Ne) of humans to be larger than the current estimates, with a foci of increased genetic diversity in east Africa, and a population size of east Africans being at least 2-6 fold larger than other populations. Both phylogenetic and network analysis indicate that east Africans possess more ancestral lineages in comparison to various continental populations placing them at the root of the human evolutionary tree. Our results also affirm east Africa as the likely spot from which migration towards Asia has taken place. The study reflects the spectacular level of sequence variation within east Africans in comparison to the global sample, and appeals for further studies that may contribute towards filling the existing gaps in the database. The implication of these data to current genomic research, as well as the need to carry out defined studies of human genetic variation that includes more African populations; particularly east Africans is paramount.Human genetic variation particularly in Africa is still poorly understood. This is despite a consensus on the large African effective population size compared to populations from other continents. Based on sequencing of the mitochondrial Cytochrome C Oxidase subunit II (MT-CO2), and genome wide microsatellite data we observe evidence suggesting the effective size ( Homo sapiens sapiens origin, exodus and dispersal out of an African birth place, was based on the mitochondrial control region variation in a global sample of 147 females The fact that Africa is the cradle of modern humans and the scene of its major biological and demographic events is not of much dispute. This is manifested in various population measures including the significantly higher effective size of Africans compared to populations from other continents. One feature of this is the marked elevated heterozygosity across the genome and low LD FST for mtDNA sequences and RST for microsatellite data were carried out for subsequent MDS plotting and a population by population correlation comparison using Mantel Test. The result showed no correlation with a P value of 0.66 similar to comparison between mitochondria and Y chromosome variations reported earlier Ne values using different loci and mutation rates to emphasize the relation of the Ne values to both parameters. The estimated Ne based on MT-CO2 in east and other parts of Africa and the rest of the world based on three different mutation rates of 9.2×10−7, 3×10−6−7Ne estimates based on microsatellites loci also using three different mutation rates Ne was lower than that obtained by MT-CO2  between the Ne and expected census size, usually taken to be ten times the effective size A line graph  plots thNe. As mitochondria are non-recombining the number of mutations and haplotypes is quite correlated. In the MT-CO2 sequence 68 haplotypes were estimated using Arlequin ver3.11 and assigned numbers from 1 to 68. Haplotype relative and absolute frequencies in the studied populations were also calculated. Strikingly, of the total 68 haplotypes, 43 occurred solely in east Africa (http://dspace.nwu.ac.za/handle/10394/4221). All published haplogroups associated with the mutations are indicated in The sheer number of haplotypes, a basic measurement of genetic diversity, is also taken as an indication of t Africa  of whicht Africa  in Sudant Africa , 31 wereThe most ancestral haplotypes as judged by Neanderthal and Chimpanzee was a group of sequences that diverged early in human history forming a cluster denoted as (A) that includes 1 individual sequence from the Rift Valley Kenya (Mkamba), in addition to Eritreans, Ugandans and Sudanese; another branch differentiate encompassing solely east African, predominantly Eritreans, Ugandans and Sudanese and denoted as cluster (B). The third cluster (C), includes members of almost all world populations particularly non-Africans who share a major haplotype that seems to have originated within an east African gene pool . The staThis was also manifested in a neighbor-joining (NJ) tree of all MT-CO2 sequences . In agreGiven the uncertainty in the value of divergence between Humans and Chimpanzees reported in the literature, and the wide range of dates for the divergence of Humans from Neanderthal and Humans MRCA and the differentiation of major mtDNA and Y chromosome haplogroups FST based distance matrix (Ne is prominent. In the FST based MDS the 2nd coordinate differentiates between Sudanese and the rest of Africa (except San), and between Asians and Europeans. Interestingly Beja population from Sudan maintains an intermediate position between Africans and non-Africans in both plots. The 3rd and 4th coordinates of the IBS plot appears to display subsequent demographic events where Africans particularly East Africans contributed independently to Europeans and Asian ancestry  plots. The first plot employs an e matrix  while the matrix . Both MDancestry  and S3.An MDS of the MT-CO2 data set  which inThe tenet of the human population genetic structure is best viewed in the light of the inherent diversity within the human genome. Humans are believed to have expanded from small founder demes somewhere in eastern Africa during the past 75,000-100,000YBP Historically, the MT-CO2 gene has been employed in species and phylogenetic inference for the various merits mentioned earlier, and inference made from such sequence information are found to be highly correlated with large data sets and whole genome analysis et al., unpublished) usually taken as an indication of populations that have passed through “recent” demographic expansion in situ evolution where alleles are exchanged between neighboring demes According to the current data East Africa is home to nearly 2/3 of the world genetic diversity independent of sampling effect. Similar figure have been suggested for sub-Saharan Africa populations et al. We compare and contrast the MT-CO2 sequence data to analysis of 848 microsatellites of the Marshfield dataset partly published by Tishkoff et al. Ne being estimated over different points in time et al. Ne of east Africans was consistently maintained, microsatellites produced values in the upper extremities of Ne. The size of difference for microsatellites was much narrower possibly due to the fact that higher mutation rates may lead to convergence, in addition to admixture, both elements that are known to affect the resolution of these markers in coalescence estimates. This should also be considered against arguments that estimates based on mitochondrial genome, tend to be biased towards low inbreeding effective size because of unique features pertaining to the mitochondrial genome Ne of 7,000 for long term effective size, in tally with our figures. Interestingly the two expansions suggested by the BSP coincide with major putative demographic events: one the critical biological changes that might have taken place around 50, 000YBP subsequently leading to the out of Africa expansion; and the other is advent of pastoralism and agriculture believed to have occurred around 10, 000–20, 000YBP, as the latter date was also attested by Y-chromosome Our estimate of the population size is more in agreement with LD and coalescent dates as in Tenesa Ne has been about three times larger in African populations than in non-Africans Previous anthropological and genetic evidence have suggested that long-term et al. A relatively low effective size and the drift like nature of the event characterizing the group that made the exodus out of Africa, is manifested in the fact that it originates in a spectacularly large female source in east Africa. This is in concordance with previous data including X chromosome. In a recent report by Keinan The complex relationship between the census and effective size is influenced by both genetic drift and inbreeding. The drift nature of the out of Africa event and the subsequent demographic expansion is portrayed in the relationship between the census and effective size in east Africa and the rest of the world particularly Asia and Europe. Both drift and inbreeding are unlikely to have influenced east Africans in a substantial way due to the limited geographic range where those groups evolved and in-situ evolution of its population.et al., Ne originally carried by the ancestral group that left Africa and seen in the number of haplotypes that survived in their gene pool. This may suggest that both census and effective size of the group that made it to Australia was large enough to counteract the effect of drift and permit survival of relics of these original haplotypes.Fluctuation in population size might be a mark of the out of Africa group because migration and challenges of adapting to new environments subject the population to both influences of drift and inbreeding. Cases of low census size and a larger inbreeding effective size are known in mammalian populations and attributed to recent population reductions It is not only genetic data that lends support to an east African origin of humans but the unparalleled ethnic and linguistic diversity that remains one of the highest worldwide. Interestingly the two most ancestral sequences in the NJ tree figure refer to Nubian individuals. Nubia is currently identified with one of the most ancient human settlements, the Say culture. Recently, a related compound associated with a lithic middle Stone Age industry was discovered in Dhofar Oman and taken as an evidence of human migration out of Africa through an Arabian route Overall, the various genetic markers used in the current analysis support the observation of human effective population size larger than previously estimated, and emphasize the importance of sampling populations of putative deep ancestry.http://www.healthresearchweb.org/br/sudan/ethics; Reference: 28/10/03etiend) and Eritrea  for the sample sets analyzed in the Khartoum laboratory. Other data has been obtained from online sources. All past and present research adopts good practices in ethics and local and international guidelines for genetic research. This includes obtaining of written informed consents from all participants, keeping the data anonymity from the beginning and communicating research results to populations concerned.Ethical approval has been obtained from regulatory bodies in Sudan (Ethics Review committee (ERC) Institute of Endemic Diseases, University of Khartoum: