Dynamic evolution of the mTHF gene family associated with primary metabolism across life.

BACKGROUND: The folate cycle of one-carbon (C1) metabolism, which plays a central role in the biosynthesis of nucleotides and amino acids, demonstrates the significance of metabolic adaptation. We investigated the evolutionary history of the methylenetetrahydrofolate dehydrogenase (mTHF) gene family, one of the main drivers of the folate cycle, across life. RESULTS: Through comparative genomic and phylogenetic analyses, we found that several lineages of Archaea lacked domains vital for folate cycle function such as the mTHF catalytic and NAD(P)-binding domains of FolD. Within eukaryotes, the mTHF gene family diversified rapidly. For example, several duplications have been observed in lineages including the Amoebozoa, Opisthokonta, and Viridiplantae. In a common ancestor of Opisthokonta, FolD and FTHFS underwent fusion giving rise to the gene MTHFD1, possessing the domains of both genes. CONCLUSIONS: Our evolutionary reconstruction of the mTHF gene family associated with a primary metabolic pathway reveals dynamic evolution, including gene birth-and-death, gene fusion, and potential horizontal gene transfer events and/or amino acid convergence.

Subgenome dominance shapes novel gene evolution in the decaploid pitcher plant Nepenthes gracilis.

Subgenome dominance after whole-genome duplication generates distinction in gene number and expression at the level of chromosome sets, but it remains unclear how this process may be involved in evolutionary novelty. Here we generated a chromosome-scale genome assembly of the Asian pitcher plant Nepenthes gracilis to analyse how its novel traits (dioecy and carnivorous pitcher leaves) are linked to genomic evolution. We found a decaploid karyotype and a clear indication of subgenome dominance. A male-linked and pericentromerically located region on the putative sex chromosome was identified in a recessive subgenome and was found to harbour three transcription factors involved in flower and pollen development, including a likely neofunctionalized LEAFY duplicate. Transcriptomic and syntenic analyses of carnivory-related genes suggested that the paleopolyploidization events seeded genes that subsequently formed tandem clusters in recessive subgenomes with specific expression in the digestive zone of the pitcher, where specialized cells digest prey and absorb derived nutrients. A genome-scale analysis suggested that subgenome dominance likely contributed to evolutionary innovation by permitting recessive subgenomes to diversify functions of novel tissue-specific duplicates. Our results provide insight into how polyploidy can give rise to novel traits in divergent and successful high-ploidy lineages.

Genomic insights into the evolution of plant chemical defense.

Plant trait evolution can be impacted by common mechanisms of genome evolution, including whole-genome and small-scale duplication, rearrangement, and selective pressures. With the increasing accessibility of genome sequencing for non-model species, comparative studies of trait evolution among closely related or divergent lineages have supported investigations into plant chemical defense. Plant defensive compounds include major chemical classes, such as terpenoids, alkaloids, and phenolics, and are used in primary and secondary plant functions. These include the promotion of plant health, facilitation of pollination, defense against pathogens, and responses to a rapidly changing climate. We discuss mechanisms of genome evolution and use examples from recent studies to impress a stronger understanding of the link between genotype and phenotype as it relates to the evolution of plant chemical defense. We conclude with considerations for how to leverage genomics, transcriptomics, metabolomics, and functional assays for studying the emergence and evolution of chemical defense systems.

The digestive systems of carnivorous plants.

To survive in the nutrient-poor habitats, carnivorous plants capture small organisms comprising complex substances not suitable for immediate reuse. The traps of carnivorous plants, which are analogous to the digestive systems of animals, are equipped with mechanisms for the breakdown and absorption of nutrients. Such capabilities have been acquired convergently over the past tens of millions of years in multiple angiosperm lineages by modifying plant-specific organs including leaves. The epidermis of carnivorous trap leaves bears groups of specialized cells called glands, which acquire substances from their prey via digestion and absorption. The digestive glands of carnivorous plants secrete mucilage, pitcher fluids, acids, and proteins, including digestive enzymes. The same (or morphologically distinct) glands then absorb the released compounds via various membrane transport proteins or endocytosis. Thus, these glands function in a manner similar to animal cells that are physiologically important in the digestive system, such as the parietal cells of the stomach and intestinal epithelial cells. Yet, carnivorous plants are equipped with strategies that deal with or incorporate plant-specific features, such as cell walls, epidermal cuticles, and phytohormones. In this review, we provide a systematic perspective on the digestive and absorptive capacity of convergently evolved carnivorous plants, with an emphasis on the forms and functions of glands.

