Modular Evolution Research Articles

Small body size is associated with increased evolutionary lability of wing skeleton proportions in birds

Birds are represented by 11,000 species and a great variety of body masses. Modular organisation of trait evolution across birds has facilitated simultaneous adaptation of different body regions to divergent ecological requirements. However, the role modularity has played in avian body size evolution, especially small-bodied, rapidly evolving and diverse avian subclades, such as hummingbirds and songbirds, is unknown. Modularity is influenced by the intersection of biomechanical restrictions, adaptation, and developmental controls, making it difficult to uncover the contributions of single factors such as body mass to skeletal organisation. We develop a novel framework to decompose this complexity, assessing factors underlying the modularity of skeletal proportions in fore-limb propelled birds distributed across a range of body masses. We demonstrate that differences in body size across birds triggers a modular reorganisation of flight apparatus proportions consistent with biomechanical expectations. We suggest weakened integration within the wing facilitates radiation in small birds. Our framework is generalisable to other groups and has the capacity to untangle the multi-layered complexity intrinsic to modular evolution.

Correlated evolution of beak and braincase morphology is present only in select bird clades.

Complex morphological structures, such as skulls or limbs, are often composed of multiple morphological components (e.g., bones, sets of bones) that may evolve in a covaried manner with one another. Previous research has reached differing conclusions on the number of semi-independent units, or modules, that exist in the evolution of structures and on the strength of the covariation, or integration, between these hypothesized modules. We focus on the avian skull as an example of a complex morphological structure for which highly variable conclusions have been reached in the numerous studies analyzing support for a range of simple to complex modularity hypotheses. We hypothesized that past discrepancies may stem from both the differing densities of data used to analyze support for modularity hypotheses and the differing taxonomic levels of study. To test these hypotheses, we applied a comparative method to 3D geometric morphometric data collected from the skulls of a diverse order of birds (the Charadriiformes) to test support for 11 distinct hypotheses of modular skull evolution. Across all Charadriiformes, our analyses suggested that charadriiform skull evolution has been characterized by the semi-independent, but still correlated, evolution of the beak from the rest of the skull. When we adjusted the density of our morphometric data, this result held, but the strength of the signal varied substantially. Additionally, when we analyzed subgroups within the order in isolation, we found support for distinct hypotheses between subgroups. Taken together, these results suggest that differences in the methodology of past work (i.e., statistical method and data density) as well as clade-specific dynamics may be the reasons past studies have reached varying conclusions.

YEvo: A modular eukaryotic genetics and evolution research experience for high school students.

The resources for carrying out and analyzing microbial evolution experiments have become more accessible, making it possible to expand these studies beyond the research laboratory and into the classroom. We developed five connected, standards-aligned yeast evolution laboratory modules, called "yEvo," for high school students. The modules enable students to take agency in answering open-ended research questions. In Module 1, students evolve baker's yeast to tolerate an antifungal drug, and in subsequent modules, investigate how evolved yeasts adapted to this stressful condition at both the phenotype and genotype levels. We used pre- and post-surveys from 72 students at two different schools and post-interviews with students and teachers to assess our program goals and guide module improvement over 3 years. We measured changes in student conceptions, confidence in scientific practices, and interest in STEM careers. Students who participated in yEvo showed improvements in understanding of activity-specific concepts and reported increased confidence in designing a valid biology experiment. Student experimental data replicated literature findings and has led to new insights into antifungal resistance. The modules and provided materials, alongside "proof of concept" evaluation metrics, will serve as a model for other university researchers and K - 16 classrooms interested in engaging in open-ended research questions using yeast as a model system.

Molecular architecture and electron transfer pathway of the Stn family transhydrogenase

The challenge of endergonic reduction of NADP+ using NADH is overcome by ferredoxin-dependent transhydrogenases that employ electron bifurcation for electron carrier adjustments in the ancient Wood-Ljungdahl pathway. Recently, an electron-bifurcating transhydrogenase with subunit compositions distinct from the well-characterized Nfn-type transhydrogenase was described: the Stn complex. Here, we present the single-particle cryo-EM structure of the Stn family transhydrogenase from the acetogenic bacterium Sporomusa ovata and functionally dissect its electron transfer pathway. Stn forms a tetramer consisting of functional heterotrimeric StnABC complexes. Our findings demonstrate that the StnAB subunits assume the structural and functional role of a bifurcating module, homologous to the HydBC core of the electron-bifurcating HydABC complex. Moreover, StnC contains a NuoG-like domain and a GltD-like NADPH binding domain that resembles the NfnB subunit of the NfnAB complex. However, in contrast to NfnB, StnC lost the ability to bifurcate electrons. Structural comparison allows us to describe how the same fold on one hand evolved bifurcation activity on its own while on the other hand combined with an associated bifurcating module, exemplifying modular evolution in anaerobic metabolism to produce activities critical for survival at the thermodynamic limit of life.

