Green land: Multiple perspectives on green algal evolution and the earliest land plants.

Green plants, broadly defined as green algae and the land plants (together, Viridiplantae), constitute the primary eukaryotic lineage that successfully colonized Earth's emergent landscape. Members of various clades of green plants have independently made the transition from fully aquatic to subaerial habitats many times throughout Earth's history. The transition, from unicells or simple filaments to complex multicellular plant bodies with functionally differentiated tissues and organs, was accompanied by innovations built upon a genetic and phenotypic toolkit that have served aquatic green phototrophs successfully for at least a billion years. These innovations opened an enormous array of new, drier places to live on the planet and resulted in a huge diversity of land plants that have dominated terrestrial ecosystems over the past 500 million years. This review examines the greening of the land from several perspectives, from paleontology to phylogenomics, to water stress responses and the genetic toolkit shared by green algae and plants, to the genomic evolution of the sporophyte generation. We summarize advances on disparate fronts in elucidating this important event in the evolution of the biosphere and the lacunae in our understanding of it. We present the process not as a step-by-step advancement from primitive green cells to an inevitable success of embryophytes, but rather as a process of adaptations and exaptations that allowed multiple clades of green plants, with various combinations of morphological and physiological terrestrialized traits, to become diverse and successful inhabitants of the land habitats of Earth.

A phylogenomically informed five-order system for the closest relatives of land plants.

The evolution of streptophytes had a profound impact on life on Earth. They brought forth those photosynthetic eukaryotes that today dominate the macroscopic flora: the land plants (Embryophyta).1 There is convincing evidence that the unicellular/filamentous Zygnematophyceae-and not the morphologically more elaborate Coleochaetophyceae or Charophyceae-are the closest algal relatives of land plants.2-6 Despite the species richness (>4,000), wide distribution, and key evolutionary position of the zygnematophytes, their internal phylogeny remains largely unresolved.7,8 There are also putative zygnematophytes with interesting body plan modifications (e.g., filamentous growth) whose phylogenetic affiliations remain unknown. Here, we studied a filamentous green alga (strain MZCH580) from an Austrian peat bog with central or parietal chloroplasts that lack discernible pyrenoids. It represents Mougeotiopsis calospora PALLA, an enigmatic alga that was described more than 120 years ago9 but never subjected to molecular analyses. We generated transcriptomic data of M. calospora strain MZCH580 and conducted comprehensive phylogenomic analyses (326 nuclear loci) for 46 taxonomically diverse zygnematophytes. Strain MZCH580 falls in a deep-branching zygnematophycean clade together with some unicellular species and thus represents a formerly unknown zygnematophycean lineage with filamentous growth. Our well-supported phylogenomic tree lets us propose a new five-order system for the Zygnematophyceae and provides evidence for at least five independent origins of true filamentous growth in the closest algal relatives of land plants. This phylogeny provides a robust and comprehensive framework for performing comparative analyses and inferring the evolution of cellular traits and body plans in the closest relatives of land plants.

Using RDNA sequences to define dinoflagellate species.

Dinoflagellate species are traditionally defined using morphological characters, but molecular evidence accumulated over the past several decades indicates many morphologically-based descriptions are inaccurate. This recognition led to an increasing reliance on DNA sequence data, particularly rDNA gene segments, in defining species. The validity of this approach assumes the divergence in rDNA or other selected genes parallels speciation events. Another concern is whether single gene rDNA phylogenies by themselves are adequate for delineating species or if multigene phylogenies are required instead. Currently, few studies have directly assessed the relative utility of multigene versus rDNA-based phylogenies for distinguishing species. To address this, the current study examined D1-D3 and ITS/5.8S rDNA gene regions, a multi-gene phylogeny, and morphological characters in Gambierdiscus and other related dinoflagellate genera to determine if they produce congruent phylogenies and identify the same species. Data for the analyses were obtained from previous sequencing efforts and publicly available dinoflagellate transcriptomic libraries as well from the additional nine well-characterized Gambierdiscus species transcriptomic libraries generated in this study. The D1-D3 and ITS/5.8S phylogenies successfully identified the described Gambierdiscus and Alexandrium species. Additionally, the data showed that the D1-D3 and multigene phylogenies were equally capable of identifying the same species. The multigene phylogenies, however, showed different relationships among species and are likely to prove more accurate at determining phylogenetic relationships above the species level. These data indicated that D1-D3 and ITS/5.8S rDNA region phylogenies are generally successful for identifying species of Gambierdiscus, and likely those of other dinoflagellates. To assess how broadly general this finding is likely to be, rDNA molecular phylogenies from over 473 manuscripts representing 232 genera and 863 described species of dinoflagellates were reviewed. Results showed the D1-D3 rDNA and ITS phylogenies in combination are capable of identifying 97% of dinoflagellate species including all the species belonging to the genera Alexandrium, Ostreopsis and Gambierdiscus, although it should be noted that multi-gene phylogenies are preferred for inferring relationships among these species. A protocol is presented for determining when D1-D3, confirmed by ITS/5.8S rDNA sequence data, would take precedence over morphological features when describing new dinoflagellate species. This protocol addresses situations such as: a) when a new species is both morphologically and molecularly distinct from other known species; b) when a new species and closely related species are morphologically indistinguishable, but genetically distinct; and c) how to handle potentially cryptic species and cases where morphotypes are clearly distinct but have the same rDNA sequence. The protocol also addresses other molecular, morphological, and genetic approaches required to resolve species boundaries in the small minority of species where the D1-D3/ITS region phylogenies fail.