Acid or base? How do plants regulate the ecology of their phylloplane?

Plants interface with and modify the external environment across their surfaces, and in so doing, can control or mitigate the impacts of abiotic stresses and also mediate their interactions with other organisms. Botanically, it is known that plant roots have a multi-faceted ability to modify rhizosphere conditions like pH, a factor with a large effect on a plant's biotic interactions with microbes. But plants can also modify pH levels on the surfaces of their leaves. Plants can neutralize acid rain inputs in a period of hours, and either acidify or alkalinize the pH of neutral water droplets in minutes. The pH of the phylloplane-that is, the outermost surface of the leaf-varies across species, from incredibly acidic (carnivorous plants: as low as pH 1) to exceptionally alkaline (species in the plant family, Malvaceae, up to pH 11). However, most species mildly acidify droplets on the phylloplane by 1.5 orders of magnitude in pH. Just as rhizosphere pH helps shape the plant microbiome and is known to influence belowground interactions, so too can phylloplane pH influence aboveground interactions in plant canopies. In this review, we discuss phylloplane pH regulation from the physiological, molecular, evolutionary, and ecological perspectives and address knowledge gaps and identify future research directions.

Primary Metabolism co-Opted for Defensive Chemical Production in the Carabid Beetle, Harpalus pensylvanicus.

Of the approximately one million described insect species, ground beetles (Coleoptera: Carabidae) have long captivated the attention of evolutionary biologists due to the diversity of defensive compounds they synthesize. Produced using defensive glands in the abdomen, ground beetle chemicals represent over 250 compounds including predator-deterring formic acid, which has evolved as a defensive strategy at least three times across Insecta. Despite being a widespread method of defense, formic acid biosynthesis is poorly understood in insects. Previous studies have suggested that the folate cycle of one-carbon (C1) metabolism, a pathway involved in nucleotide biosynthesis, may play a key role in defensive-grade formic acid production in ants. Here, we report on the defensive gland transcriptome of the formic acid-producing ground beetle Harpalus pensylvanicus. The full suite of genes involved in the folate cycle of C1 metabolism are significantly differentially expressed in the defensive glands of H. pensylvanicus when compared to gene expression profiles in the rest of the body. We also find support for two additional pathways potentially involved in the biosynthesis of defensive-grade formic acid, the kynurenine pathway and the methionine salvage cycle. Additionally, we have found an array of differentially expressed genes in the secretory lobes involved in the biosynthesis and transport of cofactors necessary for formic acid biosynthesis, as well as genes presumably involved in the detoxification of secondary metabolites including formic acid. We also provide insight into the evolution of the predominant gene family involved in the folate cycle (MTHFD) and suggest that high expression of folate cycle genes rather than gene duplication and/or neofunctionalization may be more important for defensive-grade formic acid biosynthesis in H. pensylvanicus. This provides the first evidence in Coleoptera and one of a few examples in Insecta of a primary metabolic process being co-opted for defensive chemical biosynthesis. Our results shed light on potential mechanisms of formic acid biosynthesis in the defensive glands of a ground beetle and provide a foundation for further studies into the evolution of formic acid-based chemical defense strategies in insects.

The Evolution of the Mammalian ABCA6-like Genes: Analysis of Phylogenetic, Expression, and Population Genetic Data Reveals Complex Evolutionary Histories.

