From cacti to carnivores: Improved phylotranscriptomic sampling and hierarchical homology inference provide further insight into the evolution of Caryophyllales

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143660/1/ajb21069.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143660/2/ajb21069_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143660/3/ajb21069-sup-0002-AppendixS2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143660/4/ajb21069-sup-0005-AppendixS5.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143660/5/ajb21069-sup-0001-AppendixS1.pd

Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales

The evolution of l-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. To address this, we functionally characterised 23 distinct DODA proteins for l-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l-DOPA 4,5-dioxygenase activity. We find that low l-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny. In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales. Keywords: Caryophyllales; anthocyanins; betalains; convergent evolution; gene duplication; l-DOPA 4, 5-dioxygenase (DODA); metabolic operon; plant pigments; specialised metabolism

Convergent evolution of betalains in Caryophyllales

In this thesis, I investigate the evolution of betalains in Caryophyllales. Betalains are a group of red and yellow pigments, which in plants occur only in the order Caryophyllales. Here, betalains replace the otherwise ubiquitous anthocyanin pigments, and the two pigment types appear mutually exclusive. Some lineages of Caryophyllales maintain anthocyanins, and the distribution of the two pigments with respect to relationships within the order suggests a complex evolutionary history, that allows for the possibility of repeated convergent shifts from anthocyanin to betalain pigmentation. First, I begin by framing my thesis with a review of the concepts of convergence and parallelism, including the connection between these terms and concepts of adaptation and constraint. I then discuss convergence at the level of molecular sequence data, and explore ideas of convergence at multiple hierarchies and their integration in the evolution of complex traits. Second, I conduct a reanalysis of the evolution of betalains, using the latest phylogenomic hypothesis of Caryophyllales relationships and up to date pigmentation data. I show that these data support up to four origins of betalains. I re-examine the phylogeny of DODA, a key gene in the betalain biosynthesis pathway, and show that the distribution of betalain-related enzyme activity is consistent with multiple origins of high activity following repeated gene duplications, consistent with inferred origins of betalains. I argue that multiple transitions to high activity in this enzyme have facilitated multiple origins of betalain pigmentation. Third, given this scenario, I assess the molecular signal for convergent evolution in DODA lineage. I reconstruct ancestral sequences and show that experimental testing of their activity supports multiple origins of high activity. I infer historical substitutions and show that a complex history of convergent molecular evolution has occurred, across multiple branches of the gene tree and in different order depending on origin. I support my findings with a suite of statistical methods and discuss how these approaches differ and how they can be used to interrogate betalain evolutionary history. Fourth, I explore convergence at the level of genome structure. I sequence and assemble three new Caryophyllales genomes with long reads. Two key genes of the betalain pathway are clustered in the Beta vulgaris genome, and I use comparative genomics to explore the evolutionary history of this clustering. Strikingly, I show that a pattern dispersed synteny of betalain-implicated DODA paralogues, has occurred multiple times via different mechanisms. I discuss the potential significance of this finding with respect to syntenic evolution and the evolution of betalains. Finally, I use comparative expression to explore betalain origins. I design pigment induction experiments in diverse taxa representing multiple origins and measure associated gene expression with RNA-seq. I interrogate these data with coexpression techniques and provide new insights into comparative gene networks and betalain regulation.Woolf Fisher Trust Cambridge Trust NSFDEB-NERC 193922

Compositional shifts associated with major evolutionary transitions in plants.

Funder: University of Michigan; doi: http://dx.doi.org/10.13039/100007270Funder: Woolf Fisher TrustHeterogeneity in gene trees, morphological characters, and composition has been associated with several major plant clades. Here, we examine heterogeneity in composition across a large transcriptomic dataset of plants to better understand whether locations of shifts in composition are shared across gene regions and whether directions of shifts within clades are shared across gene regions. We estimate mixed models of composition for both nucleotide and amino acids across a recent large-scale transcriptomic dataset for plants. We find shifts in composition across both nucleotide and amino acid datasets, with more shifts detected in nucleotides. We find that Chlorophytes and lineages within experience the most shifts. However, many shifts occur at the origins of land, vascular, and seed plants. While genes in these clades do not typically share the same composition, they tend to shift in the same direction. We discuss potential causes of these patterns. Compositional heterogeneity has been highlighted as a potential problem for phylogenetic analysis, but the variation presented here highlights the need to further investigate these patterns for the signal of biological processes

Characterizing gene tree conflict in plastome-inferred phylogenies.

