Ne) as well as the time since population establishment. Because the Ne estimation helps to explore population demographic history, and allows one to predict the behavior of genetic diversity through time, Ne is a key parameter for the genetic management of small and isolated populations. Here, we explored an Ne-based approach using a bighorn sheep population on Tiburon Island, Mexico (TI) as a model. We estimated the current (Ncrnt) and ancestral stable (Nstbl) inbreeding effective population sizes as well as summary statistics to assess genetic diversity and the demographic scenarios that could explain such diversity. Then, we evaluated the feasibility of using TI as a source population for reintroduction programs. We also included data from other bighorn sheep and artiodactyl populations in the analysis to compare their inbreeding effective size estimates. The TI population showed high levels of genetic diversity with respect to other managed populations. However, our analysis suggested that TI has been under a genetic bottleneck, indicating that using individuals from this population as the only source for reintroduction could lead to a severe genetic diversity reduction. Analyses of the published data did not show a strict correlation between HE and Ncrnt estimates. Moreover, we detected that ancient anthropogenic and climatic pressures affected all studied populations. We conclude that the estimation of Ncrnt and Nstbl are informative genetic diversity estimators and should be used in addition to summary statistics for conservation and population management planning.The amount of genetic diversity in a finite biological population mostly depends on the interactions among evolutionary forces and the effective population size ( Ne) [Ne has recently become a commonly used parameter in population genetics studies of endangered species [Ne, allowing the inference of the population history by estimating changes in historical Ne [Ne allows one to predict the behavior of genetic diversity under different demographic scenarios using computational simulations [It has been recognized that management strategies should consider non-genetic factors  as well as genetic factors  ,2. AlthoNe) ,8–10. Th species . Recent ical Ne –17. Thisical Ne ,19. Moreulations ,18.Ne) is defined as the size of a simple Wright-Fisher population that would have the same increase in homozygosity and the same random drift in allele frequencies as in the actual population considered [Ne will depend on the feature or genetic property of interest, such as the loss of genetic diversity due to genetic drift or inbreeding [NeV, which measures the variance of change in gene frequency through time due to genetic drift, and the inbreeding effective population size NeI, which is defined in terms of the probability that two individuals have the same parent  [NeI measures the loss of expected heterozygosity due to relatedness [Ne have been proposed [The effective population size  ,22. In oatedness . Based oproposed –15,22,23proposed ,12,15–17Ovis canadensis) population on Tiburon Island (TI) was founded in 1975 by a single introduction event of 16 individuals (14 ewes and 2 rams) from Punta Chueca Sonora, Mexico [The bighorn sheep  and found a lower effective number of founders than the actual number of individuals introduced (14 founders vs. 16 introduced). Recently, another study was published estimating the genetic diversity of the TI population [HE, HO and F' statistics.Hedrick et al.  conductepulation , reportiNeI and HE of using the TI population as a source of individuals for founding new populations. Finally, to compare our approach, we re-analyzed previously published microsatellite allelic data from seven O. canadensis populations and three other artiodactyl species.In this study, we explored an inbreeding effective population-size-based approach to assess the genetic diversity of the TI population, and the demographic scenarios that could explain such diversity, including its foundation and historical events. In addition, we estimated the possible outcome in terms of The work met the Mexican legal requirements about animal welfare and field work, and was supervised and approved by Dirección General de Vida Silvestre (DGVS), Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT). The hair sampling was performed in accordance with the Mexican official standard NOM-062-ZOO-1999 . Because hair sampling is a non-invasive technique, it was not necessary to ask for the Institutional SICUAE  approval.The sampling in Tiburon Island was conducted under the Seri Government permission, as well as with permission of the owners of "Rincón de la Madera" and "La Mesa" UMAs . The special permit for sample collection was issued under Mexican law by DGVS and SEMARNAT.Ovis canadensis), including mountain ranges and typical continental vegetation dominated by deciduous shrubs and succulent cacti [Tiburon Island is located in the Gulf of California near the coast of Sonora, Mexico . The islnt cacti ,38. Hairnt cacti ,40.Ovis aries or Bos taurus , observed heterozygosity (HO), and number of alleles (na), as well as its respective standard deviation (S.D.), using Arlequin version 3.5 [FIS statistic , so all LnP(D) values were plotted against k to visually inspect its behavior and choose the k value with higher likelihood and lower variance. The genetic diversity analysis of the mitochondrial sequences were carried out estimating the number of haplotypes, nucleotide diversity (π), and haplotype diversity using DnaSP version 5.10 [To estimate the genetic diversity of the microsatellite data of the TI population, we calculated the expected heterozygosity  weren index, ; the sigsion 2.3  with 500sion 2.3 . The numo et al.  method wo et al. . Howeverion 5.10 .NeI) [r, defined as Ncrnt /Nstbl, where Ncrnt is the current inbreeding effective size and Nstbl is the ancestral stable inbreeding effective size . The r ratio is expressed in log10. Thus, if r is negative, the population has declined; if r = 0, the population has remained stable; and if r is positive, expansion is indicated. MVSVAR version 0.4.1 also estimates θ defined as 2Ncrntμ, where μ is the mutation rate and tf is the time interval of the inbreeding effective population size change in generations scaled by Ncrnt. The software was run for 2x109 steps recording 20,000 points from the posterior distribution. The upper and lower bounds for the parameters θ, r, and tf were , , and , respectively. The limits for r comprised both population reduction and population expansion. If 95% of the High Density Interval (HDI) of the posterior distribution did not reach zero, we considered this as a signal of reduction on inbreeding effective population size. To quantify the time in years since the population size started to change (Tfa) and the ancestral stable and current inbreeding effective population sizes (hereafter Nstbl and Ncrnt), we used the software MSVAR version 1.3 [Nstbl, Ncrnt and Tfa were , , . The generation time for each species was considered as the mean time for female sexual maturity; all values were taken from AnAge Database [Nstbl, Ncrnt and Tfa) was reported as the mode of the posterior distribution, using the first and third quartiles as a measure of distribution dispersion.We used the method proposed by Beaumont  and StorNeI) . This meNeI)  and martNeI) . FinallyNeI) . In ordeNeI)  to estimsion 1.3 . The numDatabase . For botDatabase  was usedDatabase  implemenDatabase . The lasDatabase  and hdrcDatabase . In ordeDatabase , we analNcrnt and Nstbl estimates, we used the Robust Bayesian Estimate (RBE) [To test for differences among populations in the te (RBE) . This apte (RBE)  implemenNcrnt of the source population  that could explain the actual genetic diversity observed in the TI population , with Sonora Ncrnt=385; strong bottleneck (Scenario C) using Sonora Ncrnt=500; and severe bottleneck (Scenario D) at Sonora Ncrnt=918  as well as the actual number of founders . All loci had a 2 bp motif tandem repeat varying in range from of 20 to 40 bp in length. All summary statistics available were calculated and each scenario had 4 million simulations, from which 0.1% was used for parameter estimation. The scenario selection was carried out using the statistical tests implemented by the software.The ABC analysis was performed using the DIY-ABC software, version 1.0.4.37 . The sceividuals ) . The parameters recorded were HO, HE, and na. To evaluate the effects on Ncrnt on the simulated populations, one data set was selected at random and was analyzed using MSVAR version 1.3 using the settings reported above. All graphics and statistical analysis were performed with R.In addition, to infer the possible impact on the genetic diversity using TI as a source of individuals for founding new populations, we simulated the possible outcome if new populations were founded from the TI population using 8, 16, 32 and 64 individuals . The simNeI-based approach with other populations and species, we re-analyzed seven populations of bighorn sheep ; one population from New Mexico, USA (RRNM) ; and one from Alberta, Canada (SRA)  [Bison bison) populations [HE, census size, and Ncrnt (Oryx leucoryx), from a protected area in Saudi Arabia [Muntiacus crinifrons) of the Cervidae [Ncrnt estimation for all data sets was carried out with MSVAR using the scheme previously described for the TI population.In order to compare the rn sheep : four porn sheep ; one popadensis) ,30. We aulations : one popd Ncrnt . In ordei Arabia  and fromCervidae  . The overall values of HE and HO were 0.501 (S.D. 0.155) and 0.472 (S.D. 0.159)  and BM1818 had significant values . The overall locus analyses showed a slight, but significant, positive FIS value of 0.058, indicating a departure from H-W proportions for heterozygote deficit (p=0.008). Tests for linkage disequilibrium showed that 7 of 66 tests had a significant value (p < 0.05). It is worth mentioning that the loci used for this study are located on different chromosomes, except for OarFCB128 and BM2113, which are located in chromosome 2 (but have a distance > 250 cM). However, these loci did not show significant linkage values. We interpret this result as a signal of a population bottleneck, as linkage disequilibrium can be associated with population size reduction and genetic drift [k=1; hence, the TI population was considered as a single population for the MSVAR runs.We found 40 alleles for the 12 microsatellite loci analyzed. The number of alleles ranged from 2 to 7, with an average of 3.33 (S.D. 1.435)  . There w alleles . Althougent loci , only Oar estimate was -2.192, while the estimates of the current (Ncrnt) and ancestral stables (Nstbl) were 271  and 10,522  individuals. Nevertheless, the date of the event of population decrease did not correspond to the TI foundation, as the Tfa estimate was 3,155  years ago  was 0.125 (S.D. 0.055), and the nucleotide diversity (π) was 0.00249 (S.D. 0.0011). We recovered the two TI haplotypes previously reported for the species in Genbank . The haplotype AY116621 was predominant (present in 97% of the sample). There were 11 segregating sites, the haplotype diversity  and na =3.639 (S.D. 0.3465), while the Sonora-simulated population had HE =0.5136 (S.D. 0.0128) and na of 4.168 (S.D. 0.317). In accordance with the real data set, we observed the presence of null alleles in a random sample of 100 simulations taken from the TI-simulated data . A g-test performed against a binomial distribution [OarFCB266 locus in the real TI population could be explained by a deficiency of heterozygotes on this locus. This deficiency could be caused by the population size reduction, due to the TI foundation, more than an artifact of the PCR amplification. As expected, the Sonora-simulated population showed higher diversity (t-test p=0.0002) than the TI-simulated population. In the hypothetical populations, we observed a steady decrease in HE and na as the number of founders was reduced . Nevertheless, as the 95% HDI of the Ncrnt and Nstbl posterior distributions did not overlap, we take this result as a significant signal of population size change.As shown in Ncrnt estimates exhibited differences among species and contrasting values within bighorn sheep and bison populations  individuals for SMO to 388  for SRA , 103 , and 220  for SUH, WCNP, and YNP, respectively. The Ncrnt estimates for the black muntjac (HWCH) were 487  and 361 individuals  for the Arabian oryx (MNWSA). According to the Robust Bayesian Estimate (RBE) analysis, all paired comparisons of the Ncrnt estimates between populations showed significant differences  estimates also had large ranges  for SRA to 56,865  individuals for HMO. The SUH and WCNP bison populations showed 17,853  and 19,006  individuals, and the YNP population showed 24,980  individuals. The estimates for MNWSA and HWCH were 77,821  and 77,357 . All paired comparisons of Nstbl estimates and their significance are reported in The ancestral stable (e ranges . The O. Tfa) are shown in O. canadensis populations ranged from 1,404 years  for SMO to 3,211 years  for RRNM. The bison SUH and WCNP populations showed recent Tfa values , while YNP had 1,803  years. HWCH showed a similar value to bighorn sheep populations  while MNWSA was rather ancient . Except for MNWSA, most of the Tfa results were similar and their interquartile intervals overlapped. Even more, some populations presented almost the same Tfa estimate  but was higher than the estimates for other managed populations [HE values (0.501 vs 0.420). However, this difference could be due to the difference in sample size that led to a sub-sampling of alleles and, therefore, a genetic diversity underestimation . Nevertheless, our analyses indicate that the TI population is not free from a genetic bottleneck. The simulations showed that TI suffered a bottleneck that reduced its Ncrnt to about half of the source population, and that the use of TI as a source for translocation of individuals could lead to a severe Ncrnt reduction, especially when a low number of founders are used. For example, using 16 founders the genetic diversity loss for HE was 3% and for Ncrnt was 25%; meanwhile, using 8 founders we observed a reduction of 8% in HE, and Ncrnt was reduced by about 82%  [Ne caused by the mating system of the species and the isolation of the population [Ne due to an uneven male allelic contribution by polygynous behavior [FIS could be due not only to TI foundation, but also to the Wahlund effect caused by the social structure of the herds in TI. Another factor promoting high levels of genetic diversity is natural selection. Kaeuffer et al. [Ovis aries) population, genetic diversity could be influenced by natural selection, thus overtaking the effects of genetic drift. In this sense, it has been recognized that natural selection plays an important role in maintaining genetic diversity in bighorn sheep [So, what could explain the apparent success of the TI population and the relatively high levels of genetic diversity? It has been argued that the lack of predators, as well as the suitable habitat, promoted a steady growth of the population ,74. Howericanus) . The autpulation . In the pulation , and thapulation . This mibehavior ,78. Hower et al.  observedrn sheep .HE and Ncrnt. However, the results also showed deviations from this pattern   . The HE for SRN , while t for SRN . In the  for SRN , the repthan YNP . Insteadof 0.604 .Ncrnt estimates, yet they have small census sizes, especially the black muntjac. In the case of HWCH, the population was founded from a single wild source [Ncrnt could correspond to the source population and not necessarily to the actual captive population. MNWSA is similar; however, since this population was founded using several sources instead of a single source, MNWSA could offer a better representation of the source or original genetic pool. For both populations (HWCH and MNWSA), it is likely that not enough time has passed to decrease the “effective size signal” by drift or inbreeding depression and, consequently, to erode the genetic diversity [The black muntjac (HWCH) and Arabian oryx (MNWSA) populations had the largest d source ; thus, tiversity ,10.HE and Ncrnt estimates could be attributed to the fact that genetic variation could be restored more rapidly than the Ncrnt. In this sense, the introduction of alleles can promote an increase of HE but not necessarily of Ncrnt. This could be the case for bighorn populations, such as SRN, SUH and WCNP, that showed relatively high levels of HE but lower Ncrnt when compared with other populations. For MNWSA and HWCH, it is clear that the populations could not retain such genetic diversity, so it is imperative to increase the census number of both populations in order to avoid a future reduction of Ncrnt and the associated genetic diversity loss. The contrasting patterns between the Nstbl and Ncrnt were of at least one order of magnitude  the analyzed populations could have historical genetic flow with other populations, and ii) the antiquity of the ancestral stable population size. Historical interconnections with other populations could lead to an Nstbl overestimation, as MSVAR assumes that the sample represents a closed population [Ne obtained from genetic data  could approach the global Ne for the species, yielding historical Ne estimates very different from actual local census size.It is necessary to consider the factors that influence and possibly bias our pulation ,57. In apulation  and Beaupulation  stated tTfa estimates suggested that other factors besides the European settlement could have affected the bighorn sheep populations  [NeI reduction in this species could reflect a severe influence of climatic changes and/or could be partly due to human activities.Booth et al.  reportedars ago) . This evars ago)  that couNcrnt and Nstlb estimates allows a glimpse of the possible fate of genetic diversity in the future . Thus, the effective size estimation, along with classic summary statistics, allows us to make better decisions for planning management and conservation strategies.The estimation of the effective population size provides additional information about genetic diversity. In this sense, our results showed that past human influences and possibly climatic changes played a major role in demographic trends on the artiodactyls studied here. Moreover, the use of Figure S1Schematic representation of the ABC-analysis scenarios. From past to present, the simulations are as follows: Begin with an ancestral population that had a constant effective size (Nstlb); the population had a size change at an exponential rate during the Tfa years . Fifteen generations before the present, the TI foundation occurred, represented as the continental (Sonora) and TI population split. Finally, in the present (Ncrnt), 63 samples were taken from TI. We considered the actual information on the TI foundation , and parameters Nstlb, Ncrnt and Tfa were obtained from the MSVAR analysis. To determinate the most likely Ncrnt of the Sonora source population, four scenarios were considered: (A) no genetic bottleneck fixing size at 271 individuals; (B) reduced considering 385 individuals; (C) strong 500; and (D) severe bottleneck 918. Finally, in order to predict the effects on genetic diversity of using TI as source for founding new populations, we simulated the outcome for 64, 32, 16, and 8 founders using the most likely scenario obtained with the ABC-analysis.(TIF)Click here for additional data file.Protocols S1Protocols for the amplification and genotyping of the molecular markers used in this study.(DOC)Click here for additional data file.Table S12 concentration, alignment temperature, and references of the microsatellite loci used in this study.Chromosomal position, primer sequences, MgCl(DOC)Click here for additional data file.Table S2Summary statistics of microsatellite markers used in this study.(DOC)Click here for additional data file.Table S3Allele size and frequencies of microsatellite loci used in this study.(DOC)Click here for additional data file.Table S4HE and na values obtained from the ABC-analysis.(DOC)Click here for additional data file.Table S5Ncrnt) obtained form the ABC-analysis simulations.Current effective inbreeding population size ((DOC)Click here for additional data file.Table S6Ncrnt) estimates using Robust Bayesian Estimates (RBE).Paired comparisons of current inbreeding effective population size ((XLS)Click here for additional data file.Table S7Nstbl) estimates using Robust Bayesian Estimates (RBE).Paired comparisons of ancestral stable effective population size ((XLS)Click here for additional data file.


