Parallel Evolution Research Articles

Lilium liangiae, a new species in the genus Lilium (Liliaceae) that reveals parallel evolution within morphology.

The former genus Nomocharis, which has been merged as a clade within the genus Lilium (Liliaceae), represents one of the most complicated and unclear groups included in the latter. Research on members of the Nomocharis clade has been quite limited due to the sampling difficulties caused by its selective environmental preferences. In this study, we propose a new species within this clade, Lilium liangiae, as a further bridge connecting the former genus Nomocharis with other members of the genus Lilium. We conducted morphological clustering, phylogenetic, and comparative genomics analyses of nuclear internal spacers and the newly generated complete chloroplast genome, in conjunction with previously published sequences, and performed ancestral state reconstruction to clarify the evolutionary pattern of important traits in Lilium. The clustering results of 38 morphological traits indicated that the new species is allied to Nomocharis, further increasing the morphological polymorphism in the latter. The phylogenetic results and morphological clustering both supported L. liangiae belonging to the subclade Ecristata in Nomocharis, its closest affinity being Lilium gongshanense. Inconsistencies in phylogenetic relationships were detected between nuclear and plastid datasets, possibly due to ancient hybridization and ongoing introgression. Comparative genomics revealed the conservation and similarity of their chloroplast genomes, with variations observed in the expansion and contraction of the IR regions. A/T and palindromic repeat sequences were the most abundant. Seven highly variable regions (Pi≥0.015) were identified as potential molecular markers based on the chloroplast genomes of 47 species within Lilium. Both nuclear and plastid genes exhibited very low variability within the Nomocharis clade, contrasting with their highly variable morphological appearance. The ancestral state reconstruction analysis suggests that the campanulate flower form, as in L. liangiae, arose at least three times within the genus Lilium, revealing parallel evolution in the latter. Overall, this study adds important genetic and morphological evidence for understanding the phylogenetic relationships and parallel evolution patterns of species within the genus Lilium.

Abstract 3951: Timing the development of chemoresistance in relapsed pediatric cancer

Abstract Survivors of pediatric cancer face lifelong battles with severe morbidities, which includes a significant risk of recurrence. Pre-existing genetic variation within primary tumors may offer certain cell populations an evolutionary advantage, increasing the likelihood that some will be resistant to treatment. But the inherent complexity of the tumor means finding these markers of resistance has yet to be fully explored across childhood cancer. To address this, we developed a pipeline that combines clonal evolution reconstruction with mutational signature extraction at the subclonal level to elucidate changes in mutational processes. Using this pipeline to analyze 1,743 pediatric tumor genomes we found a common pattern of parallel evolution in most pediatric cancers, irrespective of their tissue of origin. Further, we detected therapy-associated mutational signatures linked to specific treatments such as temozolomide, platinum, fluorouracil, and thiopurine in post-treatment tumor subpopulations. Of 235 samples with known treatment exposure, 37.9% displayed one or more treatment-associated signatures. In the context of the phylogenetic tree, we identified specific subpopulations with therapy signatures. In multi-sample cases, resistant subpopulations had ancestors present in primary tumors at the time of diagnosis, suggesting certain subpopulations possessed the ability to withstand chemotherapy-induced pressures from an early stage and then expanded following treatment. Finally, we observed differences in the timing of mutational signatures between antimetabolite and alkylating agent treatments, reflecting the mechanisms of these drugs. Antimetabolite agents showed subclonal mutational signatures occurring in later branches, likely due to dependence on cell cycling. In contrast, alkylating agents displayed signatures in both early and late subpopulations, as they are unaffected by cell cycle state. Thus, investigating mutational signatures at the subclonal level unveils new insights into the clonal dynamics of pediatric cancers and the development of chemoresistance. This research offers valuable insights for identifying pediatric cancer patients at greater risk of recurrence, enabling the development of more effective treatment plans to enhance their quality of life and overall survival rates. Citation Format: Sasha Blay, Mehdi Layeghifard, Scott Davidson, David Chen, Astra Schwertschkow, Vijay Ramaswamy, Michael Taylor, Elli Papaemmanuil, Anita Villani, David Malkin, Ludmil Alexandrov, Mark Cowley, Adam Shlien. Timing the development of chemoresistance in relapsed pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3951.

