Nuculaatacellana) to test for population divergence. Multilocus analyses indicated a sharp discontinuity across a narrow depth range, with extremely low gene flow inferred between shallow and deep populations for thousands of generations. Phylogeographical discordance occurred between nuclear and mitochondrial loci as might be expected during the early stages of species formation. Because the geographic distance between divergent populations is small and no obvious dispersal barriers exist in this region, we suggest the divergence might reflect ecologically driven selection mediated by environmental correlates of the depth gradient. As inferred for numerous shallow-water species, environmental gradients that parallel changes in depth may play a key role in the genesis and adaptive radiation of the deep-water fauna.Ecological speciation probably plays a more prominent role in diversification than previously thought, particularly in marine ecosystems where dispersal potential is great and where few obvious barriers to gene flow exist. This may be especially true in the deep sea where allopatric speciation seems insufficient to account for the rich and largely endemic fauna. Ecologically driven population differentiation and speciation are likely to be most prevalent along environmental gradients, such as those attending changes in depth. We quantified patterns of genetic variation along a depth gradient (1600-3800m) in the western North Atlantic for a protobranch bivalve ( How species form is one of the most fundamental questions in evolutionary biology. Over the past two decades considerable progress has been made in identifying the scales, mechanisms, and driving forces of species formation in terrestrial and shallow-water ecosystems e.g. -7). Howe. Howe7])Geographic patterns of population genetic structure provide one of the primary lines of evidence for identifying the forces that might isolate gene pools. Marine organisms with pelagic dispersal were originally thought to disperse widely and show little population divergence , but recA growing body of evidence suggests that ecological speciation, defined as “the process by which barriers to gene flow evolve between populations as a result of ecologically based divergent selection between environments” , may be While considerable evidence exists for each of these mechanisms influencing population structure of shallow-water organisms, evidence of them operating in the deep sea is limited, apart from hydrothermal vents and other chemosynthetic ecosystems that have been more intensively studied . SevThese emerging phylogeographic patterns suggest that the environmental gradients paralleling changes in depth likely play an important role in the formation of new species in deep-water ecosystems . IncreasNuculaatacellana Schenck 1939 (formerly Deminuculaatacellana) distributed across a depth gradient (1600-3800 m) in the western North Atlantic. Previous work using mtDNA (16S) identified strong genetic divergence among populations above and below 3000m [Natacellana. ’s demersal pelagic larvae. Several explanations might account for the divergence including idiosyncrasies of mtDNA , selection due to environmental changes associated with depth, or the presence of bathymetrically separated cryptic species. To better evaluate these alternative explanations, we quantify phylogeographic patterns using five loci, including both mitochondrial and nuclear markers. Recent work has stressed the importance of using multiple loci because mutational and coalescent stochasticity can lead to incongruent patterns among independent loci , and, andNuci e.g. ,70). Our. OurNuNo specific permissions were required for collection of specimens, because they were collected in international waters below the continental shelf. Collection of specimens did not involve endangered or protected species.Endeavor, specimens were collected along a transect closely following the Gay Head—Bermuda transect sampled by Hessler and Sanders [Nuculaatacellana specimens sorted on board were either preserved by flash-freezing or by placing in 95% ethanol, and stored at –80°C. Additional specimens were sorted from the bulk samples after the cruise, also using chilled ethanol to slow DNA degradation. Natacellana.  was collected at 9 of 20 stations , using the standard animal tissue protocol with 2 sequential elutions of 100µL. PCR amplifications of mitochondrial COI and four noncoding nuclear loci (an actin intron (MAC), a calmodulin intron (CAL), and two noncoding anonymous fragments (DAC3 and DAC6)) were performed separately. Anonymous DNA markers were obtained by digestion of genomic DNA with the restriction enzyme AluI , agarose gel selection of 1.0-1.5 kb fragments, cloning and sequencing; candidate markers were screened for polymorphism by sequencing a subset of the above specimens. Nuclear introns were selected based on a previous survey of introns in protobranch bivalves . StandarAll successful amplifications produced single PCR bands as seen by gel electrophoresis, except for some individuals heterozygous at CAL for a 68bp indel that allowed separation of the two alleles in the gel. These alleles were gel purified and sequenced separately; all other single PCR bands were sequenced regardless of heterozygous status. Bi-directional sequencing was performed by Agencourt . The two reads for each individual were trimmed, aligned, and manually edited using Sequencher 5.0.1 .Individual base pairs were considered heterozygous if a clear double peak of near-equal height existed in both chromatograms, in the context of otherwise low or nonexistent background. Heterozygotes possessing alleles of different lengths (polymorphic indels) were ascertained by initially clear, single-peaked chromatograms that became almost-totally double-peaked (except for runs of a single nucleotide) while maintaining well-shaped peaks and regular spacing. The two juxtaposed sequences were deconvolved with the online program Indelligent ; each stTo ensure the noncoding status of nuclear loci, they were checked for the potential presence of coding sequences by BLAST searches against the GenBank nucleotide database, GenBank’s ORF finder, the Gene Ontology database BLAST2GO , and AUGArlequin 3.5  was used2 statistic and p-value for the 2-by-2 contingency table of differences: Fixed vs. Variable and Synonymous vs. Nonsynonymous [As an additional test of selection, the McDonald-Kreitman test was performed by hand on COI, between the shallow and deep groups. Statistical significance was determined by computing the χnonymous .distruct [Structure v2.3.4  was useddistruct .th base  and increasing the connection limit until all haplotypes were incorporated into a single network. For CAL, a large 68-bp indel (see Results) required two networks, one for the four “deletion haplotypes” and one for the remaining “insertion haplotypes”.Haplotype networks were constructed for individual loci in TCS , treatinST and Φ’ST values  the log of geographic great-circle distance between stations and (2) the log of depth difference between stations, separately within the shallow and deep groups identified by Structure. Regression was performed via partial Mantel tests in Arlequin [The population clustering determined by Structure (see Results) was tested in Arlequin by AMOVA on all five loci, nesting individuals within stations, and stations within the shallow and deep groups. AMOVAs on multilocus and locus-by-locus pairwise differences were calculated with significance assessed from a null distribution of 1000 randomizations; multilocus pairwise Φle value ) were alArlequin , removin8) employed 10 Metropolis-coupled chains with a two-step increment model  and a burnin of 100,000, and were continued until all ESSs > 70. Three replicate runs with different starting seeds were performed to assess convergence. Parameters were converted to “demographic units” using a heuristic generation time of 10 years. The inferred demographic history was plotted using IMfig. An Extended Bayesian Skyline Plot .The demographic history of populations was reconstructed in IM v.12.17.09 , using tt  was prot , indicating that COI is not under selection.Simple tests of neutrality revealed a few significantly negative values for Tajima’s D at several loci , with deStructure runs tended to exhibit small variance at the highest and lowest Ks (K≤3 and K≥8), with larger variance at intermediate K ; howeverThe differential admixture of individuals at mitochondrial vs. nuclear loci was apparent in haplotype networks . Particularly among deep stations, significant ΦST’ s likely reflect small sample sizes. The significant separation of shallow and deep lineages was highly supported in the starBEAST genealogy  results, indicating significant divergence between shallow and deep populations for each locus independently and when all loci were analyzed together, with little divergence within populations . The conep pairs ; the stae~121,000), and a much larger shallow population , comparable to the relative population sizes produced by the starBEAST population analysis (-7 -10-8) and asymmetric with greater dispersal from the shallow to the deep population. Translation of these estimates into demographic units indicates that, of the 4 million individuals in the shallow population, approximately 5 migrate to the deep population each generation. Results were qualitatively similar for the IM analyses excluding COI (-6 -10-7), with overall migration still extremely low. All three replicate IM runs for each analysis (with and without COI) produced very similar estimates with 95% HPD overlapping extensively for all parameters (not shown).Coalescent reconstruction of demographic history as estimated in IM using all 5 loci revealed an ancestral population that split approximately 95,000 generations in the past, resulting in largely independent shallow and deep populations along the sampled depth gradient . Althouganalysis , inset. ding COI . Effectie of 2 to 3 million, over approximately the last 1MY . ,97. 65,9Natacellana. , especially in the shallow population  were detected for some samples and can indicate purifying selection; however, these tests are highly sensitive to fluctuations in demographic parameters such as population size ,103. In pulation  where moNatacellana.  the four nuclear loci analyzed display high allelic diversity and some differentiation by depth; however, full sequences of the nuclear small ribosomal subunit (18S) and a 718 bp fragment of the large subunit (28S) were 100% identical among shallow and deep individuals (data not shown). Although not conclusive evidence, these results do suggest that populations have not been isolated long enough for divergence to accumulate in these more slowly evolving genes, indicating that populations of Natacellana.  may be at a very early stage of species formation.Significant fixed differences in COI are often ascribed to cryptic species, which are commonly revealed when morphologically identified species are analyzed genetically e.g. ,106,107),107106,1Natacellana. , which has demersal pelagic larvae that likely spend days to weeks dispersing [Natacellana.  [The distance between the shallow and deep groups  is almost certainly within the dispersal window of spersing ,108. Thecellana  and the cellana  suggest cellana ,111. If sensu [Natacellana. .The Deep Western Boundary Current (DWBC) flows south/southwestward in the immediate vicinity and depth of our sampled region, underneath and counter to the Gulf Stream ,113, prosensu ,31). It sensu ,117 and The lack of a clear isolating barrier and the extremely small scale over which divergence occurs suggest that selection might play an important role. A number of environmental gradients parallel changes in depth including temperature, oxygen, salinity, POC-flux, pressure, sediment characteristics, flow regimes, and topographic complexity as well as a suite of faunal characteristics such as the diversity, composition and trophic complexity of sediment communities -63. Any Identifying the precise environmental forces that shape bathymetric patterns of genetic variation will require considerably more research, but the greater divergence in COI compared to the nuclear loci is consistent with depth-related selection on mitochondrial variants. Metabolic processes might be especially sensitive to various depth-related environmental gradients  leading to selection for different mitochondrial variants along the depth gradient. If this selection was strong enough to impede gene flow by selecting against migrants from contrasting depths (e.g. immigrant inviability ) it coulEuniceaflexuosa appears to be related to strong environmental selection against ecophenotypes from contrasting depths that reduces gene flow and may ultimately lead to speciation [Faviafragum [Seriatoporahystrix, reciprocal transplants of depth-segregated, genetically distinct ecotypes implicated post-settlement selection against migrants from parts of the reef formation at different depths [A consensus is emerging for both shallow and deep organisms that strong differences among populations from different depths may be caused by environmental gradients that parallel depth e.g. ,124-130)-130124-1eciation . Similarfragum ,129,131.t depths ,132. Evet depths . A rapidt depths ).Eurythenesgryllus was much greater across a 3.6 km depth gradient than across 4000km at the same depth [Consistent with depth and its attendant environmental gradients playing an important role in diversification of deep-sea species, numerous studies have documented strong bathymetric divergence suggestive of cryptic species ,57-60. Ime depth  or even me depth . Finallyme depth ,133,134)me depth .The environmental gradients imposed by increasing depth in the oceans make an intriguing parallel to altitudinal gradients in terrestrial systems, with greater depth analogous to greater altitude. Numerous studies have documented gradients of lower genetic diversity with increased altitude in plants  andNatacellana.  is widely distributed in the Atlantic, with virtually no divergence between the eastern and western North Atlantic and only modest divergence between the North and South Atlantic [Another possible explanation for the depth divergence is that it formed in allopatry and the shallow and deep groups experienced secondary contact in the western North Atlantic, resulting in differential introgression of mitochondrial and nuclear genes. However, Atlantic . In addiThe deep ocean is a vast semi-continuous ecosystem that supports a highly diverse and largely endemic fauna. The evolutionary processes that gave rise to this distinctive fauna, the spatial and temporal scales over which they operate, and the geography and bathymetry of divergence are poorly understood. Given the limited ecological opportunity and the lack of obvious mechanisms that would allow population differentiation and speciation, it is unclear how new species form, especially at a rate sufficient to explain the high levels of diversity. Unraveling how and where evolution unfolds is critical for explaining biogeographic patterns of diversity , predictFigure S1Haplotype network for COI.Circle size indicates number of individuals possessing that haplotype. Small circles represent unsampled haplotypes required to connect the network. Squares indicate the most likely ancestral haplotype. Haplotypes are shaded shallow and deep as in Figure 1.(TIF)Click here for additional data file.Figure S2Haplotype network for CAL.Haplotype shape, size, and coloring are as in (TIF)Click here for additional data file.Figure S3Haplotype network for MAC.Haplotype shape, size, and coloring are as in (TIF)Click here for additional data file.Figure S4Haplotype network for DAC3.Haplotype shape, size, and coloring are as in (TIF)Click here for additional data file.Figure S5Haplotype network for DAC6.Haplotype shape, size, and coloring are as in (TIF)Click here for additional data file.Figure S6Population demographic history and migration estimates from IM for nuclear loci.The gray box indicates the estimated effective population size (Ne) of the ancestral population. Estimated splitting time is indicated by the horizontal line. Descendant shallow and deep populations are represented above the line by polygons whose starting width is the estimated Ne just after the split and whose upper width is the estimated contemporary Ne. Curved dotted arrows represent estimated migration rates per generation, forward in time from source to destination. Coloring of shallow and deep is as in (TIF)Click here for additional data file.Table S1PCR reaction mixtures and thermocycler conditions for amplified loci.PCRs were performed in 50µL reactions consisting of 1X GoTaq Flexi buffer with loading dye , 2.5mM MgCl2, 2pmol dNTPs, 1.2pmol of each primer, 2µL genomic DNA, and 1 U of Taq polymerase (Promega). Conditions specific to each locus are given below; all protocols had an initial denaturation of 94°C for 3 min., 35 cycles of , final extension at 72°C for 3 min., and a final hold at 4°C.(DOC)Click here for additional data file.


Sebastes ruberrimus, YR) are widely distributed across the northeastern Pacific Ocean, inhabiting coastal rocky reefs from the Aleutian Islands in Alaska through southern California. This species exhibits an extended pelagic larval duration and has the capacity for long distance larval transport. We assayed 2,862 YR individuals from 13 general areas in the northeast Pacific Ocean for allelic variation at nine microsatellite loci. Bayesian model-based clustering analyses grouped individuals from the Strait of Georgia (SG) into a distinct genetic cluster, while individuals from outer coastal water locations (OCLs) were partitioned equally across two genetic clusters, including the cluster associated with the SG fish. Pairwise FST values were consistently an order of magnitude higher for comparisons between the SG and OCLs than they were for all OCL-OCL comparisons (∼0.016 vs. ∼0.001). This same pattern was observed across two time points when individuals were binned into an “old” and “young” group according to birth year . Additionally, mean allelic richness was markedly lower within the SG compared to the OCLs (8.00 vs. 10.54–11.77). These results indicate that the Strait of Georgia “deep-basin” estuary oceanographic domain acts as a dispersal barrier from the outer coastal waters via the Juan de Fuca Strait. Alternatively, selection against maladapted dispersers across this oceanographic transition may underlie the observed genetic differentiation between the Georgia basin and the outer coastal waters, and further work is needed to confirm the SG-OCL divide acts as a barrier to larval dispersal.The boundaries between oceanographic domains often function as dispersal barriers for many temperate marine species with a dispersive pelagic larval phase. Yelloweye rockfish ( Population replenishment for a large number of marine populations depends upon the input of exogenously derived individuals Oceanography is an important driver of population structure in temperate reef fishes, as ocean currents largely dictate larval trajectories, planktonic survival and delivery to settlement habitat Sebastes spp.) constitute a diverse group of nearshore fishes that are distributed in temperate waters around the world S. mystinus) S. miniatus) S. caurinus) and brown rockfish (S. auriculatus) from the Puget Sound basin exhibit significant genetic divergence from populations located along the outer coast Rockfishes  in the northeast Pacific Ocean. We predicted that population structure would occur across oceanographic features that likely function as barriers to dispersal, resulting in three genetically differentiated subdivisions. These predicted genetic subdivisions include (i) the separation of the Georgia basin from the outer coast via the Juan de Fuca Strait and (ii) isolation of the Bowie Seamount location. We also predicted an isolation by distance signal would be detected across the outer coast locations, which span over 1,500 km of coastline. We detected subtle genetic structure, partitioning the Strait of Georgia population from a panmictic outer coast population. No signal of isolation by distance was detected across the outer coast sampling locations.In this study, we used microsatellite genetic markers to assess the population genetic structure of yelloweye rockfish , southeast Alaska, Washington and Oregon , Table 1Total genomic DNA was extracted with Qiagen Dneasy extraction kits . Nine microsatellite loci  according to Rousset Deviations from Hardy-Weinberg equilibrium were assessed for each locus-population combination with GENEPOP version 3.1 ST, as well as with the genetic clustering program STRUCTURE version 2.3 ST values were estimated with θ ST values, 420 individuals (which have accompanying age data) from eight sample locations were binned into a “young” or “old” age group according to birth year. Individuals born before and after 1980 were binned into the “old” and “young” group, respectively. The year 1980 was chosen as the cutoff as it is the median birth year, which also maximizes sample size similarity between the sample locations for each age group (Table S2 in K) among the sample locations, and to estimate individual assignment probabilities for each fish to each resolved cluster. The number of putative genetic clusters assessed ranged from 1 to 7. Each run consisted of a 500,000 step burn-in plus an additional 1,000,000 steps, and 20 iterations were run for each K-value. Due to low overall genetic structuring , the number of genetic clusters was evaluated under both an admixture model including a location prior K) as an estimator of K, due to the limited success of this method observed when overall differentiation is low Population structure was assessed with estimates of the summary statistic, FA total of 2862 individuals from 13 general sample locations (sample sizes ranged from 46 to 779) were assayed at nine microsatellite loci. Only individuals with allele scores from a minimum of seven loci were retained for analysis. The majority of individuals (66.8%) had genotype scores across all nine loci, 628 (21.9%) contained missing data for one locus, and 323 (11.3%) individuals had missing data for two loci. Missing data, therefore, constitutes only 4.9% of the total dataset. There was no evidence for selection acting at any of the microsatellite loci using LOSITAN .Sal3) to 39 (Sme3) with an average of 18.9 alleles per locus. Expected heterozygosity ranged from 0.488 (Sru9) to 0.877  and averaged 0.702 across loci and sample locations . Pairwise FST values range from less than 0 to 0.0193, and were consistently an order of magnitude higher for all comparisons between the Strait of Georgia (SG) sample location and outer coastal water locations (OCLs) than for comparisons between OCLs  among our samples. By contrast, results from the model including a location prior had equal support for a K of 1 and a K of 2  and one with the Strait of Georgia and Bowie Seamount sample locations removed (Mantel P = 0.268).No significant association between genetic distance and geographic distance was detected with the Mantel tests . Two IBDWe detected subtle population genetic structure that separates a putative Strait of Georgia population from a panmictic outer coast population. The results presented here constitute a re-analysis of YR population genetic structure originally investigated by Yamanaka et al. One interpretation of the genetic structure observed between the SG and the OCLs is that the oceanographic transition between these areas functions as a physical barrier to larval dispersal. Alternatively, dispersal may occur across this oceanographic divide and natural selection against maladapted dispersers reduces connectivity and promotes genetic differentiation Bowie Seamount (58.23°N 135.74°W) is approximately 200 kilometers west of Haida Gwaii and rises to within 30 meters of the surface from depths over 3,000 meters creating rockfish habitat that is isolated from coastal areas ST values The apparent lack of genetic structure or a significant IBD relationship between the OCLs should not be taken, on its own, as evidence supporting