Variation In Selection Pressures Research Articles

Similar evolutionary potentials in an obligate ant parasite and its two host species

The spatial structure of host–parasite coevolution is shaped by population structure and genetic diversity of the interacting species. We analysed these population genetic parameters in three related ant species: the parasitic slavemaking ant Protomognathus americanus and its two host species Temnothorax longispinosus and T. curvispinosus. We sampled throughout their range, genotyped ants on six to eight microsatellite loci and an MtDNA sequence and found high levels of genetic variation and strong population structure in all three species. Interestingly, the most abundant species and primary host, T. longispinosus, is characterized by less structure, but lower local genetic diversity. Generally, differences between the species were small, and we conclude that they have similar evolutionary potentials. The coevolutionary interaction between this social parasite and its hosts may therefore be less influenced by divergent evolutionary potentials, but rather by varying selection pressures. We employed different methods to quantify and compare genetic diversity and structure between species and genetic markers. We found that Jost D is well suited for these comparisons, as long as mutation rates between markers and species are similar. If this is not the case, for example, when using MtDNA and microsatellites to study sex-specific dispersal, model-based inference should be used instead of descriptive statistics (such as D or GST). Using coalescent-based methods, we indeed found that males disperse much more than females, but this sex bias in dispersal differed between species. The findings of the different approaches with regard to genetic diversity and structure were in good accordance with each other.

Evolution of Synonymous Codon Usage in Neurospora tetrasperma and Neurospora discreta

Neurospora comprises a primary model system for the study of fungal genetics and biology. In spite of this, little is known about genome evolution in Neurospora. For example, the evolution of synonymous codon usage is largely unknown in this genus. In the present investigation, we conducted a comprehensive analysis of synonymous codon usage and its relationship to gene expression and gene length (GL) in Neurospora tetrasperma and Neurospora discreta. For our analysis, we examined codon usage among 2,079 genes per organism and assessed gene expression using large-scale expressed sequenced tag (EST) data sets (279,323 and 453,559 ESTs for N. tetrasperma and N. discreta, respectively). Data on relative synonymous codon usage revealed 24 codons (and two putative codons) that are more frequently used in genes with high than with low expression and thus were defined as optimal codons. Although codon-usage bias was highly correlated with gene expression, it was independent of selectively neutral base composition (introns); thus demonstrating that translational selection drives synonymous codon usage in these genomes. We also report that GL (coding sequences [CDS]) was inversely associated with optimal codon usage at each gene expression level, with highly expressed short genes having the greatest frequency of optimal codons. Optimal codon frequency was moderately higher in N. tetrasperma than in N. discreta, which might be due to variation in selective pressures and/or mating systems.

Environmental Uncertainty and the Global Biogeography of Cooperative Breeding in Birds

Understanding why organisms as different as amoebas, ants, and birds cooperate remains an important question in evolutionary biology. Although ecology can influence cooperation and conflict within animal societies and has been implicated in species differences in sociality, the environmental predictors of sociality across broad geographic and taxonomic scales remain poorly understood. In particular, the importance of temporal variation in selection pressure has been underestimated in most evolutionary studies. Environmental uncertainty resulting from climatic variation is likely to be an important driver of temporal variation in selection pressure and therefore is expected to impact the evolution of behavioral, morphological, and physiological traits, including cooperation. Using a data set of over 95% of the world's birds, we examine the global geography and environmental, biotic, and historical biogeographic predictors of avian social behavior. We find dramatic spatial variation in social behavior for which environmental and biotic factors--namely, among-year environmental variability in precipitation--are important predictors. Although the clear global biogeographic structure in avian social behavior carries a strong signal of evolutionary history, environmental uncertainty plays an additional key role in explaining the incidence and distribution of avian cooperative breeding behavior.

