Although mtDNA and the non-recombining Y chromosome (NRY) studies continue to provide valuable insights into the genetic history of human populations, recent technical, methodological and computational advances and the increasing availability of large-scale, genome-wide data from contemporary human populations around the world promise to reveal new aspects, resolve finer points, and provide a more detailed look at our past demographic history. Genome-wide data are particularly useful for inferring migrations, admixture, and fine structure, as well as for estimating population divergence and admixture times and fluctuations in effective population sizes. In this review, we highlight some of the stories that have emerged from the analyses of genome-wide SNP genotyping data concerning the human history of Southern Africa, India, Oceania, Island South East Asia, Europe and the Americas and comment on possible future study directions. We also discuss advantages and drawbacks of using SNP-arrays, with a particular focus on the ascertainment bias, and ways to circumvent it. Studies of the genetic history of human populations have relied largely on variation in the single-locus, uniparentally inherited mtDNA and non-recombining Y chromosome (NRY). While mtDNA and the NRY continue to provide valuable insights (as reviewed elsewhere in this issue), especially with the advent of new sequencing methods based on next-generation platforms, genome-wide data are increasingly supplementing and supplanting single-locus studies. Genome-wide data generally provide more reliable insights into population history in that they are based on analyses of many independent loci, whereas the history of a single locus may depart from that of the population as a whole because of chance events or selection influencing that locus. Genome-wide data are particularly useful for inferring population divergence times, migration and admixture , changes in population size, and other aspects of demographic history. In this review, we focus on some of the stories, that is, aspects of human population history as revealed by analyses of genome-wide data from contemporary human populations that we find of particular interest, rather than providing a comprehensive overview of methods and results. There are certainly other interesting studies which we do not discuss in this review -9; otheret al.) [Whole genome sequencing is, at the time, we write this, still too costly (in terms of time and money) to be applied to large numbers of individuals from large numbers of populations - although the situation is rapidly changing. For now, most genome-wide data comes from the so-called ‘SNP chips’, which are microarrays containing probes to hundreds of thousands (or even millions) of SNPs. DNA samples can be genotyped quickly and reliably at relatively low cost; however, SNP chips are not without their drawbacks, the main one being ascertainment bias. Ascertainment bias refers to how the SNPs were chosen for inclusion on the chips and inevitably arises because, by definition, only sites known to be polymorphic in at least one population are interrogated by the microarray. And since European populations  are the most studied, most SNPs on the commercial SNP chips were ascertained to be polymorphic in Europeans. This has several important consequences. First, heterozygosity in European populations will be over-estimated relative to non-European populations  . Second,et al.) -21) cannet al.) ,23. Thiset al.) , which cet al.) . Nonetheet al.) .Figure 1MtDNA and Y-chromosome analyses have shown that Khoisan-speaking groups  from Southern Africa harbor some of the deepest rooting lineages among extant human populations ,28, and As the data depicted in Figure A subsequent re-analysis of the data in this study  was carrA reasonable test of this hypothesis would be to determine if the amount of Eastern African ancestry is correlated with the amount of Western Eurasian ancestry in Southern African groups. Unfortunately, it was not possible to carry out this test, because with the SNP chip data, Eastern African ancestry cannot be reliably distinguished from Western African ancestry. This is because the detection of ancestry from a specific population relies on the existence of sufficient genetic drift since the divergence of that population from other populations to create different allele frequencies, and thus a distinct genetic signature for that ancestry. Eastern and Western African populations have not experienced sufficient drift since their divergence to create distinctive genetic signatures of their ancestry, whereas the bottleneck associated with the migration of modern humans out of Africa has created a distinctive genetic signature for non-African populations, making it very easy to detect Western Eurasian ancestry in African populations. All of the Khoisan-speaking groups studied carry recent Western African ancestry from Bantu-speaking groups  languages are predominant in northern India and are related to languages elsewhere in Eurasia, while Dravidian languages are predominant in southern India and are restricted to South Asia. Also, agriculture seems to have spread into India from elsewhere in western Asia, possibly concomitantly with IE languages . Was theA study of genome-wide SNP data in 25 groups from across India found strong support for two distinct sources of genetic ancestry . The firvs. how far apart they are on the same chromosome and fitting an exponential equation that can then be used to estimate the number of generations that have elapsed since admixture  are the largest ethnic minority in Europe, numbering an estimated 10 to 12 million people. There are a wide variety of Romani dialects, religions, and social practices, but the Romani are united by a shared history of having migrated from India around 1,000 to 1,500 years ago. Linguistics, cultural practices, and limited genetic studies support this view of an Indian origin of the Romani, but many details  remain unknown. Two studies of genome-wide SNP data have recently provided additional insights into the origins of the Romani ,56. ThesWhen applied to genome-wide data for the Romani and reference populations from Western Eurasia and South Asia, both the admixture LD and the ABC approaches come to broadly similar conclusions. The Romani likely originated from somewhere in northwestern India Figure , even thHelicobacter pylori [Oceania holds a unique place in the human history of the world, as the genetic diversity in this region has been shaped by at least two major human migrations - the first out-of-Africa migration and the last pre-European dispersal of people, known as the Austronesian expansion. Australia and New Guinea, which up until 8,000 years ago, were joined into a single landmass called Sahul and were first settled during the expansion of modern humans out of Africa; the earliest sites documenting the presence of anatomically modern humans are dated to approximately 50,000 years ago in Australia  and apprr pylori  - providP = 0.02).Two recent studies which analyzed dense SNP genotyping data from aboriginal Australians and New Guineans ,68, althet al. estimated that Eurasians and the populations of greater Australia diverged from African populations 66 kya, while the split between Australians and New Guineans from the Eurasians was dated to around 43 kya, and the divergence between the Australians, New Guineans, and the Mamanwa Negrito group was estimated to have occurred 36 kya [H. pylori [Although genome-wide data made it possible to reject an ‘early’ dispersal hypothesis, identifying a possible route of the dispersal remains a challenging task, as any archeological evidence for the southern coastal route out of Africa would have been swallowed by rising sea levels at the end of last glaciation, and the genetic record erased by subsequent migrations. In addition to the Australian aboriginals and the highlanders of New Guinea, the so-called Negrito groups of Malaysia and the Philippines and the Andamanese Islanders are thought to be the only direct descendants of the out-of-Africa diaspora via a southern route, while the other populations who live in Southeast Asia today have been shown to have arrived later by a separate dispersal from the north -71. Gened 36 kya . This daThe next chapter in the history of Oceania started tens of thousands years later with a large-scale Austronesian expansion, which began about 4,500 years ago from Taiwan ,74-77, pWhile the first Paleolithic expansion into Near Oceania brought modern humans to Australia, New Guinea, and the nearby archipelagos (together known as Melanesia), the latter Holocene dispersal was of people who must have been in possession of more advanced seafaring skills and technologies, which enabled them to venture further into Remote Oceania, and colonize islands scattered over the Pacific Ocean and often separated from each other by thousands of kilometers of open water. Earlier mtDNA and NRY studies provided evidence that once they reached Melanesia, Austronesian speakers started mixing with the indigenous Papuan-speaking populations and that this newly admixed population subsequently expanded into Remote Oceania -85. ThisVery important insights into the origins of Polynesians were recently made possible by a study of nearly 1 million SNPs genotyped in populations of New Guinea, Fiji and seven different islands in Polynesia, as well as a population from Borneo . This stQuite remarkably, despite the scope of their expansion, the Austronesians have left no genetic traces in mainland Australia. In fact, before genome-wide data became available, it was widely believed that following the initial colonization event, aboriginal Australians remained completely isolated from the rest of the world, until the arrival of the Europeans late in the eighteenth century. Studies of mtDNA ,92 and NSince the sample of aboriginal Australians analyzed in this study came from the northwestern part of the continent, it would be interesting to investigate to what extent the Indian connection is shared throughout the Australian continent. The only other genome-wide study of aboriginal Australians was based on samples from the southeastern part of Australia   and failvs. Angal-Kewa, both from the Engan branch of the Trans-New Guinea languages) both in the PCA and in the STRUCTURE-like clustering algorithm Frappe. Fine structure was also evident in Polynesia, as PCA of just the Polynesian samples revealed a separation between the Cook Islanders and the others along the first principal axis, while PC2 roughly differentiated non-Cook-Island samples according to their island of origin. In this case, the presence of fine-scale structure is probably best explained by geography and inter-island isolation.In addition to the aforementioned insights into the history of past migrations that have shaped the history of Oceania, genome-wide data were useful in revealing finer population structure in Polynesia and in the highlanders of Papua New Guinea . Unlike By the time of the out-of-Taiwan migration, Island Southeast Asia had already been populated for tens of thousands of years. The first anatomically modern