Giant Genome Research Articles

Telomere-to-telomere gapless genome assembly of the giant grouper (Epinephelus lanceolatus)

The giant grouper (Epinephelus lanceolatus) is a large coral reef fish distributed in the Indian Ocean and the Pacific Ocean. With a high market value, this species can grow up to approximately 2.7 meters in length and weigh 440 kilograms. With the rapid development of bioinformatics, higher standards of genome analysis are now required compared to previous reference genomes. This study presents a gapless assembly of the giant grouper genome, which has a length of 1.03 Gb. The sequences were assembled onto 24 chromosomes with a coverage of over 99% (1.02 Gb), and telomeres were detected on 24 chromosomes. Analysis using Merqury indicated a high level of accuracy, with an average consensus quality value of 59.24. The ONT ultralong and PacBio HiFi data were aligned with the assembly using minimap2, resulting in a mapping rate of 99.9%. The study inferred 25,815 protein-coding genes. These results lay a foundation for exploring the evolution and biology of the giant grouper, and advancing molecular breeding techniques.

Proteome-scale structural prediction of the giant Marseillevirus reveals conserved folds and putative homologs of the hypothetical proteins.

A significant proportion of the highly divergent and novel proteins of giant viruses are termed "hypothetical" due to the absence of detectable homologous sequences in the existing databases. The quality of genome and proteome annotations often relies on the identification of signature sequences and motifs in order to assign putative functions to the gene products. These annotations serve as the first set of information for researchers to develop workable hypotheses for further experimental research. The structure-function relationship of proteins suggests that proteins with similar functions may also exhibit similar folding patterns. Here, we report the first proteome-wide structure prediction of the giant Marseillevirus. We use AlphaFold-predicted structures and their comparative analysis with the experimental structures in the PDB database to preliminarily annotate the viral proteins. Our work highlights the conservation of structural folds in proteins with highly divergent sequences and reveals potentially paralogous relationships among them. We also provide evidence for gene duplication and fusion as contributing factors to giant viral genome expansion and evolution. With the easily accessible AlphaFold and other advanced bioinformatics tools for high-confidence de novo structure prediction, we propose a combined sequence and predicted-structure-based proteome annotation approach for the initial characterization of novel and complex organisms or viruses.

Exploring the Evolutionary History and Phylogenetic Relationships of Giant Reed (Arundo donax) through Comprehensive Analysis of Its Chloroplast Genome.

Giant reed (Arundo donax) is widely distributed across the globe and is considered an important energy crop. This study presents the first comprehensive analysis of the chloroplast genome of giant reed, revealing detailed characteristics of this species' chloroplast genome. The chloroplast genome has a total length of 137,153 bp, containing 84 protein-coding genes, 38 tRNA genes, and 8 rRNA genes, with a GC content of 39%. Functional analysis indicates that a total of 45 photosynthesis-related genes and 78 self-replication-related genes were identified, which may be closely associated with its adaptability and growth characteristics. Phylogenetic analysis confirmed that Arundo donax cv. Lvzhou No.1 belongs to the Arundionideae clade and occupies a distinct evolutionary position compared to other Arundo species. The findings of this study not only enhance our understanding of the giant reed genome but also provide valuable genetic resources for its application in biotechnology, bioenergy crop development, and ecological restoration.

Function and Structure of a Terpene Synthase Encoded in a Giant Virus Genome.

Giant viruses are nonstandard viruses with large particles and genomes. While previous studies have shown that their genomes contain various sequences of interest, their genes related specifically to natural product biosynthesis remain unexplored. Here we analyze the function and structure of a terpene synthase encoded by the gene of a giant virus. The enzyme is phylogenetically separated from the terpene synthases of cellular organisms; however, heterologous gene expression revealed that it still functions as a terpene synthase and produces a cyclic terpene from a farnesyl diphosphate precursor. Crystallographic analysis revealed its protein structure, which is relatively compact but retains essential motifs of the terpene synthases. We thus suggest that like cellular organisms, giant viruses produce and utilize natural products for their ecological strategies.

