genetic homogenisation can be counteracted by the occurrence of geographical and oceanographical barriers to dispersal that determine restriction to gene flow, promoting the onset of genetically differentiated populations .i) the Messinian Salinity Crisis, that separated the Mediterranean Sea from the Atlantic Ocean  ky and 892 [95% HPD: 598–1121], respectively.r and FS indices  and consistent with those observed in previous work on chthamalids with wide geographical distribution, such as C. stellatus  and C. 312, 79%,).C. montagui in the study area had a clear biogeographical meaning, with individuals grouped in three main genetic clusters that corresponded to north-eastern Atlantic (NEA), western-central Mediterranean Sea (WCM) and Aegean Sea-Black Sea (ABS). This result corroborated the three COI haplogroups found by Shemesh et al. [N = 1, threshold value under which, as a rule of thumb, isolation between populations occurs [Chthamalus spp. was estimated to be approximately 200 kilometres [C. montagui, two additional factors should be taken into account. Firstly, we cannot exclude the occurrence of false positivity of this test due to the presence of significant genetic structuring [The deep genetic structuring observed in h et al. . As prevh et al. , Dando &h et al. , Pannacch et al. , and Sheh et al. , the WCMs occurs . An intes occurs . Given tlometres ,49, the ucturing . Secondlucturing .C. montagui. The Messinian closure of the Strait of Gibraltar (around 5.6 Ma), which determined extensive desiccation of the Mediterranean basin [C. montagui .Dando and Southward  firstly an basin , is ofteC. montagui. Dando and Southward [C. montagui can be placed in proximity of the AOF. This is the reason why we chose to analyse a sample from Portman, located in the eastern proximity of the AOF. Portman sample exhibited typical Mediterranean COI haplotypes, whereas the individuals from Tangier, located in correspondence of the Strait of Gibraltar, were characterised by the Atlantic ones . The relatively short duration of the larval stage of C. montagui, coupled with the dislike for very hydrodynamic environments typical of islands [C. montagui is not present on islands [C. montagui on Malta.Unique genetic characteristics had already been observed in individuals from Malta and were attributed to local environmental conditions ,54. The  islands , make la islands . However islands , may accC. montagui needs regular exposure to air to consolidate shell plates [Finally, the option of species’ passive transport through rafting and shipping could be excluded, as l plates . On the C. montagui can be interpreted as a result of the interplay of historical and current events. During the Pleistocene, the Black Sea was subject to periodical isolation and partial desiccation. In the glacial periods, it became an isolated freshwater basin, due to the separation from the Mediterranean Sea and the input of the great continental rivers; in contrast, during the interglacial transgressions it was repeatedly colonised by the Mediterranean marine fauna [C. montagui could have not survived in glacial Black Sea environment, so undergoing recurrent extinction-recolonisation phenomena in this basin. The ABS lineage observed in this study could have originated from coastal refugia of the Aegean Sea where these barnacles survived during the Last Glacial Maximum. The current distribution of C. montagui in the Black Sea derives from the last refilling and recolonisation of the basin, which occurred approximately 7 ka [As we reported for the Atlantic-Mediterranean break, the finding of the ABS cluster of ne fauna –58. C. mely 7 ka .C. montagui, in agreement with what observed by Shemesh et al. [vice versa. In addition, an anticyclonic front present in the south-western part of the Peloponnese peninsula [Cerastoderma glaucum [Sepia officinalis [The isolation of the ABS lineage of h et al. , could bh et al. . These ch et al.  that acteninsula  could fueninsula . Isolati glaucum  and the icinalis .C. montagui from this area. This outcome suggests that differences in salinity and water temperature do not play an important role in shaping species genetic architecture.Despite the much lower salinity and the wider range of variation of the surface temperature of the Black Sea compared to the Aegean Sea, no genetic divergence was found between the samples of C. montagui, even though it is difficult to suggest mechanisms that underlie divergence between the two population pairs.The very close estimates of divergence time obtained for the two population pairs (approximately 900 ka) lays between the lower and middle Pleistocene, more precisely in correspondence of the Calabrian-Ionian transition, which was characterised by magnetic reversal and switch from the dominance change of the 40 ky glacial/interglacial cycles to the 100 ky ones . From a C. montagui underwent past demographic expansion [FS test. The latter implying population expansion and the r index suggesting, contrarily, demographic stability  displayed the entire expansion curve for the NEA and WCM areas and a truncated curve for the ABS . Despite–200 kyr . This da Pacific .C. montagui. This statement is also supported by results from several investigations carried out on modern populations, in which an increment in abundance was observed following the warming from the late 1980s [During the MIS11, the increase of sea-water temperature could have triggered population expansion of te 1980s ,68,69. Mte 1980s  and O'Rite 1980s  found thC. montagui that requires large amounts of calcium carbonate for the construction of its robust shell.Howard  also argS1 TableDistribution of the 130 haplotypes across locations and biogeographical areas.(PDF)Click here for additional data file.


A. fumigatus have been the subject of many studies. However, most previous studies had relatively limited sample ranges and sizes and/or used genetic markers with low-level polymorphisms. In this paper, we characterize a global collection of strains of A. fumigatus using a panel of 9 highly polymorphic microsatellite markers. Using these markers, we analyze 2,026 isolates, which is ~3 times the number of isolates reported so far in previous studies. Our analyses suggest that A. fumigatus contains historically differentiated genetic populations but that its evolution is significantly impacted by contemporary forces such as widespread gene flow and local antifungal drug pressure. In the wake of a global rise in resistance to azoles in fungal pathogens, our findings should aid in developing management strategies to mitigate current increases to azole resistance.The genetic diversity and geographic structure of the human fungal pathogen  Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen capable of causing invasive aspergillosis, a globally distributed disease with a mortality rate of up to 90% in high-risk populations. Effective control and prevention of this disease require a thorough understanding of its epidemiology. However, despite significant efforts, the global molecular epidemiology of A. fumigatus remains poorly understood. In this study, we analyzed 2,026 A. fumigatus isolates from 13 countries in four continents using nine highly polymorphic microsatellite markers. Genetic cluster analyses suggest that our global sample of A. fumigatus isolates belonged to eight genetic clusters, with seven of the eight clusters showing broad geographic distributions. We found common signatures of sexual recombination within individual genetic clusters and clear evidence of hybridization between several clusters. Limited but statistically significant genetic differentiations were found among geographic and ecological populations. However, there was abundant evidence for gene flow at the local, regional, and global scales. Interestingly, the triazole-susceptible and triazole-resistant populations showed different population structures, consistent with antifungal drug pressure playing a significant role in local adaptation. Our results suggest that global populations of A. fumigatus are shaped by historical differentiation, contemporary gene flow, sexual reproduction, and the localized antifungal drug selection that is driving clonal expansion of genotypes resistant to multiple triazole drugs.IMPORTANCE The genetic diversity and geographic structure of the human fungal pathogen A. fumigatus have been the subject of many studies. However, most previous studies had relatively limited sample ranges and sizes and/or used genetic markers with low-level polymorphisms. In this paper, we characterize a global collection of strains of A. fumigatus using a panel of 9 highly polymorphic microsatellite markers. Using these markers, we analyze 2,026 isolates, which is ~3 times the number of isolates reported so far in previous studies. Our analyses suggest that A. fumigatus contains historically differentiated genetic populations but that its evolution is significantly impacted by contemporary forces such as widespread gene flow and local antifungal drug pressure. In the wake of a global rise in resistance to azoles in fungal pathogens, our findings should aid in developing management strategies to mitigate current increases to azole resistance. Aspergillus fumigatus is a globally distributed opportunistic human fungal pathogen. While its primary ecological niche is decomposing organic matter, A. fumigatus is ubiquitous in the environment and can be found in a broad range of ecological niches. In humans, it can colonize the lung and several other body sites, causing infections collectively known as aspergillosis. A. fumigatus is a major source of morbidity and mortality in immunocompromised patients causing approximately 4 to 5 million cases of allergic bronchopulmonary aspergillosis globally, approximately 10% of which become chronic  typing, PCR-RFLP, RFLP detected through Southern hybridization, and AFLP, have been used for genotyping umigatus , 12. Howumigatus . For exaumigatus .A. fumigatus isolates from 13 countries in 4 continents. We aimed to (i) critically examine the genetic relationships of the isolates within and among geographic populations of A. fumigatus, (ii) investigate how antifungal drug susceptibility patterns are related to genetic variations, and (iii) evaluate the roles of sexual and asexual reproduction in shaping the evolution of azole resistance globally. We tested the Baas Becking hypothesis that “everything is everywhere, the environment selects”  , India and Italy , and India and Australia . Similarly, we observed relatively limited but statistically significant genetic differentiations based on ecological niches. Among the 2,026 isolates, 434 had unspecified ecological origins and they were excluded from this analysis. The remaining 1,592 isolates were classified into four ecological niches , and only 3% of the total genetic variation was contributed by such ecological niche separation (P = 0.001)  showed that 6% of the total genetic variation was contributed by geographic separation, with the majority of genetic variation found within individual geographic populations (001) see .10.1128/mSphere.00019-17.1FIG S1 Aspergillus fumigatus isolates. Pie charts show percentages of variations among and within populations that can be explained by grouping isolates by geographic origin. PhiPT values are in the lower diagonal of the pairwise comparison table. Probabilities based on 999 permutations are shown above the diagonal. Download FIG S1, PDF file, 0.2 MB.Contribution of geographic separation to genetic variation in the global sample of 2,026 Copyright © 2017 Ashu et al.2017Ashu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00019-17.2FIG S2 FIG S2, PDF file, 0.2 MB.Contribution of ecological niches to the total genetic variation. A total of 1,592 isolates from our data set had known ecological  origins. Pie charts show percentages of variations among and