humans came to this region as part of the ‘southern-route’ out-of-Africa migration. Genetic evidence based on mtDNA, NRY, and autosomal markers suggests that there were additional dispersals into ISEA, possibly from mainland Asia, before the arrival of the Austronesians -103. AusGeographically, western Indonesia  lies on the Sunda Shelf, which was exposed during the last ice age , linking the islands of western Indonesia to the Asian continent. Eastern Indonesia is separated from the western Indonesia by a deep water channel known as Wallace’s Line which runs between the islands of Borneo and Sulawesi. Island Sulawesi and two archipelagos, Nusa Tenggara and the Moluccas, lie between the Sunda and Sahul  shelves.vs. Austronesian origins of the Asian ancestry in Indonesia. The pattern that has emerged from the analysis is that the Papuan ancestry gradually increased (while the Asian ancestry decreased) from west to east across Indonesia , and thus, the Austro-Asiatic ancestry observed in western Indonesia could be related to the indigenous population of Sundaland. Further studies of correlations in ancestry, and dating of admixture signals, should shed light on the origins of the Austro-Asiatic ancestry in western Indonesia. For additional reading on the population history of the region, we provide the reader with the references to other interesting and relevant studies -110.North and South America were the last continental regions to be colonized by humans. Current evidence suggests that humans first entered the New World via the Bering land bridge about 15,000 years ago , but queMost of the genetic evidence that has been used to investigate the colonization of the New World comes from either mtDNA and Y chromosome studies or from ancient DNA and hence is discussed elsewhere in this issue. However, there is some relevant genome-wide data from contemporary Native American groups. A study of genome-wide SNP data from 52 Native American populations  found evWhile the results of this study are consistent with previous genetic evidence suggesting three major migrations to the New World, there are some important caveats. The sampling of North American populations was limited to just one Na-Dene group and three Amerind groups, so it remains to be seen if the admixture graph depicted in Figure There are other questions of interest concerning Native American populations that are being addressed with genome-wide data. For example, since the arrival of Europeans and with the introduction of the African slave trade, European and/or African ancestry can be detected in many Native American populations. Over what time periods was such ancestry contributed, what were the source populations, and how much of an impact does this recent European and/or African ancestry have on Native American populations? Two studies have recently addressed these questions, one analyzing genome-wide SNP data in Caribbean populations , and theThe origins of modern Europeans remain contentious; for decades, anthropologists have tried to answer the question to what extent the Paleolithic hunter-gather populations known in Europe since around 45,000 years ago were replaced, assimilated, or have adopted the way of life of farmers, as agricultural practices and/or farmers started spreading across Europe from the southeast ca. 8,500 years ago. The most informative insights into the history of Europe have come from recent ancient DNA work -119, whiet al. [Two comprehensive studies of genome-wide variation that densely sampled across a geographic continuum of Europe ,121 reveThis geographic structure of recent relatedness was further explored by a subsequent study which used the same dataset to infer genomic segments inherited from a recent common ancestor identical by descent (IBD). The study applied a new methodology based on the estimated lengths of these IBD blocks to relate these lengths to the ages of the most recent common ancestors . As befoIn conclusion, the studies of genetic variation in Europeans show little overall genetic differentiation between populations, which could be the result of the homogenizing effect of recent migrations across Europe, yet reveal startling correspondence between genes and geography, even on a regional scale -127. GivIn this review, we have focused on a few of what we find to be the most interesting stories concerning human population history that have been illuminated by studies of genome-wide SNP data. One of the main messages is that while ascertainment bias is always an important concern with such data, there are ways to account for ascertainment bias in demographic analyses . Another main message is that as we get better and better at detecting and dating admixture signals in genome-wide data , we find


TAS2R loci. In addition, humans carry 11 TAS2R pseudogenes, some of which display evidence for substantial diversification among species, showing lineage-specific loss of function. Since bitter taste is thought to help prevent the intake of toxic substances, diversity at TAS2R genes could reflect the action of natural selection on the ability to recognize some bitter compounds rather than others. Whether species-specific variation in TAS2R pseudogenes is solely the result of genetic drift or whether it may have been influenced by selection due to different feeding behaviors has been an open question.Human bitter taste receptors are encoded by a gene family consisting of 25 functional TAS2R pseudogenes in both African and non-African populations, and compared them to those observable in nonhuman primates and archaic human species. Our results showed a similar worldwide distribution of allelic variation for most of the pseudogenes, with the exception of the TAS2R6P and TAS2R18P loci, both of which presented an unexpected higher frequency of derived alleles outside Africa. At the TAS2R6P locus, two SNPs were found in strong linkage disequilibrium (r2 > 0.9) with variants in the functional TAS2R5 gene, which showed signatures of selection. The human TAS2R18P carried a species-specific stop-codon upstream of four polymorphic insertions in the reading frame. SNPs at this locus showed significant positive values in a number of neutrality statistics, and age estimates indicated that they arose after the homo-chimp divergence.In this study, we analyzed patterns of variation at human TAS2R6P derived alleles outside Africa as the effect of the balancing selection acting on the closely linked TAS2R5 gene. In contrast, TAS2R18P displayed a more complex history, suggesting an acquired function followed by a recent pseudogenization that predated the divergence of human modern and archaic species, which we hypothesize was associated with adaptions to dietary changes.The similar distribution of variation of many human bitter receptor pseudogenes among human populations suggests that they arose from the ancestral forms by a unidirectional loss of function. However we explain the higher frequency of The online version of this article (doi:10.1186/s12862-014-0198-8) contains supplementary material, which is available to authorized users. TAS2Rs or T2Rs. In humans, this gene family is encoded by 25 functional TAS2R loci, which reside on chromosomes 5, 7 and 12 [TAS2R pseudogenes [TAS2R genes and pseudogenes.The perception of bitter taste is thought to have evolved as a protection from toxic and harmful foods ,2. Howev7 and 12 . In addiudogenes -10, and udogenes -13, highTAS2Rs genes and pseudogenes in different lineages, suggesting that the ability to perceive bitterness may be mostly due to the repertoire of TAS2R genes [TAS2Rs loci but only three pseudogenes, and the evolutionary relationships between human and mouse genes were shown to fall into three categories, depending on their orthology: 1) one-to-one orthology; 2) one-to-multiple orthology; 3) multiple-to-one orthology [TAS2R loci, respectively [TAS2Rs had a higher ratio of nonsynonymous/synonymous substitutions and a lower selective pressure on this gene family compared to rodents [Pseudogenes are considered genomic fossils, classically defined as genomic loci with sequence similarity to functional genes, but lacking coding potential ,15, ofte2R genes ,22. For rthology ,15,7. Inectively ,5. Compa rodents ,26.To better understand the evolutionary mechanisms underlying taste receptor pseudogenization events in the human lineage, we performed a survey of the human genetic variation at nine human TAS2RP loci. We then compared these to the homologous pseudogenes present in archaic human forms , as well as in other five primate species, Chimpanzee, Gorilla, Orangutan, Gibbon and Mouse Lemur. We examined the intra- and inter-species variation patterns to search for possible footprints of natural selection at these loci, with the goal of understanding the potential adaptive role of taste receptor pseudogenes in the evolutionary history of modern humans.TAS2R pseudogenes were selected from the literature and are shown in Table Homo sapiens), five other primates  and one rodent (Mus musculus), were obtained from the Ensembl Genome Browser [Nine human  Browser ,32. ThisHaploview  was usedST values, were performed with STATISTICA v. 6.0 .Arlequin v.3.5  was usedMUSCLE v.3.3   showed significantly increased frequency (0.5) in non-African populations compared to African populations (0.053) . Similarly, the derived alleles of TAS2R6P polymorphisms rs1859645 and rs11761380 showed a significantly different distribution (0.24 vs. 0.57) between Africa and other continents . Both Neandertal and Denisovan genomes carried the derived alleles of the TAS2R18P SNPs rs2290318, rs2290319, rs61928604 and rs61928603. For the TAS2R6P SNPs rs1859645 and rs11761380, the Denisovan genome showed ancestral alleles, while the Neandertal genome was heterozygous at both SNPs. In addition, derived alleles of two SNPs located in TAS2R67P (rs319269 and rs34648613) and one in TAS2R64P (rs68071847) were more frequent in African populations with respect to non-African ones , and were also present in both Neandertal and Denisovan genomes. Finally, the derived allele of one SNP located in TAS2R63P (rs2597986) was present only in a few African individuals (1.58%), as well as in the two archaic species.A total of 47 single nucleotide polymorphisms (SNPs)  and differed significantly across human populations , with a smaller percentage attributed to differences among (13.2% and 27.12%) and within groups (0.13% and 0.81%). The global FST values were 0.13 for TAS2R6P and 0.28 for TAS2R18P, while those related to H5 and H6 haplotypes were 0.14 and 0.31, respectively.To test the significance of the observed structure, the distribution of the H5 and H6 haplotypes in human populations was also investigated by means of Analysis of Molecular Variance (AMOVA). For both TAS2R6 and TAS2R16 pseudogenes in order to better understand the relationships between the inferred haplotypes. The resultant topologies  or ancestral  alleles at the H5 and H6 haplotypes.We constructed median-joining networks for the TAS2R6P and TAS2R18P. African groups  showed lower values of both nucleotide diversity (π) and estimated heterozigosity (EH) at theTAS2R18P locus. With the exception of the ASW