Natural Selection Research Articles

Evolution of plant metabolism: the state-of-the-art.

Immense chemical diversity is one of the hallmark features of plants. This chemo-diversity is mainly underpinned by a highly complex and biodiverse biochemical machinery. Plant metabolic enzymes originated and were inherited from their eukaryotic and prokaryotic ancestors and further diversified by the unprecedentedly high rates of gene duplication and functionalization experienced in land plants. Unlike prokaryotic microbes, which display frequent horizontal gene transfer events and multiple inputs of energy and organic carbon, land plants predominantly rely on organic carbon generated from CO2 and have experienced relatively few gene transfers during their recent evolutionary history. As such, plant metabolic networks have evolved in a stepwise manner using existing networks as a starting point and under various evolutionary constraints. That said, until recently, the evolution of only a handful of metabolic traits had been extensively investigated and as such, the evolution of metabolism has received a fraction of the attention of, the evolution of development, for example. Advances in metabolomics and next-generation sequencing have, however, recently led to a deeper understanding of how a wide range of plant primary and specialized (secondary) metabolic pathways have evolved both as a consequence of natural selection and of domestication and crop improvement processes. This article is part of the theme issue 'The evolution of plant metabolism'.

Genetic polymorphism of Duffy binding protein in Pakistan Plasmodium vivax isolates

Plasmodium vivax Duffy binding protein (PvDBP) is crucial for erythrocyte invasion, interacting with the Duffy Antigen Receptor for Chemokines (DARC) on the erythrocyte surface. The amino-terminal cysteine-rich region II of PvDBP (PvDBPII) is a promising blood stage vaccine candidate, yet the genetic polymorphisms of this protein in global P. vivax isolates complicate the design of effective vaccines against vivax malaria. This study analyzed the genetic polymorphism of PvDBPII in Pakistan P. vivax isolates. A total of 29 single nucleotide polymorphisms (SNPs), including 22 nonsynonymous SNPs, were identified in 118 Pakistan PvDBPII. Most amino acid substitutions occurred in subdomains II and III, with six commonly observed in the global PvDBPII population. The amino acid change patterns in Pakistan PvDBPII generally mirrored those in global PvDBPII, although the frequencies of amino acid changes varied by country. Nucleotide diversity in Pakistan PvDBPII was comparable to that found in global PvDBPII. Evidence of natural selection and recombination in Pakistan PvDBPII aligned with observations in global PvDBPII. Analysis of the haplotype network of global PvDBPII revealed a complexed network of 167 haplotypes, but no geographical clustering was observed. The findings are crucial for understanding the genetic characteristics of Pakistan PvDBPII. A comprehensive analysis of nucleotide diversity and evolutionary trends in the global PvDBPII population offers valuable insights for the development of vivax malaria vaccines based on this antigen.

How does urbanization affect natural selection?

Abstract Urbanization is one of the most significant contributors to the Anthropocene, and urban evolutionary ecology has become an important field of research. While it is commonly assumed that cities impose new and stronger selection, the contradictory assertion that selection may be relaxed in cities is also frequently mentioned, and overall, our understanding of the effects of urbanization on natural selection is incomplete. In this review, we first conduct a literature search to find evidence for patterns of natural selection on phenotypic traits including morphology, physiology, behaviour and life history, in urban and non‐urban populations of animals and plants. This search reveals that coefficients of natural selection in the context of urbanization are scarce (n = 8 studies providing selection gradients/differentials that include a total of n = 200 coefficients) and a lack of standardized methods hinders quantitative comparisons across studies (e.g. with meta‐analysis). These studies, however, provide interesting insight on the agents shaping natural selection in cities and improve our mechanistic understanding of selection processes at different spatial scales. We then perform a second literature search to review genomic studies assessing selection intensity in cities, on the genome of non‐human natural populations. While this search returns 383 articles, only 34 of these truly investigate footprints of selection associated with urbanization, and only one study provides urban genetic selection coefficients. Here again, we find highly heterogeneous approaches, yet some studies provide strong evidence of genomic footprints of urban adaptation. In neither the phenotypic nor genomic literature review were we able to quantitatively assess natural selection across urban versus non‐urban habitats. Thus, we propose a roadmap of how future studies should provide standardized metrics to facilitate mega‐ or meta‐analyses and explore generalized effects of urbanization on selection. Read the free Plain Language Summary for this article on the Journal blog.