Stability of parallel evolution in shell traits between the littoral and sublittoral ecotypes of the Antarctic limpet Nacella concinna along a 17-year time series

Abstract Ecotypes constitute valuable markers for understanding the phenotypic responses influenced by evolutionary forces and environmental variations. Assessing the role of these processes in establishing and maintaining ecotype divergence is essential for anticipating evolutionary responses to future climate shifts. We studied the evolutionary dynamics of phenotypic population structure between Nacella concinna ecotypes through a morphometric and temporal analysis (2004–2021) in Antarctica. This approach enables the assessment of contemporary evolutionary dynamics in the presence of biotic and abiotic factors, facilitating the inference of interactions between the acting forces. Using generalized Procrustes analysis, ecotypes were successfully discriminated by their lateral shell traits consistently persisting over the analysed period. Multivariate linear and univariate mixed models revealed a significant temporal effect on shell phenotypic variation, surpassing the effect of inter-ecotype differences. However, according to phenotypic trajectory analysis, this temporal effect did not alter the direction of the phenotypic trajectory of ecotype shell shape, which evolved in parallel during the time series. Despite the environmental variation, the evidence of parallel evolution might indicate the incidence of natural selection in ecotype divergence rather than relying solely on phenotypic plasticity. This work enhances our understanding of ecotype temporal changes and microevolution, underscoring the importance of long-term phenotypic monitoring.

Digital humanities: Mission accomplished? An analysis of scholarly literature

The field of digital humanities (DH) has evolved throughout the parallel evolution of computers, software and networking techniques, as well as the different attitudes of interested scholars. Since the earliest historical phases of this research field, scholars have been debating whether it can be considered as a new academic discipline and whether it is revolutionary in nature. About 20 years ago, the early denotation of ‘humanities computing’ evolved to the present label of DH, and deep changes occurred in digital information technologies, as well as in their humanities applications. Meanwhile, dedicated academic curricula were launched, thus adding an argument in favor of the debated disciplinarity of DH. This paper gives an account of the relevant scholarly debate, distinguishing between the early period and the most recent years; it then tries to frame this process in a model of scientific revolution.

Parallel differential evolution paradigm for multilayer electromechanical device optimization

Design optimization of multilayer piezoelectric transducers is intended for efficient and practical usage of wideband transducers for fault diagnosis, biomedical, and underwater applications through adjusting layer thicknesses and volume fraction of piezoelectric material in each layer. In this context, we propose a parallel differential evolution (PDE) algorithm to mitigate the complexities of multivariate optimization as well as the computation time to achieve an optimized wideband transducer for the particular application. For lead magnesium niobate-lead titanate (PMN PT)- and PZT5h-based piezoelectric materials, the fitness function is formulated based on uniformity of mechanical pressure at the first three harmonics to achieve wide bandwidth in the required functional frequency range. It is carried out using a one-dimensional model (ODM), while input layer thicknesses and volume fractions of active material are evaluated using PDE. The simulation is performed on a parallel computing platform utilizing three different host machines to reduce computational time. Results of the proposed methodology for PDE are statistically represented in the form of minimum, maximum, mean, and standard deviation of fitness value, while graphically represented in terms of speedup and time. It can be observed that the execution time for parallel DE decreases with the increasing number of cores.

Parallel functional reduction in the mitochondria of apicomplexan parasites

Extreme functional reduction of mitochondria has taken place in parallel in many distantly related lineages of eukaryotes, leading to a number of recurring metabolic states with variously lost electron transport chain (ETC) complexes, loss of the tricarboxylic acid (TCA) cycle, and/or loss of the mitochondrial genome. The resulting mitochondria-related organelles (MROs) are generally structurally reduced and in the most extreme cases barely recognizable features of the cell with no role in energy metabolism whatsoever (e.g., mitosomes, which generally only make iron-sulfur clusters). Recently, a wide diversity of MROs were discovered to be hiding in plain sight: in gregarine apicomplexans. This diverse group of invertebrate parasites has been known and observed for centuries, but until recent applications of culture-free genomics, their mitochondria were unremarkable. The genomics, however, showed that mitochondrial function has reduced in parallel in multiple gregarine lineages to several different endpoints, including the most reduced mitosomes. Here we review this remarkable case of parallel evolution of MROs, and some of the interesting questions this work raises.

