Massive Variation Research Articles

New Era for Health Care and Genomics

We witness the tremendous advances in sequencing technologies and dramatic reduction of the sequencing costs. Fast and cheaper Next Generation Sequencing (NGS) technologies will generate unprecedentedly massive (millions of individuals) and highlydimensional (dozens or even hundreds of millions) genomic and epigenomic variation data that allow nearly complete evaluation of genomic and epigenomic variation including common and rare variants, insertion/deletion, CNVs, mRNA by sequencing (RNA-seq), microRNA by sequencing (mRNA-seq), methylation by sequencing (methylationseq) and Chip-seq [1]. This will provide not only invaluable information on fully understanding the role of human genomic and epigenomic variation and its role in complex clinical phenotypes and evolution, but also powerful tools for clinical genomics: diagnosis of disease, classification of disease subtypes, prediction of clinical outcomes, characterization of disease progression, management of health care and development of treatments, and morphological evolution.

Editors' pick: transcriptomes of 1000 genomes.

Next generation sequencing (NGS) technologies facilitate massive human DNA sequence variation data to be produced in a remarkable manner and speed. The functional effects of these variants can now also be analyzed by high-throughput RNA sequencing (RNA-seq) of the transcriptome. Prior to RNA-seq, analysis of human gene expression was performed by expression arrays with more limited coverage. In the recent issue of Nature, Lappalainen et al.[1] have, for the first time, used the full capacity of RNA-seq to create the largest existing catalog of potential causative functional variants in the genome and to characterize transcriptome variation in human populations in a subset of individuals from the 1000 Genomes Project. They reported a deep analysis of high-quality mRNA and miRNA sequences from lymphoblast cell lines from > 450 individuals belonging to five populations (CEPH-CEU Europeans, Finns, British, Toscani Italians, and Yoruba from Nigeria). The authors addressed several questions with their dataset. First, they investigated human transcriptome variation at the population level, which can manifest in overall expression levels or in splicing (in one gene). In a genome-wide perspective, Lappalainen et al. showed that population differences account for about 3% of the total variation, but they also identified 263–4.379 genes with differential expression and/or transcript ratios between population pairs. In the pairwise analysis, they observed something interesting: the African-European population pairs had a higher contribution of genes with differential splicing (75 to 85%) than the European populations had between each other (6 to 40%). Although this phenomenon has not been previously observed in humans, it is in agreement with phylogenetic differences between species being better captured by splicing rather than expression levels. Lappalainen et al. analyzed 644 autosomal miRNAs that could be quantified from > 50% of the individuals. Sixty of those miRNAs (9.3%) had significant cis-expression QTLs (eQTLs) for miRNA expression levels, indicating that genetic effects on miRNA expression are more widespread than had been known. The authors also provide evidence for the existence of feedback loops for mRNA and miRNA genes to have an effect on each other’s expression supporting the idea that miRNAs offer robustness in the expression programs. Altogether, Lappalainen et al. have produced the largest and most diverse catalogue of cis-regulatory variants in a single tissue to date. The authors used their data to analyze the functional properties of the newly found set of regulatory variants and transcriptome effects of protein-truncating loss-of-function variants. This collaborative effort further used the potential of RNA-seq to discover regulatory variants that affect not only expression levels but also splicing. Of the 7.825 genes with expression QTLs (eQTLs), 34% have a second, independent eQTL for any of their exons. The authors concluded that in the transcriptome there exists allelic heterogeneity for regulatory effects on a single gene and independence of exons within the same gene. As an ancillary benefit, the authors demonstrated that RNA-seq data is consistent even when produced in different laboratories, with more detailed analysis in a companion paper in Nature Biotechnology[2]. Namely, in the replicate analyses of RNA-seq data from seven laboratories, there was a smaller amount of variation among the laboratories than seen among the individuals. The study is a great example of integration of genomic sequencing and cellular phenotype data. Obviously, the amount of data produced in this study is enormous, and it can easily be expected that the data will also be used to support other pivotal work. Meanwhile, it will take some time to completely digest the magnitude of data and appreciate the impact it will have on functional genomics.

