While research on the impact of global climate change (GCC) on ecosystems and species is flourishing, a fundamental component of biodiversity – molecular variation – has not yet received its due attention in such studies. Here we present a methodological framework for projecting the loss of intraspecific genetic diversity due to GCC.Trochulus villosus) and a caddisfly (Smicridea (S.) mucronata).The framework consists of multiple steps that combines 1) hierarchical genetic clustering methods to define comparable units of inference, 2) species accumulation curves (SAC) to infer sampling completeness, and 3) species distribution modelling (SDM) to project the genetic diversity loss under GCC. We suggest procedures for existing data sets as well as specifically designed studies. We illustrate the approach with two worked examples from a land snail (T. villosus and the second coarsest for S. mucronata. For both species, a substantial species range loss was projected under the chosen climate scenario. However, despite substantial differences in data set quality concerning spatial sampling and sampling depth, no loss of haplotype clades due to GCC was predicted for either species.Sampling completeness was diagnosed on the third coarsest haplotype clade level for The suggested approach presents a feasible method to tap the rich resources of existing phylogeographic data sets and guide the design and analysis of studies explicitly designed to estimate the impact of GCC on a currently still neglected level of biodiversity. Within the scientific community the evidence for a rapid and profound global climate change (GCC) is now widely accepted. Temperatures are predicted to rise by up to 4°C by 2100, as are severe changes in precipitation patterns ,4. GCC wWhile detailed predictions of the effect of random drift-based processes on genetic diversity are inherently difficult if not impossible to make, it should at least be possible to forecast the potential loss of genetic diversity associated with projected population extinctions or range shifts. However, to date, only few rigorous methodological frame works exists to perform such projections . SeveralFollowing a different approach, we here present a methodological framework focussing on projecting the potential loss of neutral intraspecific genetic variation based on the projected extinction of populations resulting from GCC induced changes in species distribution ranges. It consists of 1) identifying an appropriate hierarchical level of genetic diversity for inference from a sample of individual genotypes or haplotypes by 2) assessing the completeness of sampling these hierarchical levels, 3) choosing the appropriate spatial scale for SDM modelling and finally 4) projecting the loss of currently occupied species range and the associated loss of genetic variation. The suggested approach combines existing, established methods in an innovative fashion. As we will show, there is no straightforward “one-strategy-fits-all” solution, but we offer suggestions how to optimize the GCC projections for individual species depending on the intended scope of the study and previous knowledge in an iterative process. In addition, the problems are different for studies explicitly designed for this purpose and the post-hoc analysis of existing data sets e.g. of phylogeographic studies. We will address the problems in turn, then suggest solutions and finally illustrate the approach in two worked examples.In order to quantify a relative loss from a set of objects, we necessarily need to know how large the set initially was. For example, the statement that we lost half our money requires knowing how much we initially had. And, of course, we must have a clear definition of the set of objects we want to quantify and their value. To remain with the example, the extent of our economic disaster depends not only on the amount initially in our purse, but also on the values of the respective currency units lost.Genetic variation is a very broad, fuzzy concept that may refer to very different scales within an individual, population or species. For example in a population genetic variation may relate to heritable phenotypic variation such as flower colour or body size, multilocus allozyme genotypes or to haplotypes made up of SNPs on a non-recombining DNA stretch. These different levels of genetic variation are not easily compared, even though they may all be traced back to their basis, i.e. to differences on the DNA and how these differences are combined in alleles and their combinations, at least in principle. The number of DNA variations and their potential combinations within a sexually reproducing species is almost infinitely large . Basicalevolutionary units, lineages, clades or clusters (these terms will be used interchangeably hereafter) are usually successfully inferred using supposedly neutral genetic markers, like SNPs, AFLPs, microsatellites or mitochondrial sequences in animals, and chloroplast sequences in plants [hierarchical levels, depending on the level of genetic distinctness and on the resolution of the molecular marker systems applied. Often the former is a function of the latter. For example, a particular combination of SNPs along a stretch of non-recombining DNA makes up a haplotype. Depending on their ancestry, these haplotypes form monophyletic groups that can be joined with other such groups to higher level monophyletic groups. Likewise, multilocus genotypes may be most similar among individuals within families, a group of families forms a population distinguishable from other such populations by their genetic make-up and groups of populations within a species may be genetically more similar to each other than to populations of other such groups. In a study that sampled and genetically characterized individuals of a species each haplotype or genotype that is revealed represents an instance of each of the hierarchical levels it belongs to. Due to increasingly older shared common ancestry, each of those hierarchical levels thus consists of a decreasing, ever more inclusive number of genetic entities with increasingly higher position in the hierarchy. For example haplotypes that differ by a single base pair (bp) change along an analysed stretch of DNA can be nested into 1-step haplotype clades, and these 1- step clades can then be nested into 2-step haplotype clades etc. [Studies dealing with range wide neutral genetic diversity losses, however, are usually not interested in the fate of particular alleles or haplotypes (but see ) but ratn plants . Evolutides etc. . In suchdes etc. .While we cannot hope to account for the fate of every single base pair substitution, it should equally not be the goal to assess only the coarsest hierarchical level of genetic variation in any given intraspecific study. Additionally, a sensible projection of the loss of genetic diversity is only possible with a robust estimation of the quantitative and spatial extend of currently existing diversity. We therefore argue that from a statistical point of view the highest resolution level of genetic variation where all genetic entities were or can be sampled at least once represents the level that was exhaustively sampled and is thus the appropriate level for projection. Identifying the level that is biologically most meaningful for any given species on the other hand remains the task of the researcher, is likely to vary from species to species, and depends on the marker system used.The sampling design for genetic studies should always be adequate for the intended purpose ,32. IndeIt is important to assess to which degree the samples from each individual sampling site contain all relevant genetic entities actually present at this site. Otherwise, the current extent of the spatial distribution of an entity may be seriously underestimated. This problem is similar to certain sampling issues in population genetics . Here, ta priori  should thus give a conservative expectation of the distribution range of a clade at the given hierarchical level. If one or more spatially coherent unsampled areas of this or larger size exists within the species range, sampling these areas should be considered.We suggest that the The next issue we address is the spatial scale at which projections of genetic diversity loss can be reasonably made. Please note that this is not a discussion of the accuracy or methodology of SDM modelling, which depends on the spatial accuracy of the species occurrence data used  should be adequately covered, it is less clear at the beginning of a study at which spatial resolution the sampling should be performed, i.e. which distance between sampling sites in adequate.a priori knowledge of the home ranges, genetic population structure or the extent of spatial autocorrelation of the appropriate clade level of the focal organism. However, the complex interplay of spatially varying processes like life-history, demography, and dispersal but also contingent factors like population history, landscape features and natural selection will usually prevent any a priori predictability of the spatial distribution of genetic variability. It is thus likely that the sampling density needs to be adjusted in an iterative process of sampling successively more sites within the grids until saturation at the desired resolution is reached  can be determined as The situation is different if one wants to make use of the large body of phylogeographic data already available as recently proposed ,39. Heresam denotes the number of actually sampled grid cells and Nmin the minimum number of sampled grid cells necessary to reach the desired coverage. The latter can be determined with the resampling technique described above but with sampled grid cells as the unit and not the individual sampling sites. The maximum number of grid cells sampled cannot exceed the number of sites sampled. The value derived from the latter is thus a good starting point from which the optimal cell size can be empirically determined in an iterative process by successively decreasing Ncell and repooling the data according to their distribution in the grid cells. Such a rescaling towards a coarser grain size does not influence the SDM performance [whereby Nformance . The expformance .We could not find a published data set that comprehensively met the criteria outlined above in terms of spatial coverage and sampling depth. Therefore, we used two published data sets that are representative for very well and more poorly sampled phylogeographic data sets: one deeply and methodologically sampled phylogeography data set on a land snail species  and one Trochulus villosus is a land snail from the Hygromiidae family currently distributed at altitudes between 400 and 2300 metres in a relatively confined area that includes Switzerland North of the main ridge of the Alps and adjacent areas in France, Germany and Austria [ Austria . The spe Austria . From th Austria . It is tIn total, 455 individuals were sampled from 52 sampling sites  mucronata Flint 1989 is a net-spinning caddisfly of the family Hydropsychidae distributed from close to sea level to above 1000 m a.s.l. in the coastal ranges and Andes of central Chile [Smicridea (S.) S. mucronata revealed shallow but distinct clades within the coastal