High-quality assembly and methylome of a Tibetan wild tree peony genome (Paeonia ludlowii) reveal the evolution of giant genome architecture.

Tree peony belongs to one of the Saxifragales families, Paeoniaceae. It is one of the most famous ornamental plants, and is also a promising woody oil plant. Although two Paeoniaceae genomes have been released, their assembly qualities are still to be improved. Additionally, more genomes from wild peonies are needed to accelerate genomic-assisted breeding. Here we assemble a high-quality and chromosome-scale 10.3-Gb genome of a wild Tibetan tree peony, Paeonia ludlowii, which features substantial sequence divergence, including around 75% specific sequences and gene-level differentials compared with other peony genomes. Our phylogenetic analyses suggest that Saxifragales and Vitales are sister taxa and, together with rosids, they are the sister taxon to asterids. The P. ludlowii genome is characterized by frequent chromosome reductions, centromere rearrangements, broadly distributed heterochromatin, and recent continuous bursts of transposable element (TE) movement in peony, although it lacks recent whole-genome duplication. These recent TE bursts appeared during the uplift and glacial period of the Qinghai-Tibet Plateau, perhaps contributing to adaptation to rapid climate changes. Further integrated analyses with methylome data revealed that genome expansion in peony might be dynamically affected by complex interactions among TE proliferation, TE removal, and DNA methylation silencing. Such interactions also impact numerous recently duplicated genes, particularly those related to oil biosynthesis and flower traits. This genome resource will not only provide the genomic basis for tree peony breeding but also shed light on the study of the evolution of huge genome structures as well as their protein-coding genes.

A scaffolded and annotated reference genome of giant kelp (Macrocystis pyrifera)

Macrocystis pyrifera (giant kelp), is a brown macroalga of great ecological importance as a primary producer and structure-forming foundational species that provides habitat for hundreds of species. It has many commercial uses (e.g. source of alginate, fertilizer, cosmetics, feedstock). One of the limitations to exploiting giant kelp’s economic potential and assisting in giant kelp conservation efforts is a lack of genomic tools like a high quality, contiguous reference genome with accurate gene annotations. Reference genomes attempt to capture the complete genomic sequence of an individual or species, and importantly provide a universal structure for comparison across a multitude of genetic experiments, both within and between species. We assembled the giant kelp genome of a haploid female gametophyte de novo using PacBio reads, then ordered contigs into chromosome level scaffolds using Hi-C. We found the giant kelp genome to be 537 MB, with a total of 35 scaffolds and 188 contigs. The assembly N50 is 13,669,674 with GC content of 50.37%. We assessed the genome completeness using BUSCO, and found giant kelp contained 94% of the BUSCO genes from the stramenopile clade. Annotation of the giant kelp genome revealed 25,919 genes. Additionally, we present genetic variation data based on 48 diploid giant kelp sporophytes from three different Southern California populations that confirms the population structure found in other studies of these populations. This work resulted in a high-quality giant kelp genome that greatly increases the genetic knowledge of this ecologically and economically vital species.

The Role of Chromatin Modifications in the Evolution of Giant Plant Genomes.

Angiosperm genome sizes (GS) range ~2400-fold and comprise genes and their regulatory regions, repeats, semi-degraded repeats, and 'dark matter'. The latter represents repeats so degraded that they can no longer be recognised as repetitive. In exploring whether the histone modifications associated with chromatin packaging of these contrasting genomic components are conserved across the diversity of GS in angiosperms, we compared immunocytochemistry data for two species whose GS differ ~286-fold. We compared published data for Arabidopsis thaliana with a small genome (GS = 157 Mbp/1C) with newly generated data from Fritillaria imperialis, which has a giant genome (GS = 45,000 Mbp/1C). We compared the distributions of the following histone marks: H3K4me1, H3K4me2, H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me2, and H3K27me3. Assuming these histone marks are associated with the same genomic features across all species, irrespective of GS, our comparative analysis enables us to suggest that while H3K4me1 and H3K4me2 methylation identifies genic DNA, H3K9me3 and H3K27me3 marks are associated with 'dark matter', H3K9me1 and H3K27me1 mark highly homogeneous repeats, and H3K9me2 and H3K27me2 mark semi-degraded repeats. The results have implications for our understanding of epigenetic profiles, chromatin packaging and the divergence of genomes, and highlight contrasting organizations of the chromatin within the nucleus depending on GS itself.