http://www.sciencemag.org/content/324/5930/1035/suppl/DC1 and MT-CO2 DNA sequences comprising 23 Ugandans obtained from http://dspace.nwu.ac.za/handle/10394/4221), 75 other Africans, 20 Asians, 18 Australians, 45 Europeans and 20 Americans making a total of 180 sequences obtained from public mtDB database (http://www.mtdb.igp.uu.se/) in addition to 81 Eritreans and 46 Sudanese sampled and genotyped following appropriate ethical consent and documentations. PCR amplification of MT-CO2 gene for the Eritreans and Sudanese samples was carried out at the Institute of Endemic Diseases, University of Khartoum and outsourced for commercial sequencing at BGI Hong Kong, China. MT-CO2 sequences were identified by blasting against Yoruba reference sequence (accession number AF347015) and sequences with mutations were deposited at GenBank with accession number KC753688-KC753760.Data sets used in this study include genotype information on 848 nuclear microsatellites obtained from individuals of various world populations including Sudanese, Ethiopians and Kenyans available at A total of 330 MT-CO2 sequences were aligned using the software program BioEdit FSTs, in addition Tajima's D test to check for selective neutrality were calculated for the studied populations. Fisher's exact test was used to test the probability of association between the expected census size being ten times the effective size and observed census size for current population of aboriginal Australians. The correlation between the two markers was evaluated by Mantel Test within Arlequin program.Arlequin suite ver3.11 FST intermediate values  as outlined by Bandelt et al. Phylogenetic tree was constructed for the MT-CO2 sequences using Neighbor-Joining (NJ) method. The NJ tree was calculated in MEGA5 Ne) was calculated using an MLE (maximum likelihood estimate) of theta value generated by LAMARC version 2.1.8 Ne and the census size was used to plot the chart. Current and archaic Ne values  further, we used the BEAST v1.8.0 −7 substitutions per site per generation as used in LAMARC. We used a Bayesian Skyline coalescent tree prior with 10 groups under a piecewise-constant model. The analysis was run for 10 million generations with parameters logged every 1000 generations, and Tracer 1.6 (http://tree.bio.ed.ac.uk/software/tracer/) was used to inspect chain convergence and conduct the skyline reconstruction . NJ tree of the world populations based on MT-CO2 sequences. The evolutionary relationship of 171 sequences and evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.20401570 is shown. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated from the dataset. There were a total of 543 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4. Red dots: east Africa, Blue: Africa, Green: Asia, Yellow: Australia, Pink: Europe and gray: America.(TIF)Click here for additional data file.Figure S2Multidimensional Scaling Plot (MDS). The 2nd and 3rd coordinates of an MDS plot of 848 nuclear microsatellite loci from 469 individuals of 24 world populations. MDS uses pairwise IBS data based on the 848 loci generated by PLINK software and plotted using R version 2.15.0. The figure, besides a separate clustering of east Africans, indicates the substantial contribution of Africans and east Africans to the founding of populations of Europe and Asia.(TIF)Click here for additional data file.Figure S3Multidimensional Scaling Plot (MDS). The 3rd and 4th coordinates of an MDS plot of 848 Microsatellite loci, across the human genome in 469 individuals from 24 populations from Africa, Asia and Europe. MDS uses pairwise IBS data based on the 848 loci generated by PLINK software and plotted using R version 2.15.0. The central position of east Africans and some other Africans emphasizes the founding role of east African gene pool and the disparate alignment on coordinates along which the world populations were founded including populations of Aftica aligning along the 4th dimension.(TIF)Click here for additional data file.Figure S4Multidimensional Scaling Plot (MDS). First and second coordinates of an MDS plot based on MT-CO2 data set constructed from pairwise differences FST generated by Arlequin v3.11. Population code as follows: Nara: Nar, Kunama (Kun), Hidarb (Hid), Afar (Afa), Saho (Sah), Bilen (Bil), Tigre (Tgr), Tigrigna (Tig), Rashaida (Rsh), Nilotics (Nil), Beja (Bej), Ethiopians(Eth), Egyptians (Egy), Moroccans (Mor), Southern Africans (Sth), Pygmy (Pyg), Saudi Arabia (Sdi), Asia (Asi), Europe (Eur), Native Americans (NA), Australians (Ast), Nubians (Nub), Nuba (Nba)(TIF)Click here for additional data file.Figure S5Bayesian Skyline Plots (BSP). BSP for individual population to clarify the demographic events each populations. A. Global populations, B. Eritrean populations and C. Sudanese populations.(TIF)Click here for additional data file.Table S1Synonymous, non-synonymous and novel mutations identified against Yoruba reference sequence from Sudanese and Eritrean MT-CO2 sequences. The Ugandan mutations are previously reported at http://dspace.nwu.ac.za/handle/10394/4221).(XLSX)Click here for additional data file.Table S2Frequencies of 68 MT-CO2 haplotypes resolved within continental populations, the haplotype Number 1 include 164 individuals from all world populations. The rest, shared or unique haplotypes shows the name of the group possessing the haplotype.(PPTX)Click here for additional data file.Table S3FST matrix containing intermediate values generated from Arlequin version 3.11 using Marshfield microsatellite data set.(XLSX)Click here for additional data file.


The management of livestock breeds and threatened natural population share common challenges, including small effective population sizes, high risk of inbreeding, and the potential benefits and costs associated with mixing disparate gene pools. Here, we consider what has been learnt about these issues, the ways in which the knowledge gained from one area might be applied to the other, and the potential of genomics to provide new insights. Although there are key differences stemming from the importance of artificial versus natural selection and the decreased level of environmental heterogeneity experienced by many livestock populations, we suspect that information from genetic rescue in natural populations could be usefully applied to livestock. This includes an increased emphasis on maintaining substantial population sizes at the expense of genetic uniqueness in ensuring future adaptability, and on emphasizing the way that environmental changes can influence the relative fitness of deleterious alleles and genotypes in small populations. We also suspect that information gained from cross-breeding and the maintenance of unique breeds will be increasingly important for the preservation of genetic variation in small natural populations. In particular, selected genes identified in domestic populations provide genetic markers for exploring adaptive evolution in threatened natural populations. Genomic technologies in the two disciplines will be important in the future in realizing genetic gains in livestock and maximizing adaptive capacity in wildlife, and particularly in understanding how parts of the genome may respond differently when exposed to population processes and selection. The effective population size, Ne, is a measure of fundamental importance for understanding the potential of species and populations to evolve and adapt to natural and artificial selection pressures. Quantitative genetic theory predict that Ne is positively associated with the level of additive genetic variation and that the capacity of a population to respond to selection depends on the level of genetic variation for the trait(s) undergoing selection see , 1996. HIn natural populations, there are numerous examples showing that a small Ne can reduce adaptive potential as a consequence of reduced genetic variation e.g.,  and alsoPopulations of domestic animals with a small Ne can also exhibit reduced genetic variation compared to ancestral ones . For exaHowever, a small Ne in domesticated animal populations helps produce phenotypic uniformity within populations (and even more so in domesticated crops) that results in products that can be more easily processed and marketed . IntenseAlthough the implications of small Ne in natural populations and domestic breeds are somewhat different, there are insights to be gained from combining knowledge of these disparate areas. Genetic studies on small and fragmented populations in nature, such as island populations or populations at the brink of extinction, provide ideas and concepts that could be applied to the management of small domestic populations. This includes applying genetic rescue to boost the adaptive potential of small populations, and using genotype by environment interactions to ensure populations maintain a high fitness across environments . On the While Ne is important from a genetic perspective, the census size of domestic and natural populations is obviously also important for predicting extinction risk. Populations at a small census size are more likely to go extinct due to factors such as demographic and environmental stochasticity even if genetic considerations are not taken into account. However, in this paper we focus only on the consequences of low Ne, providing examples of genetic rescue in natural populations, and considering prospects of using genomics as a tool that could advance long-term conservation management in both natural and domestic populations.Figure 1). Likewise a large and increasing number of wild populations are threatened and also have low Ne . When estimates of inbreeding depression from Mating of related individuals is unavoidable in populations of finite sizes but occurs also when there is preferential mating of relatives in large populations. Inbreeding leads to a reduced number of heterozygotes and ultimately to complete homozygosity in the genome . Inbred Drosophila and bird populations with a long history of inbreeding  highly adapted to local conditions but with limited genetic variation. Recommendation: Despite currently being successful in its environment some gene flow from populations is recommended to increase Ne and genetic variation. Conservation priority: High.Scenario 3: A population that is unique but maladapted and has high levels of genetic variation. Recommendation: Characterize why this population is maladapted and start to select (if the population is managed) for increased local adaptation. Gene flow from populations adapted to similar environments is recommended if the population does not respond to selection. Conservation priority: Intermediate.Scenario 4: A population that is locally adapted, but not genetically unique and with low levels of genetic variation. Recommendation: Management should prioritize increasing Ne and genetic variation. Gene flow from populations adapted to similar environments is recommended. Conservation priority: Low assuming that there are other conspecific populations available.Scenario 5: A population that is not unique, maladapted, and with low levels of genetic variation. Recommendation: Gene flow from populations adapted to similar environments is needed. Conservation priority: Low.Box 1).Rules of thumb that have influenced management of domestic and wild populations since the early 1980s state that (1) an Ne of at least 50 is needed for avoiding inbreeding depression in the short term (five generations), and (2) an Ne of at least 500 are sufficient to retain long-term evolutionary potential . It has For domesticated animals, Oncorhynchus tshawytscha) estimated Ne from RAD-seq data and found surprisingly high Ne estimates given the census size of these populations . Intense and structured directional selection is typically not performed in these breeds, and many of them have been through more extreme bottlenecks compared to the commercial breeds, with documented low levels of genetic variation .There will sometimes be good cultural, historical and also genetic reasons for managing indigenous  breeds as separate and closed breeds. However, this will rarely be the most efficient management practice if the goal is to conserve breeds, adaptive genetic variation and the scope for local adaptation in the long run. In the literature it is sometimes taken for granted that a population in the wild or an indigenous livestock breed is locally adapted to the habitat or geographical region in which it is present. Although some well-documented cases exists, especially from the tropics , 2013, iBox 1).Maladaptation can have many genetic causes, including mutation, inbreeding, random genetic drift, gene flow leading to breakdown of co-adapted gene complexes, heterozygote advantage and pleiotropy . Space dGene flow and genetic drift might prevent or disrupt local adaptation and lead to maladaptation or outbreeding depression. Due to genetic drift, genes of adaptive value may behave neutrally, potentially leading to maladaptation in small populations. Gene flow may prevent local adaptation , but thiThe merits and challenges of genetic rescue – augmenting genetic variation and limiting inbreeding depression – have been evaluated for over a decade in wildlife biology e.g., . The lesCanis lupus) into the isolated and highly inbred populations on Isle Royale, MI, USA  from Sinai and collared lizards (Crotaphytus collaris) from the US Ozark mountains  and reduce general life expectancy  implied relocating individuals from the closest wild population, a subspecies from Texas (Puma concolor stanleyana), which was a controversial decision . The remaining individuals have extremely low genetic diversity and are highly inbred (1. Although the genetic background for the condition(s) remains unknown, the Lundehund is affected by serious gastrointestinal problems that seem particularly prevalent for this breed  locus in California condor which is being aided by comparisons with the DNA sequence of the domestic chicken . Such ex2, and will be evaluated with respect to factors such as morphology and behavior. Careful selection of future animals used for breeding should be possible through genomic analyses of hybrid profiles, by following backcrosses between F1-hybrids and Lundehund. An important objective will be to retain physical characteristics of the Lundehund breed whereas there is urgent need to reduce the prevalence and severity of the breed’s gastrointestinal problems. The methods proposed by Another example is the work currently underway to cross the dog breeds Lundehund and Buhund to augment the genetic diversity and conservation  of the Lundehund. Hybrid pups were born in 2014Box 2).With domestic species it is relatively easy to perform controlled experimental breeding and evaluate offspring characteristics, helping to assess the consequences of mixing two breeds, and optimizing strategies for cross-breeding. Thus genomic data can help expand conservation management of farm animals by permitting a careful, adaptive process with a long-term perspective whereby breeds are explicitly considered as an evolutionary work-in-progress .  cattle  .Perspectives: The use of frozen semen or embryos in breeding plans for small populations can decrease inbreeding and increase genetic variation  genome-wide profiles comprising neutral and adaptive genetic variation enabling selection for specific adaptive alleles and genetic variation, (2) estimates of Ne that are accurate and comparable, and (3) description of the demographic history of populations.• Genomes of most species can now be sequenced for relatively reasonable costs . PerspecPerspectives: Generalizations are problematic but simple guidelines suggest that Ne >50 is sufficient to avoid significant inbreeding depression in livestock . More generally, The above considerations indicate that genomic data provide highly useful information about the applicability and likely success of strategies aimed at increasing and maintaining the adaptability of threatened breeds and natural populations. By providing information on the genetic architecture of complex traits, these approaches provide tools that are changing the way we approach conservation of genetic variation in livestock and natural populations. Whereas genomics is not a magic bullet that will instantly permit full overview of the genome and the interactions among its parts, the use of an increased number of genetic markers through next generation sequencing approaches will augment the accuracy of estimating diversity and population demographic parameters of conservation relevance . FurtherBox 1). Current and past performance of a population may represent poor predictors of future performance due to factors such as genotype by environment interactions, inbreeding, genetic drift, changed breeding objectives, and environmental changes. In the long run genetic variation will be depleted in populations with small Ne. These populations will become constrained in their evolutionary responses and are likely to go extinct in environments that change rapidly and may become stressful, although the experience with livestock suggests that as long as environments are relatively constant phenotypic changes remain possible under strong selection. Nevertheless, threatened populations should not be regarded as museum specimens, and an increased focus on Ne and genetic variation in populations of conservation concern should help promote the potential for adaptive evolution. For small and threatened breeds one way to achieve this is through limited cross-breeding and active use of genomic data to guide breeding decisions.For conservation of livestock breeds and small populations in nature, we advocate that long-term genetic and phenotypic monitoring is needed, and based on such data management decisions are taken (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The degree of genetic differentiation among populations experiencing high levels of gene flow is expected to be low for neutral genomic sites, but substantial divergence can occur in sites subject to directional selection. Studies of highly mobile marine fish populations provide an opportunity to investigate this kind of heterogeneous genomic differentiation, but most studies to this effect have focused on a relatively low number of genetic markers and/or few populations. Hence, the patterns and extent of genomic divergence in high-gene-flow marine fish populations remain poorly understood.Gasterosteus aculeatus) distributed across a steep salinity and temperature gradient in the Baltic Sea, by utilizing >30,000 single nucleotide polymorphisms obtained with a pooled RAD-seq approach. We found that genetic diversity and differentiation varied widely across the genome, and identified numerous fairly narrow genomic regions exhibiting signatures of both divergent and balancing selection. Evidence was uncovered for substantial genetic differentiation associated with both salinity and temperature gradients, and many candidate genes associated with local adaptation in the Baltic Sea were identified.We here investigated genome-wide patterns of genetic variability and differentiation in ten marine populations of three-spined stickleback  contains supplementary material, which is available to authorized users. While local adaptation is likely to be of commonplace occurrence, demonstrating its occurrence can be difficult and take substantial research efforts -3. TradiOutlier detection methods have become particularly popular in identifying population structuring and adaptive differentiation in marine fishes, which generally show very low levels of genetic differentiation in neutral marker genes -22 and iGasterosteus aculeatus; e.g.,  and log-transformed geographic distances separating sampling locations.To compare the patterns of genetic variability and differentiation in SNP markers with those in microsatellite markers, we retrieved data from 40 microsatellite loci genotyped for these same populations . A simplTo validate estimates of allelic frequency from the pool-seq data, we genotyped a subset of 30 SNPs from each of the individual fish used for the pooled DNA analyses using the iPlex Gold® assay on the MassARRAY® platform (Sequenom) system. This genotyping was performed by the Technology Centre of the Institute for Molecular Medicine Finland at the University of Helsinki. Allele frequencies from this data were estimated with a custom Perl script and compared to estimates from pooled data as obtained using the procedures above.Sequences underlying this study have been deposited in NCBI’s Sequence Read Archive and accession numbers are SRR1596320, SRR1596321, SRR1596322, SRR1596323, SRR1596324, SRR1596325, SRR1596326, SRR1596327, SRR1596328, and SRR1596329.


