Significant effects of recent global climate change have already been observed in a variety of ecosystems, with evidence for shifts in species ranges, but rarely have such consequences been related to the changes in the species genetic pool. The stretch of Atlantic coast between North Africa and North Iberia is ideal for studying the relationship between species distribution and climate change as it includes the distributional limits of a considerable number of both cold- and warm-water species.Fucus vesiculosus with historical sea surface temperature (SST) patterns to draw links between range shifts and contemporary climate change. Moreover, we genetically characterized with microsatellite markers previously sampled extinct and extant populations in order to estimate resulting cryptic genetic erosion.We compared temporal changes in distribution of the canopy-forming alga Over the past 30 years, a geographic contraction of the southern range edge of this species has occurred, with a northward latitudinal shift of approximately 1,250 km. Additionally, a more restricted distributional decline was recorded in the Bay of Biscay. Coastal SST warming data over the last three decades revealed a significant increase in temperature along most of the studied coastline, averaging 0.214°C/decade. Importantly, the analysis of existing and extinct population samples clearly distinguished two genetically different groups, a northern and a southern clade. Because of the range contraction, the southern group is currently represented by very few extant populations. This southern edge range shift is thus causing the loss of a distinct component of the species genetic background.We reveal a climate-correlated diversity loss below the species level, a process that could render the species more vulnerable to future environmental changes and affect its evolutionary potential. This is a remarkable case of genetic uniqueness of a vanishing cryptic genetic clade (southern clade). Global climate change has a profound influence on distributional patterns of a wide variety of taxa causing species range shifts and population extinctions ,2. PolewAlthough several studies have addressed phylogeographic responses to millennial-scale climate variability , surprisCurrent geographical distribution of genetic diversity reflects both contemporary and historical events. Populations that are currently geographically peripheral may be particularly vulnerable to loss of genetic diversity because they are often restricted to decreasing habitat areas within unsuitable landscape. Their small size and prolonged isolation is accompanied by loss of within-population genetic diversity and increased inter-population genetic divergence due to increased random genetic drift, probability of suffering bottleneck effects, and reduced gene flow ,13. PrevThe stretch of Atlantic coast between North Africa and Iberia is ideal for the study of the relationship between marine species distribution and possible effects of climatic warming because a considerable number of both cold- and warm-water species reach their distributional limits within this stretch of coast . In Surveys carried out in the last decade along the Atlantic Iberian Peninsula have correlated increasing sea surface temperature (SST) to striking expansion or contraction events -33. SST Fucus vesiculosus, an intertidal canopy-forming brown alga. The availability of records on the species distribution along North African and Iberian Atlantic shores and of DNA from recently extinct populations provides an ideal opportunity to investigate the impact of climate-driven range reduction on the overall species genetic diversity.In this study, the model species selected was F. vesiculosus during the last three decades along this stretch of coast. Resulting biogeographic dynamics are compared with SST warming trends in order to correlate range shifts with the intensity of climatic change in the region. We further draw on genetic data from extant populations and others that have recently become extinct (previously sampled) in a phylogeographic approach to test the extent to which unique genetic variation may be lost as a result of climate change and range contraction.We investigate shifts in the southern distribution endpoints of F. vesiculosus in Southern Morocco at Khnifiss Lagoon , although the previously permanent and attached populations have totally disappeared from this coastal lagoon.The southern endpoint is now positioned on the Portuguese west coast in the Tejo estuary , representing an 11° latitude shift .Historically, F. vesiculosus. In contrast to North African shores, upwelling waters off north-west Iberian shores contribute to the SST gradient.Temperature isotherms shifted polewards to higher latitudes Figure . This grOver the last 30 years, a significant increase of coastal SST  varied between 6.28 and 3.08 with lowest values for ML, RF, TV and LX, while unbiased heterozygosity (HE) ranged between 0.381 and 0.587 with highest gene diversity values for RE, VN, RL and RM  was significantly different from HE in nine locations where heterozygote deficiency was detected, resulting in significant positive values of the inbreeding coefficient FIS , populations were grouped into two major clusters corresponding to the northern and the southern subgroups Figure . The firThe northern group had many unique alleles, but the southern cluster including the now extinct populations also displayed unique alleles. This cluster suffered diversity loss with the recent extinctions, but still retains unique diversity . While F. vesiculosus were still present along the entire Moroccan and Iberian Atlantic coasts, while in the early 2000s extensive populations of this alga still persisted in southern Portugal. When these records are compared with our more recent surveys (2009 to 2011), a major contraction of the southern range margin of this keystone intertidal species  is revealed. Taken together with past studies, there is a strong indication that the northward distributional shift along the Portuguese coast occurred very recently, probably within the last decade. This range contraction is an order of magnitude larger than the range shifts recorded for other species over the last 50 years along the Northeast Atlantic .F. vesiculosus is correlated with warming of North African and southwest Iberian coastal waters, with steady year-round increases averaging >0.2°C/decade over the last three decades. Seasonal thermal extremes might also play a crucial role in defining the distribution of species through sudden population declines or die-offs [Trends in SST indicated that the large scale disappearance of southern populations of die-offs . Howeverdie-offs ,40. Sevedie-offs ) and neadie-offs ,42). Mordie-offs ,44. Howedie-offs ,45-47) adie-offs ,49 and bdie-offs  capacitidie-offs ,52.F. vesiculosus populations affected by cold upwelled waters have also undergone extinction. In Cape Ghir, warming rates show variation at monthly timescales, with a significant SST increase of about 0.28°C/decade in March . Howeverming.com) and maxrming.com. Althougrming.com. While nF. vesiculosus persist within the Bay of Cadiz, along the retreating front. This represents a restricted  population confined to very sheltered areas within the bay . The forecasted warming trend suggests that this small and isolated relict population faces a high risk of extinction, and this prediction is further supported by the extinction of nearby (less than 10 km distant) populations .Interestingly, patches of F. vesiculosus extends its northern distributional limit in Norway, also inhabiting the northernmost and brackish regions of the Baltic Sea and in the White Sea into brackish tidal marshlands. Within the range of F. vesiculosus, there is a well-documented gap along the SW French coast (French Basque Country and Gascony) due to lack of rocky substratum [F. vesiculosus individuals from other regions in Europe are genetically closer to the northern Iberian clade identified in our study by the same markers [F. vesiculosus, will disappear completely from the Bay of Biscay.Along eastern Atlantic shores, bstratum . In cont markers . A graph markers ) scenari markers . This moFucus serratus [Himanthalia elongata [F. vesiculosus in Northern Spain is lower than that at the southern limit in Portugal. This suggests that the two genetic lineages have different thermal tolerances or that distinct factors are setting their distribution limit. Indeed, different genetic lineages or populations inhabiting different areas within a species' distributional range can show diverse resistance or resilience to abiotic stresses . Forulations . Future F. vesiculosus was clearly established using a large number of distinct genetic markers including 13 protein coding genes [F. vesiculosus is older than the divergence between other more recently evolved species within the genus Fucus [Reduction in genetic variation gives independent evidence of the severity of population and habitat contraction. Estimating losses in genetic diversity associated with habitat contraction and environmental change is one of the key challenges in biodiversity research. The intensity of genetic loss largely depends on the geographical distribution of genetic diversity within the species, including cryptic diversification. Our analyses revealed two clusters, coinciding with more northern and southern locations, distinguished by several analyses based on allele frequencies, and containing unique alleles within each group, in the five microsatellite loci here used. The differentiation between northern and southern lineages in ng genes  and 35 Sng genes . The sepus Fucus , highligus Fucus ,71 couldFucus ceranoides, distant (but relatively similar) populations do not necessarily exchange more migrants than closer (but relatively distinct) populations [The divergence between northern and southern lineages does not appear to be absolute, since a few individuals appear to occur in mixed populations . Additionally, individuals from RE, not only diverge from southern populations, but emerge as the most distinct within the northern clade  with no apparent geographical explanation. This appears geographically unexpected, but patterns of genetic structure and differentiation are not necessarily maintained by prominent ecological/oceanographic barriers to dispersal . For exaulations ; historiPresently, the distributional range of the southern clade is restricted within approximately two degrees of latitude  and southern intra-specific genetic diversity is represented by few extant populations under threat from climatic warming. Given that predictions of future climate indicate a further rise in SST , there iF. virsoides, F. spiralis and the