Human influence typically impacts on natural populations of conservation interest. These interactions are varied and sometimes complex, and may be negative and unintended or associated with conservation and management strategy. Understanding the details of how these interactions influence and are influenced by natural evolutionary processes is essential to the development of effective conservation strategies. In this study, we investigate a species in Britain that has experienced both negative impact through overhunting in historical times and management efforts through culls and translocations. At the same time, there are regional populations that have been less affected by human influence. We use mtDNA and nuclear microsatellite DNA markers to investigate patterns of connectivity and diversity and find multiple insular populations in Britain that probably evolved within the Holocene (when the habitat was free of ice). We identify three concurrent processes. First, surviving indigenous populations show highly provincial patterns of philopatry, maintaining and generating population structure on a small geographic scale. Second, founder populations into habitat extirpated of native populations have expanded, but remained largely insular. Third, introductions into established populations generate some admixture. We discuss the implications for the evolution of diversity of the integration of natural processes with anthropogenic influences on population size and distribution. Natural processes associated with vicariance, habitat dependence, local adaptation, and dispersal strategies (evolved to maximize fitness) promote the distribution of diversity among conspecific populations. These processes lead to the evolution of population structure over varying geographic scales and eventual speciation. At the same time, anthropogenic activities are often superimposed, and the impact on population structure will depend on how these different processes interact. Humans can cause fragmentation, population declines, expansions , and extirpation.For populations that have been influenced by declines, recovery in population size can be rapid, but there may be a long-term impact on diversity . Typically, population size recovery can occur as a species naturally disperses and recolonizes formerly occupied areas. This process of natural recolonization usually occurs when degraded habitats are restored and dispersal corridors are available . This set of population histories provides an opportunity to investigate the implications of the interaction between natural processes associated with population expansion and philopatry, and the influence of anthropogenic impacts on population size and distributions.Introduced populations may remain relatively isolated with low diversity following small reintroductions. This was found to be the case for the Alpine ibex, Cervus elaphus). The first postglacial records of roe deer date back to between 10,050 and 9600 YBP from a site found at Thatcham in Berkshire  from 14 main sampling areas across the United Kingdom during 2007–2009 see ; Fig. 1 Individuals were genotyped via polymerase chain reaction (PCR) at 18 previously published microsatellites shown to be polymorphic in roe deer: BM1706, BM757, BM848, CSSM39, CSSM41, CSSM43, HUJ1177, IDGVA29, IDVGA8, NVHRT48, BMC1009, OarFCB304  with cycle conditions: 95°C for 5 min; 35 cycles at 94°C for 45 sec, 51°C for 45 sec; and 72°C for 45 sec; 72°C for 5 min. PCR products were purified using Qiagen columns  and directly sequenced using an ABI 377 automated sequencer. All sequences were aligned using Clustal X . For microsatellite DNA data, allelic richness for each locus and population and FIS were calculated using the program FSTAT 2.9.3 (Goudet F-statistics (Weir and Cockerham F-statistics were used: a measure that incorporates mtDNA sequence divergence (ΦST) and a measure based on mtDNA haplotype frequencies or microsatellite allele frequencies (FST). Significance was tested using 1000 permutations and Bonferroni corrected for multiple comparisons. Distributions of mtDNA haplotypes were examined within subpopulations by plotting haplotypes (excluding singletons) onto a location map of the United Kingdom where samples were collected.For mtDNA data, the program DNA sp 10.4.9 , ranging from 2 to 10. Analyses were run twice for each value of K to check consistency between runs. For each run, 100 simulated annealing processes were performed. The composition of the K groups was identified by observing the maximum FCT index  based on microsatellite DNA data  were compared. Five independent runs for each K value (2–11) were performed at 106 Markov chain Monte Carlo (MCMC) repetitions and 105 burn-in using no prior information and assuming correlated allele frequencies and admixture. The posterior probability was then calculated for each value of K using the estimated log likelihood of K to choose the optimal K. Apparent structure not detected at the higher hierarchical level was reassessed by rerunning the program with subsets of samples.The program STRUCTURE 2.0 was used to assign putative number of populations (K = 7 and a range of migration rate (MIGPRIOR) values (0.001–0.1). Burn-in and run lengths were the same as for runs without prior population information. Spatially explicit information on population structure was assessed using the program Geneland  implemented in GENETIX 4.0  for 224 comparisons . After Bonferroni correction, six combinations were significant (P < 0.0031) at the experimental level; three of these occurred in the Norfolk population. Despite these differences, no clear patterns across samples were observed.Two microsatellites (IDVGA-29 and MCM131) were excluded from all analyses because of genotyping error revealed by Microchecker. No evidence of genotyping errors was found for any further loci. The test of genotypic disequilibrium for each pair of the 16 microsatellite loci over all populations gave 11 significant values (He was 0.65 (range 0.59–0.76) and mean Ho was 0.62 (range 0.49–0.74) with mostly small, positive FIS values .Mitochondrial DNA analyses revealed a total of 27 haplotypes and 22 variable sites (18 transitions and 4 transversions). The haplotypes occurred between one and 115 times  profile, was also at K = 7, supporting the distinction of Carlisle and Lancashire beyond those regions already supported when K = 4. A separate analysis including only samples from northern United Kingdom  indicated that these furthers divisions are robust, but at a lower level of support compared with the rest , only three potential individuals with mixed ancestry were identified, one male and two females. The male was sampled from Perthshire and assigned to this population with 25% and to the North Yorkshire/Durham population with 36% probability. One of the females was sampled from the south west (Wiltshire) and assigned to this population with 39% probability, but also to the Lancashire population with 43% probability. The other female was sampled from Ayrshire and assigned to this population with 4% probability and to the Carlisle population with 81% probability.Using prior population information in STRUCTURE , and a significant pattern of isolation by distance for the northern populations  expansion of the remnant Scottish population, founding new populations as the range expanded and establishing a pattern of isolation by distance. This would also be consistent with the results from Structure analyses where those divisions are supported at a lower hierarchical level, and by the results in Geneland showing greater admixture across the northern range .He = 0.59–0.62). In other local European roe deer populations, microsatellite diversity has ranged from low  reflecting haplotype sampling in the small founder groups, overall levels of haplotype and nucleotide diversity  were comConsistent with this study, populations of white-tailed deer that experienced bottlenecks during introductions also retained appreciable levels of nuclear genetic diversity. This was attributed to the ability of their populations to expand very quickly  out from reintroduction sites and from the remnant population in Scotland are only just now starting to overlap . This iserkshire . There werkshire . Althougerkshire , 5.In mainland Europe, various roe deer genetic studies have shown broad-scale population structure based on both mtDNA and microsatellite DNA data, defining three primary regions in western, central, and eastern Europe  introduction into Norfolk, and the translocation (or translocations) from Scotland to south western England (with this population probably differentiated by sampling effects at the time of the founder event or events). The smaller scale structure in Scotland and northern England is apparently largely due to natural processes, and suggests a strong tendency for philopatry and small natal dispersal range. Roe deer have quite small home ranges (often less than 100 ha), especially in fragmented habitat  and uniparental (mtDNA) markers did not exceed the expected fourfold reduction in genetic structure  regional populations, or a very localized admixed population with limited impact on the larger population in Britain.