Large changes in allele frequency among breeding programs suggest a major contribution of genetic drift and linked selection on genetic variation. Despite this, comparisons of 3613 modern North American cultivated barley breeding lines that differ for spike-type and growth habit permit the discovery of 142 single nucleotide polymorphism (SNP) outliers putatively linked to targets of selection. For example, SNPs within the Cbf4, Ppd-H1, and Vrn-H1 loci, which have previously been associated with agronomically adaptive phenotypes, are identified as outliers. Analysis of extended haplotype sharing identifies genomic regions shared within and among breeding populations, suggestive of a number of genomic regions subject to recent selection. Finally, we are able to identify recent bouts of gene flow between breeding populations that could point to the sharing of agronomically adaptive variation. These results are supported by pedigrees and breeders’ understanding of germplasm sharing.Barley was introduced to North America ∼400 yr ago but adaptation to modern production environments is more recent. Comparisons of allele frequencies among growth habits and spike (inflorescence) types in North America indicate that significant genetic differentiation has accumulated in a relatively short evolutionary time span. Allele frequency differentiation is greatest among barley with two-row  The evolution of breeding populations encompasses processes that reasonably mimic the evolution of natural populations, but in accelerated time  was introduced to North America by European colonists as early as 1602 as a crop essential for beer production  and the establishment of breeding programs for two- and six-rowed spike-type contribute to highly structured barley populations . Barley samples are representatives of 4 yr of germplasm enhancement (2006–2009) from 10 breeding programs. These breeding programs include most barley growing regions and market end-uses in the United States. For more information about the programs see Genotypic data from a total of 3971 barley accessions including cultivars, advanced lines, and genetic stocks from barley breeding programs that participated in the Barley Coordinated Agricultural Project (CAP) were downloaded from The Triticeae Toolbox (T3) (File S1 for more details). The current sample is divided hierarchically at the highest level into spring and winter growth habits and then within each growth habit by breeding population  using only the OPA 2011 markers. The options to download the dataset were set to minimum MAF ≥0% and to remove markers and accessions with >99.9% missing data.All 3971 accessions were genotyped with 2882 Barley Oligo Pooled Assay Single Nucleotide Polymorphisms, referred to as BOPA SNPs  and accessions where single lines represented a population. We also removed accessions with heterozygosity that exceeded the expected 6.25%, a conservative threshold intended to filter out accessions that might have genotyping errors. Finally, we removed genetic stocks or near-isogenic lines such that one accession from each near-isogenic line set was retained in the dataset.The dataset was filtered for monomorphic SNPs, and SNPs or accessions with more than 25% missing data. Additionally, we removed accessions with incomplete sample information (FIS (1 – Ho/He) using a custom Python script. The similarity between samples was estimated by the percent pairwise diversity calculated using the compute program from the libsequence library  were calculated and plotted for each of the 16 populations and for the complete dataset. To infer ancestral state, SNP states from Hordeum bulbosum as reported for BOPA SNPs were used (H. bulbosum (Cb2920/4) to the Morex draft assembly  were treated as missing data.Derived site frequency spectra . The analysis was performed using the SmartPCA program from the EIGENSOFT package (https://github.com/AnaPoets/BarleyLandraces). The 2021 SNPs shared between datasets were used to build a population tree using landraces as the outgroup. We ran 25 replicates of the tree, bootstrapping with 75 SNP windows. We used the replicate with the lowest standard error for the residuals as the base tree topology and inferred the likelihood of having between one and five migration events among breeding populations. The plot of the residuals was used to evaluate which tree best fit the data. Candidates for admixture can be identified by those population pairs with residuals above zero standard error, which represent populations that are more closely related to each other in the data than in the best-fit tree  spring vs. winter, (2) two- vs. six-rowed, and (3) among breeding populations. Owing to the high level of inbreeding in the dataset, a haploid model for FST estimation was used as this better represented the sample size in our study. Heterozygous SNPs were treated as missing data. An empirical genome-wide threshold for the top 2.5% of FST values was used to identify SNPs with large differences in frequency relative to the genome-wide average. To identify the degree of differentiation that each breeding population has with respect to other populations we report FST for all pairwise comparisons. To determine the degree to which our FST results are robust to either the specific set of SNPs or individuals used here, we used bootstrapping to remove either 20% of SNPs or samples per iteration. We ran 100 iterations in each case.To identify putative targets of long-term selection involved in the spike-type and growth habit differentiation among breeding programs we used the ierfstat . FST wasFST-like measure of c.” This estimate differs from other FST-like measures in that it attempts to estimate the degree of divergence of a population from ancestral allele frequencies. For this analysis, the dataset was divided into two groups: spring six-rowed and spring two-rowed. We do not report c values for winter barleys owing to limited sampling. For this analysis, monomorphic SNPs were removed from each group. The c value was calculated using the popdiv program from the popgen package in R (m = 10 (see File S1 for more details).To characterize average allele frequency divergence for breeding populations we used the age in R . We usedVrn-H3, Sgh3) . The phased data were only used for IBS and pairwise haplotype sharing analyses.We used an IBS analysis to identify shared genomic segments among individuals between breeding populations potentially indicative of recent introgression. The analysis used PLINK v.1.90 . MissingTo explore recent events of selection within populations, we used the pairwise haplotype sharing (PHS) approach (Results for details) was assessed using the four-population test (4f-test)  indicate that the tree evaluated does not fit the data, suggesting the presence of gene flow.The robustness of patterns of migration inferred using TreeMix (see Results for more details). Following the  notation for a tree topology with four populations , we assessed migration among the following sets of populations: ,  and , where X was replaced iteratively for any two-rowed barley and Y1 and Y2 were replaced by a pair of six-rowed barley populations. The populations UT6, OR2, and N2 were chosen because of an indication of gene flow based on TreeMix results. Pairs of populations from the same branch of the TreeMix topology were considered on the inference that these populations are more similar to each other due to shared ancestral polymorphisms. The second pair of populations was selected from the branch containing the population putatively involved in the migration event  and plots are available at https://github.com/MorrellLAB/NorthAmerica_Fst.vs. winter) or spike-type (two- vs. six-row spikes) information, 79 near-isogenic lines, and three accessions with single accessions representing a population. After quality control our dataset consisted of 3613 barley accessions (Table S1) and 2542 SNPs (Table S2).The original dataset included 3971 accessions genotyped with 2882 SNPs representative of 10 breeding programs across the United States. We removed 340 SNPs and 241 accessions with ≥25% missing data, 22 accessions with heterozygosity >6.25% across SNPs, 13 accessions with missing growth habit  revealed that the primary population structure (PC1 = 19.6% variance) is explained by differences in spike-type, which corresponds to an average FST of 0.23 . PC2 indicates that 9.1% of the variance among lines is explained by differentiation in growth habit, with an average FST of 0.17. These results are congruent with earlier analyses on a subset of these populations  were represented by an average sample size of 225 lines with a minimum of 30 lines (from UT2), and a maximum sample size of 386 for MN6 (Table S3). The average inbreeding coefficient (FIS) across populations was 0.98 as expected after four generations of self-fertilization. Percent pairwise diversity ranged from 0.15 to 0.36 and averaged 0.25 across breeding populations , reflecting minimal impact of ascertainment bias in each of these partitions. In individual breeding populations there is an excess of rare and high-frequency variants relative to neutral expectations based on Kimura’s model of a population at equilibrium . OR2, OR6, UT6, and UT2 have a higher proportion of midfrequency variants than other populations. When the breeding populations are considered jointly, the derived SFS  displays an elevated number of midfrequency variants, consistent with retention of variants segregating at an average minor allele frequency of 24% in the discovery panel . For each of the three partitions, we identified 61 SNPs in the upper ≥0.975 of FST values . Of these, 34 SNPs were outliers for both the spike-type and breeding population comparisons, seven were common between breeding populations and growth habit comparisons, and there were no common outlier SNPs between growth habit and spike-type comparisons.The distribution of FST outlier detection was dependent on the particular individual’s sampled or set of markers used. Across samples we recovered an average of 95% of SNPs outliers in any given comparison. Outliers include SNPs near or in genes mentioned below. Across SNPs we recovered an average of 87% of the genetic regions where SNPs were identified as outliers in the full dataset. This average excludes linkage group 1H where the large number of outliers precludes identification of outliers plausibly attributable to selectionBootstrapping, by removing 20% of individuals in each partition of our sample or 20% of SNPs, was