Selection and the Cell Cycle: Positive Darwinian Selection in a Well-Known DNA Damage Response Pathway

Cancer is a common occurrence in multi-cellular organisms and is not strictly limited to the elderly in a population. It is therefore possible that individuals with genotypes that protect against early onset cancers have a selective advantage. In this study the patterns of mutation in the proteins of a well-studied DNA damage response pathway have been examined for evidence of adaptive evolutionary change. Using a maximum likelihood framework and the mammalian species phylogeny, together with codon models of evolution, selective pressure variation across the interacting network of proteins has been detected. The presence of signatures of adaptive evolution in BRCA1 and BRCA2 has already been documented but the effect on the entire network of interacting proteins in this damage response pathway has, until now, been unknown. Positive selection is evident throughout the network with a total of 11 proteins out of 15 examined displaying patterns of substitution characteristic of positive selection. It is also shown here that modern human populations display evidence of an ongoing selective sweep in 9 of these DNA damage repair proteins. The results presented here provide the community with new residues that may be relevant to cancer susceptibility while also highlighting those proteins where human and mouse have undergone lineage-specific functional shift. An understanding of this damage response pathway from an evolutionary perspective will undoubtedly contribute to future cancer treatment approaches.

Plasmodium relictum infection and MHC diversity in the house sparrow (Passer domesticus).

Antagonistic coevolution between hosts and parasites has been proposed as a mechanism maintaining genetic diversity in both host and parasite populations. In particular, the high level of genetic diversity usually observed at the major histocompatibility complex (MHC) is generally thought to be maintained by parasite-driven selection. Among the possible ways through which parasites can maintain MHC diversity, diversifying selection has received relatively less attention. This hypothesis is based on the idea that parasites exert spatially variable selection pressures because of heterogeneity in parasite genetic structure, abundance or virulence. Variable selection pressures should select for different host allelic lineages resulting in population-specific associations between MHC alleles and risk of infection. In this study, we took advantage of a large survey of avian malaria in 13 populations of the house sparrow (Passer domesticus) to test this hypothesis. We found that (i) several MHC alleles were either associated with increased or decreased risk to be infected with Plasmodium relictum, (ii) the effects were population specific, and (iii) some alleles had antagonistic effects across populations. Overall, these results support the hypothesis that diversifying selection in space can maintain MHC variation and suggest a pattern of local adaptation where MHC alleles are selected at the local host population level.

Young proteins experience more variable selection pressures than old proteins

It is well known that young proteins tend to experience weaker purifying selection and evolve more quickly than old proteins. Here, we show that, in addition, young proteins tend to experience more variable selection pressures over time than old proteins. We demonstrate this pattern in three independent taxonomic groups: yeast, Drosophila, and mammals. The increased variability of selection pressures on young proteins is highly significant even after controlling for the fact that young proteins are typically shorter and experience weaker purifying selection than old proteins. The majority of our results are consistent with the hypothesis that the function of a young gene tends to change over time more readily than that of an old gene. At the same time, our results may be caused in part by young genes that serve constant functions over time, but nevertheless appear to evolve under changing selection pressures due to depletion of adaptive mutations. In either case, our results imply that the evolution of a protein-coding sequence is partly determined by its age and origin, and not only by the phenotypic properties of the encoded protein. We discuss, via specific examples, the consequences of these findings for understanding of the sources of evolutionary novelty.

The evolution of tropical adaptation: comparing taurine and zebu cattle

Beef cattle breeds consist of three major genetic subdivisions. The taurine group is adapted to temperate environments, and the zebu and Sanga groups are both adapted to tropical environments. With the advent of genotyping and sequencing technologies in agriculture, genome-wide exploration of the genetic basis for the differences in tropical adaptation has only just become possible. In this study, approximately 9000 single nucleotide polymorphism markers were genotyped on 317 animals of a selection of taurine, zebu, and composite breeds to characterize any systematic differences between these groups. We identified 91 intra-breed-class markers; 78 were polymorphic only within the zebu animals, while 13 were polymorphic only in the taurine animals. There were no fixed differences (fixed for alternate alleles between the two breed types) between zebu and taurine animals. We found 14 regions with significantly different allele frequencies between zebu and taurine animals indicative of variable selection pressure or genetic drift. We also found 12 independent regions of differential extended haplotype homozygosity (EHH), indicative of recent selection or rapid fixation of the alternate allele within a short period of time in one of the two breed classes. A preliminary functional genomics analysis of these regions pointed towards signatures of tropical attributes including keratins, heat-shock proteins and heat resistance genes. We anticipate this investigation to be a stepping-stone for future studies to identify genomic regions specific to the two cattle groups, and to subsequently assist in the discrimination between temperate and tropically adapted cattle.