Poecilia reticulata. The latter occurs widely in northern Venezuela, including the south-eastern side of Cordillera de la Costa, where it inhabits streams belonging to the San Juan drainage. Here, we present molecular and morphological analyses of differentiation among guppy populations in the Cariaco and San Juan drainages. Our analyses are based on a 953 bp long mtDNA fragment, a set of 15 microsatellites (519 fish from 20 populations), and four phenotypic traits.Geographic barriers to gene flow and divergence among populations in sexual traits are two important causes of genetic isolation which may lead to speciation. Genetic isolation may be facilitated if these two mechanisms act synergistically. The guppy from the Cumaná region (within the Cariaco drainage) of eastern Venezuela has been previously described as a case of incipient speciation driven by sexual selection, significantly differentiated in sexual colouration and body shape from the common guppy, Both microsatellite and mtDNA data showed that guppies inhabiting the two drainages are characterised by a significant genetic differentiation, but a higher proportion of the genetic variance was distributed among populations within regions. Most guppies in the Cariaco drainage had mtDNA from a distinct lineage, but we also found evidence for widespread introgression of mtDNA from the San Juan drainage into the Cariaco drainage. Phenotypically, populations in the two regions differed significantly only in the number of black crescents. Phenotypic clustering did not support existence of two distinct groupings, but indicated a degree of distinctiveness of Central Cumaná (CC) population. However, CC population showed little differentiation at the neutral markers from the proximate populations within the Cariaco drainage.Our findings are consistent with only partial genetic isolation between the two geographic regions and indicate that the geographic barrier of Cordillera de la Costa has not played an important role in strengthening the incomplete pre-zygotic reproductive barrier between Cumaná and common guppy. Significant phenotypic differentiation between genetically similar  populations suggests that mate choice can maintain divergence at sexually selected traits despite gene flow. However, neither genetic nor phenotypic clustering supported delineation of two species within the region. Local ecological adaptations, sexual selection and genetic drift in isolated populations may lead to the emergence of reproductive isolation – a fundamental requirement for speciation -4. HowevDue to a relatively high degree of isolation, streams and rivers favour formation of highly structured populations of aquatic organisms e.g. ,10, whic, whic10,Poecilia) inhabiting streams of north-eastern Venezuela . Most mtDNA haplotypes clustered into two major well-supported lineages  but was significantly differentiated among populations .In DAPC for phenotypic traits five principal components of PCA and three discriminant functions were retained. We found no support for the presence of two phenotypically distinct clusters which could be equated with two species. Indeed, the values of BIC did not reach a plateau, providing no clear indication that the optimal number of clusters was less than 20  which is separated from San Juan drainage cluster along the first dimension  and Carpathian (L. montandoni) newts which constitute well defined species, 7.79% microsatellite variation was attributed to interspecific differentiation . The 10 μl PCR mixture contained 5 μl of Hot Start PCR Master Mix (Fermentas), 1 μM of forward and 1 μM of reverse primer  correction .The authors declare that they have no competing interests.MH took part in the study design, carried out molecular, statistical and population genetic analyses and drafted the manuscript. HA took part in the study design and molecular and statistical analyses, contributed photographs and tissue samples from Venezuela, and helped to draft the manuscript. WB supervised molecular and population-genetic analyses and helped to draft the manuscript. JM and FB took part in the study design and helped to draft the manuscript. JR took part in the study design, coordinated the study, supervised statistical analyses and helped to draft the manuscript. All authors read and approved the final manuscript.Table S2. List of primers used for microsatellite amplification. Table S3. Sequences of primers used to amplify  and to sequence (1-4) fragments of mitochondrial cytochrome b gene. Table S4. Hardy-Weinberg equilibrium tests for each locus in each population (Genepop); reported p values are not corrected; missing values are due to monomorphism of the locus in this particular population; p values significant after sequential Bonferroni correction are in red. Table S5. Null alleles frequencies for each locus in each population. Table S6. Pairwise FST values for microsatellites for all pairs of populations, values after ENA correction (FreeNA); Cariaco drainage populations in red, San Juan drainage populations in blue. Table S7. Distribution of mtDNA cytochrome b haplotypes among populations; numbers indicate number of individuals with a given haplotype; Cariaco drainage populations in red, San Juan drainage populations in blue. Table S8. Pairwise Fst values for cytochrome b for all pairs of populations, p = 0.05; significant values are followed by asterisk; Cariaco drainage populations in red, San Juan drainage populations in blue.Names and abbreviations, sample sizes, sampling year and coordinates of the sampling locations. Click here for fileFST values between all sampled populations based on microsatellite frequencies; Cariaco drainage populations are enclosed in grey lines.Multidimensional scaling. Two-dimensional scaling of the matrix of pairwise Click here for fileFST and its robustness was tested with 1000 bootstrap replicates. Cariaco drainage populations are in frames.Neighbor Joining tree showing relationships among populations. The tree was constructed from the matrix of pairwise Click here for fileFST) and geographic distances between populations; r = 0.14, P = 0.051.Isolation by distance. Relationship between genetic  on y axis.Inference of the optimal number of genetic clusters. Graph output of the function Click here for file


Ficedula hypoleuca) have insufficiently adapted their breeding phenology to the ongoing advance of food peaks within their breeding area and consequently suffered local population declines. We address the question whether this population decline led to a loss of genetic variation, using two neutral marker sets , and one potentially selectively non-neutral marker (avian Clock gene). We report temporal changes in genetic diversity in extant populations and biological archives over more than a century, using samples from sites differing in the extent of climate change. Comparing genetic differentiation over this period revealed that only the recent Dutch population, which underwent population declines, showed slightly lower genetic variation than the historic Dutch population. As that loss of variation was only moderate and not observed in all markers, current gene flow across Western and Central European populations might have compensated local loss of variation over the last decades. A comparison of genetic differentiation in neutral loci versus the Clock gene locus provided evidence for stabilizing selection. Furthermore, in all genetic markers, we found a greater genetic differentiation in space than in time. This pattern suggests that local adaptation or historic processes might have a stronger effect on the population structure and genetic variation in the pied flycatcher than recent global climate changes.Global climate change is one of the major driving forces for adaptive shifts in migration and breeding phenology and possibly impacts demographic changes if a species fails to adapt sufficiently. In Western Europe, pied flycatchers ( Natural selection may alter gene frequencies even in short time periods, resulting in higher genetic differentiation at candidate loci than at neutral loci. In this study, we tested this hypothesis by comparing spatial and temporal differentiation in mitochondrial DNA, microsatellite loci, and a candidate gene (Clock gene).Fourthly, if global climate change had a significant effect on natural populations of pied flycatchers, we expected genetic differences over time to be as large as, or even larger than those among local populations (within a given time period). Directional selection may cause the overall loss of genetic variation, resulting in fewer haplotypes and alleles or other indices of molecular diversity. An even stronger effect is expected (given that populations are reasonably isolated) in fitness-related genes, such as genes responding to environmental cues determined directly (temperature) or indirectly (food availability) by global climate changes, for example, the a priori limited availability of appropriate specimens in all collections. Samples taken in the field (1990s and later) comprised blood and tissue  and feather samples , was extracted using the DTAB method . Each reaction contained about 1–10 ng template DNA, 2.5 pmol each primer, 0.2 mmol/L each dNTP, 2 mmol/L MgCl2, 1× amplification buffer, and 0.5 U Taq DNA polymerase . The PCR started with an initial denaturation at 94°C for 1 min followed by five cycles: 94°C 40 sec, 45°C 40 sec, and 72°C 1 min; then cycling 35 times: 94°C 40 sec, 52°C 40 sec, 72°C 1 min; ending with a final extension at 72°C for 5 min. PCR products were separated by electrophoresis using 1.4% agarose gels and visualized by staining with ethidium bromide (1 ppm). In agreement with Töpfer et al. , historiFrom the first DNA extracts, all historic samples were amplified and sequenced at least twice. In all cases, both sequencing reactions yielded identical results. In order to control for the possible occurrence of single-base errors in PCR products from museum specimens , or which were genotyped with less than eight of ten microsatellites, were discarded from further analyses. Swedish pied flycatchers collected on Öland are likely to be migrants heading to northern Sweden  and those samples taken after 1995 as “extant” or “recent” (N = 201). This temporal division coincides with the first observations of decline in pied flycatcher populations in the Netherlands in the mid-1980s, as well as with a general decline of forest-dwelling long-distance migrants during the same time , and therefore, we labeled all samples collected before 1977 as “historic” or “museum samples”  for microsatellite loci and the Clock gene and ΦST for mtDNA were evaluated with GenAlEx ver. 6.5 (Meirmans and Hedrick Ho) for microsatellite loci and the Clock gene were estimated using Arlequin 3.5.1.2 (Excoffier and Lischer Fst) for neutral and non-neutral markers was also calculated with Arlequin 3.5.1.2.  based on mitochondrial and microsatellite loci as well as microsatellite loci and the Clock gene locus were tested using a Mantel test  in STATISTICA ), we defClock gene and for microsatellite analysis  were successfully used for the amplification and sequencing of the control region, analysis . For theanalysis . RegardlControl region: A total of 304 pied flycatcher individuals from nine populations Table  were anaFst estimates varied from zero  to about 0.5 for all pairwise comparisons with the Spanish population . 