recently-described F. guiryi [F. vesiculosus [F. vesiculosus lineage could end ongoing diversification (or speciation) processes and compromise the adaptive potential of the species as a whole in the face of future global warming.Past and present distributional ranges of the southern lineage are characterized by markedly higher SST than those of the northern lineage; it can be hypothesized that the existence of this lineage is explained by local adaptation or phenotypic plasticity to withstand warmer waters. Our multi-locus genetic data represent neutral genetic change and do not necessarily reflect evolutionary responses to selection. However, divergent lineages often arise in areas of speciation . Ind. guiryi  are deriiculosus . The potF. vesiculosus, as expressed by both gene diversity and allelic richness, was higher in northern Iberian populations than in the southern lineage. Higher genetic diversity in the northern Iberian region can be a signature of higher temporal stability of large populations while the opposite, in the southern region, could be the result of lower population sizes and/or temporal variability  of the trailing edge populations. Higher diversity could also result from secondary contact between distinct lineages and/or from hybridization and introgression. While F. vesiculosus co-occurs with other fucoids throughout the majority of its range, including Northern Iberia, Southern Iberian individuals occur in allopatry. Therefore, hybridization events are likely only within and to the north of the contact zone between the sympatric and allopatric ranges [F. vesiculosus would be significantly reduced should these last remaining populations also become extinct.In this study, genetic diversity of c ranges . Given tA remarkable climate-induced species range reduction is driving a cryptic genetic clade (southern clade) to extinction. This clade occurs under stressful and selective environmental conditions and is, therefore, a potential pool of adaptive traits for the species; losing it could thus reduce the adaptive potential of the species, a serious conservation concern. However, the recent loss of most of the populations in this lineage suggests a more drastic scenario where their genetic distinctiveness has not been able to rescue them from extinction. Although low diversity within small marginal populations can reduce local adaptive capacity , here, eF. vesiculosus populations is likely to have other immediate ecological consequences. This is a dominant intertidal ecosystem engineer that influences coastal species richness by modifying habitats, increasing spatial complexity and facilitating the presence of other species [In addition to genetic erosion, the elimination of  species ,80; thus species ), with pF. vesiculosus along Atlantic North African and Iberian coastlines were gathered from a comprehensive literature review and information from herbarium collections. Data for the last decade also included personal records and associated sample collections made by our team in multiple surveys of the Iberian and Moroccan coastline for genetic sampling of this species for previous studies [Fucus, vesiculosus, Morocco, Iberia, Iberian Peninsula, Portugal, Spain, France, Atlantic, Western Sahara, distribution, herbarium, Fucales, Fucophyceae. In addition, literature reported in AlgaeBase  was screened and searched up until 16 May 2012. Sampling artifacts may bias range shift studies [F. vesiculosus is a scientifically popular species with well-known biology. It has the clear distinctive trait of possessing air bladders on the thallus, diagnostic for this species, and presents separate sexes, in contrast with the only other congeneric species occurring southwards from North Portugal. In addition, museum and literature records did not show any discrepancy. Therefore, we are confident about the quality of the points of reference used in this study.Data on the past distribution and southern endpoint of  studies ,72,82,83 studies ,84,85. P studies  because F. vesiculosus. The survey was carried out during low spring tides between 2009 and 2011 on rocky intertidal shores along the Atlantic North African and Iberian coastlines. Eighty-four moderately wave exposed and estuarine locations were sampled from Hondarribia  to Dakhla , covering a latitudinal extension of more than 2,150 km. Locations of the survey were chosen based on previous reports of the presence of F. vesiculosus . Five additional locations were only surveyed in 2009 to 2011, of which two were in Western Sahara (Boujdour and Dakhla), south of the historical southern limit.Current distribution and endpoints were obtained based on an extensive field survey thoroughly covering the entire past distribution of Most of the locations were visited at least twice, covering winter and summer months, and each location included two sites from 500 m to 1 km apart. At each site, two observers performed searches lasting approximately 60 minutes across all microhabitats present.Monthly averaged SST data between January 2009 and December 2011 with a 4 km resolution were retrieved from the Moderate Resolution Imaging Spectroradiometer-Aqua (MODIS-Aqua) dataset available from the National Aeronautics and Space Administration (NASA) Goddard Earth Sciences (GES) Data and Information Services Center (DISC). Visualization was performed using Giovanni, a web-based application developed by the GES DISC .http://www.coastalwarming.com/index.html[http://www.ncdc.noaa.gov).Coastal SST warming data over the last three decades (°C/decade from January 1982 to December 2011) were obtained from the Worldwide Coastal Warming Assessment project website at ndex.html. This prNote that, although warming rates at coastal and ocean locations often depend on the selected spatial and temporal scales, our SST trends are comparable with those reported in other studies that focused on different periods and spatial resolutions ,59,87,88F. vesiculosus collected in 2001 and 2002 from 12 locations, including four extinct populations, were used for genetic analyses  and expected (HE) heterozygosities were estimated, and deviations from Hardy-Weinberg equilibrium were tested for significance with 10,000 permutations, using GENETIX 4.05 software , applying q-value correction for multiple tests [Â) was estimated for each population separately, standardized to 20 individuals to account for differences in sample size, using the StandArich package and R 2.10.1 software. Plot of allelic frequencies for each location and marker was performed with the above package.Observed  procedure on populations implemented in GENETIX 4.05  and grapST Fand confidence intervals were estimated between pairs of populations with the estimator θ [EST [imator θ , and comimator θ . Additior θ [EST  using SPr θ [EST . Signifi® Illustrator® CS4 14.0.0 (Adobe System Inc.).The Cavalli-Sforza and Edwards' chord distance  was compet al. [STRUCTURE 2.3.3. software  was usedet al. . CLUMPP et al.  was subset al. .O Â, Hand E Hwere calculated for the different clusters determined by STRUCTURE analyses by pooling all populations from each cluster.Finally, to compare the level of diversity within clusters, estimates of Â: allelic richness; CA: correspondence analyses; FIS: inbreeding coefficient; FST: genetic differentiation; FCA: factorial correspondence analysis; HE: expected heterozygosity; HO: observed heterozygosity; K: population clusters; MODIS-Aqua: Moderate Resolution Imaging Spectroradiometer-Aqua; SNP: single nucleotide polymorphism; SST: sea surface temperature.The authors declare that they have no competing interests.GIZ, KRN, EAS and GAP conceived and designed the study. EAS and GAP provided distribution records and genetic data for extinct populations. GIZ and KRN gathered and analyzed temperature and distribution data. ST and KRN analyzed genetic data and all authors interpreted the data. KRN and GIZ wrote the article, which was critically revised by EAS and GAP. All authors read and approved the final version.List of locations surveyed. Name and coordinates of locations surveyed during 2009-2011, ordered from north to south. Presence/absence of the species and latest known year of its presence is also reported.Click here for fileAllele frequencies for locus L20. Codes correspond to locations in Figure 1, locations belonging to the southern lineage are encircled and extinct populations are shown in bold. The actual values of frequencies are represented by dots of varying diameter: allele codes are indicated on the x axis and population names on the y axis.Click here for fileAllele frequencies for locus L58. Codes correspond to locations in Figure 1, locations belonging to the southern lineage are encircled and extinct populations are shown in bold. The actual values of frequencies are represented by dots of varying diameter: allele codes are indicated on the x axis and population names on the y axis.Click here for fileAllele frequencies for locus L94. Codes correspond to locations in Figure 1, locations belonging to the southern lineage are encircled and extinct populations are shown in bold. The actual values of frequencies are represented by dots of varying diameter: allele codes are indicated on the x axis and population names on the y axis.Click here for fileAllele frequencies for locus L78. Codes correspond to locations in Figure 1, locations belonging to the southern lineage are encircled and extinct populations are shown in bold. The actual values of frequencies are represented by dots of varying diameter: allele codes are indicated on the x axis and population names on the y axis.Click here for fileAllele frequencies for locus L38. Codes correspond to locations in Figure 1, locations belonging to the southern lineage are encircled and extinct populations are shown in bold. The actual values of frequencies are represented by dots of varying diameter: allele codes are indicated on the x axis and population names on the y axis.Click here for fileGenetic differentiation between pairs of populations. Codes correspond to locations in Figure 1 and are ordered from north to south, bold characters are extinct populations. Genetic differentiations (STF) were estimated with the estimator θ, and are reported above the diagonal while Jost's DEST are reported below the diagonal. All values are significant at P <0.001 after multiple test correction.Click here for fileConfidence intervals of the genetic differentiation between pairs of populations. Codes correspond to locations in Figure 1 and are ordered from north to south, bold characters are extinct populations. Confidence intervals of genetic differentiation (STF) are reported above the diagonal.Click here for file