SNP Markers: Analysis of Genetic Diversity and Identification of Genomic Regions in Pantaneiro Sheep and Texel Sheep Under Natural Selection.

Sheep farming is growing substantially in Brazil, driven by the increasing demand for sheep meat. This rising demand has heightened the focus on sheep, making them the subject of numerous studies, including those centered on genetic analysis. A notable research area involves Pantaneiro sheep, which are indigenous to the Pantanal region of Mato Grosso do Sul and other locations. These sheep are of particular interest due to their adaptation to the unique environmental conditions of the Pantanal, a floodplain characterized by its distinctive climatic and ecological features. This study primarily aimed to conduct a comprehensive genomic analysis of Pantanal sheep subjected to natural selection within the Pantanal region and compare different sample herds using methodological approaches. Genomic analysis was performed to examine genetic diversity and structure via GGP50K single nucleotide polymorphism (SNP) analysis. A sample of 192 adult sheep over 4 years old was categorized into seven populations based on location: Six populations comprised Pantaneiro sheep with one Texel sheep population. Outlier SNPs were assessed to pinpoint regions under natural selection, with comparisons between the Pantaneiro and the commercial Texel breeds. All data analyses were conducted using the R programming language, employing specialized genetic analysis packages. These outlier SNPs were detected using three methodologies, PCAdapt, OutFLANK, and FDIST2/fsthet, with false discovery rate (FDR) corrections applied to ensure result accuracy. Each method was evaluated, and the genes associated with the identified SNPs were cross-referenced with the most recent sheep genome database, focusing specifically on genes with known phenotypic traits. Analysis of a sample comprising 192 adult individuals revealed greater genetic variability within the Pantaneiro breed than the Texel breed, highlighting the adaptation of the Pantaneiro breed to the unique Pantanal environment. Conversely, the Texel breed exhibited significantly higher levels of inbreeding, attributed to its controlled breeding practices. Outlier SNPs were detected with notable variation across different methodologies, underscoring the importance of FDR correction in ensuring the reliability and concentration of identified outliers. These outlier SNPs facilitated the identification of genes associated with key phenotypic traits, including hair growth, tissue regeneration, pigmentation regulation, and muscle capacity. The integrated analysis of methodologies demonstrated significant efficiency in elucidating the genomic landscape of Pantanal sheep, highlighting the genetic richness inherent in sheep from the Pantanal region of Mato Grosso do Sul. The techniques employed effectively identified outlier SNPs associated with phenotypically relevant genes. These findings, which reveal greater genetic variability and adaptability, underscore the potential of these animals for future research and their significance within Brazilian sheep farming. The Texel breed served as a valuable comparative group, illustrating the limited genetic variability in highly controlled breeding environments.

Genomic insights into codon usage bias in Cannabis sativa and pathogenic interactions

This study delves into codon usage bias (CUB) across Cannabis sativa and its associated pathogens (Meloidogyne incognita, Alternaria alternata, Aspergillus flavus, Aspergillus niger, and Fusarium oxysporum). It finds an AT-bias in C. sativa and M. incognita, favouring AT ending codons, while fungal pathogens lean towards GC-ending codons. Analysis suggests weak codon bias overall, with host and nematode showing similar preferences, and fungal pathogens preferring GC-ending codons. In ENC/GC3 plot, parity plot analyses both natural selection and mutational pressure influence CUB, slightly favouring T-ending codons.Neutrality plot analysis demonstrated varying degrees of mutational pressure and selection across the organisms. Correspondence analysis revealed indistinguishable CUB effective in all pathogens, despite differences in evolutionary forces. Correlation analysis illustrated the impact of nucleotide composition, mutational pressure, and natural selection on CUB. Additionally, amino acid composition analysis indicated differential usage of amino acids across organisms. Codon context analysis unveiled common trends in codon pairing, emphasizing the prevalence of preferred codons in C. sativa and M. incognita. This comprehensive analysis provides insights into the factors shaping CUB and the evolutionary forces influencing the genomes of the host plant and its associated pathogens, contributing to a deeper understanding of molecular interactions in host-pathogen systems. This data informs conservation for Cannabis sativa by identifying pathogen-resistant genetic traits, guiding breeding and management for biodiversity preservation.