Studies of Natural Selection in the Era of Next-generation Sequencing

A deeper understanding of positive selection is of fundamental importance. Evolutionary pressures acting on the genome as a whole and the specific role of environmental pressures on divergence shape the natural selection. However, despite the intense interest in genome-wide scans, a coherent study of recent human evolutionary history has yet to emerge due to the limitations of data, models, and analytical tools. Fast and cheaper Next Generation Sequencing (NGS) technologies will generate unprecedentedly massive and highly-dimensional genomic variation data. NGS will offer unprecedented opportunities for population genetic and natural selection studies, but also raise great challenges. In the conventional population genetics, most researches have primarily focused on natural selection acting on a single locus. Little attention has been paid to determining how the natural selection acts on multiple interacted genes in response to environmental perturbation. In addition, the current paradigm for analysis of natural selection on gene-expression is to use a single value of summarizing statistic to represent gene expression level and overlook all information on expression difference in exons, genomic position and alleles. To address these critical limitations, we develop a unified framework and statistical methods for genome-wide scans for natural selection and investigation of natural selection on pathways. To explore observed expression variation in exons or in genomic position across the genes, we extend one dimensional diffusion process to multidimensional diffusion process and a single variate stochastic differential equation to multivariate stochastic differential equations. We then use the extended multidimensional diffusion processes to model the evolution of gene-expression acted by natural selection. We hope that the present new development of natural selection analysis for NGS data will open a new avenue for natural selection analysis with NGS data.

Structure of the Essential Diversity-Generating Retroelement Protein bAvd and Its Functionally Important Interaction with Reverse Transcriptase

Diversity-generating retroelements (DGRs) are the only known source of massive protein sequence variation in prokaryotes. These elements transfer coding information from a template region (TR) through an RNA intermediate to a protein-encoding variable region. This retrohoming process is accompanied by unique adenine-specific mutagenesis and, in the prototypical BPP-1 DGR, requires a reverse transcriptase (bRT) and an accessory variability determinant (bAvd) protein. To understand the role of bAvd, we determined its 2.69Å resolution structure, which revealed a highly positively charged pentameric barrel. In accordance with its charge, bAvd bound both DNA and RNA, albeit without a discernable sequence preference. We found that the coding sequence of bAvd functioned as part of TR but identified means to mutate bAvd without affecting TR. This mutational analysis revealed a strict correspondence between retrohoming and interaction of bAvd with bRT, suggesting that the bRT-bAvd complex is important for DGR retrohoming.

Predicting the functional effect of amino acid substitutions and indels.

As next-generation sequencing projects generate massive genome-wide sequence variation data, bioinformatics tools are being developed to provide computational predictions on the functional effects of sequence variations and narrow down the search of casual variants for disease phenotypes. Different classes of sequence variations at the nucleotide level are involved in human diseases, including substitutions, insertions, deletions, frameshifts, and non-sense mutations. Frameshifts and non-sense mutations are likely to cause a negative effect on protein function. Existing prediction tools primarily focus on studying the deleterious effects of single amino acid substitutions through examining amino acid conservation at the position of interest among related sequences, an approach that is not directly applicable to insertions or deletions. Here, we introduce a versatile alignment-based score as a new metric to predict the damaging effects of variations not limited to single amino acid substitutions but also in-frame insertions, deletions, and multiple amino acid substitutions. This alignment-based score measures the change in sequence similarity of a query sequence to a protein sequence homolog before and after the introduction of an amino acid variation to the query sequence. Our results showed that the scoring scheme performs well in separating disease-associated variants (n = 21,662) from common polymorphisms (n = 37,022) for UniProt human protein variations, and also in separating deleterious variants (n = 15,179) from neutral variants (n = 17,891) for UniProt non-human protein variations. In our approach, the area under the receiver operating characteristic curve (AUC) for the human and non-human protein variation datasets is ∼0.85. We also observed that the alignment-based score correlates with the deleteriousness of a sequence variation. In summary, we have developed a new algorithm, PROVEAN (Protein Variation Effect Analyzer), which provides a generalized approach to predict the functional effects of protein sequence variations including single or multiple amino acid substitutions, and in-frame insertions and deletions. The PROVEAN tool is available online at http://provean.jcvi.org.