Host range and molecular and ultrastructural analyses of Asparagus virus 1 pathotypes isolated from garden asparagus Asparagus officinalis L.

Asparagus samples were examined from growing areas of Germany and selected European as well as North, Central and South American countries. Overall, 474 samples were analyzed for Asparagus virus 1 (AV1) using DAS-ELISA. In our survey, 19 AV1 isolates were further characterized. Experimental transmission to 11 species belonging to Aizoaceae, Amarantaceae, Asparagaceae, and Solanaceae succeeded. The ultrastructure of AV1 infection in asparagus has been revealed and has been compared with the one in indicator plants. The cylindrical inclusion (CI) protein, a core factor in viral replication, localized within the cytoplasm and in systemic infections adjacent to the plasmodesmata. The majority of isolates referred to pathotype I (PI). These triggered a hypersensitive resistance in inoculated leaves of Chenopodium spp. and were incapable of infecting Nicotiana spp. Only pathotype II (PII) and pathotype III (PIII) infected Nicotiana benthamiana systemically but differed in their virulence when transmitted to Chenopodium spp. The newly identified PIII generated amorphous inclusion bodies and degraded chloroplasts during systemic infection but not in local lesions of infected Chenopodium spp. PIII probably evolved via recombination in asparagus carrying a mixed infection by PI and PII. Phylogeny of the coat protein region recognized two clusters, which did not overlap with the CI-associated grouping of pathotypes. These results provide evidence for ongoing modular evolution of AV1.

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9.

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.

A benchmark generator framework for evolving variant-rich software

Software often needs to exist in different variants, which account for varying customer requirements, environments, or non-functional aspects, such as energy consumption. Unfortunately, the number of variants can grow exponentially with the number of features. As such, developing and evolving variant-rich systems is challenging, since they do not only evolve “in time” as single systems, but also “in space” with new variants. Fortunately, many different methods and tools for variant-rich systems have been proposed over the last decades, especially in the field of software product line engineering. However, their level of evaluation varies significantly, threatening their relevance for practitioners and that of future research. Many tools have only been evaluated on ad hoc datasets, minimal examples, or unrealistic and limited evolution scenarios, missing large parts of the actual evolution lifecycle of variant-rich systems.Our long-term goal is to provide benchmarks to increase the maturity of evaluation of methods and tools for evolving variant-rich systems. However, providing manually curated and sufficiently detailed benchmarks that cover the whole evolution lifecycle of variant-rich systems is challenging. We present the framework vpbench, which simulates the evolution of a variant-rich system and thereby generates an evolution enriched with metadata explaining the evolution. The generated benchmarks, i.e., the evolution histories and metadata, can serve as ground truth to check the results of tools applied on it. We formalize the claims we make about the generator and the generated benchmarks as requirements. The design of vpbench comprises modular generators and evolution operators that automatically evolve real codebases. We implement simple and advanced evolution operators—e.g., relying on code transplantation to incorporate features from real projects. We demonstrate how vpbench addresses its claimed requirements, also considering multiple degrees of realism, extensibility and language-independence of the generated benchmarks.

Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition

Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER1/2 inside module 1/2 and the ketosynthase domain KS3 inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS4 of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.