within populations that can be explained by grouping isolates by ecological origin. PhiPT values are in the lower diagonal of the pairwise comparison table. Probabilities based on 999 permutations are shown above the diagonal. Download Copyright © 2017 Ashu et al.2017Ashu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Given the diverse geographic and ecological niches of the samples, the relatively limited contributions of geography and ecological niche to the total genetic variations were surprising. Among the factors contributing to the limited geographic and ecological differentiation were the high levels of allelic and genotypic diversities within most geographic and ecological niche populations see . In tota10.1128/mSphere.00019-17.3FIG S3 2)]; I, Shannon’s information index {−1 × Sum [pi × Ln(pi)]}; h, diversity [1 − Sum pi2]; uh, unbiased diversity {[N/(N − 1)] × h}, where pi is the frequency of the ith allele for the population and Sum pi2 is the sum of the squared population allele frequencies. Download FIG S3, PDF file, 0.3 MB.Mean allelic information patterns across geographical and genetic populations. Na, number of different alleles; Ne, number of effective alleles [1/  . One of = 0.001) . The inf= 0.001) . The exi10.1128/mSphere.00019-17.4FIG S4 FIG S4, PDF file, 0.2 MB.Population differentiation among STRUCTURE-inferred genetic clusters. Our clonally corrected data set of 1,230 genotypes was used to infer the number of genetic cluster. Pie charts show percentages of variations among and within populations. PhiPT values are in the lower diagonal of the pairwise comparison table. Probabilities based on 999 permutations are shown above the diagonal. Download Copyright © 2017 Ashu et al.2017Ashu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A. fumigatus. In this study, six of the eight clusters had at least five private alleles each at the nine analyzed loci. The percentages of private alleles by genetic cluster were as follows: for Pop 2, 23% (53/230); for Pop 3, 5.3% (5/95); for Pop 4, 15.8% (26/165); for Pop 5, 12.6% (26/237); for Pop 6, 25.6% (23/237); and for Pop 8, 23.9% (104/237). Pop 1 and Pop 7 had no private alleles.While STRUCTURE analysis is based on the use of allelic association patterns to separate reproductively isolated strains into genetic populations/clusters and can be used to indicate historical differentiations, other indicators can also be used to support the hypothesis of historical differentiation in A. fumigatus genetic clusters (P < 0.01). Similarly, evidence for both clonality and recombination was also found within individual geographic populations of A. fumigatus  environmental niche was unknown. Download Copyright © 2017 Ashu et al.2017Ashu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the r2 = 0.002) at the global scale.Similarly, there is abundant evidence for gene flow among geographic populations. First, pairwise PhiPT values between most geographic populations were low . Second,A. fumigatus.While the allele and genotype sharing between geographically distant countries such as those described above represents long-distance gene flow, evidence for gene flow among regions within individual countries is also abundant. For example, the samples from within India were obtained from diverse locations separated by hundreds of kilometers and several genotypes were found to be widely distributed across several locations. Taken together, our results suggest frequent gene flows among regional and continental populations of P = 0.001). Interestingly, while the triazole-susceptible populations showed relatively little genetic difference among geographic populations, the triazole-resistant isolates separated based on geographic origins showed a large amount of genetic differentiation  (see P = 0.001). Taken together, the results from these analyses suggest that clonal expansion of triazole resistance genotypes plays a significant role in geographic structuring of A. fumigatus.About 6% of the isolates in our data set showed resistance to triazole antifungal drugs. Overall, there was a small but statistically significant genetic difference between the triazole-susceptible and triazole-resistant samples  see . We furt10.1128/mSphere.00019-17.6FIG S6 FIG S6, PDF file, 0.2 MB.Contribution of geographic separation to genetic variation in the triazole-resistant sample of 102 isolates. Pie charts show percentages of variations among and within populations that can be explained by grouping resistant isolates by geographic origin. A total of 106 resistant isolates from five countries were identified; however, countries with fewer than seven isolates were excluded from our analysis of molecular variance. PhiPT values are in the lower diagonal of the pairwise comparison table. Probabilities based on 999 permutations are shown above the diagonal. Download Copyright © 2017 Ashu et al.2017Ashu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the  A. fumigatus, a result consistent with historical differentiation. However, we noted low differentiation levels and frequent sharing of alleles and genotypes among geographic and ecological populations, suggesting that contemporary gene flow is prevalent. Our analyses also identified abundant signatures of both sexual recombination and local clonal expansion. Local clonal expansions were most evident in geographic populations where resistance to triazole antifungal drugs was prevalent. Taken together, our results suggest that both historical and contemporary factors have played a significant role in shaping the global population structure of A. fumigatus.We investigated the global population structure using nine highly informative microsatellite markers to analyze a comprehensive set of isolates from four continents. Our analyses showed the existence of eight significantly differentiated genetic clusters within the global sample ofA. fumigatus genetic clusters and suggested that one of these clusters could be a cryptic Aspergillus species. In 2012, Klaassen et al.  for each of the eight genetic clusters is unresolved. Generally, the geographic center of origin should contain the highest allelic and genotypic diversities. In fungi, abundant evidence of sexual recombination has also been used as a signature for ancestral populations. However, uneven sample sizes and incomplete sampling in many countries make any conclusions at present extremely tentative . AdditioA. fumigatus, limited but statistically significant population differentiations were observed among certain geographical or ecological populations. Finlay argued that microbes smaller than about 2 µm in size are unlikely to be restricted by geographical barriers in their dispersals  for such long-distance dispersals are of utmost importance in containment and eradication of pathogens. LDD events have been previously reported to play a crucial role in fungal pathogen recolonization, invasion, and adaptation to host resistance  was likely spread from Vancouver Island to mainland British Columbia, Canada, and the Pacific Northwest of the United States in dust on cars and under shoes of travelers that took the ferry to Vancouver City , which can be assisted or not assisted by travel by humans. Assisted LDD refers to dispersal involving humans or associated with human artifacts whereby travelers can carry conidia on/in their bodies or on infested inanimate objects from one region to another far away. In contrast, unassisted LDD involves dispersals without human interventions, e.g., by air currents. Our data here show that certain microsatellite genotypes are found in countries up to 7,500 km apart. For example, genotype 115 was isolated from air in Belgium and from patients in the United States. However, whether dispersal of this genotype and other shared genotypes of sistance . For exa of 1978 . Similarver City . Owing tA. fumigatus, frequent gene flow, which is known to facilitate dissemination of alleles highly beneficial to the fungus such as those conferring triazole resistance, can be attributed to the abundance of asexual spores, their capability of long-distance dispersal, and their nonfastidious requirements for growth and reproduction. The limited geographic differentiation, the presence of multiple private alleles within individual geographic and genetic populations, and the evidence for gene flow among global populations of A. fumigatus indicate that the evolution of A. fumigatus will be continuously impacted by both local and global factors.Gene flow can have both advantageous and disadvantageous consequences with respect to the selective maintenance of genetic variation within and between microbial populations. For instance, gene flow is thought to limit population divergence and hence local selection. Gene swamping into naive populations favors fixing of alleles with the best average reproductive success, thereby counteracting the stability of local selection . HoweverCYP51A gene from the Netherlands belonged to one genetic cluster (Pop 3 in their study and Pop 8 here in our study). Interestingly, while Pop 8 contained 80% (35/44) of all triazole-resistant genotypes, triazole resistance genotypes were also found in other genetic clusters. However, our results do indicate that the frequency of triazole resistance in Pop 8  was at least four times higher than that in any other genetic population, suggesting that special attention should be paid to understanding this genetic cluster. Since the gene(s) related to resistance is not known to be tightly linked to the analyzed nine microstatellite loci, the tight clustering of most triazole-resistant isolates in Pop 8 suggests three possibilities. The first possibility is that Pop 8 is more frequently distributed in geographic regions where triazole drug use is very common. It has been previously hypothesized that extensive agricultural use of azole fungicides in A. fumigatus in India and elsewhere leads to resistance (P = 0.001). Using Escherichia coli as a model organism, it has been experimentally demonstrated that the strength of antibiotic exposure plays a significant role in the evolution of antibiotic resistance (Puccinia graminis f. sp. tritici and Pyrenophora tritici-repentis (A. fumigatus genetic populations could have significant implications in the initiation and dissemination of resistant/virulent strains capable of causing aspergillosis outbreaks (Our analyses suggest that genetic Pop 2 and Pop 8 are the most widely distributed and are recombining. They also contained the most private alleles. The previous study by Klaassen et al.  revealedined 80% /44 of alsistance . Althougsistance . The difsistance . The secrepentis , 40. Therepentis  did not utbreaks .A. fumigatus populations which may allow faster adaptation to host resistance, it can, however, be costly in terms of energy and time and may have no measurable advantage in new ecological niches (21–A. fumigatus population structure. Indeed, the clonal spread of adaptive recombinant progeny resistant to multiple triazole drugs in India was the main cause of the significant genetic differentiations between the Indian A. fumigatus population and those isolated elsewhere in the world.Although sexual reproduction can generate genotypic variation among l niches . This col niches , 21–23. A. fumigatus. Our findings go beyond those previously identified by other studies. We report limited but statistically significant genetic differentiations among geographic and ecological populations of A. fumigatus. The identification of eight genetically differentiated clusters is consistent with historical differentiation, but contemporary gene flows are blurring the historical patterns. Interestingly, unlike the triazole-susceptible samples, where geographic populations were largely undifferentiated, triazole-resistant samples were significantly differentiated according to geographic region. The differences in triazole usage among the countries have likely contributed to the genetic differences among the triazole-resistant samples. Though evidence for clonality was found in all geographic, ecological, and genetic populations, we also found evidence for recombination in all analyzed populations, a result different from those reported in earlier studies (–A. fumigatus migration patterns. From a practical perspective, our findings should aid in better tracking and management of aspergillosis outbreaks.In conclusion, using a large number of isolates from geographically and ecologically diverse regions, our study allowed us to address several fundamental questions about the global population of  studies –10. ThisSamples used in this study were obtained as part of collaborative studies between Canisius Wilhelmina Hospital and research centers in several countries , 43–49. 43–4 iterations, thereby validating the run length. Two approaches were used to identify the optimal number of clusters. The ad hoc statistic DK, based on the rate of change in the log probability of data between successive runs of K, was calculated as previously recommended (In order to investigate the existence of distinct genetic clusters, we used both multivariate and model-based Bayesian clustering as implemented in the ADEGENET package in R version 3.0 and STRUCTURE software version 2.3 , 51. Mulommended . The secIn order to identify the potential contributors to the observed genetic variation, the pairwise samples grouped based on different criteria were compared using the GenAlEx version 6.5 . In thesNei’s genetic diversity corrected for sample size was calculated for independent populations using GenAlEx version 6.5 . The mul


