Chromis verater, based on 319 shallow specimens and 153 deep specimens. The mtDNA markers cytochrome b and control region were sequenced to analyze genetic structure: 1) between shallow (<30 m) and mesophotic (30–150 m) populations and 2) across the species' geographic range. Additionally, the nuclear markers rhodopsin and internal transcribed spacer 2 of ribosomal DNA were sequenced to assess connectivity between shallow and mesophotic populations. There was no significant genetic differentiation by depth, indicating high levels of vertical connectivity between shallow and deep aggregates of C. verater. Consequently, shallow and deep samples were combined by location for analyses of horizontal connectivity. We detected low but significant population structure across the Hawaiian Archipelago  and a larger break between the archipelago and Johnston Atoll . The population structure within the archipelago was driven by samples from the island of Hawaii at the southeast end of the chain and Lisianski in the middle of the archipelago. The lack of vertical genetic structure supports the refugia hypothesis that deep reefs may constitute a population reservoir for species depleted in shallow reef habitats. These findings represent the first connectivity study on a mobile organism that spans shallow and mesophotic depths and provide a reference point for future connectivity studies on mesophotic fishes.Understanding vertical and horizontal connectivity is a major priority in research on mesophotic coral ecosystems (30–150 m). However, horizontal connectivity has been the focus of few studies, and data on vertical connectivity are limited to sessile benthic mesophotic organisms. Here we present patterns of vertical and horizontal connectivity in the Hawaiian Islands-Johnston Atoll endemic threespot damselfish,  The majority of coral reef ecosystems studied to date occur at depths shallower than 30 m, yet zooxanthellate corals can extend to depths of over 150 m Studies on vertical and horizontal connectivity have been highlighted as priorities in MCE research Vertical connectivity has been the primary emphasis of mesophotic genetic studies to date, with less focus on horizontal connectivity. Kahng et al. Chromis veraterC. verater is not known but has been postulated to last as long as three months C. verater may recruit in deeper habitats, migrating into shallower water later in life Here we apply mitochondrial and nuclear markers to assess connectivity in the threespot chromis, C. verater and 2) horizontal connectivity across mesophotic populations and also across the geographic range of this species. With respect to vertical connectivity, we predict exchange between shallow reefs and MCEs based on the abundance of juveniles at depth. Regarding horizontal connectivity, most reef fishes show no structure across the Hawaiian Archipelago, but the exceptions tend to be endemics Dascyllus albisella and Stegastes marginatus demonstrated genetic structure across the Hawaiian Archipelago and between the archipelago and Johnston Atoll Our study addresses two primary issues: 1) vertical connectivity between shallow and mesophotic populations of C. verater specimens (fin clips) were collected. While the majority of specimens were adults, 1 shallow and 58 deep individuals were juveniles, based on field observations of body size. Collections at 12 shallow sites were made with pole spears or hand nets with SCUBA or while snorkeling  and control region (CR). Cytb was amplified with primers GLUDG-5′ http://nmg.si.edu/bermlab.htm). CR was amplified with primers Pro-L b.Tissue specimens were preserved in salt-saturated water with 20% DMSO N = 49) and mesophotic (N = 45) specimens from the Hawaiian Archipelago were amplified for two nuclear markers: rhodopsin and internal transcribed spacer 2 of ribosomal DNA (ITS2). Rhodopsin was amplified according to published nested amplification protocols, using RHO-30F and RHO-319R for the first set of primers and Rod-F2x and Rod-R4n in the second To verify that observed patterns were not restricted to the mitochondrial genome, subsets of the shallow (Polymerase chain reactions (PCRs) were performed in 14 µl reactions containing 1 µl of diluted DNA extract (one part DNA to 49 parts of nanopure water), 0.29 µl of each 10 µM primer, 7.14 µl of premixed PCR solution MangoMix™ , and 5.28 µl of nanopure water. PCR amplification of cytb consisted of an initial denaturation at 94°C for 3 min, followed by 35 cycles of 45 s at 94°C, 1 min 15 s at 50°C, and 1 min 15 s at 72°C, with a final extension for 5 min at 72°C. PCR amplification of CR consisted of an initial denaturation at 94°C for 5 min, followed by 35 cycles of 30 s at 94°C, at 49°C, and at 72°C, with a final extension for 7 min at 72°C. After purification of PCR products following the manufacturer's protocol , sequencing was performed with the forward PCR primers on an ABI 3730xl DNA analyzer  at the University of California Berkeley's DNA Sequencing Facility. Sequences were aligned and edited using Geneious R6 . Alignments of cytb and rhodopsin were unambiguous, while CR and ITS2 each contained multiple indels, which varied from 1–2 bp (CR) and 1–28 bp (ITS2) in length. For the nuclear markers, IUPAC ambiguity codes were used to score heterozygous individuals. Unique haplotypes for each marker were identified in Arlequin and were uploaded to GenBank (accession numbers KP183329-KP183902).h) and nucleotide diversity (π) were calculated in Arlequin 3.5 ST comparisons in Arlequin. The ΦST fixation index incorporates genetic distance and ranges from 0 to 1, with low values indicating a lack of genetic structure and high values indicating genetic differentiation. Significance of pairwise ΦST comparisons and AMOVA calculations was tested with 10,000 permutations, and to correct for multiple comparisons, a modified false discovery rate method was implemented N = 2) and Necker deep (N = 1) had small sample sizes, they were included in adjacent populations of Pearl and Hermes deep and French Frigate Shoals deep respectively for most population genetic analyses, after establishing that their haplotypes were closely related to those at these adjacent sites. Parsimony-based haplotype networks for each marker were constructed in R using haploNet in the package pegas 0.5–1 Haplotype diversity (N = 296) was overwhelming population structure due to the mesophotic specimens (N = 129). To accomplish this, we ran AMOVAs with equal sample sizes for the shallow and mesophotic specimens. The shallow dataset was randomly subsampled for 129 individuals to match the number of mesophotic specimens, and subsampling was replicated ten times. Then for both mitochondrial markers, AMOVAs were run with the full set of Hawaiian mesophotic specimens and each of the shallow subsample sets to determine whether there was significant genetic structure between shallow and deep.For comparisons within the Hawaiian Archipelago, we wanted to rule out the possibility that the large sample size of shallow specimens  with a matrix of geographic distances. A posteriori AMOVAs subsequently were performed on population groupings inferred by Barrier output.To avoid making ST values and geographic distances as calculated by the Geographic Distance Matrix Generator To determine whether any observed genetic structure was due to isolation by distance, Mantel tests were performed to test for a correlation between genetic distance and geographic distance. Mantel tests were run in the vegan package in R with 10,000 permutations, using matrices of pairwise Φθ (4Neµ) and M (m/µ) were generated using slice sampling. We employed the recommended Bayesian inference search strategy with a single 500,000 or 700,000 step chain, discarding the first 25% as burn-in θ and M informed priors for subsequent runs. Once parameters were optimized, runs were replicated three times, utilizing four short heated chains. We estimated the number of migrants between regions per generation (Nm) by multiplying estimates of M and θ of the destination population. Migrate was run to test for both horizontal connectivity and vertical connectivity. For horizontal connectivity, the program was run among Johnston Atoll, the NWHI, and the MHI, using 47 individuals randomly sampled from each of those groups. For vertical connectivity, the program was run for individual sampling locations where shallow and mesophotic specimens were available and had N>10 for each depth zone. Also, the program was run with two groups: one with all shallow Hawaiian individuals and one with all deep Hawaiian individuals. Since the shallow Hawaiian Archipelago and deep Hawaiian Archipelago runs did not have unimodal normally distributed posterior distributions, we do not present these data. Though our Migrate estimates are based on one locus, they are still useful for finding relative differences within this dataset.To infer directionality of gene flow, we used Migrate 3.6.4 b and 394 bp of CR were resolved for 319 shallow and 153 mesophotic C. verater specimens, including those from Johnston Atoll. Summary statistics for number of haplotypes (H), haplotype diversity (h), and nucleotide diversity (π) are listed in h = 0.9041 to 0.9066 for cytb and h = 0.9994 to 0.9997 for CR  and deep Johnston Atoll (h = 0.7645) were lower than that of any site in the Hawaiian Archipelago (h = 0.8182–0.9722). Nearly every CR sequence was a unique haplotype, so haplotype diversity was even higher for this marker and had a narrower range across the various sites (h = 0.9833–1.0000). Haplotype diversity across shallow sites was similar to that across mesophotic sites for both markers . Overall, haplotypes do not appear to group by geographic location, except for some clustering of Johnston Atoll haplotypes in the CR haplotype network.The haplotype networks for cytby depth . In the C. verater specimens from the Hawaiian Archipelago. Summary statistics for number of haplotypes (H), haplotype diversity (h), and nucleotide diversity (π) for the nuclear markers are listed in S1 Table. Nucleotide diversity across shallow sites was higher than that across mesophotic sites for both nuclear markers (S1 Table). Haplotype diversity across shallow sites was similar to that across mesophotic sites.A total of 442 bp of rhodopsin and 401 bp of ITS2 were sequenced for 49 shallow and 45 mesophotic Similar to the haplotype networks for the mtDNA markers, the networks for ITS2 and rhodopsin do not show clustering of haplotypes by depth . The netC. verater, first we ran an AMOVA separating all of the specimens into two groups: shallow and mesophotic. Neither cytb nor CR demonstrated significant genetic structure between the shallow and mesophotic groups  . Likewise, AMOVAs indicated no significant genetic structure between shallow and deep populations for either nuclear marker . For the mtDNA markers, none of the individual locations that had shallow and mesophotic individuals demonstrated significant population structure across depth (To determine if there was significant genetic differentiation by depth in = 0.103) . In addiss depth .b  and significant for control region . Since there was a lack of significant vertical genetic structure in the majority of these runs, we did not perform additional subsampling runs.For the mtDNA comparisons within the Hawaiian Archipelago where we employed the subsampling procedure for the shallow specimens, nine out of ten runs showed no evidence of population structure by depth . One runNm = 305.27, 95% CI = 37.96–945.22) than from deep to shallow . Likewise, the estimated number of migrants per generation was much higher from shallow to deep for Maui and Oahu. Conversely, at Pearl and Hermes and Niihau, the migration rate was higher from deep to shallow. No general pattern was apparent from these data.Effective migration rates using Bayesian methods revealed that migration between shallow and deep populations varied by sampling location . At Johnb and CR    . JohnstoP<0.001) . Also, BP<0.001) .b: ΦST = 0.035, P = 0.002; CR: ΦST = 0.032, P = 0.003) .Specifically to test for connectivity across mesophotic sites, an AMOVA was performed across all Johnston Atoll and Hawaiian mesophotic sites, revealing low but significant structure (cyt= 0.003) . Again, b: ΦST = 0.009, P = 0.020; CR: ΦST = 0.012, P = 0.009)  but remained significant for CR . For both cytb and CR, the island of Hawaii was significantly different in at least half of the comparisons . In the analysis of the archipelago, Barrier identified a genetic break between the island of Hawaii and the rest of the Hawaiian populations. Grouping individuals into these two regions in an AMOVA confirmed a significant break   . Populat= 0.009) . For cyt= 0.005) .b: r = 0.022, P = 0.400; CR: r = 0.010, P = 0.427). When Johnston Atoll was included in the analysis, there was no significant correlation between ΦST and geographic distance .Mantel tests were performed combining shallow and deep specimens by location both with Johnston Atoll individuals and with only Hawaiian locations. There was no evidence for isolation by distance across the Hawaiian Archipelago  than from the archipelago to Johnston Atoll. Within the Hawaiian Archipelago, there was greater gene flow from the NWHI into the MHI  than from the MHI into the NWHI.Coalescent-based estimates of migration provided insight into the direction of gene flow between Johnston Atoll, the NWHI, and the MHI . With reC. verater to make broad generalizations about connectivity patterns in mesophotic fishes, and caution should be exercised in extending these results to other types of fishes that occur at depth. Nevertheless, the results presented here portray a reef fish species that spans shallow and mesophotic depths and provide an initial reference point for understanding connectivity in mobile mesophotic organisms.This study represents the first attempt to assess: 1) horizontal connectivity across mesophotic populations and 2) vertical connectivity between shallow and mesophotic reefs in a species of reef fish. We acknowledge the shortcomings of low mesophotic sample sizes and uneven geographic sampling, which are due to the difficulty of collecting specimens at mesophotic depths. It would be premature to use these data on b and CR, we found high levels of genetic connectivity between shallow and mesophotic populations of C. verater in the Hawaiian Archipelago. At individual locations where shallow and mesophotic individuals had been collected, there was no significant genetic differentiation by depth. For the Hawaiian Archipelago, the large number of shallow specimens (N = 296) was not obscuring a signal of genetic structure from the mesophotic specimens (N = 129). When analyses were run with equivalent sample sizes of shallow and mesophotic individuals, nine out of ten runs exhibited high levels of vertical connectivity. We dismissed the possibility that this trend was limited to the mitochondrial genome by sequencing a subset of specimens for nuclear markers rhodopsin and ITS2, which also failed to demonstrate genetic differentiation by depth. For individual sampling locations where shallow and deep individuals were collected, coalescent-based Migrate estimates revealed that migration tended to be biased in one direction, but the direction varied by sampling location, with greater migration from shallow to deep at Johnston Atoll, Maui, and Oahu and greater migration from deep to shallow at Pearl and Hermes and Niihau.Using the mitochondrial markers cytExplicit collection depths were not available for most specimens, raising the possibility that the lack of vertical genetic structure is due to uncertainty in categorizing specimens as shallow or mesophotic. However, most mesophotic specimens were collected during expeditions to explore deep reefs (50–150 m) with open-circuit technical diving, rebreather diving, or submersibles, so we believe that this potential for error is minimal. To address this concern, future connectivity studies that span shallow and mesophotic reefs may want to consider a sampling approach that targets three depth categories, such as shallow (<20 m), middle (20–40 m), and deep (40+ m). This would allow for comparison of the shallowest and deepest individuals, as well as a separate comparison of specimens that were collected near the threshold depth of 30 m. Execution of such a sampling strategy would be more difficult but could perhaps reveal fine-scale vertical connectivity patterns. Additionally, future studies may benefit from the application of more variable loci.C. verater contrasts with a number of mesophotic studies that demonstrate limited vertical connectivity in sedentary benthic organisms C. verater. Furthermore, C. verater is suspected to have a life history trait that would explain connectivity between populations at different depths: C. verater larvae may settle on deep reefs, gradually migrating inshore with age The lack of genetic structure between shallow and mesophotic C. verater are capable of replenishing shallow populations. This is supported by the migration estimates for Pearl and Hermes and Niihau, which indicate greater gene flow from deep to shallow populations. However, other locations showed that the opposite is also true for C. verater, with higher migration estimates from shallow to deep. So far, it appears that the ability for mesophotic populations to serve as “deep reef refugia” varies by site and by organism. For example, at Scott Reef in northwestern Australia, there was evidence of migration from deep (31–43 m) to shallow (25–27 m) colonies in the scleractinian coral Seriatopora hystrix. Meanwhile, there was no evidence to support the “deep reef refugia” hypothesis at Yonge reef in northeastern Australia, where this species did not exhibit migration from deep to shallow colonies Montastraea cavernosa, regional differences in patterns of vertical connectivity suggest that the likelihood for deep reefs to restock shallow reefs varies among and within geographic locations due to local hydrology With respect to the “deep reef refugia” hypothesis, the extensive vertical connectivity revealed by our results implies that mesophotic populations of b. Since there were no shallow individuals available for this location, it is not certain that this pattern is unique to mesophotic individuals.For the analyses of horizontal connectivity using the mitochondrial markers, we made no distinction between shallow and mesophotic individuals, combining them per location. Nevertheless, an AMOVA run with only the mesophotic individuals did not indicate significant genetic structure across the Hawaiian Archipelago. The only anomaly with the mesophotic specimens was that the Lisianski population was significantly different in most pairwise comparisons for cytC. verater. Genetic studies on the submesophotic Hawaiian Grouper Hyporthodus quernus and deepwater snappers C. verater because those species are not tightly associated with MCE habitat. Studies on mesophotic corals reveal mixed patterns of horizontal connectivity. The coral S. hystrix exhibited more genetic structure between depths than horizontally across geographic locations Corallium rubrum demonstrated significant geographic genetic differentiation at multiple spatial scales, from tens of meters to hundreds of kilometers, illustrating limited horizontal connectivity M. cavernosa demonstrated low horizontal connectivity as well as genetic differentiation by depth There are limited genetic connectivity studies on mesophotic reef fishes with which to compare the results from b: ΦST = 0.068, P<0.001; CR: ΦST = 0.116, P<0.001). This trend remained significant regardless of whether shallow, mesophotic, or shallow/mesophotic specimens were analyzed. The Johnston Atoll population was significantly differentiated from almost all of the Hawaiian locations in pairwise comparisons for both mitochondrial markers. Furthermore, Migrate analyses demonstrated that gene flow was biased from Johnston Atoll toward the Hawaiian Islands.When shallow and mesophotic individuals were combined, the results indicate limitations to horizontal connectivity across the 860 km that separate Johnston Atoll and the Hawaiian Archipelago  as hypothesized, then they may complete their pelagic larval duration in these eddies, retained near the island of Hawaii. Notably, this explanation does not apply to Lisianski, the only other location in the archipelago to show a low but significant level of population differentiation. Lisianski, a small (1.5 km2) flat outpost of coral reef habitat, lies 1676 km northwest of Oahu (2) lies directly southeast of Lisianski. Instead, it is more likely that oceanographic conditions unknown to us are driving this trend at Lisianski.Within the Hawaiian Archipelago, Barrier identified a significant genetic break between the island of Hawaii and the rest of the archipelago, and this was supported by low but significant AMOVAs with both mitochondrial markers. This genetic break is concordant with one of the strongest marine barriers previously identified in the Hawaiian Archipelago and is believed to be based on oceanographic conditions  of Oahu . ExplanaC. verater, a reef fish occupying both shallow and mesophotic reefs, constitutes the first glimpse of connectivity patterns for mobile organisms that inhabit MCEs. This species exhibits high connectivity between shallow (<30 m) and mesophotic reefs (>30 m) in the Hawaiian Archipelago and Johnston Atoll, while maintaining weak population structure across this range. This dichotomy between vertical and horizontal connectivity provides an interesting perspective on dispersal in endemic species. The restricted range sizes of endemic reef fishes is thought to be a reflection of their limited dispersal abilities C. verater in terms of vertical connectivity, a scale of 7–113 m in this study. However, vertical connectivity is on a much smaller scale than horizontal connectivity, which show some limitations within the Hawaiian Archipelago (2600 km) and between the archipelago and Johnston Atoll (860 km).This genetic survey of C. verater is relevant to emerging conservation issues for MCEs. Biodiversity hotspots are a focus for conservation efforts, and endemic species are a large component of regional biodiversity C. verater, highlighting the link between these deep reefs and other parts of coral reef ecosystems. The high levels of vertical connectivity observed in our study lend support to the argument that MCEs serve an important ecological role as habitat and refugia for populations that may be depleted in shallow habitats. Marine protected areas that encompass shallow habitats and adjacent mesophotic reefs will allow for the protection of refugia that may replenish shallow reefs.Our study on connectivity in S1 FigureCytb haplotype network for Chromis verater. Parsimony-based network using cytb sequence data and color-coded by sampling location.(EPS)Click here for additional data file.S2 FigureCR haplotype network for Chromis verater. Parsimony-based network using CR sequence data and color-coded by sampling location.(EPS)Click here for additional data file.S3 FigureITS2 haplotype network for Chromis verater. Parsimony-based network using ITS2 sequence data for subsample of 94 specimens and color-coded by sampling location.(EPS)Click here for additional data file.S4 FigureRhodopsin haplotype network for Chromis verater. Parsimony-based network using rhodopsin sequence data for subsample of 94 specimens and color-coded by sampling location.(EPS)Click here for additional data file.S1 TableNuclear molecular diversity indices for shallow and mesophotic samples of Chromis verater. Number of individuals (N), number of haplotypes (H), nucleotide diversity (π), and haplotype diversity (h) are listed for subsample of 94 individuals sequenced for rhodopsin and ITS2.(PDF)Click here for additional data file.