used to determine whether FST comparison for growth habit located in the vernalization sensitivity locus   (Cbf4)  in the outliers . Both ofper se (EPS) loci; one characterized example is the early maturity 6 locus  (Eam6) (FST (average FST= 0.87)  responsible for flowering time variation   study  outlier . Additioke-types .Vrn-H3)  that promotes flowering time under long days . In a study by Figure S7B), showing no association between these SNPs and spring vs. winter growth habit (maximum FST = 0.04).Other SNPs occurring in well-characterized genes did not appear as outliers despite the previously reported contribution to function. There were three SNPs in our dataset  occurring within the vernalization sensitive locus 3  in Vrs1. However, none of the SNPs reached the empirical cutoff for FST in this comparison, with a maximum observed FST of 0.45. The “G” nucleotide state at SNP 12_30900 results in an amino acid substitution associated with the six-rowed phenotype ; however, the alternative nucleotide state “C” can be found in either six- or two-rowed accessions  the “G” state always resulted in a six-rowed phenotype. In our sample of North American breeding programs 2% of individuals that carry this variant state were reported as two-rowed barleys, which is similar to previous results that found this SNP significant for spike-type differentiation segregating in two-rowed accessions with an allele frequency of 1% . In a small number of cases, focal SNPs in the PHS analysis were identified as outliers in more than one breeding population. Sharing of PHS outliers is greatest for OR6 and UT6 (22), BA6 and WA6 (17), and AB2 and OR2 (14) (Table S8). The number of focal SNPs that were PHS outliers shared among populations were an average of three SNPs shared within two-rowed populations and four SNPs within six-rowed populations. An average of two focal SNPs were shared between two- and six-rowed populations. Out of the 775 SNPs, 77 were in genes with known function. The haplotypes for these significant PHS values varied in length from 19.7 cM to 139.6 cM with mean 55.9 cM across breeding populations . The frequency of the SNP state with significant PHS ranged from 10%  to 61% with mean 24% .The PHS analysis permits the identification of genomic regions that are putatively involved in more recent selection. PHS analysis within individual breeding populations identified a total of 775 SNPs in the upper ≥0.975 of the PHS distribution  in the Vrs1 gene. The SNPs in Vrn-H3 and Vrs1 were at an average frequency of 22.25% in each of these populations, with an average length of shared haplotype of 66.98 cM (Table S7 and Table S9). There are three SNPs  with significant PHS value in OR2 (frequency 11% and haplotype length 138.9 cM). SNPMeta annotations identified SNP 12_20593 within the nicotianamine synthase 2 (nashor2) gene in barley, which is a key enzyme for iron homeostasis (TaGolS 2), which in turn is orthologous to the characterized gene TaGolS 2 in A. thaliana  at an average frequency of 24%, significantly exceeding genome-wide similarities. These regions were putatively subject to recent selection. Among outliers for PHS, BA2 showed significant PHS surrounding three SNPs  with all three SNPs occurring within c . Among the two-rowed populations, Utah two-rowed has diverged the most while Idaho two-rowed resembles ancestral allele frequencies, suggesting reduced effects of drift or linked selection in the latter population. Among the six-rowed populations, Utah six-rowed was the closest to ancestral allele frequencies while Minnesota six-rowed has experienced the most divergence. Utah two-rowed diverged more from ancestral allele frequencies than its six-rowed counterpart (contrasting the pattern observed in other spike-type pairs of populations). This is expected because the two-rowed individuals came from crosses between two-rowed and six-rowed varieties (data not shown).Quantification of allele frequency divergence based on Table S11, Table S12, Table S13, Figure S10, and Figure S11). There was a high frequency of shared haplotypes among two-row populations at 50 SNP windows, but this was reduced when 100 SNP windows were considered. At 100 SNP windows BAI2 and N2 presented the lowest degree of shared haplotypes with other two-rowed populations, while BA2 shared the most haplotypes . For both 50 and 100 SNP windows, high-frequency shared haplotypes were common for all six-rowed populations – see for example Figure S10, Figure S11, Table S14, and Table S15). This contrasted with VT6, which showed a very low degree of haplotype sharing, particularly with the various spring breeding populations . These results are consistent with differentiation in allele frequency observed in FST comparisons between breeding populations (Table S16).To identify genomic segments subject to recent introgression, we tested for regions with a high degree of IBS between breeding populations. Window sizes of 50 (0.63–44.96 cM) and 100 (13.0–62.25 cM) SNPs were used, and we permitted up to 10% mismatch among haplotypes. As expected, populations within growth habit and spike-type had a higher degree of haplotype sharing than between these partitions. The degree of haplotype sharing was lower within spike-type than within growth habit (ulations . HaplotyTable S13 and Table S14). An exception to this was the comparison between OR2 and OR6, where shared haplotypes were quite common. Sharing occurred at every 100 SNP window, with shared haplotypes at frequencies as high as 90% in the two populations  with an average frequency within populations of 0.46 and 0.58  (Table S15). IBS with 50 SNP windows for N2 and N6 spanned 94% of all windows, with shared haplotypes occurring at average frequencies of 22% and 50%, respectively . Increasing the window size to 100 SNPs identified many fewer shared haplotypes between N2 and N6, leaving 70% of the genome shared at an average frequency of 8% in N2 and 35% in N6 .For 50 SNP windows, there were a high number of IBS segments shared between spike-types. However, when windows of 100 SNPs were considered, the majority of the IBS haplotypes were at low frequency (<20%) in one of the two populations compared . With three migrations, we infer exchange between spring six-rowed populations and N2; VT6/OR6, and OR2, and spring six-rowed populations and UT6 , with z-scores significantly different from zero (significance at P < 0.05). The 4f-test for introgression between N2 and any spring six-rowed population resulted in values significantly different from zero, consistent with gene flow. The 4f-test did not support introgression between OR2 and VT6 and/or OR6 since none of the possible tree topologies involving these populations were significant.Based on the FST analyses of North American breeding populations; (iii) we identify putative signals of recent and long-term selection similar in magnitude to those at previously isolated genes of known function; (iv) the average per SNP allele frequency divergence at the population level appears consistent with the dominant action of genetic drift and linked selection; and (v) we identify populations that are recently subject to exchange of genetic material. There are low levels of genetic exchange across spike-type boundaries.Comparative analyses of allele frequency differentiation and extended haplotype sharing in a sample of 3613 barley accessions representing 16 barley breeding populations result in five primary conclusions: (i) barley breeding programs in North America have strongly differentiated allele frequencies among breeding populations; across programs, inflorescence type followed by growth habit account for the greatest proportion of variance in allele frequency; (ii) a number of loci previously identified as major contributors to growth habit adaptation in barley (primarily in Europe) are readily identifiable as outliers in allele frequency in our FST outlier analysis is generally the result of long-term directional selection . Many additional FST outliers are newly identified as putative targets of selection.Allele frequency differentiation sufficient to be detected in an election . The SNPVrs1 where the six-rowed phenotype is conferred by multiple alleles .PHS analysis in the North American breeding programs detected a series of loci that are putatively targets of recent selection within barley breeding populations, comparable to findings in wheat  (Vrs1. One important contributing factor may be that the six-rowed phenotype can result from multiple disruptions of the Vrs1 gene (Despite having five SNPs positioned in the pikelet) , includirs1 gene  and can Vrn-H3 where we see limited correlation between SNPs in this gene and allele frequency difference in the growth habit comparison. It should also be noted that the resequencing panel in which Vrn-H3 alleles were found to be associated with the phenotype included only five spring and eight winter individuals  accounts for inconsistencies in the phenotype–genotype associations reported in previous studies and our study using a panel of >3000 accessions.The BOPA SNP platform used here is largely derived from variants identified from cDNA libraries, from cultivated accessions  for discFigure S5) with respect to neutral model of a population at equilibrium. During selection, alleles linked to the target variant can be carried to high frequencies. Therefore, an excess of rare and high-frequency derived variants can be associated with the effects of linked selection (Oryza sativa (Vrs1 gene contributing to spike-type (identified by SNP 12_30901). Two of the SNPs that permitted the identification of the outlier haplotype occur within an iron homeostasis (nashor 2) and drought-stress tolerance genes (TaGolS 2).The derived SFS in individual breeding populations identifies an excess of rare and high-frequency derived variants (election  as has bTable S16) suggest that genetic drift (and likely linked selection) plays a major role in differentiation among these populations. One major factor that can reduce effective population size, and thus increase the role of drift in breeding populations, is the frequent reuse of popular cultivars as parents in breeding programs. Historically, this included founders such as Betzes, Hanna, Lion, Manchuria, and Trebi .The cumulative effects of selection and genetic drift over several generations of breeding may result in reduced response to selection . The magFST) across the genome between populations of similar inflorescence type, we speculate that these segments could