73.72% of the total variation was found within populations. Pairwise pulation . SignifiHo) covered a range from 0.681 to 0.783 with a mean of 0.722  or stuttering (2 of 90), and no evidence for large allelic dropout (12 of 90) was found. Private alleles were found in all populations . Overall, we observed a tendency of less genetic variation  in the current Dutch population than in the historic Dutch population  to 0.05 (Spain/historic Netherlands). In concordance with control region divergence, we found that the Spanish population was significantly differentiated from all other populations at nuclear loci . In contion Fig. .In the control region alignment, we did not find any bias with respect to an increased frequency of C to T substitutions in the historic sampling. The network based on recent and historic samples (61 haplotypes) contained nine unique tip haplotypes (not shown); however, among these, we found no bias with respect to our historic sampling. In fact, eight unique tip haplotypes occurred in the extant populations.The network based on recent samples only  displayeIn order to describe the distribution of haplotypes among current and historic populations, we reconstructed a network using control region sequences of Dutch, German, and Swedish populations. Like the other analyses (microsatellites and candidate genes), this revealed very little temporal differentiation Fig. A and agaEmploying a mode-based clustering method for microsatellite data of all recent samples, individuals were probabilistically assigned into K = 4 populations . IndividClock gene. We identified five length variants ranging from 193 bp (10 poly-Q repeats) to 205 bp (14 poly-Q repeats). At least three different alleles were found in each population, and all five alleles were detected in the current Dutch as well as in the historic German population. No pied flycatcher population exhibited private alleles. However, allele 205 was only found once in a historic German sample and in one recent sample from the Netherlands, and allele 193 was not found in extant Norwegian and Swedish samples. The remaining alleles were found in all populations with allele 199 being much more abundant in all populations than the other two. The observed heterozygosity (Ho) was consistent over the populations and ranged from 0.400 to 0.563 , were genotyped for length variation at the poly-Q locus of the Fst-values ranging from zero to 0.0174. Only the pairwise comparison between the recent populations from the Netherlands and Spain showed low , but significant differentiation at the Clock gene locus  nor any interaction of population x gender  or males .A subset of 205 samples was further analyzed for sex-specific differentiation at the poly-Q locus. GLM analysis performed in STATISTICA yielded no significant differences between geographical regions  compared with nuclear markers (microsatellites and Clock gene). However, there was a significant association between pairwise Φst values based on mtDNA and G′′ST values based on microsatellite loci  which is in concordance with the finding that populations showed hardly any genetic divergence at the Clock gene locus.Regardless of the marker system used, geographical differentiation was much more pronounced than differentiation between historic and recent samples from the same country Fig. . This waClock gene locus among our study populations, the overall pattern of greater genetic differentiation on the spatial scale compared with the temporal scale was similar at this candidate gene and neutral loci.Contrary to our expectations, genetic differences on the temporal scale within populations were much weaker than spatial differentiation, and only a slight temporal decline of genetic variation was found in the Dutch population. Thus, any postulated negative effect of climate change on local genetic variation, particularly in declining Western European populations, would have been counteracted or mitigated by gene flow. Second, despite very little genetic variation in the Ficedula flycatcher species has been largely shaped by historic processes such as Pleistocene range fragmentation  and nuclear loci matches well the findings by Haavie et al.  could be related to differences in rate and patterns of mutations  is similar to that observed in other bird species , great tits (Parus major), and bluethroats (Luscinia svecica)  and the barn swallow (Hirundo rustica) (6 to 8)  of pied flycatchers is in the same order of magnitude as values measured in blue tits (Ho = 0.489) although slightly higher than in bluethroats (Ho = 0.213) and differs considerably from the low values reported for great tits (Ho = 0.077) and barn swallows (Ho = 0.030).Variation in the number of poly-Q repeats at the Clock gene variation with both breeding latitude and breeding phenology of birds is scarce and inconsistent so far. A latitudinal cline of avian Clock ploy-Q variation was detected only in a nonmigratory species , Clock poly-Q variation is significantly correlated with migration distance, and in the same study, species as well as in the blackcap (Sylvia atricapilla), the ADCYAP1 locus, another gene of the ‘migratory gene package’  or quantitative (QST) differentiation, lower differentiation in potentially selected traits or genes than in neutral loci suggests that natural selection must be favoring the same mean phenotype in different populations (Merilä and Cnorkrak Clock gene locus.Among blue tit and bluethroat populations, significant associations between pairwise ers Fig. B. Similaers Fig. . As a coClock gene variation considered here is not a good candidate for such an adaptive process because neither between- nor within-population variation was related to variation in timing. The general lack of geographical variation in neutral genetic markers suggests that dispersal is an important process in this and other species of long-distance migrants, which would swamp most of the declines in genetic diversity in shrinking populations. This observation poses the question how much of the observed local phenotypic variation has a genetic origin. In fact, in the Spanish pied flycatcher population included in our analysis, spring arrival of males and breeding dates are shaped by a mixed-mating strategy of this species (sexual selection was suggested to be the main driving force of protandry; Canal et al. So far, it is not clear whether adaptation of migration and breeding time to rapid environmental changes is restricted through limited phenotypic plasticity and/or a lack of genetic variation on which selection can act. The increased selection for breeding date as observed in several long-term population studies (Both and Visser


Nilaparvata lugens species complex for the analysis of genetic polymorphisms. A total of 11 populations of Nilaparvata spp. were collected from 6 locations from Malaysia. Two sympatric populations of brown planthopper, N. lugens, one from rice and the other from a weed grass (Leersia hexandra), were collected from each of five locations. N. bakeri was used as an out group. Three oligonucleotide primer pairs, DALP231/DALPR′5, DALP234/DALPR′5, and DALP235/DALPR′5 were applied in this study. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on genetic distances for the 11 populations of Nilaparvata spp. revealed that populations belonging to the same species and the same host type clustered together irrespective of their geographical localities of capture. The populations of N. lugens formed into two distinct clusters, one was insects with high esterase activities usually captured from rice and the other was with low esterase activities usually captured from L. hexandra. N. bakeri, an out group, was the most isolated group. Analyses of principal components, molecular variance, and robustness also supported greatly to the findings of cluster analysis.Direct amplified length polymorphism (DALP) combines the advantages of a high-resolution fingerprint method and also characterizing the genetic polymorphisms. This molecular method was also found to be useful in brown planthopper,  Nilaparvata lugens (Stål) (Homoptera: Delphacidae), is a major pest of rice, which is widely distributed from tropical to temperate areas of Asia and Australia. The insect is a phloem-feeder and is restricted to cultivated and wild rice as host plants. It causes “hopperburn” and complete wilting and drying of rice plants [Leersia hexandra, which grows abundantly in canal near paddy fields in South East Asia [ N. lugens fails to survive on rice plants. Conversely, rice infesting population of N. lugens does not thrive on grass [Leersia infesting populations of brown planthopper (BPH) represented two distinct sympatric biological species. In recent studies, the analyses of isozymes and RAPD-PCR markers indicated that BPH with high esterase activity usually captured from rice plant, and those with low esterase activity usually captured from L. hexandra in Malaysia represent two distinct closely related sibling species [The brown plant hopper, e plants  and alsoe plants . Large-se plants . Anotherast Asia , 4. The on grass . Based oon grass  suggeste species , 7.Direct amplification of length polymorphisms (DALPs) is a technique which uses arbitrarily primed PCR (AP-PCR) to produce genomic finger prints and to enable sequencing of DNA polymorphisms in any species. Oligonucleotide pairs were designed to produce a specific multibanded pattern for each individual of a population and between populations. This strategy combines the advantages of a high-resolution fingerprint technique and also characterizing the polymorphisms . Higher N. lugens, one from rice and the other from weed grass, L. hexandra. We hypothesized that the molecular method newly applied in rice brown planthopper could be able to detect structures among the populations of brown planthopper species complex.Genetic polymorphic markers, such as isozymes, RAPD and SSR, and nuclear or organelle DNA polymorphism, have been developed for a variety of studies on genetic diversity, population structures, and subdivisions , 7, 9–11 N. lugens, one from rice and other from L. hexandra, were collected from each of five locations. The locations were Universiti Putra Malaysia (UPM), Tanjung Karang (TK), Melaka (MK), Perak (PK), and Sabah (SB). An out group, N. bakeri was also collected from Cameron Highlands (CH), Malaysia. Locations, host type, date of collection, population code are shown in A total of 11 populations were collected from 6 locations. Two sympatric populations ofThe individual insect used for DALP analysis was tested for esterase activity on a simple filter paper using the method reported by Pasteur and Georghiou .