Pagrus major, Temminck et Schlegel) in Japan. In this study, we examined the putative effects of intensive offspring releases on the genetic structure of red sea bream populations along the Japanese archipelago by genotyping 848 fish at fifteen microsatellite loci. Our results suggests weak but consistent patterns of genetic divergence . Red sea bream in Japan appeared spatially structured with several patches of distinct allelic composition, which corresponded to areas receiving an important influx of fish of hatchery origin, either released intentionally or from unintentional escapees from aquaculture operations. In addition to impacts upon local populations inhabiting semi-enclosed embayments, large-scale releases  appeared also to have perturbed genetic structure in open areas. Hence, results of the present study suggest that independent large-scale marine stock enhancement programs conducted simultaneously on one species at different geographical locations may compromise native genetic structure and lead to patchy patterns in population genetic structure.Large-scale hatchery releases are carried out for many marine fish species worldwide; nevertheless, the long-term effects of this practice on the genetic structure of natural populations remains unclear. The lack of knowledge is especially evident when independent stock enhancement programs are conducted simultaneously on the same species at different geographical locations, as occurs with red sea bream ( Spatial patterns in genetic structure of organisms in the wild reflect historical and contemporary effects of micro-evolutionary processes  acting on animal and plant populations. Intrinsic life-history features and differences in environmental regimes are also relevant to genetic population structuring. The evolutionary and adaptive potential of the species to cope with new environmental conditions demands such intra-specific diversity . While In addition to natural processes, the genetic structure and adaptive potential of marine populations can be subject to significant human-induced stressors. One of the most controversial activities is associated with the putative harmful genetic effects of translocations and hatchery-produced juvenile releases on native populations , 9–13. ABoth high survival and high recapture rates are needed to justify the economic investment in marine stock enhancement programs. Thus, they usually are conducted in semi-enclosed embayments and on species not displaying highly migratory behavior , 15. TheOcean dynamics along the Japanese archipelago is dominated by three main strong currents see , which oPagrus major) was one of the eight species selected during the early development of marine stock enhancement in Japan [Red sea bream . Stock enhancement programs for red sea bream in Japan are run regionally, usually at hatcheries operated by prefectural governments. The size, origin and replacement rate of the broodstock as well as the number of juveniles released every year are specific for each program. The largest numbers of annual juvenile releases were reported in the East China Sea, in the southern prefectures of the island of Kyushu, e.g., Kagoshima and Kumamoto , and no special permits were required for sampling the species either for research or commercially at these locations , and individual genotyping was analyzed with GENEMAPPER v. 4.1 (Applied Biosystems).Total genomic DNA was extracted from fin tissues using either the DNeasy kit (Qiagen) or the DNAzol (Invitrogen) protocol, re-suspending the DNA in TE buffer. Microsatellite polymorphism was analyzed at fifteen polymorphic markers: z et al. . AmplifiA), allelic richness (Ar) and gene diversity within samples (HS) and the average for all samples (HT), based on Nei and Chesser [FIS [Levels of genetic variation were characterized by counting observed allele  and to infer admixture proportions between wild and hatchery-reared fish in the data set without a priori information of population partition. We assumed an admixture model and correlated allele frequencies [K between 1 and 7, and the calculation was done five times for each K. 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 v. 1.1 [Spatial genetic differentiation patterns were examined by a Principal Component Analysis (PCA) based upon a covariance matrix of allele frequencies, and visualized using PCAGEN v. 1.2.1 . The Bayquencies . Each ruistic ΔK . Once K T v. 1.1 .FST estimates were linearized, as FST ⁄ (1—FST), and regressed against the natural logarithm of the shortest maritime distance connecting each sample pair (http://ibdws.sdsu.edu/~ibdws/). As sample KHR comprised only hatchery-released specimens, it was excluded in this analysis and in the subsequent landscape genetic analysis performed in BARRIER v. 2.2 [FST estimates along all pairs of localities. The analysis was performed including all loci to infer the rank of importance of the barriers. Statistical support for each barrier was evaluated by the number of loci that supported it, and by 1000-bootstrap analysis of the multilocus Weir and Cockerham’s [FST matrix generated using the DIVERSITY R package [Geographic patterns and the scale at which genetic structuring occurs were further investigated by testing putative correlations between genetic and geographic distances . Pairwisple pair . IsolatiR v. 2.2 ; this sokerham’s  FST mat package .The amount of genetic variation explained by alternative sample grouping was tested with AMOVA using ARLEQUIN v. 3.5 . As all Ne) under a sample-size bias correction [Ne and jackknife-adjusted 95% confidence intervals for each sample were estimated using Pcrit = 0.02, i.e., excluding those alleles with frequencies lower than 0.02. Waples and Do [We estimated the effective population sizes  and gene diversity within samples (HS), appeared very similar among sample localities , as expected for a locus with potential null alleles when FST estimates are small [In general, loci conformed to Hardy-Weinberg (HW) expectations . Deviatire small , 64; theLinkage disequilibrium was found to be statistically significant (at the 5% level) in 81 out of 1680 pairwise tests (4.8%). Twenty of them (1.2%) remained statistically significant after the FDR correction, distributed evenly among samples and pairs of loci. Thus, our results provided no reason to conclude that the loci were linked. The outlier test on BAYESCAN suggested no evidence for selection operating on any of the loci (results not shown).FST = 0.002, p < 0.001). Differences were significant (at the 5% level) at 11 of the 15 scored loci (FST comparisons indicated significant differentiation in 64 out of 120 pairs (53%), with 45 (37%) remaining statistically significant after FDR . Excluding the pairwise value corresponding to the proximal sampling localities from Kanagawa Prefecture (KSB and KTB) turned the sign of the slope into a positive value; nevertheless, the regression remained statistically non-significant at the 5% level (data not shown).The spatial analysis performed in BARRIER suggested three main barriers to gene flow around the genetically most divergent sampling locations . The twoNe = 48), WAK (Ne = 126) and KKB (Ne = 163), which also showed the most divergent genetic profiles. In fact, only KHR and WAK gave finite confidence intervals.Estimates of effective population sizes ranged between 48 in KHR to 614 in FKS . The lowOur results based on fifteen microsatellite loci suggests weak, but consistent patterns of genetic divergence among red sea bream samples from coastal waters of Japan. The species appeared spatially structured in several patches with differentiated allelic composition, corresponding to areas receiving an important influx of fish of hatchery origin, either released intentionally , 14, 66 FST = 0.002, p < 0.001). Disagreement with previous studies suggesting a large panmictic population in Japan [Genetic comparisons between red sea bream samples separated by hundreds of kilometers indicated low but significant levels of genetic differentiation  [Scomberomorus niphonius) in the Seto Inland Sea, and reported no significant temporal changes in the diversity indices. However, most pairwise FST comparisons involving a post-release sample from the eastern part of the Seto Inland Sea were significant, including the comparison with the pre-release sample from the same area.Current results suggest a temporally stable pattern of genetic structure of red sea bream in Wakayama Prefecture , 28, wheunctata) , populatunctata)  evaluateGirella punctata larvae drifted almost 300 km from the spawning ground. The similarity in gene pools observed among red sea bream populations along the latitudinal range contrasts with results for Pacific herring , another species subject to intensive long-term releases on the northern coasts of Japan [The strong Kuroshio Current also is likely to contribute to gene flow and genetic homogeneity among red sea bream populations along the Pacific coast Figs  and 4. Pof Japan , 76. Heaof Japan . In thisof Japan ); nevertFST = 0.001, p < 0.001; without KKB, FST = -0.000, p = 0.078), suggesting high levels of demic connectivity and more population admixture. The warm Tsushima Current can transport the early pelagic stages of several marine organisms; such as lobsters, argonauts and fishes [Similar to the Pacific coast, the genetic profiles of red sea bream in the Southwest and the Japan Sea coast appeared even less structured  and Sagami Bay in Kanagawa Prefecture (KSB), two bays with an intense juvenile release history , 14, 66.Kinko Bay presents a maximum depth of 237 m and a mouth width of 8.7 km. Oceanic drift was suggested to favor gene flow and admixture between red sea bream populations inhabiting the inner and outer part of the bay , 23 and Sagami Bay (KKB), and to a lesser extent the neighboring Tokyo Bay (KTB), appeared as another area where red sea bream showed a distinct genetic composition. In contrast to Kinko Bay, Sagami Bay reaches almost 1500 m in depth with a narrow continental shelf and sudden drop-off, and presents a wide mouth to the open sea which offers possibilities for gene flow. Meanwhile, the eastern side of Sagami Bay is connected to Tokyo Bay and likely receives an influx of fish from this semi-enclosed bay. Population dynamics of red sea bream in these interconnected bays is highly influenced by juvenile releases, which may comprise up to 74% of commercial landings . In a reTurbo (Batillus) cornutus) [Acanthopagrus schlegelii) [Disparity in the genetic profiles of populations inhabiting semi-enclosed areas and with an intensive stocking history is not exclusive to red sea bream. Studies conducted with heavily released Japanese turban shell (ornutus)  and blaclegelii)  already legelii) , 12. In legelii)  concludelegelii) , 28. Prelegelii)  and fishlegelii)  can have


