Rubisco is the primary entry point for carbon into the biosphere. However, rubisco is widely regarded as inefficient leading many to question whether the enzyme can adapt to become a better catalyst. Through a phylogenetic investigation of the molecular and kinetic evolution of Form I rubisco we uncover the evolutionary trajectory of rubisco kinetic evolution in angiosperms. We show that rbcL is among the 1% of slowest-evolving genes and enzymes on Earth, accumulating one nucleotide substitution every 0.9 My and one amino acid mutation every 7.2 My. Despite this, rubisco catalysis has been continually evolving toward improved CO2/O2 specificity, carboxylase turnover, and carboxylation efficiency. Consistent with this kinetic adaptation, increased rubisco evolution has led to a concomitant improvement in leaf-level CO2 assimilation. Thus, rubisco has been slowly but continually evolving toward improved catalytic efficiency and CO2 assimilation in plants.

The development of multiple chromosome-scale reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple genomes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation. Here, we present GENESPACE, which addresses these challenges by integrating conserved gene order and orthology to define the expected physical position of all genes across multiple genomes. We demonstrate this utility by dissecting presence–absence, copy-number, and structural variation at three levels of biological organization: spanning 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic orthology in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred, and other complex genomes.

The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes. ### Competing Interest Statement The authors have declared no competing interest.

Determining the evolutionary relationships between genes is fundamental to comparative biological research. Here, we present SHOOT. SHOOT searches a user query sequence against a database of phylogenetic trees and returns a tree with the query sequence correctly placed within it. We show that SHOOT performs this analysis with comparable speed to a BLAST search. We demonstrate that SHOOT phylogenetic placements are as accurate as conventional tree inference, and it can identify orthologs with high accuracy. In summary, SHOOT is a fast and accurate tool for phylogenetic analyses of novel query sequences. It is available online at www.shoot.bio .

Abstract The Orthology Benchmark Service (https://orthology.benchmarkservice.org) is the gold standard for orthology inference evaluation, supported and maintained by the Quest for Orthologs consortium. It is an essential resource to compare existing and new methods of orthology inference (the bedrock for many comparative genomics and phylogenetic analysis) over a standard dataset and through common procedures. The Quest for Orthologs Consortium is dedicated to maintaining the resource up to date, through regular updates of the Reference Proteomes and increasingly accessible data through the OpenEBench platform. For this update, we have added a new benchmark based on curated orthology assertion from the Vertebrate Gene Nomenclature Committee, and provided an example meta-analysis of the public predictions present on the platform.

Stomata evolved as plants transitioned from water to land, enabling carbon dioxide uptake and water loss to be controlled. In flowering plants, the most recently divergent land plant lineage, stomatal pores actively close in response to drought. In this response, the phytohormone abscisic acid (ABA) triggers signalling cascades that lead to ion and water loss in the guard cells of the stomatal complex, causing a reduction in turgor and pore closure. Whether this stimulus-response coupling pathway acts in other major land plant lineages is unclear, with some investigations reporting that stomatal closure involves ABA but others concluding that closure is passive. Here we show that in the model fern Ceratopteris richardii active stomatal closure is conditional on sensitisation by pre-exposure to either low humidity or exogenous ABA and is promoted by ABA. RNA-seq analysis and de novo transcriptome assembly reconstructed the protein coding complement of the C. richardii genome with coverage comparable to other plant models, enabling transcriptional signatures of stomatal sensitisation and closure to be identified. In both cases, changes in abundance of homologs of ABA, Ca 2+ and ROS-related signalling components were observed, suggesting that the closure response pathway is conserved in ferns and flowering plants. These signatures further suggested that sensitisation is achieved by lowering the threshold required for a subsequent closure-inducing signal to trigger a response. We conclude that the canonical signalling network for active stomatal closure functioned in at least a rudimentary form in the stomata of the last common ancestor of ferns and flowering plants. Significance Statement Stomata are valve-like pores that control the uptake of CO 2 and the loss of water vapour in almost all land plants. In flowering plants, stomatal opening and closure is actively regulated by a stimulus-response coupling network. Whether active stomatal responses are present in other land plant lineages such as ferns has been hotly debated. Here we show that stomatal responses in the fern Ceratopteris richardii are active but depend on their past growth environment, and demonstrate that fern stomatal closure and sensitisation are associated with the altered expression of genes whose homologs function in the canonical stomatal regulatory network of flowering plants. Genetic pathways for active stomatal regulation therefore most likely evolved before the divergence of ferns and flowering plants.