Accelerated biological evolution in outer space: Insights from numerical analysis

As humanity continues its space exploration, understanding biological evolution in extraterrestrial environments will become crucial. On Earth, organisms have adapted to new environments, and some genetic data indicate positive natural selection. This paper investigates the impact of space environments, such as high radiation and microgravity, which may lead to high mutation rates and positive selection, on biological evolution, using numerical analysis. It quantifies the evolutionary rates and the time until a new mutation reaches fixation (100 % frequency within population) beyond Earth. The findings reveal accelerated evolution rates, 1,000 to 10,000 times faster than on Earth for beneficial mutations, with the time until fixation being 0.002 to 0.004 times shorter, assuming mutation rates are 10–100 times higher. These results offer insights into various areas, including space facility design, space agriculture, astrobiological exploration, and life sustainability beyond Earth and Solar System, illuminating the potential for a ‘Big Bang of Evolution’ in outer space.

Limited Variation in Codon Usage across Mitochondrial Genomes of Non-Biting Midges (Diptera: Chironomidae).

The codon usage patterns of mitochondrial genomes offer insights into the evolutionary and phylogenetic studies of species. Codon usage analysis has been conducted in a few Chironomidae species, and the codon usage patterns in other species remain ambiguous. We aim to reveal the codon usage differences in the mitochondrial genomes across this family. We sequenced the first mitochondrial genome of the genus Conchapelopia and the third mitochondrial genome of the subfamily Tanypodinae. Then, we analyzed its relative synonymous codon usage and effective number of codons with registered mitochondrial genomes from 28 other genera. The results indicated that there was limited variation in codon usage across five subfamilies, Chironominae, Orthocladiinae, Diamesinae, Prodiamesinae and Tanypodinae. While Parochlus steinenii from Podonominae presented a weaker codon bias, P. steinenii possessed the most genes experiencing natural selection. Additionally, ND1, ND2 and ND3 were found to be the most frequently selected genes across all species. Our findings contribute to further understanding the evolutionary and phylogenetic relationships of Chironomidae.

Natural selection on floral volatiles and other traits can change with snowmelt timing and summer precipitation.

Climate change is disrupting floral traits that mediate mutualistic and antagonistic species interactions. Plastic responses of these traits to multiple shifting conditions may be adaptive, depending on natural selection in new environments. We manipulated snowmelt date over three seasons (3-11 d earlier) in factorial combination with growing-season precipitation (normal, halved, or doubled) to measure plastic responses of volatile emissions and other floral traits in Ipomopsis aggregata. We quantified how precipitation and early snowmelt affected selection on traits by seed predators and pollinators. Within years, floral emissions did not respond to precipitation treatments but shifted with snowmelt treatment depending on the year. Across 3 yr, emissions correlated with both precipitation and snowmelt date. These effects were driven by changes in soil moisture. Selection on several traits changed with earlier snowmelt or reduced precipitation, in some cases driven by predispersal seed predation. Floral trait plasticity was not generally adaptive. Floral volatile emissions shifted in the face of two effects of climate change, and the new environments modulated selection imposed by interacting species. The complexity of the responses underscores the need for more studies of how climate change will affect floral volatiles and other floral traits.

Natural selection and the evolution of plant functional traits: an appreciation of Geber and Griffen (2003)

Natural selection and the evolution of plant functional traits: an appreciation of Geber and Griffen (2003)

Phylogenetic exploration, codon usage bias, and genomic divergence in Hydrocotyle: a comparative plastome study across different geographical locations.

Hydrocotyle himalaica from Bhutan, a perennial herb that thrives from 1500 to 2600m, possesses both ecological importance and medicinal properties. The plastome analysis revealed a length of 153,383bp, showing variation from conspecific taxa in China. Its standard structure comprises two IR regions (18,336bp IRa and 18,336bp IRb), an LSC region of 97,944bp, and an SSC region of 18,767bp, with a GC content of 37.63%. Non-coding regions showed higher mutation susceptibility, with Pi values from 0.006 to 0.107. An AT-rich codon bias was consistent across all 18 Hydrocotyle species. Nucleotide composition and GC% in coding sequences differed among the species. The codon preference in Hydrocotyle is shaped by multiple factors, with natural selection being the primary influence, as indicated by the ENC-plot, PR2-plot, and Neutrality-plot. Codon usage patterns varied, with RSCU values from 0 to 2.23. Codons ending in A or U had RSCU > 1, while those ending in C or G had RSCU < 1. GC2 content surpassed GC3 and GC1 in most genes. The phylogenetic analysis placed H. himalaica, sourced from Kanglung, Bhutan, within the monophyly of the Hydrocotyloideae subfamily. However, the species showed weaker bootstrap support (BS < 50) with H. javanica and H. hookeri subsp., a deviation from a prior report on the same species from Jiangkou, Guizhou, China. This analysis highlighted the genomic characteristics and evolutionary relationships of H. himalaica from Bhutan, underscoring the need for a comprehensive phylogenetic, ecological, and botanical characterization to confirm intra-specific variation within Hydrocotyle species.