Perna perna  is a dominant constituent of intertidal communities and a strong invader with multiple non-native populations distributed around the world. In a previous study, two polymorphic microsatellite loci were developed and used to determine population-level genetic diversity in invasive and native P. perna populations. However, higher number of microsatellite markers are required for reliable population genetic studies.The brown mussel P. perna origins and history of invasion and to compare population genetic structure in native versus invaded areas, we developed 10 polymorphic microsatellite markers.In this context, in order to understand P. canaliculus .Described microsatellite markers were developed from an enriched genomic library. Analyses and characterization of loci using 20 individuals from a population in Western Sahara revealed on average 11 alleles per locus (range: 5–27) and mean gene diversity of 0.75 (range: 0.31 - 0.95). One primer pair revealed possible linkage disequilibrium while heterozygote deficiency was significant at four loci. Six of these markers cross-amplified in P. perna, including dispersal scales, genetic variation and population structure, in both native and invaded areas.Developed markers will be useful in addressing a variety of questions concerning  Perna belongs to the Mytilidae , the family of “true mussels” which includes green and brown shell mussels from tropical, subtropical, warm and cold temperate regions[The genus  regions. Species regions,3. They  regions.Perna perna is a subtropical/tropical species widely distributed along the west coast of Madagascar, east African coast , extending through the Gulf of Aden into the Red Sea, west coast of Africa (apart from the upwelling-influenced Benguela region on the west coast of South Africa)[P. perna has been reclassified as an old introduction, most likely dating from the sixteenth century[.The brown mussel  Africa),6; and f Africa). It is a Africa)-9. In Br century. Moreove century and in T century. It has  century.P. perna, only two microsatellite markers have previously been published for this species[P. perna.Despite the economic and ecological impacts of  species. These m13, (GT)13, (TAC)10 and (GTAT)7 oligonucleotide repeats[P. perna individuals from one population from Western Sahara .Total genomic DNA was extracted from 5 individuals collected in South Africa  using a phenol-chloroform extraction method. An enri repeats,18. Of 52, 3.0 μl of 5x PCR Buffer and 0.75 U of GoTaq Polymerase . Cycling conditions consisted of an initial denaturing step of 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at annealing temperature (see Tablehttp://www.vgl.ucdavis.edu/informatics/STRand), binned using the R package MsatAllele[O) and expected (HE) heterozygosities were estimated, and deviations from linkage and Hardy-Weinberg equilibria were tested using GENETIX software[Polymerase chain reaction (PCR) was performed in volumes of 10 μl containing ±10 ng of DNA, 0.5 μM of each primer labelled with a florescent marker, 0.2 mM dNTPs (Bioline), 1.5 mM MgClatAllele, and mansoftware. The numsoftware.O = 1; TableE; TableIS not significantly differing from zero. All markers were then tested for cross-amplification in five individuals of P. canaliculus from New Zealand, of which several amplifications were positive  showingP. perna. In this case, we cannot exclude species-specific, locus specific or population specific explanations for the results. Further genetic population structure analyses could shed light to this phenomenon in P. perna. Moreover, these ten microsatellite loci provide a useful tool to understand processes influencing species boundaries, such as range expansions outside the native distribution of P. perna populations, and compare diversity and differentiation scales in invasive and native populations.Deficiency in the number of heterozygotes observed (relative to Hardy-Weinberg expectation), with both allozyme and microsatellite studies have been documented in many marine molluscs e. g.-28, inclhttp://www.ncbi.nlm.nih.gov/). The accession numbers on the repository are the following (see also TableThe microsatellite sequences are available through the National Center for Biotechnology Information (seePerper-01- JX183697; Perper-02 -JX183698; Perper-05 -JX183699; Perper-08 -JX183700; Perper-11 -JX183701; Perper-16 -JX183702; Perper-20 -JX183703; Perper-26 -JX183704; Perper-27 -JX183705 and Perper-29 -JX183706.The authors declare they have no competing interests.All authors participated in the design and implementation of the study, supervision of the work and processing interpretation of the results. CNC, NKR and ZGI participated in data analysis, microsatellite marker validation and drafted the manuscript. All authors read and approved the final manuscript.


With the recent revolution in sequencing, we revisit the unresolved question of what influences the range and values of genetic diversity across taxa. Understanding why some species have more genetic diversity than others is central to the study of ecology and evolution, and carries potentially important implications for conservation biology. Yet not only does this question remain unresolved, it has largely fallen into disregard. With the rapid decrease in sequencing costs, we argue that it is time to revive it. As a first step towards reviving these questions, we compile existing estimates of nuclear sequence diversity. These data are highly preliminary, but they underscore how little is known about the narrow span of diversity levels across species or why some species maintain more genetic variation than others genetic drift”) N and u, where u is the rate of mutation per generation. In reality, populations fluctuate in size over time and individuals can vary greatly in their reproductive success. Often, these and other deviations can be accommodated by substituting a much smaller “effective population size,” Ne, for the census population size NeN), leading them to maintain greater levels of neutral genetic diversity.According to the neutral theory of molecular evolution, genetic diversity levels at neutral sites reflect a balance between mutational input and the loss of genetic variation due to the random sampling of gametes in a finite population  can maintain it At sites on which natural selection acts, however, the rate of loss of genetic variation will depend in more complex ways on the population size. Population genetic theory indicates that selection will be more effective in large, random-mating populations selection at linked sites; that is, they will depend on the frequency and strength of selection and the distribution of selected loci throughout the genome balancing selection is common These considerations matter in predicting diversity levels at sites directly under selection but also at nearby neutral sites, because selection at one site impacts variation at neighboring positions in the genome through linkage Drosophila species, notably, the analysis of polymorphism and divergence suggests that half of the amino acid substitutions between species have been fixed by selection  than do chordates (0.26%), and plants fall in the middle (1.48% for outcrossing Magnoliophyta and 0.52% for Pinophyta) , a few intriguing observations emerge: the species in the four most diverse phyla live mainly in marine or freshwater environments . This pattern might reflect a larger variance in male reproductive success relative to females or other demographic effects p = 0.016), in principle consistent with a ratio of 3∶4 and a (much) lower mutation rates in females, where the X is found two-thirds of the time p = 0.001), even though the Z chromosome spends most of its time in males, who are thought to have a higher mutation rate Finally, across taxa with heterogametic sexes, sex chromosomes show different patterns relative to autosomes . Making Lynx lynx to 8.01% in Ciona savignyi, a span of only 800-fold , whereas in Drosophila buzzatii, a species distributed worldwide, they are only ∼10 times higher (1.94%) when population size estimates are on the same order per hectareD. buzzatii, the census population sizes are unlikely to have ever been within an order of magnitude.In addition to these crude patterns, the compilation of silent diversity levels makes evident the same puzzle as seen in the allozyme data 800-fold . While c−10 per site in Saccharomyces cerevisiae to 3.5×10−9 in Drosophila melanogaster, 1.3×10−8 in Homo sapiens, and 3.8×10−8 in Mus musculusS. cerevisiae). If across taxa there is a systematic relationship between mutation rate and population size, it would lead large populations to have relatively low diversity levels and thus to a smaller range of diversity levels than population sizes Ficedula albicollis is estimated to have an approximate population size of four to seven million and the sparrow Zonotrichia albicollis of 140 million (http://www.birdlife.org). Mutation rates in these two bird species are unknown but presumably similar, yet average diversity levels differ by less than 2-fold . As another illustration, within Drosophila, there is a surprisingly small  difference between the extremes of narrow endemics and cosmopolitan species  Why then are neutral diversity levels and allozyme variation contained within such a narrow range? If neutral diversity levels are indicative of the ability of a species to adapt to novel selective pressures, then, as argued in the context of conservation biology, there may be a lower limit beyond which a species cannot maintain the variation necessary to respond to a change in environment and so is rapidly driven to extinction e.g., . In turngenetic draft”) at linked neutral sites Alternatively, the narrow range of diversity may be due to the effects of selection at linked sites. That habitat and range are predictive of diversity is consistent with a neutralist scenario in which aquatic species, species with larger ranges, or outcrossers have greater and more stable population sizes and therefore maintain higher neutral diversity, but it may also be consistent with models in which positive selection is ubiquitous. Under certain assumptions, widespread adaptation can constrain the range of neutral diversity across species: when adaptation is limited by the input of new mutations, larger populations experience a greater influx of beneficial mutations and therefore greater effects of variation-reducing selection (“Nes|<1) and therefore under more effective purifying selection in larger populations, diversity levels may increase much more slowly with Nenearly neutral model could in principle help to account for a reduced range of diversity levels, such an explanation raises the question already voiced by Gillespie and Ohta 15 years ago: “Why should nature conspire to have the value of 2Nes