Symphodus melops), a rocky shore species at the northern limit of its distribution range in Scandinavia. Our results revealed a major genetic break separating populations inhabiting the western and southern coasts of Norway. This genetic break coincides with the longest stretch of sand in the whole study area, suggesting habitat fragmentation as a major driver of genetic differentiation of this obligate rocky shore benthic fish in Scandinavia. The complex fjords systems extending along the western coast of Norway appeared responsible for further regional genetic structuring. Our findings indicate that habitat discontinuities may lead to significant genetic fragmentation over short geographical distances, even for marine species with a pelagic larval phase, as for this rocky shore fish.Habitat fragmentation has been suggested to be responsible for major genetic differentiations in a range of marine organisms. In this study, we combined genetic data and environmental information to unravel the relative role of geography and habitat heterogeneity on patterns of genetic population structure of corkwing wrasse ( Connectivity between geographically separated populations plays a pivotal role in populations dynamics and genetic diversity . The preBiological attributes of species, such as dispersal ability and reproductive mode, also play an important role on population demography, connectivity and the location of genetic discontinuities or breaks –12. In sSymphodus melops) is a small rocky shore fish inhabiting coastal areas of the Northeast Atlantic and reaches its northern limit in Scandinavia. Previous population genetic studies on this species revealed a major genetic break across the North Sea, and ascribed the significant reduction in genetic diversity of the northern populations to the postglacial colonization of the Scandinavian Peninsula [Corkwing wrasse  is a small benthic fish inhabiting the first few meters of the rocky shorelines of the Northeast Atlantic from southern Portugal to Norway and the western part of the Mediterranean Sea [Corkwing wrasse . After nean Sea ).et al. [The present study focuses on populations inhabiting the Norwegian coastline of North Europe. This area stands as the northern limit of distribution of many temperature species, including the corkwing wrasse, as well as the southern limit of some cold water species , with a et al. –32). Theet al. –37.http://www.lovdata.no. Accessed 2016 July 30). Practices for sampling and handling of fish for this study were approved by the Norwegian Animal Research Authority and were performed by experienced personnel. The species is not protected by Norwegian law , and no special permits were required either for research or commercially at sampled locations. Muscle tissue was taken from fresh or frozen specimens and stored in 96% ethanol prior to DNA extraction.The sampling design aimed at a geographically fine scale coverage of the southern and the western coast of Norway, to complement earlier investigations ,23. The Tm-600 Liz as size standard on an ABI 3130XL automated sequencer. As a guard against potential genotyping errors, all samples were run with the same size standard and on the same machine as those previously used by Knutsen et al. [Total genomic DNA was extracted from ethanol-preserved flesh using either the Viogene Inc. extraction kit  or the DNeasy kit , re-suspending the DNA in TE buffer. Microsatellite polymorphism was screened at nine polymorphic markers and following the same PCR protocols as described earlier for the species ,38. DNA n et al. . CapillaA), allelic richness (Ar) and gene diversity within samples (HS) and the total over all samples (HT), based on Nei & Chesser [Levels of genetic variation were characterized by counting observed alleles . Deviat Chesser ). Here,  Chesser  when int Chesser ). The pr Chesser ).FST, using Weir & Cockerham’s [θ applied to all samples, to pairs of sample localities and also within and among geographic regions. The statistical significance of observed genetic differentiation was estimated by 10 000 permutations in GENETIX . We adoon 4.05;  to correK between 1 and 10 and with Markov chain Monte Carlo (MCMC) resampling using 100 000 repetitions after a burn-in of 100 000. The most likely number of clusters, K, was estimated as the value which maximized the averaged log-likelihood, log Pr(X|K) and the ad hoc statistic ΔK [K was determined, individuals were assigned to the respective clusters and plotted with DISTRUCT . This aistic ΔK . Once K ion 1.1; ).Second, in order to visualize spatial patterns of population structure and locate discontinuities among corkwing wrasse populations, we used GENELAND  in R 5252. The aThird, a neighbour-joining (NJ) tree, based on the modified Cavalli-Sforza’s distance Da of Nei et al. , was conFST estimates, FST ⁄ (1—FST)). Pairwise geographic distances between sample locations were calculated following the coastline (range of distances between 12 and 1259 km). Patterns of IBD were investigated comparing genetic and geographic distance matrices by Mantel tests with 10 000 permutations in IBDWS [http://kart.naturbase.no/) and imported in QGIS [We examined the relative importance of geographic distance  and habitat discontinuity , on spatial patterns of genetic differentiation (linearized pairwise in IBDWS . For the in QGIS . The IBE in QGIS  controll in QGIS  with 10  in QGIS . This apHT = 0.253) to high for SMB11 .Results were based on 1437 individuals genotyped at nine microsatellite markers with a successful coverage rate > 99% . A total of 162 alleles were scored . Levels FST = 0.064, P < 0.001, FST estimates averaged over loci ranged greatly among sample localities, from -0.002 to 0.150  than did western samples . Genetic differentiation within the western region appears to be driven largely by the two most northern samples  compared to southern samples .Genetic variability varied greatly among sample localities in the southern and the western regions . Omitting one of the three locus at a time  overall FST yielded similar results ; therefore, we decided to proceed the downstream analyses keeping all loci.Deviations from Hardy-Weinberg (HW) expectations were significant in 11 of 144 (5.5%) cases. Four of them (2.8%) remained statistically significant at the 5% level also after FDR correction  were found to be significant (at the 5% level) in 30 of 576 pairwise tests (5.2%) before correction for multiple tests. Significant outcomes appeared randomly distributed among samples and only one pair (SMB11-SMD112 in SMO) remained statistically significant after the FDR approach (at the 5% level). Therefore, our results are consistent with the loci being independent.The STRUCTURE analysis uncovered two distinct clusters (K = 2) of sample localities . One clu; i.e. Boknafjord, Hardangerfjord and northern samples (SMO and VES), with relatively high bootstrap support (64–79%). In the Skagerrak, EGE clustered apart from the rest of the samples (94% bootstrap support); nevertheless, branch lengths separating Skagerrak samples were usually shorter than those separating western Scandinavian samples.The NJ tree based on Da genetic distance corroborated the existence of two major clusters (100% bootstrap support) comprising populations inhabiting the west coast of Scandinavia and the Skagerrak coast . The ana2 = 0.399, P < 0.01, 2 = 0.428, P < 0.01) between geographical distance and genetic differentiation, but gave considerably higher score to IBE as an explanatory variable for the spatial patterns of genetic structure . Genetic distance remained significantly correlated to both geographic distance  and environmental distance  when the influence of one of the other factor was controlled in partial Mantel tests  was a much stronger predictor of the observed genetic patterns than was geographic distance  , whereas no correlation between genetic and environmental matrices was detected when controlling for geographic distances . The MMRR analysis confirmed that geographic distance  was a stronger predictor of the observed genetic patterns than environmental distance . Both factors together explained nearly 70% of the genetic variability in the west coast  distance showed moderate correlation (Rel tests . Integra < 0.01) . The inf < 0.01) . In the st coast .FST = 0.107, P < 0.001, The current study performed on corkwing wrasse collected along the Norwegian coast revealed the existence of a major genetic discontinuity or “break” in this rocky shore fish species. The genetic break was located in the south-western part of the Scandinavian Peninsula, separating populations inhabiting the western and southern  coast of the Scandinavian Peninsula , with higher levels in the west cf. . These fOur analyses suggested environmental distance in the form of long stretches of sandy areas to be a better predictor of current patterns of genetic population structure in Scandinavia than is geographical distance , Table 4Genetic breaks have usually been concordant with the location of strong historical oceanographic features and biogeographic breaks, reflecting the parallelism between the processes governing geographic and genealogical boundaries of the species ,61–63. HRegionally, corkwing wrasse revealed further patterns of genetic substructure c.f. . WesternCurrent findings have important implications for fisheries management and conservation, considering that corkwing wrasse is intensively exploited for use as a cleaner fish in the salmon industry ,30. A maS1 FigThe size of the bubble corresponds to the frequency of the respective allele in the sample.(PDF)Click here for additional data file.S2 FigK = 2 to 5). The right graph shows ΔK for different numbers of genetic clusters, suggesting K = 2 as the most likely outcome.Each vertical bar in the left graph denotes an individual fish, whilst colours denote inferred clusters ((PDF)Click here for additional data file.S1 TableA = number of alleles, HT = gene diversity in the total material; FST = estimate of θ (Weir & Cockerham 1984). Numbers in bold indicate statistical significant tests at 1% level after the False Discovery Rate approach (Benjamini & Hochberg 1995).(PDF)Click here for additional data file.