Parallel evolution in an island archipelago revealed by genomic sequencing of Hipposideros leaf-nosed bats.

Body size is a key morphological attribute, often used to delimit species boundaries among closely related taxa. But body size can evolve in parallel, reaching similar final states despite independent evolutionary and geographic origins, leading to faulty assumptions of evolutionary history. Here, we document parallel evolution in body size in the widely distributed leaf-nosed bat genus Hipposideros, which has misled both taxonomic and evolutionary inference. We sequenced reduced representation genomic loci and measured external morphological characters from three closely related species from the Solomon Islands archipelago, delimited by body size. Species tree reconstruction confirms the paraphyly of two morphologically designated species. The nonsister relationship between large-bodied H. dinops lineages found on different islands indicates that large-bodied ecomorphs have evolved independently at least twice in the history of this radiation. A lack of evidence for gene flow between sympatric, closely related taxa suggests the rapid evolution of strong reproductive isolating barriers between morphologically distinct populations. Our results position Solomon Islands Hipposideros as a novel vertebrate system for studying the repeatability of parallel evolution under natural conditions. We conclude by offering testable hypotheses for how geography and ecology could be mediating the repeated evolution of large-bodied Hipposideros lineages in the Solomon Islands.

Parallel gene size and isoform expansion of ancient neuronal genes

How nervous systems evolved is a central question in biology. A diversity of synaptic proteins is thought to play a central role in the formation of specific synapses leading to nervous system complexity. The largest animal genes, often spanning hundreds of thousands of base pairs, are known to be enriched for expression in neurons at synapses and are frequently mutated or misregulated in neurological disorders and diseases. Although many of these genes have been studied independently in the context of nervous system evolution and disease, general principles underlying their parallel evolution remain unknown. To investigate this, we directly compared orthologous gene sizes across eukaryotes. By comparing relative gene sizes within organisms, we identified a distinct class of large genes with origins predating the diversification of animals and, in many cases, the emergence of neurons as dedicated cell types. We traced this class of ancient large genes through evolution and found orthologs of the large synaptic genes potentially driving the immense complexity of metazoan nervous systems, including in humans and cephalopods. Moreover, we found that while these genes are evolving under strong purifying selection, as demonstrated by low dN/dS ratios, they have simultaneously grown larger and gained the most isoforms in animals. This work provides a new lens through which to view this distinctive class of large and multi-isoform genes and demonstrates how intrinsic genomic properties, such as gene length, can provide flexibility in molecular evolution and allow groups of genes and their host organisms to evolve toward complexity.

Parallel evolution of gravity sensing.

Omnipresent gravity affects all living organisms; it was a vital factor in the past and the current bottleneck for future space exploration. However, little is known about the evolution of gravity sensing and the comparative biology of gravity reception. Here, by tracing the parallel evolution of gravity sensing, we encounter situations when assemblies of homologous modules result in the emergence of non-homologous structures with similar systemic properties. This is a perfect example to study homoplasy at all levels of biological organization. Apart from numerous practical implementations for bioengineering and astrobiology, the diversity of gravity signaling presents unique reference paradigms to understand hierarchical homology transitions to the convergent evolution of integrative systems. Second, by comparing gravisensory systems in major superclades of basal metazoans (ctenophores, sponges, placozoans, cnidarians, and bilaterians), we illuminate parallel evolution and alternative solutions implemented by basal metazoans toward spatial orientation, focusing on gravitational sensitivity and locomotory integrative systems.

Convergent and/or parallel evolution of RNA-binding proteins in angiosperms after polyploidization.