SUMMARY The colonization of land by plants was one of the most transformative events in the history of life on Earth. The transition from water, which coincided with and was likely facilitated by the evolution of 3-dimensional (3D) growth, enabled the generation of morphological diversity on land. In many plants, the transition from 2-dimensional (2D) to 3D growth occurs during embryo development. However, in the early divergent moss Physcomitrium patens (formerly Physcomitrella patens ), 3D growth is preceded by an extended filamentous phase that can be maintained indefinitely. Here, we describe the identification of the cytokinin-responsive NO GAMETOPHORES 2 ( PpNOG2 ) gene, which encodes a shikimate o- hydroxycinnamoyltransferase. In mutants lacking PpNOG2 function, transcript levels of CLAVATA and SCARECROW genes are significantly reduced, excessive gametophore initial cells are produced, and buds undergo premature developmental arrest. Our results suggest that PpNOG2 functions in the ascorbic acid pathway leading to cuticle formation, and that NOG2-related genes were co-opted into the lignin biosynthesis pathway after the divergence of bryophytes and vascular plants. We present a revised model of 3D growth in which PpNOG2 comprises part of a feedback mechanism that is required for the modulation of gametophore initial cell frequency. We also propose that the 2D to 3D growth transition in P. patens is underpinned by complex auxin-cytokinin crosstalk that is regulated, at least in part, by changes in flavonoid metabolism.

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analyzed. Thus, it is possible that the correlations observed were an artefact of the presence of phylogenetic signal in rubisco kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically resolved analysis of rubisco kinetics and show that there is a significant phylogenetic signal in rubisco kinetic traits. We re-evaluated the extent of catalytic trade-offs accounting for this phylogenetic signal and found that all were attenuated. Following phylogenetic correction, the largest catalytic trade-offs were observed between the Michaelis constant for CO2 and carboxylase turnover (∼21-37%), and between the Michaelis constants for CO2 and O2 (∼9-19%), respectively. All other catalytic trade-offs were substantially attenuated such that they were marginal (<9%) or non-significant. This phylogenetically resolved analysis of rubisco kinetic evolution also identified kinetic changes that occur concomitant with the evolution of C4 photosynthesis. Finally, we show that phylogenetic constraints have played a larger role than catalytic trade-offs in limiting the evolution of rubisco kinetics. Thus, although there is strong evidence for some catalytic trade-offs, rubisco adaptation has been more limited by phylogenetic constraint than by the combined action of all catalytic trade-offs.

Organelle biogenesis and function is dependent on the concerted action of both organellar-encoded (if present) and nuclear-encoded proteins. Differences between homologous organelles across the Plant Kingdom arise, in part, as a result of differences in the cohort of nuclear-encoded proteins that are targeted to them. However, neither the rate at which differences in protein targeting accumulate nor the evolutionary consequences of these changes are known. Using phylogenomic approaches coupled to ancestral state estimation, we show that the plant organellar proteome has diversified in proportion with molecular sequence evolution such that the proteomes of plant chloroplasts and mitochondria lose or gain on average 3.6 proteins per million years. We further demonstrate that changes in organellar protein targeting are associated with an increase in the rate of molecular sequence evolution and that such changes predominantly occur in genes with regulatory rather than metabolic functions. Finally, we show that gain and loss of protein target signals occurs at a higher rate following gene duplication, revealing that gene and genome duplication are a key facilitator of plant organelle evolution.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