Giant Fern Genomes Show Complex Evolution Patterns: A Comparative Analysis in Two Species of Tmesipteris (Psilotaceae).

Giant genomes are rare across the plant kingdom and their study has focused almost exclusively on angiosperms and gymnosperms. The scarce genetic data that are available for ferns, however, indicate differences in their genome organization and a lower dynamism compared to other plant groups. Tmesipteris is a small genus of mainly epiphytic ferns that occur in Oceania and several Pacific Islands. So far, only two species with giant genomes have been reported in the genus, T. tannensis (1C = 73.19 Gbp) and T. obliqua (1C = 147.29 Gbp). Low-coverage genome skimming sequence data were generated in these two species and analyzed using the RepeatExplorer2 pipeline to identify and quantify the repetitive DNA fraction of these genomes. We found that both species share a similar genomic composition, with high repeat diversity compared to taxa with small (1C < 10 Gbp) genomes. We also found that, in general, characterized repetitive elements have relatively high heterogeneity scores, indicating ancient diverging evolutionary trajectories. Our results suggest that a whole genome multiplication event, accumulation of repetitive elements, and recent activation of those repeats have all played a role in shaping these genomes. It will be informative to compare these data in the future with data from the giant genome of the angiosperm Paris japonica, to determine if the structures observed here are an emergent property of massive genomic inflation or derived from lineage specific processes.

A giant virus genome is densely packaged by stable nucleosomes within virions

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

Genome of a giant isopod, Bathynomus jamesi, provides insights into body size evolution and adaptation to deep-sea environment

BackgroundThe deep-sea may be regarded as a hostile living environment, due to low temperature, high hydrostatic pressure, and limited food and light. Isopods, a species-rich group of crustaceans, are widely distributed across different environments including the deep sea and as such are a useful model for studying adaptation, migration, and speciation. Similar to other deep-sea organisms, giant isopods have larger body size than their shallow water relatives and have large stomachs and fat bodies presumably to store organic reserves. In order to shed light on the genetic basis of these large crustaceans adapting to the oligotrophic environment of deep-sea, the high-quality genome of a deep-sea giant isopod Bathynomus jamesi was sequenced and assembled.ResultsB. jamesi has a large genome of 5.89 Gb, representing the largest sequenced crustacean genome to date. Its large genome size is mainly attributable to the remarkable proliferation of transposable elements (84%), which may enable high genome plasticity for adaptive evolution. Unlike its relatives with small body size, B. jamesi has expanded gene families related to pathways of thyroid and insulin hormone signaling that potentially contribute to its large body size. Transcriptomic analysis showed that some expanded gene families related to glycolysis and vesicular transport were specifically expressed in its digestive organs. In addition, comparative genomics and gene expression analyses in six tissues suggested that B. jamesi has inefficient lipid degradation, low basal metabolic rate, and bulk food storage, suggesting giant isopods adopt a more efficient mechanism of nutrient absorption, storage, and utilization to provide sustained energy supply for their large body size.ConclusionsTaken together, the giant isopod genome may provide a valuable resource for understanding body size evolution and adaptation mechanisms of macrobenthic organisms to deep-sea environments.

Chinese pine (Pinus tabuliformis Carr.)