ABC membrane transporters are a large and complex superfamily of ATP-binding cassette transporters that are present in all domains of life. Both their essential function and complexity are reflected by their retention across large expanses of organismal diversity and by the extensive expansion of individual members and subfamilies during evolutionary history. This expansion has resulted in the diverse ABCA transporter family that has in turn evolved into multiple subfamilies. Here, we focus on the ABCA6-like subfamily of ABCA transporters with the goal of understanding their evolutionary history including potential functional changes in, or loss of, individual members. Our analysis finds that ABCA6-like genes, consisting of ABCA6, 8, 9, and 10, are absent from representatives of both monotremes and marsupials and thus the duplications that generated these families most likely occurred at the base of the Eutherian or placental mammals. We have found evidence of both positive and relaxed selection among the ABCA6-like genes, suggesting dynamic changes in function and the potential of gene redundancy. Analysis of the ABCA10 genes further suggests that this gene has undergone relaxed selection only within the human lineage. These findings are complemented by human population data, where we observe an excess of deactivating homozygous mutations. We describe the complex evolutionary history of this ABCA transporter subfamily and demonstrate through the combination of evolutionary and population genetic analysis that ABCA10 is undergoing pseudogenization within humans.

Pygidial glands of Harpalus pensylvanicus (Coleoptera: Carabidae) contain resilin-rich structures.

The pygidial gland system is a key innovation in adephagan beetles, producing, storing, and spraying defensive chemical compounds. As the source of defensive chemical production and storage, the pygidial gland system experiences severe chemical stress which challenges the integrity of the entire gland system. Here, we utilize autofluorescence-based confocal laser scanning microscopy to examine the morphology of pygidial gland secretory lobes and collecting ductules in a common Pennsylvanian harpaline species, Harpalus pensylvanicus. The glandular units are composed of type-III exocrine cells which empty into resilin-rich ductules, which themselves lead into a larger resilin-rich collecting duct, and ultimately the pygidial reservoir pump. We also utilize histological staining with toluidine blue and brightfield imaging to provide additional support for the presence of resilin in the collecting duct, as toluidine blue has been shown to stain resilin without metachromasia. We hypothesize that the high resilin content of the collecting ducts might be a widespread key evolutionary adaptation to prevent damage caused by physical and chemical stress generated in pump-containing insect exocrine gland systems.

Carabidae Semiochemistry: Current and Future Directions.

Ground beetles (Carabidae) are recognized for their diverse, chemically-mediated defensive behaviors. Produced using a pair of pygidial glands, over 250 chemical constituents have been characterized across the family thus far, many of which are considered allomones. Over the past century, our knowledge of Carabidae exocrine chemistry has increased substantially, yet the role of these defensive compounds in mediating behavior other than repelling predators is largely unknown. It is also unclear whether non-defensive compounds produced by ground beetles mediate conspecific and heterospecific interactions, such as sex-aggregation pheromones or kairomones, respectively. Here we review the current state of non-exocrine Carabidae semiochemistry and behavioral research, discuss the importance of semiochemical research including but not limited to allomones, and describe next-generation methods for elucidating the underlying genetics and evolution of chemically-mediated behavior.

Patterns of alternative splicing vary between species during heat stress.

Plants have evolved a variety of mechanisms to respond and adapt to abiotic stress. High temperature stress induces the heat shock response. During the heat shock response a large number of genes are up-regulated, many of which code for chaperone proteins that prevent irreversible protein aggregation and cell death. However, it is clear that heat shock is not the only mechanism involved in the plant heat stress response. Alternative splicing (AS) is also important during heat stress since this post-transcriptional regulatory mechanism can produce significant transcriptome and proteome variation. In this study, we examine AS during heat stress in the model species Arabidopsis thaliana and in the highly thermotolerant native California mustard Boechera depauperata. Analyses of AS during heat stress revealed that while a significant number of genes undergo AS and are differentially expressed (DE) during heat stress, some undergo both AS and DE. Analysis of the functional categories of genes undergoing AS indicated that enrichment patterns are different in the two species. Categories enriched in B. depauperata included light response genes and numerous abiotic stress response genes. Categories enriched in A. thaliana, but not in B. depauperata, included RNA processing and nucleotide binding. We conclude that AS and DE are largely independent responses to heat stress. Furthermore, this study reveals significant differences in the AS response to heat stress in the two related mustard species. This indicates AS responses to heat stress are species-specific. Future studies will explore the role of AS of specific genes in organismal thermotolerance.

Long-read sequencing uncovers the adaptive topography of a carnivorous plant genome.