Increasing studies suggest that the biased retention of stress-related transcription factors (TFs) after whole-genome duplications (WGDs) could rewire gene transcriptional networks, facilitating plant adaptation to challenging environments. However, the role of posttranscriptional factors (e.g. RNA-binding proteins, RBPs) following WGDs has been largely ignored. Uncovering thousands of RBPs in 21 representative angiosperm species, we integrate genomic, transcriptomic, regulatomic, and paleotemperature datasets to unravel their evolutionary trajectories and roles in adapting to challenging environments. We reveal functional enrichments of RBP genes in stress responses and identify their convergent retention across diverse angiosperms from independent WGDs, coinciding with global cooling periods. Numerous RBP duplicates derived from WGDs are then identified as cold-induced. A significant overlap of 29 orthogroups between WGD-derived and cold-induced RBP genes across diverse angiosperms highlights a correlation between WGD and cold stress. Notably, we unveil an orthogroup (Glycine-rich RNA-binding Proteins 7/8, GRP7/8) and relevant TF duplicates (CCA1/LHY, RVE4/8, CBF2/4, etc.), co-retained in different angiosperms post-WGDs. Finally, we illustrate their roles in rewiring circadian and cold-regulatory networks at both transcriptional and posttranscriptional levels during global cooling. Altogether, we underline the adaptive evolution of RBPs in angiosperms after WGDs during global cooling, improving our understanding of plants surviving periods of environmental turmoil.

Blast Phase of Myeloproliferative Neoplasm Resembles Acute Myeloid Leukemia, Myelodysplasia-Related, in Clinical Presentation, Cytogenetic Pattern, and Genomic Profile, and Often Undergoes Reversion to Second Chronic Phase Status After Induction Chemotherapy.

BCR::ABL-negative myeloproliferative neoplasm (MPN) has a prolonged clinical course, and some cases eventually undergo transformation to blast phase; its pathogenesis remains to be elucidated. To evaluate the clinicopathologic characteristics of MPN in blast phase. The study aimed to retrospectively analyze the clinical and laboratory data of 24 cases. Median latency to blast phase was 48 months (range, 7-384 months). Complex karyotypes were seen in 12 of the 24 cases (50%). Overall, 16 cases (66.7%) exhibited high allele burdens of MPN driver mutations along with increased blasts, consistent with linear clonal evolution, whereas the remainder (8; 33.3%) showed loss or partial loss of the driver mutation suggestive of a parallel evolution. Additional mutations were noted in 23 cases (100%), including TP53 mutations in 10 of 24 cases (41.7%). Following chemotherapy, 15 of the 24 patients (62.5%) reverted to a second chronic phase while retaining or regaining MPN driver mutations and losing blast-related mutations, although 9 of the 15 patients (60%) later died of disease progression. Median overall survival was 10 months (CI, 4.6-15.4), with those harboring complex karyotypes demonstrating decreased survival (6 versus 29 months; P = .004). MPN-blast phase resembles acute myeloid leukemia, myelodysplasia-related, in cytogenetic pattern, mutation profile, and clinical outcome. Two patterns of clonal evolution are inferred by dynamic analysis of mutation profiles: linear and parallel evolutions. Although overall survival was dismal, 62.5% of our cases achieved second chronic phase, and they showed better survival than those without second chronic phase.

Parallel Multi-Qubit Evolution in the Quantum Non-Demolition Interaction Protocol

Parallel Multi-Qubit Evolution in the Quantum Non-Demolition Interaction Protocol

Genomic underpinnings of head and body shape in Arctic charr ecomorph pairs.

Across its Holarctic range, Arctic charr (Salvelinus alpinus) populations have diverged into distinct trophic specialists across independent replicate lakes. The major aspect of divergence between ecomorphs is in head shape and body shape, which are ecomorphological traits reflecting niche use. However, whether the genomic underpinnings of these parallel divergences are consistent across replicates was unknown but key for resolving the substrate of parallel evolution. We investigated the genomic basis of head shape and body shape morphology across four benthivore-planktivore ecomorph pairs of Arctic charr in Scotland. Through genome-wide association analyses, we found genomic regions associated with head shape (89 SNPs) or body shape (180 SNPs) separately and 50 of these SNPs were strongly associated with both body and head shape morphology. For each trait separately, only a small number of SNPs were shared across all ecomorph pairs (3 SNPs for head shape and 10 SNPs for body shape). Signs of selection on the associated genomic regions varied across pairs, consistent with evolutionary demography differing considerably across lakes. Using a comprehensive database of salmonid QTLs newly augmented and mapped to a charr genome, we found several of the head- and body-shape-associated SNPs were within or near morphology QTLs from other salmonid species, reflecting a shared genetic basis for these phenotypes across species. Overall, our results demonstrate how parallel ecotype divergences can have both population-specific and deeply shared genomic underpinnings across replicates, influenced by differences in their environments and demographic histories.