Sturnidae sensu lato Mitogenomics: Novel Insights into Codon Aversion, Selection, and Phylogeny.

The Sturnidae family comprises 123 recognized species in 35 genera. The taxa Mimidae and Buphagidae were formerly treated as subfamilies within Sturnidae. The phylogenetic relationships among the Sturnidae and related taxa (Sturnidae sensu lato) remain unresolved due to high rates of morphological change and concomitant morphological homoplasy. This study presents five new mitogenomes of Sturnidae sensu lato and comprehensive mitogenomic analyses. The investigated mitogenomes exhibit an identical gene composition of 37 genes-including 13 protein-coding genes (PCGs), 2 rRNA genes, and 22 tRNA genes-and one control region (CR). The most important finding of this study is drawn from CAM analyses. The surprisingly unique motifs for each species provide a new direction for the molecular species identification of avian. Furthermore, the pervasiveness of the natural selection of PCGs is found in all examined species when analyzing their nucleotide composition and codon usage. We also determine the structures of mt-tRNA, mt-rRNA, and CR structures of Sturnidae sensu lato. Lastly, our phylogenetic analyses not only well support the monophyly of Sturnidae, Mimidae, and Buphagidae, but also define nine stable subclades. Taken together, our findings will enable the further elucidation of the evolutionary relationships within Sturnidae sensu lato.

Fluctuating selection in a monkeyflower hybrid zone

Abstract While hybridization was viewed as a hindrance to adaptation and speciation by early evolutionary biologists, recent studies have demonstrated the importance of hybridization in facilitating evolutionary processes. However, it is still not well-known what role spatial and temporal variation in natural selection play in the maintenance of naturally occurring hybrid zones. To identify whether hybridization is adaptive between two closely related monkeyflower species, Mimulus guttatus and Mimulus laciniatus, we performed repeated reciprocal transplants between natural hybrid and pure species’ populations. We planted parental genotypes along with multiple experimental hybrid generations in a dry (2021) and extremely wet (2023) year in the Sierra Nevada, CA. By taking fine-scale environmental measurements, we found that the environment of the hybrid zone is more similar to M. laciniatus’s seasonally dry rocky outcrop habitat than M. guttatus’s moist meadows. In our transplants hybridization does not appear to be maintained by a consistent fitness advantage of hybrids over parental species in hybrid zones, but rather a lack of strong selection against hybrids. We also found higher fitness of the drought-adapted species, M. laciniatus, than M. guttatus in both species’ habitats, as well as phenotypic selection for M. laciniatus-like traits in the hybrid habitat in the dry year of our experiment. These findings suggest that in this system, hybridization might function to introduce drought-adapted traits and genes from M. laciniatus into M. guttatus, specifically in years with limited soil moisture. However, we also find evidence of genetic incompatibilities in second generation hybrids in the wetter year, which may balance a selective advantage of M. laciniatus introgression. Therefore, we find that hybridization in this system is both potentially adaptive and costly, and that the interaction of positive and negative selection likely determines patterns of gene flow between these Mimulus species.