Blastocystis subtypes (STs) recovered from symptomatic children by analysing partial sequences of the small subunit rDNA gene region (SSUrDNA) and internal transcribed spacers (1 and 2) plus the 5.8S region  and comparing with isolates from other countries.The purpose of this study was to assess the genetic variation and differentiation of Blastocystis-infected children with gastrointestinal symptoms and negative for pathogenic enterobacteria were analysed. PCR was performed on DNA from all the samples to identify Blastocystis STs, amplifying a fragment of SSUrDNA and the ITS region. The amplicons were purified and sequenced, and consensus sequences were submitted to GenBank; afterwards, SSUrDNA sequences were analysed for genetic diversity according to geographic area. Regarding the Blastocystis STs found, 51% were ST1, 23% ST2, 19% ST3 and 2% ST7. For ITS, a haplotype network tree and Bayesian inference revealed the presence of two novel variants of ST1, clustering some sequences into ST1A and ST1B. The values of nucleotide diversity (π) and haplotype polymorphism (θ) for ST1, ST2 and ST3 ranged from 0 to 1, whereas the ratio of genetic differentiation (FST)/migration index (Nm) showed the highest differentiation between Libya and Thailand-Philippines for ST2 (0.282/0.63). In contrast, a high flow gene was observed between Czech Republic-Denmark-Holland-Spain and USA-Mexico-Colombia for ST1 (0.003/84).Faecal samples from 47 Blastocystis STs. Moreover, the ITS region could be used as a genetic marker to assess genetic variation in this parasite.Our data on genetic differentiation and gene flow might explain the differences for the prevalence of  Blastocystis spp are very common intestinal parasites of humans and animals, with a prevalence that varies from country to country and among various communities within the same country [Blastocystis based on the analysis of its small subunit rDNA (SSUrDNA) [Blastocystis [ country -3. In adSSUrDNA) ,4-8. In tocystis ,10.Blastocystis STs recovered from symptomatic children from Michoacan state, Mexico, by analysing partial SSUrDNA sequences and internal transcribed spacers (1 and 2) plus the 5.8S region  and comparing with isolates from other countries.As there is scarce knowledge of the evolution, ecology, and population genetics in this parasite, the purpose of the present study was to assess the genetic variation of Blastocystis , who were treated at the Hospital Infantil de Morelia “Eva Samano de Lopez Mateos” due to gastrointestinal symptoms and were negative for pathogenic enterobacteria, were analysed. Clinical and demographic data were obtained from medical records, and information about the Blastocystis load (reported as “more than” or “less than” five Blastocystis cells per 40X field) was obtained from laboratory files; variables were analysed by χ2 tests. The Ethics and Research Committee of the hospital approved the study, and informed consent was obtained from one parent of each child.Frozen faecal samples from 47 children infected by et al. [Blastocystis sequences and other related organisms in GenBank . The designed forward and reverse primers were ITS_Blas_F (5′-GGAAGGTGAAGTCGTAACAAG-3′) and ITS_Blas_R (5′-CAGCAGGTCTTCTTRCTTGA-3′).Stool DNA was extracted from approximately 250 mg faeces using QIAamp DNA Stool Mini Kit . A partial SSUrDNA sequence (~500 bp) was obtained using the primers reported by Santín et al.  A new sp2), 1X dNTPs, 0.01 mg BSA, and 1 U Taq DNA Polymerase (Promega). After the first denaturation step at 94°C for 5 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 45 s, and extension at 72°C for 30 s were performed, followed by a final extension step at 72°C for 10 min. The amplicons were subjected to electrophoresis in 1.2% agarose gels and then purified and sequenced on both strands by a commercial service.DNA (2 μL) was used to amplify the genomic sequences in a 25-μL PCR reaction. For ITS1-5.8S-ITS2 region amplification, the reaction contained 200 pmol of each nucleotide, 1X PCR buffer . These indexes refer to the following: π, the average proportion of nucleotide differences between all possible pairs of sequences in the sample; θ, the proportion of nucleotide sites that are expected to be polymorphic in any suitable sample from this region of the genome. Both indexes are used to assess polymorphisms at the DNA level and to monitor diversity within or between ecological populations and examine the genetic variation in related species or their evolutionary relationships. FST is a typical genetic statistic used to measure differentiation between or among populations. The common used values for genetic differentiation are as follows: 0 to 0.05, small; 0.05 to 0.15, moderate; 0.15 to 0.25, great; values above 0.25 indicate huge genetic differentiation. The gene flow or migration index (Nm) refers to the movement of organisms among subpopulations; those strongly differentiated have an Nm < < 1, whereas an Nm > 4 behaves as a single panmictic unit [All sequences were subjected to a BLAST search in the GenBank database; multiple alignments were performed using the CLUSTAL W  and Musctic unit .Endolimax nana (19%), Entamoeba coli (15%) and Entamoeba hartmanni (13%). Only 10 samples (21%) presented >5 Blastocystis stages per 40X field, with the vacuolar form being the most frequent stage found. Regarding Blastocystis STs, 43 sequences were obtained for SSUrDNA and 46 for ITS. For five samples, we did not have a sufficient amount of DNA with adequate quality to obtain amplicons, most likely due to the activity of several DNases that degrade nucleic acids and therefore decreased the DNA concentration in the samples [Blastocystis. All amplicons for SSUrDNA were ~500 bp. For ITS, the amplicons ranged from 530 to 620 bp, showing products of ~550, ~530, ~620 and ~590 bp for ST1, 2, 3 and 7, respectively. Regarding Blastocystis STs, 51% were ST1, 23% ST2, 19% ST3 and 2% ST7 by SSUrDNA and/or ITS. Only two cases (samples 17 and 34) did not match, which might have been due to a phenomenon of co-infection between STs because the sequencing of several clones is required to detect potential co-infections by different STs when SSUrDNA markers are used. No statistical associations between the symptoms, parasite load and Blastocystis STs were found.Table  samples . A BLASTBlastocystis ST1, −2 and −3 from Libya (Africa), Thailand-Philippines-Japan (Asia), Czech Republic-Denmark-Holland-Spain (Europe), and USA-Colombia (North and Latin America) deposited in the GenBank databases, as well as our sequences (accession numbers from KM213425 to KM213513), were analysed for their genetic variability. The average nucleotide diversity (π) and haplotype polymorphism (θ) for SSUrDNA was 0.0179 ± 0.0112 and 0.0355 ± 0.0076, respectively .Figure  to 0.07 ; and forST)/migration index (Nm) for SSUrDNA showed the highest differentiation between the Libyan and Thailand-Philippines populations for ST2 (0.282/0.63); in contrast, a high flow gene was observed between the European and American populations for ST1 (0.003/84)  and thus is a good marker for research on genetic diversity, as for other parasites ,24,27,28quencing . However [et al.  analysedet al. [Blastocystis STs in populations from Africa and UK and compiled and contrasted the relative distribution of Blastocystis STs across continents, finding a significant variation in ST prevalence between populations. Our results support the analysis of Alfellani et al. [ST/Nm data, the gene flow (Nm) of ≈ 4 for America-Europe and America-Africa as well as for America-Asia (Nm ≈ 9) suggest a high migrant flow, but less than Europe-Asia (Nm ≈ 12) and Africa-Asia (Nm ≈ ∞), indicating a vast migrant flow among these areas. For ST2, America shows the highest frequency (up to 30%), with low values of Nm <4 for America-Africa and America-Asia but Nm = 33 for America-Europe; these results could mean that ST2 from America is more isolated than in other major geographic regions. Finally, ST1 shows contrasting values because it was more frequent in America and East/Southeast Asia and lower in Europe and Africa. The FST/Nm data showed Nm ≤ 4 among Africa vs Europe-America-Asia, whereas Nm ranged from 4 to 84 between Europe and America-Asia-Africa. Therefore, due to the current globalisation, the variable frequencies observed in ST prevalence might indirectly show the genetic differentiation and gene flow of each ST population around the world.The SSUrDNA analysis revealed that ST1 and ST2 showed the lowest genetic differentiation, with a high gene flow between the American and European populations, whereas the match between ST1 and ST2 for the Libyan and American populations showed a higher genetic differentiation with little gene flow. Interestingly, ST3 exhibited the most gene flow (migrants) for Thailand-Philippines vs Libya, America and Europe. Libya had a low number of sequences (ranging from 4 to 7); thus, differences in the variation of the genetic values were also large (ranging from 0 to 1). Alfellani et al. [Blastocystis ST7 and Stensvold et al. [Blastocystis. Considering that we used part of SSUrDNA in our genetic analyses, we can speculate that these biological features may be a secondary effect due to variation in other genes; most likely by a direct genetic hitchhiking phenomenon [Poirier et al.  described et al.  emphasisenomenon  or by anenomenon .