ranges and the Andes, though clades were shared among regions [T. villosus.al Chile ,44 . Futureate.org/,). The biThe potential present distribution of both species was computed with a maximum entropy approach  in MaxenT. villosus, the nesting comprised 5 levels, including the haplotypes  database [2. This was completed with distribution estimates for France, Germany and Austria on the basis of [2. The distribution area of S. mucronata estimated from [2. Area estimates were calculated in GRASS (GRASS Development Team 2011).The distribution area of database , which cbasis of  and the ted from ,44 was 62 at 47 degrees latitude) for the systematically sampled T. villosus. The area of grid cells was 30.8 arc-minutes for S. mucronata . This is under the assumption that sampling was performed in a way that each grid cell harbours at maximum a single site. However, when plotting the sampling localities over the upscaled bioclimatic layers, it became clear that the effective number of sampled grid cells was lower than the calculated thresholds. In the case of T. villosus the number of informative grid cells was 71, because three pairs of the 74 sampling localities fell into the same grid cell. In the case of the less densely sampled S. mucronata 4 of the 16 sampling localities fell into a grid cell with one or more other sampling sites, so the species’ range should be divided into 15 grid cells instead of 24. We therefore downscaled the 10 arc-minute grid cells once again. The final area of grid cells were only slightly larger  for T. villosus  for S. mucronata  was larger than the entire distribution range (see above).The sites furthest apart harbouring individuals from the least widely distributed clade on the 3-step level were 96 km apart in T. villosus . However, the resulting niche estimate is always a modelling-technique-dependent, more or less additive composite of the spatial distribution of all underlying entities, encompassing the respective niches of these subunits . Using sIt is recommended that the spatial scale of SDM should be consistent with the information content of the data . This alIt is well known that the quality of the projection depends strongly on the quality of the SDM, with all the known issues of e.g. microrefugia below SDM resolution where the genetic variation is at least partially preserved and local adaptation to the new conditions is possible. These problems and pitfalls also pertain to the forecasting of intraspecific genetic diversity, but we will not discuss them here, because they have been discussed at length elsewhere .Predicting the fate of intraspecific genetic variation has additional issues that should be kept in mind when interpreting the results. For example, GCC may alter dispersal behaviour and in some cases provoke increased dispersal , which mS. mucronata it was only possible to make inferences on the highest clade level, while in T. villosus, the second most coarse clade level could be used . Whethec scales ,70. In T valleys ,72,73. IT. villosus data set allowed for a higher prediction accuracy, because it was possible to find rarer occurrences. For example, the inference that none of the 3-step clades are threatened by extinction is based on two occurrences at a single sampling site in this species. The number of sites sampled and their spatial distribution also determined the spatial resolution for SDM. The projection grain in T. villosus was both absolutely (10.9 vs. 39.8 arc minutes) and relatively (1% vs. 7% of respective species range size) finer than in S. mucronata. The more thoroughly sampled T. villosus example thus gives much more confidence in the validity of the achieved results.The higher spatial coverage and the sampling depth of the However, despite the substantial quantitative differences in the data sets, the presented data is a conservative prediction in the sense that we have, inherently to the approach, rather under- than overestimated the spatial distribution of the respective clades. Intraspecific genetic diversity is thus probably even less threatened than suggested here. These inferences may well differ under different climate scenarios, but the aim here was rather to illustrate the approach than to exhaustively analyse the data.The presented approach presents a feasible, sound methodological framework to 1) tap the rich resources of existing phylogeographic studies and 2) guide the design and analysis of studies explicitly aimed to estimate the impact of GCC on a currently largely neglected level of biodiversity.The authors declare to have no competing interests.MP had the conceptual idea, MP and SP collected and analysed the data, MB performed the SDM modelling, MP drafted the manuscript and all authors contributed to writing the manuscript. All authors read and approved the final manuscript.Determining the probability of having sampled an entity with a given frequency at least once.Click here for file


Calonectris borealis).Ancient DNA has revolutionized conservation genetic studies as it allows monitoring of the genetic variability of species through time and predicting the impact of ecosystems’ threats on future population dynamics and viability. Meanwhile, the consequences of anthropogenic activities and climate change to island faunas, particularly seabirds, remain largely unknown. In this study, we examined temporal changes in the genetic diversity of a threatened seabird, the Cory’s shearwater  together with modern DNA sequences representative of the entire breeding range of the species. Our results show high levels of ancient genetic diversity in the Canaries comparable to that of the extant population. The temporal haplotype network further revealed rare but recurrent long-distance dispersal between ocean basins. The Bayesian demographic analyses reveal both regional and local population size expansion events, and this is in spite of the demographic decline experienced by the species over the last millennia.Our findings suggest that population connectivity of the species has acted as a buffer of genetic losses and illustrate the use of ancient DNA to uncover such cryptic genetic events. Puffinus holeae), the lava shearwater (P. olsoni), and an unidentified species of petrel (Pterodroma sp.) [Over the last millennia island faunas have been progressively decimated by human exploitation, biological invasions and habitat destruction . Determioma sp.) -8.Calonectris borealis). The breeding population of the species has been estimated as more than 270,000 pairs, however it underwent an acute decline between 1970–1990 and currently it continues to decline [One of the most abundant Procellariform species inhabiting the Canary archipelago is the Atlantic Cory´s shearwater ( decline ). In the decline . This haAncient DNA (aDNA) has revolutionised biodiversity and conservation studies because it provides an excellent opportunity to monitor the genetic variability of species or populations through time . HoweverCalonectris species, the Cory’s and the Scopoli’s (C. diomedea) shearwaters, we aim: (i) to estimate levels of genetic diversity and relationships between ancient and modern Cory’s shearwater populations, and (ii) to reconstruct the population demographic history of the Cory’s shearwater and estimate population size changes within the Canary archipelago through time.Using an heterochronous sampling consisting of Cory’s shearwater bone samples from late-Holocene from the Canary Islands together with current sequences representative of the entire breeding range of two sister 14C dates show that the three samples analyzed are from the last millennium. The 2σ confidence interval of two samples fall between 1004–1530 cal yr AD being the third younger than 1499 cal AD  and modern samples, and agrees with the marked genetic differentiation between the two r Figure . These fThe demographic analysis on the Cory’s shearwater indicated significant departures from a constant population model with the majority of credible sets containing non-zero population size changes. The Extended Bayesian Skyline Plot (EBSP) reconstruction of changes in genetic diversity through time shows a population expansion event at about 50,000 yr Figure A. At a lCalonectris shearwaters share the typical and extreme life history traits of Procellariiform seabirds: long-lived, low fecundity and high survival, and thereby the species is particularly susceptible to threats increasing adult mortality [Overall, these findings would not support the initial hypothesis of a loss in genetic variability in the Cory’s shearwater, in spite of its marked demographic population decline over the last millennium in the Canaries . Severalortality . In thisCalonectris shearwaters had been conducted. Ancient bone fragments were collected in Montaña Clara and Alegranza islets in the northeastern part of the Canaries . The rest of the bones were subsequently used for genetic analysis. Due to the fragmentary nature of the remains, the bones of different specimens could not be individualized. Subsequently, we generated fourteen subsamples (one for each of the fourteen sampling places) of pooled skeletal remains of similar weight (around 150 mg) aiming to identify a posteriori the different mitochondrial (mtDNA) haplotypes present on each subsample analysed. This pooling strategy was imposed by the limitation in size of the remains, and although it does not allow determination of the precise number of individuals analysed , it is still a useful inexpensive way to estimate the genetic diversity within a population [All experimental procedures on ancient samples were performed in a dedicated aDNA laboratory , where no previous work with extant s Figure . All sampulation . The foupulation . We ampl around 10 mg aimiThe modern samples included 240 mtDNA control region sequences published in previous studies from across the entire distribution range of the two sister species, the Cory’s and the Scopoli’s shearwaters ,17. A suGenetic statistics for both modern and ancient samples were performed in the program DnaSP, v. 5.10.01 . To depiTo investigate the demographic history of Cory’s shearwaters from the Canary archipelago, we estimated the shape of the population growth function through time by constructing Extended Bayesian Skyline Plots (EBSP) as implemented in BEAST v1.7.4   from Montaña Clara Islet . The lower value of each interval must be considered a maximum the age of each bone.Conventional radiocarbon ages (yr BP) and 2σ calibration intervals  from 3 bones of the Cory's Shearwater . The majority of the modern samples (240 out of 282); are part of a genetic study previously published by the authors [ authors . The GenClick here for fileSupplementary Methods.Click here for filePrimers used for the amplification of a fragment corresponding to the mtDNA control region.Click here for file