high connectivity between these locations. High effective population sizes (Ne), commonly observed in marine fishes, may resist the effects of drift and contribute to low FSebastes spp.) in the late 1990's Conservation concerns surrounding Pacific rockfishes  Our analyses support a population boundary between the inshore waters of the Strait of Georgia and the outer coastal waters that coincides with the transition between two oceanographic domains via the Juan de Fuca Strait. Several important implications can be drawn from this result. Principally, the genetic data suggest that dispersal is restricted regionally by major oceanographic features. This scale of dispersal seems to match the scale at which YR stocks are managed, although more detailed studies are needed to fully elucidate the complex metapopulation dynamics observed. If dispersal between the Strait of Georgia and the outer coastal waters is indeed rare or demographically insignificant, it is unlikely that outside areas will function as substantial larval sources for the inshore population.File S1Supporting figures and tables. Figure S1. Rockfish Conservation Areas. The distribution of rockfish conservation areas (RCAs) in British Columbia (figure reproduced with permission from Yamanaka & Logan http://www.pac.dfo-mpo.gc.ca/fm-gp/maps-cartes/rca-acs/index-eng.htm. Table S1. Primer information. The forward (F) and reverse (R) primer sequences, PCR annealing temperatures (TA), Genbank accession number (GB AC #), and reference for each microsatellite locus are shown below. Table S2. Temporal population structure comparisons. The sample size for the “old” and “young” datasets , as well as the pairwise FST value for each within location, “old” vs. “young” comparison is shown below. None of the pairwise FST values is statistically significant. Table S3. Individual locus descriptive statistics. Mean number of alleles (NA), total number of alleles (NT), observed heterozygosity (HO), expected heterozygosity (HE), inbreeding coefficient (FIS), theta, and standard error of theta (S.E.) for each locus are shown. Table S4. “Old” pairwise FST values. Pairwise FST values for the “old” dataset are shown below. Statistically significant values are shown in bold. Table S5. “Young” pairwise FST values. Pairwise FST values for the “young” dataset are shown below. Statistically significant values are shown in bold. Table S6. STRUCTURE mean log likelihood results. Mean log likelihood of each K value (LnP(K)) and standard deviation (S.D.) are shown for both models evaluated in STRUCTURE: an admixture model without a location prior, and an admixture model with a location prior. The most likely value of K is shown in bold under each model.(DOC)Click here for additional data file.


The review covers the analysis of our own and published data pertaining topopulation and genetic consequences in various mammalian species underconditions of high levels of ionizing radiation as a result of the Chernobylaccident. The findings indicate that these conditions have promoted thereproduction of heterozygotes in polyloci spectra of molecular genetic markersand animals with a relatively increased stability of the chromosomal apparatus.The prospects of using the reproductive “success” of the carriers of thesecharacteristics as an integral indicator of the selective influence ofenvironmental stress factors are discussed. Homo sapiens, whose core elementsinclude the simultaneous combination of biological, socio-cultural andtechnological adaptations. The Chernobyl accident revealed the incompatibilityof these elements. Technological adaptation being the most mobile of the listedadaptations, it determines the general direction of the history of humankind.Domestication of plants and animals as a condition of the transition tosedentary life characterized the first phase of the formation of the agrariancivilization and, hence, the creation of novel technologies capable of adaptingto dynamic environmental factors. Culture itself (including the productionculture) must play a major role in the coordination of all three components ofthe adaptive strategy. The Chernobyl accident and the attitude towards itsconsequences and the consequences of numerous technogenic accidents anddisasters of the 20th century indicate that culture is developing significantlyslower than technological process and its biospheric consequences. Thepersistent controversies regarding the evaluation of the consequences for thehealth of the human population not only after the Chernobyl disaster, but alsothose of the atomic bombings of the cities of Hiroshima and Nagasaki serve asan illustrative example of the latter fact.It has been 26 years since the largest technogenic catastrophe of the 20thcentury – the Chernobyl disaster – which highlighted many of the global issuesfacing industrial and post-industrial societies. From the point of view of asystemic-evolutionary approach, the processes of anthropogenesis andsociogenesis are the results of employing unique strategies of survivalpertaining exclusively to Thus far, the consequences of ecological changes for living objects are merelydeclarative: species on the brink of extinction are counted, their reproductivefunction is assessed, and changes in communities are evaluated. Theinconsistency in the evaluation of the consequences of the Chernobyl accidentfor biota yet again brings forward an even more global problem; that is a lackof relatively reliable and consistent evaluations of biological safety in humanhabitats. The urgent need to develop novel approaches for the assessment ofbiological safety has two reasons: the abrupt increase in the spread ofcontaminants and the complexity of their composition. The conventional methodsfor the detection of toxic agents in the air, ground and water do not take intoaccount the constantly emerging novel contaminants and neglect their combinedeffects, thus requiring an additional analysis of the complex of livingorganisms which act as a target for toxic agents. It is evident that theselection of indicator species in which population genetic changes can be usedas an objective indicator of the biological safety of the region undersurveillance remains among the key issues today.However, the genetic consequences of genotoxic effects are most commonlyconsidered exclusively from the point of view of the risk of emergence ofmutant organisms, i.e., the carriers of constitutional mutations that arepresent in all the cells of a multicell organism. The frequency of occurrence can be used as an indirect measure of the “sensitive” part of thegene pool which does not participate in the reproduction of the populationanymore, since the carriers of constitutive mutations in the populations ofhigher organisms are typically less fertile than the normal individuals.The main reasons complicating an objective assessment of the damaging effectsof ionizing radiation include the following ones: heterogeneity in theradiosensitivity of the investigated groups of organisms at the level ofspecies, populations, individual organisms, various tissues and organs;volatility of the radiosensitivity of the cells isolated from the same organismduring the ontogenesis and under the influence of environmental factors  the individual sensitivity of a given organismto the factor under investigation , 2) the genotypical characteristics of the activity of theenzymes participating in the repair of the induced defects, 3) the enzymesfacilitating the detoxification of the toxic agents that enter the cells andare formed during the intracellular metabolism, and 4) the rate of removal ofthe damaged cells.The polygenic nature of the listed functions presumably leads to the widespreadindividual variability in mammalian groups in response to genotoxic impacts ofidentical intensity.It should be emphasized that the underdeveloped conceptions related to themultiplicity of targets for the damaging effect of ionizing radiation in thecytoplasm and nucleus, in various DNA segments and to the no less complex waysof formation of these damages in the form of mutations, and to the protectionsystems of a multicellular organism, which prevent the accumulation of mutantcells, results in the fact that the predicted damaging effects (for humans inparticular) are drastically different from empirically collected data. ;Taking into account the lack of information regarding the cascades ofmutagenesis-related molecular events, the problematic nature of predicting theconsequences of an ionizing radiation impact becomes apparent in case thisprognosis is based on the assessment of the actual mutational events. Thespecific features of the mutagenesis of various genomic elements, whosemechanisms, initiation rate, and biological consequences significantly differfrom each other, usually are not discussed.Meanwhile, without changing our views regarding the mechanism of the effect ofionizing radiation on the genetic material of biological objects, it isimpossible to expect that the contradictions between the experimental data andpredictions based on simplified conceptions and models of the mutagenic actionof ionizing radiation on living objects will be resolved.Heterogeneity of target sensitivity in livingobjects. Molecular, cellular, organ, species diversityof the damaging effects of ionizing radiation-11 per base per round of cell division[9 nucleotides, and roughly10% of those are found in the coding regions; i.e., this frequency is typicalof 6 ・108 genomic nucleotides. Hence, approximately1・10-3 mutations emerge in the coding regions of the structuralgenes in each genome in each round of replication.One of the reasons behind the existing difficulties is the fact that mutationalevents also occur at a relatively high frequency due to endogenous events.Thus, the frequency of spontaneous mutations in the structural genes in humancells is 5 ・10division. The fre9 nucleotides) using a comparative analysis of thecompletely sequenced genomes of parents and two monozygotic twins. Thefrequency of spontaneous substitutions estimated using this method reaches 1.1・10–8 per nucleotide per round of replication [Today the frequency of mutations is determined per nucleotide in the humanhaploid set .It is clear that such lesions occur 6–7 orders of magnitude more frequently as comparedto the nucleotide substitutions in the structural genes. The typical AP sites inducenucleotide substitutions  or may result in frameshift mutations. Thesemutations have been found in the microsatellite loci of a plasmid treated withН2О2. The number of AP sites increases when the cells aretreated with oxidizing or methylating agents.However, these mutations are preceded by DNA lesions at the potential mutationsites, since they can either be easily repaired or become actual mutations.They usually emerge due to hydrolysis, oxidation, or electrophilic influence onDNA molecules. These reactions occur as a result of an exogenous impact,including the impact of ionizing radiation; they can also result fromendogenous metabolic processes. The endogenous events cause a large number ofvarious DNA lesions . Thus, fIt has been known for a relatively long time that the action of oxygen radicalsleads to the emergence of a large number of oxidized bases in DNA, as well asto DNA breaks. Base modifications that occur due to the other oxidizingprocesses are discussed less frequently. Thus, polyunsaturated fatty acids, oneof the main components of membrane phospholipids, are characterized by highsensitivity to oxidation and are the main target of oxygen radicals. During 2,3-εG) havebeen identified in the DNA isolated from various human tissues; their levelincreases with an increase in the concentration of oxygen radicals.N2,3-εG can be found in rodent tissues subjected to oxidizingstress. The amounts of εА, εС are increased in patients with Wilson’s diseaseand the diseases associated with accumulation of copper and iron in the liver[Exocyclic ethylene groups . Approximately 10 doublestrand DNA breaks per cell cyclein the form of blocked replication forks occur during DNA replication. It is eStudies of animals with various knock-outs