group, the same pattern was observed for the TAS2R6P gene. The number of polymorphic sites (PS) was similar in all the studied populations  metric was then used to plot the obtained FST values. This produced a clear distinction between continental populations for the observed variation. As shown in Figure TAS2R18P was separated from African and Asian populations along the first dimension, with a distinct cluster, containing the European and Latin American (admixed) populations, occupying an intermediate position between them. Variation at the TAS2R6P locus showed a similar pattern of population structure among continental clusters  and showing short branch lengths of the tree, suggesting an ancient pseudogenization event at this locus. Longer branch lengths in Prosimians and Anthropoidea after their divergence may indicate that the pseudogenization at TAS2R18P locus occurred independently in the two lineages. It should be noted that these conclusions might be affected by problems with ortholog detection, considering the high dynamism and extreme complexity of this gene family.We also created a phylogenetic tree using the nucleotide sequences of TAS2R6P identity between Hominidae and Hylobatidae (>95%) and Prosimians, Hominidae and Hylobatidae (>75%). TAS2R18P sequences showed a lower degree of similarities (<70%) between primates and Prosimians , only one unique stop-loss mutation (TAG > CAG) was found in the chimpanzee TAS2R6P. All the human species  shared a fixed stop-gain mutation (TGG > TAG) on TAS2R18P  were identified in the reading frame, upstream of the seven stop-codons shared across species and downstream of the human-specific stop codon. One of them (rs113657094) showed clear differential distribution among continental clusters of populations, with Africans displaying a significantly lower frequency of the A insertion (42%) compared to other ethnic groups  . For comparison, the TAS2R6P sequence was also examined and neither insertions nor other structural variants were found.To better understand the species-specific changes that occurred in the d Figure . Four poTAS2R6P and TAS2R18P was explored for linkage disequilibrium patterns. The TAS2R6P rs11761380 and rs1859645 polymorphisms were found to be in strong LD (r2 > 0.9) with SNPs rs62477710, rs10952507 and rs6962558 that reside in the functional TAS2R5 gene , while TAS2R18P has a high level of identity with the functional TAS2R42 gene and with the TAS2R67P pseudogene   showed a much higher sequence similarity (91.32%) with their functional forms (i.e. TAS2R48), indicating ancient pseudogenization events at the TAS2R6P and TAS2R18P loci.A 200 kb (100 kb in both 5’ and 3’ directions) region surrounding both TAS2R18P Tajima’s D and Fu’s FS. Africans and Asians also showed positive values  for these statistics, although these did not reach significance . However, rs62477710, rs10952507 and rs6962558 polymorphisms in the adjacent TAS2R5 gene showed significantly positive values of Tajima’s D and Fu’s FS , indicating that recent selection has acted on these loci. However, the p-values for this test were not significant for TAS2R6P (p > 0.05).Neutrality tests based on the analysis of frequency spectrum of variants were performed for each population. European and Latin American groups showed significantly positive values for both TAS2R18P, rs2290318 was estimated to be 1,553,750 +/− 632,500 years old, while rs2290319 was estimated to be 1,113,750 +/− 495,000 years old, with a TMRCA of 3,520,000 +/−1,198,750 years. Similar values were also obtained for the TAS2R6P SNPs rs11761380 and rs1859645, with age estimates of 1,382,462 +/− 326,856 and 1,600,637 +/− 421,558 years, and a TMRCA of 3,457,712 +/− 1,326,856 years.Finally, the ages of these variants were estimated using a coalescent-based method implemented in the GENETREE package. Estimated ages turned out to be considerably ancient. For SNPs in TAS2R6P and TAS2R18P polymorphisms, at both the allelic and haplotypic level. Genomes of the archaic Neandertal and Denisova species also differed in the distribution of these derived alleles compared to modern human populations. In contrast to the usual pattern of human variation, the highest genetic diversity was found in non-African populations. Similarly, heterozygosity was higher in non-African populations. In addition, results from both population structure analyses and AMOVA demonstrated that most of variation was due to differences within populations. In fact, Europeans and Latin Americans fell into the same cluster and did not differ in allelic distribution, suggesting a similar pattern of SNP distribution in these two populations and highlighting the admixed nature of the 1000 Genomes American populations.A small number of studies have examined genes encoding functional bitter taste receptors and their corresponding pseudogenes from an evolutionary perspective ,23,25,26TAS2R6P is very old, predating the divergence of Antrhopoidea from Prosimians. The TAS2R18P pseudogenization event occurred later in the primate genealogy, but still prior to the appearance of the Hominidae family.Previous work ,7,23 indTAS2R6P variation patterns enabled the identification of a unique cluster grouping Prosimians, Hylobatidae and Hominidae families and high level of sequence similarity among primates (>95%), as well as between primates and Prosimians (>75%). TAS2R18P sequence similarity was lower between the mouse lemur and other primates (<70%) and the reconstructed phylogenetic trees showed longer branch lengths after the divergence of the two lineages. In addition, sequence similarity confirmed this view, showing a lower percentage (<70%) of identity between these two pseudogenes and their functional forms, when compared to the similarities between very recent pseudogenes and their functional forms (>90%).Our results were consistent with these findings: TAS2R6P polymorphisms rs11761380 and rs1859645 were in strong linkage disequilibrium (r2 > 0.9) with rs62477710, rs6962558 and rs6962558 SNPs of the functional TAS2R5 gene, suggesting that evolutionary forces acting on this gene could have driven the allele differentiation at the TAS2R6P locus. To test this assumption, Tajima’s D and Fu’s FS tests were performed: these analyses showed that TAS2R6P did not show evidence of departure from neutral expectations while the neighboring TAS2R5 gene, which shared more than 60% sequence similarity with TAS2R6P, showed significant positive values for both these statistics. Since positive values of these tests may indicate balancing selection or may be the result of the confounding effect due to demographic history [TAS2R6P has not undergone recent selective pressures, whereas TAS2R5 showed significant signatures of selection. This is consistent with balancing selection maintaining multiple alleles for long evolutionary times, and extending to maintain allele frequencies at closely linked neutral sites [TAS2R18 also showed significant signatures of selection and both Tajima’s D and Fu’s FS values were significantly positive at this locus in European and Latin American populations. This suggests that balancing selection potentially acted on the genes in these groups. In addition, these populations showed increased values of nucleotide diversity and heterozygosity in the TAS2R18P pseudogene compared to neighboring loci. Moreover, global FST values were unusually high compared to the typical range of 0.10 to 0.16 for estimated FST values in global populations [TAS2R18P alleles and enhancing genetic diversity at this locus in European and Latin American populations.The analysis of the genomic regions surrounding these pseudogenes showed that  history ,44, the al sites . The appulations , suggestTAS2R18P showed no evidence of association with adjacent functional genes. This pseudogene carries a human-specific stop-codon that is shared among H. sapiens Neandertal and Denisova. In addition, the exonic region of this pseudogene carried four polymorphic insertions in the reading frame, upstream of the shared stop-codons and downstream of the human-specific one. These data suggest that these insertions may have shifted the human TAS2R18P reading frame, with a consequent acquired function of this gene, shortly after the homo-chimp divergence. Such an event, followed by balancing selection operating outside Africa, would produce the observed TAS2R18P rs2290318 and rs2290318 different allele distributions in human populations. The estimated ages of these mutations indicate that they arose after the divergence of humans from chimpanzee that occurred 7–8 million years ago [Variants at ears ago . We hypoTAS2R18P in primate evolution, suggesting that this locus may have acquired its function during the evolution of the human lineage, shortly after the homo-chimp divergence. This was followed by a much more recent deactivation due to the stop-gain mutation which was shared among modern humans, Neandertals and Denisovans. We speculate that this event may have been due to the disappearance of some bitter compound only found in Africa, which was specifically recognized by the product of this pseudogene, and that therefore the functionality of TAS2R18P was useful only in a given stage of human evolution.Our results provide evidence for a dynamic role for The article does not report new empirical data since the analyzed sequences were already deposited at public databases, including the 1000 Genomes Project, the UCSC Genome Browser and the Ensemble Genome Browser (see the


Senecio madagascariensis , a native from southern Africa, is a strong competitor in agricultural activities and has toxic alkaloids that may result in high cattle mortality. In Brazil, this weed was collected for the first time in 1995 and has since spread quickly throughout the Pampas region. To better understand the invasion of the fireweed in South America, we used a genetic characterization with internal transcribed spacer (ITS) and microsatellite markers. Based on the ITS data, the southern Brazil populations of S. madagascariensis shared genetic homology with samples taken from the Hawaiian Islands and South Africa. Microsatellite analysis showed the genetic diversity split in two clusters, perhaps intimating the independent introduction of each species into South America. Although fireweed was introduced recently in southern Brazil, the considerable levels of genetic diversity, gene flow, and inbreeding may indicate success in the species establishment in this environment.Non-indigenous plants exhibit different attributes that make them aggressive competitors with indigenous plants and serious threats to biodiversity. Invasive plants have different attributes and mechanisms that allow their dispersal and establishment in new locations . They maTrade and travel are primarily responsible for the introduction of alien organisms across the globe , facilitSenecio madagascariensis Poir., widely known as fireweed, is a member of the Asteraceae family, native to southern Africa . In other parts of Brazil, the weed does not appear to grow below the southern latitude of 29°S. More recently, there is evidence that fireweed, together with otherSenecio species, has caused great economic