Oculina patagonica, first recorded in the Mediterranean Sea in 1966, is believed to have been introduced in anthropogenic times and expanding in a west to east direction. However, its present identification and status as a recently introduced species remain to be explored. In this study, we used multi-locus genetic data to test whether O. patagonica in the Mediterranean has been recently introduced from the western North Atlantic.Effective policies, management, and scientific research programs depend on the correct identification of invasive species as being either native or introduced. However, many species continue to be misidentified. O. patagonica from the western North Atlantic or an expansion across the Mediterranean. Instead, Mediterranean and Atlantic populations are genetically distinct and appear to have begun diverging about 5 Mya. We also found evidence of a fossil record of Oculina spp. existing in the eastern North Atlantic millions of years before the present.We found no genetic or historical demographic evidence to support a recent introduction of O. patagonica have long been isolated from the western Atlantic, either in undetectable numbers or overlooked and undersampled sites and habitats, and have only recently been expanding to invasive levels as a result of environmental changes. Accurate identification of species’ invasive statuses will enable more effective research programs aimed at better understanding the mechanisms promoting the invasive nature of species, which can then lead to the implementation of efficient management plans.Our results suggest that Mediterranean populations of The online version of this article (doi:10.1186/s12862-015-0356-7) contains supplementary material, which is available to authorized users. The number of introduced and invasive species is rising , rapidlyIntroduced species can be mislabeled as native due to taxonomic misidentification ,6. NativInvasive species are often assumed to have been introduced ,10, howeAn introduced invasive population can be distinguished from native invasive populations in several ways. An expanding introduced population is expected to be genetically similar to an external source population where it originated . In contOculina were originally described from the southeastern coast of North America )  Figure . In factOculina spp. in the eastern Atlantic and/or the Mediterranean. We found 16 fossil specimens of Oculina spp. in two independent museum collections: the Smithsonian National Museum of Natural History’s Department of Invertebrate Zoology  of this species existing outside the Mediterranean resides [O. patagonica in South America are lacking, and a recent survey of fouling communities in Argentinian ports failed to find any evidence of this species [O. patagonica are not present in the western South Atlantic, they could not have been recently introduced into the Mediterranean from this region. Although it is possible that O. patagonica still resides in the western South Atlantic in low undetected numbers or habitats, there are no grounds to suggest O. patagonica recently originated from South America until (if) those specimens are found.The original hypothesis for the origin of Mediterranean  resides . However species . If liviO. patagonica has not recently travelled east across the Atlantic to the Mediterranean, then where did it originate? We found records for 16 fossil specimens of Oculina spp. from France  [Oculina has long been present in the eastern North Atlantic.If ce Table . Along w 64 Mya)  and sout 64 Mya) . AlthougOculina spp.  due largely to a unique way in which polyps bud [Schizoculina africana and Oculina patagonica are in fact conspecific, and whether O. patagonica originated from the northwestern coast of Africa.Another hypothesis for the origin of pe Verde ,59. Origlyps bud ,60. Howelyps bud  and may Oculina is more closely related to some members of different families  than to some members of its own family. Thus, representatives from some extra-familial genera with which Oculina has sometimes been allied  [Oculina patagonica.Oculinidae is a taxonomically confused family ,62 in nelliidae)  should aO. patagonica’s increase in abundance at shallow depths at many localities in the Mediterranean over the past 20 years [O. patagonica were first reported from a single site in the Saronikos Gulf of the Aegean Sea [O. patagonica could be found in 45 of 54 surveyed sites in this region [Direct observations testify to 20 years ,34. Alon20 years . In 2005gean Sea . By 2009O. patagonica has been expanding west to east, just as first reports of its presence have. Our tests, however, did not detect a genetic signal of expansion across the Mediterranean. While this may result from low power, the proposed west to east spread is also opposite to most other range expansions in the Mediterranean, which have occurred in a north-westward direction in response to increasing sea temperature [O. patagonica [O. patagonica was first reported from the Levant prior to Greece [O. patagonica was first established in the western Mediterranean and only more recently in the east, then the western populations would likely harbor more genetic diversity; however, we found similar levels of genetic diversity across the Mediterranean. Alternatively, O. patagonica could be moving into the Mediterranean from elsewhere in the eastern North Atlantic, but in sufficient numbers to not leave a genetic signature of expansion.Fine et al.  proposedperature . Becausetagonica , a west tagonica , and O. o Greece ,29,35. FO. patagonica’s invasive behavior may have “expanded” west to east. Serrano et al. [Despite lack of evidence for a demographic expansion from west to east, it appears that o et al.  report ao et al.  reportedo et al.  that popo et al. .O. patagonica has always existed somewhere in the eastern Atlantic and has recently become invasive in the Mediterranean, expanding in local regions in response to environmental change [Carijoa riisei, was believed to have been recently introduced from its native range in the Caribbean. However, Concepcion et al. [Carijoa and found that the Hawaiian populations were not genetically similar to the Caribbean and therefore did not originate from there. The originally misidentified native diatom Didymosphenia geminata remained undetected in its native range for decades before blooms were documented in the 1990s [Littorina littorea, has long been thought to have been recently introduced to North American from Europe. However, both mitochondrial and nuclear sequence data indicated that the North American and European populations diverged thousands of years ago. This native gastropod is also believed to have begun expanding along the coast of New England as a result of environmental changes [It seems most likely that l change , likely l change ,34. In an et al.  used mithe 1990s . Today, he 1990s ,64. The O. patagonica will require work aimed at better identifying and characterizing the source populations and population dynamics of well documented locally expanding O. patagoncia populations within the Mediterranean, such as along the coasts of Spain [O. patagonica exits but is not to date invasive to better understand the mechanisms driving its expansion. Such studies may also aid in assessing the future of the newly discovered coral species, Oulastrea crispate, in the Mediterranean as it too is expected to rapidly expand its range [Identifying additional mechanisms that facilitate invasiveness in of Spain  and Greeof Spain . The conts range .Oculina patagonica. While marine range shifts may occur at a slower rate than marine introductions, their potential effects on the community are likely to be just as significant [O. patagonica has been shown to successfully compete with bryozoan Watersipora sp. [O. patagonica dominance in the Mediterranean. Given that macroalgae are important primary producers, this shift may result in significant changes in ecosystem functioning.Additional studies are also needed to better understand the ecological consequences of the expansion of nificant . O. patapora sp. . Serranopora sp.  have repO. patagonica in the midst of environmental change may shed light into assessing and managing the long-term success of corals that are currently at risk.Corals are currently facing worldwide declines as a result of stresses, including increasing sea temperatures, disease, and other anthropogenic disturbances -71. UndeOculina patagonica is a recently introduced coral species in the Mediterranean, we found no genetic or historical demographic evidence to support that claim. Our results suggest that Mediterranean populations of O. patagonica have long been isolated from WA Oculina spp., and have only recently become invasive in the Mediterranean, most likely due to environmental changes. We advise against hastily identifying a previously unknown species as being introduced without detailed genetic analyses and comparisons to potential source populations. Accurate identification of species’ invasive statuses will enable more effective research programs aimed at better understanding the mechanisms promoting the invasive nature of species, which can then lead to the implementation of efficient management plans.Despite years of maintaining that Oculina patagonica were collected from Spain, Italy, Greece, Lebanon, and Israel during the summers of 2011–13  of Oculina spp. samples (n = 56) consisted of a subset of populations along the coast of the eastern United States from Eytan et al. [O. arbuscula, O. varicosa, and O. diffusa, although Eytan et al. [O. diffusa and O. varicosa from Bermuda. We will refer to these samples collectively as “Western Atlantic (WA) Oculina spp”. All sampling was conducted by or with local collaborators in accordance with local and CITES regulations.Western North Atlantic n et al. . The foun et al.  found noCOI) gene and the coding region of five nuclear genes  were previously developed to assess subdivision in WA Oculina spp. populations [crystalline and S-adenosylmethionine synthetase) were developed using an expressed sequence tag (EST) library [We extracted genomic DNA using QIAGEN DNeasy Kit following the manufacturer's protocols with the following modifications. We lysed tissues at 56°C overnight. We added 200 μl elution buffer and incubated at room temperature for an hour prior to the final centrifuge step. All individuals were genotyped, either previously or in this study, for the mitochondrial cytochrome oxidase I  amplifications were conducted in 25 μl reactions consisting of 2.5 μl of 10x buffer, 10 μM of dNTPs and each primer, and 0.2 μl of One TaqTM DNA polymerase (New England Biolabs Inc.). Amplifications were performed in a Bio-Rad T100 thermocyler under the conditions outlined by Eytan et al. . SamplesTo prevent clonal reproduction from skewing subdivision and genetic diversity measures, we removed individuals that shared a multilocus genotype with another individual in the same population. The final nuclear data set contained 105 individuals , genetic diversities  and migration rates (m1 and m2) for two populations. To convert divergence time (t), which is scaled by mutation in IMa, to years, we used the average nuclear substitution rate for Porites corals of 0.138% per Ma [Oculina corals is unavailable, which was converted to a rate per locus per year for each marker . IMa analyses were conducted with high performance computational resources provided by Louisiana State University .We recorded the maximum-likelihood estimate from the posterior probability distribution for divergence time, adjusted with a two year generation time  and its Oculina spp. in the eastern Atlantic and/or the Mediterranean. First, we explored the Smithsonian National Museum of Natural History’s Department of Invertebrate Zoology records by performing a keyword search of Oculina on the IZ collections database website [Oculina on the collections website [Oculina spp. from eastern Atlantic and Mediterranean countries and recorded the catalog number, species name, location, and geologic age. Several of the specimens from the Smithsonian National Museum of Natural History were observed during a visit to the museum in January 2013.We searched museum collections for evidence of a fossil record of  website . We also website . From boTo test for expansion in the Mediterranean, we used LAMARC 2.0 , which eCOI, p14, p62, and p302 for western North Atlantic Oculina spp. populations obtained by Eytan et al. [Haplotypes for n et al.  are avai