All representations are not equal: Differential activation of words and phonological variants across accents

Given the massive variation in natural speech, how listeners recognize words is a central issue for linguistic theory. Listener sensitivity to acoustic fluctuations in speech has provided us with one piece of the puzzle: detailed, specific representations. We provide another piece: differential activation of these representations. We conducted a semantic priming with General American listeners of positively viewed non-rhotic British English accents and negatively viewed non-rhotic New York City accents (slender'THIN; slend-UH(BE)'THIN; slend-UH(NYC)'THIN). Controlling participant experience and self-reported familiarity with both accents, only the words produced in a British accent primed semantically related targets. There is no a priori reason to expect this pattern, as the phonological variant and lexical items are controlled. We suggest that two variants equally experienced in number (raw frequency) are perceived differentially because positive and negative attitudes influence the activation lexical representations. Additionally, by examining particular participant groups from non-rhotic regions, we show an oscillation between effects of raw frequency and listener attitudes. This work (1) provides a broader understanding of factors influencing activation, (2) increases our understanding of how frequency effects are modulated by other factors (subjective listener attitudes), and (3) illuminates the interactive nature of linguistic and social factors in speech perception.

International Neuropathy Workshop of 2009: Introduction to the final reports

Neuropathies are amongst the most common of the long-term complications of diabetes, affecting up to 50% of patients. Their clinical features vary immensely and patients may present with a wide spectrum of specialties, from neurology to urology, for example, or from cardiology to podiatry. Neuropathies are typically characterized by a progressive loss of nerve fibres which may affect both of the principal divisions of the peripheral nervous system. The epidemiology and natural history of the diabetic neuropathies remain poorly defined. The International Consensus Workshop in Toronto in 2009 arose from the fact that at the moment there are no clear, universally accepted guidelines regarding the definition of diabetic neuropathies. This has resulted in a massive variation in how neuropathy is diagnosed in different centres and countries. A preliminary summary report of the Toronto meeting was published in 2010. The series of papers published in this issue of Diabetes/Metabolism Research and Reviews are the detailed reports that came from each sub-group of this Consensus panel. These reviews cover the problems with definitions and classification of neuropathy, the management of painful neuropathy and then the sub-group of small fibre neuropathies. There are also 3 papers on the autonomic neuropathies, covering cardiovascular autonomic neuropathies, as well as other areas of the autonomic neuropathies including gastrointestinal, urogenital and pseudomotor neuropathies. This series of papers will give the reader detailed information on the diverse aspects of diabetic neuropathies, their measurement and management, and will also assist in the selection of appropriate measures of both autonomic and somatic nerve function in clinical trials. This is clearly work in progress as diagnostic criteria for diabetic neuropathies are likely to evolve with developments in the field.

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10(20) potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

To Know Behavior Is to Know Ecology and Physiology: Integration and the Flexible Phenotype