losses to livestock in southern Brazil, with 45,500 animals lost annually  to clarify the invasion history including the origin of introduced genotypes and the possibility of multiple introduction events; b) to determine the genetic diversity and the structure of the invasive populations; and c) to examine the gene flow among populations.In Argentina, the first specimen of  diploid , and in S. madagascariensis individuals were sampled from 15 sites (hereafter referred to as populations) covering the entire geographical range of the distribution for this species in Brazil and Uruguay , and young leaves were carefully collected in the field for genetic analysis. After drying on silica gel, the leaves were frozen in liquid nitrogen and ground into a fine powder. Subsequently, DNA was extracted from the powdered leaves as described by A total of 307  Uruguay . We collFor these analyses, we used 139 samples belonging to 15 populations  and 24 Gh) and nucleotide diversity (π) (STF(pairwise differences) were obtained in Arlequin 3.5.1.2 (We used DnaSP 5  to detersity (π) , and the 3.5.1.2 . We estiS. madagascariensis microsatellite loci used in this study can be found in the study by Genetic Profiler 2.2 software (Amersham Biosciences). All microsatellite loci were screened for null alleles and large allele dropouts using MICRO-CHECKER 2.2.3  and allelic richness (RA); expected (EH) and observed (oH) heterozygosity, deviations of genotype frequencies from those expected under Hardy-Weinberg equilibrium (HWE); and the inbreeding coefficient (ISF) using Arlequin and FSTAT 2.9.3 (STF (pairwise differences) in Arlequin.Genetic diversity was estimated for each population in terms of the number of alleles (AT 2.9.3 . AdditioSTF) distances among populations, with the statistical significance assessed using a Mantel test with 10,000 permutations. To test the structure and to assess the scale of geographic differentiation among populations, we used STRUCTURE 2.3.3 (Markov Chain Monte Carlo (MCMC), and a model allowing for admixture and correlated allele frequencies. We did not use a prioriinformation on population origin. Five independent runs were performed by setting the number of populations (K) from 1 to 15, and an average likelihood value, L(K), was calculated for each K across all runs. Additionally, we calculated ΔK  was used to test the relationships between geographic and genetic .During the fieldwork, it was possible to observe the rapid spread of STF = 0.312;P <0.001) in the global dataset when the populations were clustered into five geographical regions: South America, South Africa, Madagascar, Swaziland, and the Hawaiian Islands. We analyzed the 15 populations from South America independently from the other global populations (STFdecreased to 0.146 (P <0.001), showing a reduction in the genetic structure. Median-joining network analysis verified the absence of a geographic structure in South American populations  in all analyzed samples, with 31 polymorphic sites in the alignment and one insertion/deletion event and 30 substitutions (11 transitions and 19 transversions). Among the 26 sequence types inferred by DNAsp, nucleotide diversity (π) was 0.0023 ± 0.002, while genetic diversity was 0.763 ± 0.017 . Of the ulations . FSTdecrulations . Among tulations .EH) ranged from 0.577 to 0.771 (mean = 0.715), observed heterozygosity (OH) from 0.352 to 0.597 (mean = 0.456) and ISF ranged from 0.071 to 0.490 (mean = 0.334) across all loci  to 44 (Se-136); within populations, the number of alleles ranged from 2 to 17 (mean = 7.03) and allelic richness ranged from 2.71 to 3.75 (mean = 3.39) . Expecteall loci . Except STF = 0.103), similar to the results from the sequence data (ITS). Across fireweed populations, the Mantel test showed a moderately significant positive correlation between genetic and geographic distances  . The results of Bayesian cluster analyses revealed the presence of structured genetic diversity among groups. Following the method of e.g., POA, NSR, GLO, TOR; STF = 0.387). On average, populations from the Central Group differed significantly from Coastal Group populations in terms of heterozygosity (Ho), inbreeding (ISF) and levels of differentiation among populations (STF). However, there were no significant differences in observed allelic richness (RA) or unbiased gene diversity (Hs) between the two groups (Student's pairedt-test) (Diversity within population explained ca. 90% of the genetic diversity based on microsatellite (dt-test) .P = 0.01). However, given the low genetic structure observed between pairwise populations, the results based on the identification of immigrants should be interpreted with caution because a decrease in genetic differentiation and limited number of loci reduces the ability to correctly identify immigrants. Therefore, we focused on overall patterns of potential migration. Not surprisingly, many of the migrants between Central and Coastal groups correspond with those individuals that show a distinct population pattern in the dendrogram , constituting a secondary invasion. Several works have attributed the arrival of invasive plants via cargo ships from South Africa , while samples from Madagascar and Swaziland were unique and distant from the others . This su Islands . Their iS. madagascariensis populations. Although the ITS sequences of S. madagascariensis have exhibited low variability when compared with other groups of plants , and this information is not available. Therefore, due to insufficient variability, the ITS analysis did not allow us to explore the network relationships among South American populations. Instead, the relationships were analyzed on a global scale, allowing the differentiation of the indigenous populations in southern Africa  (The ITS data showed many sequences in South American populations of fireweed. Over 75% of the sampled sequence types (20 of 26) were found in South America. However, the most thorough comparison is the quantification of the genetic variability of indigenous agascar) .Alliaria petiolata (Bieb.) Cavara and Grande analyzed by Phalaris arundinacea L. in Microsatellite analysis revealed levels of genetic diversity similar  to those obtained from the Hawaiian populations  and the S. madagascariensis in the Pampas region is the considerable level of inbreeding observed here. This reproductive feature facilitates rapid population increase and spread. Additionally, e.g.,S. madagascariensis in the Pampas region. In contrast, the gene flow among populations can influence a rapidly increasing population in preventing genetic drift and inbreeding depression (Although fireweed was introduced recently into southern Brazil, the field observations in recent years suggest that this weed has already reached the current spreading stage. This rapid success may be related to high production, seed dispersal and preadaptation rather than to early aliens . AnotherSenecio madagascariensis has been very effective in its dispersal and establishment in new locations in the Pampas region. The considerable levels of genetic diversity, gene flow and inbreeding indicate that this species has great potential for successful establishment in new environments in southern Brazil. Fireweed is quickly expanding geographically and in population density. Additionally, fireweed is an increasingly significant threat to indigenous species and livestock production in the Pampas region. Researchers aim to control this and other Senecio species in the Pampas region (s region  due to tSenecio madagascariensis into the Pampas region it is still important to investigate more molecular markers and other populations from Uruguay and Argentina. In this regard, the EST collection performed by Thus, for a better understanding of the invasion of


We monitored the last remaining Asian elephant populations in China over the past decade. Using DNA tools and repeat genotyping, we estimated the population sizes from 654 dung samples collected from various areas. Combined with morphological individual identifications from over 6,300 elephant photographs taken in the wild, we estimated that the total Asian elephant population size in China is between 221 and 245. Population genetic structure and diversity were examined using a 556-bp fragment of mitochondrial DNA, and 24 unique haplotypes were detected from DNA analysis of 178 individuals. A phylogenetic analysis revealed two highly divergent clades of Asian elephants, α and β, present in Chinese populations. Four populations  carried mtDNA from the α clade, and only one population (Nangunhe) carried mtDNA belonging to the β clade. Moreover, high genetic divergence was observed between the Nangunhe population and the other four populations; however, genetic diversity among the five populations was low, possibly due to limited gene flow because of habitat fragmentation. The expansion of rubber plantations, crop cultivation, and villages along rivers and roads had caused extensive degradation of natural forest in these areas. This had resulted in the loss and fragmentation of elephant habitats and had formed artificial barriers that inhibited elephant migration. Using Geographic Information System, Global Positioning System, and Remote Sensing technology, we found that the area occupied by rubber plantations, tea farms, and urban settlements had dramatically increased over the past 40 years, resulting in the loss and fragmentation of elephant habitats and forming artificial barriers that inhibit elephant migration. The restoration of ecological corridors to facilitate gene exchange among isolated elephant populations and the establishment of cross-boundary protected areas between China and Laos to secure their natural habitats are critical for the survival of Asian elephants in this region. Elephas maximus) is the largest terrestrial animal in Asia. It occurs in Bangladesh, Bhutan, Cambodia, China, India, Indonesia, Laos, Malaysia, Myanmar, Nepal, Sir Lanka, Thailand, and Vietnam. The total population size of Asian elephants was approximately 34,470–53,720 across their range [The Asian elephant , including size, shape, and presence of broken tips; and other morphological characteristics . From 19http://www.esri.com/software/arcgis/arcview).654 elephant dung samples were collected from December 2003 to September 2007 (over 100 samples each year) from five regions, including Mengla, Shangyong and Mengyang in Xishuangbanna, Nangunhe in Lincang and Simao in Pu'er. Up to 5grams of dung was collected for each sample peeled from outer layer, and stored in ≥75% ethanol. New gloves and collection tubes were used for collecting each sample to avoid potential cross contamination between different samples. All samples were stored at ambient temperature and transported to the laboratory within eight weeks of collection. The locations of all the samples were recorded via GPS and mapped using Arcview3.2 (Loxodonta africana and L. cydotis) had been published[DNA extraction was conducted twice for each fecal sample following previously described protocols . Prior tpublished–23. We tpublished. Touch-dpublished, Nyakaanpublished, Eggert published and Cai published , including the 3′ end of the cytochrome