Aeschynomene, some have the unique property of being root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions.Among semi-aquatic species of the legume genus ITS and low-copy nuclear genes, along with cytogenetic experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance, two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem nodulation.A combination of phylogenetic analyses, comprising Aeschynomene species can be efficiently nodulated in a Nod-independent fashion.A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some The online version of this article (10.1186/s12870-018-1260-2) contains supplementary material, which is available to authorized users. Leguminosae) accounts for ~ 27% of the world’s primary crop production and is second only to cereals in economic and nutritional value. It includes many crops of agronomic importance for grain production, pasture and agroforestry. Many legumes are pioneers plants improving soil fertility and moderating harsh environments. Such economic and ecological success of the legume family is, in large part, due to the ability of the vast majority of its 20,000 species to develop symbiotic interactions with nitrogen-fixing bacteria collectively referred as rhizobia . This must facilitate the identification and distinction of the different subgenomic components of the polyploid genomes and this indicated that these polyploid taxa must behave genetically as diploids. Segregation of molecular markers, however, has not been performed to date at a genome-wide scale and we therefore cannot exclude that they might behave as segmental allopolyploids just as recently reported for peanut and chrysanthemum [A. pratensis for which the two expected homeologous versions of the tested low-copy nuclear genes were not always detected.This in-depth characterization of a Nod-independent anthemum , 28. SucAeschynomene species, our analyses were used to identify different taxa and to delimit their taxonomic boundaries. Indeed, the approach of genotyping a germplasm collection already has shown to be a powerful tool resolving taxonomic issues and providing the basis of a good taxonomic classification in the legume genus Lens that includes the cultivated lentil [Aeschynomene pluriarticulata and A. magna were found to have ITS and low-copy nuclear gene sequences very similar to those of A. evenia and A. denticulata, respectively  but are clearly distinct entities in the SSR tree ; this taxon was consistently found to be divergent in the ITS, low-copy nuclear gene and the SSR trees , others in the production of small white infected nodules proved to be defective in nitrogen fixation (Fix−) [All these data taken together make our germplasm collection a valuable genetic resource for the Nod-independent  species , 18. Theogenesis , 4. But n (Fix−) –38. ThisLYK3 in Medicago as corresponding to the SYM2 gene that controls the symbiotic infection in a Nod factor structure dependent manner in the pea ‘Afghanistan’ ecotype [Sinorhizobium meliloti Rm41 to form functional or non-nitrogen fixing nodules depending on the Medicago accession used, two genes, NFS1 and NFS2, were identified and shown to code for NCR peptides [Rj2/Rfg1 encoding a TIR-NBS-LRR resistance (R) gene and Rj4 that codes for a thaumatin-like protein [It is noteworthy that the genetic analysis of natural variation shed light on the function of some key symbiotic genes. The LysM-RLK receptors, which were identified using the mutant approach to be the probable Nod factor receptors, represent a well-known example. Indeed, a synteny-based positional cloning identified  ecotype , 38. The ecotype . More repeptides –40. This protein . This reAeschynomene group. To screen our germplasm collection, many Bradyrhizobium strains are available, including photosynthetic and non-photosynthetic ones [Aeschynomene species to be nodulated in a T3SS-dependent fashion by the non-photosynthetic Bradyrhizobium strains STM6978 and USDA61 [A. evenia s.s., providing the basis for a genetic analysis of this differential susceptibility. Variations in symbiotic traits with naturally-nodulating photosynthetic Bradyrhizobium strains have not been thoroughly surveyed yet, but the observations made in the present study using the strains ORS278 and BTAi1 yield promising preliminary results. By evaluating different genotype-rhizobial combinations, we predict that symbiotic polymorphisms will be found, notably in the induction of nodulation, nitrogen fixation and also in stem nodulation. Crossable accessions exhibiting polymorphic symbiotic phenotypes can then be selected for hybridization experiments, subsequent genetic analysis and locus mapping. This will pave the way for the identification of the genes underpinning these symbiotic responses through forward or reverse genetics. To assist in these studies, genomic and genetic data, together with a number of molecular tools, are being accumulated for the model species A. evenia and it thus represents the easiest system to work with [A. evenia, the number of available germplasm samples remains relatively modest since it has not been as extensively sampled as other legumes of interest [A. sensitiva, represent a good complement. In addition, the expected high level of microsynteny with A. evenia will facilitate a synteny-based positional approach, as has been successfully performed between Pea and Medicago to identify the gene underlying the SYM2 locus [Aeschynomene clade can be fully exploited for the search of natural variation in nodulation with Bradyrhizobium.These illuminating examples show that there is a mileage to be gained from exploring the natural variation in nodulation in the Nod-independent tic ones , 43, 44.d USDA61 . Such maork with , 20. Desinterest . TherefoM2 locus , 45. As Aeschynomene legumes, we developed a large collection of 226 accessions that spans the geographical distribution of the different taxa in this group, including the model species A. evenia. These accessions were subjected to combined analyses of gene sequencing, cytogenetics, hybridization experiments and SSR genotyping. This work resulted in the delineation of taxon boundaries and in the discovery of new genotypes. Taxa displaying a significant genetic diversity were characterized by a clear geographically-based genetic structure. In addition, low-copy nuclear genes and patterns of microsatellite diversity illuminated the genetic basis of the Aeschynomene diploid and polyploid taxa that are all predominantly autogamous and have a predicted simple disomic inheritance, two attributes favorable for genetics. Such a well-characterized collection of accessions constitutes a major genetic resource for exploring the natural variation of nodulation in response to different Bradyrhizobia strains and for searching for their underlying genetic determinants. Discoveries of alternative functions in symbiotic genes identified in other model legumes or of new genes involved in the recognition of the still unknown non-Nod bacterial signal, nodule functioning and in the restriction of compatibility would add a new dimension to our understanding of the genetic control of nodulation in the Nod-independent symbiosis.With the goal of discovering natural variation in the Nod-independent Aeschynomene used in this study, their geographical origin and source data are listed in Additional file All the accessions of Bradyrhizobium sp. strains ORS278 and BTAi1 [Nodulation tests were carried out using nd BTAi1 . The strnd BTAi1 .Lycopersicum esculentum (Solanaceae) cv “Roma” (2C = 1.99 pg) as the internal standard. For chromosome number counts, metaphasic chromosomes were prepared from root-tips, spread on slides and stained with DAPI . Chromosomes were counted from images obtained with a fluorescent microscope as previously described [Flow cytometry measurement was performed on leaf material to estimate genome sizes of various accessions as already explained . These eescribed .Genomic DNA was extracted from fresh leaves using the CTAB (Cetyl Trimethyl Ammonium Bromide) method improved by the addition of β-mercaptoethanol 2% and PVPP 2%. For herbarium material, a protocol adapted for fragmented DNA was used, with increased length of the incubation (90 min), centrifugation (20 min) and precipitation (15 min) steps was used. DNA quantity was evaluated by spectrophotometer and DNA samples normalized to a uniform concentration of 10 ng/μL.CYP1, eiF1α, Sucrose Synthase, SUI1 and a gene homolog to Glyma.07G136800 and Glyma.18G187300 identified in Glycine max were amplified with the primers listed in Additional file The nuclear ribosomal internal transcribed spacer region (ITS: ITS1-5.8S rDNA gene-ITS2) and five low copy nuclear genes A. evenia  and the observed heterozygosities Ho for each SSR locus and for different accession groups Additional file 2:Table S2. Nuclear genes used for the phylogenetic analyses. (XLSX 11 kb)Additional file 3:Table S3. GenBank numbers for the sequences used in the phylogenetic analyses. (XLSX 12 kb)Additional file 4:Doc. S1.ITS sequences obtained for the Nod-independent Aeschynomene accessions. (DOCX 30 kb)Additional file 5:Figure S1. Chromosome numbers in new Aeschynomene taxa. Root tip metaphase chromosomes stained in blue with DAPI . Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 1008 kb)Additional file 6:Figure S2. Schematic representation of the different steps of the genotyping process from marker selection to data treatment. (PPTX 64 kb)Additional file 7:Table S4. Origin, location and primer sequences for the SSR markers used for genotyping. (XLSX 13 kb)Additional file 8:Table S5. Repeat motif and allelic amplification profiles of the SSR selected for genotyping. (XLSX 13 kb)Additional file 9:Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file Additional file 10:Table S6. Allelic diversity of the SSR markers (XLSX 22 kb)Additional file 11:Table S7. Cross-species transferability of the SSR markers (XLSX 13 kb)Additional file 12:Table S8. Scoring of mean allele number per SSR and species (XLSX 15 kb)Additional file 13:Table S9. Observed heterozygosity (Ho) in Aeschynomene species (XLSX 14 kb)Additional file 14:Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb)