reflect a history of shared ancestry among these populations, possibly predating the separation of breeding programs  linkage disequilibrium . ShorterFigure S13).Gene flow that involves adaptive variation can result in differential retention of genomic segments in the recipient population, and can also be evident as shared genomic regions with reduced diversity , and theFigure S10). Pairwise FST with other breeding populations and genetic differentiation as measured by PCA  suggest that this isolation has been maintained over many generations. Large IBS segments (100 SNP windows) shared between VT6 and OR2 and OR6  . Isolated populations, like VT6, can carry adaptive variants absent in other populations  since 2001  and the genetic history shared between six-rowed barleys. This may also account for the elevated pairwise diversity in the Utah populations. Additionally, there is evidence of introgression between N2 and two six-rowed populations (WA6 and AB6), but not with N6. Introgression between the six-rowed populations and N2 are uncommon , thus this result needs more exploration.The large number of IBS segments shared between the Utah populations and most of the six-rowed populations yielded a significant signal of introgression based on the 4f-test, forces populations of the same spike-type to be assumed as related by shared history. Therefore, gene flow within spike-types is not investigated. Although crosses across spike-type are not common in barley breeding programs due to concerns of recombining desirable alleles with less favorable ones, introgression across spike-type could increase diversity  by each breeding program. Given that one of the goals of the Barley Coordinated Agricultural Project (http://www.barleycap.org) was to evaluate the diversity in the North American barley breeding program, each program was encouraged to submit representative lines. We assume that the data represent the diversity within each breeding program and that the sampling scheme across breeding programs is comparable. Deviations from these assumptions could influence the results in four primary ways: (i) underestimation of the diversity within breeding populations; (ii) overestimation of the role of drift in breeding populations due to reduced representation of the parental lines used in the breeding programs; (iii) over- or underestimation of allele frequency differentiation between partitions of the data; and (iv) excess of shared haplotypes due to accessions highly related by pedigree . With these caveats in mind, we made the best use of the dataset to uncover the underlying genetic response to breeder’s efforts. We encourage readers of this paper and barley breeders to evaluate the results with an understanding of the limitations of the sampling.It is important to note that the results presented here are dependent on the samples submitted for genotyping (FST outliers reported here provides a reasonable first step toward the discovery of genes underlying agronomic adaptation or potential markers linked to these genes.Comparative population genetic approaches have the potential to uncover breeding history at a level of detail not previously possible. Our applications of allele frequency differentiation analyses in barley breeding populations are able to identify candidate genes or linked markers controlling major traits. Though the nature of the target of selection identified by individual SNPs is not always readily apparent, the identification of FST analyses also reveal the effects of linked selection and drift on breeding populations. Linkage has the potential to reduce the efficacy of selection by forcing selection to act on the net effect of linked variants (variants . Furthervariants . Gains fvariants  suggeste


Lynx rufus) distributed across a highly fragmented urban landscape. We genotyped more than 300 bobcats, sampled from 1996 to 2012, for variation at nine neutral and seven immune gene-linked microsatellite loci. We found that two freeways are significant barriers to gene flow. Further, a 3-year disease epizootic, associated with secondary anticoagulant rodenticide exposure, caused a population bottleneck that led to significant genetic differentiation between pre- and post-disease populations that was greater than that between populations separated by major freeways for >60 years. However, balancing selection acted on immune-linked loci during the epizootic, maintaining variation at functional regions. Conservation assessments need to assay loci that are potentially under selection to better preserve the adaptive potential of populations at the urban–wildland interface. Further, interconnected regions that contain appropriate habitat for wildlife will be critical to the long-term viability of animal populations in urban landscapes.Urbanization profoundly impacts animal populations by causing isolation, increased susceptibility to disease, and exposure to toxicants. Genetic effects include reduced effective population size, increased population substructure, and decreased adaptive potential. We investigated the influence that urbanization and a disease epizootic had on the population genetics of bobcats ( Anthropogenic barriers reduce habitat connectivity, impeding gene flow between populations and accelerating the loss of genetic diversity due to drift  living in and around Santa Monica Mountains National Recreation Area (SMMNRA). This region comprises a collection of protected park areas near downtown Los Angeles. Bobcats inhabiting SMMNRA have been monitored by National Park Service (NPS) biologists since 1996. Within a localized region of SMMNRA, the NPS has demonstrated that a major freeway  populations, but potentially also as a social barrier. Specifically, the small percentage of bobcats (11.5%) and coyotes (4.5%) that successfully crossed the 101 Freeway were unlikely to establish territories and reproduce , western fence lizards , western skinks (Plestiodon skiltonianus), and wrentits (Chamaea fasciata)  impede gene flow and promote population isolation and genetic drift; consequently, populations separated by freeways should be highly differentiated at neutral loci. In addition, we hypothesize that the population decline associated with the disease outbreak should enhance genetic drift; thus, samples collected after the disease outbreak are expected to show reduced genetic diversity at neutral loci compared to samples collected before the disease outbreak and to be genetically differentiated from each other. With respect to immune-related loci, we expect that heterogeneity in disease prevalence and in the severity of population isolation would lead to differential patterns of selection and genetic drift. Specifically, we predict that the population severely impacted by disease would experience directional or balancing selection at immunologically important genes, as genetic variation at these loci may confer resistance to disease . Route I-405, established in 1962 (www.cahighways.org), is the most travelled highway in the country with an average of 374 000 vehicles daily (www.fhwa.dot.gov). Many secondary roads are also found throughout SMMNRA. Human land uses within SMMNRA include commercial and residential development.Bobcats were sampled in and around Santa Monica Mountains National Recreation Area  highly fragmented habitat east of I-405 and including an area east of the 101 Freeway in Griffith Park (GP) sampled from 2010 to 2011 (E405 population); (ii) a region west of the I-405 and south of the 101 Freeway (S101 population) that is comprised largely undeveloped protected parkland intersected by secondary roads and pockets of urban development; and (iii) an area north of the 101 Freeway (N101 population) consisting largely of habitat patches interspersed with roads and development  and from fecal samples using QIAamp® DNA Stool Mini Kits (Qiagen) according to the manufacturer's protocols. Individuals were genotyped using nine supposed neutral dinucleotide microsatellite markers  previously used in bobcat studies  (Rice α = 0.002). Genotyping error, the presence of null alleles, scoring errors, and allele dropout were evaluated using micro-checker  and linkage disequilibrium (LD) were tested using 05) Rice . Global HO) and expected (HE) heterozygosities  (Mousadik and Petit FIS) were estimated using fstat 2.9.3 . Because theory predicts changes in heterozygosity when a significant population decline occurs, allelic richness, observed and expected heterozygosity, and the inbreeding coefficient were also tested for differences between the N101 before mange (1996–2001) and the N101 postmange (2006–2012). Genetic diversity values for the N101 population before, during, and after the mange epizootic are presented in the Supporting Information (t-tests (α = 0.05). All across-group and pairwise analyses were conducted in r  across populations using a LD method , the performance of the program was nearly equivalent to that for sample sizes of N ≥ 30.We obtained estimates of effective population size . Cornuet and Luikart (N = 30) and 2006–2012 (N = 26) to determine whether a population bottleneck occurred regionally across the study area. We ran 1000 iterations of the two-phase model (TPM) recommended for microsatellite data, with a variance (ps = 90%)  Rienzo . RecentlM-ratio method implemented in the program m_p_val  and the average size of multistep mutations (Δg = 3.5). As above, we also tested ps = 78%. Garza and Williamson (θ (4Neμ) based on a range of effective population size estimates calculated for bobcat populations in the study area using LDNe as described above. These values corresponded with a Ne of 25 (θ = 0.05), 50 (θ = 0.1), and 150 (θ = 0.3), with a fixed mutation rate (μ) of 5 × 10−4 per locus per generation. Statistical significance of the M-ratio was evaluated with 10 000 iterations.In addition, we tested for a bottleneck signature using Garza and Williamson's lliamson  recommenstructure 2.3.4 infers population assignments without any a priori assumptions about sample location , as well as the spatial distribution of, and genetic discontinuities between, assigned populations  without any a priori assumptions about the sample location. The stability of the inferred clusters was evaluated using 10 independent runs at K = 1–10 with a burn-in period of 50 000 iterations followed by 500 000 MCMC cycles. We examined both raw probability values of LnP(K) and the ΔK estimate , we performed analyses to evaluate the individual cluster assignments using capture location as prior