b gene, the transfer RNAs for threonine and proline, and partial control region, the latter of which included the hyper variable left domain of the d-loop ,24,27–29et al. .http://www2.unil.ch/biomapper) to analyze the suitability of elephant habitats across the entire study area [3S technology  was used to analyze the data obtained from both field surveys and satellite images. Landsat images of elephant range area from the years 1975, 1990, 2005, and 2009 in the Worldwide Reference System (WRS) were used to analyze vegetation cover changes based on the transition matrix method. We used the 90-m resolution Digital Elevation Model (DEM) derived from Shuttle Radar Topography Mission (SRTM) data to generate a 10-m DEM of our study area, given our plot size, through ordinary Kriging Interpolation processes . Elevatiudy area . Based oudy area .Vanderploge and Scavia’s selectivity index was used to evaluate the habitat selection of elephants according to different environmental parameters ,15,16. BThe research based on fecal DNA lab studies and behavioral observation in the field. The research did not have any animal welfare problem to those elephants during the research period. The corresponding author LZ is a member of the ethic committee of the International Union of Biological Sciences. Although there is not ethics committee in the Beijing Normal University during the study period, the corresponding author is leading a research professor group to develop an animal research protocol for the future study now.Yunnan Forestry Department approved our research in all elephant ranges in China including Lincang, Pu'er and Xishuangbanna . MOUs signed between Beijing Normal University and Xishuangbanna National Nature Reserve management authority, and with Nangunhe National Nature Reserve management authority. All field studies did not have any interaction with endangered or protected animals. An ethic committee of Xishuangbanna National Nature Reserve was consulted regarding interviewing the villagers. We only interviewed those villagers who agreed to provide elephant information to the nature reserve's community monitoring network. No any identifying or demographic information was obtained from the villagers. The villagers would be asked whether they were willing to answer our questions, and they could decide to refuse to answer any question during the interview. The Xishuangbanna National Nature Reserve signed and renewed agreements with those villagers working for the community monitoring network annually.Using traditional transects surveys in the field, combined with morphological identification of individuals from photos obtained over the past decade, we estimated the elephant population number in China to be between 221 and 245 .A total of 479 DNA samples  were successfully extracted and amplified. The mean number of alleles per population ranged from 2.33 (SM) to 3.67 (ML and MY). Expected heterozygosity varied from 0.3167 (SM) to 0.4198 (ML), and observed heterozygosity varied from 0.3565 (NGH) to 0.4132 (ML) . In geneet al. [et al. [A 556-bp fragment of mtDNA was sequenced from 178 genotype individuals. MtDNA diversity in the ML, MY, SM and SY populations was lower than in the other South and Southeast Asian populations, but the NGH population had similar diversity as others . Twenty-et al. . There wet al. . An addiet al. –28. Two  [et al. . The NanIn our ten independent replicates of the Bayesian Structure analysis for the estimation of K, values of LnP(D) sharply increased as K increased from 1 to 4, and delta K had a prominent peak at K = 4. Under the optimal cluster number (K = 4), the elephant populations separated into four clusters . IndividSince 1960, much of the original forests in Mengla County had been cut down to plant rubber trees, and since 1980, the affected area had continued to grow. Due to the rapid increase in the price of rubber in the international market in recent years, more original forest had been destroyed to plant rubber trees in Xishuangbanna. This had ecologically isolated Mengxing-Xiangming-Yiwu, including the large and small Kongming Hills and Yao areas, and formed geographical barriers between Shangyong-Mengla (SY-ML) and Pu’Er-Mengyang (SM-MY), driving elephants to withdraw their traditional home ranges. There had been no migration between elephant populations from the two areas over the past several decades. Genetic drift among wild Asian elephant populations is expected to proceed slowly because elephants are large mammals with large territories, long reproductive cycles , and low birth rates . Over the past five decades, extensive removal of forests and reclamation of farmland had fragmented elephant habitat. The original forests had contracted and became fragmented by rubber plantations and farmland, resulting in a lack of gene flow between the two isolated populations. However, as these events were recent, no significant genetic differences and no obvious genetic divergence had yet arisen between the two populations.The optimal solution for enhancing gene flow among the geographically isolated populations is to establish ecological corridors. The feasibility and necessity of building ecological corridors among the Mengyang (MY), Shangyong (SY) and Mengla (ML) nature reserves had been evaluated ,16. SuchST test of haplotype frequencies revealed a significant difference between the Nangunhe population and the other four geographic populations in Pu’Er and Xishuangbanna , we found that the marginality value of the Asian elephant in Pu’Er was 0.992, which was very high and approached 1. This result indicated a deviation of their preferable niche from the average value of the background environment and suggested that the selection of environmental variables was not random. The tolerance value was 0.315, indicating that the niche breadth of the Asian elephant was narrow and that elephant survival was restricted by environmental conditions.Based on Habitat Suitability Index (HSI) from the ENFA model, we divided the habitat of the Asian elephant in into four different categories: unsuitable habitat (0<HSI≤12), marginal habitat (12<HSI≤38), optimal habitat (38<HSI≤64) and suitable habitat (65<HSI≤100).2 of optimal elephant habitat, 574.32 km2 suitable habitat, 2,909.48 km2 marginal habitat and 38,722.32 km2 unsuitable habitat. The unsuitable habitat accounted for 90.86% of the total area, while optimal habitat regions only 0.96% of the total area. According to the habitat distribution map, both optimal and suitable habitats, which comprised small areas, occurred in all counties of Pu’Er, except Ximeng County. The largest area of optimal habitat  and suitable habitat  were found in Jiangcheng County. Landscape analysis showed that optimal, suitable, and marginal habitats all suffered from severe fragmentation, low connectivity, and high human disturbance.The results showed that Pu’Er provided 409.32 kmBased on these results, we recommend that new protected areas be established as soon as possible in the range of the Asian elephant, coupled with effective and legal protection measures in Pu’Er. Additionally, it is important to establish ecological corridors to link all the small family groups in the region and to enhance the connection with those elephants in Xishuangbanna.Based on the separated vegetation and administrative boundaries, we divided the Xishuangbanna Prefecture into two parts: Jinghong-Menghai and Shangyong-Mengla. The marginality values of the two parts were both greater than 1, and the tolerance values were both approximately 0.5, suggesting that the elephants were selective with respect to environmental variables, although they had some tolerance and adaptability to the environment. Of all of the environmental factors, vegetation, particularly the distribution of bamboo, was the primary factor affecting habitat quality in both areas, indicating that food was the most important factor influencing habitat selection by the Asian elephant.2 of optimal elephant habitat, 1,965.25km2 of suitable habitat, and 2,589.25km2 of marginal habitat; these values were predicted from all of the distribution points of all elephants, including both farmland and non-farmland. In contrast, unsuitable habitats occupied 71.2% of Xishuangbanna. We found that 30.7% of suitable habitats were farmland, which was the result from the software “Biomapper” analysis based on those plots with elephant occupancy inside farmlands. Analysis of the distribution points within non-farmland areas alone identified 436.75km2 of optimal habitat, 1,443.75km2 of suitable habitat, and 974.75km2 of marginal habitat. These results showed that the amount of suitable habitat decreased sharply after farmland sites were excluded. Farmland had always been regarded as components of the elephants’ foraging habitat and migration path, rather than true suitable habitat. However, loss and fragmentation of the elephant habitat by degradation and establishment of rubber plantations had resulted in crop raiding and the dispersion of elephants out of that nature reserves, leading to severe human-elephant conflicts.Currently, Xishuangbanna provided 952.75kmThe Asian elephant population was distributed over three main areas in Xishuangbanna Prefecture: Mengyang, Mengla, and Shangyong. Suitable habitats outside of the nature reserves contained the elephants’ migration routes. This result suggested that ecological buffers should be established in these key regions to connect the Mengyang-Lancang (Corridor 1), Mengyang-Simao (Corridor 2), Mengyang-Jiangcheng (Corridor 3) and Mengla-Shangyong (Corridor 4) habitats as elephants migrate through the corridors (see S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Laminaria digitata is an important component of the ecosystem in Northern European rocky intertidal habitats. We studied four L. digitata populations for the effects of marginality on genetic diversity and sexual reproduction. Two populations were marginal: One  was well within the geographic range, but was genetically isolated from other populations by large stretches of sandy beaches. Another population was at the range limits of the species  and was exposed to much higher seasonal temperature changes. Microsatellite analyses confirmed that these populations showed decreased genetic and allelic diversity, consistent with marginality and genetic isolation. Sporophytes from both marginal populations showed greatly diminished spore-production compared to central populations, but only the southern-limit population (Quiberon) showed a high propensity for producing unreduced (2N) spores. Unreduced 2N spores formed phenotypically normal gametophytes with nuclear area consistent with ≥2N DNA contents, and microsatellite studies suggested these were produced at least in part by automixis. However, despite this being the dominant path of spore production in Quiberon sporophyte individuals, the genetic evidence indicated the population was maintained mostly by sexual reproduction. Thus, although spore production and development showed the expected tendency of geographical parthenogenesis in marginal populations, this appeared to be a consequence of maladaptation, rather than an