fall within such a narrow window for most creatures?” Selection on silent sites themselves may also be a factor contributing to the narrow range of diversity across species. It is well established that codon bias and other selective pressures constrain the evolution of synonymous sites, and that many sites in non-coding regions are subject to purifying selection e.g., . If a suThe central puzzle remains: both allozyme and diversity levels at sites less likely to be directly affected by selection vary surprisingly little among species, and mostly in ways that we still do not understand. This puzzle has persisted for close to half a century because it is a difficult one, and simply gathering more data will not resolve it. However, characterizing diversity levels along the genomes of thousands of species is a necessary first step, and now a feasible one. In fact, data collection is already on its way, with hundreds of genome sequences now available, and the proposal to scale up to 10,000 species, sampled throughout the plant and animal kingdoms In addition to enabling population-level sequence data, the revolution in sequencing will also permit the estimation of de novo mutation rates Click here for additional data file.Figure S1Drosophila. The estimate of 0 for Drosophila sulfurigaster bilimbata (based on five loci) is not shown.Nucleotide diversity estimates for sex chromosomes (X or Z) across species. Each estimate represents the mean of at least three loci on the X or Z chromosome and is based on silent sites or the entire chromosome in all but four cases. The estimates are colored by the phylum to which each species belongs and within phylum are ordered by diversity level; phyla are ordered by their median diversity level, shown as a horizontal bar. Crosses indicate estimates for individual populations when population structure was reported in the original study. Within Chordata, open circles denote mammals and triangles birds; within Arthropoda, open circles denote (TIF)Click here for additional data file.Figure S2p = 0.068). Open circles represent Drosophila (within Arthropoda) and mammals (within Chordata). In panel (B), the distribution of nucleotide diversity (left) and allozyme heterozygosity (right) across species are shown, with the medians represented at the same level as a black bar. The number given at the bottom is the coefficient of variation.Comparison of nucleotide diversity and allozyme heterozygosity. Autosomal nucleotide diversity estimates are from the current compilation and allozyme heterozygosity estimates are from (TIF)Click here for additional data file.Figure S3Autosomal nucleotide diversity by mating system in flowering plant species. Genetic diversity estimates for species in the phylum Magnoliophyta, colored according to whether the mating system allows for self-fertilization. Horizontal lines indicate the median genetic diversity for each of the two categories.(TIF)Click here for additional data file.Table S1The median nucleotide diversity within a phylum considering estimates based on all site types versus only synonymous sites. Listed are phyla in which at least three species have a synonymous diversity estimate and estimates for multiple types of sites are represented.(DOC)Click here for additional data file.Table S2The median nucleotide diversity within a phylum considering estimates based on sampling a single population versus sampling multiple populations with no observed population structure. Listed are phyla with at least two species in each group.(DOC)Click here for additional data file.Text S1Supporting information and methods.(DOC)Click here for additional data file.


BMC Genetics, Albuquerque de Moura et al. asked whether such a general pattern of population structure can be observed in Heliconius species, which could have strong implication in the evolution of colour pattern diversification in these butterflies. In this commentary we discuss the potential roles of these three processes  on the evolution of Heliconius wing patterns in regard to the findings of a common fine-scale population structure within the co-mimetic species H. melpomene and H. erato. Indeed, a general pattern of population subdivision in the history of these two species may have provoked the major phenotypical shifts observed in their wing colour patterns. The suggestion that coupled environmental pressures  could be responsible for identical genetic differentiation profiles in H. erato and H. melpomene clearly merits further investigations using both detailed population genetic (including landscape genetic) and ecological studies.To what extent population structure favours the establishment of new phenotypes within a species remains a fundamental question in evolutionary studies. By reducing gene flow, habitat fragmentation is a major factor shaping the genetic structuring of populations, favouring isolation of small populations in which drift may rapidly change frequencies of new variants. When these variants provide advantages to individuals, the combined effect of selection and drift can lead to rapid shifts in phenotypes. In a study published in  Rapid changes of phenotypes can be observed in small genetically isolated population because drift may enhance the rate of fixation of new variants. As a result, the long-term evolution of traits with ecological importance is thought to be tightly linked to the historical population structure of species for which strong genetic isolation is a common feature. Of course, species living in highly fragmented landscapes are more prone to exhibit strong genetic subdivision because gene flow will tend to be reduced in patchily distributed habitats.et al. [BMC Genetics aimed at determining if such a general pattern of population structure can be observed in Heliconius species, which could have strong implications in the evolution of colour pattern diversification in these butterflies. Using a panel of genetic markers, they effectively found a widespread genetic differentiation of populations on unusually small geographic distances in the Mullerian co-mimetic butterflies Heliconius erato and H. melpomene, but no isolation by distance [Heliconius butterflies ranges from 3 to 10 km [et al. study [The study of Albuquerque de Moura et al.  publishedistance . Populatdistance ,3. Howevdistance , such a to 10 km ,6. Thus,et al. [H. erato and H. melpomene and 1144 AFLP for both species to properly infer the genetic structuring of each species. If all marker types revealed the presence of a fine-scale population structure within both species, inter-marker patterns within species were not correlated (Mantel tests non significant at the 5% level). This discrepancy between mitochondrial and nuclear markers may reflect different dispersal behaviours between males and females as proposed by the authors. However, the discrepancy between genetic structure inferred from microsatellites and AFLP markers is probably the result of an insufficient number of microsatellite loci preventing high enough statistical power, which is especially true when populations have not reached genetic equilibrium [The use of different marker types to infer genetic population structure has now become a widespread and powerful strategy. Although dominance effects, as well as mutational processes, involve fundamental differences in marker evolution, they should all reflect the same population history , provideet al.  have genilibrium . In contH. erato and H. melpomene . In other words, pairs of populations harbour similar genetic divergence levels in the two species. We suggest that such strong parallel patterns of genetic isolation can be observed when species face the same environmental pressures.Thus, we think that the more reliable results concerning the nuclear genome are those obtained with AFLP. Comparing patterns of genetic differentiation obtained with AFLP, we found a strong correlation between Heliconius species due to selection pressures acting on similar genomic regions. Among potential candidate characters on which selection could generate such genomic patterns, the coloration of wing patterns, which serves as a warning signal for predators, is noteworthy. It has been shown that wing patterns in co-mimetic Heliconius species are driven by several homologous genomic regions functioning as "supergenes" [Heliconius lineages (among which H. melpomene and H. erato) with parallel changes in gene regulation across co-mimetic species [H. melpomene and H. erato share the same wing pattern phenotype in the studied region . Nonetheless, we encourage a detailed morphological analysis that could reveal subtle co-evolved changes in the wing colour patterning of populations in both species. Besides, we propose the careful investigation of ecological mechanisms that would allow the maintenance of the observed genetic differentiation, like assortative mating between individuals of the same populations or local specialization on particular host plants. Indeed, the genetic determinism of such ecological mechanisms is probably complex.An interesting point is the possibility of a convergent pattern of genetic isolation between co-distributed ergenes" -12. Thos species ,14. TheyHeliconius species. This finding could be of great importance when trying to retrace the evolutionary history of Heliconius species. As mentioned by the author, the additional effect of drift in these small populations of Heliconius can rapidly increase the frequency of new benefit alleles, enhancing the rate of change in the genetic structuring and phenotypes in populations. In what measure drift and selection have influenced shifts in Heliconius wing patterns remains an exciting question that needs to be raised in the context of habitat fragmentation, taking advantage of the new advances in landscape genetics.Overall, it seems that adaptive responses under particular environmental constraints may involve the same genomic regions in populations of co-distributed et al. [H. melpomene and H. erato populations at a local scale. Finally, extending this population genetic study using selected loci responsible for wing pattern expression needs to be considered to determine the putative role of 'selection on the wing' in this system, and to go further in our comprehension of the long-term processes underlying co-mimicry in Heliconius.To conclude, we think that an additive or an interaction effect of 1) dispersal counter-selection in response to habitat fragmentation and (2) selection on important ecological traits  might be responsible for the genetic structuring we observe here. In this sense, the study of Albuquerque de Moura  dispersaDL, VMS and MB conceived of the manuscript, DL wrote the manuscript, all authors read and approved the final manuscript.