Orthobench is the standard benchmark to assess the accuracy of orthogroup inference methods. It contains 70 expert-curated reference orthogroups (RefOGs) that span the Bilateria and cover a range of different challenges for orthogroup inference. Here, we leveraged improvements in tree inference algorithms and computational resources to reinterrogate these RefOGs and carry out an extensive phylogenetic delineation of their composition. This phylogenetic revision altered the membership of 31 of the 70 RefOGs, with 24 subject to extensive revision and 7 that required minor changes. We further used these revised and updated RefOGs to provide an assessment of the orthogroup inference accuracy of widely used orthogroup inference methods. Finally, we provide an open-source benchmarking suite to support the future development and use of the Orthobench benchmark.

Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While some convergent traits are caused by simple sequence changes, many are associated with extensive changes to the sequence and regulation of large cohorts of genes. It is unknown how organisms traverse this expansive genotype space to assemble such complex convergent phenotypes. C 4 photosynthesis is a paradigm of large-scale phenotypic convergence. Conceptual and mathematical models propose that C 4 photosynthesis evolved from ancestral C 3 photosynthesis through sequential adaptive changes. These adaptive changes could have been rapidly assembled if modifications to the activity and abundance of enzymes of the C 4 cycle was neutral in C 3 plants. This neutrality would enable populations of C 3 plants to maintain genotypes with expression levels of C 4 enzymes analogous to those in C 4 species and thus enable rapid assembly of a functional C 4 cycle from naturally occurring genotypes given shared environmental selection. Here we show that there is substantial natural variation in expression of genes encoding C 4 cycle enzymes between natural accessions of the C 3 plant Arabidopsis thaliana . We further show through targeted transgenic experiments in the C 3 crop Oryza sativa , that high expression of the majority of C 4 cycle enzymes in rice is neutral with respect to growth, development, biomass and photosynthesis. Thus, substantial variation in the abundance and activity of C 4 cycle enzymes is permissible within the limits of operation of C 3 photosynthesis and the emergence of component parts of this complex convergent trait can be facilitated by neutral variation.

Rhizomorphic lycopsids are the land plant group that includes the first giant trees to grow on Earth and extant species in the genus Isoetes. Two mutually exclusive hypotheses account for the evolution of terminal rooting axes called rootlets among the rhizomorphic lycopsids. One hypothesis states that rootlets are true roots, like roots in other lycopsids. The other states that rootlets are modified leaves. Here we test predictions of each hypothesis by investigating gene expression in the leaves and rootlets of Isoetes echinospora. We assembled the de novo transcriptome of axenically cultured I. echinospora. Gene expression signatures of I. echinospora rootlets and leaves were different. Furthermore, gene expression signatures of I. echinospora rootlets were similar to gene expression signatures of true roots of Selaginella moellendorffii and Arabidopsis thaliana. RSL genes which positively regulate cell differentiation in roots were either exclusively or preferentially expressed in the I. echinospora rootlets, S. moellendorffii roots and A. thaliana roots compared to the leaves of each respective species. Taken together, gene expression data from the de-novo transcriptome of I. echinospora are consistent with the hypothesis that Isoetes rootlets are true roots and not modified leaves.

The identification of orthologs—genes in different species which descended from the same gene in their last common ancestor—is a prerequisite for many analyses in comparative genomics and molecular evolution. Numerous algorithms and resources have been conceived to address this problem, but benchmarking and interpreting them is fraught with difficulties (need to compare them on a common input dataset, absence of ground truth, computational cost of calling orthologs). To address this, the Quest for Orthologs consortium maintains a reference set of proteomes and provides a web server for continuous orthology benchmarking (http://orthology.benchmarkservice.org). Furthermore, consensus ortholog calls derived from public benchmark submissions are provided on the Alliance of Genome Resources website, the joint portal of NIH-funded model organism databases.