Pinus tabuliformis, commonly called Chinese pine, is a drought- and cold-tolerant heliophilous, indigenous conifer species, widespread in China. It has a giant genome comprising 25.4 Gb, with 12 huge chromosomes. The recently released nearly complete genome of P. tabuliformis provides new perspectives on genome expansion and adaptative and reproductive evolution of conifers. With an expansion of the genome, a multitude of long introns were observed in the P. tabuliformis genome. Interestingly, large genes with ultra-long introns tend to be expressed at higher levels, which suggests that intron length may serve as an evolutionary force in gymnosperms, though the underlying molecular mechanism is still unknown.

Effective prediction of biosynthetic pathway genes involved in bioactive polyphyllins in Paris polyphylla

The genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.

The long road to bloom in conifers.

More than 600 species of conifers (phylum Pinophyta) serve as the backbone of the Earth's terrestrial plant community and play key roles in global carbon and water cycles. Although coniferous forests account for a large fraction of global wood production, their productivity relies largely on the use of genetically improved seeds. However, acquisition of such seeds requires recurrent selection and testing of genetically superior parent trees, eventually followed by the establishment of a seed orchard to produce the improved seeds. The breeding cycle for obtaining the next generation of genetically improved seeds can be significantly lengthened when a target species has a long juvenile period. Therefore, development of methods for diminishing the juvenile phase is a cost-effective strategy for shortening breeding cycle in conifers. The molecular regulatory programs associated with the reproductive transition and annual reproductive cycle of conifers are modulated by environmental cues and endogenous developmental signals. Mounting evidence indicates that an increase in global average temperature seriously threatens plant productivity, but how conifers respond to the ever-changing natural environment has yet to be fully characterized. With the breakthrough of assembling and annotating the giant genome of conifers, identification of key components in the regulatory cascades that control the vegetative to reproductive transition is imminent. However, comparison of the signaling pathways that control the reproductive transition in conifers and the floral transition in Arabidopsis has revealed many differences. Therefore, a more complete understanding of the underlying regulatory mechanisms that control the conifer reproductive transition is of paramount importance. Here, we review our current understanding of the molecular basis for reproductive regulation, highlight recent discoveries, and review new approaches for molecular research on conifers.

Modern Approaches to Molecular Genetics of Viruses in the Study of the Members of the Family Coronaviridae

The existence of certain suspicions about the artificial origin of the COVID-19 pandemic and about the possible use of reverse genetics technology to create the SARS-CoV-2 virus require an understanding of its capabilities in the design of new viruses. The aim of this work is to show how the use of reverse genetics allows the design of previously non-existent coronaviruses, technologies and the main achievements in their creation. Only the information in the public domain was used for the preparation of this article. The technology is called «reverse genetics» because when obtaining RNA viruses capable of replication, the process is going not from DNA to RNA, as usual, but on the contrary, from the RNA of the virus to its complementary DNA (cDNA), and from it with the help of T7 RNA polymerase – «back» to the infectious RNA. Since the resulting plus-RNA of the coronavirus genome mimics cellular messenger RNA (mRNA), it is immediately recognized by the cell's translation machine and triggers the formation of its own infectious viral particles. Two systems of reverse genetics have been developed, involving the production of an infectious plusRNA, in vitro and in vivo. The problem of obtaining a full-length cDNA of the giant genome of coronaviruses is solved by fragmentation and subsequent stitching of fragments using standard molecular biology approaches. The article provides the examples of how this technology makes it possible to obtain synthetic coronaviruses that are indistinguishable from those isolated from nature, to change the range of their hosts, to enhance virulence and resistance to specific antibodies, and to influence the pathogenesis of the disease. The article also shows the prospects for the use of recombinant viruses in cellular screening analyses and infection models in vivo for the identification of preventive and therapeutic approaches to the virus disease treatment

Investigation of the activity of transposable elements and genes involved in their silencing in the newt Cynops orientalis, a species with a giant genome