Utricularia gibba, the humped bladderwort, is a carnivorous plant that retains a tiny nuclear genome despite at least two rounds of whole genome duplication (WGD) since common ancestry with grapevine and other species. We used a third-generation genome assembly with several complete chromosomes to reconstruct the two most recent lineage-specific ancestral genomes that led to the modern U. gibba genome structure. Patterns of subgenome dominance in the most recent WGD, both architectural and transcriptional, are suggestive of allopolyploidization, which may have generated genomic novelty and led to instantaneous speciation. Syntenic duplicates retained in polyploid blocks are enriched for transcription factor functions, whereas gene copies derived from ongoing tandem duplication events are enriched in metabolic functions potentially important for a carnivorous plant. Among these are tandem arrays of cysteine protease genes with trap-specific expression that evolved within a protein family known to be useful in the digestion of animal prey. Further enriched functions among tandem duplicates (also with trap-enhanced expression) include peptide transport (intercellular movement of broken-down prey proteins), ATPase activities (bladder-trap acidification and transmembrane nutrient transport), hydrolase and chitinase activities (breakdown of prey polysaccharides), and cell-wall dynamic components possibly associated with active bladder movements. Whereas independently polyploid Arabidopsis syntenic gene duplicates are similarly enriched for transcriptional regulatory activities, Arabidopsis tandems are distinct from those of U. gibba, while still metabolic and likely reflecting unique adaptations of that species. Taken together, these findings highlight the special importance of tandem duplications in the adaptive landscapes of a carnivorous plant genome.

Molecular characterization and evolution of carnivorous sundew (Drosera rotundifolia L.) class V ß-1,3-glucanase.

MAIN CONCLUSION: A gene for ß-1,3-glucanase was isolated from carnivorous sundew. It is active in leaves and roots, but not in digestive glands. Analyses in transgenic tobacco suggest its function in germination. Ancestral plant ß-1,3-glucanases (EC 3.2.1.39) played a role in cell division and cell wall remodelling, but divergent evolution has extended their roles in plant defense against stresses to decomposition of prey in carnivorous plants. As available gene sequences from carnivorous plants are rare, we isolated a glucanase gene from roundleaf sundew (Drosera rotundifolia L.) by a genome walking approach. Computational predictions recognized typical gene features and protein motifs described for other plant ß-1,3-glucanases. Phylogenetic reconstructions suggest strong support for evolutionary relatedness to class V ß-1,3-glucanases, including homologs that are active in the traps of related carnivorous species. The gene is expressed in sundew vegetative tissues but not in flowers and digestive glands, and encodes for a functional enzyme when expressed in transgenic tobacco. Detailed analyses of the supposed promoter both in silico and in transgenic tobacco suggest that this glucanase plays a role in development. Specific spatiotemporal activity was observed during transgenic seed germination. Later during growth, the sundew promoter was active in marginal and sub-marginal areas of apical true leaf meristems of young tobacco plants. These results suggest that the isolated glucanase gene is regulated endogenously, possibly by auxin. This is the first report on a nuclear gene study from sundew.

Independent Co-Option of a Tailed Bacteriophage into a Killing Complex in Pseudomonas.

UNLABELLED: Competition between microbes is widespread in nature, especially among those that are closely related. To combat competitors, bacteria have evolved numerous protein-based systems (bacteriocins) that kill strains closely related to the producer. In characterizing the bacteriocin complement and killing spectra for the model strain Pseudomonas syringae B728a, we discovered that its activity was not linked to any predicted bacteriocin but is derived from a prophage. Instead of encoding an active prophage, this region encodes a bacteriophage-derived bacteriocin, termed an R-type syringacin. This R-type syringacin is striking in its convergence with the well-studied R-type pyocin of P. aeruginosa in both genomic location and molecular function. Genomic alignment, amino acid percent sequence identity, and phylogenetic inference all support a scenario where the R-type syringacin has been co-opted independently of the R-type pyocin. Moreover, the presence of this region is conserved among several other Pseudomonas species and thus is likely important for intermicrobial interactions throughout this important genus. IMPORTANCE: Evolutionary innovation is often achieved through modification of complexes or processes for alternate purposes, termed co-option. Notable examples include the co-option of a structure functioning in locomotion (bacterial flagellum) to one functioning in protein secretion (type three secretion system). Similar co-options can occur independently in distinct lineages. We discovered a genomic region in the plant pathogen Pseudomonas syringae that consists of a fragment of a bacteriophage genome. The fragment encodes only the tail of the bacteriophage, which is lethal toward strains of this species. This structure is similar to a previously described structure produced by the related species Pseudomonas aeruginosa. The two structures, however, are not derived from the same evolutionary event. Thus, they represent independent bacteriophage co-options. The co-opted bacteriophage from P. syringae is found in the genomes of many other Pseudomonas species, suggesting ecological importance across this genus.