Bertholletia excelsa, is a monotypic genus, endemic to upland forests and distributed along almost the entire expanse of the Amazon [Amazonian upland forests are expansive and can comprise large continuous tracts. There have been several studies on the population genetic structure of species in this kind of forest, but there are few studies that aim to understand genetic structure throughout the Amazon . The Brae Amazon -5.Genetic diversity distribution through Amazon forest is an understudied issue, specially in plants. Quantifying and understanding population genetic structure, associated to gene flow and mating system studies are recognized as important tools for the development of strategies for conservation and management. Such information can also be helpful in identifying effects of habitat fragmentation ,7.B. excelsa populations in the Amazon and to verify if the structuring is influenced by distance between them.This study aimed to evaluate genetic structure of Seven polymorphic microsatellite markers were developed from a dinucleotide-enriched genomic library. Material from 379 individuals was collected from nine Amazonian subpopulations distributed in five Brazilian states . All seven microsatellite markers developed for the species were used with four others previously published markers , for genAnalyses within and among populations were performed to evaluate genetic diversity and population structure. All nine populations were characterized with 11 microsatellite loci for number of alleles per locus, allele frequency and observed and expected heterozygosity under Hardy-Weinberg expectation. Wright fixation indices were also estimated. Genetic distance estimates were correlated with different potential factors to find possible causes of genetic structure.ISF, non-significantly different from zero, were found. The observed high heterozygote proportions in most subpopulations and absence of inbreeding support the existence of self-incompatibility mechanisms and selection in favor of heterozygotes. Maintenance of genetic variability is favored by the alogamous breeding system, which is an important feature to be considered in conservation and management strategies, in order to avoid fertility deficits.A few alleles were found in each subpopulation and considerable variation was observed in alleles found in each subpopulation and in their allele frequencies, especially when compared with very distant subpopulations. Heterozygote excess was observed in five subpopulations while in the other four subpopulations, estimates of q= 0,02) to high . B. excelsa genetic structure can be analyzed on three different scales: (i) within population; (ii) among moderately distant populations (<500km); and (iii) among very distant subpopulations. At all scales, significant correlations were found between genetic structure and geographic distance between pairs of individuals or populations. This may indicate that distance is an important factor in this population’s genetic structure, but probably there are other factors acting in conjunction, especially on a large geographical scale.Fine-scale structure, when present, was small. Estimates of inter population genetic structure varied from low (


Melipona quadrifasciata, 61 colonies were collected in eight locations in the state of Minas Gerais, Brazil. By means of PCR analysis, 119 ISSR bands were obtained, 80 (68%) being polymorphic. He and H B were 0.20 and 0.16, respectively. Two large groups were obtained by the UPGMA method, one formed by individuals from Januária, Urucuia, Rio Vermelho and Caeté and the other by individuals from São João Del Rei, Barbacena, Ressaquinha and Cristiano Otoni. The Φst and θB values were 0.65 and 0.58, respectively, thereby indicating high population structuring. UPGMA grouping did not reveal genetic structuring of M. quadrifasciata in function of the tergite stripe pattern. The significant correlation between dissimilarity values and geographic distances  implies possible geographic isolation. The genetic differentiation in population grouping was probably the result of an interruption in gene flow, brought about by geographic barriers between mutually close geographical locations. Our results also demonstrate the potential of ISSR markers in the study of Melipona quadrifasciata population structuring, possibly applicable to the studies of other bee species. For a study of diversity and genetic structuring in  Melipona quadrifasciata Lepeletier, 1836 is a stingless bee distributed along the eastern Brazilian coast, from Rio Grande do Sul to Paraíba, extending westwards inland towards Misiones, in Argentina, and southeastern Paraguay  that was present in M. q. anthidioides, but absent in M. q.quadrifasciata. Similarly, M. q.anthidioides and M. quadrifasciata from northern Minas Gerais, northeastern Bahia and Sergipe, both with a tergite stripe pattern similar to that of M. q. quadrifasciata.Paraguay . Traditiidioides . The sube do Sul , whereas Paraiba . PopulatM. quadrifasciata, the difference between the two subspecies could be emphasized  detected specific ISSR markers for the subspecies Apis mellifera carnica and Apis mellifera caucasica that permitted genetic differentiation between the two.Through population analysis of  species . AlthougM. quadrifasciata from different localities in Minas Gerais, thereby constituting the first appraisal of populations with bees of the Meliponini tribe using the PCR-ISSR approach.In this study, ISSR polymorphism was assessed and used for characterizing the population structure of Melipona quadrifasciata colonies from eight locations in the state of Minas Gerais, Brazil  and 50 pmoles primers. Amplification conditions included initial denaturation at 94 °C for 3 min, followed by 40 cycles of 1 min at 92 °C, 2 min at primer annealing temperature, 2 min at 72 °C, and a final step of 7 min at 72 °C. The reactions were accompanied by a negative control containing all the components, with the exception of genomic DNA. The PCR products were separated by electrophoresis in 1.5% (p/v) agarose gel, and visualized by staining with ethidium bromide (0.2 μg/mL) and photodocumented using the AlphaDigiDoc system.The reaction mixture (25 μL) contained 10 ng of DNA, 2.0 μL of dNTPs at 100 μM, 2.5 μL of a 10X buffer, 0.5 U free f model. In this analysis, the HB value was analogous to He, and the θB value to Φst of the AMOVA. The spatial distribution pattern based on genetic distance was assessed through the Mantel test using the GENES program v. 2007.0.0 (Amplification products were codified as binary traits according to band presence (1) and absence (0). The percentage of polymorphic loci and genetic diversity (He) were estimated using the TFPGA program 1.3 version . Groupin2007.0.0 .Melipona quadrifasciata was considered high and fairly close to that estimated for the subspecies Apis. mellifera. carnica and Apis. mellifera. Caucásica (66.7%), also when applying ISSR polymorphism .Amplifications with clear and reproducible band patterns were available from only nine of the 93 initially assessed ISSR primers (UBC Kit). PCR reactions involving these nine primers resulted in 119 DNA bands, of which 80 were polymorphic (68%). The number of bands per primer ranged from 8 to 16, with a mean of 13 bands per primer, the reactions with UBC-807 and UBC-834 primers presenting the highest number of bands (16). The value of 67% ISSR polymorphism detected in e), assuming Hardy-Weinberg equilibrium was 0.20, and the HB estimate based on Bayesian analysis, that does not assume the Hardy-Weinberg equilibrium, was 0.16. Low diversity values have also been reported in studies on other Hymenoptera and were justified by the haplodiploidy system associated to the effective size of the population, social behavior and environmental variation  at the level of 0.75 genetic similarities separated the specimens into two large groups. The first group supported by bootstrap value of 99% comprised the samples from Januária, Urucuia, Rio Vermelho and Caeté, whereas the second supported by a bootstrap value of 55% consisted of those from São João Del Rei, Barbacena, Ressaquinha and Cristiano Otoni . The sigM. q. anthidioides is associated with higher altitudes throughout mountain ranges in Minas Gerais, Espirito Santo and Bahia and absent in the lowlands in northern Espirito Santo, southern Bahia and areas in the upper São Francisco river.Genetic differentiation within populations was probably the outcome of interrupted gene flow, caused by geographic barriers between otherwise geographically close neighbors. This population isolation, as reflected in local genetic structuring, probably arose from the rough geographical topography of Minas Gerais. This hypothesis is supported in studies undertaken by M. quadrifasciata from Januária and Urucuia with continuous tergite stripe pattern and individuals from Rio Vermelho and Caeté in which this pattern is discontinuous. Retained ancestral polymorphism could possibly cause incongruence between ISSR profiles and tergite stripe patterns. Similar results were reported by UPGMA grouping revealed no genetic structuring associated with the tergite stripe pattern in these bees. The first group supported by a high bootstrap value (bootstrap = 99%) included M. quadrifasciata in the state of Minas Gerais  showed that the percentage of variation among locations (58.81%) was higher than that within locations (41.19%). The Φst (0.59) showed a high population structuring. The values of the Φst matrix pair by pair between locations confirmed the high differentiation of s Gerais . AnalysiB = 0.58) also showed that the two groups were highly structured. Evidence that M. quadrifasciata is found structured in the localities assessed in the present study was also reported by AMOVA (Φst = 0.59) and Bayesian analysis (θM. quadrifasciata genetic structuring, while not expressed in the tergite stripe pattern, could be related to geographic isolation. Furthermore, the potential of the ISSR marker in studies of population structuring in M. quadrifasciata was also demonstrated, this possibly constituting a useful aid in the studies of other bee species.In short, it was shown that