Eukaryotic genomes harbor diverse families of repetitive DNA derived from transposable elements (TEs) that are able to replicate and insert into genomic DNA. The biological role of TEs remains unclear, although they have profound mutagenic impact on eukaryotic genomes and the origin of repetitive families often correlates with speciation events. We present a new hypothesis to explain the observed correlations based on classical concepts of population genetics.The main thesis presented in this paper is that the TE-derived repetitive families originate primarily by genetic drift in small populations derived mostly by subdivisions of large populations into subpopulations. We outline the potential impact of the emerging repetitive families on genetic diversification of different subpopulations, and discuss implications of such diversification for the origin of new species.Several testable predictions of the hypothesis are examined. First, we focus on the prediction that the number of diverse families of TEs fixed in a representative genome of a particular species positively correlates with the cumulative number of subpopulations (demes) in the historical metapopulation from which the species has emerged. Furthermore, we present evidence indicating that human AluYa5 and AluYb8 families might have originated in separate proto-human subpopulations. We also revisit prior evidence linking the origin of repetitive families to mammalian phylogeny and present additional evidence linking repetitive families to speciation based on mammalian taxonomy. Finally, we discuss evidence that mammalian orders represented by the largest numbers of species may be subject to relatively recent population subdivisions and speciation events.The hypothesis implies that subdivision of a population into small subpopulations is the major step in the origin of new families of TEs as well as of new species. The origin of new subpopulations is likely to be driven by the availability of new biological niches, consistent with the hypothesis of punctuated equilibria. The hypothesis also has implications for the ongoing debate on the role of genetic drift in genome evolution.This article was reviewed by Eugene Koonin, Juergen Brosius and I. King Jordan. Eukaryotic genomes contain multiple copies of TEs historically known as "interspersed repetitive DNA." This repetitive DNA originates from different classes of TEs, multiplying and integrating themselves or other DNA in the host genomes at different evolutionary periods. TEs play an intrinsic role in genome evolution -5 and arSequence studies point to a large diversity among TE families originating in different species. For example, mammals tend to harbor a small number of large families with few recently active elements whereas plants, insects and fish frequently harbor numerous small families composed of very young TEs . FurtherIn this paper we propose that the observed species-specific differences between families of TEs are determined, at least to a large extent, by the population structure of the host. Fixation of TEs can take place in small populations by genetic drift -20, and A large population with a heterogeneous pool of active TEs can subdivide into small subpopulations (demes), each carrying a random set of active elements. If the subpopulations are small enough and gene flow among them slow enough, they will be subject to genetic drift . Some ofWe assume that most eukaryotic populations include individuals that carry a variety of active TEs (source genes), not fully suppressed by the silencing mechanisms. Active TEs that survive the silencing are likely to be outliers in terms of their mosaic structure (e.g. new SINE elements), or sequence divergence. We also assume that the fixation of repetitive families takes place primarily in small populations by genetic drift and that the transposed copies of TEs are neutral or slightly harmful. The number of TEs fixed under relaxed selection in small populations is determined primarily by the rate of transposition and the time during which the elements were active -29.Figure Most subpopulations will remain connected to their metapopulations through migrations of their genetic material (gene flow), and the distribution of different families of TEs in metapopulations is likely to be determined by a balance between genetic drift and migrations, analogously to the distribution of neutral genes . Over tiThe potential involvement of TEs in speciation has been discussed since the early eighties based on the contributions of Barbara McClintock (reviewed in ), and thThe CASP hypothesis proposes that the population subdivision triggers fixation of repetitive families by genetic drift. Therefore, new repetitive families are likely to coincide with the origin of a new species from small subpopulations whether or not TEs contribute to the process of speciation. At the same time, accumulation of TEs in different subpopulations may eventually help them to "drift apart" due to their mutagenic impact driven by the rate of transposition, which can increase the probability of reproductive isolation due to genetic diversification. Such mechanistic scenarios were already explored in the early eighties , but theThe TE-mediated diversification is most likely to cause productive speciation if TEs can expand to large families in a relatively short time and increase the chances of reproductive isolation. Probably only a few subpopulations can survive large bursts of mutations and only a few active TEs can reach such bursts of activity. Therefore, large repetitive families are probably derived from a small number of the most active TEs, which is consistent with the "master gene" hypothesis . Slowly The rapid TE-mediated diversification is likely to be evolutionarily significant in populations that cannot easily subdivide into isolated subpopulations for any extended period of time. Highly dispersible populations are less likely to produce species with large species-specific repetitive families. Furthermore, the critical mass of mutations that contribute to the effective separation of different subpopulations is expected to positively correlate with genome size. As new species emerge, and their populations continue to grow, the rate of fixation of new TEs by genetic drift is likely to decline. It can be revived again by new cycles of subdivisions producing small subpopulations and new families of TEs.A. gambiae and A. aegypti) differ by a factor of magnitude. Analogous, although less dramatic differences in the diversity of TEs can be seen in vertebrates and plants. Among the four plants listed, the most "primitive" ones (S. moellendorffii and P. patens) have the lowest numbers of different families of TEs.Table A. aegypti has been more subdivided in its recent evolutionary history than that of A. gambiae. This is consistent with the apparent adaptability of A. aegypti as it spread to new geographical locations in recent history  there are more than 40 specific families of L2 LINEs and associated Mon-SINEs. These families are not present in other sequenced genomes of birds and mammals. In several Mon-SINE families there is less than 2% divergency, which would indicate young age (Supplementary Notes S20 in the aforementioned citation).Response: Many families of TEs from the species listed above were discovered and entered to Repbase by the authors of this manuscript. Nevertheless, most of them are more than 5% divergent from their consensus sequences (even though they are species- or lineage-specific), and we decided not to detail them in this hypothesis. They deserve a separate in-depth analysis and we stand by this decision. However, we gratefully acknowledge a large underlying point made by the reviewer: we did not take advantage of a large body of excellent phylogenetic studies involving old families of TEs, due to the original focus on response to the "genomic drive hypothesis". This omission is remedied and phylogenetic analyses are prominently cited in the revised manuscript.3) CASP also proposes that small subpopulations resulting from the subdivisions are primary incubators of new repetitive families originally fixed by genetic drift, and subsequently undergoing purifying selection. I doubt that most new repetitive families are under purifying selection. There are a few exceptions where novel RNAs, namely BC1 and BC200 RNA, itself generated by retroposition in rodents and primates, respectively, were exapted into a function. Now, they are under purifying selection and also happened to be master genes for repeats .Response: This must be a misunderstanding since we certainly did not mean to imply that "most new repetitive families are under purifying selection." However, a certain fraction of elements will eventually become a conserved part of the genome. We rephrased the sentence and included ref. 83 to broaden the context.4) "However, the lack of young families in genome is less informative because it may be caused by random loss of active source genes in founder populations."In contrast to the mosquito genome, the honeybee (Apis mellifera) has no active TEs except for two families of DNA transposons [Weinstock et al (2006) Insights into social insects from the genome of the honeybee Apis mellifera. Nature 443: 931-949]. There are 32 known Apis species, 24 of which are close to Apis mellifera. This would contradict CASP. Perhaps CASP does not apply to all phylogenetic branches?Another observation is the paucity of TEs in birds despite a tremendous speciation activity during neoavian radiation . An additional problem is that one cannot conclude from extant species to species diversity in the various lineages over the past dozens of million years.Response: The CASP hypothesis predicts that at least some species including those living in marginal niches (e.g. primitive plants), or species such as the honeybee do not encounter enough opportunities to explore new alternative niches and to split into many small subpopulations for any extended period of time. The same may apply to "fine-tuned" parasites and deserves a separate analysis. On the other side