information, as previously described  all individuals; and (ii) without the observed four migrants identified by structure (see Results).We used FST using fstat 2.9.3.2 -Relate  using the program Maximum-likelihood  or drift dominates the observed patterns of variation at immune regions , and if the genotyped population is not completely representative of the focal population, null allele frequencies may be artificially inflated  during the period 1996–2012. Opportunistic samples were collected postmortem from 39 mortalities from 1996 to 2012, and 39 scats were collected from 2008 to 2012. A total of 365 samples  were genotyped for nine neutral loci, and 299 individuals were genotyped for seven immune-linked loci  in LD across the four bobcat populations. DRB and DRA Class II genes are found within 18.3 Mb of each other on chromosome B2 and have related functions. Specifically, DRB1 and DRB3 are separated by 24 kb, DRB1 and DRB4 are separated by 79 kb, and DRB1 and DRA1 are separated by 18.3 Mb. Consequently, because the linkage of these loci may have influenced the results of our analyses, we performed the tests with and without DRB1 and found no apparent change in the results or significance tests .Seven neutral and three immune-linked loci showed significant deviation from HWE, although none deviated from equilibrium across all four populations . SignifiHO) ranged from 0.55 to 0.72 for neutral loci, and 0.60 to 0.65 for immune-linked loci (TablesHE) ranged from 0.64 to 0.73 for neutral loci and from 0.63 to 0.71 for immune-linked loci. Mean per-locus allelic richness (AR) ranged from 5.22 to 6.88 for neutral markers, and 4.71 to 6.21 for immune-linked markers , observed heterozygosity , and expected heterozygosity  did not significantly vary across the four populations for either class of marker. These measures also did not significantly vary for immune-linked loci when DRB1 was excluded . However, values for each diversity measure and class of marker were lowest in the most isolated, fragmented eastern population of bobcats (E405).All loci were polymorphic with 6–15 alleles observed per neutral locus and 3–10 alleles observed per-immune-linked locus. Observed heterozygosity  after the mange outbreak (2006–2012) compared with before mange  values measured using neutral loci were found in E405, the most isolated and fragmented population, and in N101 postmange, the population that suffered a sharp decline as a result of the disease outbreak  was 14 times greater than N101 before/during mange , the population with the lowest FIS. The inbreeding coefficient measured with neutral loci increased 10-fold for the N101 population post-disease  . Neutral FIS pairwise comparisons between E405 and N101 before/during mange, and N101 before/during mange and postmange, differed significantly . Neutral loci FIS values were also significantly different from zero for E405, S101, and N101 postmange (P = 0.001 for each population).The highest inbreeding coefficient  and when DRB1 was excluded  , and thus, the lack of significant difference across the four populations for immune-linked loci may be due to insufficient power or Type II error. Immune FIS values were significantly greater than zero for S101 and N101 before/during mange (P = 0.002 for each population).In contrast, 7) Tables. HoweverF3,131 = 3.21, P < 0.001). The significant results reflect the high number of individual pairwise relatedness observations, but the relatedness values are very similar. Thus, the biological significance of the differences is questionable. Within population, relatedness was highest for N101 postmange  and E405  while lowest for S101  and N101 before/during mange . In pairwise comparisons, E405 was significantly higher than S101  and N101 before/during mange . S101 and N101 before/during mange had significantly lower R compared with N101 postmange . Relatedness did not significantly differ between E405 and N101 postmange  or S101 and N101 before/during mange .Relatedness Table was signbottleneck, we detected significant heterozygote excess in the N101 population from 2004 to 2005 (P = 0.007) and 2006 to 2008 (P = 0.02) using both 78% and 90% single-step mutation parameters, evidence that a bottleneck occurred as a result of the disease outbreak , 2006–2008 (M = 0.71), and 2009–2012 (M = 0.67).For neutral markers, using the program rs Tables and S15.Ne, for bobcats found north of the 101 Freeway (N101) before mange was 47 (95% CI: 25.9–130.7) individuals during 1996–1998 and 17.6 (95% CI: 12.1–27.6) individuals during 2000–2001. The cause of the pre-disease effective population size variation is unknown. However, limited field observations made by NPS do not support that a population decline occurred from 1996 to 2001. Further, the effective population size estimates have overlapping confidence intervals across most years for the N101 population. Therefore, the differences in effective population size pre-disease may be due to stochastic differences in sampling. However, during the final years of the mange outbreak, Ne was nine (95% CI: 5.6–13.0) individuals (TableNe was estimated to be 97.7 (95% CI: 71–143.6).The effective population size, K value  clusters had the highest posterior probability assignments for neutral loci in structure  values generated in structure for each K value plateau at K = 9 and do not point to a discrete number of population clusters  and Geneland included individuals captured east of I-405 (E405), south of the 101 Freeway (S101), and two clusters north of the 101 Freeway (N101) divided temporally. The first N101 population included individuals sampled from 1996 to 2005 (structure) or 1996 to 2001 (Geneland) in the mange-affected region, along with the Moorpark satellite group of individuals (N = 10) sampled northwest of the mange-affected region in 2010–2011. The second population north of the 101 Freeway included all animals sampled postepizootic (2006–2012), with the exception of the Moorpark individuals that clustered with N101 before/during mange population. Interestingly, nine samples from GP assign with the E405 population although the park is isolated from this population by the 101 Freeway  were resolved that corresponded with pre- and postbottleneck cluster assignments  with a global Dest = 0.10. All probability calculations for neutral Dest were significant at P ≤ 0.001. The greatest Dest values were observed between the E405 and N101 populations that are geographically separated by two major freeways and the greatest geographical distance. For the N101 before/during mange and N101 postmange populations Dest = 0. 08, a value greater than for those populations separated by a major freeway (TableBoth freeways and disease influenced population differentiation Tables and S10.est Table showed gDest (TableDest = 0.02–0.17) with a global Dest = 0.09. All probability calculations for immune Dest were significant at P ≤ 0.002. Using immune markers, overall genetic differentiation between populations was lowest between N101 before/during mange and postmange (Dest = 0.02) and highest between E405 and N101 postmange (Dest = 0.17).Using immune markers, all pairwise comparisons of est Table also shoDest values were significant, using structure and Geneland, no clear population structure was resolved when tested using immune-linked loci    Table. This fi = 0.03) . Consequsix loci . Thus, isix loci  or allelture Fig..Dest = 0.00 , while DRB3 and DRB4 had Dest values similar to the overall neutral value (Dest = 0.08 and 0.10). Using probability tests, none of the per-locus Dest values were significant . Further, despite the 10-fold increase in neutral FIS in N101 post-disease, immune-linked loci FIS decreased by more than half. The decrease was not significant when comparing N101 before/during mange and postmange , but when N101 before and postmange were compared . Per-locus decreases in FIS were also observed for multiple immune-linked loci when comparing N101 before/during mange with postmange , but decreased in N101 postmange and no longer differed significantly from zero. DRB4, in contrast, increased significantly from 0.06 to 0.17.Inbreeding coefficients showed evidence of both balancing selection and genetic drift acting on immune-linked loci in SMMNRA. The S101 population had the highest inbreeding coefficient at immune-linked loci, greater than for neutral loci and equivalent to the E405 neutral inbreeding coefficient. In contrast, the E405 population had the greatest inbreeding coefficient using neutral loci Table, and yetcompared , the difK = 4 in structure analysis and Geneland. These clusters were comprised of populations separated by freeways or before and after a disease outbreak. Neutral measures of genetic diversity were lowest in the most isolated, fragmented population (E405) and in N101 postmange, a population that suffered a population bottleneck caused by a disease outbreak. These findings indicate the freeways and disease are important contributors to genetic diversity and differentiation in bobcat populations in SMMNRA.Urban development is increasingly recognized to have important genetic impacts on wildlife populations  cohorts than expected at random was attributed to strong directional selection favoring specific MHC alleles  may increase survival of male Alpine chamois (Rupicapra rupicapra) during rutting season . Thus, balancing selection may also be an important regulator of genetic variation at immune-linked loci in the E405 population in response to mange parasitism.Differentiation in neutral and immune-linked loci is similar and nonsignificant across other populations, suggesting the latter are not experiencing divergent natural selection or under balancing selection in other areas of SMMNRA. We also observe an absence of population structure for immune-linked loci that may reflect the limited power of seven loci, qualitative differences in variation of the neutral and immune markers, as well as differential patterns of drift and selection acting on individual immune-linked loci across the study area. One suggestive finding is that Our data highlight the importance of increased urbanization, as manifest by increased exposure to anticoagulants and the presence of freeways, on the genetic structure and diversity of animal populations and suggest that efforts to ameliorate these effects are critical to long-term conservation and effective management. Bottlenecks can reduce genetic variation and thereby threaten the long-term viability of wildlife populations (e.g., Roelke et al.