TEMPORAL VARIATION AT THE MHC CLASS IIB IN WILD POPULATIONS OF THE GUPPY (POECILIA RETICULATA)

Understanding genetic diversity in natural populations is a fundamental objective of evolutionary biology. The immune genes of the major histocompatibility complex (MHC) are excellent candidates to study such diversity because they are highly polymorphic in populations. Although balancing selection may be responsible for maintaining diversity at these functionally important loci, temporal variation in selection pressure has rarely been examined. We examine temporal variation in MHC class IIB diversity in nine guppy (Poecilia reticulata) populations over two years. We found that five of the populations changed significantly more at the MHC than at neutral (microsatellite) loci as measured by F(ST), which suggests that the change at the MHC was due to selection and not neutral processes. Additionally, pairwise population differentiation measures at the MHC were higher in 2007 than in 2006, with the signature of selection changing from homogenizing to diversifying selection or neutral evolution. Interestingly, within the populations the magnitude of the change at the MHC between years was related to the change in the proportion of individuals infected by a common parasite, indicating a link between genetic structure and the parasite. Our data thereby implicate temporal variation in selective pressure as an important mechanism maintaining diversity at the MHC in wild populations.

Associations of PCR markers with freezing tolerance and photosynthetic acclimation to cold in winter barley

Associations of PCR markers with freezing tolerance and acclimation of photosynthetic apparatus to cold were tested on 28 winter barley cultivars and advanced breeding forms to select alleles for practical application in marker assisted selection (MAS). We found significant associations between freezing tolerance evaluated with field-laboratory method (FLM) and markers located on 5H chromosome in region of gene Fr-H2 (bin9-10: Xbmag812, Xmwg2230) and region of gene Fr-H1 (bin11: Xmwg514, HvBM5, Xmwg644). Additionally, significant associations with photochemical quenching of chlorophyll a fluorescence (qP) were found for PCR markers Xpsr115 and Xmwg2062. In our study variation in the promoter region of Vrn-H1 (HvBM5) was directly connected with freezing tolerance of plants partially de-acclimated in the field. The results obtained here showed that different loci of freezing tolerance may play role in variable selection pressure of winter conditions.

Antagonistic coevolution across productivity gradients: an experimental test of the effects of dispersal

Coevolution commonly occurs in spatially heterogeneous environments, resulting in variable selection pressures acting on coevolving species. Dispersal across such environments is predicted to have a major impact on local coevolutionary dynamics. Here, we address how co-dispersal of coevolving populations of host and parasite across an environmental productivity gradient affected coevolution in experimental populations of bacteria and their parasitic viruses (phages). The rate of coevolution between bacteria and phages was greater in high-productivity environments. High-productivity immigrants ( approximately 2% of the recipient population) caused coevolutionary dynamics (rates of coevolution and degree of generalist evolution) in low-productivity environments to be largely indistinguishable from high-productivity environments, whereas immigration from low-productivity environments ( approximately 0.5% of the population) had no discernable impact. These results could not be explained by demography alone, but rather high-productivity immigrants had a selective advantage in low-productivity environments, but not vice versa. Coevolutionary interactions in high-productivity environments are therefore likely to have a disproportionate impact on coevolution across the landscape as a whole.

Soil salinity as a selection pressure is a key determinant for the evolution of salt tolerance in Blue Panicgrass ( Panicum antidotale Retz.)

To assess the role of selection pressure in plant adaptation to saline environment, a hydroponic experiment was conducted on six Panicum antidotale Retz. populations collected from a wide range of habitats with varying selection pressure in the form of soil salinity. The soil electrical conductivity of six different habitats ranged from 3.39 to 19.23 dS m −1 and pH from 5.86 to 7.65. Plants of all populations collected from varying habitats were established in pots containing normal soil and allowed to grow for 6 months. Newly grown tillers from each plant were separated and 10 of them each formed a composite sample for a particular population. They were then transplanted in plastic containers each containing 10 l of half strength Hoagland's nutrient solution alone or with 150 mol m −3 NaCl. After 42 days growth in salt treatment, the populations collected form highly saline habitats proved to be more salt-tolerant compared with those from mild or non-saline habitats in terms of growth performance. The populations adapted to high salinity showed less decrease in leaf K +/Na + and Ca 2+/Na + ratios under salinity stress. Moreover, under stress the salt-tolerant populations showed less reduction in photosynthetic capacity than the salt-sensitive populations. In addition, hyper-accumulation of organic solutes such as glycinebetaine and proline and thereby higher osmotic adjustment seemed to be associated with the higher degree of adaptability of the salt-tolerant populations to salt stress. From the data presented, it is plausible to conclude that selection pressure (soil salinity) must have been one of the important determinants bringing about the evolution of salt-tolerance trait in Blue Panic grass.