of the spectrum are birds that can disperse more readily than mammals into local subpopulations (with the possible exception of bats). Therefore, in birds high activity of TEs might have been less relevant for speciation. According to the hypothesis, mammalian populations certainly preserved many smaller bird-like families of TEs, but they are not necessarily associated with speciation. For example, there are multiple families of LTR retrotransposons in mammals, but they probably didn't proliferate fast enough to sufficiently affect the divergence of different subpopulations, which is a prerequisite for the origin of new species with large families.1) The manuscript would gain a lot by including more references.Just to give a few examples, such as earlier publications on the role of TEs in speciation: . Or, publications on the source, founder, master gene concepts: . Or, a reference concerning the proposal that Alu elements were probably removed from the male sex chromosome over time (page 10).In support to some suggestions, fixation of individual TEs in connection to the population structure was proposed based on experimental data in the following articles: . These findings should be discussed in connection to CASP hypothesis.Response: All the references under this point and many listed above are added and discussed in the context of the revised manuscript.2) The TINE1 element is not a good example to illustrate a "new family" of retroelements. TINE1 is not specific for the tarsier family as it is found in other primates, and hence a "master gene" appeared prior to tarsier speciation. In addition, characterization of TINE1 as a "processed pseudogene" is questionable, because there is no processing except polyadenylation at a solitary LTR.Response: Deleted.3) The sentence: " The hypothesis predicts that repetitive families represent only the most visible and probably most abundant portion of all types of mutations be fixed in small populations in addition to TEs." is unclear: Repetitive families consist of TEs?Response: Rephrased and clarified. Throughout the paper "repetitive families" and "families of TEs" are used interchangeably.4) Not clear why the AluYa5 and AluYb8 families of repeats are of particular interest? Why are the ratios of chromosomal densities of AluYa5 elements consistent with the model of paternal transmission, i.e. inheritance of active Alu elements through male germ line only?Response: These families are the largest among the potentially active ones and as such they are subject to meaningful statistics. The second point: apparently Alu elements are activated in male germ line. This may be related to de-methylation of chromosome Y (see reference 37 in publication 61 cited in this paper).5) "... that Alu elements were probably removed from the male sex chromosome over time." - citation is necessary, and explanation of the removal process would be useful.Response: See ref. 61 page 1271 (bottom) and page 1272 (top).6) CSINE2 - is a family present in pika and all Leporidae and were active before pika and rabbit speciation [Kriegs JO, Zemann A, Churakov G, Matzke A, Ohme M, Zischler H, Brosius J, Kryger U, Schmitz J 2010) Retroposon insertions provide insights into deep lagomorph evolution. Mol Biol Evol 27:2678-81, Figure 0 RetropoResponse: We added Figure 3with cumulative numbers of all families and for phylogenetic details we included the reference to Kriegs et al. (70).I. King Jordan, Georgia Institute of Technology, USAIn this compelling hypothesis paper, Jerzy Jurka and colleagues lay out their vision for the relationship between the genome dynamics of transposable elements (TEs) and the process of speciation. They present the 'carrier subpopulation (CASP)' hypothesis, which emphasizes that species-specific differences in TE family composition are best explained by differences in species' population structure. The basic idea of the CASP hypothesis is that subdivided populations will inherit distinct sets of active TEs, or TE subfamilies, and furthermore different subsets of these TEs will be randomly fixed by genetic drift among the divided subpopulations. Meanwhile, given sufficient time and the availability of distinct niches, the divided subfamilies will diverge into new species. Together, this will lead to a greater relative diversity of young TE families for lineages that include numerous species. The CASP hypothesis is notable for the fact that it posits a passive, rather than a causal, role for TE activity and accumulation in the process of speciation. As such, the CASP hypothesis serves as a counter-point to the 'genomic drive' hypothesis for the significance of TEs with respect to speciation, which holds that amplification of TEs leads to speciation by increasing genomic variability.The presentation of the CASP hypothesis in this paper is interesting, thoughtful and timely, and I expect that this work will be quite thought provoking to investigators working on TEs, evolution and genomics. Thus, I certainly support publication of the work in Biology Direct. Below, I provide a number of comments, questions and suggestions that the authors may wish to consider prior to finalization of their manuscript.1. There are assumptions of the CASP model that need to be critically interrogated. One critical assumption of the model is that fixation of repetitive families takes place primarily in small populations by genetic drift. Of course, it is entirely reasonable to posit that random fixation any genetic element would occur preferentially in small populations. However, this assumption seems to imply that TE fixation dynamics are dominated, or even exclusively shaped, by population level forces. In fact, the population dynamics of repetitive elements can be considered to take place at two levels - there are indeed population level dynamics predicated upon differential reproductive success of individual organisms but there are also genome level dynamics based upon differential reproductive success of individual TE copies or subfamilies. This understanding was articulated in the early formulation of the selfish DNA theory when it was theoretically demonstrated that TEs could increase in copy number even if their replication was deleterious to the host (Hickey 1982 Genetics 101: 519). In other words, the genome level replication dynamics of TEs could overcome the population level effects of selection. This may be an extreme view, but there is certainly an interplay between population dynamics and genome level dynamics when it comes to TE replication and fixation. Any model that only treats one or the other of these two important levels may be missing a critical component.Response: The CASP hypothesis is based on neutral theories (refs. 27-29) that include slightly deleterious mutations under relaxed selection in small populations. Under relaxed selection the mutation rate by TEs (the rate of transposition) and the fixation rate are the same, which may leave the impression that one is missing.2. Another important aspect of the model is the idea that randomly divided subpopulations will inherit different sets of active TE copies Figure . On its Response: The CASP hypothesis permits subpopulations without active TEs. However, such subpopulations may be less likely to diversify fast enough to become foundations for new species (see section "Carrier subpopulations and the origin of species"). The hypothesis implies that very active TEs are most likely to contribute to productive speciation. Nevertheless, they can be very destructive and only a few of them left behind viable subpopulations that were foundations for new species. They also left behind large repetitive families in primates that were the basic evidence for the master gene hypothesis (refs. 10-15). Slowly replicating TEs are much less likely to affect speciation. They are also less destructive and probably less frequently suppressed by the silencing mechanisms. Therefore, they are more likely to be represented by multiple active copies as proposed by the "transposon model" (refs. 47-48).3. There is one slightly troubling (or perhaps simply confusing) aspect of the CASP hypothesis articulated in the conclusion (implications) section of the article. Here, the authors mention that the 'genomic record of young TEs can be a powerful indicator of... subdivisions in the population that underlie speciation events' but then go on to state that the opposite pattern of a lack of young TE families in a genome may not be informative because it could be due to other factors. This would seem to suggest that the hypothesis lacks discriminating power with respect to the relationship between the extent of young TE families in a genome and speciation rates along a lineage. Is this really the case? Does this mean that one would not be able to systematically relate the extent, or lack, of young TE families to high or low levels of speciation?Response: The CASP hypothesis links speciation to population subdivision, which is driven by the availability of biological niches. There are three basic categories of subpopulations: (1) those that do not carry any active TEs but still evolve into new species, due to geographical factors allowing both survival and reproductive isolation; (2) subpopulations that carry moderately active TEs that have little or no impact on potential speciation events and (3) subpopulations that are rapidly mutated by very active TEs and, if they survive, they are likely to become founding populations for new species. Given the ubiquitous nature of TEs, the first category of subpopulations is probably rare. We think that the most common is category 2 and majority of the diverse families of TEs originate in such subpopulations (see "Origin of diverse families of TEs"). The last category is the "extreme version" of the category 2.i.e. the role of genetic drift, in shaping genome architecture (see his book The Origins of Genome Architecture). Similar to what is proposed here, Lynch holds that TEs are able to accumulate to high copy numbers owing to the reduced efficacy of natural selection in small populations. The CASP