GalCEM. I. An Open-source Detailed Isotopic Chemical Evolution Code

This is the first of a series of papers that will introduce a user-friendly, detailed, and modular Galactic Chemical Evolution Model, GalCEM, that tracks isotope masses as a function of time in a given galaxy. The list of tracked isotopes automatically adapts to the complete set provided by the input yields. The present iteration of GalCEM tracks 86 elements broken down into 451 isotopes. The prescription includes massive stars, low-to-intermediate-mass stars, and Type Ia supernovae as enrichment channels. We have developed a preprocessing tool that extracts multidimensional interpolation curves from the input yield tables. These interpolation curves improve the computation speeds of the full convolution integrals, which are computed for each isotope and for each enrichment channel. We map the integrand quantities onto consistent array grids in order to perform the numerical integration at each time step. The differential equation is solved with a fourth-order Runge–Kutta method. We constrain our analysis to the evolution of all light and intermediate elements from carbon to zinc, and lithium. Our results are consistent up to the extremely metal-poor regime with Galactic abundances. We provide tools to track the mass rate change of individual isotopes on a typical spiral galaxy with a final baryonic mass of 5 × 1010 M ⊙. Future iterations of the work will extend to the full periodic table by including the enrichment from neutron-capture channels as well as spatially dependent treatments of galaxy properties. GalCEM is publicly available at https://github.com/egjergo/GalCEM.

Patterns and Temporal Dynamics of Natural Recombination in Noroviruses.

Noroviruses infect a wide range of mammals and are the major cause of gastroenteritis in humans. Recombination at the junction of ORF1 encoding nonstructural proteins and ORF2 encoding major capsid protein VP1 is a well-known feature of noroviruses. Using all available complete norovirus sequences, we systematically analyzed patterns of natural recombination in the genus Norovirus both throughout the genome and across the genogroups. Recombination events between nonstructural (ORF1) and structural genomic regions (ORF2 and ORF3) were found in all analyzed genogroups of noroviruses, although recombination was most prominent between members of GII, the most common genogroup that infects humans. The half-life times of recombinant forms (clades without evidence of recombination) of human GI and GII noroviruses were 10.4 and 8.4-11.3 years, respectively. There was evidence of many recent recombination events, and most noroviruses that differed by more than 18% of nucleotide sequence were recombinant relative to each other. However, there were no distinct recombination events between viruses that differed by over 42% in ORF2/3, consistent with the absence of systematic recombination between different genogroups. The few inter-genogroup recombination events most likely occurred between ancient viruses before they diverged into contemporary genogroups. The recombination events within ORF1 or between ORF2/3 were generally rare. Thus, noroviruses routinely exchange full structural and nonstructural blocks of the genome, providing a modular evolution.

Domain Evolution of Vertebrate Blood Coagulation Cascade Proteins

Vertebrate blood coagulation is controlled by a cascade containing more than 20 proteins. The cascade proteins are found in the blood in their zymogen forms and when the cascade is triggered by tissue damage, zymogens are activated and in turn activate their downstream proteins by serine protease activity. In this study, we examined proteomes of 21 chordates, of which 18 are vertebrates, to reveal the modular evolution of the blood coagulation cascade. Additionally, two Arthropoda species were used to compare domain arrangements of the proteins belonging to the hemolymph clotting and the blood coagulation cascades. Within the vertebrate coagulation protein set, almost half of the studied proteins are shared with jawless vertebrates. Domain similarity analyses revealed that there are multiple possible evolutionary trajectories for each coagulation protein. During the evolution of higher vertebrate clades, gene and genome duplications led to the formation of other coagulation cascade proteins.

Analysis of the Evolution of the MoxR ATPases.

MoxR proteins comprise a family of ATPases Associated with diverse cellular Activities (AAA+). These proteins are widespread and found across the diversity of prokaryotic species. Despite their ubiquity, members of the group remain poorly characterized. Only a few examples of MoxR proteins have been associated with cellular roles, where they have been shown to perform chaperone-like functions. A characteristic feature of MoxR proteins is their association with proteins containing the von Willebrand factor type A (VWA) domain. In an effort to understand the spread and diversity of the MoxR family, an evolutionary approach was undertaken. Phylogenetic techniques were used to define nine major subfamilies within the MoxR family. A combination of phylogenetic and genomic approaches was utilized to explore the extent of the partnership between the MoxR and VWA domain containing proteins (VWA proteins). These analyses led to the clarification of genetic linkages between MoxR and VWA proteins. A significant partnership is described here, as seven of nine MoxR subfamilies were found to be linked to VWA proteins. Available genomic data were also used to assess the intraprotein diversification of MoxR and VWA protein sequences. Data clearly indicated that, in MoxR proteins, the ATPase domain is maintained with high conservation while the remaining protein sequence evolves at a faster rate; a similar pattern was observed for the VWA domain in VWA proteins. Overall, our data present insights into the modular evolution of MoxR ATPases.