Emmenopterys henryi. We found that geography played a predominant role at all levels – phylogeographic clades are broadly geographically structured, the deepest levels of divergence are associated with major geological or pre-Quaternary climatic events, and isolation by distance (IBD) primarily explained population genetic structure. However, environmental factors are clearly also important – climatic fluctuations since the Last Interglacial (LIG) have likely contributed to phylogeographic structure, and the population genetic structure (in our AFLP dataset) was partly explained by isolation by environment (IBE), which may have resulted from natural selection in environments with divergent climates. Thus, historical and contemporary geography and historical and contemporary environments have all shaped patterns of genetic structure in E. henryi, and, in fact, changes in the landscape through time have also been critical factors.Examining how historical and contemporary geographic and environmental factors contribute to genetic divergence at different evolutionary scales is a central yet largely unexplored question in ecology and evolution. Here, we examine this key question by investigating how environmental and geographic factors across different epochs have driven genetic divergence at deeper (phylogeographic) and shallower (landscape genetic) evolutionary scales in the Chinese Tertiary relict tree  Understanding the contemporary and historical ecological  factors shaping population genetic diversity, structure, and divergence is of great interest to molecular ecology, evolutionary biology and conservation biology1245678941112131416For example, climatic fluctuations during the Quaternary which resulted in population isolation in multiple refugia are considered major drivers of population divergence and broad phylogeographic patterns1819521Cathaya, Ginkgo, Metasequoia, Davidia, Emmenopterys) are thought to represent remnants of the so-called ‘boreotropical flora’ that likely formed a belt of vegetation around the Northern Hemisphere during the Early Tertiary/Eocene2728Emmenopterys henryi Oliv. (Rubiaceae).So, how do historical and contemporary geographic and environmental factors contribute to genetic divergence at different evolutionary scales? This is a central yet largely unexplored question in ecology and evolution16232425Emmenopterys henryi is a particular suitable species for addressing these issues. This deciduous tree, which is the only extant species of its genus, is native to subtropical China, where it occurs in disjunct montane valleys of mainly warm-temperate deciduous (WTD) forests, at elevations ranging from c. 400–1600 (2000) m above sea levelE. henryiEmmenopterys species are known from the Eocene of North America and GermanyE. henryi. However, there is dated molecular evidence to suggest that the origin of this species dates back to the Early MioceneE. henryi exhibits significant population genetic structure and divergenceE. henryi across subtropical China. Our main objectives were to: (i) estimate the timing and pattern of divergence among populations of E. henryi; (ii) investigate how climatic and geographical variation over space and time explain patterns of phylogeographic and population genetic structure; and (iii) explore the specific environmental variables that may underlie local adaptation through natural selection in divergent environments.In this study, we integrate genetic markers that capture signatures from historical (chloroplast DNA) and contemporary  divergenceE. henryi were aligned along a total length of 2163 bp with 26 single-site mutations (including two 1-bp indels), 18 length polymorphisms (2–78 bp) and one inversion (27 bp) observed  was estimated to be 0.928 and within-population diversity (hS) was 0.332 , but πs was weakly related to latitude .The three cpDNA-IGS regions surveyed across the 443 individuals of observed . A totalal China ; Fig. 1Aal China ; Fig. 1Aas 0.332 . Regressbeast supported the monophyly of E. henryi [posterior probability (PP) = 1], and two main (‘Northern’ vs. ‘Southern’) lineages were recognized with weak support . The four cpDNA groups identified are populations S1–S6 in the east (Southeast group), S7–S23 in the middle , N1–N6 in the northeast (Northeast group), and N7–N15 in the northwest (Northwest group). This grouping is mostly consistent with the phylogenetic analyses . Hierarchical AMOVA, however, revealed that of the total genetic variation, 36.48% was distributed between the Northern lineage and the Southern lineage (ΦCT = 0.365), 45.43% was explained by variation among populations within regions (ΦSC = 0.715), and only 18.10% was found within populations  when compared to the Northern lineage  and in the Southern lineage , but no such structure was found in the Northern lineage  and the region scale .Non-hierarchical AMOVA  revealedE. henryi were aligned with a total of length of 767 bp, exhibiting 10 nucleotide substitutions  of otype R1 .E. henryi and P. bracteata diverged at c. 22 Ma . The onset of lineage diversification was estimated as 3.42 Ma  and 3.64 Ma . For this cpDNA-IGS chronogram, we recovered an average substitution rate of 6.225 × 10−10 s/s/y by the beast analysis, which is much lower than the average values generally reported for noncoding regions of the chloroplast genome (e.g. 1.2–1.7 × 10−9 s/s/y)373839Based on the assumption that E. henryi, at least one of the estimates of Tajima’s D and Fu’s FS was generally significant, except for the Central-Southwest group , with the exception of the Northeast group , we dated the three spatial expansions to the last glacial cycle(s)  were polymorphic. Different primer pairs amplified variable numbers of fragments, from 38 to 69, with an average of 50.8 ± 10.1 fragments per primer combination  varied widely , with PPF = 57.77%, I = 0.035, and HE = 0.023. Regression analyses showed that HE was weakly related to latitude .The nine primer combinations employed with samples from 37 populations  of bination . The perbination . Becaused widely . The higbaps identified nine different clusters , levels of population subdivision still remained high (ΦSC = 0.364), but were markedly higher in the Southern lineage (ΦST = 0.416) than in the Northern one  produced by clusters . All popclusters . In the clusters . The NJ clusters . With feclusters  and S2B.maxent model for E. henryi had high predictive power and did not overfit the presence data (AUC = 0.804 ± 0.003). The current distributional predictions  in this species. In both analyses, IBD explained more of the variation in genetic differentiation than IBE  and estimated that IBD (βD = 0.360 ± 0.037) contributed about twice as much as IBE (βE = 0.181 ± 0.151) to genetic distance. Similarly, MMRR also estimated the contribution of IBD  to be larger than that of IBE . Each analysis detected low levels of covariation between geographical and environmental distance (0.133 ± 0.028 in SEM and 0.488 in MMRR), although the covariation based on the MMRR analysis was approaching the reliability limit of 0.5  and multiple matrix regression with randomization (MMRR) analyses both revealed significant effects of IBD and IBE on neutral (non-outlier) AFLP divergence  and bio7  were the primary contributors, followed by bio1 , bio5 (max temperature of warmest month), bio9 (mean temperature of driest quarter), and bio15 . By contrast, slope, soil, and bio12  had very minor contributions.For the SEM analysis, we also quantified the contributions of the individual environmental variables to the composition of the environmental dissimilarity latent variable . The resfdist, 67 of 457 loci (14.66%) showed high probability (99.5%) for divergent selection among the nine baps groups, including 21 loci under directional selection and 46 loci under balancing selection  associated with at least two of the eight selected environmental variables , bio4 , bio5 (maximum temperature of the warmest month), bio12  and bio15  were most highly associated with the potential loci under selection.Using election . Additiobayescan . Eventua . The boundary between these lineages is located in the region of the Yangtze River and next to the Three Gorges Mountain Region  of E. henryi, at approximately the Mio-/Pliocene boundary [c. 5.06 Ma (1.68–8.91) Ma; see node 2 in 4345c. 3.4 Ma)46E. henryi through habitat fragmentation and the formation of physical barriers to gene flowTaxus wallichianaCercidiphyllum japonicumKalopanax septemlobusE. henryi during the last glaciations, indeed, is supported by our ENM analysis, which indicates larger distribution ranges of this lineage at the LGM compared to both the LIG and present  of its range. These groups  harbor lower genetic diversity (E. henryi phylogeny (tcs networks (c. 0.19 Ma (95% CI: 0.111–0.314 Ma) and c. 0.26 Ma , although, due to the broad confidence intervals, this interpretation requires some caution. In fact, the species is not predicted to have occurred in the northern Sichuan Basin and the Daba/Qinling Mts. during the LIG  once more coincides with the penultimate (Riss) glacial (see above). In addition to the ENM and MDA analyses . Thus, we found evidence of both isolation by distance (IBD)E. henryi populations, and broad-scale geographic and climatic factors appear to have shaped the spatial distribution of genetic variation in this species at both deeper evolutionary and more recent ecological time scales.Clearly, at broad phylogeographic scales, our results demonstrate distinct geographic genetic structure across the range of baps clusters of E. henryi along environmental clines. All four loci identified by both fdist and bayescan as undergoing putative diversifying selection . Altogether, these results are consistent with population genetic divergence under natural selection in divergent environments, suggesting this may have been the process generating IBE in this system.To examine the potential mechanism driving the pattern of IBE, we tested for loci under selection, in our AFLP dataset, and their associations with environmental/climatic variables. In identifying outlier AFLP loci, we sought to determine how selection might play a role in shaping genetic differentiation of the nine election  were asselection , suggestE. henryi, and, in fact, changes in the landscape through time have also been critical factors.In conclusion, by using phylogeographic, landscape genetic, and ecological niche modeling analysis together, we were able to identify the many factors, both historical and contemporary, that have shaped spatial genetic structure in this Tertiary relict species. At various spatial and evolutionary scales, we found that both geographical and environmental/climatic factors contribute to patterns of genetic structure. Geography played a predominant role at all levels – phylogeographic clades are broadly geographically structured, the deepest levels of divergence are associated with major geological or pre-Quaternary climatic events, and IBD primarily explained population genetic structure. However, environmental factors are clearly also important – climatic fluctuations since the LIG have likely contributed to phylogeographic structure, and the population genetic structure (in our AFLP dataset) was partly explained by IBE, which may have resulted from natural selection in environments with divergent climates. Thus, historical and contemporary geography and historical and contemporary environments have all shaped patterns of genetic structure in Emmenopterys henryi throughout its range (n = 212) was also sequenced at the entire internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Of these 433 individuals, 394  were surveyed for AFLPs (Pinckneya bracteata (Bartram) Raf., collected from the JC Raulston Arboretum , was selected as an outgroup for the phylogenetic analyses based on a previous molecular phylogenetic study of RubiaceaeE. henryi are stored at the Herbarium of Zhejiang University .We obtained silica-dried leaf material from 38 populations of or AFLPs ; Fig. 1ApsbA–trnH, trnL–trnF, trnT–trnL) and the ITS region (i.e. ITS1 + 5.8S + ITS2). The primers and methodology for amplification of these four DNA regions were described in Shaw et al.et al.geneious v.4.8.5For the phylogeographic DNA analyses, we sequenced three cpDNA-IGS regions  and nucleotide (π) diversities were calculated for each population and the species overall using dnasp v.5.10tcs v.1.21permut was employed to compare parameters of population differentiation with unordered and ordered alleles  based on 1000 random permutationssamova v.1.0FST/(1 − FST) against the logarithm (log10) of geographic distance for all pairs of populations, or subsets thereof, following RoussetFor both cpDNA and ITS, haplotype (FT), the percentage of fragments that are polymorphic within each population (PPF), Nei’s (1973) gene diversity (HE), and Shannon’s information index I following the method of Lynch & Milliganaflpsurv v.1.0DW; according to Schönswetter & TribschK) in the AFLP dataset, we used three