of the genes encoding the enzymesused to repair doublestrand breaks have demonstrated the significance of thesemodifications for the occurrence of mutational events at the nucleotide andchromosome levels.Deinococcus radiodurans, which are capable ofwithstanding 5000 Gy [It is obvious that the high frequency of spontaneous mutational events leads tothe fact that mutagenic effects are attributed not to the occurrence of thelesions but to the activity of their repair .The investigation into the mechanisms of spontaneous lesions in the geneticmaterial  indicates that these events are multistage by their nature.Supplementing this cascade with ionizing radiation will be an additional factorincreasing the probability of conversion of potential DNA lesions intomutations.The next level of control of the genetic stability in a multicellular organism,which prevents the accumulation of mutant cell clones, is based on the variousoptions of cell death available to genetically deficient cells, as well as theparticipation of immune system cells in the elimination of mutant clones. It is oThe reproduction of mutant cell clones of the germinative line and theemergence of mutant progeny are controlled by a cascade of events, each ofwhich can be modified by ionizing radiation.in vitro inСС57W/Mv mice under the influence of absorbed ionizing radiation at a dose of0.4–0.5 Gy (with respect to the control) as was confirmed in our experiments[It is interesting to point out that embryonic preimplantation mortality isincreased and embryonic fission is slowed down eriments. It shouThe analysis of the events of mutagenesis at the gene, cell and organism levelsdemonstrate that the characteristics of its alterations under the influence oflow-dose ionizing radiation depend on many factors, which are connected bothwith genetically determined processes and with the modifying effect of theenvironmental factors. The complexity of the interactions between the geneticand environmental components prevents the examination of individualcharacteristics of mutagenesis as an integral indicator of the effect of lowdoses of ionizing radiation on multicellular organisms. A decrease inpopulation fertility seems to be the most objective indicator.Conceptions of the radiation doses capable of causing damage to living objectsSignificant amounts of experimental observations of the consequences ofincreasing the level of ionizing radiation in various species, including thehuman one, have been accumulated. The most commonly used methods for assessingthe health consequences of increased levels of ionizing radiation are asfollows: determining the growth in the incidence of oncological diseases; thefrequency of occurrence of dividing cells containing lesions in the genetic apparatus; and the proportion of childrenborn with congenital anomalies .Today aLaboratory and field studies have demonstrated that an increase in the level ofionizing radiation in a number of cases  is accompanied by an increase in the frequency of individual speciescharacterized by an increased proportion of somatic cells with various mutations, 29.MeaLeukemia was the first oncological disease whose incidence was connected to theconsequences of the atomic bombings in Hiroshima and Nagasaki. The maximumincidence was observed 5 years after the bombings. Ten years later, an increasein the growth of solid tumors was recorded. Chronic lymphocytic leukemia,pancreatic and prostate cancer, and endometrial cancer were the most frequentlyidentified types. Even 55 years after the atomic bombings, 40% of the peopleinitially included in the Atomic-bomb Survivor Research Program are stillalive; this fact makes it possible to assess the longterm consequences of theexposure. It was determined that the relative risk of developing variousconditions per dose unit is higher among survivors of the bombings than amongthose who were subjected to medical radiation exposure. The risk of developinga disease among employees of atomic stations and miners is on averagecomparable to that observed among some population groups which survived theatomic bombings. The risk of somatic conditions among those who were subjectedto primary exposure of identical doses decreases with increasing age at whichthe exposure occurred. The frequency of various conditions of respiratory,gastrointestinal, and vascular systems is increased among survivors of the atomic bombings, 32.The connection between the doses absorbed by the embryos and the incidence ofleukemia in children was analyzed in England, Scotland, Greece, Germany, andBelarus following the Chernobyl accident. The total doses absorbed by theembryos varied from 0.02 mSv in England to 0.06 mSv in Germany, 0.2 mSv inGreece and 2 mSv in Belorus. A statistically significant increase in the riskof developing leukemia in newborns was identified at the peak of the exposure,between July 1, 1986, and December 31, 1987, as compared with children bornbetween January 1, 1980, and December 31, 1985, and between January 01, 1988,and December 31, 1990. In certain countries, the risk increases nonmonotonouslywith respect to the dose absorbed: it increases abruptly at low doses, followedby a decrease at high doses. The findings have been discussed in connectionwith the mechanisms of embryo/cell death at high doses and dose-dependentinduction of DNA repair. The accumulated results show the need forreconsidering our views on the absorbed doses that could be harmful to embryos. In chilet al. [Meanwhile, there are data available that indicate that the incidence ofcongenital developmental anomalies among children born to fathers whoparticipated in the cleanup effort after the Chernobyl accident exceeds theaverage frequency recorded in the Russian Federation. An incret al. .It was demonstrated that high and low doses of ionizing radiation have anonthreshold effect on the cardiovascular system. At least two mechanisms areinvolved in the formation of these effects: the impact on the formation ofmacrophage-enriched atherosclerotic plaques due to inflammation processes onthe vessel wall and a decrease in the cardiac muscle blood supply as a result.The manifestation of the pathology of the cardiovascular system following theirradiation exhibits a large lag-phase, especially after exposure to a low dose.It was discovered that the number of boys born in Bavaria and Denmark in 1987after the Chernobyl Nuclear Power Plant accident was higher than that of girls. The morin utero had considerably lower IQ scores as compared to those in the controlgroup of the same age. The differences were limited to the verbal IQ score; thenonverbal IQ score was not affected. These effects were not identified in thegroup of people who were exposed to radiation after 16 weeks of prenataldevelopment [Young people who received doses of ionizing radiationelopment .The incidence of cytogenetic anomalies in cells isolated from the peripheralblood and bone marrow at various laboratory and wild-type small murine rodentsthat reproduced in the alienation zone of the Chernobyl Nuclear Power Plant (CNPP) was determined. This zone is a unique system model to study the populationgenetic transformations caused by a change in the direction and intensity ofnatural selection. An abrupt change in the entire complex of ecological factorsoccurred on a limited territory following the accident. Representatives ofvarious taxonomic groups, including higher mammals, dwell successfully in spiteof these changes. It is important to emphasize that the main culprit in theecological catastrophe (the emission of radionuclides) has been wellestablished. It should be highlighted that it is impossible to simulate theaction of interdependent and interrelated changes in various conditions ofpopulation reproduction under the influence even of a single environmentalstress factor in a laboratory , whereas it is typically impossible to analyze the inheritanceof induced changes in generations of families under field conditions.In order to evaluate the possible direction of population genetic changes underthe influence of an increase in ionizing radiation in the alienation zone ofthe CN PP, with allowance for the genetic heterogeneity of bioindicator speciesin field conditions, a comparative analysis of long-term changes in generationsof genetically homogenous laboratory СС57W/Mv mice and in species of fieldvoles captured between 1994 and 2001 in the alienation zone of the CN PP inlocations with various levels of radionuclide contamination was carried out inthe present study. The cytogenetic variability in generations of thegenetically homogenous population was analyzed using two populations ofСС57W/Mv mice bred at the Institute of Molecular Biology and Genetics of theNational Academy of Sciences of Ukraine: the Chernobyl group  and the control group (avivarium in Kiev). These populations were kindly provided by S.S. Malyuta, anacademician of the National Academy of Sciences of Ukraine. The bone marrowcells isolated from mice of first and second generations of the Kiev population and the first, second, fifth, seventh, and tenth generations of theChernobyl population  reproducing in thespecialized vivarium at a rated dose of absorbed radiation of approximately 0.6Gy per animal were analyzed.Microtus arvalis andClethrionomys glareolus) captured in the alienation zoneof the CN PP at locations with lowlevels of radionuclide contamination (< 5 Cu/km2), which were consideredspontaneous  under conditions of an intermediate level ofradionuclide contamination  and with high levels .The mutational spectra were determined in representatives of the field volespecies (n± 1 (A2). The numbers of binuclear leukocytes (BL) and leukocytes withmicronuclei (LM) were determined in cells with an intact cytoplasm using thesame preparations. The mitotic index (MI) and the frequency of occurrence of BLand LM were determined per 1,000 cells.The following cytogenetic characteristics were taken into consideration in theanimal bone marrow cells: two types of aneuploidy, polyploidy, the frequency ofoccurrence of metaphases with chromosomal aberrations (CA), centric fusionsknown as Robertsonian translocations (RB) and asynchronous separation ofchromosomal centromeric regions at the end of the metaphase (ASCR ). Thepercentage of aneuploid cells was determined in two different variants: thecells with a loss or gain of the chromosome number, more than one  and aneuploid cells with a number of chromosomes2Table 1, 2) and in generations of the experimental population(Table 3).A significant increase in the frequency of cytogenetic anomalies was identifiedin the mutational spectra of the Chernobyl population of СС57W/Mv mice . Meanwhile, the responses toidentical levels of increased ionizing radiation were different to thestatistically significant level in groups of linear mice of various ages gave grounds toassume that the age-related changes are accompanied by a decrease inerythroblast sensitivity to damaging effects. It can be also expected that inthis case the processes of physiological adaptation in “old” animals ascompared to the ”young” group are connected to the long-lasting action ofchronic low-dose exposure to radiation. It was determined that thisphysiological adaptation was accompanied by an increase in the number ofdividing cells and a certain acceleration of the cell cycle, if thedetermination is based on the number of binuclear leukocytes per round ofmitosis in bone marrow cells isolated from the groups of the “old” control andthe Chernobyl animals (Table 2).The data in Table 3).It turned out that the incidence of such cytogenetic characteristics as RB, CA, and ASCR , which are directlyconnected with intra-chromosomal aberrations, decreases in the 5th generation,increases in the 7th generation, and decreases again in the 10th generation. Asimilar pattern of variability in the generations was identified for a fractionof the second type of aneuploid cells (2n = 40 ± 1chromosome). The incidence of other types of cytogenetic anomalies was higheronly in the first generation of Chernobyl mice as compared to that in thecontrol populations (Table 3).The