Although genetic diversity ultimately determines the ability of organisms to adapt to environmental changes, conservation assessments like the widely used International Union for Conservation of Nature (IUCN) Red List Criteria do not explicitly consider genetic information. Including a genetic dimension into the IUCN Red List Criteria would greatly enhance conservation efforts, because the demographic parameters traditionally considered are poor predictors of the evolutionary resilience of natural populations to global change. Here we perform the first genomic assessment of genetic diversity, gene flow, and patterns of local adaptation in tropical plant species belonging to different IUCN Red List Categories. Employing RAD-sequencing we identified tens of thousands of single-nucleotide polymorphisms in an endangered narrow-endemic and a least concern widespread morning glory (Convolvulaceae) from Amazonian savannas, a highly threatened and under-protected tropical ecosystem. Our results reveal greater genetic diversity and less spatial genetic structure in the endangered species. Whereas terrain roughness affected gene flow in both species, forested and mining areas were found to hinder gene flow in the endangered plant. Finally we implemented environmental association tests and genome scans for selection, and identified a higher proportion of candidate adaptive loci in the widespread species. These mainly contained genes related to pathogen resistance and physiological adaptations to life in nutrient-limited environments. Our study emphasizes that IUCN Red List Criteria do not always prioritize species with low genetic diversity or whose genetic variation is being affected by habitat loss and fragmentation, and calls for the inclusion of genetic information into conservation assessments. More generally, our study exemplifies how landscape genomic tools can be employed to assess the status, threats and adaptive responses of imperiled biodiversity. Species conservation assessments have largely neglected measures of genetic diversity, even though genetic diversity ultimately determines the ability to adapt to environmental changes and therefore underpins species long-term persistence  from Amazonian savannas, one of the least known, highly threatened and under-protected tropical ecosystems  Gene flow is probably affected by the structure of natural landscapes in both species, but landscape modifications by mining are more likely to hinder gene flow in the narrowly-distributed I. cavalcantei. Additionally, landscape effects on gene flow are expected to show time-lags determined by the generation time of each species ; (iii) Although similar adaptations to the extreme montane savanna environments are expected in both species, genetic drift is likely stronger in the narrowly-distributed species, thus slowing adaptive responses. We therefore expect to find weaker adaptive signals (a lower proportion of candidate loci) in I. cavalcantei.We hypothesized that extent of occurrence , observing a minimum distance of 10 m between samples to avoid re-sampling the same individuals   for sequencing and bioinformatic analyses. Briefly, nextRAD genotyping-by-sequencing libraries were prepared  and a de novo reference was created by collecting 10 million reads evenly from the samples, excluding reads that had counts fewer than 10 or more than 400. The remaining loci were then aligned to each other to identify alleles. All reads were mapped to the reference with an alignment identity threshold of 95% using BBMap tools (bbmap). Genotype calling was done using Samtools and bcftools , and the resulting set of genotypes was filtered to remove loci with a minor allele frequency of <2.5%. Loci with more than 30% missing genotypes, or that were heterozygous in all samples or contained more than 2 alleles in a sample  were removed. The absence of artifacts was checked by counting SNPs at each read nucleotide position and determining that SNP number did not increase with reduced base quality at the end of the read. This pipeline was carried out separately for each species, with a de novo reference created separately for each .Reads were trimmed using https://github.com/nspope/r2vcftools) - a wrapper for VCFtools , and strong deviations from the Hardy Weinberg Equilibrium . Additionally, we removed any potential loci under selection detected through environmental association and FST outlier tests (see details below). The resulting sets of neutral and independent loci were then used to calculate genetic relatedness , inbreeding coefficients (F), mean per-site nucleotide diversity (π) and Tajima's D (D). Effective population size (Ne) was also estimated employing the linkage disequilibrium approach of NeEstimator 2.0.1 and a threshold lowest allele frequency value of 0.05  package  was allowed to vary between 1 and 10, with 10 replicate runs for each k-value, and the best k was chosen based on cross-entropy and cross-validation errors .To address our first prediction we estimated genetic diversity and assessed spatial genetic structure. The R package r2vcftools (2) and isolation (distance to the nearest highland in meters) using a pre-mining (1984) mapping of montane savanna highlands  as response variables, area and isolation as predictors, and highland ID as a random effect   using the dredge function from the MuMIn (v1.4) package  of pairwise relatedness to pairwise geographic distance , but not for LD or HWE. Adaptive signals were evaluated across all SNPs, even though many contigs contained more than one SNP. We thereby avoided the arbitrary exclusion of SNPs and the loss of any potentially adaptive signal. Linkage disequilibrium was nevertheless low within our 150 bp contigs . These were minimum temperature of coldest month, precipitation of warmest quarter, and precipitation of wettest quarter for I. cavalcantei; and minimum temperature of coldest month, precipitation of wettest quarter, and precipitation of coldest quarter for I. maurandioides. Correlations between these variables were weak (r < 0.3), except for minimum temperature of coldest month and precipitation of wettest quarter in I. maurandioides (r = 0.67). We then used these variables to run LFMM using the LEA R package. Since incorrect assumptions about underlying demographic structure can increase both Type I and Type II errors , and then intersected candidate loci detected across all runs. Models were ran for k ± 2 latent factors . We used 10,000 iterations, a burn-in of 5,000 and five runs per environmental variable  and false discovery rates were set using the Benjamini-Hochberg algorithm . In addition, we ran genome scans for selection using the snmf function from the LEA package, to identify outlier loci based on FST only  and false discovery rates were set using the Benjamini-Hochberg algorithm. Full R scripts of LFMM and genome scans for selection can be found in the supporting information of François et al. (http://membres-timc.imag.fr/Olivier.Francois/LEA/index.htm). To evaluate if adaptive signals differed between species we used a Chi-square test to compare the number of contigs containing candidate SNPs in relation to all the tested contigs.Finally, we employed environmental association and s Figure , so mosts Figure . We firss Figure , and sels et al.  and the http://www.ebi.ac.uk/Tools/st/emboss_transeq/) to obtain its corresponding protein sequences. We used all six frames with standard code (codon table), regions (start-end), trimming (yes), and reverse(no). We then ran a functional analysis using InterPro , searching for go terms and pathways along the respective annotation databases .In order to search for the proteins coded by the genes contained in the flanking regions of our candidate SNPs, contig sequences containing candidate loci were first submitted to the EMBOSS Transeq  we obtained sets of neutral markers containing 14,145 and 7,259 loci respectively. While heterozygosity and inbreeding levels were similar between I. cavalcantei and the analyzed populations of I. maurandioides, nucleotide diversity, Tajima's D and effective population size were higher in I. cavalcantei , and 3,065 contigs containing candidate SNPs out of 9,143 analyzed contig sequences in I. maurandioides . While the panmictic population of I. cavalcantei occurs both in preserved and degraded montane savanna highlands  were equally informative in the case of I. maurandioides  in the narrow-endemic species, thereby suggesting that stronger genetic drift is slowing adaptive responses in  1 Table , indicatFST outlier tests in both species  was found associated to all environmental variables and was also identified through es Table . This pres Table . Indeed es Table , while Les Table . On the es Table , and deaes Table . Finallyes Table . Interests Table . Our rests Table . These pts Table .Ne of 50 is deemed necessary to avoid short-term inbreeding depression, while an Ne of 500 is recommended to maintain long-term evolutionary potential. More recently, Willoughby et al. . On the other hand, precautionary conservation measures should aim at maintaining Ne > 50 within each population to avoid short term inbreeding depression in I. maurandioides. Although land cover was found to influence gene flow in the the narrow-endemic species, overall population panmixia indicates that a substantial level of genetic connectivity is maintained across the fragmented habitat of I. cavalcantei. Additionally, we identified possible adaptations for life in Amazonian savannas, which could inform future conservation and restoration programs.Most conservation geneticists have used the 50/500 rule to define minimum viable populations . We are well aware this will be no easy endeavor, as funding limitations, country-specific conservation policies and communication barriers between scientists and practitioners are among the issues to overcome (Hoban et al., Our study exemplifies how landscape genomic tools can be applied to assess the conservation status, susceptibility to global change, and adaptive responses of imperiled biodiversity. More specifically, our genomic assessment revealed unexpected patterns of genetic diversity, spatial genetic structure and gene flow in tropical plant