approaches. First, we used baps v.6.0q) with regard to the detected clusters. Second, we utilized genalex v.6.4phylip package 3.6 with 1000 bootstraps  selected by jmodeltest v.2.0ucld) model, and a coalescent model assuming constant population size were used to model the tree prior. Since no reliable fossils of E. henryi are currently known (see Introduction), we used the divergence time between E. henryi and P. bracteata, inferred from a fossil-calibrated cpDNA phylogeny of Rubiaceae, as a secondary calibration point to calibrate the stem node of E. henryitracer v.1.5 (http://tree.bio.ed.ac.uk/software/tracer/) to ensure that parameter values were fluctuating at stable levels. Based on these results, the first 5000 trees were discarded as burn-in, and the remaining samples were summarized as a maximum clade credibility (MCC) tree with mean divergence times and 95% highest posterior density (HPD) intervals of age estimates in treeannotator v.1.8.0figtree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).To relate differentiation among cpDNA haplotypes of DFSarlequin to infer the demographic history of two main lineages identified  from their combined cpDNA-IGS sequences by mismatch distribution analysis (MDA) (further details in Tajima’s E. henryi has not changed (and will not change) its climatic preference over at least the last glacial/interglacial cycle and into the futureE. henryi across its range under present (1950–2000), past [the Last Glacial Maximum 80c. 130–114 kya)maxent v.3.2.1. In addition to the 38 distribution records included in this study, 76 herbarium-recorded presences were sourced from the Chinese Virtual Herbarium (www.cvh.org.cn) and the National Specimen Information Infrastructure of China (www.nsii.org.cn). Based on a total of 114 records, a current distribution model was developed using six bioclimatic data layers  available from the WorldClim database (www.worldclim.org)39http://gisweb.ciat.cgiar.org/GCMPage/) with 30 arc-second resolution.Assuming maxent. Values of the area under the ROC curve (AUC) between 0.7 and 0.9 indicate good fitModel performance was evaluated using receiver operating characteristic (ROC) analyses in FST) with arlequin using all putatively neutral (non-outlier) AFLP loci (i.e. 451 loci) described above. We obtained a total of 21 environmental and geographical variables for our study area, including 19 bioclimatic variables with 30 arc-second resolution (http://www.bioclim.org), a slope layer based on a digital elevation model with 1-km resolution from the USGS EROS database (http://eros.usgs.gov), and soil type from the Chinese soil taxonomy recordTo quantify the roles of geography and ecology in spatial genetic divergence, we quantified isolation by distance (IBD)588FST). MMRR provides a straightforward method for estimating linear regressions among distance matrices, while SEM allows for flexibility in defining the relationships among variables and utilizes latent variable modelling. For the MMRR analysis, we constructed an environmental distance matrix by taking the population pairwise Euclidian distances across all 21 environmental variables. We then analyzed the effects of environmental distance and geographic distance, as explanatory variables, on genetic distance, as the response variable, using the ‘MMRR’ function in Rhttp://cran.r-project.org/web/packages/lavaan/). Prior to analysis, all environmental, geographic, and genetic distance matrices were standardized. In each case, the analysis was used to test three alternative hypotheses: (1) only geographical distance contributes significantly to genetic distance, (2) only environmental distance contributes significantly to genetic distance, and (3) geographical distance and environmental distance each contribute significantly to genetic distance. We performed both analyses on AFLP pairwise genetic distances (FST) for all populations.We then employed two related methods to quantify IBD and IBE: multiple matrix regression with randomization (MMRR)FST comparisons to identify the loci that are significantly different than expected under neutrality and a given demographic model; and (ii) correlative methods that look for associations between genetic variation and environmental variables. The key to using these approaches together is that they identify links between putative loci under selection and individual environmental variables. Hence, our primary purpose for using these tests was to identify the specific environmental variables that are potential drivers of genetic differentiation via divergent natural selection (e.g. IBE resulting from selection in divergent environments).Two different basic approaches allow screening for AFLP loci that are putatively under selection: (i) a genome scan test procedure based on fdistbayescanbaps analysis (K = 9). Then, to detect associations between allele frequencies and environmental variables, we used Multiple Linear Regression (MLR)fdist and bayescan, we computed their population pairwise frequencies of AFLP alleles at the 37 sampling sites. We extracted values for the geographical coordinates of each population from 19 bioclimatic GIS data layers with 30 arc-second resolution . We then regressed the allele frequencies of the retained outlier loci (dependent variables) on the selected environmental variables  using the MLR model . Sci. Rep. 6, 24041; doi: 10.1038/srep24041 (2016).


The study was conducted at Southern Regional Research Center, ICAR-Central Sheep and Wool Research Institute (CSWRI), Mannavanur, Kodaikanal, Tamil Nadu to estimate genetic trends for birth weight (BWT), weaning weight (3WT), 6 months weight (6WT), and greasy fleece weight (GFY) in a Bharat Merino (BM) flock, where selection was practiced for 6WT and GFY.The data for this study represents a total of 1652 BM lambs; progeny of 144 sires spread over 15 years starting from 2000 to 2014, obtained from the BM flock of ICAR-SRRC (CSWRI), Mannavanur, Kodaikanal, Tamil Nadu, India. The genetic trends were calculated by regression of average predicted breeding values using software WOMBAT for the traits BWT, 3WT, 6WT and GFY versus the animal’s birth year.The least square means were 3.28±0.02 kg, 19.08±0.23 kg, 25.00±0.35 kg and 2.13±0.07 kg for BWT, 3WT, 6WT and GFY, respectively. Genetic trends were positive and highly significant (p<0.01) for BWT, while the values for 3WT, 6WT and GFY though positive, were not significant. The estimates of genetic trends in BWT, 3WT, 6WT and GFY were 5 g, 0.8 g, 7 g and 0.3 g/year gain and the fit of the regression shows 55%, 22%, 42% and 12% coefficient of determination with the regressed value, respectively. In this study, estimated mean predicted breeding value (kg) in BWT and 3WT, 6WT and GFY were 0.067, 0.008, 0.036 and −0.003, respectively.Estimates of genetic trends indicated that there was a positive genetic improvement in all studied traits and selection would be effective for the improvement of body weight traits and GFY of BM sheep. During the last 40 years, extensive efforts have been made to improve the production potential of indigenous sheep through various research and developmental efforts by different agencies. The earlier efforts were aimed at improving sheep for fine wool production through introducing exotic fine wool inheritance or up-gradation using the indigenous improver wool or dual-purpose breeds on a limited scale. Though, considerable achievements have been made in developing new strains like Bharat Merino (BM). The flock of BM sheep has undergone 16 generations of multiple trait selection so far. The reliable estimates of genetic trends, i.e. the annual rate of the genetic change allow comparison of expected and realized genetic change in the experimental situations and assessment of progress in a particular trait.A study of genetic trend in important economic traits is, therefore, necessary for knowing its performance status over the years to develop the breeding strategies for further genetic improvement of productivity of this flock of sheep. The estimation of genetic trends over time is problematic because of the difficulty in conducting experiments in uniform conditions over a period of several generations so that changes in performance of a selected population may reflect, in part, both environmental and genetic changes . TheoretThe objective of this study was to estimate genetic trends for birth weight (BWT), 3WT, 6WT and GFY out of which the traits, 6WT and GFY were under selection in the BM sheep.All the experiments used in the study were carried out as per the approval from institutional animal ethics committee.Mannavanur is located at an altitude of 2030 m above MSL in the Western Ghats of southern India. The minimum and maximum ambient temperature ranges from 0°C to 5°C and 26°C to 30°C, respectively in this sub-temperate region, with the mean relative humidity between 15% and 90%. The rainfall is erratic and round the year with an annual mean rainfall of 1055 mm.Pennisetumclandestinum) having 11.35% crude protein (CP); 23.09% crude fiber (CF) and spear grass (Heteropogoncontortus) having 7.90% CP; 29.38% CF on dry matter basis. The ewes were housed in sheds during night. The animals were also provided concentrate mixture  at 400 g/day/ewe during late pregnancy and lactation. In the remaining period, ewes were provided with concentrate mixture at 3000 g/day/ewe. Breeding was carried out in two seasons viz. spring breeding (March-April) and autumn breeding (September-October). The animals were shorn once in a year during March.The data for the present study represents a total of 1652 BM lambs; progeny of 144 sires spread over 15 years starting from 2000 to 2014 was obtained from the BM flock of SRRC, Mannavanur. In addition to two different economic traits 6WT and GFY under selection, BWT and 3WT were used for analysis in BM genetic improvement project. The traits under study were BWT, weaning weight (3WT), 6 months weight (6WT) and grease fleece wool yield (GFY). A flock of around 400 BM sheep was maintained at the ICAR-SRRC (CSWRI), under semi-intensive with standard management system. The animals were allowed to graze from morning 8 am to evening 5 pm on the grazing area having grass species mostly kikuyu  value, respectively (2) value could be that these traits being quantitative, there are several environmental factors affecting them. The possible independent variables based on availability on data were included in the model. Estimated MPBV (kg) in BWT, 3WT, 6WT and GFY were 0.067, 0.008, 0.036 and −0.003, respectively. The magnitude of genetic trends estimated illustrate that there has been a highly significant and positive genetic improvement in BWT. However, the values for other traits though positive, were not significant.The genetic trends express the outcome of a given genetic improvement program in a herd in terms of the rate of change in performance level of the herd a unit of time which is commonly referred to as “trends” of performance level of the herd. The evaluation of genetic trend gives an indication of genetic direction of the breed as well as the rate of genetic improvement from the time of application of the breeding program . The estectively . Reason et al., Di et al. [et al. [Means of predicted breeding values of BWT in each year of birth calculated from the four-trait analysis are shown in i et al. ,13 for Mi et al. ,15. Howe [et al. . When co [et al. , Kermani [et al.  and Dorm [et al.  was highet al. [et al.  reportedet al. ,18,20 weet al. [In et al.  reportedet al. ,21 were et al. .et al. [The genetic trend for GFY indicated that the genetic gain was 0.3 g/year, which was low, although in positive direction . But in et al.  reportedUsually, the genetic gain correspondents to the estimate of additive genetic variance of the breed and also the selection practices being followed. However, not only this but also the environmental determinants of variation have a significant impact on most of the production traits on which we have a very little control such as erratic rain fall, decrease in grazing area, and disease occurrence. There is a need to also unravel factors which increase the predictability of these variables for better management solutions. Estimates of genetic trends indicated that there was positive genetic improvement in all studied traits and selection would be effective for the improvement of body weight traits and GFYof BM sheep.PKM and SMKT designed the experiment. PKM, SRP, and GM conducted the experimental work and collected data. PKM, RV and SMKT were involved in analysis of the data. PKM, RP and GN were involved in scientific discussion, PKM, RP, GN and RV drafted and revised the manuscript. ASR monitored the overall work. All authors read and approved the final manuscript.