μL extraction buffer  0.05 M EDTA, 0.5% SDS). The glass rod was washed with an additional 40 μL of extraction buffer and the homogenate was incubated at 65°C for 40 min. An amount of 10 μL of 8 M potassium acetate was added and the tube was placed on ice for 40 min. The tube was spun at 14000 rpm for 20 min. The supernatant was transferred into a fresh 1.5 mL tube. One hundred microliters of chilled (−20°C) 100% ethanol was added and the DNA was allowed to precipitate at room temperature for 10 min. The tube was spun for 20 min and the ethanol was carefully removed with a pipette. The DNA pellet was washed with 100 μL of chilled 70% ethanol and spun for 10 min. The DNA pellets were dried by pouring off the ethanol. The tubes were kept for 10 min at room temperature. The dried DNA pellet was suspended in 50 μL TE  and gently mixed for 10 min. The DNA concentration was measured using LKB-Ultrastep III UV/visible spectrophotometer at the absorbance of 260 nm and 280 nm. The DNA was considered pure if the ratio of OD260/OD280 was within the range of 1.6–1.9 [DNA from individual insect was extracted by grinding single frozen adult insect with a glass rod in a 1.5 mL tube containing 20  1.6–1.9 .33 PATP (10 m Cie/mL) [A total of three forward sequencing primer denoted as DALP231, DALP234, DALP235 and a universal reverse primer, DALPR (5′TTTCACACAGGAAACAGCTATGAC-3′), were used for the PCR amplification . Primer  Cie/mL) .+, 0.15 μM of oligonucletide primer, 200 μM of each dNTP, 1 unit Taq DNA polymerase (Promega), and 1x PCR buffer in a total volume of 25 μL. Amplification reactions were carried out in a programmable thermal cycler  programmed as follows: predenaturation at 95°C for 2 min; followed by 30 cycles of denaturation at 91°C for 30 sec, annealing temperature at 55°C for 45 sec, and extension at 70°C for 30 sec. After the last cycle, final extension was at 70°C for 5 min. The protocols were modified from [The PCR reaction mixture contained 60 ng of insect DNA, 1.8 mM of MgμL 100% formamide loading dye and then heated for 10 min at 96°C before loading. The gel was run at 55 W for 3 hours.Electrophoresis was performed on 6% denaturing polyacrylamide gels and run on a 50 cm long gel apparatus. The samples were mixed with 5 After electrophoresis, the gel was transferred to a Whatman paper and dried and developed after 5 days of exposures to X-ray film.DALP-PCR band profiles were scored visually for each DNA sample for each primer pair. The data was recorded according to the presence/absence criterion .mxy is the number of bands showed by sample x and sample y and mx and my are the number of bands in sample x and sample y, respectively. The value produced by this index ranges from 0 (representing no band sharing) to 1 (representing complete identity). The within or between population values are based on pairwise comparisons between individuals for a particular primer. The values obtained are then averaged over primers.The Dice algorithm was used for similarity index. The similarity index was calculated between two samples from within or between populations according to :(1)Sxy=D = 1 − S [The between population similarity indices were also converted to distance values using the relationship  = 1 − S , 17. The = 1 − S  to find  = 1 − S ).The test of robustness or bootstrapping was performed using the Phylogeny Inference Package  developed by Felsenstein . The booA principal component analysis was performed based on the distance matrix among the populations using the NTSYS-PC software. The relationship among the populations is expressed in a three-dimensional graph based on the first three components.N is the total number of bands and Nxy is the number of bands shared by two samples. The resulting distance matrix was used in an AMOVA [The distance between two samples was calculated according to the formula of Excoffier et al. :(2)D=N{an AMOVA . In the Leersia infesting populations of N. lugens using primer DALP235/DALPR. A hundred percent marker frequency represented monomorphism while 0% showed complete absence of the particular marker. The data showed a range of 28.3–42.9% polymorphic markers for rice infesting populations of N. lugens while Leersia infesting populations and an out group, N. bakeri showed 31.9–45.5% and 17.1% polymorphic markers, respectively. The overall data for 10 populations of N. lugens based on three primers showed 29 (42.6%) polymorphic markers. Frequency of DALP markers, total number of markers, number of polymorphic markers, % polymorphic markers for each population are shown in Band or marker frequency was calculated for each marker pair for each population for DALP primers. N. lugens ranged from 0.112487 to 0.285200 (average 0.2245843) while distances within Leersia infesting populations ranged from 0.152379 to 0.235396 (average 0.2078274). The genetic distances between two sympatric populations of N. lugens, one from rice and the other from grass, ranged from 0.24019 to 0.390182 (average 0.31672).All data from three pairs of DALP primers were incorporated for cluster analysis. In this analysis, pairwise genetic distances were calculated between all individuals in order to make comparison. The distances within rice infesting populations of N. lugens and Leersia infesting populations of N. bakeri (out group) ranged from 0.555389 to 0.564963 (average 0.540276) but it was ranged from 0.499403 to 0.578171 (average 0.539168) between the populations of Leersia infesting N. lugens and N. bakeri  was far away from either rice or Leersia infesting populations of N. lugens  population. Within rice and Leersia infesting populations, confidence level ranged from 37–56% to 35–51%, respectively  (26.90%), followed by DALP231 (10.10%), and DALP234 (9.68%). The percentage of the variance component among groups (rice versus Leersia) was greater than the percentage of the variance component among the populations detected by the three primers DALP235, DALP234 and DALP231. The results of AMOVA as well as dendrogram confirmed that genetic variation exists between the brown plant hopper of rice and Leersia. Three level nested structures for each pair of primer of DALP are shown in Nilaparvata species. Three major clusters were observed in the dendrogram as well as in the graph. The results showed that population of N. bakeri formed the most isolated cluster from populations of either rice or Leersia infesting populations of N. lugens. The rice infesting populations of UPM, Tanjung Karang, Melaka, Perak, and Sabah, Malaysia clustered together as a group. On the other hand, Leersia infesting populations of the same localities formed another distinct cluster. Leersia infesting populations with low esterase activities seem to be formed, a different structure from rice infesting populations of brown planthopper, N. lugens. These results were also confirmed by bootstrapping analysis as described by Felsenstein [Leersia infesting populations of N. lugens and also for the genetically isolated group, N. bakeri.Cluster and principal component analyses revealed the genetic relationships among the different populations of senstein  and Latisenstein . BootstrN. lugens. Saxena and Barrion [Leersia infesting populations. Despite their morphological similarities, a distinct cytological incongruity and a certain degree of genetic isolation between the two populations were inferred. Species differentiation in early stages of a species formation may not be associated with substantial genetic change [Lethe eurydice L. and Lethe appalacia L., although the two species were found to be good species [N. lugens complex might have developed through insecticide exposures that were heavier in rice-infesting populations than in grass populations, through RAPD-PCR analysis.In DALP fingerprinting method, we did not get any diagnostic markers between two sympatric populations of  Barrion  reportedc change –27. Manyc change , 29. The species . Latif e species  reportedLeersia infesting populations (low esterase activities) of brown planthoppers indicated that these sympatric populations represented two distinct but closely related biological species. The genetic distance indicates the magnitude of genetic variation between populations. Genetic distance commonly ranged from nearly 0.01 for populations within species, 0.1 for different subspecies, and 1.0 for different species . So, theLeersia) and among population components. The total genetic variation, an average 15.56% was attributable to group divergence (Rice versus Leersia), 13.01% to population differences and 77.37% to individual differences within a population. The percentage of variance component among groups (rice versus Leersia) was larger than the percentage of variance component among populations for bands detected by three DALP primers and these were tested by random permutation. These results revealed that there was genetic differentiation between the brown planthopper of rice versus Leersia (two sympatric populations of N. lugens). AMOVA was performed and was confirmed the differentiation into two groups of Aphid gossypii [Acorus gramineus [Oryza rufipogon [ L. hexandra in Malaysia, represent two distinct closely related species and supported previous results as reported by Latif et al. [The results of AMOVA in single primer yielded highly significant variance among group (rice versus gossypii , five grramineus , and twoufipogon . Therefof et al. . Althoug


Lippia origanoides, a species of the Verbenaceae family that shows promise as a crop plant, was investigated along an altitudinal gradient in the basin of the Chicamocha River in northeastern Colombia. The economic importance of the species, quality of its essential oils, and the fact that it is restricted to some few semiarid areas in northern South America may put the species at risk in a scenario of uncontrolled harvest of natural populations. Lippia origanoides was sampled along an altitudinal gradient from 365 to 2595 m.a.s.l. throughout Chicamocha River Canyon, a semiarid area in northeastern Colombia. Genetic diversity was assessed by means of AFLP markers. The number of AFLP loci (355) and the number of individuals sampled (173) were sufficient to reliably identify four populations at contrasting altitudes , two populations in the lower basin, one population in the medium basin, and one population in the upper basin, with a low level of admixture between them. In average, genetic diversity within populations was relatively high ; nevertheless, diversity was significantly reduced at higher altitude, a pattern that may be consistent with a scenario of range expansion toward higher elevations in an environment with more extreme conditions. The differences in altitude among the basins in the Chicamocha River seem to be relevant in determining the genetic structure of this species.The genetic diversity and population structure of  Plant species that show tolerance or resistance to extreme conditions, such as those associated with arid and semiarid areas and that, additionally, are important for agricultural and/or industrial applications, are of interest for population genetics studies, not only because these species may be pre-adapted to the consequences of climate change  but also because their uncontrolled exploitation from the wild may impose a risk for their survival.Lippia origanoides H.B.K., an aromatic plant species of the Verbenaceae family that has promise for the quality and potential industrial use of its essential oils. The species occurs in northern South America , the south (departments of Cauca and Nariño), and the Atlantic coast   .Lippia origanoides has an important phytochemical variation represented in three chemotypes according to the major compounds present in their essential oils   . The bioterranea , Sclerotaseolina , Rhizocts maydis , Mycosphijiensis , Mycobacrculosis , Candidama cruzi , Escheri zeamais , antivir zeamais ,b, and D zeamais . These