Jamunapari, a dairy goat breed of India, has been gradually declining in numbers in its home tract over the years. We have analysed genetic variation and population history in Jamunapari goats based on 17 microsatellite loci, 2 milk protein loci, mitochondrial hypervariable region I (HVRI) sequencing, and three Y-chromosomal gene sequencing. We used the mitochondrial DNA (mtDNA) mismatch distribution, microsatellite data, and bottleneck tests to infer the population history and demography. The mean number of alleles per locus was 9.0 indicating that the allelic variation was high in all the loci and the mean heterozygosity was 0.769 at nuclear loci. Although the population size is smaller than 8,000 individuals, the amount of variability both in terms of allelic richness and gene diversity was high in all the microsatellite loci except ILST 005. The gene diversity and effective number of alleles at milk protein loci were higher than the 10 other Indian goat breeds that they were compared to. Mismatch analysis was carried out and the analysis revealed that the population curve was unimodal indicating the expansion of population. The genetic diversity of Y-chromosome genes was low in the present study. The observed mean M ratio in the population was above the critical significance value (Mc) and close to one indicating that it has maintained a slowly changing population size. The mode-shift test did not detect any distortion of allele frequency and the heterozygosity excess method showed that there was no significant departure from mutation-drift equilibrium detected in the population. However, the effects of genetic bottlenecks were observed in some loci due to decreased heterozygosity and lower level of M ratio. There were two observed genetic subdivisions in the population supporting the observations of farmers in different areas. This base line information on genetic diversity, bottleneck analysis, and mismatch analysis was obtained to assist the conservation decision and management of the breed. Genetic diversity, the primary component of adaptive evolution, is essential for the long-term survival probability of a population –4. GenetIn this study, we sample the Jamunapari goat population to analyse the genetic variation due to locus-specific events (selective sweep) as well as genome wide events (bottlenecks). Microsatellite markers are highly polymorphic and have been extensively used for breed diversity analysis. Mitochondrial DNA (mt DNA) and Y-chromosome region are usually sensitive to genetic drift and can be useful for detecting effects of bottlenecks in the population. Nonneutral markers are also being used to analyse population diversity, and milk protein gene has been used as the region is directly involved for the survival of the individual and under strong selective pressure. By integrating data from multiple markers, we provide the possible factors affecting the genetic consequence of population reduction in this breed.Fifty blood samples were collected in 10 villages in which the breed has a major concentration. Samples were collected from the individuals exhibiting typical breed characteristics such as white colour, Roman nose, and pendulous ear (farmers are not selecting for these traits) and at least two samples were collected from each village. An effort was made to collect samples from unrelated individuals based on information provided by farmers. The breeding buck is available with one or two farmers in every village, and some farmers also maintain breeding bucks during breeding season, disposing of them after the breeding season. Blood samples were collected from each animal using EDTA vacutainer and stored at –20°C till further use.rst [E) based on Hardy-Weinberg assumptions. We tested genotypic linkage disequilibrium between all pairs of loci in each population with GENEPOP  based oβ-LG gene and CSN1S1 (αs1-casein) were analysed using PCR-RFLP to observe genetic variability in 35 individuals. The αs1-casein (CSN1S1) gene produced an amplified fragment of 223 bp which was digested with the XmnI restriction enzyme. The β-LG gene produced an amplified product of 426 bp, and RFLP analysis was carried out with the SacII restriction enzyme. The PCR-RFLP analysis was carried out as described by Kumar et al. [Milk protein genes, which are expected to be nonneutral markers, were also used to analyse the population variability. Two milk protein genes, namely, r et al. , 19, andr et al. .mtDNA HVRI sequencing was carried out as described by Joshi et al. . Four huμL reaction volume containing 100 ng of DNA, 20 pM of each primer, 200 μM of dNTP, 2 mM Mgcl2, and %U of Taq DNA polymerase . The samples were subjected to sequencing after purifying the PCR product by gene elute PCR clean up kit. Individual PCR amplified products were subjected to sequencing in 12 samples. PCR products were sequenced on both the strands directly using 50 ng (2.0 μL) of PCR product and 4 pM (1.0 μL) of primer, 4 μL of Big Dye Terminator ready reaction kit , and 3.0 μL of double distilled water to adjust the volume to 10.0 μL. Cycle sequencing was carried out in a Gene Amp 9600 thermal cycler (Perkin Elmer) employing the PCR conditions. Extended products were purified by alcohol precipitation followed by washing with 70% alcohol. Purified samples were dissolved in 10 μL of 50% Hi-Di formamide and analysed in an ABI 3700 automated DNA Analyzer . Nucleotide diversity, expected heterozygosity, Tajima's D, and Fu's Fs values were estimated in ARLEQUIN 3.1 [Genetic divergence was analysed by selecting three primers from ovine male-specific region  . PCR wasQUIN 3.1 .rst [ Genetic bottleneck was detected using microsatellite data by three approaches, heterozygote excess, mode-shift, and M ratio test. We first used the M ratio   as implerst , becauserst .The BOTTLENECK programme  was usedk) in the Jamunapari populations. STRUCTURE software sorts individual genotypes into clusters that maximize the fit of the data to theoretical expectation. Based on preliminary analyses, we evaluated the likelihood of k = 2 and k = 3, with 5 runs performed for each k, and a burn-in length of 500,000 and 100,000 MCMC replicates for each run. We assumed an admixture model and correlated allele frequencies among populations [We used an individual-based clustering approach  to deteulations .P value <0.05. The LD was significant in 13.21% of the locus pair combinations in the population. The overall excess of homozygosity for the population as a whole varied from 0.089 to 0.785 over the loci and the average was 0.500. The high levels of allelic diversity are coupled with very high Fis indicating that the population is experiencing high levels of nonrandom mating in the breeding tract but simultaneously maintaining allelic diversity over the entire range of the breed. Gour et al. [The markers with their chromosome number, number of alleles identified, and allele size range have been described in r et al.  also obsCSN1S1 and β-LG was 0.395 and 0.107. The effective number of alleles was 1.653 and 1.20 at CSN1S1 and β-LG loci, respectively. The β-LG locus showed significant departure from HW equilibrium. The gene diversity and effective number of alleles at milk protein loci were higher than for 10 other Indian goat breeds, supporting the fact that the breed maintains higher genetic variability [Genetic variation at iability , 19.The population was examined for allele frequency distribution for gaps, and M ratios are presented in P = 0.8487, one tail for heterozygosity excess), and there was no significant departure from mutation-drift equilibrium detected in the population. Under the two-phase mutation model equilibrium, each individual has a roughly equal chance of having heterozygote deficiency or excess. The analysis indicated that the population has not suffered any bottleneck recently and was a constant size population. The present study agrees well with the observation of Gour et al. [Bottleneck detection in Jamunapari goat was presented in r et al. .The sequence analysis of the 457 bp mitochondrial HVRI region identified 34 mtDNA haplotypes, and the overall haplotype diversity was 0.984. The mtDNA variation detected relatively high number of haplotypes from a total 50 individuals, and haplotype diversity was quite high. Fu's Fs value is based on the probability of recovering a number of haplotypes greater than or equal to the observed number in a sample drawn from a stationery population with the same mean number of pairwise differences as the observed sample. Fu's Fs value was −15.53. The significant negative Fs values indicated the large and sudden expansion in the population at geographical locations. The population showed a significant negative Fs value indicating excess of rare mutations, a pattern commonly attributed to a normal growing population.Mismatch distribution analysis revealed the genetic signature of population expansion of Jamunapari goat. The tau value with 95% confidence interval was 8.20 (5.22–10.68), and the Sum of Square Difference (SSD) value was 0.0048. The SSD value showed large population expansion in Jamunapari goats. The amplified product for amelogenin gene (AMELY), SRY gene, and ZFY gene was 733 bp, 632 bp, and 584 bp, respectively. No diversity was observed in SRY and AMELY genes. Nucleotide diversity and expected heterozygosity were 0.135 ± 0.089 and 0.1384, respectively, for ZFY gene. Similarly Tajima's D value and Fu's Fs value were zero for SRY and AMELY gene. Tajima's D value and Fu's Fs value were 0.6797 and 2.539, respectively and non-significant for ZFY gene. Transition and transversion ratio was 47 : 90 for ZFY gene. ZFY gene showed low gene diversity and positive Tajima's D value and Fu's Fs value. Y-linked nucleotide diversity was found low in human, wolf, cattle, reindeer and Lynx indicating that reduced levels of Y-chromosome polymorphism may be a generalized feature of mammalian genome [k = 2 or 3). The cluster defined by STRUCTURE showed clear membership of individuals in two clusters indicating a genetic subdivision within the breed. There was a difference between expected and observed heterozygosity supporting the genetic subdivision within the breed. It has been also observed during survey that there are two strains of Jamunapari locally known as “Kathey” and “Kandhan”. Kathey-type goats have long thick broad and less folded ears with thick and flat neck. Kandhan-type goats have long, soft, and folded ears, and the neck is thin and cylindrical. Kandhan is restricted to river banks of Chambal and Kathey is distributed throughout the breeding tract. (CIRG Report).The proportion of membership of individuals in the Jamunapari population placed 12.6% of population into one cluster group in both STRUCTURE analyses  in the population suggesting that it has not suffered severe or long-lasting genetic bottlenecks , 36. Het


Abies cilicica currently persists in Lebanon in geographically isolated forest patches. The impact of demographic and evolutionary processes on population genetic diversity and structure were assessed using 10 nuclear microsatellite loci. All remnant 15 local populations revealed a low genetic variation but a high recent effective population size. STF-based measures of population genetic differentiation revealed a low spatial genetic structure, but Bayesian analysis of population structure identified a significant Northeast-Southwest population structure. Populations showed significant but weak isolation-by-distance, indicating non-equilibrium conditions between dispersal and genetic drift. Bayesian assignment tests detected an asymmetric Northeast-Southwest migration involving some long-distance dispersal events. We suggest that the persistence and Northeast-Southwest geographic structure of Abies cilicica in Lebanon is the result of at least two demographic processes during its recent evolutionary history: (1) recent migration to currently marginal populations and (2) local persistence through altitudinal shifts along a mountainous topography. These results might help us better understand the mechanisms involved in the species response to expected climate change.The threatened conifer  Geographic patterns of genetic variation in conifers have long been studied at the species and populations levels The genetic imprint of geographic isolation, or fragmentation, on natural populations can vary with respect to their demographic structure. Relict populations in fragmented landscapes predictably show low genetic variation but high genetic differentiation due to spatial isolation The biogeographic position within a geographic range or a biome may impact genetic diversity within and among populations. In the Mediterranean basin, genetic diversity is higher than other regions of the world for conifers Abies cilicica Carr. in