Patterns of selection of two North American native and nonnative populations of monkeyflower (Phrymaceae)

To better understand invasion dynamics, it is essential to determine the influence of genetics and ecology in species persistence in both native and nonnative habitats. One approach is to assess patterns of selection on floral and growth traits of individuals in both habitats. Mimulus guttatus (Phrymaceae) has a mixed mating system and grows under variable water conditions across its native and nonnative range in North America. Field investigations of patterns of selection of floral and plant size traits were conducted in two native and two nonnative populations. Field-collected seed was grown and crossed in the glasshouse using a paternal half-sib design. The resulting offspring were grown in saturated and dry-down low-water conditions and the same traits were measured in both environments. Patterns of selection varied across years in the native range. Nonnative populations exhibited selection for increased floral size, consistent with the hypothesis that selection favors larger size in nonnative habitats. In the glasshouse, we detected genetic variation for traits across population/treatment combinations. However, size hierarchy in the glasshouse was dependent on water conditions. Our results suggest that both variable selection pressures and local adaptation probably influence the persistence of both native and nonnative populations.

MIGRATION-SELECTION BALANCE AND LOCAL ADAPTATION OF MITOCHONDRIAL HAPLOTYPES IN RUFOUS-COLLARED SPARROWS (ZONOTRICHIA CAPENSIS) ALONG AN ELEVATIONAL GRADIENT

Variable selection pressures across heterogeneous landscapes can lead to local adaptation of populations. The extent of local adaptation depends on the interplay between natural selection and gene flow, but the nature of this relationship is complex. Gene flow can constrain local adaptation by eroding differentiation driven by natural selection, or local adaptation can itself constrain gene flow through selection against maladapted immigrants. Here we test for evidence that natural selection constrains gene flow among populations of a widespread passerine bird (Zonotrichia capensis) that are distributed along an elevational gradient in the Peruvian Andes. Using multilocus sequences and microsatellites screened in 142 individuals collected along a series of replicate transects, we found that mitochondrial gene flow was significantly reduced along elevational transects relative to latitudinal control transects. Nuclear gene flow, however, was not similarly reduced. Clines in mitochondrial haplotype frequency were strongly associated with transitions in environmental variables along the elevational transects, but this association was not observed for the nuclear markers. These results suggest that natural selection constrains mitochondrial gene flow along elevational gradients and that the mitonuclear discrepancy may be due to local adaptation of mitochondrial haplotypes.

Molecular and morpho-physiological characterization of sea, ruderal and cultivated beets

Beta vulgaris genetic resources are essential for broadening genetic base of sugar beet and developing cultivars adapted to adverse environmental conditions. Wild beets (sea beets, B. vulgaris spp. maritima and their naturalized introgressions with cultivated beets known as ruderal beets) harbor substantial genetic diversity that could be useful for beet improvement. Here, we compared molecular and morpho-physiological traits of wild beets collected on the Adriatic coast of Italy with sugar beet using eight primer-pairs amplifying 194 polymorphic fragments and four root traits (glucose and fructose content in the root tip, root elongation rate, number of the of root tips, total root length and its distribution among diameters ranges). Genetic diversity was higher in the sea beet accession, which may be due to the highly variable selection pressures that occur in heterogeneous ecological niches, compared with the ruderal and cultivated beets. Sea and sugar beet accessions showed contrasting root patterns in response to sulfate deprivation: sugar beet showed an increase of reducing sugars in the root tips and higher root elongation rate, and the sea beet accession showed an increase in root tip number, total root length and fine root length (average diameter < 0.5 mm). The ruderal beet showed intermediary responses to sea and sugar beet accessions. AFLP and morpho-physiological cluster analyzes showed sea, ruderal and cultivated beets to be genetically distinct groups. The results of this study indicate variability in response to sulfate deprivation is present in undomesticated beets that could be deployed for sugar beet improvement.