species belonging to different IUCN Red List Categories. Given the level of resolution of our genetic diversity and gene flow estimates, our findings emphasize that the IUCN Red List Criteria approach does not systematically prioritize the conservation of species with low genetic diversity (Rivers et al., RJ: conceived, designed and coordinated the project; RJ and VI-F: initiated the project; RJ, ÉL, and AG: coordinated the field work and sampling; ÉL, NC, WM, and SV: performed laboratory work; RJ, ÉL, NP, RA, WM, AS, and PS-F: performed the data analysis. The first draft of the paper was written by ÉL with input from RJ. All authors contributed to discussing the results and editing the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FADS1) gene changed omega-3 fatty acid (FA) intake and blood levels in young female adults (18–25 years). Participants were randomized into Genetic (intervention) and Non-Genetic (control) groups, with measurements taken at Baseline and Final (12 weeks). Dietary intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was assessed using an omega-3 food frequency questionnaire. Red blood cell (RBC) FA content was quantified by gas chromatography. Implications of participation in a nutrigenetics study and awareness of omega-3 FAs were assessed with online questionnaires. Upon completion of the study, EPA and DHA intake increased significantly (p = 1.0 × 10−4) in all participants. This change was reflected by small increases in RBC %EPA. Participants in the Genetic group showed increased awareness of omega-3 terminology by the end of the study, reported that the dietary recommendations were more useful, and rated cost as a barrier to omega-3 consumption less often than those in the Non-Genetic group. Providing participants FADS1 genetic information did not appear to influence omega-3 intake during the 12 weeks, but did change perceptions and behaviors related to omega-3 FAs in this timeframe.Nutrigenetics research is anticipated to lay the foundation for personalized dietary recommendations; however, it remains unclear if providing individuals with their personal genetic information changes dietary behaviors. Our objective was to evaluate if providing information for a common variant in the fatty acid desaturase 1 ( The field of nutritional genomics, or nutrigenetics, aims to unravel the genetic basis for why individuals respond differently to the same nutrients and/or foods ,2,3. TheTo date, the vast majority of nutrigenetic studies have examined and assessed perceptions of genetic and health information in various populations ,4,5. When-3) and docosahexaenoic acid , is beneficial for cardiovascular, metabolic, developmental and cognitive health [Omega-3 fatty acids (FAs) represent an ideal nutrient to examine in the context of a nutrigenetics intervention for several reasons. From a global health perspective, it is widely recognized that increased intake of omega-3 FAs, in particular eicosapentaenoic acid . The fatty acid desaturase 1 and 2 genes (FADS1 and FADS2) play critical roles in this pathway [FADS genes influence the degree of endogenous conversion of ALA into EPA and DHA [FADS1 and/or FADS2 have been reported to have reduced desaturase activity, resulting in lower levels of EPA [FADS genotype information may yield a new approach to encourage increased intake and optimize dietary recommendations for omega-3 FAs.From a genetic perspective, EPA and DHA are endogenously produced to a limited extent through a well-characterized pathway that desaturates and elongates the essential omega-3 FA, alpha-linolenic acid  were recruited through emails and poster advertisements displayed around the University of Guelph campus. Individuals completed an online screening questionnaire prior to acceptance into the study. Individuals were ineligible to participate in the study if they regularly consumed omega-3 FA supplements and/or fish more than two times per week. Baseline and Final (week 12) study visits were scheduled when participants were menstruating to minimize variability in lipid levels within an individual, as recommended by Mumford et al. . TherefoParticipants attended three in-person study visits. Participants signed the consent form and provided a saliva sample to be used for DNA analysis at visit #1. Once all DNA samples were analyzed, participants were randomized into either a Genetic group (intervention) or Non-Genetic group (control) using a random number generator by two individuals external to the study intervention. The randomization process used also ensured that each group would have an equivalent number of major and minor allele carriers. The lead investigator for the study intervention was blinded to this assignment. Participants were informed of which group they were in at Visit #2.FADS1 and omega-3 metabolism. Specifically, this document provided information about omega-3 FAs, including general nutritional information, foods and supplements that have these FAs, and possible health effects associated with their consumption; compiled from the Dietitians of Canada fact sheets [FADS1 (rs174537). Specifically, it was indicated that individuals who are homozygote GG allele carriers have been reported to have more EPA in their bodies and an increased ability to convert ALA into EPA and DHA [FADS1.Visit #2 corresponded to the baseline study visit. Participants provided fasted blood samples, had a one-on-one information session, and received a copy of all study intervention materials. At this visit, all participants were also given a generic information document that provided details of omega-3 FAs, and an overview of the association between SNPs in t sheets ,26. Furt and DHA ,28,29, w and DHA ,22. ThisFADS1 genotype. If the participants were in the Non-Genetic group, the letter indicated that they would receive their genetic information at the end of the study. Importantly, we did not provide any dietary advice in a genotype-specific manner, as the primary goal of the study was to assess if individuals who were given their personal FADS1 genetic information changed their omega-3 dietary habits or not.At the end of Visit #2, each participant was given a security-style sealed envelope to open after their appointment that made them aware if they had been randomly selected to the Genetic Group or the Non-Genetic Group. If participants were assigned to the Genetic group, the letter identified them as either a major (GG) or minor (GT + TT) allele carrier, according to their personal FADS1 genetic information at this point.Visit #3 corresponded to the final week 12 study visit. All participants provided a fasted blood sample. Participants in the Non-Genetic group were given their personal Participants were contacted by email throughout the study for survey distribution and appointment scheduling. Participants were not provided with additional omega-3 FA or genetic information throughout the intervention; however, we acknowledge it is possible that participants may have sought out more information independently (not monitored by the research team).Participants completed online food frequency questionnaires (FFQs) at Baseline and Final (week 12) to assess dietary intake of omega-3 FAs. Data on the awareness of omega-3 FAs, perceptions of receiving genetic information, and overall perceptions of the study intervention was also assessed through online questionnaires at Final (week 12). Qualtrics software  was used to host the online surveys. The online surveys were pilot-tested to ensure ease of completion. Participants could complete the surveys within a week of their distribution, on an electronic device of their choosing. There were questions of various types . Survey questions are provided in Dietary intake of omega-3 FAs  was assessed at Baseline and Final (week 12) using a validated Canadian FFQ  that wasThis survey was comprised of researcher-generated and literature-generated questions. We examined the participants’ awareness of terminology used to describe omega-3 FAs, including the full scientific names  and their corresponding abbreviations , at the start and end of the intervention. After the final study visit, participants completed a questionnaire regarding perceived dietary changes and perceptions of the study intervention . This SNP was first reported in a large genome wide association study [rs174537 and estimated desaturase activity was confirmed in the present study [DNA was extracted from saliva using the Oragene DNA collection kit, according to manufacturer’s instructions . Participants were genotyped for the rs174537 SNP in on study , and hason study ,35 and oon study ,36,37,38nt study .v/v), using the methodology established by Folch et al. [n-6) and arachidonic acid . The Omega-3 Index was calculated by summing %EPA and %DHA [Following an overnight fast, venous blood samples were collected from participants at Baseline and Final (week 12) visits. Serum and red blood cells (RBCs) were separated by centrifugation and frozen at −80 °C for subsequent analyses. RBC FAs were extracted with chloroform:methanol  visits. Fasted serum samples were sent to LifeLabs Medical Laboratory Services  for the analysis of triglycerides (TAG), total cholesterol, LDL-cholesterol (LDL-c), and HDL-cholesterol (HDL-c).R software  was used to determine a sufficient sample size prior to commencing the study. A minimum sample size of n = 25 individuals per group was calculated using changes in RBC %EPA reported in previous omega-3 FA intervention studies, and differences in RBC %EPA based on SNPs in FADS1. The %EPA values used to calculate the effect sizes were based on past work from our lab [ our lab ,43 and o our lab ,44. Samp2 tests were used for questions requiring a determination of difference in proportions between the Genetic and Non-Genetic groups. An independent samples t-test was used to determine differences in the means between the Genetic and Non-Genetic groups when analyzing Likert-style questions .All survey data including FFQs, and diet and genetics questionnaires were analyzed using SPSS . Descriptive statistics were completed for all questions to determine trends and potential issues before individual question analysis. A repeated