hypothesis should be considered in light of the previous work of Lynch along with the ensuing discussion (controversy) that his work engendered.4. Michael Lynch has written extensively on the importance of non-adaptive aspects of evolution, Response: In the last section we discuss the relationship between the CASP hypothesis and the Lynch & Conery hypothesis in the context of the ongoing debate on the role of drift in evolution of genomic complexity.5. The authors make a very clear and strong statement in the abstract regarding the relationship between the numbers of young TEs in genomes and the number of species in a lineage. In the body of the manuscript, they go on to provide data on the diversity of TE families among several vertebrate, plant and insect genomes Table  and addiResponse: The main points can be summarized as follows (with some oversimplification): (1) Multiple families of TEs in a genome are associated with multiple subpopulations in the historical population from which the genome has emerged (younger multiple families are associated with more recent subdivisions). (2) Speciation events are likely to correlate with the cumulative number of subpopulations that originated (and vanished) during the history of a lineage and indirectly with the number of families of TEs generated in those subpopulations (recent speciation events are likely to correlate indirectly with recent fixations of repetitive families). (3) On average, there should be higher proportions of surviving species from recent speciation events than from the old ones.Currently, there is not enough data to correlate the number of species or speciation events with the population structure over geologic time. However, we found a way to indirectly support the first point above by showing a significant positive correlation between two unrelated families  based on the prediction that they both correlate with the number of subpopulations in a population.6. A corollary to comment #5 is that the best hypotheses make very specific predictions that can be empirically tested - or in the case of evolutionary hypotheses at least interrogated via direct observations on standing variation. The CASP hypothesis directly contradicts the genomic drive hypothesis (Oliver and Greene 2009 Bioessays 31:703) with respect to the agency of TEs in the process of speciation. Here, it would help if the authors could set up some mutually exclusive predictions that would clearly distinguish these two hypotheses. Oliver and Greene have recently published additional evidence for their genomic drive hypothesis (Oliver and Greene 2011 Mobile DNA 2: 8). Consideration of this work could be relevant to the authors' efforts to distinguish the two hypotheses.Response: The basic premise of the genomic drive hypothesis is that TEs constitute the main engine of the process that can result in the generation of "widely divergent new taxa, fecund lineages, lineage selection, and punctuated equilibrium." We find this premise untenable without rooting it in a broader context of the population structure, which is the frame of reference for the definition of species and other taxonomic units in a lineage (see ref. 81). Furthermore, in their papers Oliver and Greene ignored the fundamental problem of reproductive isolation. In the most recent version of the hypothesis published in Mobile DNA, the authors moved closer to the population genetics perspective by invoking "environmental and ecological factors." However, they ended up with enumeration of changes introduced by TEs in primates to support their premise. Therefore, we find little theoretical overlap between the CASP hypothesis based on the fundamental concepts of the population genetics and the genomic drive hypothesis except that both hypotheses attempt to make sense of the undeniable evidence linking TEs and speciation. In the revised manuscript we comment only on the concept of "fecundity" in the context of large mammalian orders and briefly address the main premise of the hypothesis in the Implications section.7. The authors work at the Genetic Information Research Institute - the home of Repbase - and thus would seem to have a uniquely close perspective and deep insight into the distribution of TE diversity among evolutionary lineages. However, the rationale behind the choice of species/lineages represented among the primary data presented in Tables Response: First, we focused on young families of TEs to minimize uncertainties associated with annotations of older repeats. In Table 1we tried to focus on well annotated species to illustrate the proposed correlation between population structure and the diversity of TEs. In Figure inspired by this review, we use a more extensive dataset from Repbase to verify the predicted correlation. In Table 2we focus on mammalian species that are not only well annotated in Repbase, but they also have been extensively studied from the evolutionary point of view. This helped to combine multiple lines of published evolutionary evidence in support of the hypothesis.


The State of Israel that was declared independent in 1948 is a country that is ∼450 km in length and 180 km at its maximum width Fig. . In 2012Most of the population lives in the coastal area and, in particular, in the Tel Aviv region. The Jewish population is located primarily in cities, whereas the Arab population mostly resides in small towns and villages.The Jewish people originate from the Middle East. After the destruction of the First Temple, part of the community remained in Palestine while another part moved eastward founding the Babylonian Jewry, ancestors of the Iraqi and Iranian Jews. With the rise of the Greco-Roman empires, Jews began to move also westward as far as Spain and Portugal, where a large Jewish community developed in the Middle Ages. Sephardic Jews are descendants of the Jews who were forcibly expelled during the Inquisition in Spain and settled mostly in the countries along the Mediterranean Sea and in the Netherlands and the New World. The major movement of Jews toward Eastern Europe was in the middle Ages to France and Germany. Later on, there were two important waves of migrations of the Ashkenazi Jews in central Europe: the first in 15th to 16th century eastward to Bohemia and eventually to Poland-Lithuania and then from the end of the 18th century back to the west .The Jewish communities differed in their cultural outlook and way of life, in their spoken language and their traditions. While differences existed between the various regions where the Ashkenazi Jews were living, it is difficult to delineate subgroups among them. On the other hand, other Jewish communities remained geographically separated one from the other and developed as distinct identities. Therefore, the non-Ashkenazi Jews are best delineated as communities according to their country/region of origin. While the non-Ashkenazi communities represented more than 90% of the Jews in the 12th century, because of the size expansion of Ashkenazim, the non-Ashkenazi Jews represented only 10% of the world Jewry in 1930. The total number of Jews, in particular Ashkenazi Jews, was dramatically reduced by the holocaust but today, the estimated number of Jews worldwide is 13–14 millions. In 2012, there were 5,999,600 Jews living in Israel, ∼50% were of Ashkenazi origin , or G6PD deficiency found with a relatively high frequency among some of the Jewish communities are also frequent in the non-Jewish local population. However, these are exceptions and in most cases the high frequency of the genetic diseases is unique to the Jewish community as a result of its religious isolation. In most of the cases a relatively high frequency is found in a single Jewish community due to a unique founder mutation. There are few diseases in which the same mutations are found in several communities either since they were ancient mutations and existed already before the dispersion of the Jews or because of migration form one community to another , was launched in 2006  and not participating in pregnancy screening or testing. In this community, preimplantation genetic diagnosis (PGD) is the preferred procedure for couples at risk for genetic diseases, since the embryo is considered an individual only 40 days after conception. The majority of the Israeli Jewish population, even if religious (but not ultra orthodox as mentioned above), will envisage termination of pregnancy at almost every stage of the pregnancy if the fetus is abnormal. The Israeli law allows for termination of pregnancy at all stages of pregnancy on the condition that the fetus is affected with a severe disease as determined by a medical committee. As a result of this duality, in the last decade most Jewish children with Down syndrome are born to women from the ultraorthodox community  have been issued in various countries Hessini . The AraSince the enactment of universal health care law in 1995, the entire population benefits from a comprehensive health insurance plan that included curative and preventive out-patient care as well as hospital care ; each unit is a component of the IVF division and of the genetic department and offers molecular and cytogenetic diagnosis. The indications for PGD are delineated by the Ministry of Health. Using PGD for sex selection for nonmedical purposes is forbidden in Israel. A special committee exists and is entitled to make exceptions in rare cases.Most health funds have genetic clinics run by medical geneticists that provide genetic consultations which numbers are rapidly increasing. In addition, several genetic clinics in the community are affiliated with the regional hospital genetic department, and are run by a member of the genetic department team.The cytogenetics laboratories provide two types of services: clinical diagnosis and prenatal diagnosis. All but one of the cytogenetics laboratories are integral parts of the Medical genetic departments and are found in hospitals. The exception is a large laboratory of the Maccabi Health fund that provides public tests as well an important portion of the private prenatal tests in Israel. The directors