Galaxias zebratus  that occurs across two currently isolated river systems close to the Joubertina area in the eastern CFR. Results from both analyses revealed that observed genetic differentiation cannot be explained by isolation between the two river systems. No genetic differentiation was found between the Krom River system and a population from one of the Gamtoos tributaries. Shallow genetic differentiation was found between the Krom and the other Gamtoos populations. Historical river capture events and sea-level changes do not explain the present distribution of Galaxias zebratus “Joubertina” across the Krom and Gamtoos River systems. Interbasin dispersal during pluvial periods, recent river capture, or recent human-mediated translocation seems to be the most plausible explanations.Range expansion of obligate freshwater fishes in the Cape Floristic Region (CFR) of South Africa has mostly been attributed to river capture events and confluence of rivers following sea-level regression. The role of low drainage divides and interbasin water transfers has received less attention. This study analyzed mitochondrial and nuclear DNA sequences to assess the processes that could have influenced the phylogeographic patterns of a newly discovered lineage of  Freshwater taxa tend to display higher levels of genetic structuring among populations than marine taxa, because they generally have smaller effective population sizes and populations are often isolated by terrestrial or marine barriers , a tributary of the Krom River system (the present day Kouga catchment) was pirated by the Baviaanskloof River, a tributary of the Gamtoos River system . Waters and Cambray   did not allow them to test this hypothesis. It is possible that the low drainage divide between the Twee and Krom may represent a recently abandoned river course due to river capture . Recent reports suggest that some of these dams are connected by a series of canals that divert water from the upper Krom to the Kouga catchment  and upper Kouga (Gamtoos River system) catchments which are located in the Langkloof valley in the Eastern Cape Province of South Africa  was amplified using the polymerase chain reaction (PCR) with the primers Gcyt-Glu (5′ GAA AAA CCA CCG TTG TTA TTC A – 3′) and Gcyt-Thr (5′ – CGA CTT CCG GAT TAC AAG ACC – 3′) , 0.1 μL of 5 U Super-Therm DNA polymerase (Taq), and 3–5 μL of DNA template (and negative controls with no DNA template to test for contamination). Volumes were adjusted to a final volume of 25 μL with ddH2O. All PCR products were purified and sequenced by Macrogen Inc. (South Korea).Total genomic DNA was isolated from muscle tissue using the Wizard® Genomic DNA purification kit  following the manufacturer's protocol. A section of the mitochondrial cytochrome cyt b sequences and 36 S7 sequences were generated for this study. Sequences were checked manually and edited using SeqMan (DNASTAR Lasergene 9 Core Suite). Corrected sequences were aligned using ClustalX  (Nei π)  Nei  and nuclGalaxias zebratus “Joubertina”: (1) two groups were defined to test the river systems, and groups were therefore defined as the populations in the Krom and Gamtoos river systems; (2) four groups were defined according to the catchments, namely the Krakeel, Wabooms (including Granaat and Diepkloof), Twee, and Krom catchments; (3) six groups were defined as the individual tributaries, namely the Krakeel, Wabooms, Granaat, Diepkloof, Twee, and Krom, and finally, (4) two groups were defined differently to the first structure by combining the Twee and Krom populations, because they were sharing haplotypes, and compared to all the other populations in the Gamtoos. The significance of the variance components was determined with 1000 permutations , Krakeel (N = 25), Diepkloof (N = 30), and Wabooms (N = 30) (referred to in this study as the western Kouga tributaries) and the Twee (N = 30) in the eastern part of the Kouga catchment. The species was only found in one tributary of the Krom River system, namely the upper Krom (N = 40). Two of these tributaries represent new distribution localities (Krakeel and Diepkloof)  each in the Granaat and Wabooms and four localities in the Krom . Low numbers of individuals were found in the remaining tributaries. To reduce the impact on these populations and to increase the probability of capturing more of the population's genetic diversity, four localities each were sampled for the Twee , Krakeel , and Diepkloof  tributaries. Overall, Galaxias zebratus “Joubertina” was only found in 22 localities of the 72 localities that were surveyed (31%).Specimens of keel N = 5, Diepklb resulted in seven haplotypes defined by nine variable sites .Analysis of 121 individuals for 616 base pairs of cytochrome HD) ranged from 0.468 to 0.695 for the Wabooms, Granaat, and Krakeel, but was 0 for the Krom, Twee, and Diepkloof populations.All the individuals from the Twee and Krom populations shared haplotype 1, but did not share this haplotype with any individuals from other populations. The Diepkloof also had only one haplotype (haplotype 2), but shared it with all the western Kouga tributaries . Haplotype 6 was restricted to the Granaat and Krakeel tributaries . Divergence between the Krom and Twee compared to the western Kouga populations ranged from 0.2 to 0.7%. The largest divergence was between the Wabooms and Granaat populations (0–1%) and the Wabooms and Krakeel populations (0–1%). Estimates of divergence times between the Krom and Gamtoos populations ranged between zero and 33 000 years ago. There were no significant differences in gene diversity between the Wabooms, Granaat, and Krakeel populations, but the first two showed relatively higher values compared to the Krakeel population (Tableπ = 0.003) within populations and did not differ significantly between the Wabooms, Granaat, and Krakeel populations   and FSC: 0.689 (68.9%), P < 0.001) (TableFCT: −0.005 (−0.5%)). This result was due to the sharing of the same haplotype between individuals from the Twee and Krom populations. The overall ФST values were all significantly large for all four structures suggesting high levels of differentiation .Analysis of 36 individuals for 492 base pairs of S7 revealed 5 unique haplotypes defined by 4 variable sites. Only one heterozygotic position was found; therefore, 72 sequences were used for analysis after separating the maternally and paternally derived sequences. No significant deviations from those expected under predictions of neutrality were observed when all S7 haplotypes were analyzed together (Tajima : D = −0.u and Li : F = −1.D) ranged from 0.143 (Krom) to 0.750 (Diepkloof), and overall haplotype diversity was 0.512. Gene diversity was high for the Granaat (0.546 ± 0.062) and Diepkloof (0.750 ± 0.097) populations compared to the other four populations (Tableπ = 0.001)  with haplotypes 2 and 4, which are restricted to the Krom and Twee tributaries, respectively. Clade 1–2 is restricted to the western Kouga tributaries and comprises of haplotype 3  and haplotype 5 (restricted to the Diepkloof tributary)  between the Krom and Gamtoos populations of Galaxias zebratus “Joubertina” are consistent with the proposed connection of the Krom and Gamtoos River systems during the last glacial maximum (LGM) about 18,000 years ago . Dry conditions, which prevailed after the Holocene Altithermal, could have caused the dissociation of low-gradient connections between the river systems, leaving too little time for loss of diversity or divergence of haplotypes. It is also possible that the low drainage divide was once the drainage line for the Twee flowing into the upper Krom catchment. Although possible, it seems unlikely that the Krom would have flowed into the Kouga catchment, because the upper Krom flows into a deeply incised gorge below the present distribution of Galaxias zebratus “Joubertina”. The genetic similarity between fish from the adjacent Twee and Krom compared to the western Kouga tributaries is consistent with the “Intermittent Connections hypothesis.”Swartz et al.  proposedGalaxias zebratus “Joubertina” in two isolated river systems. Such drainage re-arrangements would need to have occurred over the timescales relevant to the present phylogeographic patterns (which coincides with the Holocene and Peistocene). Alternatively, the lack of differentiation between the Krom and Twee populations could be an indication of ongoing exchange of individuals between the two populations across the drainage divide between the Gamtoos and Krom river systems due to the constructed canals that connect the upper Krom and upper Kouga catchments. The lower reaches of the Twee and the lower sections of the canals are not suitable for the permanent occurrence of the Galaxias zebratus “Joubertina,” because of the occurrence of Micropterus salmoides, habitat destruction, and unsustainable water abstraction. However, these canals could allow gene flow between the Krom and Twee populations during winter high flows or floods. The Interbasin Transfer Hypothesis can therefore also not be rejected.Recent geological evidence suggests that the landscape around the areas encompassed by the present study (Kouga and Bainskloof) has undergone remarkably little change for a very long period of time, probably since the Miocene , but this could be due to insufficient time for mutations to accumulate. Connection of the rivers through the Interbasin Transfer (IBT) canal system, natural low-gradient areas, or recent river capture could have then allowed colonization of the Krom River. Alternatively, the Krom was connected to some or all Gamtoos populations during wet periods and later became isolated following onset of contemporary dry conditions. Assuming the Twee population went extinct or never existed in the first place, a unique haplotype could have become isolated in the Krom, which later established in the Twee through dispersal across the IBT, low-gradient connections, or recent river capture. The first scenario seems to be the most plausible explanation, because Galaxias zebratus “Joubertina” does not occur elsewhere in the Krom River system, but it occurs in five tributaries of the Gamtoos River system. It is therefore unlikely that the Twee tributary did not have Galaxias zebratus “Joubertina” when the Krom population became established. The most likely scenario is that the occurrence of this lineage in the Krom is due to the series of canals (IBT) that connect the two river systems, but the present data cannot reject the possibility of intermittent dispersal across low gradients during wet periods. Both nuclear and mitochondrial DNA revealed relatively higher levels of genetic structuring between Galaxias zebratus “Joubertina” populations within the Gamtoos River system. The geographic distribution of genetic diversity within river systems can be influenced by stream structure, physical barriers, isolation by distance, and historical processes on population structure (Shaw et al. Galaxias zebratus “Joubertina” showed high levels of differentiation between the Twee and western Kouga populations and between the Krakeel and the three populations of the Wabooms catchment which could be due to isolation by distance. In-stream barriers have been demonstrated to be effective in affecting population genetic structure (Currens et al. Galaxias zebratus “Joubertina” has limited dispersal ability, a small home range, and/or a preference for certain habitat types.The direction of movement between the river systems is uncertain, but there are two possible scenarios that most likely explain why recent gene flow has occurred between these two systems. Firstly, haplotypes may have evolved from a common ancestral haplotype in the Gamtoos River system. The Twee River population would have become isolated relatively recently from the other Gamtoos populations, and a mutation would have caused a unique mitochondrial cytochrome Recent impacts such as the spread of alien fishes, over abstraction of water, and construction of weirs within the Gamtoos river system are causing population fragmentation which can cause loss of diversity, genetic drift, and inbreeding in populations previously connected by gene flow. This may appear like historical low gene flow when the fish were actually able to migrate historically. Loss of genetic diversity through fragmentation by weirs has been demonstrated (Meldgaard et al. Galaxias zebratus “Joubertina” individuals to move between populations, and the use of faster evolving genetic markers such as microsatellites, is required to resolve finer scale genetic patterns and shed more light on the evolutionary history of Galaxias zebratus “Joubertina.” Such information will also help provide clearer recommendations to conserve the genetic diversity of this taxon.Genetic structuring within the Gamtoos River system was probably influenced by isolation by distance, physical barriers, a natural tendency to have a small home range facilitated by a preference for upper mountain stream habitats, a natural inability to disperse between catchments, and/or recent alien fishes and human impacts. More detailed assessment of the capabilities of