measures two-way analysis of variance (ANOVA) assuming unequal variance was used to analyze dietary intakes of EPA and DHA using Group (Genetic vs. Non-Genetic), Time , and the Group × Time interaction. As a secondary outcome, major (GG) and minor (GT + TT) allele carriers within the Genetic group were also compared to determine any differences related to genotype. A repeated measures two-way ANOVA was also used to analyze awareness of omega-3 terminology, using Group and Time effects as described above. Pearson’s χp ≤ 0.05 was considered statistically significant. R software  was used for correction of multiple comparisons using the Benjamini Hochberg approach [GraphPad Prism  was used to assess normality of the data, and to evaluate differences in anthropometric, clinical, and FA data at Baseline and Final with a repeated measures two-way ANOVA, using Group, Time and Group × Time effects, as described above. A approach ,46.The age of participants ranged from 19–25 years (mean = 22.0 ± 1.5 years). On average, participants rated interest in their personal health as 8.5 ± 1.3, out of a possible score of 10. Self-selected ethnicity, school or employment status, field of study/area of work, and contraceptive use were not different between the Genetic and Non-Genetic groups .rs174537 in FADS1. Our population consisted of 26 homozygous major (GG), 23 heterozygous (GT), and 8 homozygous minor allele (TT) carriers (All participants were genotyped for carriers . The SNPp = 0.27) (p = 1.0 × 10−4).At baseline, participants were consuming an average of 200 ± 29 mg EPA and DHA/day. There were no significant differences in EPA and DHA intake between Genetic and Non-Genetic groups at Baseline . As indi = 0.27) ; howeverFADS1 genotype. Dietary intake of EPA and DHA tended to be higher in GT + TT individuals (272 ± 71 mg/day) than GG individuals (141 ± 38 mg/day) at Baseline (p = 0.06). The same trend was seen at final (week 12), where dietary intake of EPA and DHA was higher in GT + TT individuals  than GG individuals  (p = 0.08). However, the Group × Time interaction revealed there was no significant difference in EPA and DHA intake between GG and GT + TT individuals at Final (week 12) (p = 0.29). The Genetic group was further investigated to determine if there were any differences in EPA and DHA consumption when stratified according to There were 10/56 (~18%) participants (four in the Genetic Group and six in the Non-Genetic Group) who reported initiating supplement use after the first study visit and who continued taking these supplements up to week 12. Out of these 10 participants, nine participants took fish oil supplements and one participant took an algae oil supplement. These participants were amongst those with the highest EPA and DHA intakes measured with the FFQ at Final (week 12).p = 0.02)  . The Ome = 0.02) .There were no differences between the Genetic and Non-Genetic groups at Baseline for any of the clinical parameters measured . As indiThe Genetic and Non-Genetic groups were well matched for their level of awareness of omega-3 terminology at Baseline : Q1. A gp = 0.05) and that this information was useful when they considered their diet throughout the study (p = 0.03), in comparison to the Non-Genetic group  : Q2. Speic group .yes”, “no” and “sometimes” to making changes to overall omega-3 consumption in their diet, respectively. Participants who said “yes” or “sometimes” were then asked a subsequent question about the factors contributing to their dietary changes.At Final (week 12), we asked participants to self-report if they had made changes in their consumption of omega-3 foods, fortified products, or supplements over the course of the study : Q3–Q6. p = 0.09) (Omega-3 foods are expensive” compared to the Non-Genetic group (61%).There were 8/28 (28.6%) in the Genetic Group and 5/28 (17.9%) in the Non-Genetic group who reported that the reason for their dietary changes were related to the generic omega-3 information document provided to them at the start of the study ( = 0.09) : Q7. How = 0.09) : Q8. Res = 0.09) , althougFADS1 genetic information on the consumption of omega-3 FAs. We found that individuals in both the Genetic and Non-Genetic groups increased their intake of EPA and DHA by the end of the study, and this was reflected by significant increases in RBC %EPA. Both the Genetic and the Non-Genetic groups were meeting the minimum dietary reccomendation of 300 mg EPA and DHA/day (according to the Dietitians of Canada) by the end of the study. Providing individuals with their personal FADS1 genetic information did not lead to significant differences in dietary intake or blood levels of omega-3 FAs compared to controls. However, the results suggest that providing individuals with genetic information can increase awareness of omega-3 FA terminology, render generic omega-3 nutritional information more useful in the context of their genetic information, and minimize barriers to the consumption of omega-3 FAs. Consequently, providing individuals with their personal FADS1 genetic information may have an impact on longer-term omega-3 FA intake.This study examined the effect of providing individuals with their personal Our FFQ analysis revealed that Baseline intake of EPA and DHA was ~200 mg/day in our study participants, which is slightly higher than recent global reports of EPA and DHA intake (~100 mg DHA/day in developed countries) ,18,47, aAPOE) genetic make-up [n = 93, 46 ± 12 years, 43% women) enrolled in a weight loss program [FADS1 genetic information on omega-3 intake may have been more pronounced in the Genetic group compared to the Non-Genetic group.Our findings regarding the limited impact of providing personal genetic information on dietary behavior appears to conflict with previous investigations; however, important differences exist between our trial and these previous studies. For example, Hietaranta-Luoma et al. gave adults  information about their risk for cardiovascular disease (CVD) in relation to their personal apolipoprotein E  the generic omega-3 FA information document we provided at the onset of the study as new and useful (5.7/7) more often than those in the Non-Genetic group (4.9/7). This aligns with findings by Nielsen and El-Sohemy, who found that young adults who received personal genetic information related to four dietary components  reported to have a greater understanding and utility for the dietary advice provided to them . AdditioFADS1 genetic information on dietary behavior changes in different subgroups of the population, such as those outside academia. Third, the FFQ used in this study was validated for EPA and DHA [FADS1 genotype. Fourth, increasing the sample size and expanding the analysis to other age groups will provide independent validation of our results. Finally, providing personalized genetic information represents a new area of investigation, therefore future research should focus on thoroughly assessing the qualitative effect of genetic information  using focus groups.The present study has some limitations that warrant consideration. First, the current study only included female participants who were primarily of Caucasian/European descent. Gender and ethnicity may influence dietary behavior changes upon receiving personal genetic information; therefore, a more diverse participant population is needed in future studies. Second, our population consisted solely of well-educated young adults. Future studies should examine the role of personal  and DHA , but notFADS1 genetic information and subsequent changes in omega-3 FA intake. We found little evidence that providing personal FADS1 genetic information affected EPA and DHA intake and circulating blood levels compared to the control group. However, we did find that individuals who received their genetic information had greater awareness of omega-3 terminology, rated cost as a barrier to omega-3 consumption less often, and found their genetic information to be useful in the context of generic nutritional information pertaining to omega-3 FAs compared to the control group. Therefore, providing personal FADS1 genetic information to young adults may provide an additional factor to help motivate behavior changes to increase the consumption of omega-3 FAs.The present study represents the first of its kind to explore the provision of


Sargassum thunbergii, which can drift on surface water, leading to long-distance dispersal.Long-term survival in isolated marginal seas of the China coast during the late Pleistocene ice ages is widely believed to be an important historical factor contributing to population genetic structure in coastal marine species. Whether or not contemporary factors  continue to shape diversity gradients in marine organisms with high dispersal capability remains poorly understood. Our aim was to explore how historical and contemporary factors influenced the genetic diversity and distribution of the brown alga Sargassum thunbergii populations sampled along the China coast. Population structure and differentiation was inferred based on genotype clustering and pairwise FST and allele-frequency analyses. Integrated genetic analyses revealed two genetic clusters in S. thunbergii that dominated in the Yellow-Bohai Sea (YBS) and East China Sea (ECS) respectively. Higher levels of genetic diversity and variation were detected among populations in the YBS than in the ECS. Bayesian coalescent theory was used to estimate contemporary and historical gene flow. High levels of contemporary gene flow were detected from the YBS (north) to the ECS (south), whereas low levels of historical gene flow occurred between the two regions.We used 11 microsatellites and the plastid RuBisCo spacer to evaluate the genetic diversity of 22 S. thunbergii along the China coast may result from long-term geographic isolation during glacial periods. The dispersal of S. thunbergii driven by coastal currents may facilitate the admixture between southern and northern regimes. Our findings exemplify how both historical and contemporary forces are needed to understand phylogeographical patterns in coastal marine species with long-distance dispersal.Our results suggest that the deep genetic divergence in The