Within most multicellular species, it is variation within individuals in how the genome is expressed, not variation between individuals in the genome itself that produces the larger, more rapidly induced phenotypic variability. For example, in many temperate-zone bird species, the testes increase in size by ,100-fold over the course of a few weeks with the annual onset of the breeding season, a rate and magnitude of change within individuals that vastly exceeds the variation between individuals of the same species examined at the same stage of breeding. Feeding in some snakes elicits a change in digestive physiology and morphology that can include a doubling in liver, pancreas, and intestine mass and changes in stomach pH from 7–8 to 1–2, all within a week or so. Massive phenotypic variation. Zero genomic variation. These examples of phenotypic change are impressive in their magnitude as well as their speed, both of which influence the accessibility of selection. But these rates of change are relatively modest compared to the speed with which behavior can drive phenotypic change in animals. Behavior, as a field of study, has provided a perspective on phenotypic variation that few other disciplines, including epigenetics, can match, yet it has been through its integration with other levels of biological organization that has enabled the most insight. Just as architects consider both the surrounding physical environment as well as the eventual occupants in designing a building, behaviorists must consider both the ecological context and the developmental and physiological mechanisms that give rise to the behavior. As Niko Tinbergen suggested in 1963, behavior cannot be completely understood in the absence of understanding the forces that ultimately and proximately influence it, and it is therefore no wonder why behavioral research that integrates across multiple levels of biological organization has been so well received by the larger biological research community in recent times. The integration of multiple levels of biological organization combined with the importance of intra-individual phenotypic flexibility together serve as general themes throughout The Flexible Phenotype: A BodyCentred Integration of Ecology, Physiology, and Behaviour, a new book by Theunis Piersma and Jan A. van Gils. Piersma and van Gils take the reader through a loose history of their research program on the migration stop-over ecology, behavior, and physiology of the red knot (Calidris canutus), a migratory shorebird that has served as the foundation for their long-time collaboration. Although they load their story with examples and anecdotes from numerous other species, it is primarily the focus on the red knot that has enabled these authors to understand not only migratory stopover biology, but more generally behavior, ecology, and digestive physiology as a whole. The writing style is more conversational than that to which we are typically accustomed as readers of the scientific literature. But beneath such whimsical section titles as ‘‘Thermometers Do Not Measure Feelings’’ and ‘‘It Takes Guts to Eat Shellfish’’ is a foundation of scientific rigor. Part I of the book, ‘‘Basics of Organismal Design,’’ details the principles of water, heat, nutrient, and energy balance and explores the concept of symmorphosis, which posits that organisms are economically designed. Among the several intriguing ideas discussed in this section was one on the evolution of endothermy raised by Marcel Laassen and Bart Nolet in 2008. The adaptive basis for the evolution of endothermy has confounded researchers for decades, but, over the years, many converged on the idea that the elevated activity levels enabled by endothermy allowed better exploitation of relatively less active herbivores as a food resource. In other words, endothermic carnivores could more easily prey on herbivores. Taking an integrative approach by combining digestive physiology with foraging ecology, Laassen and Nolet hypothesized that endothermy evolved as a mechanism for burning the excess carbon consumed as part of a primarily herbivorous diet. Thus, endothermy may have first evolved in herbivores, not carnivores. Although researchers are sure to debate the evolutionary origins of endothermy for years to come, the utility of an integrative approach in this example was clear. Part II, ‘‘Adding Environment,’’ begins by discussing the relationships between

Agronomic phosphorus imbalances across the world's croplands.

Increased phosphorus (P) fertilizer use and livestock production has fundamentally altered the global P cycle. We calculated spatially explicit P balances for cropland soils at 0.5° resolution based on the principal agronomic P inputs and outputs associated with production of 123 crops globally for the year 2000. Although agronomic inputs of P fertilizer (14.2 Tg of P·y(-1)) and manure (9.6 Tg of P·y(-1)) collectively exceeded P removal by harvested crops (12.3 Tg of P·y(-1)) at the global scale, P deficits covered almost 30% of the global cropland area. There was massive variation in the magnitudes of these P imbalances across most regions, particularly Europe and South America. High P fertilizer application relative to crop P use resulted in a greater proportion of the intense P surpluses (>13 kg of P·ha(-1)·y(-1)) globally than manure P application. High P fertilizer application was also typically associated with areas of relatively low P-use efficiency. Although manure was an important driver of P surpluses in some locations with high livestock densities, P deficits were common in areas producing forage crops used as livestock feed. Resolving agronomic P imbalances may be possible with more efficient use of P fertilizers and more effective recycling of manure P. Such reforms are needed to increase global agricultural productivity while maintaining or improving freshwater quality.