Lebanon (Cilician fir). Abies cilicica is a long-lived and outcrossed conifer species. It is endemic to the Eastern Mediterranean in the Taurus, Anti-Taurus and Amanus mountains of Turkey, the Alawi mountains in Syria and the Northern Mount Lebanon range in Lebanon  Is biogeographic position affecting genetic diversity within and among populations?, (2) Is habitat fragmentation increasing spatial genetic structure?, (3) Is geographic isolation limiting dispersal?, and (4) Is geographic marginality affecting persistence?Because genetic diversity is potentially high in the Eastern Mediterranean and for mountain species Abies cilicica in Lebanon was carried out after obtaining mission permits from CNRS in France and NCSR in Lebanon  and following the approval in Lebanon of corresponding municipalities and nature reserves organizational teams. Sampling of Abies marocana Trab.  was permitted by the Talassemtane National Park, Morocco. Needle samples of Abies bornmuelleriana Mattf., Abies cephalonica Loud. and Abies alba Mill. were provided by INRA-URFM.All the field sampling of Abies cilicica is currently distributed in Lebanon across 15 remnant populations located between ca. 1000–1800 m.a.s.l. on the western slopes of northern Mount Lebanon. Moist airstreams off the Mediterranean approaching over Mount Lebanon result in a classic orographic precipitation pattern. Rain falling on the western side of Mount Lebanon becomes snow at higher elevations during winter. The species currently occupies humid bioclimatic zones with a mean annual precipitation over 800 mm. The varied topography of Mount Lebanon allows it to inhabit both the Supra-Mediterranean (1000–1500 m.a.s.l.) and Mountain-Mediterranean (1500–2000 m.a.s.l.) vegetation zones. Currently, it occupies a patchy distribution covering 1613 ha equivalent to 1.2% of the total forest cover of Lebanon, compared to 1.58% for Cedrus libani A. Rich. (Cedar of Lebanon) A. cilicica in Lebanon  at INRA Avignon, with the GeneScan™ - 500 LIZ™ size standard (Applied Biosystems) run with each sample. In order to decrease the risks of genotyping errors related to automated or arbitrary decisions in allelic binning genepop 4.1.3 genepop 4.1.3 by using the log likelihood ratio statistic test (G-test). Exact P-values for the individual population or locus tests were estimated using the Markov Chain algorithm implemented in genepop 4.1.3 with 10000 dememorization steps, 100 batches and 5000 iterations and were then corrected both using the sequential Bonferroni procedure freenafreena was used to compute the STF statistic ENA (Excluding Null Alleles) correction method, i.e. by excluding or not excluding null allelic states from the STF computation STF values were calculated using 50000 replicates over loci.Exact tests for deviations from Hardy–Weinberg equilibrium (HWE) were performed using the heterozygote deficiency and heterozygote excess tests implemented in genalex v6 fstat version 2.9.3.2 OH, expected heterozygosity EHRAISFISF value over loci was calculated in fstat version 2.9.3.2 using 15000 replicates. Rarefied private allelic richness sП was computed using the rarefaction method hp-rare 1.1 OH and EH across all samples for all loci was verified using a Student's t-test. Differences in the within-population genetic diversity and allelic richness among A. cilicica, A. marocana, A. bornmuelleriana, A. cephalonica and A. alba were assessed using a permutation test implemented in fstat version 2.9.3.2 spagedi STFSTRSTR computed before randomization was larger than STR obtained after allele size permutation (STpR). The randomization test also infers whether or not strict stepwise-like mutations contributed to population differentiation The differentiation between populations was computed in structure 2.3.3 K) and probabilistically assigns individual multilocus genotypes to these clusters. To infer posterior probabilities of K, we ran a series of independent runs with different user-defined values of K clusters ranging from 1 to 15. Assuming that individuals may have mixed ancestry and that the different populations have correlated allele frequency 6 iterations of MCMC after a burn-in period of 30000. No prior information on the sampling locations of each individual was used as recommended in the software documentation structure results were then processed in a web-based program, structureharvesterK. The second Bayesian approach, implemented in the software baps 6 K clusters ranging from 1 to 15. To find the optimal partition, the program uses a stochastic optimization algorithm, which is less computationally intensive than the MCMC algorithm used in structurePopulation genetic structure was also assessed using two Bayesian clustering approaches with different underlying assumptions concerning the units of clustering , the types of clustering , and the computation algorithm (Markov chain Monte Carlo (MCMC), stochastic optimization). The first Bayesian clustering approach, implemented in the software fstat version 2.9.3.2 OH) and expected (EH) proportion of heterozygotes and the overall gene diversity (TH) according to ISF and STF according to RA) for a sample size of n = 102 gene copies or 51 diploid individuals STRspagedi geneclass2 0) migrants. The likelihood (L) that an individual originates from a given population sample was computed using the statistical criterion A. cilicica in Lebanon have been extensively sampled, this criterion was considered to be informative on recent dispersal Direct estimates of recent migration rates within and between the current clusters were generated in the software ibdws version 3.23 Isolation-by-distance (IBD) A. cilicica in Lebanon had experienced recent and severe genetic bottlenecks. The first method, implemented in the software m-p-val and critical_mk) to the range in allele size (r), from a population sample of microsatellite loci sp (proportion of one-step mutations) = 0.88 and gΔ (average size of non one-step mutations) = 2.8 as reviewed in m-p-val, the average observed value of M calculated from the microsatellite dataset was compared with a simulated equilibrium distribution of M for different values of the population parameter critical_m, values of the critical M threshold (Mc), below which a bottleneck is evident, were also calculated for the different θ values. Considering an average mutation rate per locus per generation equal to 0.0005 θ Є {10; 1; 0.5; 0.1} corresponding respectively to a pre-bottleneck effective population size of 5000, 500, 250 and 50.Three complementary methods were used to detect whether or not bottleneckThe second method makes use of a Wilcoxon's signed rank test to compare the distribution of the heterozygosity expected under mutation-drift equilibrium and under HWE and can detect recent genetic bottlenecks over the past 4 STF both using and without using the ENA correction method gave equal results; STFENAnot using   =  STFENAusing   =  0.0524 with the respective 95% CI [0.039–0.072] and [0.038–0.073]. Classical measures of population differentiation are only slightly biased with a null allele frequency ranging between 5% and 8% on average across loci STF did not vary after excluding null alleles, all loci were kept for further statistical analyses.Out of a total of 149 tests for heterozygote deficiency, only the population KCHB for the locus SFb5 showed heterozygote deficiency (P<0.05), after controlling both the FWER and the FDR. Heterozygosity excess was never detected in any population and any locus. Further, the LD test showed no statistical significance for all the 666 tests between pairs of loci indicating an independent segregation of loci. The locus SFb5 for the population KCHB showed a relatively high percentage of null alleles (21%). However, the percentage of null alleles for SFb5 on average across the 15 local populations was only 4.5%. In total, null allele frequency estimates ranged from 0.6% to 4.5% with 2.5% on average across loci. The estimation of OH = 0.578±0.017 SE) was not significantly different from the mean expected heterozygosity under HWE (EH = 0.595±0.016 SE) (P>0.05) (ISF = 0.029) was not significantly different from zero with the 99% CI [−0.024–0.090]  and had significantly less allelic richness (P<0.001) than the studied northern Mediterranean fir species     for a standardized sample size of n = 20 gene copies and 2.77 (±0.01 SE) alleles per locus and per population for a standardized sample size of n = 6 gene copies . Mean ra(P>0.05) . The ove4–0.090] . Permutaalonica) , Table 2(P>0.05) , Table 2(P>0.05) , Table 2STF) and on allele sizes (STR) averaged 0.0536 (P = 0) and 0.0374 (P = 0), respectively, across all loci. Allele size permutation tests showed that the observed value of STR computed before randomization was not significantly different from the STR obtained after allele size permutation (STpR) (P = 0.43). Therefore, strict stepwise mutations did not contribute to among-population differentiation and no phylogeographic pattern was detected.The among-population genetic differentiation based on allele identities  were grouped into a single deme (deme 1) while the other 4 populations  formed another deme (deme 2). Because of their geographic distribution, throughout this paper, the demes 1 and 2 are referred to as “Northeastern ridge” and “Southwestern ridge” respectively.The Bayesian analysis of population structure in fstat revealed significant differences among demes. Notably, the Southwestern ridge had significantly lower allelic richness (P<0.001), had significantly lower gene diversity (P<0.01), but significantly higher differentiation (P<0.05) than the Northeastern ridge (ISF (P>0.05) (STR was not significantly different from STpR for each deme (P>0.05), thus, no phylogeographic pattern was detected within the Northeastern ridge and within the Southwestern ridge (The permutation test of populations performed in rn ridge . The tot(P>0.05) . Additiorn ridge .geneclass2 allowed the detection of 11 first-generation migrants among the 15 remnant populations of A. cilicica in Lebanon (P<0.01)  and less gene flow from the Southwestern ridge towards the Northeastern ridge (1 F0 migrant) (The likelihood-based Bayesian assignment test performed within (P<0.01) . Gene flmigrant) . Dispers2 = 0.05) (Isolation-by-distance (IBD) assessed over all populations was significant . However, the strength of the IBD was weak, with the RMA explaining only 5% of the variance in the whole distribution area  , thus imM-ratios were not significantly lower than the simulated equilibrium distribution of M for all the different pre-bottleneck θ values (P>0.05). Second, the bottleneck threshold values, calculated for all the pre-bottleneck θ values, were significantly lower than the observed M-ratios (P<0.05)  . The Wil(P<0.05) . We concA. cilicica in Lebanon revealed low within-population genetic diversity (0.595±0.016 SE), low allelic richness (2.77±0.01 SE), compared to other Abies species, and low among-population genetic differentiation . Populations were not inbred and showed no signal of a recent and severe genetic bottleneck. Despite the low spatial genetic structure inferred from classical genetic differentiation parameters, populations showed a significant genetic divergence, forming two demes, the Northeastern and