Modular Grammatical Evolution for the Generation of Artificial Neural Networks.

This article presents a novel method, called Modular Grammatical Evolution (MGE), toward validating the hypothesis that restricting the solution space of NeuroEvolution to modular and simple neural networks enables the efficient generation of smaller and more structured neural networks while providing acceptable (and in some cases superior) accuracy on large data sets. MGE also enhances the state-of-the-art Grammatical Evolution (GE) methods in two directions. First, MGE's representation is modular in that each individual has a set of genes, and each gene is mapped to a neuron by grammatical rules. Second, the proposed representation mitigates two important drawbacks of GE, namely the low scalability and weak locality of representation, toward generating modular and multilayer networks with a high number of neurons. We define and evaluate five different forms of structures with and without modularity using MGE and find single-layer modules with no coupling more productive. Our experiments demonstrate that modularity helps in finding better neural networks faster. We have validated the proposed method using ten well-known classification benchmarks with different sizes, feature counts, and output class counts. Our experimental results indicate that MGE provides superior accuracy with respect to existing NeuroEvolution methods and returns classifiers that are significantly simpler than other machine learning generated classifiers. Finally, we empirically demonstrate that MGE outperforms other GE methods in terms of locality and scalability properties.

Evolutionary modularity, integration and disparity in an accretionary skeleton: analysis of venerid Bivalvia.

Modular evolution, the relatively independent evolution of body parts, may promote high morphological disparity in a clade. Conversely, integrated evolution via stronger covariation of parts may limit disparity. However, integration can also promote high disparity by channelling morphological evolution along lines of least resistance-a process that may be particularly important in the accumulation of disparity in the many invertebrate systems having accretionary growth. We use a time-calibrated phylogenetic hypothesis and high-density, three-dimensional semilandmarking to analyse the relationship between modularity, integration and disparity in the most diverse extant bivalve family: the Veneridae. In general, venerids have a simple, two-module parcellation of their body that is divided into features of the calcium carbonate shell and features of the internal soft anatomy. This division falls more along developmental than functional lines when placed in the context of bivalve anatomy and biomechanics. The venerid body is tightly integrated in absolute terms, but disparity appears to increase with modularity strength among subclades and ecologies. Thus, shifts towards more mosaic evolution beget higher morphological variance in this speciose family.

Modular evolution of secretion systems and virulence plasmids in a bacterial species complex

BackgroundMany named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes.ResultsWe utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins.ConclusionsWe demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

Development and function explain the modular evolution of phalanges in gecko lizards.

Selective regimes favouring the evolution of functional specialization probably affect covariation among phenotypic traits. Phalanges of most tetrapods develop from a conserved module that constrains their relative proportions. In geckos, however, biomechanical specializations associated with adhesive toepads involve morphological variation in the autopodium and might reorganize such modular structures. We tested two hypotheses to explain the modular architecture of hand bones in geckos, one based on developmental interactions and another incorporating functional associations related to locomotion, and compared the empirical support for each hypothetical module between padded and padless lineages. We found strong evidence for developmental modules in most species, which probably reflects embryological constraints during phalangeal formation. Although padded geckos exhibit a functional specialization involving the hyperextension of the distal phalanges that is absent in padless species, the padless species are the ones that show a distal functional module with high integration. Some ancestrally padless geckos apparently deviate from developmental predictions and present a relatively weak developmental module of phalanges and a strongly integrated distal module, which may reflect selective regimes involving incipient frictional adhesion in digit morphology. Modularity of digit elements seems dynamic along the evolutionary history of geckos, being associated with the presence/absence of adhesive toepads.

Modular Evolution of the Drosophila Metabolome.

Comparative phylogenetic studies offer a powerful approach to study the evolution of complex traits. Although much effort has been devoted to the evolution of the genome and to organismal phenotypes, until now relatively little work has been done on the evolution of the metabolome, despite the fact that it is composed of the basic structural and functional building blocks of all organisms. Here we explore variation in metabolite levels across 50 My of evolution in the genus Drosophila, employing a common garden design to measure the metabolome within and among 11 species of Drosophila. We find that both sex and age have dramatic and evolutionarily conserved effects on the metabolome. We also find substantial evidence that many metabolite pairs covary after phylogenetic correction, and that such metabolome coevolution is modular. Some of these modules are enriched for specific biochemical pathways and show different evolutionary trajectories, with some showing signs of stabilizing selection. Both observations suggest that functional relationships may ultimately cause such modularity. These coevolutionary patterns also differ between sexes and are affected by age. We explore the relevance of modular evolution to fitness by associating modules with lifespan variation measured in the same common garden. We find several modules associated with lifespan, particularly in the metabolome of older flies. Oxaloacetate levels in older females appear to coevolve with lifespan, and a lifespan-associated module in older females suggests that metabolic associations could underlie 50 My of lifespan evolution.