Caudata is an order of amphibians with great variation in genome size, which can reach enormous dimensions in salamanders. In this work, we analysed the activity of transposable elements (TEs) in the transcriptomes obtained from female and male gonads of the Chinese fire-bellied newt, Cynops orientalis, a species with a genome about 12-fold larger than the human genome. We also compared these data with genomes of two basal sarcopterygians, coelacanth and lungfish. In the newt our findings highlighted a major impact of non-LTR retroelements and a greater total TE activity compared to the lungfish Protopterus annectens, an organism also characterized by a giant genome. This difference in TE activity might be due to the presence of young copies in newt in agreement also with the increase in the genome size, an event that occurred independently and later than lungfish. Moreover, the activity of 33 target genes encoding proteins involved in the TE host silencing mechanisms, such as Ago/Piwi and NuRD complex, was evaluated and compared between the three species analysed. These data revealed high transcriptional levels of the target genes in both newt and lungfish and confirmed the activity of NuRD complex genes in adults. Finally, phylogenetic analyses performed on PRDM9 and TRIM28 allowed increasing knowledge about the evolution of these two key genes of the NuRD complex silencing mechanism in vertebrates. Our results confirmed that the gigantism of the newt genomes may be attributed to the activity and accumulation of TEs.

Modern Approaches to Molecular Genetics of Viruses in the Study of the Members of the Family Coronaviridae

The existence of certain suspicions about the artificial origin of the COVID-19 pandemic and about the possible use of reverse genetics technology to create the SARS-CoV-2 virus require an understanding of its capabilities in the design of new viruses. The aim of this work is to show how the use of reverse genetics allows the design of previously non-existent coronaviruses, technologies and the main achievements in their creation. Only the information in the public domain was used for the preparation of this article. The technology is called «reverse genetics» because when obtaining RNA viruses capable of replication, the process is going not from DNA to RNA, as usual, but on the contrary, from the RNA of the virus to its complementary DNA (cDNA), and from it with the help of T7 RNA polymerase – «back» to the infectious RNA. Since the resulting plus-RNA of the coronavirus genome mimics cellular messenger RNA (mRNA), it is immediately recognized by the cell's translation machine and triggers the formation of its own infectious viral particles. Two systems of reverse genetics have been developed, involving the production of an infectious plusRNA, in vitro and in vivo. The problem of obtaining a full-length cDNA of the giant genome of coronaviruses is solved by fragmentation and subsequent stitching of fragments using standard molecular biology approaches. The article provides the examples of how this technology makes it possible to obtain synthetic coronaviruses that are indistinguishable from those isolated from nature, to change the range of their hosts, to enhance virulence and resistance to specific antibodies, and to influence the pathogenesis of the disease. The article also shows the prospects for the use of recombinant viruses in cellular screening analyses and infection models in vivo for the identification of preventive and therapeutic approaches to the virus disease treatment.

The giant axolotl genome uncovers the evolution, scaling, and transcriptional control of complex gene loci

Vertebrates harbor recognizably orthologous gene complements but vary 100-fold in genome size. How chromosomal organization scales with genome expansion is unclear, and how acute changes in gene regulation, as during axolotl limb regeneration, occur in the context of a vast genome has remained a riddle. Here, we describe the chromosome-scale assembly of the giant, 32 Gb axolotl genome. Hi-C contact data revealed the scaling properties of interphase and mitotic chromosome organization. Analysis of the assembly yielded understanding of the evolution of large, syntenic multigene clusters, including the Major Histocompatibility Complex (MHC) and the functional regulatory landscape of the Fibroblast Growth Factor 8 (Axfgf8) region. The axolotl serves as a primary model for studying successful regeneration.