Southwestern ridges. Additionally, despite their isolation-by-distance, populations were capable of long-distance dispersal. We discuss in the following sections how these contradictory features may have emerged and their effect on the persistence of A. cilicica in Lebanon. We finally discuss the implications of these results for conservation.Abies species in the Mediterranean, using allozymes and chloroplast microsatellites A. cilicica in Turkey and Syria harbor high values of heterozygosity. High levels of genetic variation have also been found for A. cephalonica and A. bornmuelleriana in numerous studies A. cilicica in Lebanon revealed unexpected low within-population genetic diversity and low allelic richness. Particularly, this low genetic variation was not significantly different from the genetic variation of A. marocana in the Moroccan Rif and A. alba in the French Pyrenees. Previous studies have also reported low genetic variation in A. marocanaA. alba in the French Pyrenees with respect to A. alba in the French Southern Alps is also consistent with a previous study A. alba in the Pyrenees is isolated within a glacial refugium and highly differentiated from the rest of its range A. alba in the Pyrenees might be the result of their exposure to a historic severe genetic bottleneck Previous comparative studies between A. cilicica in Lebanon might be explained by the historical timber use during the Ancient Egyptian New Kingdom's rule over Phoenicia A. cilicica in Lebanon. The low genetic variation might have been shaped over time by a rather long-term than a short-term low effective population size. One probable cause of this might be the temporal fluctuation in population sizes The low genetic variation of STF, the Bayesian clustering methods detected two genetically distinct, but highly admixed demes, the Northeastern and Southwestern ridges. Populations of the Southwestern ridge inhabited the smallest habitat patches and occupied the southernmost part of the distribution area. Populations of the Southwestern ridge were significantly less genetically diverse, had significantly less allelic richness, but were significantly more genetically differentiated than populations of the Northeastern ridge. Despite these differences, the Northeastern and Southwestern ridges shared similar ecological attributes. Both demes occupied a similar elevation range, between ca. 1000 and 1800 m.a.s.l. Neither deme showed inbreeding or sub-population structure. Both demes preserved the same amount of total genetic diversity (TH), and were capable of short-distance and long-distance dispersal. The recent large effective population size and the weak IBD pattern suggest an outperformance of migration over genetic drift. Consequently, genetic drift may not be the leading evolutionary force for population differentiation in A. cilicica from Lebanon. In the latter case, FST may not be accurate Despite the low observed value of A. cilicica in Lebanon may result from rapid colonization where both long-distance and short-distance dispersal are key demographic processes A. cilicica from Lebanon as a recent rear-edge migration with both recent colonization balancing local extinctions and recent range expansion along altitudinal gradients. Considering that brief episodes of range expansions are thought to leave strong and persistent signature on the genetic structure of natural populations A. cilicica in Lebanon suggests two plausible demographic processes promoting persistence.Similarly to many tree species, the genetic structure of First, the Northeast-Southwest structure might result from recent colonization balancing local extinctions. The likelihood-based Bayesian assignment test detected an emigration rate from the Northeastern ridge exceeding three-fold that from the Southwestern ridge . AlthougSecond, the Northeast-Southwest structure might also result from recent range expansion along altitudinal gradients. Private alleles in both the Northeastern and Southwestern ridges could indicate the presence of different glacial microrefugia in both demes . In the A. cilicica in Lebanon, and have shaped current diversity via long-distance migration and altitudinal range shifts, all local populations of the Northeastern ridge as well as QARS of the Southwestern ridge have disproportionate conservation importance. The very low census size of QARS and its location on the lowest elevation warrant special attention. Conserving other Southwestern ridge populations (such as EHDN) would also be useful as they are stepping stones for gene flow between the two demes. EHDN is an interesting example for highlighting conservation challenges. A mixed fir/cedar population, EHDN has been managed as a nature reserve since 1992. The low number of surviving fir trees in this nature reserve as well as their low genetic variation suggest that a change of strategy is needed if conserving the genetic diversity of A. cilicica is a target for management. Dynamic conservation of genetic diversity in situ requires that a particular attention should be given to regeneration and to avoid introducing seeds or seedlings from exotic locations outside demes or farther away. Attention should be given to keeping gene flow between the Northeastern and Southwestern ridges at reasonable rates. This can be potential through maintaining historic-likewise levels of gene flow by (1) preserving the natural ecological corridor between the Northeastern and Southwestern ridges, and (2) preserving a buffer zone across each local population to facilitate the natural recolonization of local populations over unoccupied habitat patches. For local populations with very low census size and genetic diversity, ex situ conservation  or assisted migration to suitable locations where future climate should be favorable to A. cilicica is strongly advisable.Because they contain most of the genetic diversity of A. cilicica in Lebanon is at odds with biogeographical and life history trait expectations. Rather, demographic processes linked to current habitat fragmentation and geographic range structure, and to original historical range contraction and recolonization better explain genetic diversity. A. cilicica in Lebanon is made of two demes moderately connected by geneflow shaping a weak isolation-by-distance pattern. Persistence of A. cilicica in Lebanon was linked to two plausible demographic processes, occurring concurrently or sequentially, which we called rear-edge type migration: (1) alternating colonization and extinction events and (2) range shifts along altitudinal gradients. These two demographic events might help us better understand the species response to expected climate change. Populations of the Northeastern and Southwestern ridges might harbor a large evolutionary potential, and should be consequently prioritized for conservation.The population genetic diversity and structure of Table S1Origin, location sample size of Abies cilicica and other Mediterranean Abies species. Legend: Origin, location, and sample size of the 15 populations of Abies cilicica in Lebanon and other Mediterranean Abies species. Altitude is in meters above sea level ( m.a.s.l.) and the surface of the sampled populations of Abies cilicica in Lebanon is in hectares. N.B. Surface is not indicative of population density.(XLS)Click here for additional data file.


Programs to rebuild imperiled wild fish populations often include hatchery-born fish derived from wild populations to supplement natural spawner abundance. These programs require monitoring to determine their demographic, biological, and genetic effects. In 1990s in Washington State, the Summer Chum Salmon Conservation Initiative developed a recovery program for the threatened Hood Canal summer chum salmon Evolutionarily Significant Unit (ESU) (the metapopulation) that used in-river spawners (wild fish) for each respective supplementation broodstock in six tributaries. Returning spawners (wild-born and hatchery-born) composed subsequent broodstocks, and tributary-specific supplementation was limited to three generations. We assessed impacts of the programs on neutral genetic diversity in this metapopulation using 16 microsatellite loci and a thirty-year dataset spanning before and after supplementation, roughly eight generations. Following supplementation, differentiation among subpopulations decreased (but not significantly) and isolation by distance patterns remained unchanged. There was no decline in genetic diversity in wild-born fish, but hatchery-born fish sampled in the same spawning areas had significantly lower genetic diversity and unequal family representation. Despite potential for negative effects from supplementation programs, few were detected in wild-born fish. We hypothesize that chum salmon natural history makes them less vulnerable to negative impacts from hatchery supplementation. Ne) in supported populations  whose parents arose from a supplementation-style hatchery in comparison with wild-origin steelhead with no hatchery ancestry  and Chinook salmon  and occurred regardless of number of generations in the hatchery or the origin  of the hatchery broodstock. However, Sharma et al.  have a typical anadromous Pacific salmon life history, negative impacts from a supplementation hatchery program may be lower for chum salmon than for other salmonids. Chum salmon juveniles migrate to estuaries within days of emergence , chum salmon juveniles spend little time in freshwater, migrating downstream to estuaries and salt water a few weeks after emergence. Chum salmon stray at similar rates to other Pacific salmon . A recovery program started in the early 1990s, prior to the ESA listing, enacted crucial harvest reductions and supplementation programs. In 2000, state and tribal co-managers completed the Summer Chum Salmon Conservation Initiative  in 1992 and for three more subpopulations  in 1997, 1999, and 2000, respectively (each supplementation program used wild-origin broodstock collected from its respective tributary). Summer chum salmon from three streams with supplementation programs  were also the donor stocks for programs used to reintroduce summer chum salmon in one tributary each  where native populations had gone extinct. Two subpopulations, Dosewallips and Duckabush, were not deliberately supplemented. Supplementation programs were designed to reduce extinction risk and to speed recovery and recolonization while minimizing risks of deleterious genetic, ecological, and demographic effects to supplemented and unsupplemented subpopulations , and samples from unsupplemented tributaries and evaluated changes following supplementation using tests described below. The software COLONY2.0.0.1 Wang  calculatNe; Ne is a fundamental parameter determining the evolutionary potential of a population, yet different techniques employ different assumptions and yield somewhat different results. Tracking changes in Ne may indicate changes associated with supplementation  giving rise to the collection, rather than the actual Ne. To calculate actual Ne, the Nb is multiplied by the generation time, which is 3–5 years in chum salmon. Because generation time is variable in chum salmon , we simply referred to the values calculated for a single-year mixed-age collection by LD and SA as Ne