rspectrum , with pospectrum . NowadayL. origanoides as n = 12. Nevertheless, additional studies are needed to confirm its monoploid number and ploidy , in a topography of steep slopes (>60°) and shallow soils . At low asticity , and othasticity . In contL. origanoides along an elevation gradient (365–2595 m.a.s.l.) at the lower, medium, and upper basins of the Chicamocha River, a semiarid area in Colombia, using AFLP molecular markers. Our ultimate aim is to investigate the processes that influenced the current distribution of L. origanoides in the canyon, mainly in those populations at higher elevations. For this purpose, we analyzed a sample of 173 individuals from the lower, medium, and upper basins of the canyon, using 355 AFLP loci, and applied a variety of approaches for assessing genetic structure and diversity with dominant markers.The objective of this study was to evaluate the genetic structure of Lippia origanoides is an aromatic shrub, erect and branched, which grows to 3 m in height and belongs to the Verbenaceae family  that according to preliminary results are informative in L. origanoides . The AFLP amplifications were carried out as recommended by the manufacturer. The visualization of the products was performed following the manufacturer's instructions, although the staining of 6% PAGE was performed in silver nitrate. The gels were documented with a scanner Epson Perfection® 4490 Photo ; each image was optimized and scored using GIMP 2.6 and EasyPen i405 . This procedure of digital-scoring was performed by painting a black line on the bands indicating their presence and a white line indicating the absence of the fragment, which improved automated processing for obtaining the presence/absence or 1/0 matrix with Cross Checker 2.91  was used to amplify five selective primer combinations  using the distance matrix under the method proposed by  library  in the p library . Additio library  to calcuIn most genetic studies, the number of markers and the number of individuals sampled are defined either arbitrarily or by available resources. However, in genetic structure studies, this may have a great effect on the validity and reliability of the inferred clusters. For this reason, we used the method reported by We estimated the appropriate number of groups or partitions (populations or subpopulations), in addition to the level of admixture between the groups, and assigned each individual to each of the inferred populations using two Bayesian approaches implemented in the programs STRUCTURE 2.2  and BAPShttp://www.bioportal.uio.no). Similarity among runs was estimated according to ΔK as the mean of the absolute values of the second order rate of change of L(K) (Ln P(D) in STRUCTURE output) divided by the standard deviation according to the methodology proposed by ΔK were calculated using an R-script “Structure-sum-2011” available from http://uit.no/ansatte/organisasjon/ansatte/person?p_document_id=41186&p_dimension_id=88165  approach, in which there are K populations and the individuals are assigned to these populations probabilistically. We used a model of ancestry with no admixture and a model of allele frequencies uncorrelated or independent, suitable for dominant molecular markers . Ten runid=88165 .Similarly, we used BAPS  to infer the genetic structure in our data considering the geographic information collected in the field. Briefly, BAPS treats both K and allele frequencies as random variables, and returns one optimal partitioning and the probability of the optimal K. In order to strengthen the inferences, we used as prior the geographic coordinates of the sampled individuals. Spatial clustering and admixture of individuals based on mixture clustering were calculated using a vector of values of K from 2 to 20 and 20 replicates. Voronoi tessellation and admixture clustering graphics were obtained to indicate the optimal partitioning or potential population structure and the level of admixture within the clusters inferred from the canyon of the Chicamocha River.full model was also used for estimating genetic diversity, defined as the average panmictic heterozygosity within each population or cluster, without assuming HWE  and Shannon's information index (I)   was calcndex (I)  and theyII, a measure of the amount of genetic differentiation among contemporaneous populations and estimated by HICKORY, and (2) AMOVA, analysis of molecular variance among populations using pairwise difference as distance method and 100,000 permutations for support, implemented in ARLEQUIN 3.5 (Two approaches for determining the level of genetic differentiation among populations or clusters were used: (1) θQUIN 3.5 .Lippia origanoides is an aromatic and medicinal species promising for the quality of its essential oils and the broad activity spectrum that these have shown against pathogenic microorganisms, relevant to agriculture and human health  and Betéitiva in the upper basin . The elevation range covered during the sampling was between 365 and 2595 m.a.s.l. for the entire canyon. On average, elevations for each basin were: (1) 532 m.a.s.l. for the lower basin, (2) 1440 m.a.s.l. for the medium basin, and (3) 2350 m.a.s.l. for the upper basin.N criterion as 1−(3/N), where N is the number of samples)  on a digital image ; 244 (68.7%) and 227 (63.9%) markers were polymorphic at 0.99 and 0.95, respectively. According to samples) . Howeversamples) .A pairwise individual-by-individual genetic distance matrix was built following the method of L. origanoides , with low level of admixture between clusters or populations  was not a suitable criterion for determining the optimal K, given that L(K) increased after reaching the real value of K in our data  with a reduction of 20% compared with the other populations of the canyon. Lippia origanoides is considered to be a habitat specialist species, with high levels of plasticity and stress tolerance  as an alternative measure to quantitate diversity (see I = 0.44) and significantly higher than UB (I = 0.348), which is in accordance to the results presented above. Interestingly, the genetic diversity in L. origanoides seems to follow a pattern previously reported for species inhabiting elevation gradients, which are characterized by exhibiting a marked decrease in genetic variation in marginal populations compared with populations centrally situated , as suggested by k) was estimated as Ct = (Ht−Ht/k)/Ht .The individual contribution of each population to genetic diversity was determined using Ht/k)/Ht , where HFull loci show the values of Nei's genetic diversity and Shannon's information index estimated using the full set of markers scored in this study, 355 AFLP loci. The estimators of the diversity were reduced in approximately 30% compared with the data sets that fulfill the parameters of polymorphism, usually 0.99 level of polymorphism.The effect of monomorphic markers on estimation of diversity was evaluated. In I = 0.453 and hs = 0.484. Although the value of I reported for LBb in our study (I = 0.425) was similar to that obtained by hs presented here is significantly different (hs = 0.484 vs. hs = 0.288), probably due to the use of different molecular marker systems  was previously reported by  systems .FST = 0.17916, P-value < 0.0000). In general, 17.9% of the variation was due to differences between populations of the canyon, while the remaining variation was due to differences between individuals within populations. The Bayesian estimate of differentiation among contemporary populations θII and implemented in HICKORY showed a similar level of population subdivision (θII = 0.15592 ± 0.02311). Pairwise FST estimates (see FST = 0.306), and moderate differentiation between nearby populations .The distribution of genetic diversity within and among populations in the canyon of the Chicamocha River was determined using two-level analysis of molecular variance see . AMOVA sates see  indicateL. origanoides in the canyon and at contrasting elevations , and probably to the difference in altitude among the basins. The population that contributed the most to the genetic diversity was LBa, which corresponds to the municipalities of Juntas and Jordan, where further research and conservation activities should be focused. However, these previous results encourage further sampling and research in areas that have not been sampled yet in order to understand more of the adaptation and distribution of this important species in Colombia.In summary, with the results obtained in this study, we conclude that the genetic structure of


Sula granti) populations are substantially differentiated, even within the small geographic scale of this archipelago. On the other hand, Galapagos great frigatebird (Fregata minor) populations do not show any genetic structure. We characterized the genetic differentiation by sampling five colonies of both species in the Galapagos archipelago and analyzing eight microsatellite loci and three mitochondrial genes. Using an F-statistic approach on the multilocus data, we found significant differentiation between nearly all island pairs of Nazca booby populations and a Bayesian clustering analysis provided support for three distinct genetic clusters. Mitochondrial DNA showed less differentiation of Nazca booby colonies; only Nazca boobies from the island of Darwin were significantly differentiated from individuals throughout the rest of the archipelago. Great frigatebird populations showed little to no evidence for genetic differentiation at the same scale. Only two island pairs  were significantly differentiated using the multilocus data, and only two island pairs had statistically significant φST values  according to the mitochondrial data. There was no significant pattern of isolation by distance for either species calculated using both markers. Seven of the ten Nazca booby migration rates calculated between island pairs were in the south or southeast to north or northwest direction. The population differentiation found among Galapagos Nazca booby colonies, but not great frigatebird colonies, is most likely due to differences in natal and breeding philopatry.Seabirds are considered highly mobile, able to fly great distances with few apparent barriers to dispersal. However, it is often the case that seabird populations exhibit strong population genetic structure despite their potential vagility. Here we show that Galapagos Nazca booby ( Spheniscus mendiculus)  (Conolophus sp.)    and Galagoensis)  have higphus sp.), Galapaggoensis) , and Galharrisi)  show higFregata minor], blue-footed booby [Sula nebouxii], red-footed booby [S. sula], magnificent frigatebird [F. magnificens]), as well as endemic species . The great frigatebird breeds throughout the Pacific, the South Atlantic, and the Indian Oceans. The Nazca booby (S. granti) is a common, resident Galapagos seabird throughout the archipelago that was elevated to species status in 2002 after morphological (S. dactylatra), with breeding colonies located primarily on oceanic islands on the Nazca tectonic plate, namely the Revillagigedo Islands in Mexico, Clipperton and Malepo Islands in Colombia, the Galapagos Islands and La Plata Islands in Ecuador  sequence data from three genes to describe the population genetic structure of Galapagos great frigatebirds and Nazca boobies. There is some indication that