Gigantic Genomes Provide Empirical Tests of Transposable Element Dynamics Models

Transposable elements (TEs) are a major determinant of eukaryotic genome size. The collective properties of a genomic TE community reveal the history of TE/host evolutionary dynamics and impact present-day host structure and function, from genome to organism levels. In rare cases, TE community/genome size has greatly expanded in animals, associated with increased cell size and changes to anatomy and physiology. Here, we characterize the TE landscape of the genome and transcriptome in an amphibian with a giant genome — the caecilianIchthyophis bannanicus, which we show has a genome size of 12.2 Gb. Amphibians are an important model system because the clade includes independent cases of genomic gigantism. The I. bannanicus genome differs compositionally from other giant amphibian genomes, but shares a low rate of ectopic recombination-mediated deletion. We examine TE activity using expression and divergence plots; TEs account for 15% of somatic transcription, and most superfamilies appear active. We quantify TE diversity in the caecilian, as well as other vertebrates with a range of genome sizes, using diversity indices commonly applied in community ecology. We synthesize previous models that integrate TE abundance, diversity, and activity, and test whether the caecilian meets model predictions for genomes with high TE abundance. We propose thorough, consistent characterization of TEs to strengthen future comparative analyses. Such analyses will ultimately be required to reveal whether the divergent TE assemblages found across convergent gigantic genomes reflect fundamental shared features of TE/host genome evolutionary dynamics.

Giant lungfish genome elucidates the conquest of land by vertebrates

Lungfishes belong to lobe-fined fish (Sarcopterygii) that, in the Devonian period, ‘conquered’ the land and ultimately gave rise to all land vertebrates, including humans1–3. Here we determine the chromosome-quality genome of the Australian lungfish (Neoceratodus forsteri), which is known to have the largest genome of any animal. The vast size of this genome, which is about 14× larger than that of humans, is attributable mostly to huge intergenic regions and introns with high repeat content (around 90%), the components of which resemble those of tetrapods (comprising mainly long interspersed nuclear elements) more than they do those of ray-finned fish. The lungfish genome continues to expand independently (its transposable elements are still active), through mechanisms different to those of the enormous genomes of salamanders. The 17 fully assembled lungfish macrochromosomes maintain synteny to other vertebrate chromosomes, and all microchromosomes maintain conserved ancient homology with the ancestral vertebrate karyotype. Our phylogenomic analyses confirm previous reports that lungfish occupy a key evolutionary position as the closest living relatives to tetrapods4,5, underscoring the importance of lungfish for understanding innovations associated with terrestrialization. Lungfish preadaptations to living on land include the gain of limb-like expression in developmental genes such as hoxc13 and sall1 in their lobed fins. Increased rates of evolution and the duplication of genes associated with obligate air-breathing, such as lung surfactants and the expansion of odorant receptor gene families (which encode proteins involved in detecting airborne odours), contribute to the tetrapod-like biology of lungfishes. These findings advance our understanding of this major transition during vertebrate evolution.

Mind thenumt: Finding informative mitochondrial markers in a giant grasshopper genome

The barcoding of the mitochondrial COX1 gene has been instrumental in cataloguing the tree of life, and in providing insights in the phylogeographic history of species. Yet, this strategy has encountered difficulties in major clades characterized by large genomes, which contain a high frequency of nuclear pseudogenes originating from the mitochondrial genome (numts). Here, we use the meadow grasshopper (Chorthippus parallelus), which possesses a giant genome of ~13 Gb, to identify mitochondrial genes that are underrepresented as numts, and test their use as informative phylogeographic markers. We recover the same full mitochondrial sequence using both whole genome and transcriptome sequencing, including functional protein-coding genes and tRNAs. We show that a region of the mitogenome containing the COX1 gene, typically used in DNA barcoding, has disproportionally higher diversity and coverage than the rest of the mitogenome, consistent with multiple insertions of that region into the nuclear genome. By designing new markers in regions of less elevated diversity and coverage, we identify two mitochondrial genes that are less likely to be duplicated as numts. We show that, while these markers show high levels of incomplete lineage sorting between subspecies, as expected for mitochondrial genes, genetic variation reflects their phylogeographic history accurately. These findings allow us to identify useful mitochondrial markers for future studies in C. parallelus, an important biological system for evolutionary biology. More generally, this study exemplifies how non-PCR-based methods using next-generation sequencing can be used to avoid numts in species characterized by large genomes, which have remained challenging to study in taxonomy and evolution.