and did not multiply by generation time. Because assumptions and results varied somewhat between methods, we calculated a harmonic mean (and variance) of Ne values (HmNe) from the two methods, following Waples and Do  and after supplementation for the latest collected wild-born samples . The metapopulation Ne was first calculated according to the Stepping Stone model  based on the Stepping Stone and Island models. For the Stepping Stone model the average immigration rate was estimated from the global FST values for the two sets of six collections , and the same global FST values were used in denominators for the Island model.Effective population size analyses were conducted from a metapopulation perspective , we used Wilcoxon sign rank tests to test for significant differences in these genetic attributes among collection categories; original, supplementation hatchery-born, and wild-born  was used to test for significant differences in global FST values among categories of samples  implemented in GenAlEx 6.5 ] as a genetic similarity measure  patterns Slatkin , using t Rousset . Geograp Bohonak . To asseSupplementation programs boosted abundance of spawners and contributed from 5% to 96% of spawners to escapements in supplemented HC and SJF subpopulations Table . The proOne-106) to 97% (Oki-1) per locus over all individuals (N = 2086). Because samples  were from spawner carcasses of varying freshness, genotyping success varied among samples, regardless of contemporary or historical status. In tests for Hardy–Weinberg equilibrium (HWE) at individual loci , two tests out of 832 were significant after corrections for multiple tests and all samples except 08Salmon_W were in HWE in tests over all loci .Genotypic coverage averaged 95% and ranged from 84% One-06 to 97%uals N = 086. BecaP = 0.019). The collections from 02Lilli_H, 03_04Lilli_H, and 03_04JCL_H were notable for high linkage disequilibrium: COLONY calculated a single family with 14 full-siblings in 02Lilli_H (12% of pairwise relationships were full-siblings), seven families with between three and six full-siblings in 03_04Lilli_H, and six families with between three and eight full-siblings in 03_04JCL_H (4% of pairwise relationships were full-siblings). Siblings in collections were noted but not removed.Linkage disequilibrium and pairwise sibship analyses suggested most collections included some family groups, ranging from a pair of siblings to large families (data not shown). There were 120 locus pairs examined per collection, and most collections had six or more locus pairs in linkage disequilibrium at the 5% level and 12 collections had six or more locus pairs in linkage disequilibrium at the 1% level  and pairwise sibship analysis (SA-Ne). In most calculations, the two values were similar and the 95% confidence intervals overlapped (see data for both methods for each collection in Supplementary Information Table S4 ‘All_Ne’). Differences arose for collections with high linkage and small samples sizes, which biased LD-Ne downward  varied over time and space, both within and between tributaries (Table Ne (e.g. 03Union_H and 03Union_W), except for samples from Lilliwaup Creek: in two comparisons of hatchery-and wild-born samples for that creek, the HmNe for the hatchery-born samples was equal to or greater than the HmNe for the wild-born sample. In the original Lilliwaup samples, the HmNe values were similar to those of Union River, but they declined to less than half by 1997. We lack wild-born samples from Lilliwaup Creek after 2006, but in the most recent hatchery-born samples, the HmNe values were similar to original values, suggesting that diversity may be recovering in the Lilliwaup Creek subpopulation. The HmNe in the original Jimmycomelately Creek sample was roughly half the value of the most recent wild sample, suggesting that diversity is also recovering in the Jimmycomelately Creek subpopulation. The calculated ratios of HmNe to census size , likely reflecting increases in census size (N) throughout the restoration program. Further, in an anova the ratio of HmNe to census size for the wild-born fish was negatively correlated with the number of years of supplementation .We calculated es Table . Where tFST values for the six original and six wild-born samples were 0.029 and 0.021, respectively (P = 0.2414), resulting in calculated immigration rates of 8.37 and 11.65, respectively. The meta-HmNe for the original and wild-born samples was 488 and 591, respectively, with the Stepping Stone model, and 502 and 603, respectively, with the Island model . Paired t-tests for LD-Ne and SA-Ne values indicated no significant differences between values for original and wild-born fish . Although meta-HmNe increased following supplementation, the difference was not significant.The global Ne, we computed the harmonic mean of the calculated Ne values over all collections with hatchery-born and wild-born spawners combined in collections and separated . The calculated value for the wild-born spawners (78.95 ± 33.86) was larger than the value calculated for the hatchery-and wild-born spawners combined (55.19 ± 37.44), but the confidence intervals overlapped. Also, within some single-year examinations, the ratios of HmNe/N were higher in uncombined than in combined collections, suggesting Ryman–Laikre effects from combining hatchery-and wild-born fish into single collections.In one further consideration of FST tests were mostly congruent . Pairwise tests indicated temporal stability within most subpopulations, with the exception of Lilliwaup Creek . Among Lilliwaup Creek samples, 37 of 66 pairwise genotypic comparisons were significant and 34 of 66 pairwise FST comparisons were significant. Union River samples also differed from all other HC samples and are the most isolated geographically .Assignment tests supported a metapopulation structure with straying among close subpopulations, especially along the west side of HC  of the genetic variance among samples . In comparisons among sample categories, because the tests for homogeneity among regressions in the ancova indicated significant differences between regressions, we were unable to compare regressions statistically. However, the IBD slope was steeper for comparisons among original samples  in wild-born salmon returning to spawning areas throughout the ESU. However, genetic distances within the metapopulation decreased and assignments back to collection of origin decreased following supplementation, suggesting higher gene flow within the metapopulation and lower genetic drift in subpopulations. Hatchery-born spawners collected in the same locations as wild-born spawners usually had lower genetic diversity and smaller HmNe values, suggesting that supplementation hatchery programs sampled a subset of genetic diversity in the target subpopulations and that factorial matings of hatchery broodstocks may have decreased HmNe. Higher linkage disequilibrium in hatchery-born fish indicated potential for Ryman–Laikre effect, and combining hatchery-and wild-born fish into single collections lowered ratios of effective population sizes to census sizes. However, with the exception of the Lilliwaup Creek subpopulation, suspected Ryman–Laikre effects did not increase genetic drift in wild-born spawners. Thus, possible negative impacts of supplementation appeared minimal and likely diminished as wild-born spawner abundance increased, suggesting that population recovery in this summer chum salmon ESU was unimpaired by supplementation.This study joins a growing body of research on the genetic effects of supplementation hatchery programs on wild fish populations. We examined a time series of a summer chum salmon metapopulation (a designated ESU) from before, during, and after supplementation, providing a temporal perspective of supplementation and responses by threatened subpopulations. We found that after several years of hatchery supplementation, there was little change in genetic diversity and harmonic means of effective population sizes , coho and Chinook salmon  diminished. We suspect that negative genetic impacts may be less or shorter-lived for chum salmon because their juveniles spend little time in the hatchery environment. We will continue to monitor this metapopulation to assess the long-term impacts of supplementation and success of recovery efforts.In summary, our results show that, although changes were not significant, in the HC summer chum salmon ESU contemporary genetic diversity and effective population sizes increased from original levels, suggesting that as natural production increased, possible negative impacts from supplementation (such as depressed


Replicate experiments are often difficult to find in evolutionary biology, as this field is inherently an historical science. However, viruses, bacteria and phages provide opportunities to study evolution in both natural and experimental contexts, due to their accelerated rates of evolution and short generation times. Here we investigate HIV-1 evolution by using a natural model represented by monozygotic twins infected synchronically at birth with an HIV-1 population from a shared blood transfusion source. We explore the evolutionary processes and population dynamics that shape viral diversity of HIV in these monozygotic twins.Despite the identical host genetic backdrop of monozygotic twins and the identical source and timing of the HIV-1 inoculation, the resulting HIV populations differed in genetic diversity, growth rate, recombination rate, and selection pressure between the two infected twins.Our study shows that the outcome of evolution is strikingly different between these two "replicates" of viral evolution. Given the identical starting points at infection, our results support the impact of random epigenetic selection in early infection dynamics. Our data also emphasize the need for a better understanding of the impact of host-virus interactions in viral evolution. Over the past few decades, evolutionary biology has had an increasing impact on biomedical research -3. Evolupro), Reverse Transcriptase (rt), and Envelope (env) in each monozygotic twin. Using these data, we estimated relevant population genetic parameters, including recombination rates, genetic diversity, growth rates, and selection pressure to compare between the two host individuals. While these twins have identical genetic backgrounds and were infected with the same source of HIV (a common transfusion source collected from one donor and administered simultaneously to both twins at birth), they have remarkable differences in their clinical courses. Twin A is almost normal in terms of immune system  and growth, whereas twin B is almost 5 years delayed in terms of size and had many complications including a rare neoplasm.A few complications in the study of host-virus coevolution are the different genetic backgrounds of the host and the genetic diversity of the infecting viral population. The model of monozygotic twins infected synchronically through exposure to the same viral population, therefore, provides an opportunity to examine the population dynamics of HIV while holding a number of confounding variables constant. Here we exploit HIV-1 as a model system  to examiRecent studies focusing on monozygotic twins are conflicting