New reports on the association between eggs and sclerotium-forming fungi in Neotropical termites with insights into this mutualistic interaction

Abstract Over many years of co-evolution, termites and fungi have developed varied interactions. Sclerotia, termed termite balls, are fungal resting structures often mixed with termite eggs, with many reports in temperate areas. Here, we show that this interaction is more widespread than suggested, by reporting the occurrence of fungal sclerotia harboured in the nests of four Neotropical termitids: Cornitermes cumulans, Silvestritermes euamignathus (Syntermitinae), Velocitermes heteropterus, and Nasutitermes corniger (Nasutitermitinae). We performed termite–fungus recognition bioassays and carried out morphometrical, histological, and histochemical analyses on the sclerotia, assuming that they vary in their stored materials according to the maturation stage. Morphometrical and morphological features varied among the sclerotia associated with the different termite species. Moreover, workers preferentially carried related sclerotia, suggesting a species-specific interaction, and did not mistake them for eggs. Immature and mature sclerotia varied in the concentration of stored proteins, polysaccharides, and melanin. This facultative mutualism is advantageous for the sclerotia because they are kept in a competitor-free environment and, in turn, are likely to serve as an additional source of termite digestive enzymes. We provide new findings concerning the geographical distribution, termite species, and nesting habits related to this termite–fungus association, reinforcing its parallel evolution.

Dissecting the genetic architecture of quantitative traits using genome-wide identity-by-descent sharing.

Additive and dominance genetic variances underlying the expression of quantitative traits are important quantities for predicting short-term responses to selection, but they are notoriously challenging to estimate in most non-model wild populations. Specifically, large-sized or panmictic populations may be characterized by low variance in genetic relatedness among individuals which, in turn, can prevent accurate estimation of quantitative genetic parameters. We used estimates of genome-wide identity-by-descent (IBD) sharing from autosomal SNP loci to estimate quantitative genetic parameters for ecologically important traits in nine-spined sticklebacks (Pungitius pungitius) from a large, outbred population. Using empirical and simulated datasets, with varying sample sizes and pedigree complexity, we assessed the performance of different crossing schemes in estimating additive genetic variance and heritability for all traits. We found that low variance in relatedness characteristic of wild outbred populations with high migration rate can impair the estimation of quantitative genetic parameters and bias heritability estimates downwards. On the other hand, the use of a half-sib/full-sib design allowed precise estimation of genetic variance components and revealed significant additive variance and heritability for all measured traits, with negligible dominance contributions. Genome-partitioning and QTL mapping analyses revealed that most traits had a polygenic basis and were controlled by genes at multiple chromosomes. Furthermore, different QTL contributed to variation in the same traits in different populations suggesting heterogeneous underpinnings of parallel evolution at the phenotypic level. Our results provide important guidelines for future studies aimed at estimating adaptive potential in the wild, particularly for those conducted in outbred large-sized populations.

Porous borders at the wild-crop interface promote weed adaptation in Southeast Asia

High reproductive compatibility between crops and their wild relatives can provide benefits for crop breeding but also poses risks for agricultural weed evolution. Weedy rice is a feral relative of rice that infests paddies and causes severe crop losses worldwide. In regions of tropical Asia where the wild progenitor of rice occurs, weedy rice could be influenced by hybridization with the wild species. Genomic analysis of this phenomenon has been very limited. Here we use whole genome sequence analyses of 217 wild, weedy and cultivated rice samples to show that wild rice hybridization has contributed substantially to the evolution of Southeast Asian weedy rice, with some strains acquiring weed-adaptive traits through introgression from the wild progenitor. Our study highlights how adaptive introgression from wild species can contribute to agricultural weed evolution, and it provides a case study of parallel evolution of weediness in independently-evolved strains of a weedy crop relative.