Evolutionary relationships among plants have been inferred primarily using chloroplast data. To date, no study has comprehensively examined the plastome for gene tree conflict. Using a broad sampling of angiosperm plastomes, we characterize gene tree conflict among plastid genes at various time scales and explore correlates to conflict (e.g., evolutionary rate, gene length, molecule type). We uncover notable gene tree conflict against a backdrop of largely uninformative genes. We find alignment length and tree length are strong predictors of concordance, and that nucleotides outperform amino acids. Of the most commonly used markers, matK, greatly outperforms rbcL; however, the rarely used gene rpoC2 is the top-performing gene in every analysis. We find that rpoC2 reconstructs angiosperm phylogeny as well as the entire concatenated set of protein-coding chloroplast genes. Our results suggest that longer genes are superior for phylogeny reconstruction. The alleviation of some conflict through the use of nucleotides suggests that stochastic and systematic error is likely the root of most of the observed conflict, but further research on biological conflict within plastome is warranted given documented cases of heteroplasmic recombination. We suggest that researchers should filter genes for topological concordance when performing downstream comparative analyses on phylogenetic data, even when using chloroplast genomes

Characterizing gene tree conflict in plastome-inferred phylogenies

Evolutionary relationships among plants have been inferred primarily using chloroplast data. To date, no study has comprehensively examined the plastome for gene tree conflict. Using a broad sampling of angiosperm plastomes, we characterize gene tree conflict among plastid genes at various time scales and explore correlates to conflict (e.g., evolutionary rate, gene length, molecule type). We uncover notable gene tree conflict against a backdrop of largely uninformative genes. We find alignment length and tree length are strong predictors of concordance, and that nucleotides outperform amino acids. Of the most commonly used markers, matK, greatly outperforms rbcL; however, the rarely used gene rpoC2 is the top-performing gene in every analysis. We find that rpoC2 reconstructs angiosperm phylogeny as well as the entire concatenated set of protein-coding chloroplast genes. Our results suggest that longer genes are superior for phylogeny reconstruction. The alleviation of some conflict through the use of nucleotides suggests that stochastic and systematic error is likely the root of most of the observed conflict, but further research on biological conflict within plastome is warranted given documented cases of heteroplasmic recombination. We suggest that researchers should filter genes for topological concordance when performing downstream comparative analyses on phylogenetic data, even when using chloroplast genomes

The evolution of betalain biosynthesis in Caryophyllales

Timoneda A, Feng T, Sheehan H, et al. The evolution of betalain biosynthesis in Caryophyllales. New Phytologist. 2019;224(1):71-85

Are seven amino acid substitutions sufficient to explain the evolution of high l-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018).

This work revisits a publication by Bean et al. (2018) that reports seven amino acid substitutions are essential for the evolution of l-DOPA 4,5-dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Bean et al. (2018). Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Bean et al. (2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Bean et al. (2018) to re-observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2-mut3 variant. Multiple in vivo assays, in both Saccharomyces cerevisiae and Nicotiana benthamiana, did not result in visible DODA activity in BvDODAα2-mut3, with betalain production always 10-fold below BvDODAα1. In vitro assays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2-mut3 proteins, explaining their differing performance in vivo. In summary, we were unable to replicate the in vivo analyses of Bean et al. (2018), and our quantitative in vivo and in vitro analyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Bean et al. (2018)

Multiple mechanisms explain loss of anthocyanins from betalain‐pigmented Caryophyllales, including repeated wholesale loss of a key anthocyanidin synthesis enzyme

Publication status: PublishedFunder: Woolf Fisher Cambridge ScholarshipFunder: BBSRC High Value Chemicals from Plants NetworkSummary In this study, we investigate the genetic mechanisms responsible for the loss of anthocyanins in betalain‐pigmented Caryophyllales, considering our hypothesis of multiple transitions to betalain pigmentation. Utilizing transcriptomic and genomic datasets across 357 species and 31 families, we scrutinize 18 flavonoid pathway genes and six regulatory genes spanning four transitions to betalain pigmentation. We examined evidence for hypotheses of wholesale gene loss, modified gene function, altered gene expression, and degeneration of the MBW (MYB‐bHLH‐WD40) trasnscription factor complex, within betalain‐pigmented lineages. Our analyses reveal that most flavonoid synthesis genes remain conserved in betalain‐pigmented lineages, with the notable exception of TT19 orthologs, essential for the final step in anthocyanidin synthesis, which appear to have been repeatedly and entirely lost. Additional late‐stage flavonoid pathway genes upstream of TT19 also manifest strikingly reduced expression in betalain‐pigmented species. Additionally, we find repeated loss and alteration in the MBW transcription complex essential for canonical anthocyanin synthesis. Consequently, the loss and exclusion of anthocyanins in betalain‐pigmented species appear to be orchestrated through several mechanisms: loss of a key enzyme, downregulation of synthesis genes, and degeneration of regulatory complexes. These changes have occurred iteratively in Caryophyllales, often coinciding with evolutionary transitions to betalain pigmentation. </jats:p