analysis of the incidence of cytogenetic anomalies in the bone marrow cellsof the “young” СС57W/ Mv mice in the sequential generations  reproducing under conditions of chronic exposure to increasedlevels of ionizing radiation allowed to identify a nonlinearity of the changesin a number of cytogenetic characteristics in generations of geneticallyhomogenous animals .An analysis of genetically heterogeneous populations of field voles captured invarious years at locations with various levels of radionuclide contaminationallowed to obtain data supporting the fact that the selection forradioresistant animals became clearly evident in 1999 and 2001 amongpopulations of bank voles and common vole which inhabit regions characterizedby high levels of radionuclide contamination (Table 4).It was determined that an increase in the number of radioresistantspecies among the bank voles was most prominently evident in the “Red Forest”population (1000 Cu/km2). This selection was not observed at thelocations characterized by a significantly lower level of radionuclidecontamination .Thus, the bank vole populations captured in 1999 and 2001 at locations with thehighest level of radionuclide contamination mainly contained animals whose bonemarrow cells were characterized by an incidence of cytogenetic anomalies thatnot only was higher than that in the conditional control population, but wassometimes lower with respect to certain characteristics(Table 5).Similar data were obtained when studying common vole populations that each strain under control conditions has its ownspectrum of spontaneous mutations in bone marrow cells, and that only certaincharacteristics of this spectrum varied with animal age and the season duringwhich the research was carried out .Thus, aThe same tendency was revealed for the voles captured in the areas withelevated levels of radionuclide contamination. Only cytogenetic abnormalities,whose increased variability is species-specific for voles dwelling inunpolluted areas, accumulate in bone marrow cells: metaphases with Robertsonianinterchromosomal fusions for the bank voles and aneuploidy for the commonvoles. The data obtained suggest that elevated levels of ionizing radiation(within the investigated range) increase the incidence of cytogenetic anomaliesin laboratory strains of mice and in voles, which are characterized by highstrain- and species-specific variability (in mice) under control conditions.The analysis of these mutational spectra is further complicated by the factthat individual chromosomes of the experimental animals exhibit a pronouncedpredisposition to a particular type of cytogenetic abnormalities.A comparative analysis of the incidence of various chromosomal breaks in bloodcells extracted from 14- to15-year-old children was carried out. One groupconsisted of children who received ionizing radiation at a dose ofapproximately 30 mSv during their prenatal development; the second groupconsisted of children who received approximately the same dose, but in thecourse of their entire lives as they lived in the contaminated areas(approximately 1.5 mSv/year) ,51. TheEscherichia coli). The absolute record holder in this respectis the bacterium D. radiodurans, whose individual cellssurvive and retain their reproductive capacity after being exposed to 5000 Gy[It is interesting to mention that the tendency towards increasing sensitivityto ionizing radiation with an increase in the complexity of an organism hasbeen established a long time ago; i.e., the more ancient the species the moreresistant it is to ionizing radiation. The mean half-lethal dose of radiationis approximately 4–6 Gy for mammals; it reaches 30 Gy for colibacillus of thegenetic methods for assessing chromosomal aberrations at low-dose exposureswill ensure a more accurate determination of the relationship between low-doseradiations and induced mutational events. This assumption was supported by thedata pertaining to the occurrence of new mutations in highly polymorphic DNAsequences in children born to irradiated parents, 53.Howet al., in whicAttempts to assess the genetic consequences of the action of ecotoxic factorshave been made for an appreciably long period; two main trends can bedistinguished in this development. One consists in searching for the moleculargenetic systems associated with detoxification and antioxidant enzymes; thesecond one is the population genetic investigation of the dynamics of allelicvariants in the genetic systems associated with resistance to these factors.The biomarkers of xenobiotic metabolism are usually subdivided into genes whoseproducts are involved in the metabolic activation of promutagens(procarcinogens) with the occurrence of short-lived, highly toxic derivatives and genes whose products control theirdetoxification . Thedirect link between the genetically determined polymorphism of these enzymesand the induction of cytogenetic abnormalities by a number of xenobiotics,reproductive function disorders, and the development of certain types of tumorsin humans has recently been discovered using PCR–56.The second area is being investigated less successfully. It is based onresearch into the changes in the structure of the populations that aresubjected to environmental stress. The distribution of allelic variants and thegenotypes of the structural genes and anonymous (in terms of their function),highly polymorphic DNA, whose involvement in the formation of sensitivity togenotoxic agents has not been established, are being investigated.Associations have been discovered between human resistance to ionizingradiation and the occurrence of certain genotypes, mainly with respect to thetransferrin and haptoglobin loci ,58. JapSuccess in searching for the biomarkers of resistance to genotoxic effectslargely depends on the quality and adequacy of the models used to study thepopulation genetic consequences of various types of environmental stress.It is obvious that the high frequency of spontaneous mutational events leads tothe fact that mutagenic effects are attributed not to the occurrence of thelesions but to the activity of their repair .The identification of the genes and gene ensembles whose inheritance ispreferable and is associated with the selection for resistance to newenvironmental conditions may result in the development of the so-calledindividual “genetic passports” of resistance to physical and chemicalenvironmental pollution. These data enable to control the population geneticstructure of the species and facilitate its transformation in the desireddirection.The basic data obtained during our investigations were thoroughly described inthe monograph  and perf137Cs): a) reduced fertility and increasedmortality in newborn calves; b) disruption of the equiprobable inheritance ofindividual allelic variants – elimination of some and preferable inheritance ofthe other allelic variants; c) displacement of the genetic structure of theparent generation typical of dairy cattle towards less specialized forms; d)changes in the genetic structure that coincided with the population geneticeffects of such biotic and abiotic stress factors as the selection forresistance to bovine leukemia infection and introduction into new reproductionconditions. Therefore, the findings suggest that the main response to aprolonged exposure to low-dose ionizing radiation consists not in the inductionof the emergence of new genes but in the preferential selection of new genecombinations in the generations. Hence, this concept is consistent with theprinciples of the evolution theory elaborated by I.I. Schmalhausen[A shift in the spectra of organ-specific isoenzymes occurs in a number of farmanimal species under the influence of elevated levels of ionizing radiation (anabsorbed dose of 0.6–0.8 Gy/year), mostly in kidney and heart tissues. Theheart tissue starts to express a number of isoenzymes, which are typicallyfound in the poorer specialized muscle tissue. No significant increase in thenumber of individuals with constitutive mutations has been identified duringpopulation-based studies of various mammal species. The following observationshave been made with respect to generations of cattle that received absorbeddoses of approximately 0.8 Gy/year  were also employed. The informative nature of utilizing additionalcharacteristics of the genetic structure of the species was also assessed: ananalysis of the patterns of interloci gene associations was carried out. The invThe following data were obtained during the investigation of the geneticstructure of a number of generations of cattle bred under conditions of highradionuclide contamination in the alienation zone of the CN PP. Only one animalwith a mutation in the locus encoding transferrin was identified in the secondgeneration. Disruption of the equiprobable transfer of the allelic variantsfrom parent to offspring was detected in certain loci; changes in the resultsof the assessment of disequilibrium with respect to the linkage in a number ofloci were also recorded. It was demonstrated that, regardless of consanguineousmating under conditions of increased radionuclide contamination, theheterozygosity in the generations did not decrease.Therefore, the occurrence of population genetic changes in animals underconditions of environmental stress was initially established using this model,and these changes were shown to be directed towards the preferentialreproduction of heterozygotes with respect to the structural genes and“anonymous” DNA segments (ISSR-PCR markers).2, an abiotic factor). The population geneticstudies were conducted using a variety of markers, including electrophoreticvariants of proteins, restriction site polymorphism, and ISSR-PCR markers.Next, we compared the population genetic consequences of elevated levels ofionizing radiation and the influence of the other biotic and abioticenvironmental stress factors on the genetic structure, which was evaluatedusing a variety of molecular genetic markers, inbreeding groups of differentbreeds of cattle. The following mechanisms of the influence of environmentalfactors were considered. The analysis of the Red Steppe breed included twogroups of animals that differed in terms of their resistance to the influenceof a biotic stress factor (infected and uninfected with the bovine leukemiavirus) – from farms in the Kherson region, which was relatively unaffected bytechnogeneous pollution; and from farms in Kirovograd and Donetsk, which werecharacterized by elevated levels of chemical contamination (an abiotic factor).Three groups of animals belonging to the Pinzgau breed were examined inconnection with their reproduction in the plains, mountains, and underhigh-altitude conditions (an abiotic factor). The gray Ukrainian breed wasrepresented by two groups of animals: those from the Kherson region  and from the Altai region and Siberia (new conditions – anabiotic factor). In the Holstein breed, the effect of the abiotic factor wasdetermined by comparing the genetic structure of two groups, one of whichreproduced at farms in the relatively uncontaminated Kherson region and theother  reproduced on the “Novoshepelichi” farm located inthe alienation zone of the CN PP . This suggests theexistence of universal characteristics of the population genetic response ofcattle to the influence of various environmental stress factors.An analysis of the reproductive characteristics of experimental herds of cattlecan reveal the mechanisms behind the changes in the genetic structure whichoccurr in the descendants of parents exposed to ecotoxic factors and deletionsof a number of alleles and genotypes associated with increased sensitivity.Thus, the experimental herd exposed to high levels of ionizing radiation(absorbed dose of 0.8–1.1 Gy/year) included the parental generation (F0)consisting of three cows  and a bull named Uranus capturedin 1987 near the CN PP and of 13 cows imported between 1990 and 1993 to the“Novoshepelichi” farm (Pripyat) from relatively uncontaminated areas. The F0generation of cows  from the experimental herd born in theuncontaminated area gave birth to a total of 96 calves , 20 of which (21%) did not