on whether HIV evolution can be predictable ,15 or unenv revealed clear evolutionary differences between the viral populations present in each twin , suggesting that selection is acting with more force on twin B compared to twin A. Both phylogenetic and network reconstruction analyses showed similar results for all genes analyzed  (data not shown). Growth rates were also different between the HIV populations infecting the two twins with twin A showing a rate, at least, two times higher than twin B (Table rt where the recombination rate (C) was three times higher in twin A. In both twins the substitution rate was higher than the recombination rate, as indicated by the low estimates of r (c/μ). The ratio of the per-site rate of recombination to the per-site rate of mutation (c/μ) in all genes was less than one, indicating that mutation plays a more significant role in producing novel genetic combinations than recombination and is, therefore, the major force driving the evolution of these viral populations . Moreovrt from twin B (presumably associated with drug resistance) and in env from twin A (presumably associated with immune avoidance) using PAML (Table P > 0.95) under model M2 and 13 positively selected sites under model M8 in env from twin A. All of the 11 sites detected under model M2 were also found by model M8. Models M2 and M8 also detected one site under selection in rt from twin B (S162D). This site is not documented as a drug resistance mutation site in rt. Even though they are not detected as positively selected sites in rt, we found, in both twins, M184V mutation that is associated with conferring resistance to 3TC. Also we detected T215F and K219Q mutations in rt in twin B, which are associated with conferring resistance to AZT. There are 26 positions known to be associated with protease inhibitors [pro in both twins, but none of these changes are known to confer drug resistance. A recent study by Nozawa et al. [et al. [env for both twins and in rt for twin B. The REL (Random Effect Likelihood) selection analyses, that took into account the presence of recombination inferred through GARD, clearly indicated that in env, positive selection was stronger for twin A, while no difference was detected in the other genes . Thus, the higher genetic diversity and higher number of selected sites in env appear to be associated with slower disease progression, results concordant with that found in a broad study of disease progression in infants [rt and env regions showed the viral population in the healthier twin A with higher levels of genetic diversity throughout the history of infection even when there were significant shifts in overall levels of diversity. On the other hand, pro showed the viral population in twin B with a gradual increase in diversity post drug therapy with a more rapid increase in twin A that was delayed by the same time period as the delay in the RT inhibitor (2 years). This result suggests that the shape of the response to drug therapy in terms of the HIV population diversity might be diagnostic of future disease progression, but further study with larger sample sizes are needed to better test this response as predictive of disease progression. Nevertheless, these twins clearly show very different responses to infection.Our goal was to investigate HIV-1 evolution in identical twins infected synchronically at birth with the same blood transfusion. We found compelling distinctions between the viral populations from each twin with respect to their population dynamics, phylogenetic structure, growth rates, recombination rates, genetic diversity, and selection pressures. These results were unexpected due to a combination of having identical starting points with respect to both the infecting viral population and the host genetic background. That coupled with the seemingly limited pathways of evolution for both immune evasion and evolution of drug resistance ,23 would infants . SimilarThis difference in viral population dynamics is concordant with the differences observed in the clinical courses in each twin. The immune system in twin A shows CD4 T cells at an almost normal rate. The immune system in twin B is depressed, hence no strong selective pressure is acting upon its virus population to evolve fast . All theIndeed, some studies in monozygotic twins revealed increasing epigenetic differences with age ,26. Addirt. We also found differences in population dynamics across all three gene regions. These differences suggest that epigenetic factors are important in disease progression and can impact viral genetic population dynamics.We used monozygotic twins infected at birth from the same blood transfusion contaminated with HIV-1 to study the association of population genetic and phylogenetic diversity with disease progression and clinical outcomes. We documented that these twins had very different clinical outcomes with twin A being relatively healthy compared to twin B. Associated with this slower disease progression in twin A, we found phylogenetic differences, higher growth rates, and higher genetic diversity in the HIV population and higher recombination rates in Subjects A and B are monozygotic twins. Due to intrapartum blood loss, they simultaneously received transfusion shortly after birth from a common blood transfusion source in 1983. The parents were notified that the twins were infected with HIV when they were 2 years old and the twins were declared HIV positive in the same year (1985). At that time, they were both asymptomatic. No superinfection was reported during the first five years of age of the twins. Also the twins did not receive additional transfusions since their birth. The twins came to NIH when they were 5 years old (1988). Since then, they have been closely monitored at NIH. The twins started receiving nucleoside analog reverse transcriptase inhibitors at ages 5 (Twin B) and 7 (Twin A). In 1988, twin B started AZT therapy because he was sicker than his brother. Twin A started ddI therapy two years later (1990). Twin A is normal in terms of immune system and growth. In contrast, twin B is 5 years delayed in terms of size and he presented a rare neoplasm, more likely due to HIV infection. Blood samples and clinical information were obtained 15 years after their birth at the NIH Clinical Center, Bethesda, MD (1998). The twins subsequently showed extremely different clinical courses. Samples were collected under protocols approved by the institutional review boards at the National Cancer Institute. Written informed consent was obtained from participants. This research complied with all relevant federal guidelines and institutional policies. Permission was obtained from the parents of the patients for utilization of samples and data for research, analysis and publication.g for 30 min, and the cell pellets were stored in liquid nitrogen until use. DNA was extracted from PBMCs using the PureGene genomic DNA isolation kit  and prepared at optimal concentration for nested polymerase chain reaction (PCR) method. Nested PCR amplification of the V1-V5 region of envelope coding gene, the protease gene, and a part of reverse transcriptase gene was performed with the Expand High Fidelity PCR System  in a 50 μ l reaction containing 1× Expand High Fidelity buffer 3, 200 μ M dNTPs, 2 mM MgCl2, 400 nM primers, and 1.75 U of Expand High Fidelity PCR System enzyme mix. Primer sets used to amplify a 1.1-Kbp fragment, encompassing the env V1-V5 region were: +6559 (sense) 5'-GGGATCAAAGCCTAAAGCCA-3' and -7648 (antisense) 5'- ACTTCTCCAATTGTCCCTCA-3' in a first round reaction; +6586 (sense) 5'- AATTAACCCCACTCTGTGTTA-3' and -7627 (antisense) 5'- TATCTCCTCCTCCAGGTCTGA-3' in a second round reaction. Amplification of the HIV-1 pro was done with the following primer sets: +2165 (sense) 5'- CAGAAGAGAGCTTCAGGTTTGGG-3' and -2588 (antisense) 5'- ACTTTTGGGCCATCCATTCCTGGY-3' in a first round reaction; +2208 (sense) 5'-TCAGAAGCAGGAGCCGATAGAC-3' and -2550 (antisense) 5'-TGGTACAGTCTCAATAGGACTAATGGG-3' in a second round reaction. The part of rt was amplified with: +1882 (sense) 5'-GAAGCAATGAGCCAAGTAACAAAT-3' and -3544 (antisense) 5'-GATATGTCCATTGGCCTTGCCCCT-3' in a first round reaction; +1966 (sense) 5'-TTCAATTGTGGCAAAGAAGGGCAC-3' and -3501 (antisense) 5'-TAAGTCTTTTGATGGGTCATAATA-3' in a second round reaction. Each round of PCR consisted of 25 cycles, with the initial denaturation at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 15 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min, with the final extension at 72°C for 7 min. The PCR products were purified with the QIA quick PCR purification kit , and then cloned into pCR2.1-TOPO vector using the TOPO TA Cloning Kit . The clones for each gene were sequenced using the ABI BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit . Sequences were then analyzed on an ABI PRISM 3130 × l Genetic Analyzer . GenBank accession numbers for the sequences determined in this study are GQ118464 to GQ118640.Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by Ficoll-Hypaque gradient density at 650 7 cycles, sampling every 1000th generation. In order to confirm that the chains converged and mixed well, we monitored the likelihood scores and compared means and variances of all likelihood parameters from independent runs using Tracer v1.4.1 [Sequences from each twin were aligned using MAFFT v5.3  and manur v1.4.1 . The relr v1.4.1 , as implr v1.4.1 . This apθ = 4eμN; where e Nis the effective population size and μ is the mutation rate per site), recombination rates , and growth rates (g) were estimated for each set of genes using the maximum likelihood coalescent approach implemented in LAMARC v2.0.2 [C) was obtained by multiplying r and θ [Genetic diversity  was set up to 1/4 of the sequences in each dataset. All the Bayesian MCMC output generated by BEAST was then analyzed in Tracer v1.4.1 [HIV-1 past population dynamics in each twin was inferred using the Bayesian skyline plot  and relar v1.4.1  to test ω = dN/dS) per site and per gene using the codon-based nested models M1 /M2 (selection), and M7 (beta)/M8 (beta and ω), as implemented in PAML v4 [P > 0.95) [ω estimates were also calculated in DataMonkey [The extent of natural selection was inferred by estimating the ratio of nonsynonymous to synonymous substitutions ( PAML v4 . Model l > 0.95) . In ordetaMonkey . PositivtaMonkey . RecombitaMonkey .LT participated in the conception and design of the study, carried out the phylogenetic, haplotype network reconstructions, LAMARC and PAML analyses and drafted the manuscript. HI participated in the conception and design of the study, generated nucleotide sequence data and helped to draft the manuscript. SH supervised the sample collection. JAM and SO helped to collect clinical specimens and medical records. MP-L carried out the BEAST analyses and helped to draft the manuscript. DP carried out the GARD and HYPHY analyses and helped to draft the manuscript. HCL participated in the conception, design and funding of the study, supervised the generation of nucleotide sequence data and helped to draft the manuscript. KAC participated in the conception, design, and funding of the study and helped to draft the manuscript. All authors read, discussed the results and approved the final manuscript.