online version of this article (10.1186/s12862-017-1089-6) contains supplementary material, which is available to authorized users. The ECS and South China Sea (SCS) were thus isolated due to a land bridge extending from eastern China to Taiwan Island. The Sea of Japan (SOJ) also became an enclosed inland sea [Chthamalus challengeri [Sargassum horneri [Cellana toreuma [Sargassum fusiforme [The past decade has witnessed extensive research regarding genetic diversity and the evolutionary history of marine species on the coast of China –5. This land sea –8  coastlines, especially in tropics [S. muticum is one of the most well-known invasive species [trnW–I marker: one is widely spread in both native (e.g. Seto Inland Sea) and introduced area , and the other is restricted along the east coast of Honshu, Japan [S. horneri and S. hemiphyllum in the ANP also showed deep genetic split corresponding to the biogeographic basins, and homogeneous populations along the China coasts which ascribe to the founder effects and recent expansion [S. aquifolium and S. polycystum in Southeast Asia demonstrated shallow genetic structure with genetic diversity decline from south to north, implying surviving in a single refugium during glacial period. tropics . It is o species , 27, andxpansion . In contSargassum thunbergii Kuntze is a perennial brown macroalga occurring in the intertidal and sublittoral habitats of the ANP, ranging from Sakhalin, Russia, to Hainan Island, China [Sargassum that serve as spawning, nursery, and feeding grounds for marine animals [S. thunbergii is well adapted to diverse temperatures and salinities and thus has been proposed as a promising candidate for restoring the structure and function of impacted intertidal zones [S. thunbergii is one of the most heavily used natural feed for aquaculture animals , which has led to its near eradication in the past decade. Effective conservation of S. thunbergii will depend on detailed biogeographic knowledge of evolutionarily significant units [S. thunbergii across its entire range, and found that the Kuroshio Current played a significant role in shaping population genetic connectivity [S. horneri [S. fusiforme in the ANP [S. thunbergii along the coast of China.d, China . This sp animals . S. thunal zones , 31. In nt units . We receectivity . Similar horneri  but not  the ANP . Hyperva the ANP , 34–36, S. thunbergii along the coast of China. Our main goals were to (i) explore fine-scale population genetic connectivity in S. thunbergii along the China coast; (ii) determine if the genetic subdivision was influenced by long-distance dispersal at different time scales, and (iii) test which biogeographic scenario summarized above matches the genetic diversity patterns observed in S. thunbergii along the China coast.In this study, we integrated microsatellite genotyping and a plastid marker to analyse the diversity and distribution of Sargassum thunbergii specimens (n = 661) were collected from 22 sites along the coast of China, ranging from 39.04°N to 25.07°N  was amplified using the primer set 3F , which were included in Additional file S. thunbergii. Details on the 11 microsatellites are also summarized in Additional file Genomic DNA was extracted using the Plant genomic DNA kit  according to the manufacturer’s instructions. The plastid RuBisCo spacer  and S97CTAC-3′; ) PCR, elCTAC-3′; . The PCRrbc spacer, number of haplotypes (Nh), haplotype (h) and nucleotide (π) diversity were estimated for each locality using ARLEQUIN 3.5 [FST). All results for significance of covariance components were tested using 105 permutations. A median-joining network among plastid haplotypes was constructed using NETWORK 4.5 [For the plastid QUIN 3.5 . This prWORK 4.5 .Ne), private allelic richness (Ap), inbreeding coefficients (FIS), mean expected (He) and observed (Ho) heterozygosity were calculated in GENETIX 4.05 [FST and Jost’s D  was conducted to detect the proportion of genetic differentiation among regions, and within and among populations [For microsatellite loci, allelic richness  were pe  using GENALEX [We used two approaches to infer the number of distinct genetic clusters and to assign individuals to a given cluster. First, a Bayesian clustering analysis was implemented in STRUCTURE 2.3.4 , 48. The GENALEX . To visu GENALEX  was cons GENALEX . BootstrS. thunbergii along the coast of China was influenced by long-distance dispersal of floating thalli at different time scales, we estimated contemporary and historical gene flow. BAYESASS 3.0 [In order to determine if the population genetic differentiation in SASS 3.0  could idSASS 3.0  considerm) were calculated with BAYESASS 3.0 [6 iterations, with a burn-in of 106 iterations, and a sampling frequency of 2 × 104. Five replications were performed to ensure consistency between runs.Recent migration rates . Our analyses included 3 long chains and 5 replicates. Burn-in was set in 4 × 104 with a sampling increment of 40 and a total of 5 × 104 recorded steps. Heating was set with four temperatures  with a static scheme. We calculated the effective number of migrants using the relation  to keep them comparable to contemporary estimates.Mutation-scaled migration rate (M) estimates were calculated using a Bayesian coalescent approach implemented in MIGRATE-n 3.6 . MIGRATErbc spacer sequences with an aligned length of 795 bp, which contained seven polymorphic sites and yielded seven haplotypes (GenBank accession numbers: MF767271-MF767277). Of these haplotypes, five  were unique and two (R1 and R5) were present in most of the sampling locations  and Yingzuishi (POP2). The number of private alleles ranged from 0.000 to 0.182 in the YBS and from 0.091 to 0.455 in the ECS  showed slightly higher genetic diversity than those in the south coast (POP 15–22)  showed little evidence for significant deviations from equilibrium expectations  and ECS (average Dest = 0.047), respectively . Pairwise FST values based on rbc spacer also indicated deep genetic divergence between the north and south coasts , accounting for 89.03% of the total variance   and the lowest was from Dongtou (POP16) to Qingdao (POP14) (m = 0.0112). In addition, strong asymmetric gene flow was detected from Qingdao (POP14) to Shengsi (POP15) (m = 0.0241) and Dongtou (POP16) (m = 0.0227), respectively, which was almost two times larger than gene flow in the opposite direction  and the lowest from Qingdao (POP14) to Dongtou (POP16) , followed by Qingdao-Shengsi . All historical gene flow between populations in the ECS were lower than contemporary gene flow except in the pairwise Shengsi-Dongtou  and Beihuangcheng POP5) (29) Fig. . For ECSion Fig. . Histori (29) FigSargassum thunbergii along the coast of China, and their geographical distributions are in accordance with two marginal seas: the Yellow-Bohai Sea (YBS) and East China Sea (ECS). In addition, contemporary gene flow driven by coastal currents could cause a low yet significant connectivity between populations in two marine regimes.In this study, microsatellites and plastid DNA consistently revealed two genetically diverged clusters in Sargassum thunbergii populations were divided into two genetic clusters that correspond to the YBS and ECS  than the northern populations (POP15–18) (Ap = 0.091–0.182), suggesting that S. thunbergii populations in the ECS may have survived in the southern Okinawa Trough or the SCS during glacial periods. Populations in the YBS, on the other hand, were possibly recolonized from the northern Okinawa Trough or the Sea of Japan. In addition, our simulations of gene flow revealed limited historical gene flow between populations from the YBS and ECS  could be transported to fringe areas of the continental shelf and waters influenced by the Kuroshio Current after they become detached off Zhejiang Province, China [S. thunbergii populations in the YBS and ECS respectively, an assumption that was further supported by shared haplotypes  which e–34 psu; ), thus ee, China , 64. We pes Fig. . Similarignature . Thus diS. thunbergii along the coast of China may indicate different evolutionary processes. We detected substantial population subdivision in the YBS, but no clear genetic divergence among populations in the ECS  had higher genetic diversity than in the ECS (southern area) (Table Cellana toreuma in this region [S. thunbergii into the YBS from the west coast of the Korean Peninsula (e.g. the Jeju Island), leading to high levels of genetic admixture as reported in the red alga Gelidium elegans [Interestingly, our results indicated that a) Table . Similars region . This pas region . High ge elegans .S. thunbergii in the YBS and the ECS suggest long-term isolation. Besides, the comparison of contemporary and historical gene flow indicate that the Changjiang diluted water could not restrict the gene flow of S. thunbegii populations along the Chinese coasts. These findings contrast with some previous studies on invertebrates with similar phylogeographic structure [The genetic divergence of tructure , 15. FutAdditional file 1: Table S1.rbc spacer) haplotype distribution in each Sargassum thunbergii population. (DOCX 15 kb)Sampling locality, code, coordinates, sample size and RuBisCo spacer Sequences of 7 Additional file 3: Table S3.p) for each population and each locus. (DOCX 19 kb)Probability of deviation from Hardy-Weinberg equilibrium  and FST based on plastid rbc spacer (lower left), respectively. (DOCX 113 kb)The average Additional file 5: Table S4.Sargassum thunbergii populations base on microsatellites. (DOCX 18 kb)Pairwise differentiation of 22 Additional file 6: Table S5.FST values between 22 Sargassum thunbergii populations based plastid rbc spacer. (DOCX 16 kb)Additional file 7: Table S6.Sargassum thunbergii based on rbc spacer and SSR. (DOCX 14 kb)Analysis of molecular variance (AMOVA) to partition genetic variance in the Northwest Pacific Additional file 8: Figure S2.Sargassum thunbergii individuals using 11 microsatellites from all the localities along the coast of China. (DOCX 51 kb)Most likely number of genetic clusters using the Evanno method for Additional file 9: Figure S3.Sargassum thunbergii populations based on microsatellites. (DOCX 53 kb)UPGMA tree of 22