Plastic parasites: sophisticated strategies for survival and reproduction?

Adaptive phenotypic plasticity in life history traits, behaviours, and strategies is ubiquitous in biological systems. It is driven by variation in selection pressures across environmental gradients and operates under constraints imposed by trade-offs. Phenotypic plasticity has been thoroughly documented for multicellular taxa, such as insects, birds and mammals, and in many cases the underlying selective pressures are well understood. Whilst unicellular parasites face many of the same selective pressures and trade-offs, plasticity in their phenotypic traits has been largely overlooked and remains poorly understood. Here, we demonstrate that evolutionary theory, developed to explain variation observed in the life-history traits of multicellular organisms, can be applied to parasites. Though our message is general – we can expect the life-histories of all parasites to have evolved phenotypic plasticity – we focus our discussion on malaria parasites. We use an evolutionary framework to explain the trade-offs that parasites face and how plasticity in their life history traits will be expressed according to changes in their in-host environment. Testing whether variation in parasites traits is adaptive will provide new and fundamental insights into the basic biology of parasites, their epidemiology and the processes of disease during individual infections.

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas

BackgroundThe bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species.ResultsWe sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an ω ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence.ConclusionThe models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection.ReviewersThis article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

Microgeographic life history variation in an alpine caddisfly: plasticity in response to seasonal time constraints

Summary1. Temporally constrained environments, such as habitats with short growth seasons or short hydroperiods, cause potentially strong selection on life histories. Depending on the predictability of these events and the extent of spatial and temporal heterogeneity, local populations could become adapted either via a fixed phenotype or via life history plasticity in response to these environmental cues.2. We used a common garden experiment to investigate microgeographic variation in life history responses to combined changes in photoperiod (ambient/late) and hydroperiod (constant/drying) time constraint cues in an alpine caddisfly (Trichoptera). We compared six populations (three permanent/three temporary streams) originating from a small, alpine floodplain and which spanned an expected gradient in growth period duration (GPD) with distance from glaciers.3. We made two main predictions in relation to locally varying selection pressures: (i) populations nearest glaciers (shorter GPD and strongest time constraints) should have the fastest development rates and (ii) populations from permanent streams should be less able to respond to drying hydroperiods than populations from temporary streams.4. All populations and both sexes accelerated development in response to late season photoperiod cues. However, only permanent stream populations showed an increase in development time with increasing GPD, suggesting that other factors were influencing populations in temporary streams.5. Permanent stream populations showed countergradient variation (genetic and environmental influences were in opposition) in development time, and under‐compensation of growth rates resulted in a converse Bergmann cline in body size (smaller body size along gradients of declining season length). The extent of plasticity in response to hydroperiod, and the combined effects of both time constraints, differed between populations and sexes, but were not consistent among populations.6. Taken together, our results suggest adaptive plasticity in response to season length. The lack of a predictable pattern in response to hydroperiod may be due to gene flow or weak selection. We conclude that spatially structured populations can strongly differ in phenotypic plasticity even at microgeographic scales.

Selection on an Antimicrobial Peptide Defensin in Ants

Ants live in crowded nests with interacting individuals, which makes them particularly prone to infectious diseases. The question is, how do ants cope with the increased risk of pathogen transmission due to sociality? We have studied the molecular evolution of defensin, a gene encoding an antimicrobial protein, in ants. Defensin sequences from several ant species were analyzed with maximum likelihood models of codon substitution to infer selection. Positive selection was detected in the mature region of defensin, whereas the signal and pro regions seem to be evolving neutrally. We also found a significantly higher rate of nonsynonymous substitutions in some phylogenetic lineages, as well as dN/dS >1, suggesting varying selection pressures in different lineages. Earlier studies on the molecular evolution of insect antimicrobial peptide genes have focused on termites and dipteran species, and detected positive selection only in duplicated termicin genes in termites. These findings, together with our present results, provide an indication that the immune systems of social insects (ants and termites) and dipteran insects may have responded differently to the selection pressure caused by microbial pathogens.