Cytogenetic analysis of 2554 samples of the products of conception from early reproductive losses

Aim. Early pregnancy losses (EPL), to some extent, can be considered a mechanism of natural selection that prevents the development of defective fetuses, since it is believed that even up to 80 % of the causes have genetic disorders. Among the genetic factors of EPL karyotype disorders should be included first of all. Overall, up to 50 % of early miscarriages are caused by karyotype abnormalities. The purpose of this study was studied the contribution of chromosomal abnormalities to the genesis of EPL. Methods. Banding cytogenetic and interphase mFISH with the centromeric probe panel for chromosomes 13, 14, 15, 16, 17, 18, 20, 21, 22, X and Y were used. Results. Were examined 2554 cases of material of early reproductive loss. A normal karyotype was established in 1638 cases (64.1 %), and a pathological karyotype in 916 (35.9 %). Karyotype abnormalities are as follows: autosomal aneuploidy – 478 (52.2 %), polyploidy – 256 (27.9 %), gonosomal aneuploidy – 182 (19.9 %). Conclusions. Among autosomal aneuploidies prevailed trisomy 16 (26.9 %), among polyploidy – triploidy (24.6 %) and among gonosomal aneuploidy – monosomy X (18.4 %).

Non-darwinian theories of biological evolution

The article presents material related to evolutionary theories such as Berg’s nomogenesis, Kimura’s theory of neutral evolution, and neo-Lamarckism, the content of which is opposed to Darwin’s doctrine of natural selection as a leading factor in biological evolution. Nomogenesis, a hypothesis according to which the evolution of organisms is based on internal laws. The general thesis of Berg’s worldview was that evolution occurs naturally, in accordance with a pre-existing – pre-established – law of development. The neutral theory of molecular evolution was put forward by Motoo Kimura. It argues that the vast majority of mutations at the molecular level are neutral in nature with respect to the action of natural selection. Thus, the theory of neutral evolution contradicted Darwinism, pointing to the fixation of neutral mutations in the population without the participation of natural selection. Neo-Lamarckism is a combination of three heterogeneous trends in evolutionary doctrine. Despite the fact that neo-Lamarckists have deviated from the basic postulates of Lamarck, most of their concepts contain two provisions that correspond to Lamarck's laws. The first is the recognition of adequate variability that occurs under the direct or indirect influence of the environment and creates well-acquired adaptive traits. The second is the inheritance of similar traits in individual life.

Inbreeding and its effect on the gene fund in dog breeding

Aim. Identifying problems and approaches to dog breeding that emphasize the importance of the quality of parents and hereditary factors in determining the offspring's quality. Methods. Theoretical review of inbreeding and its impact on the gene pool in dog breeding, comparing it with outbreeding, and exploring natural selection mechanisms and their influence on animal reproduction. Results. The paper discusses the principles of selecting breeders in dog breeding and provides recommendations for avoiding mating dogs with deficiencies in a particular trait. It also highlights the study of genetic mechanisms and their impact on the inheritance of traits in dogs. Two main breeding methods are considered in animal husbandry – inbreeding and outbreeding. The study findings indicate types of inbreeding and their influence on the genetic homogeneity of the offspring. Conclusions. The breeder must clearly define the type of dog he wants to obtain, specifying desired characteristics such as appearance, health, and temperament. In the initial stages, the breeder must achieve stability in breeding offspring with desired characteristics, creating animals with a typical temperament. After achieving stability, one can begin fine-tuning specific traits by selecting for improvements in breed aspects. A dog should be viewed as a holistic system, striving for overall correctness and balance, paying attention to the interrelation between different aspects. The breeder must ensure the preservation of unique breed characteristics during selection. Effective selection involves careful planning, observation, evaluation, and collaboration with experienced veterinarians and breed specialists.

The selfish machine? On the power and limitation of natural selection to understand the development of advanced AI

The selfish machine? On the power and limitation of natural selection to understand the development of advanced AI

Limited population structure but signals of recent selection in introduced African Fig Fly (Zaprionus indianus) in North America.

Invasive species have devastating consequences for human health, food security, and the environment. Many invasive species adapt to new ecological niches following invasion, but little is known about the early steps of adaptation. Here we examine population genomics of a recently introduced drosophilid in North America, the African Fig Fly, Zaprionus indianus. This species is likely intolerant of subfreezing temperatures and recolonizes temperate environments yearly. We generated a new chromosome-level genome assembly for Z. indianus. Using resequencing of over 200 North American individuals collected over four years in temperate Virginia, plus a single collection from subtropical Florida, we tested for signatures of recolonization, population structure, and adaptation within invasive populations. We show founding populations are sometimes small and contain close genetic relatives, yet temporal population structure and differentiation of populations is mostly absent across recurrent recolonization events. Although we find limited signals of genome-wide spatial or temporal population structure, we identify haplotypes on the X chromosome that are repeatedly differentiated between Virginia and Florida populations. These haplotypes show signatures of natural selection and are not found in African populations. We also find evidence for several large structural polymorphisms segregating within North America populations and show X chromosome evolution in invasive populations is strikingly different from the autosomes. These results show that despite limited population structure, populations may rapidly evolve genetic differences early in an invasion. Further uncovering how these genomic regions influence invasive potential and success in new environments will advance our understanding of how organisms evolve in changing environments.