survive 3 months after birth. Thefirst generation (F1) born on the experimental “Novoshepelichi” farm wassignificantly different from the parent generation with respect to this figure.Thus, 21 cows of the 36 cows from the F1 generation were sterile (58%), andonly 15 of them yielded offspring ; 13 calves diedbefore reaching three months of age (26%). Four cows from the F2 generationgave birth only to 10 calves (F3) in 2–4 years, i.e. 0.94 ± 0.06 calves per cowper year. It should be mentioned that most of the calves from the F1generation, which did not survive, were bulls ; thesex ratio amongst 13 dead calves from the F2 generation was approximately thesame (seven heifers and six bulls).Therefore, the changes in the genetic structure of the offspring received fromparents exposed to the influence of ecotoxic factors can be attributed to adecrease in fertility and an increase in infant mortality .There are many “radioactive” areas located in different parts of the earth;various population genetic characteristics of the populations inhabiting themhave been identified in some of them. Thus, extensive studies of thepopulations living in areas characterized by a highly radioactive backgroundwere conducted , where theexposure of the population to radiation ranges from 0.6 to 10 cGy per year. Noincrease in the number of human congenital diseases was revealed during theseinvestigations ,65. TheRamsar County in Iran is best known for its annual absorbed dose of 260 mSv;the global average dose is equal to 3.5 mSv/year. The residents of RamsarCounty are not characterized by elevated rates of mortality or birth ofchildren with congenital developmental abnormalities.Meanwhile, distinctive differences in the radioresistance of blood cellsisolated from the local population living in this area are observed, incontrast to residents of areas characterized by a low natural radioactivebackground. Thus, the exposure of the cell cultures of peripheral bloodisolated from the inhabitants of Ramsar County to a dose of 1.5 Gy resulted ina significantly smaller increase in the number of cells with cytogeneticanomalies as compared to the blood cells of the control group.The published results of studies of populations living in “radioactive” areassuggest that the selection process for increased radioresistance occurs inthese locations from generation to generation. Thus, 125,079 residents of the“radioactive” province were examined in China during the period between 1979and 1995; 10,415 deaths and 1,003 cancer cases have been analyzed. It was foundthat the cancer mortality rate in the “radioactive” province was lower thanthat among residents of the control area .In anotNo significant differences in the incidence of congenital defects have beenidentified among newborns  from the “radioactive” province in India(approximately 35.0 mSv/year) and among newborns  in the control group. ScreeniAnnual detection of oncological conditions per 100,000 people in thepopulations inhabiting Indian regions, which vary from each other by only 0.03mSv/ year with respect to the external radiation exposure proportionatelydecreases from one area to another one simultaneously with an increase in thebackground level of ionizing radiation by 0.03 mSv/year – from the hypotheticalincidence of oncological conditions being equal to 79 : 100,000 people, underconditions of “zero” level of external exposure. The authors arrived atconclusion that an increase in ionizing radiation decreases the risk ofdeveloping cancer .It should be emphasized that among the 116,000 people evacuated from theChernobyl zone, only approximately 5% of them received a dose of ionizingradiation exceeding 100 mSv/year, and this very dose  is considered to be the limit exceeding which leads toan increase in the incidence of oncological conditions.Thus, the actual danger is not the received dose ofionizing radiation but its “novelty” to this particularpopulation, species, or species community. It is obviousthat an annual increase in the absorbed dose by 3.5mSvwill not result in any health consequences among theresidents of Ramsar Country, but for most Europeanpopulations whose previous generations have not beenexposed to doses exceeding 1 mSv/year such an increasecan lead to the elimination of radiosensitive speciesfrom the gene pool, thus resulting in a change inthe genetic structure of the populations.Hence, there is a wide range of doses of ionizing radiation within the naturalconditions, which are compatible with the ability of various organisms(including humans) to survive and reproduce. This complicates the evaluation ofthe biological (including genotoxic) effects of low-dose ionizing radiation.Our own experimental data suggest that a chronic exposure to elevated levels ofionizing radiation causes no increase in the number of mutant individuals amongthe investigated species. An increased frequency of somatic cells withcytogenetic anomalies was not accompanied by qualitative changes in comparisonto the spontaneous mutational spectra, since the increase was only observed forthe indicators of the instability of the chromosomal apparatus that possessedgenotypic features in the strains of mice and field vole species.A comparative analysis of the mutational spectra in field voles conducted indifferent years showed that the number of individuals of different species witha high frequency of mutant cells in the bone marrow decreased gradually overtime despite the persistence of high levels of radioactive contamination at thecapture locations. The frequency of occurrence of individual species with highlevels of cytogenetic anomalies among the common voles and bank voles wassignificantly higher in 1996 than that in the relatively ”uncontaminated areas,and in animals captured at the same locations but during the later years, in1999 and 2001. Thus, metaphases with chromosomal aberrations in the commonvoles were observed in the control group at a frequency of 2.5 ± 1.5%, and inChistogalovka at a frequency of 3.6 ± 0.8%, 5.0 ± 2.3%, and 2.5 ± 0.3% in 1996,1999, and 2001, respectively. These metaphases were encountered in the bankvole species of the control group at a frequency of 1.2 ± 0.7%; in the «RedForest » in 1996, 1999, and 2001 at a frequency of 7.3 ± 3.4%, 3.5 ± 0.8%, and0.9 ± 0.3%, respectively.2), in contrast to the animals inhabiting the areas with lowerlevels of radionuclide contamination . Thus, therate of selection for radioresistance is higher when the level of radionuclidecontamination is higher. It is noteworthy that even in the areas of thealienation zone characterized by a high level of radionuclide contamination(i.e. “Red Forest”), an accumulation of radioresistant species was detectedonly in 1999; i.e., 13 years after the Chernobyl accident, after 26 generationsof voles (voles breed twice a year).It is important to emphasize that this decrease indicating the gradualaccumulation of radioresistant species among the bank vole species was onlyobserved in the animals captured in the “Red Forest” area, which wascharacterized by a very high level of radionuclide contamination  and in the area of risk-associated livestockfarming in the southern part of the Gobi Desert. A comparThe disruption of equiprobable transmission of allelic variants of severalmolecular genetic markers and an increase in heterozygosity were observed amonggenerations of the experimental “Novoshepelichi” herd of the black and whiteHolstein cattle. The frequency of occurrence of leukocytes containingmicronuclei in the parental generation was higher at a statisticallysignificant level (P < 0.05) as compared to these values in the first,second and third generations of animals born in the zone with an elevatedradionuclide contamination. However, this parameter was significantly lower inthe third generation (P < 0.01) as compared to that in the secondgeneration. The frequency of occurrence of binucleated leukocytes in theperipheral blood smears was also significantly higher in the parentalgeneration than those in the first and second generations of animals. Thus, theradioresistance of animals born under conditions of the elevated levels ofionizing radiation increases over generations as evidenced by the incidence ofcytogenetic abnormalities in the peripheral blood smears. No carriers ofRobertsonian translocations were identified, which are often found inuncontaminated areas among representatives of species with acrocentricautosomes.A comparative analysis with respect to a complex of molecular genetic markersin the experimental herd of black and white Holstein cattle from the relativelyuncontaminated breeding areas, as well as the representatives of the ancientprimitive breed known as the Ukrainian gray breed allowed to observe aconvergence of the genetic structure of animals from the experimental herd bornunder conditions of elevated levels of radionuclide exposure with the gene poolof this ancient breed in contrast to the parental group of individuals. Thisshift in the genetic structure of the gene pool of the experimental“Novoshepelichi” herd originally belonging to a specialized dairy breed towardsa more primitive species was observed with respect to the allelic variants ofthe structural genes and DNA fragments flanked by inverted microsatelliterepeats. It can be expected that these changes are a universalpopulation-genetic response of cattle to the influence of various environmentalstress factors.The following data are common for our own findings and to the published data.The main problem that populations of different species (including humansinhabiting the areas contaminated with radionuclides after the Chernobylaccident) face is not the absolute value of the dose of ionizing radiation, butthe novelty of these doses to the populations. The main genetic effects forpopulations of various species consist not in the increase in the number ofmutant organisms but in the fact that some of the genes are excluded from theirreproduction as a result of selection against radiosensitive organisms. Inother words, new genes do not emerge, but the ”old” ones associated with highsensitivity of the organisms to new conditions of reproduction abandon thepopulation. There is some indirect evidence suggesting that the lessspecialized organisms of the species turn out to be more adapted to newconditions.in utero exposure resulted in a decrease exclusively in theverbal IQ score among young people [This ”reversion” to the more primitive, developmentally and evolutionarilyearlier forms of life is observed at various levels of organization ofbiological material: a shift in the organ-specific isozymatic spectra todevelopmentally earlier versions; displacement of the population-geneticstructure in generations to the predominance of less specialized forms;g people ,which iIt is interesting to note that data have been obtained indicating that thefirst-priority destruction of the youngest biological systems in evolutionaryterms is a relatively universal biological rule .The accumulated data allow one to formulate four major laws of the Chernobyldisaster. We believe they can be universally applied to the consequences of allfundamental environmental changes associated with natural and man-madedisasters and crises. These laws are as follows: 1) not everyone who wassupposed to be born is being born after the Chernobyl accident; 2) theselection against specialized forms of life and preferential reproduction ofless specialized forms characterized by higher resistance to adverseenvironmental factors occurs; 3) the response to the same dose of ionizingradiation depends on its “novelty” for the population of preceding selectionfor resistance to such doses in the ancestral generations; and 4) the actualconsequences of the Chernobyl accident for human populations will be availablefor analysis no earlier than 20 years from now, since the generation subjectedto direct damaging influence has only recently entered its reproductive period.It should be emphasized that the increase in the incidence of even the thyroidpathology resulting from the Chernobyl accident reduces the chances ofreproductive success in its carriers.