of the cytogenetics laboratories are either acreditated Ph.D. medical cytogeneticists or Israeli board certified physician medical geneticists. These laboratories are regulated by the Ministry of Health.Chromosomal microarray analysis (CMA) is performed using different platforms in several of the cytogenetics laboratories. All these laboratories offer CMA for clinical diagnosis; a few of them also perform CMA for prenatal diagnosis.Cytogenetics for oncology/hematology are done either in general cytogenetics laboratories or in separate specialized laboratories found within the oncology/hematology departments. The set up is different from one hospital to the other.While almost all the genetic departments/units have a molecular laboratory, many laboratories are independent and found outside of the genetic departments. As a rule the laboratories that are found in other hospital departments perform tests for diseases specific to that department (such as hematology or endocrinology). There are two private molecular genetic laboratories providing molecular testing including genetic sequencing.The directors of the molecular laboratories are either acreditated Ph.D. medical molecular geneticists or Israeli board certified physician medical geneticists or Israeli board certified physicians specialized in the field of the laboratory. These laboratories are regulated by the Ministry of Health and the list of tests provided must be listed in the Israeli genetic database.As of January 2014 next-generation sequencing is not yet performed as a clinical service in any of the Israeli accredited laboratories. Next-generation sequencing tests that are performed for medical diagnosis are done in laboratories outside of Israel including whole exome sequencing.Since 1986 genetics is a medical specialty recognized by the Israeli Medical Association and the Ministry of Health. A two and a half year training program for medical genetics in an approved medical genetic department is available for specialists (physicians after at least 5 years training and board examination). The syllabus includes 24 months in the genetic clinics and 6 months in a genetic laboratory. After completion of training there is a national board examination including a written and an oral test.To become a genetic counselor in Israel one must have completed a master in human genetics or in genetic counseling. In both cases after obtaining the master degree the candidate needs to perform a 2-year training syllabus in a medical genetic department and pass a board examination.There are two Master's degree genetic counseling training programs in Israel. The Hebrew University – Hadassah Medical School, Jerusalem program was established in 1997 and the other program that was established in 2009 in the Rappaport medical school, Technion Haifa. Both are 2-year programs with one class of six students at a time. Most of the graduates have remained in Israel to provide genetic counseling services .β-thalassemia has been implemented for the Arab population and some of the Jewish communities in which the disease is relatively frequent .PGD is available free of leftge for couples at risk for a severe genetic disease. Non invasive prenatal test (NIPT) is available privately, partially covered by complementary insurance being performed in the different laboratories outside of Israel.The tests recommended by the Association of Israeli Medical Geneticists are available free of leftge. Other screening tests may be done as part of the complementary insurance or privately.In the debate regarding disability rights and prenatal diagnosis, the majority of the Israeli population is in favor of prenatal genetic testing as well as selective abortion Raz . In IsraAnother field that has been in continuous expansion is the use of prenatal screening and testing with the aim of prevention. The use of prenatal screening tests including those recommended or those done privately is very high in particular in the nonorthodox Jewish population and the non-Jewish population  patients have been active trying to introduce these diseases in the neonatal screening in the newborn screening program. Screening for cystic fibrosis was not included in the expansion of the program in 2009 because of the relatively small number of children born affected with the disease as a direct consequence of the population carrier screening (Zlotogora and Israeli


Alfalfa is an autotetraploid, allogamous and heterozygous forage legume, whose varieties are synthetic populations. Due to the complex nature of the species, information about genetic diversity of germplasm used in any alfalfa breeding program is most beneficial. The genetic diversity of five alfalfa varieties, involved in progeny tests at Institute of Field and Vegetable Crops, was characterized based on RAPD markers. A total of 60 primers were screened, out of which 17 were selected for the analysis of genetic diversity. A total of 156 polymorphic bands were generated, with 10.6 bands per primer. Number and percentage of polymorphic loci, effective number of alleles, expected heterozygosity and Shannon’s information index were used to estimate genetic variation. Variety Zuzana had the highest values for all tested parameters, exhibiting the highest level of variation, whereas variety RSI 20 exhibited the lowest. Analysis of molecular variance (AMOVA) showed that 88.39% of the total genetic variation was attributed to intra-varietal variance. The cluster analysis for individual samples and varieties revealed differences in their population structures: variety Zuzana showed a very high level of genetic variation, Banat and Ghareh were divided in subpopulations, while Pecy and RSI 20 were relatively uniform. Ways of exploiting the investigated germplasm in the breeding programs are suggested in this paper, depending on their population structure and diversity. The RAPD analysis shows potential to be applied in analysis of parental populations in semi-hybrid alfalfa breeding program in both, development of new homogenous germplasm, and identification of promising, complementary germplasm. Total genomic DNA was isolated from leaves according to the protocol of Somma .2.2.®GmbH kits X, Y and Z were initially tested [In order to test amplification profiles for polymorphism, readability and reproducibility, sixty decamer primers from ROTHy tested  from whi2.3.Each fragment amplified using RAPD primers was treated as binary unit character and scored “0” for absence and “1” for presence. In order to measure informativeness of the markers, the polymorphism information content (PIC) for each primer was calculated . PIC proJaccard’s coefficient of similarity was calculated and dendograms for varieties and individual samples were constructed by using Unweighted Pair Group Method of Arithmetic Mean (UPGMA), using software package NTSYS-PC Version 2.11 . The sam3.Out of 60 tested RAPD primers, 17 primers generated stable and reproducible bands in all samples during the preliminary primer testing, and later in genetic diversity investigation . A totalI = 0.375). The mean value within varieties was 0.322 and total genetic diversity across populations was 0.426. Among these five varieties, variety Zuzana exhibited the highest level of variation  = 66.67, Ne = 1.453, He = 0.256, I = 0.375). A positive Pearson correlation was detected between percentage of polymorphism and Shannon’s diversity index  and between effective number of alleles per locus and expected heterozygosity , suggesting that polymorphism was unevenly distributed among populations. Most of the genetic variability estimated by AMOVA was attributed to variation among individuals within varieties (88.39%) and only 11.61% was found between varieties  than might be expected based solely on the decrease in heterozygosity . The larGenetic diversity determined in this study was high, which agrees with previous research on tetraploid alfalfa ,34. The The dendogram of cluster analysis of individuals clearly illustrated population structure of the tested varieties. It showed the presence of subpopulations in Banat and Ghareh and different levels of similarity between them. It also showed that varieties Pecy and RSI 20 are more uniform, forming tight, clearly defined clusters. As the estimates of genetic diversity indicated, variety Zuzana showed very high level of variation. The obtained results are in accordance with breeding history of the analyzed varieties: Zuzana is old, very adaptable synthetic variety developed by crossing large number of parents, Banat and Ghareh were developed mainly by collecting local germplasm with a certain level of uniformity in yield and morphological traits, while Pecy and RSI 20 resulted from intensive breeding programs.In our opinion, the use of investigated germplasm in the breeding programs would depend on their population structure and diversity. The populations with high level of genetic variation like Zuzana might be used as a source for selection of desirable germplasm, for development of new varieties. Sub-populations in Banat and Ghareh could be used in development of new, unique and homogenous germplasm. The germplasms with a higher level of uniformity, like Pecy and RSI 20 in this investigation, could be used as potential parents in semy-hybrid breeding program, for producing heterozygous hybrid progenies. The previous research in alfalfa showed that selection of very diverse parental genotypes according to their molecular markers distance has not been successful in predicting heterosis –39. Howe5.The presented results confirmed that RAPD analysis can be successfully used in estimation of genetic diversity within and among alfalfa varieties. The obtained data were clear and reproducible, showing that this simple and quick molecular technique is very useful for purposes such as ours. This type of molecular marker analysis might potentially be used in development of new, uniform germplasm and identification of promising, complementary germplasm aiming at reduced number of necessary crossings and therefore making future alfalfa breeding programs more efficient.