adaptation to, life in a marginal habitat.Adaptation to marginal habitats at species range-limits has often been associated with parthenogenetic reproduction in terrestrial animals and plants. Laboratory observations have shown that brown algae exhibit a high propensity for parthenogenesis by various mechanisms. The kelp  The ubiquity of sex among eukaryotes has long been a paradox due to the high costs associated with mating and recombination Geographical parthenogenesis refers to the case where closely related sexual and asexual lineages exhibit distinct distributions Ectocarpus sp., Laminaria japonica, Undaria pinnatifida and Lessonia nigrescensEctocarpus sp. Brown algae (Phaeophyceae) are a closely-related group of multicellular organisms that form essential structural components of near-shore marine ecosystems. These organisms exhibit a complex haplo-diplontic life cycle where both haploid and diploid phases exhibit vegetative growth. Diploid sporophytes produce spores by meiosis, which then undergo mitotic division to form multicellular haploid gametophytes. Fertilization eventually restores the diploid sporophyte state, which again grows vegetatively. Multiple mechanisms to avoid or modify sexuality exist within brown algae. Haploid cells from the gametophyte can undergo endomitosis to restore diploidy without fertilization in Laminaria digitata exhibits a broad distribution along the European coast, with the southern limit clearly defined on the Atlantic coast of Southern Brittany Laminaria digitata. We studied four populations for the effects of marginality on genetic diversity and sexual reproduction. Two populations were marginal: One  was well within the geographic range, but was genetically isolated from other populations by large stretches of sandy beaches that represent a local barrier of unfavorable habitat in vitro culture and microscopic observations.The kelp Two or three sites in each of four regions along the Brittany coast  were selected according their contrasting geographical and ecological characteristics , Table 1http://www.ecoop.eu.). The full remote sensing dataset for the entire Brittany coast is described in Gallon et al. L. digitata sporophytes was directly recorded using temperature data loggers TidbiT v2-UTBI-001 Hobo Onset fixed to rocks in the Roscoff site 48°42′44″N 3°57′30″W between July 2009–March 2010, and in the Quiberon site 47°28′09″N 3°05′40″W between July 2009–January 2010. Data loggers recorded temperature every 15 minutes.Sea surface temperature (SST) data were obtained from daily High Resolution Sea Surface Temperature from 1992 to 2003 over the IBIROOS area  in a 0.044° Geographical Grid AVHRR (Advanced Very High Resolution Radiometer) from NOAA a posteriori multiple comparisons of means (α = 0.05). Comparisons of median temperature between sites recorded from data loggers were made using Mann-Whitney tests MINITAB v. 13.2 .Comparison of mean seasonal temperature between sites was tested using a 2-way ANOVA with season  and location  as factors. Differences among locations were considered in relation to season. The response variable was the mean temperature. Data was log transformed in order to fit the assumptions of normality and homogeneity of variances. Type III sum of squares was used for tests of significance. All statistical analyses were done with MINITAB v. 13.2 , by performing ANOVA and Tukey’s tests for Laminaria digitata , and two locations, Quiberon and Locquirec, were sampled, one at the southern range limit of the distribution and the other in northern Brittany. These latter locations were considered to be marginal because they represents the southern-most population of L. digitata and the other is isolated by sandy beaches. From 23 to 38 mature sporophytic individuals were collected from these four regions. This sampling was done to analyze spores by flow cytometry and culturing.A total of 135 fertile thalli were sampled along the Brittany coast between Locquirec and Quiberon  from JulIn addition, a hierarchical sampling was performed in order to compare genetic diversity within and among the four regions  and proteinase k 10 mg/mL according to Wattier et al. For the genetic analyses of the sporophyte individuals, DNA extraction and PCR amplifications of seven microsatellite loci  were performed using the same protocols as those described in Billot et al. PCR products were electrophoresed on 6% polyacrylamide denaturing gels using an automated DNA sequencer (Li-Cor 4200) along with a DNA sequence of known length to estimate allele sizes to ensure that allele sizes corresponded exactly to those estimated in other studies IS estimates of the average deviation from random mating within populations Standard measures of genetic diversity, including mean number of alleles per locus (Na), unbiased expected heterozygosity He; , and all2) were cut off from each plant, cleaned in running tap water and exposed to air for 30 minutes. Ten fragments per plant were transferred to four 50 mL plastic Falcons tubes containing 40 µL of filtered seawater at 0.2 µm, set on ice and agitated. Fertile fragments were withdrawn from the tubes after 12 h. Each spore suspension was divided in three aliquots. The first aliquot was used to estimate the number of released spores per ml by light microscopy with Neubauer chambers, the second one was used to obtain gametophyte cultures, and the third one was fixed with 1% formaldehyde for flow cytometry analyses.Fertile fragments of equal size  on a FACSCanto II flow cytometer . Exponentially growing cultures of haploid Emiliania huxleyi RCC1217, a unicellular eukaryotic phytoplankton, were fixed with the same fixative and added as an internal standard during staining. This species was selected as it could be easily distinguished from L. digitata spores based on chlorophyll, forward scatter, and side scatter properties. Early trials on isolated nuclei obtained from live spores using previously established protocols Ploidy levels (relative DNA content) of −2s−1. Culture medium was changed once a week and observations were done monthly for 10 months.Five drops of each spore suspension were inoculated in 55 mm x 14 mm plastic Petri dishes with Provasoli enriched seawater and incubated in a culture chamber set at 15°C, 12∶12 light:dark, 25–35 µmol photons mSex ratio estimations were done on 9 progenies from Quiberon. Male and female gametophytes were identified according to their morphological characteristics using a Nikon Eclipse TE300 inverted microscope . Female gametophytes are characterized by large cells and filaments with few branches whereas male gametophytes are smaller and display highly branched filaments formed by small cells. These morphological differences make them unambiguously identifiable under the light microscope. The numbers of male and female gametophytes were determined by counting their occurrence in three visual fields per slide using the 10x objective. Sex ratio was expressed as the frequency of males per progeny ).www.nih.gov).Comparative ploidy levels of 10 male and female gametophytes from 2 progenies from Quiberon and 2 gametophytes from 2 progenies from Roscoff were estimated by measuring nuclear area using the fluorescent DNA Hoechst stain  and epifluorescence inverted microscope Zeiss Observer Z1 , as described in Bothwell et al. (2010). Images of nuclei were analyzed with ImageJ software . The season by location interaction was significant   . The Qui<0.0001) .The number of alleles varied from 2  to 17 . Microsatellite analysis  showed tIS, corresponding to the deficit of heterozygotes in a population, was relatively low for all populations studied, ranging from −0.019 in Sieck (Roscoff) to 0.085 in Molène (Porspoder). No clear difference was observed between marginal and central populations. Of the 396 individuals analyzed, 390 (98.4%) had unique seven-locus genotypes and 3 genotypes were repeated two times . These individuals with the same multilocus genotype were observed only in two sites in the marginal region of Quiberon . To investigate causes, spore production was examined from over 25 individuals from all four sites. Sporophyte individuals from Quiberon and Locquirec both showed greatly diminished spore release in comparison with individuals from Roscoff and Porspoder. Average spore production was 14-fold higher in Roscoff and 25-fold higher in Porspoder in comparison to Quiberon .The spores produced by individuals from Roscoff, Porspoder, and Locquirec populations were predominantly haploid, whereas individuals from Quiberon produced high percentages of diploid spores , Fig. 4.The spores produced by the sporophyte individuals from sites in Quiberon germinated and developed normally into gametophytes. These gametophytes did not differ in color, shape or texture from gametophytes obtained from germination of spores from Roscoff individuals. In culture, these gametophytes initiated new juvenile sporophytes that for the first steps of development did not differ from those obtained from gametophytes of Roscoff.Average sex ratios of the gametophytes obtained from spores of Quiberon were balanced (average of 0.461+/−0.185). However, three of the nine studied progenies displayed female bias, as judged by comparison to the binomial law .The nuclei of filamentous gametophytes were visible under epifluorescence microscopy . The gamThe heterozygous sporophyte  sampled L. digitata when temperature reaches 18°C, whereas reproduction is maximum at 10°C. Both the Quiberon and Locquirec populations showed reduced genetic diversity (He) and allelic richness (AR), consistent with lower immigration and diminished population size . The Locquirec population was not near the geographic range limits of the species, yet this population is isolated from other populations by extensive sandy beaches. In contrast, the Quiberon population represents the southern-most limit of the range of the species distribution, and is exposed to more pronounced seasonal changes in temperature, with, in particular, a higher summer temperature that is frequently above the favorable temperature conditions in the field to produce meiospores L. digitata populations in Quiberon and/or Locquirec might also be displaying some form of geographical parthenogenesis, and that the parthenogenetic mechanism selected might help to distinguish between alternative hypotheses. For example, the production of spores by apomeiosis would preserve heterozygosity and locally adapted genotypes, while also permitting retention of a heteromorphic life cycle. Genetic data from the Quiberon populations in the field indeed detected individuals sharing the same MLGs produced by asexual reproduction at a low frequency (i.e. possibly produced by apomeiosis and/or fragmentation).A variety of mechanisms for parthenogenesis or cloning have been demonstrated to be available to Laminarian algae in laboratory studies in vitro. Three mechanisms could allow diploid gametophytes: 1) Apomeiosis, the complete replacement of meiosis with mitotic divisions, would cause the production of gametophytes that were essentially genetically identical to the sporophyte parents, 2) Automixis, where two of the four nuclear products of