The emerging fields of microbial population genetics and genomics provide an avenue to study the ecological rules that govern how communities form, function, and evolve. Our struggle to understand the causes and consequences of microbial diversity stems from our inability to define ecologically and evolutionarily meaningful units of diversity. The 16S rRNA-based tools that have been so useful in charting microbial diversity may lack sufficient sensitivity to answer many questions about the ecology and evolution of microbes. Examining genetic diversity with increased resolution is vital to understanding the forces shaping community structure. Population genetic analyses enabled by whole genome sequencing, multilocus sequence analyses, or single-nucleotide polymorphism analyses permit the testing of hypotheses pertaining to the geographic distribution, migration, and habitat preference of specific microbial lineages. Furthermore, these approaches can reveal patterns of gene exchange within and between populations and communities. Tools from microbial population genetics and population genomics can be used to increase the resolution with which we measure microbial diversity, enabling a focus on the scale of genetic diversity at which ecological processes impact evolutionary events. This tighter focus promises to improve our understanding of the causes and consequences of microbial community structure. According to Greek mythology, Proteus knew of all things past, present, and future, but only shared this knowledge if captured. When pursued he would change shape, and the closer his pursuer the more quickly he changed, his knowledge just beyond grasp. Our efforts to define the causes and consequences of microbial community structure are like grappling with Proteus. A rigorous framework for understanding microbial community structure remains beyond our grasp because our concepts of what constitutes a community, a species, or a population are ill-developed and fungible. We argue that our comprehension of microbial communities is severely hampered by our inability to recognize ecologically and evolutionarily meaningful units of diversity. We believe observations from microbial population genetics and genomics promise to change the manner in which we define microbial diversity. Population genetics and population genomics both focus on the evolutionary dynamics of populations. While these approaches employ different analytical techniques, conceptually they represent points along a continuum, the differences being the amount and type of genetic information used to make evolutionary inference. Population-based approaches represent a powerful new paradigm for exploring the fundamental units of community structure.The ribosomal RNA (rRNA) paradigm has been invaluable in charting the diversity of the microbial world by providing both a phylogenetic framework for understanding microbial diversity and tools for characterizing microbial communities without the biases imposed by cultivation. The remarkable conservation of rRNA gene sequences makes them excellent for determining phylogenetic relationships between diverse microbial assemblages. However, as a result of their high conservation these molecules are insensitive to evolutionary changes that occur in response to ecological dynamics. As the field of microbial ecology matures, we increasingly seek to understand the ecological rules that govern how communities assemble, function, and evolve. The rRNA-based tools that have fueled the growth and development of our field may be poorly suited to answering many of the ecological questions we now face.0.03; post hoc manner as a way of supporting the pre-existing species definition. Hence, the foundation that underpins the OTU0.03 does not have a firm ecological basis and we should consider carefully the range of ecological and evolutionary hypotheses that can be addressed effectively on the basis of this unit of diversity.The most common unit of diversity employed in analyses of microbial community structure is the operational taxonomic unit (OTU) based on the 16S rRNA gene. An OTU is generally defined as a group of sequences that differ by less than 3% of nucleotide positions in the 16S rRNA gene OTU0.03; . This cuEscherichia coli and Salmonella enterica, 2.8% divergence of 16S rRNA between species can take approximately 63–120 million years  or whole genome sequences, provide even greater phylogenetic resolution and the ability to explore patterns of gene exchange and signatures of selection. Population genetics and genomics now provide a solid foundation to study evolutionary dynamics at the sale of ecological interactions and a framework for addressing specific questions such as: (1) How are microbial lineages distributed spatially, and what are the roles of migration and local adaptation in defining the genetic and functional characteristics of communities? (2) How do patterns of gene flow vary with respect to the genetic and geographic distance between strains? (3) How do population dynamics influence community dynamics and ecological processes? The following is a brief overview of how our understanding of microbial ecology may be enhanced by taking a population genomics approach.A focus on genetic diversity at a smaller phylogenetic scale than the OTUBacillus anthracis provides a case study to demonstrate this point  of > 1,700 global Burkholderia isolates. Population structure of these isolates supports the existence of two geographically distinct Burkholderia pseudomallei subpopulations, originating in Australia and Southeast Asia and separated by the Wallace Line, a geographical pattern well-documented in macroorganisms. The deeply branching Australian Burkholderia pseudomallei group was determined to be the most genetically diverse lineage, thus, representative of the ancestral gene pool. Molecular clock estimates suggest these Burkholderia pseudomallei populations diverged between 16,000 and 225,000 years ago  on microbial evolution and ecology. HGT can blur the lines of ancestry between lineages, shuffling adaptive genes, and HGT may prevent the development of genetically and ecologically cohesive populations . Genomicundaries , while, undaries . Likewiseciation . These oeciation . In the  strains , suggestAt a gross level, it is clear that the composition of a microbial community has strong impacts on environmental biogeochemistry, though the biotic and abiotic mechanisms that link community structure and function remain poorly described. If we want to understand community structure–function relationships at a fundamental level, we need to start with measurements of diversity that capture adaptive differences within and between lineages. We need to understand how the genomic diversity within a lineage impacts ecological function and is distributed in the environment, how evolutionary and ecological forces regulate gene exchange, and how patterns of gene exchange within and between lineages impact community function. These inquiries can be enabled by studying populations as the fundamental units from which communities are constructed.Microbial population genetics and genomics are opening avenues for understanding the ecological and evolutionary mechanisms governing microbial diversity. However, a focus on the dynamics of populations and species still faces several formidable obstacles. Foremost is the current lack of coherent and pragmatic definitions for populations and species. Without such criteria it is difficult to objectively compare results between studies. As our understanding of microbial populations improves, it will be important to develop objective criteria for defining lineages that will be relevant and applicable across a wide range of microorganisms.Another challenge to the application of microbial population genomics is a lack of information about how to properly sample the genetic diversity of microbial populations, both in terms of spatial scale and numbers of strains. Rigorous population studies require robust genetic sampling across appropriate spatial, temporal, or habitat scales in order to achieve the ultimate goal of accurately depicting patterns of biodiversity existing in nature. Sufficient individuals must be sampled to represent the breadth of genetic diversity across an organism’s geographic range and to capture genetically informative loci that reflect its evolutionary history. What spatial scales best capture the genomic diversity of microbial populations? Over what spatial and temporal scales does a microbial cell sample genetic material from its environment? What scales are most suitable for inferring adaptive traits from an organism’s habitat distribution? How do we define a microhabitat, or an ecological niche? What environmental parameters should be measured as part of the sampling design? All of these questions are fundamental and must be addressed as we move forward.An obvious limitation to population genomic approaches is the need for strains to be cultivated in isolation. While most microorganisms have yet to be cultivated, much progress can still be made with organisms that we can cultivate now. Model systems can be developed and used to explore the evolutionary and ecological mechanisms that regulate microbial diversity. Once revealed, these mechanisms can be used to make predictions relevant to organisms more recalcitrant to cultivation. In addition, a solution to the cultivation problem is available through application of single-cell genomics. Single-cell methods of genome analysis can be used to perform genome sequencing or multilocus analysis on individual microbial cells without the need for cultivation . MetagenH. pylori, eBURST may not be appropriate (Moraxella catarrhalis (H. pylori (Streptomyces (The ongoing exponential decline in sequencing costs has made population genomics a reality, but the computational tools and theory for understanding these data still lag. While a wide variety of computational population genetics tools exist, many are based on theory developed for eukaryotic organisms. There are fundamental differences between macroorganisms and microorganisms that may impact assumptions implicit in population genetic models. In addition, not all models can equally capture the range of ecological and evolutionary dynamics that operate in the microbial world. For example, the algorithm eBURST determines founding genotypes of clonal complexes from MLSA and has been used to describe evolutionary patterns in many microbial lineages . Howeverropriate . The proropriate  and has arrhalis , H. pylo. pylori , and Strptomyces . Howeverptomyces  is basedptomyces , which mUltimately, population-level approaches promise to shed light on the forces that govern microbial diversification and evolution. By providing evidence to interpret adaptive traits and identify selective habitats, these approaches will inform our understanding of the competitive interactions within and between lineages, laying the framework for an understanding of community-level interactions. These approaches should also provide insight on the genetic and ecological forces that govern gene exchange. We should focus now on making empirical observations to inform our understanding of the vertical and horizontal components of ancestry and whether and how microbial lineages form ecologically and genetically cohesive units. A better grasp of the relevant units with which to measure microbial diversity is essential for progress in microbial ecology. Insights from microbial population studies promise to improve our understanding of microbial diversity, providing access to knowledge about the causes and consequences of microbial community structure.