Twenty years of evolution and diversification of digitaria streak virus in Digitaria setigera.

Within the family Geminiviridae, the emergence of new species results from their high mutation and recombination rates. In this study, we report the variability and evolution of digitaria streak virus (DSV), a mastrevirus isolated in 1986 from the grass Digitaria setigera in an island of the Vanuatu archipelago. Viral DNA of DSV samples was amplified from D. setigera specimens, derived from the naturally infected original plant, which were propagated in different laboratories in France and Italy for more than 20 years. From the consensus sequences, the nucleotide substitution rate was estimated for the period between a sample and the original sequence published in 1987, as well as for the period between samples. In addition, the intra-host genetic complexity and diversity of 8 DSV populations with a total of 165 sequenced haplotypes was characterized. The evolutionary rate of DSV was estimated to be between 1.13 × 10−4 and 9.87 × 10−4 substitutions/site/year, within the ranges observed in other single-stranded DNA viruses and RNA viruses. Bioinformatic analyses revealed high variability and heterogeneity in DSV populations, which confirmed that mutant spectra are continuously generated and are organized as quasispecies. The analysis of polymorphisms revealed nucleotide substitution biases in viral genomes towards deamination and oxidation of single-stranded DNA. The differences in variability in each of the genomic regions reflected a dynamic and modular evolution in the mutant spectra that was not reflected in the consensus sequences. Strikingly, the most variable region of the DSV genome, encoding the movement protein, showed rapid fixation of the mutations in the consensus sequence and a concomitant dN/dS ratio of 6.130, which suggests strong positive selection in this region. Phylogenetic analyses revealed a possible divergence in three genetic lineages from the original Vanuatu DSV isolate.

Whole lifecycle evolution control method of modular product structure oriented to personalized service

ABSTRACT The modular product structure is the orderly integration of the information generated by the modular design. With the deepening of the personalized service concept, in order to better provide personalized product customization services driven by customer demands, the research of modular product structure evolution control in the whole lifecycle is extremely necessary. Considering the different concerns of the tasks in different stages of the whole lifecycle, a 4-level modular product structure whole lifecycle evolution modelling system is proposed to transform the modular product structure into different tree structure for each lifecycle stage. By modelling the attributes for the nodes and the relationship for the edges of each stage tree, the standardized management and invocation of the information required by the personalized service process is realized. On this basis, two different personalized product customization methods led by recommendation or configuration are proposed, and the flow path and algorithm writing logic of personalized product configuration are introduced based on each stage tree. Finally, the method is applied to small agricultural machinery (SAM) products, which proves that the method can reasonably and orderly controls the evolution process, and provides a new idea for the development of whole lifecycle personalized services.

Limits to Decentralized Modular Evolution in Large Digitally Enabled Systems

Recent research on digitally enabled systems has emphasized modular approaches that lead to decentralized innovation. Yet there are reasons to believe that some digital systems cannot evolve optimally under fully decentralized modular architectures, and important opportunities for value creation may exist through building of more centralized systems. We explore the nature of such opportunity by examining the development of systems for cancer radiation therapy, where ability to cure with safety improved dramatically as a leading firm developed a more comprehensive system, taking a considerable degree of control over modular evolution. At least three characteristics of the radiation therapy industry could have slowed advance if modular evolution had not been more centrally directed: first, unmanaged, incompletely specified interfaces coupled with rapid change and the need for increasing doses of dangerous radiation could have caused injuries; second, the inability of players with key capabilities to develop their products and expand adequately could have prevented them from providing full value as independent firms, and third, difficulties in providing comprehensive customer support for a highly modularized system could have reduced customer benefits. We conclude that in today’s digital economy, opportunities for firms to achieve strong performance through creation of centrally directed modular systems is likely to be significant.