In spite of nearly 40% of variability in blood pressure being explained by genetic factors, the identification of genes associated with essential high blood pressure is difficult to determine in populations where individuals have different genetic backgrounds. In these circumstances it is necessary to determinate whether the population is sub-structured because this can bias studies associated with this disease. To determine the genetic structure of the population in Bucaramanga from genetic polymorphisms associated with the regulation of blood pressure: 448G>T, 679C>T y 1711C>T from the gene kinase 4 of the dopaminergic receptor linked to the protein G and Glu298Asp, -786T>C and the VNTR of the intron 4 of the gene of endothelial nitric oxide. A sample of 552 unrelated individuals was studied through analysis of restriction fragment length polymorphism. The allelic, haplotypic and genotypic frequencies were calculated, the Hardy-Weinberg equilibrium was determined and a molecular analysis of variance was performed to determine the genetic structure.ST= 0.0038). Thirty-eight (38) Haplotypes were identified with GCCTG4b being the most frequent (21.2%). The most diverse polymorphism was 448G>T with a frequency of 49.9% for heterozygous. The six polymorphisms were found in genetic equilibrium and a genetic structure of populations was not evidenced (F The population studied does not present a genetic sub-structure and the polymorphisms analyzed were found in genetic equilibrium. This indicates that the population mixes randomly and there are no sub-groups capable of affecting the results of the association studies. Blood pressure levels tend to aggregate in families due in part to shared genetic predispositions. In fact, about 40% of the variability in blood pressure is explained by genetic factors and the risk of developing it after age 50 doubles for each first-degree relative with a history of hypertensionGenetic variants or polymorphisms associated with the regulation of urinary excretion of sodium and vasomotor regulation are potential risk factors for the development of hypertension. Among the former are the polymorphisms 448g>T or R65L or rs2960306, 679C>T or A142V or rs1024323 and 1711C>T or A486V or rs1801058 of the gene GRK4 that encodes the kinase 4 of receptors coupled to the G protein, specifically D1 and D2 dopamine receptors, which mediate the natriuretic effect of catecholamine in the proximal convoluted tubule of the nefron-One of the obstacles in identifying genetic variants associated with hypertension is the comparison of cases and controls that come from populations with different genetic backgrounds. This problem is known as genetic structure and generates a selection bias due to cases and controls having a different distribution of alleles of the polymorphisms associated with the disease of interestTaking into account that the national and local context are minimal, the studies involving the aforementioned polymorphisms, likewise the deficiency of research to analyze participation in the genetic structure in our population led to a population genetic study being designed in Bucaramanga. It starts with genotyping of the polymorphisms 448G>T, 679C>T and 1711C>T of the gene GRK4 and Glu298Asp,-786T>C and intron 4 of the eNOS gene in order to establish the degree of genetic structure for this population. It is expected that these results will give support to subsequent association studies of these polymorphisms with HAE in the population of Santander and thus avoid potential bias regarding the possibility of finding associations that could be false.Five hundred fifty-two (552) participants were selected for the INEFAC (Incidence of Cardiovascular Disease and Risk Factors in Colombian) project, a cohort study with random sampling of residents of Bucaramanga, Colombia from the lower socio-economic strata of 2 and 3. The sample included 372 women and 180 men between the ages of 16 and 69 years with an average of 35.1 years and normotensive (systolic blood pressure <120 mmHg and diastolic blood pressure <80 mmHg). This study was approved by the ethics committee of the School of Health from the Universidad Industrial de Santander and all participants gave their written informed consent.-The sample calculations were performed taking into account the frequency of the minor allele prevalence for the six polymorphisms studied in different populationsFrom all individuals a sample was taken of peripheral blood with EDTA anticoagulant and from it DNA was extracted by the phenol-chloroform methodThe sequences of the primers for amplifying each polymorphism are noted in 2, 0.5 µM of each primer, 0.8 µM of deoxyribonucleotide triphosphate (dNTP), 1 U of enzyme Taq DNA polymerase (Promega®) and 3.9 ng of DNA in a final volume of 10 µL. The amplification protocol included an initial step of 94° C for 5 min, followed by 38 cycles as follows: denaturation at 95° C for 15 seconds, annealing at 60° C for 15 seconds and extension at 72° C for 30 seconds and a final step at 72° C for 7 min. In each PCR reaction amplification used negative controls and positives were used for the wild-type and mutant alleles. The PCR products were verified by electrophoresis in a 1% agarose gel including a molecular weight marker of 50 to 500 bp, to check the size of the amplified sample.The PCR reaction for the polymorphisms of the gene GRK4 contained 1X buffer, 3.5 mM MgClThe PCR products were subjected to restriction using the enzymes reported in 2 , 0.4 µM of each primer, 0.8 µM dNTPs, 1 U of enzyme Taq DNA polymerase (promega®) and 2.4 ng of DNA in a final volume of 25 µL. The amplification protocol included an initial step of 94° C for 4 min, followed by 35 cycles as follows: denaturation at 94° C for 30 seconds, annealing at 63° C for 30 seconds and extension at 72° C for 1 min and a final step at 72° C for 5 min. The amplification of these polymorphisms was verified by electrophoresis in a 1% agarose gel. The PCR products were subjected to enzyme restriction using the following protocol: 1X buffer, 0.1 µg/µL of acetylated BSA, 1 U of the corresponding restriction enzyme (The set-up of the Glu298Asp polymorphism PCR contained 1X Buffer-GoTaq (r) - Green Master Mix 0.4 µM of each primer and 2.4 ng of DNA, for a final volume of 25 µL. The amplification protocol consisted of an initial step of 95° C for 2 min, followed by 35 cycles as follows: denaturation at 95° C for 45 seconds, annealing at 63° C for 45 seconds and extension at 72° C for 45 seconds and a final step at 72° C for 5 min. The PCR set-up for polymorphism -786T>C containing 1X Buffer, 2.5 mM MgCln enzyme  and 0.5 The products of the enzymatic digestion of the two SNPs studied were separated by means of electrophoresis in 3% agarose gels and for visualization of the bands ethidium bromide was added. The gels were run in an electrophoresis chamber Power Pac 300 (BioRad®) in a 1X TBE buffer and 60 volts were applied for 80 min. Recognition sites for enzymes, the sizes of the expected fragments and the assigned genotypes are shown in The set-up of the PCR for intron 4 was performed using the same protocol as the polymorphism-786T>C. The sizes of the fragments of this polymorphism, amplification products were visualized by means of electrophoresis in a 3% agarose gel with a tension of 60 volts for 80 min. In Table 3 displays the size of the amplification products of this intronOnce the PCR was standardized, six samples were selected of each polymorphism to be sequenced with the Big Dye terminator kit (Applied Biosystems®) and the sequences obtained were aligned with the Clustal W software  to confirm correspondence with the expected fragments.With the results obtained from reading the electrophoresis, two databases were compiled using Microsoft Excel® 2007 validated by the Data Compare software Epi Info, version 3.5.1p values found were greater than 0.05  was evident in the analyzed sample. Additionally, it was not observed that the FST values obtained for each polymorphism studied  and with 10,000 replicates assuming a mixed modelIn the estimation of haplotype frequencies for the six polymorphisms studied, it was found that the most frequent was GCCTG4b (21.2%) from a total of 38 haplotypes detected in the 1,104 chromosomes analyzed .According to the values obtained for the allele frequencies in the study population, the most common allele for each polymorphism of the GRK4 gene was: 448g>T allele T (50.7%), 679C>T allele C (70%), 1711C>T C allele (69%); in a previous study conducted on a Hispanic population in Southern California, the results coincide for the polymorphisms 679C>T and 1711C>T and differ for polymorphism 448g>TThe most frequent allele in each polymorphism of the eNOS gene was: -786T>C allele T (76%), Glu298Asp allele G (72%) and intron 4 allele 4b (90%). These results are consistent with those found in a prior study performed on the Bucaramangan populationIn the present study 38 haplotype combinations were found, the most frequent being GCCTG4b with 21.2%. No publications were found on populations where haplotype frequencies were reported for these six polymorphisms in Hispanic or Colombian populations, making this study the first report thereof.All studied polymorphisms were found in the Hardy-Weinberg Equilibrium indicating that the population is composed of individuals that are mixed randomly. This finding agrees with that reported in previous studies on a Bucaramangan population for the gene eNOSAdditionally, no population structure was found in the analyzed sample, which coincides with that reported in a previous study conducted on the population in the city of Bucaramanga from the analysis of other genetic polymorphic markersThe results found confirm that the study population was found in HWE for all systems studied and do not present population substructure, which allows for further association study of these polymorphisms with essential hypertension since it is clear that the associations between candidate genes to develop multi-factorial diseases must be interpreted within the context of the genetic structure of the population being studied.