From each according to his need, to each according to his ability: A comparative analysis of post-communist corruption

Though venality and graft arguably presents a political and economic problems in the post-communist world tout court, the 28 countries exhibit very different levels of corruption. Interestingly, these differences follow a strictly geographic pattern. The more to the West we move, the less corrupt are the post-communist countries, something which indicates that ‘deeper’ historical constraints are important for the present levels of corruption. Building on – and qualifying – prior work of Herbert Kitschelt, we show that most of the massive variation in post-communist corruption levels can be traced back to pre-communist bureaucratic legacies. These legacies explain not only the variation across the present national borders of the post-communist world but seemingly also within ‘cleft countries’ in which more than one pre-communist bureaucratic legacy is present. Contrary to much of the literature on post-communist transitions, we, therefore, argue that these countries were not characterized by a relatively uniform point of departure in 1989–1991, but rather by deep-seated structural diversity.

Ambient Air Temperature Does Not Predict whether Small or Large Workers Forage in Bumble Bees (Bombus impatiens).

Bumble bees are important pollinators of crops and other plants. However, many aspects of their basic biology remain relatively unexplored. For example, one important and unusual natural history feature in bumble bees is the massive size variation seen between workers of the same nest. This size polymorphism may be an adaptation for division of labor, colony economics, or be nonadaptive. It was also suggested that perhaps this variation allows for niche specialization in workers foraging at different temperatures: larger bees might be better suited to forage at cooler temperatures and smaller bees might be better suited to forage at warmer temperatures. This we tested here using a large, enclosed growth chamber, where we were able to regulate the ambient temperature. We found no significant effect of ambient or nest temperature on the average size of bees flying to and foraging from a suspended feeder. Instead, bees of all sizes successfully flew and foraged between 16°C and 36°C. Thus, large bees foraged even at very hot temperatures, which we thought might cause overheating. Size variation therefore could not be explained in terms of niche specialization for foragers at different temperatures.

Evolution of the protein domain repertoire of eukaryotes reveals strong functional patterns

Background Eukaryotes exhibit massive variation in their morphological complexity, ranging from protists to basal animals, such as Trichoplax adhaerens with only four different cell types, to mammals with ~ 210 different cell types [1]. Yet, the number of protein coding genes in eukaryotic genomes remains remarkably constant. Here, we attempt to shed some light on this paradox by analyzing eukaryotic genome evolution with a protein function centric view.

Paden van paradigmatische vereenvoudiging - Morfologie, fonologie of pragmatiek?

The Dutch dialects display massive variation in their verbal paradigms. Traditionally, this variation has been explained as the result of developments in phonology, such as deletion of inflectional endings such as schwa, –t and –n, and pragmatics, such as the replacement of the 2sg. pronoun du with the honorific jij, which caused the s-ending to disappear. More recently, it has been proposed that morphology, more specifically paradigmatic structure, plays a role as well: the patterns of syncretism that are found in the verbal paradigms of the Dutch dialects, are believed to be manifestations of natural patterns of syncretism (e.g., Bennis & MacLean 2006). This article shows that these morphological accounts lack cross-linguistic support, and discusses data from Dutch that argue against paradigmatic structure as an explanatory factor. The focus lies on the 2pl.-data, where the shift of –t to –e(n) in Hollandic dialects has been argued to be the clearest example of a morphologically-driven change (Aalberse 2007). But this development too is better explained as the result of an interplay of pragmatic and prosodic factors, viz. the rise of the 2pl. pronoun jullie and the avoidance of a stresh clash occurring by the use of jullie in post-verbal position.

Gradients of the Intergenerational Transmission of Health in Developing Countries

This paper investigates the sensitivity of the intergenerational transmission of health to exogenous changes in income, education and public health, changes that are often delivered by economic growth. It uses individual survey data on 2.24 million children born to 600000 mothers during 1970-2000 in 38 developing countries. These data are merged with macroeconomic data by country and birth cohort to create an unprecedentedly large sample of comparable data that exhibit massive variation in maternal and child health as well as in aggregate economic conditions. The country-level panel is exploited to control for aggregate shocks and trends in unobservables within countries, while a panel of children within mother is exploited to control for family-specific endowments and neighbourhood characteristics. Child health is indicated by infant survival and maternal health by (relative) height. We find that improvements in maternal education, income and public health provision that occur in the year of birth and the year before birth limit the degree to which child health is tied to family circumstance. The interaction (gradient) effects are, in general, most marked for shorter women suggesting that children are more likely to bear the penalty exerted by poor maternal health if they are conceived or born in adverse socio-economic conditions.