Investigating the evolution of plant biochemistry is challenging because few metabolites are preserved in fossils and because metabolic networks are difficult to experimentally characterize in diverse extant organisms. We report a comparative computational approach based on whole-genome metabolic pathway databases of eight species representative of major plant lineages, combined with homologous relationships among genes of 72 species from streptophyte algae to angiosperms. We use this genomic approach to identify metabolic gains and losses during land plant evolution. We extended our findings with additional analysis of 305 non-angiosperm plant transcriptomes. Our results revealed that genes encoding the complete biosynthetic pathway for brassinosteroid phytohormones and enzymes for brassinosteroid inactivation are present only in spermatophytes. Genes encoding only part of the biosynthesis pathway are present in ferns and lycophytes, indicating a stepwise evolutionary acquisition of this pathway. Nevertheless, brassinosteroids are ubiquitous in land plants, suggesting that brassinosteroid biosynthetic pathways differ between earlier- and later-diverging lineages. Conversely, genes for g ibberellin biosynthesis and inactivation using methyltransferases are found in all land plant lineages. This suggests that bioactive gibberellins might be present in bryophytes, although they have yet to be detected experimentally. We also found that cytochrome P450 oxidases involved in cutin and suberin production are absent in genomes of non-angiosperm plants that nevertheless do contain these biopolymers. Overall, we identified significant differences in crucial metabolic processes between angiosperms and earlier-diverging land plants and resolve details of the evolutionary history of several phytohormone and structural polymer biosynthetic pathways in land plants.

Musashi (Msi) proteins are an evolutionarily conserved group of RNA-binding proteins, required for targeted control of mRNA translation during many important developmental processes in animals. Most notably, Msi proteins play important roles during both spermatogenesis and oogenesis. Msi proteins also exist in plants but these are largely uncharacterized. Here we report the functional characterization of an Arabidopsis Msi ortholog ABORTED GAMETOPHYTE 2 ( AOG2 ), which encodes a protein containing two RNA recognition motifs and an ER-targeting signal. AOG2-GFP translational fusions were localized to the ER in transient assays, suggesting that AOG2 most likely binds to ER-targeted mRNAs. We show that disrupted AOG2 function leads to a high rate of both ovule and seed abortion, and that homozygous loss of function mutants are embryo lethal. Furthermore, we demonstrate that AOG2 is required to establish asymmetry during pollen mitosis I, and that loss of AOG2 function leads to loss of pollen viability. Collectively the results reveal that AOG2 is required for the establishment of polarity and/or the progression of mitosis during gametophyte development in Arabidopsis, and thus Msi-related proteins have an evolutionarily conserved role in gametogenesis in both animals and plants. SIGNIFICANCE STATEMENT ABORTED GAMETOPHYTE 2 ( AOG2 ) encodes a Musashi-related RNA-binding protein that is required for gametogenesis and embryogenesis in Arabidopsis. AOG2 is required for the establishment of polarity and/or the progression of mitosis during gametophyte development in Arabidopsis, and thus Musashi-related proteins have an evolutionarily conserved role in gametogenesis in both animals and plants.

Here, we present a major advance of the OrthoFinder method. This extends OrthoFinder’s high accuracy orthogroup inference to provide phylogenetic inference of orthologs, rooted gene trees, gene duplication events, the rooted species tree, and comparative genomics statistics. Each output is benchmarked on appropriate real or simulated datasets, and where comparable methods exist, OrthoFinder is equivalent to or outperforms these methods. Furthermore, OrthoFinder is the most accurate ortholog inference method on the Quest for Orthologs benchmark test. Finally, OrthoFinder’s comprehensive phylogenetic analysis is achieved with equivalent speed and scalability to the fastest, score-based heuristic methods. OrthoFinder is available at https://github.com/davidemms/OrthoFinder.

Evaluation measures for images segmentation are suggested. The methods compare the results of automatic segmentation with ground truth. The presented methods for assessing the similarity of the segments are based on three different approaches: the number of pixels in common, the similarity of the contours, and the location of centroids. The fuzzy approach consists of considering the significance of segment differences in relation to the size of the segments. The final measures for the whole images are based on recall and precision, widely used in information retrieval tasks. The approaches presented in this paper apply the fuzzy set theory instead of classical evaluation methods.