Spiraling into History: A Molecular Phylogeny and Investigation of Biogeographic Origins and Floral Evolution for the Genus Costus.

Rapid radiations are notoriously difficult to resolve, yet understanding phylogenetic patterns in such lineages can be useful for investigating evolutionary processes associated with bursts of speciation and morphological diversification. Here we present an expansive molecular phylogeny of Costus L. (Costaceae Nakai) with a focus on the Neotropical species within the clade, sampling 47 of the known 51 Neotropical species and including five molecular markers for phylogenetic analysis (ITS, ETS, rps16, trnL-F, and CaM). We use the phylogenetic results to investigate shifts in pollination syndrome, with the intention of addressing potential mechanisms leading to the rapid radiation documented for this clade. Our ancestral reconstruction of pollination syndrome presents the first evidence in this genus of an evolutionary toggle in pollination morphologies, demonstrating both the multiple independent evolutions of ornithophily (bird pollination) as well as reversals to melittophily (bee pollination). We show that the ornithophilous morphology has evolved at least eight times independently with four potential reversals to melittophilous morphology, and confirm prior work showing that neither pollination syndrome defines a monophyletic lineage. Based on the current distribution for the Neotropical and African species, we reconstruct the ancestral distribution of the Neotropical clade as the Pacific Coast of Mexico and Central America. Our results indicate an historic dispersal of a bee-pollinated taxon from Africa to the Pacific Coast of Mexico/Central America, with subsequent diversification leading to the evolution of a bird-pollinated floral morphology in multiple derived lineages.

Inside the trap: gland morphologies, digestive enzymes, and the evolution of plant carnivory in the Caryophyllales.

The digestion of prey by carnivorous plants is determined in part by suites of enzymes that are associated with morphologically and anatomically diverse trapping mechanisms. Chitinases represent a group of enzymes known to be integral to effective plant carnivory. In non-carnivorous plants, chitinases commonly act as pathogenesis-related proteins, which are either induced in response to insect herbivory and fungal elicitors, or constitutively expressed in tissues vulnerable to attack. In the Caryophyllales carnivorous plant lineage, multiple classes of chitinases are likely involved in both pathogenic response and digestion of prey items. We review what is currently known about trap morphologies, provide an examination of the diversity, roles, and evolution of chitinases, and examine how herbivore and pathogen defense mechanisms may have been coopted for plant carnivory in the Caryophyllales.

Molecular and functional evolution of class I chitinases for plant carnivory in the caryophyllales.

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.

Virus-induced gene silencing in the culinary ginger (Zingiber officinale): an effective mechanism for down-regulating gene expression in tropical monocots.

Virus-induced gene silencing (VIGS) has been shown to be effective for transient knockdown of gene expression in plants to analyze the effects of specific genes in development and stress-related responses. VIGS is well established for studies of model systems and crops within the Solanaceae, Brassicaceae, Leguminaceae, and Poaceae, but only recently has been applied to plants residing outside these families. Here, we have demonstrated that barley stripe mosaic virus (BSMV) can infect two species within the Zingiberaceae, and that BSMV-VIGS can be applied to specifically down-regulate phytoene desaturase in the culinary ginger Zingiber officinale. These results suggest that extension of BSMV-VIGS to monocots other than cereals has the potential for directed genetic analyses of many important temperate and tropical crop species.

The value of avian genomics to the conservation of wildlife.

BACKGROUND: Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution.Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care).In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. RESULTS: Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology.Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one. CONCLUSION: Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease.In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.