Selective Ligand Recognition by a Diversity-Generating Retroelement Variable Protein

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.

Preventing kernicterus: a wake-up call

Preventing kernicterus: a wake-up call

Wetlands as landscape units: spatial patterns in salinity and water chemistry

Athalassic wetlands play a pivotal role in sediment and nutrient cycling and retention at the catchment level and are important ecosystems for local and regional biodiversity. Yet, the management of wetlands outside of riverine floodplains (non-riverine wetlands) is difficult, as there is limited understanding of these water bodies and of the processes that threaten them, like secondary salinisation. Accordingly, we describe the patterns of variation in wetland salinity and water chemistry across a regional landscape that is threatened by secondary salinisation. Spatial analyses indicated the distribution of the study wetlands was non-random and there was considerable positive spatial auto-correlation in water chemistry among wetlands—indicating a lack of independence. We detected massive variation in water chemistry among wetlands compared to minimal within-wetland variation and conductivity accounted for most of the among-wetland variation confirming its prominence in non-riverine wetland water chemistry. Wetland salinities were classified by their chemical evaporative pathway and we found a number of wetlands that may have become secondary salinised. The results reported here support the notion that the study, conservation and management of non-riverine wetlands should include assessments made at multiple spatial scales from individual waterbodies through to catchments. This is important because wetlands may not be independent units, but components of larger systems. However, we also note that the use of individual wetlands as units of replication may be problematic under some circumstances. We also argue that the identification of secondarily salinised wetlands will often require a multiple lines of evidence approach.

The C-type lectin fold as an evolutionary solution for massive sequence variation

Only few instances are known of protein folds that tolerate massive sequence variation for the sake of binding diversity. The most extensively characterized is the immunoglobulin fold. We now add to this the C-type lectin (CLec) fold, as found in the major tropism determinant (Mtd), a retroelement-encoded receptor-binding protein of Bordetella bacteriophage. Variation in Mtd, with its approximately 10(13) possible sequences, enables phage adaptation to Bordetella spp. Mtd is an intertwined, pyramid-shaped trimer, with variable residues organized by its CLec fold into discrete receptor-binding sites. The CLec fold provides a highly static scaffold for combinatorial display of variable residues, probably reflecting a different evolutionary solution for balancing diversity against stability from that in the immunoglobulin fold. Mtd variants are biased toward the receptor pertactin, and there is evidence that the CLec fold is used broadly for sequence variation by related retroelements.

Macroevolution, hierarchy theory, and the C-value enigma

For more than 60 years, evolutionary biologists have debated the issue of whether the processes of genetic change observable within populations (microevolution) can provide an adequate explanation for the large-scale patterns in the history of life (macroevolution). In general, population geneticists have argued in favor of microevolutionary extrapolation, whereas paleontologists have sought to establish an autonomous and hierarchical macroevolutionary theory based on the operation of selection at several levels of biological organization (especially species). The massive variation in eukaryotic genome sizes (haploid nuclear DNA contents, or “C-values”) has similarly been a subject of debate for more than half a century, and it has become clear that no one-dimensional explanation can account for it. In this article, the basic concepts of macroevolutionary theory are reviewed and then applied to the long-standing puzzle of genome size variation (the “C-value enigma”). Genome size evolution provides a clear example of hierarchy in action and therefore lends support to the theoretical approach of macroevolutionists. Perhaps more importantly, it is apparent that genome evolution cannot be understood without such a hierarchical approach, thereby providing an intriguing conceptual link between the most reductionistic and expansive subjects of evolutionary study.