Male advertisement call characters as phylogeographical indicators in European water frogs

Anuran vocalizations are valuable in the determination of species identity and have the potential to discriminate intraspecific variation. We developed novel bioacoustic sampling techniques, based on Fast Fourier Transforms, to increase the precision and sensitivity of male advertisement call analysis and applied the method to European water frogs. This approach robustly separated the three types of north European water frogs (Rana ridibunda, R. lessonae and their viable, fertile hybrid R. esculenta) by their call subunit characteristics. The hybrid frog exhibited a high frequency call component absent from both the parental species. Furthermore, call analysis demonstrated significant intraspecific differences among populations of all three frog types. Call characters of R. ridibunda changed systematically as a function of longitude. This trend may reflect either clinal variation in selection pressures across Europe, or the consequences of drift following postglacial colonization from eastern refugia. High resolution vocalization analysis therefore provides a potentially useful method for investigating intraspecific differentiation and the phylogeographical origins of anuran distributions.

Reply

We are grateful to Drs Grugan and Nakagawa for their careful review of our recent study (Cancer Cell 2008;13:441–453) and appreciate their detailed discussion. However, we would like to clarify a few points regarding the role of the primary tumor and the evolutionary model of cancer progression. First, we did not state “that the primary tumor cannot be used to estimate prognosis.” Primary tumors can and have often been used to predict prognosis. As mentioned by Drs Grugan and Nakagawa, predictive gene expression signatures have been identified as well as informative protein markers. However, a closer look into how these signatures are usually obtained and on the genes that the signatures comprise, provides evidence that the signatures do not describe the molecular mechanisms that are active during early systemic progression. Linking outcome with gene expression results in lists of genes that carry prognostic information, but do not define the functional processes involved in metastasis. Not surprisingly, most if not all signatures are proliferation signatures (Nat Rev Cancer 2007;7:545–553). Proliferation certainly is fundamental for metastasis, but mechanisms involved might differ between clones and depend, for example, on the microenvironment (availability of growth factors, cellular signals, oxygen, nutrients). Our data suggest that different genomes are selected during early metastasis. High proliferation at the primary site is likely to support the generation of variant cells fit to survive and thrive at distant sites. Therefore, our findings do not contradict the suitability of primary tumors to predict outcome. However, they do challenge the contention that therapy targets that are identified in primary tumors will necessarily be present and essential in disseminated cancer cells. Thus, we deem primary tumors to be less reliable surrogate markers for the selection of adjuvant therapies. Molecular processes and thereby molecular targets involved in dissemination, (ectopic) survival, and colonization apparently differ for cells being selected at the primary site and at distant sites. The divergent genetic aberrations might reflect selection processes during homing but also during survival and early colonization. Second, Drs Grugan and Nakagawa might have misunderstood our paper on early dissemination of breast cancer (Cancer Cell 2008;13:58–68). We did not observe an increase of DTCs in bone marrow of breast cancer patients with larger tumor size. In contrast, we were puzzled by the lack of such a significant association in >600 patients. Although cell numbers in primary tumors increase several hundred fold from T1 stage to stage T3/4, no such increase is observed for DTCs in bone marrow. Thus, if also true for other cancers, this finding might suggest that indeed sessile cells are selected within a colony (primary tumor or metastasis) and that dissemination peaks transiently at a certain stage of cancer development. Again, this would fit into an evolutionary model of cancer progression and would make a more careful analysis of early systemic cancer necessary. Primary tumors and metastases are important for our understanding of cancer because they arose from clones that have successfully established colonies. However, varying selection pressures (such as those exerted by different anatomic sites or adjuvant therapies) favor different clones. Thus, searching the Achilles' heel of metastatic precursor cells to prevent metastases may benefit from the direct analysis of disseminated cancer cells. HER2 Amplification in Micrometastatic Esophageal Cancer Cells Predicts PrognosisGastroenterologyVol. 135Issue 3PreviewStoecklein NH, Hosch SB, Bezler M, et al. (Department of Pathology, University of Regensburg, Regensburg, Germany). Direct genetic analysis of single disseminated cancer cells for prediction of outcome and therapy selection in esophageal cancer. Cancer Cell 2008;13:441–453. Full-Text PDF