Early menarche and childbirth accelerate aging-related outcomes and age-related diseases: Evidence for antagonistic pleiotropy in humans.

Aging can be understood as a consequence of the declining force of natural selection with age. Consistent with this the antagonistic pleiotropic theory of aging suggests that aging results from the trade-offs that promote early growth and reproduction. However, evidence for antagonistic pleiotropy in humans is largely lacking. Using Mendelian Randomization (MR), we demonstrated that later ages of menarche or first childbirth were genetically associated with longer parental lifespan, decreased frailty index, slower epigenetic aging, later menopause, and reduced facial aging. Moreover, later menarche or first childbirth were also genetically associated with a lower risk of several age-related diseases, including late-onset Alzheimer's disease (LOAD), type 2 diabetes, heart disease, essential hypertension, and chronic obstructive pulmonary disease (COPD). We validated the associations between the age of menarche, childbirth, and the number of childbirths with several age-related outcomes in the UK Biobank by conducting regression analysis of nearly 200,000 subjects. Our results demonstrated that menarche before the age 11 and childbirth before 21 significantly accelerated the risk of several diseases, and almost doubled the risk for diabetes, heart failure, and quadrupled the risk of obesity, supporting the antagonistic pleiotropy theory. We identified 128 significant single nucleotide polymorphisms (SNPs) that influenced age-related outcomes, some of which were involved in known longevity pathways, including IGF1, growth hormone, AMPK, and mTOR signaling. Our study also identified higher BMI as a mediating factor in causing the increased risk of certain diseases, such as type 2 diabetes and heart failure, in women with early menarche or early pregnancy, emphasizing the importance of the thrifty gene hypothesis in explaining in part the mechanisms behind antagonistic pleiotropy. Our study highlights the complex relationship between genetic legacies and modern diseases, emphasizing the need for gender-sensitive healthcare strategies that consider the unique connections between female reproductive health and aging.

Mechanical Characteristics of Bivalve (Mollusc) Shells from the East Coast of India: Implications and Their Significance on the Survival Strategy

Due to natural selection, an increase in biotic and abiotic factors develops an adaptive response in bivalves (molluscs) for survival. Morphological variation in a bivalve is strongly correlated with the changes in the structural and compositional properties of a shell. Therefore, the mechanical characteristics of a shell reflect its morpho-functional adaptation against predation and ecological stresses. An initial investigation into the primary mechanical attributes of predominant bivalve shells from the east coast of India, Tegillarca granosa, Meretrix meretrix, Sunetta meroe, Mactra turgida and Donax scortum, has been documented in this study. The quantitative assessment of the centreline thickness, Vickers hardness and density variation of the shells were measured and correlated along with the preliminary investigation of the microstructural patterns. The results from our study are in agreement with the most vulnerable region of a shell, located just below the umbonal zone, which has been verified through predatory boreholes in other studies. Our data shows a high correlation between shell hardness and density, which could also be the potential properties to consider in detail for future studies, apart from the shell thickness, which could provide a broader perspective on the stability of bivalve shells. Different microstructural patterns observed in the species could be phylogenetically driven based on their life modes.

Mathematical model for the distribution of DNA replication origins.

DNA replication in yeast and in many other organisms starts from well-defined locations on the DNA known as replication origins. The spatial distribution of these origins in the genome is particularly important in ensuring that replication is completed quickly. Cells are more vulnerable to DNA damage and other forms of stress while they are replicating their genome. This raises the possibility that the spatial distribution of origins is under selection pressure. In this paper we investigate the hypothesis that natural selection favors origin distributions leading to shorter replication times. Using a simple mathematical model, we show that this hypothesis leads to two main predictions about the origin distributions: that neighboring origins that are inefficient (less likely to fire) are more likely to be close to each other than efficient origins; and that neighboring origins with larger differences in firing times are more likely to be close to each other than origins with similar firing times. We test these predictions using next-generation sequencing data, and show that they are both supported by the data.