A key goal in evolutionary quantitative genetics is to understand how evolutionary trajectories are constrained by pleiotropic coupling among multiple traits. Because studying pleiotropic constraints directly at the molecular genetic level remains very difficult, several analytical approaches attempt to draw conclusions about constraints by relating the orientation of the eigenvectors of the traits' (co)variance matrix to vectors of multivariate selection. On the basis of explicit models of genetic architecture, I here argue that the value of such approaches is greatly overestimated. The reason is that eigenvector orientation can be highly unstable and lack a biologically meaningful relationship with the underlying traits' genetic architecture. Genetic constraints are more profitably explored through experimental approaches avoiding the mathematical abstraction inherent in eigenanalysis. Identifying such constraints, and quantifying their strength, is the major avenue to understanding multivariate evolution in EQG  is to understand the rate and direction of evolution in multiple traits. A pivotal concept here is that traits generally cannot evolve independently because their genetic architecture is shared to some extent  to obtain its eigenvectors (EVs). The EVs are described by their eigenvalue (quantifying magnitude) and trait loadings (quantifying orientation) and provide a representation of the total variation in G along orthogonal, variance-maximizing multivariate axes  is very difficult to obtain and highly incomplete even for model organisms  showed that genotypes at one extreme of the population had relatively short, slender bodies; narrow mouths; and numerous, long gill rakers” (Schluter gmax is the first EV of G.) A direct and interpretable connection between trait loadings on an EV, genetic (co)variance among traits, and underlying genotypes is here assumed  is compared with the orientation of the vector of multivariate selection (estimated from observed evolutionary trajectories among populations). A close directional association between the two vectors is taken as evidence that multivariate evolution has been biased by genetic constraints, although alternative interpretations are possible  axes of selection. These approaches are hereafter called hluter's  test forhluter's . Here, tSchluter . The appSchluter .G's EVs and genetic architecture, however, has been challenged. For instance, in response to an influential paper championing the use of matrix diagonalization in EQG  should be relatively stable. If this criterion is not satisfied , comparing the orientation of EVs to directions of selection will have little biological relevance. Second, trait loadings, which define an EV's orientation, should have a meaningful link to the traits' (co)variance and correlation structure, and to the underlying genetic architecture. Otherwise, interpreting the orientation of EV biologically (see quotes above) might be misleading, and forging a link between EVs and molecular genetic variation might be impossible. The analytical approaches described below are tailored to explore these two aspects, that is, the directional stability of EVs, and the relationship between EV loadings and genetic architecture.The validity of the assumption that the orientation of G's EVs in relation to different genetic architectures and to sample size was explored through a series of simulations. The default simulation approach involved the generation of a multivariate data set consisting of 100 individuals with four quantitative traits. Individual values for each trait were obtained by summing allelic states  across seven independent genetic loci . This was achieved by raising the number of loci determining the first trait (T1) to 10, 13, and 16, while reducing the number of loci driving T2–T4 to 6, 5, and 4 , I modified the default genetic architecture such that an increasing number of loci  were shared among all four traits. The strength of pleiotropic coupling among the traits thus increased gradually in this series, while the total variance again remained constant. The endpoint of this series, shown in To examine the effect of sample size on EV stability, several genetic architectures from the asymmetry and pleiotropy series were modeled with sample sizes of 50, 200, and 400 in addition to the default sample size (100). The lowest sample size modeled seems to be representative of typical empirical studies: median sample size  across 18 haphazardly chosen EV-based studies of genetic constraints was exactly 50 .G, which was subjected to singular value decomposition to obtain the EVs. Directional stability of EVs in relation to variance asymmetry, the strength of pleiotropy, and sample size was then explored by evaluating trait loadings on the EVs. Note that for these simulation series, the presentation of results is limited to EV1, as this leading axis of trait (co)variance is usually considered the most important variances Issues in the relationship between the direction of EVs and their underlying genetics are illustrated by two examples. In the first, I specified a genetic architecture involving two genetically independent traits , each of which was linked to T2 by three pleiotropic loci D. T4 wasG matrix for each of the two scenarios based on the exact parametric (co)variance contributed by a single locus with the allelic states given above.  This matrix was subjected to singular value decomposition to obtain the EVs. I then assessed qualitatively the relationship between trait loadings on the EVs and the trait's theoretical correlation structure derived from G. In addition, I plugged each G, along with a linear selection gradient vector β of 0.5, 0, 0, 0 (selection on T1 only), into the multivariate breeder equation To explore the connection between EV trait loadings and genetic architecture, I calculated the theoretical The robustness of the findings emerging from the above analyses was scrutinized in several ways. First, I modified the distribution of allelic values of the genetic loci. For instance, I here considered values drawn at random from a normal distribution, or allowed different loci to display different allelic ranges, thereby mimicking quantitative trait loci (QTL) with different effect sizes. Second, I doubled the number of both the independent and pleiotropic loci in each scenario. These modifications had no effect on the outcome of the simulations. I therefore limit the presentation of results to the allelic values and number of loci described above.Second, I modified trait space dimensionality. The 18 EV-based studies mentioned above displayed a median trait number of nine. I therefore approximated this number by doubling the number of traits in all scenarios from four to eight . These alternative analyses did not produce qualitatively novel results and are therefore not presented.Data generation, analysis, and plotting were performed by using the R language (R Development Core Team and displayed relatively similar levels of variance. The orientation stabilized as the asymmetry in variance increased (and the sphericity of G thus decreased) (N = 50), trait loadings generally proved relatively inconsistent across replicate simulations.The asymmetry series showed that trait loadings on the first EV, and hence the orientation of this vector, were poorly defined when traits were genetically independent creased) , top. Sicreased) , middle.creased) , bottom.G's EVs was misleading when subject to standard biological interpretation. For example, EV1 displayed strong loadings by T1, T2, and T3. This pattern would generally be taken as evidence for the presence of pleiotropic genetic factors shared among all three traits. In reality, however, T1 and T3 were genetically independent, but simply associated with the same third variable. Similarly, the strong but opposed loadings of T1 and T3 on EV2 might suggest the presence of an additional layer of antagonistically pleiotropic loci influencing this trait pair. Such loci, however, were not modeled.Scenario D modeled two sets of pleiotropic loci, each influencing T2 and either T1 or T3. This genetic architecture was mirrored in the traits' theoretical covariance and correlation structure , top. InIn scenario E, I specified a situation with both strong global pleiotropy and heterogeneity in the magnitude of variance among the traits. As expected, this produced strong correlations among all traits, and selection on T1 caused correlated evolution in all the other traits , bottom.I emphasize that the results obtained from the scenarios D and E are not caused by symmetries built into the genetic architecture; similar findings emerged when giving each trait a unique magnitude of variance. Also, extending these scenarios to include more traits produced qualitatively similar results (details not presented).G yields axes that can be used to infer how genetic (co)variation due to pleiotropic gene action constrains trajectories of multivariate evolution. This assumption was here investigated, leading to two main insights.Vector-based approaches in EQG rely on the assumption that the diagonalization of G imprecise enough to generate substantial fluctuation in the orientation of EVs under different genetic architectures. While estimation error has long been recognized as a general concern in EQG  is distributed relatively evenly among the (remaining) EVs. This will be the case whenever traits exhibit relatively similar magnitudes of variance, and when pleiotropy is low variance structure, the directionality of y is low , or by cresamples of the same sample due to chance, even when EV orientation is completely unstable across independent replicate samples from the same statistical population  independent trait combinations is therefore flawed, and interpreting EV loadings in genetic or even functional terms is poor practice. Conclusions regarding genetic constraints on multivariate evolution drawn in EV-based EQG studies should be taken with skepticism.The second major finding is that even when EVs are directionally stable, it cannot be taken for granted that their orientation has a meaningful relationship with the genetic architecture of the constituent traits, with the estimated (co)variance and correlation structure of the traits, and with the traits' responses to selection predicted by the multivariate breeder framework. The problem is that the diagonalization of any EV-based analysis of genetic constraints. The problem is, however, that with complex real-world data, even the evaluation of eigenvalues and/or the inspection of the genetic correlation structure among traits might provide little guidance as to whether the assumption of a meaningful association between any EV and genetic architecture is justified.I emphasize that the present investigation does not demonstrate that the identified analytical difficulties will necessarily, or equally strongly, compromise The findings presented in this study also question the value of EVs as a bridge between phenotype and genotype maps Houle , or as aG, but that avoid the additional mathematical abstraction inherent in the diagonalization of this matrix. These methods include comparing the magnitude of variance in the direction of evolution to the magnitude of variance in random directions (Hansen and Houle G matrix to the rate of adaptation predicted if all traits were genetically independent (Agrawal and Stinchcombe G, however, also face a number of potentially serious problems (reviewed in Mitchell-Olds and Rutledge G matrices can emerge from very different genetic architectures.If approaches relying on the orientation of EVs are inappropriate to explore genetic constraints, what alternatives are available? Several EQG methods have been introduced that make use of the summary statistic G's eigenvectors.To summarize, it may be debatable whether observational EQG approaches, in general, are the most effective route to understanding multivariate evolution, or whether efforts are more profitably invested in manipulative experimental approaches, such as artificial selection and the measurement of correlated responses (Mitchell-Olds and Shaw