both sexes of great frigatebirds are somewhat natally philopatric ; howeverSeabirds were sampled in July 2007, June-July 2008, June 2010, and July 2011 from six islands in the Galapagos  (780 bp) and NADH dehydrogenase subunit 2 (ND2) (566 bp), and cytochrome oxidase I (COI) (700–800 bp) for all great frigatebirds and a subset of the Nazca boobies (n = 50). The subsample of Nazca boobies was selected at random, but checked to ensure equal numbers of each sex. Primers for cyt b were B3 and B6 . Deviations from linkage equilibria were tested in Arlequin v.3.5.1.2 (ST (ST (Deviation from Hardy–Weinberg equilibrium (HWE) was tested for each locus with allele randomizations within populations (1000 permutations) and over all populations  in FSTAT v. 2.9.3.2 . Genetic.3.5.1.2  using ln.1.2  using the locprior setting, the admixture model, correlated allele frequencies, and a burn-in of 200,000 cycles followed by 500,000 additional cycles. We also performed shorter runs using different settings  to evaluate the importance of model choice. Results were averaged for the runs and the program DISTRUCT v.1.1  and aligned using BioEdit v.7.0.9.0 . The mitjima's D  tests imjima's D . Standar HapStar . Unique a), Nei's unbiased genetic diversity (h), and rarefied allelic richness (Rs) varied between different populations .All eight microsatellite loci for both species were found to be in Hardy–Weinberg equilibrium for all populations and no loci showed any signature of null alleles. Overall, we detected 40 alleles in 133 Nazca boobies  and 67 as, ranged from two to eight (mean = 3.8). Average genetic diversity per population was more uniform, ranging from 0.497 in Wolf to 0.572 in Genovesa. Recent population bottlenecks were detected in three of the five colonies: Española, Genovesa, and San Cristobal. In all three cases, seven of the eight loci showed a relative heterozygosity excess and P-values for the sign tests were 0.042, 0.048, and 0.040 for Española, Genovesa, and San Cristobal, respectively.In the Nazca booby populations genetic diversity, h, ranged from 0.071 to 0.870, with a mean of 0.58 and rarefied allelic richness, RIn the great frigatebird populations, genetic diversity ranged from 0.283 to 0.926, with a mean of 0.656. Rarefied allelic richness ranged from 2 to 14 (mean = 6.02) and average genetic diversity per population was even, ranging from 0.64 in the North Seymour and Darwin populations to 0.68 in the Wolf population. No recent bottlenecks were detected in great frigatebird populations.P > 0.05). Eighteen haplotypes were identified in great frigatebirds, using 1954 bp of mitochondrial sequence from 108 individuals. Haplotype diversity was 0.644 ± 0.051 while nucleotide diversity was 0.00054 ± 0.00048. Haplotype and nucleotide diversity per population and the mitochondrial haplotype network can be found in P > 0.05).A total of 19 mitochondrial haplotypes were detected in Nazca booby samples using 2145 bp of mitochondrial DNA sequenced from 50 individuals. Overall haplotype diversity was 0.886 ± 0.028 and overall nucleotide diversity was 0.001 ± 0.0001. Haplotype and nucleotide diversity per population were very similar, and are shown in ST and RST for Nazca booby populations to be 0.070 and 0.071, respectively. Due to the similarity of values given by both FST and RST, we will only report and discuss FST values for all subsequent comparisons. Eight of the 10 pair-wise estimates of structure between colonies using microsatellites were statistically significant (P < 0.01) (ST = 0.012), and Española and Genovesa (FST = −0.0003). In the subsample of mtDNA sequences, the global φST was 0.127 and four of the 10 pair-wise comparisons between colonies were statistically significant  . The onlnificant . All fouST for great frigatebird populations was 0.007. Only two of the 10 pair-wise comparisons between island colonies  were statistically significant .In contrast, the global Fnificant , while mr2 = −0.466, P = 0.98; mitochondrial data Mantel test: r2 = 0.396, P = 0.01). In fact, according to the multilocus data, there was weak evidence for higher differentiation between closer colony pairs, driven by the significant differentiation between Darwin and Wolf, which are separated by 38 km. For Nazca boobies, there was no relationship between FST and geographic distance using multilocus data . We did, however, detect a significant positive relationship between geographic distance and φST values for the mitochondrial data set, but the relationship explained only a very small amount of the variance and is likely driven by the significant differentiation between Darwin, a peripheral island, and all other colonies .We found no evidence for isolation by distance in the great frigatebird populations using either dataset (multilocus data Mantel test: P = <0.001). When an AMOVA was run with three defined groups , there was marginal support for this structure . Under this scenario, 8.77% of the variance was among groups and 0.15% was among populations within groups and 3.02% among individuals within populations.Hierarchical AMOVAs were run only on the Nazca booby dataset where genetic structure was detected. The AMOVA run on multilocus data showed strong support for two genetic groups  with 9.52% of the variance among groups and 2.3% of the variance among populations within groups . In the case of Nazca booby populations, the analysis run with and without locprior indicated that three genetic clusters were most likely (locprior results). One population consisted of Nazca boobies sampled from the isolated, northwestern islands of Darwin and Wolf, another included the birds from Española and Genovesa, and the third population consisted of the birds from San Cristobal  in the case of movement from Darwin to San Cristobal to 0.2912 (95% CI: 0.2249 – 0.3265) from Española to Genovesa  in the case of movement from Darwin to North Seymour to 0.2963  from Española to North Seymour. Migration rates of great frigatebirds did not have any clear directional pattern.Migration rates for Nazca boobies calculated in BayesAss had a mean of 0.037 ± 0.072 SD between all pairs of island comparisons . Rates rGenovesa , Table 5Genovesa . MigratiGenovesa . Rates rOur analyses reveal that despite short geographic distances between several of the breeding colonies of Nazca boobies, there is substantial genetic differentiation within the Galapagos archipelago and that three genetically distinct populations occur within the archipelago, based on the Bayesian clustering analysis. In contrast, very weak to no population genetic structure was found in the great frigatebird using both mitochondrial and nuclear markers and we found evidence of migration of individuals between multiple colony pairs. Most of the migration rates calculated for Nazca boobies were low, with the exception of high levels of nearly unidirectional gene flow were detected between two Nazca booby colonies, Española and Genovesa. We found that several of the larger migration rates  were from Española to other colonies, indicating that it might be a source population. The pronounced genetic differentiation in Galapagos Nazca boobies detected here corroborates previous mark-recapture studies that demonstrated very limited natal and breeding dispersal of Galapagos Nazca boobies .Mimus spp.) (35%)  and 65% (great frigatebird) heterozygosity is similar to values reported for other Galapagos taxa such as the Galapagos dove (56–65%)  and the .) (35%) . The cav.) (35%) . Additio.) (35%) . WheneveEvidence for recent bottlenecks was detected in the Española, Genovesa, and San Cristobal colonies of Nazca boobies. This could be due to the El Niño Southern Oscillation (ENSO) events that raise sea surface temperature, which can negatively affect marine life in Galapagos. The 1986–1987 ENSO event, while less severe than the one in 1982–1983, caused Nazca boobies to either suspend breeding or adjust the timing of their breeding cycle . These rMyiarchus magnirostris) (h = 0.491)  (h = 0.671) (P > 0.05).Haplotype diversities estimated from mtDNA were fairly high , compared with the Galapagos flycatcher (= 0.491)  and comp= 0.671) . Four isST, 0.0396, between Darwin and Wolf, the two islands closest in proximity. Even with the Locprior setting in STRUCTURE, we detected no population subdivision among great frigatebird colonies (k = 3 for comparison with the three groups detected in the Nazca booby). Interestingly, the mitochondrial genes provide evidence for weak differentiation between N. Seymour and Darwin and N. Seymour and Wolf. These discrepancies in overall pattern calculated using different markers could be due to the timescale on which the markers provide the best resolution  and/or any sex bias in dispersal . Although we have evidence that Galapagos great frigatebirds are genetically distinct from their non-Galapagos conspecifics , the birds breeding within the archipelago appear to be exchanging genes at a rate that swamps any effects of philopatry. The archipelago-wide average migration rate for great frigatebirds is similar to the one calculated for Nazca boobies; however, individual rates between colonies are more variable in the frigatebird while Nazca booby migration rates are all quite low aside from substantial movement of individuals from Española to Genovesa and from Wolf to Darwin. There is historical evidence from great frigatebirds breeding in the northwestern Hawaiian Islands demonstrating that the locations of breeding colonies are dynamic and the patterns may be explained by changes in vegetation important for nesting . The gene flow between Darwin and Wolf is not surprising given that they are separated by only 38 km. Gene flow between Genovesa and Española, separated by 194 km, but not between San Cristobal and either Genovesa (140 km) or Española (87 km) is a bit more puzzling. The western tip of San Cristobal is slightly east of a straight line between Española and Genovesa, but the main seabird colonies are located on the extreme northeastern tip of the island, also the most eastern point in the archipelago, with other smaller colonies along islets on the north side. Española birds dispersing in a north-northwestern direction, and therefore not passing over the colony on San Cristobal, would explain our estimates of archipelago-wide directional migration rates, and suggest that most gene flow occurs in a northern or northwestern direction. Interestingly, gene flow was also highest between Galapagos doves sampled on Española and Genovesa, although San Cristobal was omitted from the analyses due to small sample size , where peripheral populations were found to be more genetically distinct , and the relative isolation of Darwin and Wolf compared with any other islands in the archipelago. Migration rate estimates indicate that the highest level of gene flow occurs from Española to Genovesa and from Wolf to Darwin. Interestingly, there is negligible gene flow from Genovesa to Española. The majority of migration rate estimates >0.01 are in a north or northwestern direction, the direction of the prevailing winds.Sulidae species show strong phylogeographic signals and/or population genetic structure . A posswn booby . Populator males . Only onor males . Documenor males . This vaor males . We gainor males . Individor males . These dor males  where soor males . This pa