meiosis fuse, resulting in diploid spores that were genetically different and, 3) Endomitosis, that would involve haploid spores restoring diploidy by chromosome doubling The nuclear area of male and female gametophytes developed from predominantly 2N spores of Quiberon individuals was consistent with gametophytes that are at least diploid. These diploid gametophytes are able to survive and to develop normally L. digitata sporophyte parents exhibited only one allele at each locus, heterozygous gametophytes were detected, and two individuals showed a distinct diploid genotype than their parents (heterozygous at only one locus compared to parents that were heterozygous at both loci). As Quiberon parents typically produced some 1N spores , this result could be produced by a combination of 1N spores and 2N spores produced by automixis . However, nuclear area measurements suggested that the Quiberon gametophytes were apparently predominantly of diploid DNA content. This would imply that most progeny were homozygous diploids, with recombination causing heterozygosity in a small proportion. That would be consistent with failure specifically of Meiosis II after recombination between homologous chromosomes in Meiosis prophase, or automixis involving re-fusion preferentially of the two nuclear products of Meiosis II . This mechanism, which has also been proposed in animals Thalassiosira angulataAlthough most of the gametophytes successfully cultured and genotyped from Quiberon L. digitata to engage in parthenogenesis, microsatellite genetic data are not consistent with it being the major reproductive mode in natural populations. Automixis, especially terminal fusion automixis, would be expected to produce populations that are deficient in heterozygotes. Yet, although FIS values for Quiberon were two times higher than in Roscoff, these values stayed close to zero, and so there was no important heterozygote deficiency. Endomitosis would lead to completely homozygous individuals and can also be ruled out as being important in the L. digitata natural populations examined here. Finally, the small number of repeated multi-locus genotypes detected suggests that parthenogenesis by apomeiosis might indeed be more frequent in Quiberon than in the central populations, but still remains rare.Surprisingly, despite the apparent propensity for Eucypris virens that reported that triploid, but not diploid asexual clones were able to colonize higher latitudes, expanding the range. It was suggested that the wider distribution range of triploids was due to elevated ploidy rather than asexuality Polyploidy is often associated with asexuality L. digitata is sensitive to elevated temperature, and the observed change in its reproductive system might be an effect of exposure to higher and more fluctuating temperatures. The diminished spore production combined with the capacity for non-reduced spores to develop into gametophytes creates a greater proportional capacity for asexuality in marginal populations, similar to the pattern of geographical parthenogenesis frequently observed in terrestrial species. However, the production of high percentages of 2N spores by Quiberon individuals was not reflected in a restoration of total spore production capacity and gametophyte formation. Furthermore, population genetics shows no evidence that parthenogenesis is actually dominant in the maintenance of marginal populations of L. digitata.Marginality is usually associated with higher abiotic stresses, and the Quiberon habitat exhibits higher and more fluctuating regimes of temperature than central populations. As all kelps, Laminaria species) does not increase enough to compensate. Such a maladaptive response at the southern edge of its distribution means that this European kelp species is at risk of local extinction as predicted by Ecological Niche Models under global change scenarios Of the many hypotheses often invoked to explain the pattern of geographical parthenogenesis seen in terrestrial organisms, a dominant theme is that parthenogenic reproduction might provide particular genetic advantages either in avoiding inbreeding in small populations or in preserving well-adapted genotypes when dominant abiotic stressors are predictable, as opposed to biotic stressors which are highly variable and subject to co-evolution Figure S1Photo of an acrylamide gel that shows the genotyping of a sporophyte parent (lane 1) and three unambigously identifiable descendents including two homozygous gametophytes  and one clear heterozygous gametophyte (lane 4).(DOC)Click here for additional data file.Table S1Nuclear area of gametophytes from Quiberon and Roscoff.(DOC)Click here for additional data file.Table S2Genotyping results for 8 progenies of Quiberon with two microsatellite loci. Parents 1–8 are from Quiberon, parent 10 is from Roscoff. In each case the alleles are given for each locus in the parents, and the number of progeny with each combination of parental alleles is given . Gametophytes where only a single allele was detected at each loci that was successfully scored might be haploid or homozygous diploids.(DOC)Click here for additional data file.


The objectives of this study were to assess linkage disequilibrium (LD) and selection-induced changes in single nucleotide polymorphism (SNP) frequency, and to perform association mapping in popcorn chromosome regions containing quantitative trait loci (QTLs) for quality traits. Seven tropical and two temperate popcorn populations were genotyped for 96 SNPs chosen in chromosome regions containing QTLs for quality traits. The populations were phenotyped for expansion volume, 100-kernel weight, kernel sphericity, and kernel density. The LD statistics were the difference between the observed and expected haplotype frequencies (D), the proportion of D relative to the expected maximum value in the population, and the square of the correlation between the values of alleles at two loci. Association mapping was based on least squares and Bayesian approaches. In the tropical populations, D-values greater than 0.10 were observed for SNPs separated by 100-150 Mb, while most of the D-values in the temperate populations were less than 0.05. Selection for expansion volume indirectly led to increase in LD values, population differentiation, and significant changes in SNP frequency. Some associations were observed for expansion volume and the other quality traits. The candidate genes are involved with starch, storage protein, lipid, and cell wall polysaccharides synthesis. BecauseAssociation mapping refers not only to the identification of QTLs, but also to the identification of candidate genes based on statistical significance between markers and phenotype. Its main advantages relative to QTL mapping are the use of breeding population instead of population derived by crossing two inbred or pure lines and more precise identification of candidate genes . The assZea mays L.), LD analyses and association studies have been performed using inbred line panels. The LD analysis performed by 2 value of 0.20 for SNPs at a spacing of 200 kb. Based on a panel of 240 inbreds genotyped for 29,619 SNPs, 2values ranging from 0.21 to 0.25. LD blocks were observed on all chromosomes, with the LD decay occurring over regions of 200-300 kb.In maize  Zhuk.) chromosome regions containing QTLs for quality traits.To our knowledge, little information is available on LD and SNP frequency changes due to selection in maize and special maize breeding populations, nor have QTLs been identified in such populations by association mapping. Thus, our objectives were to assess LD and SNP frequency changes due to selection, and to perform association mapping in popcorn (4 (generated from four inbred progeny selection cycles applied in the Viçosa population), Beija-Flor c1 and Beija-Flor c4 , and UFV MP-1 and UFV MP-2 . The first seven populations, representing tropical germplasm, were cultivated during the 2012-2013 growing season in an experimental field at the Federal University of Viçosa (UFV), Minas Gerais, Brazil. The populations UFV MP-1 and UFV MP-2, representing temperate germplasm, were cultivated in 20-L pots in a greenhouse at UFV, in 2014. Leaf samples of 100-150 young plants were collected from each population for genotyping.The populations employed in this study were Viçosa, Viçosa cycles 1 (c1) and 4 (c4) , Viçosa cycle 2 (c2) fsf (derived from Viçosa after two full-sib selection cycles), Viçosa SThe populations derived from Viçosa and Beija-Flor c1 were obtained by progeny and plant-within-progeny selection for expansion volume. In trials of non-inbred progeny, 196 progeny were assessed using a 14 x 14 lattice design with two replications, at the UFV experimental station located in Coimbra, Minas Gerais. The 20 superior half-sib families were recombined using one male row to four female rows. In the recombination plots with the 20 superior full-sib families, at least one female per family was crossed with a male from another progeny, providing 380 full-sib families. In the half-sib progeny recombination plots, 196 plants were selected, providing the half-sib families for the next cycle. The 196 full-sib families for the next cycle were selected based on the expansion volume of the female parent.1 progeny, 309 S2progeny, 277 S3 progeny, and 268 S4 progeny. In each progeny, three to five plants were selfed. The progeny for the next cycle were obtained by selecting the best families and then the superior selfed plants. Populations Viçosa S1 to Viçosa S4 were obtained by recombining all assessed inbred progeny. The progeny tests and recombination plots were conducted during 1998-1999 to 2007-2008 growing seasons. To assess expansion volume, we used a hot air popcorn popper  or a 27-L microwave oven (900 W), and samples of 30 g per plot and 10 g per plant.The inbred progeny were assessed in an experimental field at UFV using an incomplete block design with replications only for the controls . Each incomplete block consisted of 10 progeny and the controls. The trials included 344 SDNA was extracted using KitWizard Genomic DNA Purification kit according to the manufacturer's protocol with modifications. A Qubit 2.0 fluorometer  and a NanoVue spectrophotometer  were used to assess DNA quantity and purity level, respectively. Individuals were genotyped from 50 ng/μL DNA samples using GoldenGate assays . Genotyping was performed on an Illumina BeadXpress. Individuals were genotyped for 96 SNPs located in chromosome regions containing QTLs for the following popcorn quality traits: expansion volume, flake volume, unpopped kernel number, and flake size . The SNPExpansion volume was assessed in a 27-L microwave oven (900 W) using samples of 10 or 30 g per plant. To provide an estimate of error variance for expansion volume, two measurements were obtained for most plants in the temperate populations. Hundred-kernel weight was measured with an electronic scale. Average kernel sphericity was calculated as the ratio of geometric mean diameter  to kernel length  of 10 randomly-selected kernels per plant . The numBecause no reference was available for interpreting the LD analysis results for the popcorn populations, we