Barbara Surek

Phylotranscriptomic analysis of the origin and early diversification of land plants

Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution. Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

Data access for the 1,000 Plants (1KP) project

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

The Basal Position of Scaly Green Flagellates among the Green Algae (Chlorophyta) is Revealed by Analyses of Nuclear-Encoded SSU rRNA Sequences

The prasinophytes comprise a morphologically heterogeneous assembly of mostly marine flagellates and coccoid taxa, which represent an important component of the nano- and picoplankton, and have previously figured prominently in discussions about the origin and phylogeny of the green plants. To evaluate their putative basal position in the Viridiplantae and to resolve the phylogenetic relationships among the prasinophyte taxa, we determined complete nuclear-encoded SSU rRNA sequences from 13 prasinophyte taxa representing the genera Cymbomonas, Halosphaera, Mamiella, Mantoniella, Micromonas, Pterosperma, Pycnococcus, and Pyramimonas. Phylogenetic analyses of SSU rRNA sequences using distance, parsimony and likelihood methods revealed four independent prasi.nophyte lineages (clades) which constitute the earliest divergences among the Chlorophyta. In order of their divergence these clades are represented by the genera Cymbomonas, Halosphaera, Pterosperma, Pyramimonas (clade I), Mamiella, Mantoniella, Micromonas (clade II), Pseudoscourfieldia (strain CCMP 717), Nephroselmis (clade III), and Tetraselmis, Scherffelia (clade IV). The coccoid Pycnococcus provasolii diverged after clade II, but before clade III. Since no other coccoid prasinophyte taxa were analyzed in this study, the phylogenetic status of this taxon is presently unresolved. Our analyses provide further evidence for the basal phylogenetic position of the scaly green flagellates among the Chlorophyta and raise important questions concerning the class-level classification of the Chlorophyta.

Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Algal ancestor of land plants was preadapted for symbiosis

Significance Colonization of land by plants was a critical event for the emergence of extant ecosystems. The innovations that allowed the algal ancestor of land plants to succeed in such a transition remain unknown. Beneficial interaction with symbiotic fungi has been proposed as one of these innovations. Here we show that the genes required for this interaction appeared in a stepwise manner: Some evolved before the colonization of land by plants and others first appeared in land plants. We thus propose that the algal ancestor of land plants was preadapted for interaction with beneficial fungi and employed these gene networks to colonize land successfully. Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

Ribosomal RNA sequence comparisons demonstrate an evolutionary relationship betweenZygnematales and charophytes

The nuclear-encoded small subunit ribosomal RNA (rRNA) sequences were determined forGenicularia spirotaenia, Mesotaenium caldariorum, andStaurastrum spec. (Zygnematales) to elucidate the evolutionary position of these green algae. Results of neighbour-joining and maximum parsimony phylogenetic analyses support a monophyletic origin of theZygnematales within the evolutionary assemblage defined by theCharophyceae (sensuMattox & Stewart) and land plants. TheZygnematales/Charophyceae/land plants are evolutionarily distinct from the monophyletic lineage defined by theChlorodendrales, Pseudoscourfieldiales, and theMicrothamniales/Chlorophyceae.

Visualization of antigenic proteins blotted onto nitrocellulose using the immuno-gold-staining (IGS) method.

A new and simple method for the detection of antigenic proteins blotted onto nitrocellulose was developed. After transfer of spinach stromal proteins and purified phosphoribulokinase immunolabeling was performed with phosphoribulokinase antiserum, followed by a) Protein A-labeled colloidal gold particles, and b) by horseradish peroxidase conjugated Protein A and substrate mixture. The Protein A-Gold method is at least twofold more sensitive than the Protein A-peroxidase procedure. Incubation of immunolabeled nitrocellulose replicas with 0.1 M glycine, pH 2.2, removes the antibody-Protein A-Gold complexes quantitatively without influencing the antigenicity of the immobilized proteins. The replicas can be re-used for immunostaining with other antisera. The versatile applicability of the immuno-gold-staining method suggests that it is a true alternative to the peroxidase assay.

Primary and secondary structure analyses of the rDNA group-I introns of the Zygnematales (Charophyta)

The Zygnematales (Charophyta) contain a group-I intron (subgroup ICl) within their nuclear-encoded small subunit ribosomal DNA (SSU rDNA) coding region. This intron, which is inserted after position 1506 (relative to the SSU rDNA ofEscherichia coli), is proposed to have been vertically inherited since the origin of the Zygnematales approximately 350–400 million years ago. Primary and secondary structure analyses were carried out to model group-I intron evolution in the Zygnematales. Secondary structure analyses support genetic data regarding sequence conservation within regions known to be functionally important for in vitro self-splicing of group-I introns. Comparisons of zygnematalean group-I intron secondary structures also provided some new insights into sequences that may have important roles in in vivo RNA splicing. Sequence analyses showed that sequence divergence rates and the nucleotide compositions of introns and coding regions within any one taxon varied widely, suggesting that the “1506” group-I introns and rDNA coding regions in the Zygnematales evolve independently.

Spinach ferredoxin is a calcium-binding protein.

Spinach-leaf ferredoxin was identified as a calcium-binding protein by 45Ca autoradiography on nitrocellulose membranes and with the cationic carbocyanine dye 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide (“stains-all”). Binding of 45Ca was observed at pH 6.8 and pH 7.8 and in the presence of 5 mM and 20 mM MgCl2. At the higher MgCl2 concentration the Ca2+-binding capacity is reduced. Only micromolar concentrations of LaCl3, however, are required to achieve a similar effect. Both the oxidized and reduced forms of ferredoxin bind calcium.

First records of the benthic, bloom-forming, non-toxic dinoflagellate Thecadinium yashimaense (Dinophyceae) in Europe: with special emphasis on the invasion in the North Sea

Thecadinium yashimaense was recorded for the first time in France, Great Britain, The Netherlands, and Germany. The invasion and establishment of the species in the German Bight was documented reliably and is presented here. The geographic expansion of the species from the North Pacific to the North Atlantic Ocean is discussed. This bloom-forming, marine, sand-dwelling dinoflagellate was shown to be non-toxic. Also Thecadinium kofoidii, the type species of the genus, was analyzed for potential toxin production and turned out to be non-toxic as well.