Highly dynamic evolution of the chemosensory system driven by gene gain and loss across subterranean beetles

Chemical cues in subterranean habitats differ highly from those on the surface due to the contrasting environmental conditions, such as absolute darkness, high humidity or food scarcity. Subterranean animals underwent changes to their sensory systems to facilitate the perception of essential stimuli for underground lifestyles. Despite representing unique systems to understand biological adaptation, the genomic basis of chemosensation across cave-dwelling species remains unexplored from a macroevolutionary perspective. Here, we explore the evolution of chemoreception in three beetle tribes that underwent at least six independent transitions to the underground, through a phylogenomics spyglass. Our findings suggest that the chemosensory gene repertoire varies dramatically between species. Overall, no parallel changes in the net rate of evolution of chemosensory gene families were detected prior, during, or after the habitat shift among subterranean lineages. Contrarily, we found evidence of lineage-specific changes within surface and subterranean lineages. However, our results reveal key duplications and losses shared between some of the lineages transitioning to the underground, including the loss of sugar receptors and gene duplications of the highly conserved ionotropic receptors IR25a and IR8a, involved in thermal and humidity sensing among other olfactory roles in insects. These duplications were detected both in independent subterranean lineages and their surface relatives, suggesting parallel evolution of these genes across lineages giving rise to cave-dwelling species. Overall, our results shed light on the genomic basis of chemoreception in subterranean beetles and contribute to our understanding of the genomic underpinnings of adaptation to the subterranean lifestyle at a macroevolutionary scale.

Comparative anatomy of the Sapajus sp. (bearded capuchin) hand with comments on tool use in a parallel evolution with the hominid pathway.

Introduction: Bearded capuchins display a wide variety of manipulatory skills and make routine use of tools in both captivity and the wild. The efficient handling of objects in this genus has led several investigators to assume near-human thumb movements, despite a lack of anatomical studies. Methods: Here, we performed an anatomical analysis of muscles and bones in the capuchin hand. Sapajus morphological traits were quantitatively compared with those of humans, chimpanzees, gorillas, and baboons. Results: The comparative analysis indicated that the Sapajus hand is more similar to that of baboons and least similar to that of humans according to the muscles, bones, and three-dimensional data. Furthermore, these findings suggest that bearded capuchins lack true thumb opponency. Regarding manipulatory skills, they display rather primitive hand traits, with limited resources for precision grasping using the opponens pollicis. Discussion: These findings suggest that bearded capuchins' complex use of tools depends more heavily on their high cognitive abilities than on a versatile hand apparatus. These findings offer crucial insights into the evolution of primate cognition.

Pathway Evolution Through a Bottlenecking-Debottlenecking Strategy and Machine Learning-Aided Flux Balancing.

The evolution of pathway enzymes enhances the biosynthesis of high-value chemicals, crucial for pharmaceutical, and agrochemical applications. However, unpredictable evolutionary landscapes of pathway genes often hinder successful evolution. Here, the presence of complex epistasis is identifued within the representative naringenin biosynthetic pathway enzymes, hampering straightforward directed evolution. Subsequently, a biofoundry-assisted strategy is developed for pathway bottlenecking and debottlenecking, enabling the parallel evolution of all pathway enzymes along a predictable evolutionary trajectory in six weeks. This study then utilizes a machine learning model, ProEnsemble, to further balance the pathway by optimizing the transcription of individual genes. The broad applicability of this strategy is demonstrated by constructing an Escherichia coli chassis with evolved and balanced pathway genes, resulting in 3.65gL-1 naringenin. The optimized naringenin chassis also demonstrates enhanced production of other flavonoids. This approach can be readily adapted for any given number of enzymes in the specific metabolic pathway, paving the way for automated chassis construction in contemporary biofoundries.

Host association and intracellularity evolved multiple times independently in the Rickettsiales

The order Rickettsiales (Alphaproteobacteria) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. Here, in order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built a large and phylogenetically-balanced dataset that includes de novo sequenced genomes and a selection of published genomic and metagenomic assemblies. We perform detailed functional reconstructions that clearly indicates “late” and parallel evolution of obligate host-association in different Rickettsiales lineages. According to the depicted scenario, multiple independent horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion and/or invasion.