also analyzed two simulated populations. Simulated population 1 (Pop1) was a second generation composite obtained by crossing two populations in linkage equilibrium. This population was in LD only for linked markers and/or genes. The second simulated population (Pop2) was obtained from Pop1 after 10 cycles of random crosses assuming sample sizes of 100 and 300. The effective population sizes were 200 and 600, respectively. The program used for simulating genotypes and phenotypes - REALbreeding - has been developed by the second author using REALbasic software . The ave-1. We also defined bidirectional dominance (-1.2 ≤ (d/a)i ≤ 1.2) and used a heritability of 50%. The proportion of the phenotypic variance explained by each QTL was set to 2.4%.Nineteen QTLs (candidate genes) and 81 minor genes affecting the expansion volume trait were randomly distributed along the nine chromosomes. Based on user input, which included minimum and maximum genotypic values for homozygotes, degree of dominance (d/a), direction of dominance, and broad sense heritability, the REALbreeding program provided the phenotypic values of each genotyped individual. The phenotypic values were computed from the true population mean, additive and dominance values, and error effects sampled from a normal distribution. The error variance was computed from the broad sense heritability. The minimum and maximum genotypic values of homozygotes were 5 and 50 mL gST) was computed using GenAlEx 6.5  was less than 1%. Only SNPs in Hardy-Weinberg equilibrium, as assessed using a chi-square test at the 5% significance level, were used for the LD analysis. The LD measures were D, D', and rSNP sequences in FASTA format were obtained from the NCBI Database of Single Nucleotide Polymorphisms and used to perform BLAST searches against the 'B73'RefGen_v2 reference genome at the MaizeGDB. Information on gene products, expression, and ontology  was obtained using the MaizeCyc database, the Maize eFP browser, and the Gramene database. To identify candidate genes, we searched up to 1 Mb upstream and downstream of each SNP region.2 values were observed in Beija-Flor c4 and Viçosa c2 fsf, respectively. The lowest and highest average D' values were observed in UFV MP-2 and Viçosa c2 fsf, respectively. In the simulated populations, the number of polymorphic SNPs agreed with the value expected at the 5% significance level (at least 91), the expected heterozygosity approached the maximum value, and 10 generations of random mating decreased LD values. Also as expected, average LD values for linked SNPs were greater than those for linked and unlinked SNPs. This decrease occurred only in 50% of the cases for the popcorn populations. In the tropical popcorn populations, D-values greater than 0.10 were observed for SNPs separated by 100-150 Mb. Most of the D-values relative to the temperate populations were less than 0.05  and an indirect decrease in 100-kernel weight , but were characterized by intermediate (0.4) to low (0.2) magnitudes, especially for tropical populations. The sign of the estimates was also variable depending on the population.According to a l weight . Compare2 ranged from approximately 10% to 51%, 13% to 20%, and 64% to 118%, respectively. The increments for linked and unlinked SNPs were even higher. The genetic differentiation was proportional to the number of cycles. Relative to the Viçosa population, FST ranged from 0.09 in Viçosa c1 to 0.16 in Viçosa S4. The highest FST estimates, ranging from 0.19 to 0.34, were observed between tropical and temperate populations. The lowest value (0.00) was evidence of no genetic differentiation between Beija-Flor c1 and Beija-Flor c4 populations. Interestingly, genetic differentiation between the improved Viçosa and Beija-Flor populations was negligible (less than 0.05).Selection for expansion volume was accompanied by increases in LD values in the Viçosa population , populat4 and BeijaFlor derived populations as a fifth subpopulation, and a non-existent population . Based on the Waples' test, the number of SNPs with significant (p < 0.05%) allele frequency changes relative to the base populations (Viçosa and Beija-Flor c1) ranged from 23 in Viçosa c1 to 41 in Viçosa c4, proportional to the number of cycles. The average change in SNP frequency ranged from 0.21 in Beija-Flor c4 to 0.30 in Viçosa c4 and Viçosa S4, which was also proportional to the number of cycles. Unexpected significant changes in SNP frequencies in the simulated population Pop2, with a sample size of 300, ranged from 0.10 to 0.14 (average of 0.12). It should be noted that one to seven SNPs in almost all bins showed significant frequency changes.These findings are partially consistent with results from the population structure analysis. The inferred plateau method uncovered six subpopulations corresponding to UFV MP-1, UFV MP-2, Viçosa, three Viçosa-derived populations as a fourth subpopulation, Viçosa SNot a single significant association at a FDR of 5% was observed in the popcorn populations. Assuming a FDR of 10%, we found three associations for expansion volume, two associations for 100-kernel weight, and seven associations for kernel density in distinct populations . The BayTables S1 andS2 in the Supplementary Material). In general, the identified candidate genes are involved in starch biosynthesis, lipid metabolism, cell wall polysaccharide  biosynthesis, and storage protein metabolic/catabolic processes such as α-zein synthesis. Expression levels of these candidate genes in seeds  are variable, generally ranging from intermediate to high depending on the reproductive stage (R1 to R4).We found one or more candidate genes for each SNP with a significant association at a FDR of 10% and/or a significant frequency change at 0.05% , genetic drift (due to finite population size), migration, and mutation. Migration and mutation should be irrelevant causes. The inclusion of the simulated populations evidenced that genetic drift is not a relevant cause. Notice the equivalence between the parameters estimated in the populations with sample sizes 300 (lower genetic drift) and 100 (higher genetic drift). It should be also highlighted that the average random change in SNP frequencies in the simulated populations was lower than the average changes in the popcorn populations.Selection based on expansion volume indirectly led to a decrease in the number of polymorphic SNPs and in the number of SNPs in Hardy–Weinberg equilibrium, and an increase in expected heterozygosity, Fcys2 locus, one allele showed a decrease with selection for high methionine content (from 0.25 to 0.01) and an increase with selection for low methionine content (from 0.25 to 0.74). 2 was higher for Syngenta lines (0.61) than for public lines (0.39). For SNPs separated by 1 to 10 Mb, the average r2 was 0.03 and 0.04 for public and Syngenta inbreds, respectively. In a study on the extent of LD in commercial maize germplasm, 2 values for unlinked and linked SNPs respectively ranging from 0.009 to 0.013 and 0.020 to 0.029 relative to four heterotic pools.Our results revealed greater LD for SNPs separated by more than 10 Mb in tropical populations than in both temperate and simulated populations. In general, tropical populations showed average LD values greater than those of temperate populations. However, LD in the tropical populations was lower than that observed in a secondgeneration composite and higher than that in the composite after 10 generations of random crosses. 2:3, BC1S1, or BC2F2designs. From an analysis of RILs in four environments, The differing efficacy of least squares association mapping and Bayesian GWAS to detect true associations, as evidenced by the analysis of the simulated data, can be best attributed to the reduced proportion of phenotypic variance explained by the QTLs. In QTL mapping studies performed by To identify candidate genes for expansion volume and other popcorn quality traits, we based our analysis on kernel physiochemical characteristics affecting expansion volume, such as kernel size, shape, and density as well as kernel moisture, starch, protein, and fatty acid contents. The endosperm is the most important kernel component affecting popping, while starch is the major polymer involved in popcorn expansion. Popcorn kernels contain both vitreous (horny or hard) and opaque (floury or soft) endosperm. During popping, starch granules in the vitreous endosperm are highly expanded and responsible for flake formation, whereas starch granules in the opaque endosperm appear to undergo little change. Acting as a pressure vessel during heating, the pericarp gives popcorn its distinct popping ability. The pericarp is the primary source of fiber in the popcorn kernel, while the germ (embryo) is the primary source of lipids. Other than fracturing the pericarp, popping does not substantially alter either the germ or pericarp. In general, small- to medium kernel size (lower 100-kernel weight) and greater kernel sphericity, kernel density, ratio of vitreous to opaque endosperm, and linoleic acid, oleic acid, and α-zein protein levels are associated with greater expansion volume. Pericarp damage and thickness also greatly affect expansion volume (One candidate gene for SNPs SYN4651 and SYN4646 is GRMZM2G058472 (Gramene ID). The gene product is a glycosyltransferase involved in synthesis of glucuronoxylan, a polysaccharide of the hemicellulose fraction of the cell wall. The gene exhibits intermediate expression level in the pericarp during the middle fruit ripening stage (R3). GRMZM2G060579 is the candidate gene for SNPs PZE-107105783 and SYN36108. This gene encodes an uncharacterized protein involved in pectin biosynthesis and shows an intermediate level of expression in the entire seed  during early to middle stages of fruit ripening (R1 to R4). The candidate gene for SNP PZE-107083429, GRMZM2G133613, encodes N-acetyllactosaminide 3-alpha-galactosyltransferase. This enzyme participates in glycoprotein synthesis, which is important for endosperm development . The cansugary1), a determinant of starch composition in maize kernels , which encodes glucose-1-phosphate adenylyltransferase large subunit 1 involved in starch biosynthesis. Mutation at this locus greatly reduces starch levels in the endosperm (ohp2). Similar to the well-known opaque-2 locus (o2), this gene also regulates the expression of many members of the zein multigene family of storage proteins (Among the SNPs with significant frequency changes, PZE-104008299, SYN33394, and SYN526 are particularly of interest. Changes in the frequency of these SNPs ranged from 0.23 to 0.38, 0.15 to 0.39, and 0.35 to 0.50, respectively. The SNP PZE-104008299 is located in a region containing at least 12 genes coding for precursors of α-zeins, which are storage proteins accounting for 70% of maize endosperm protein . All α-zTo conclude, our results confirm some previously mapped QTLs for popcorn quality traits and provide evidence for several candidate genes affecting starch, storage protein, and oil content of popcorn kernels and pericarp polysaccharide content. The highlighted candidate genes are located in bins 1.04, 3.08, 4.02, 4.05, 5.01, 6.05, 7.02, 7.04, and 10.04.