Microbial biodiversity: A newly isolated cyanobacterium sheds light on the evolution of photosynthesis.

A newly isolated cyanobacterium found growing in close association with a tropical, non-vascular plant has been cultured and its genome sequenced. Its lineage is well over a billion years old and gives insights into the evolutionary origin of oxygenic photosynthesis.

Salinity-induced Changes in Gene Expression in the Streptophyte Alga Chara: The Critical Role of a Rare Na+ -ATPase.

The primarily freshwater genus Chara is comprised of many species that exhibit a wide range of salinity tolerance. The range of salt tolerance provides a good platform for investigating the role of transport mechanisms in response to salt stress, and the close evolutionary relationship between Charophytes and land plants can provide broader insights. We investigated the response to salt stress of previously identified transport mechanisms in two species of Chara, Chara longifolia (salt-tolerant), and Chara australis (salt-sensitive): a cation transporter (HKT), a Na+ /H+ antiport (NHX), H+ -ATPase (AHA), and a Na+ -ATPase (ENA). The presence of these candidate genes has been confirmed in both species of Chara, with the exception of the Na+ -ATPase, which is present only in salt-tolerant Chara longifolia. Time-course Illumina transcriptomes were created using RNA from multiple time points (0, 6, 12, 24 and 48 h) after freshwater cultures for each species were exposed to salt stress. These transcriptomes verified our hypotheses of these mechanisms conferring salt tolerance in the two species examined and also aided in identification of specific transcripts representing our genes of interest in both species. The expression of these transcripts was validated through use of qPCR, in a similar experimental set-up used for the RNAseq data described above. The RNAseq and qPCR data showed significant changes of expression mechanisms in C. longifolia (respectively), a down-regulation of HKT and a substantial up-regulation of ENA. Significant responses to salt stress in salt-sensitive C. australis show up-regulation of NHX and AHA.

The role of ion-transporting proteins in the evolution of salt tolerance in charophyte algae.

Species within the genus Chara have variable salinity tolerance. Their close evolutionary relationship with embryophytes makes their study crucial to understanding the evolution of salt tolerance and key evolutionary processes shared among the phyla. We examined salt-tolerant Chara longifolia and salt-sensitive Chara australis for mechanisms of salt tolerance and their potential role in adaptation to salt. We hypothesize that there are shared mechanisms similar to those in embryophytes, which assist in conferring salt tolerance in Chara, including a cation transporter (HKT), a Na+ /H+ antiport (NHX), a H+ -ATPase (AHA), and a Na+ -ATPase (ENA). Illumina transcriptomes were created using cultures grown in freshwater and exposed to salt stress. The presence of these candidate genes, identified by comparing with genes known from embryophytes, has been confirmed in both species of Chara, with the exception of ENA, present only in salt-tolerant C. longifolia. These transcriptomes provide evidence for the contribution of these mechanisms to differences in salt tolerance in the two species and for the independent evolution of the Na+ -ATPase. We also examined genes that may have played a role in important evolutionary processes, suggested by previous work on the Chara braunii genome. Among the genes examined, cellulose synthase protein (GT43) and response regulator (RRB) were confirmed in both species. Genes absent from all three Chara species were members of the GRAS family, microtubule-binding protein (TANGLED1), and auxin synthesizers (YUCCA, TAA). Results from this study shed light on the evolutionary relationship between Chara and embryophytes through confirmation of shared salt tolerance mechanisms, as well as unique mechanisms that do not occur in angiosperms.

A genetic element in the SARS-CoV-2 genome is shared with multiple insect species.

SARS-CoV-2 is a member of the subgenus Sarbecovirus and thus contains the genetic element s2m. We have extensively mined nucleotide data in GenBank in order to obtain a comprehensive list of s2m sequences both in the four virus families where s2m has previously been described and in other groups of organisms. Surprisingly, there seems to be a xenologue of s2m in a large number of insect species. The function of s2m is unknown, but our data show a very high degree of sequence conservation both in insects and in viruses and that the version of s2m found in SARS-CoV-2 has unique features, not seen in any other virus or insect strains.

Neoproterozoic origin and multiple transitions to macroscopic growth in green seaweeds.

The Neoproterozoic Era records the transition from a largely bacterial to a predominantly eukaryotic phototrophic world, creating the foundation for the complex benthic ecosystems that have sustained Metazoa from the Ediacaran Period onward. This study focuses on the evolutionary origins of green seaweeds, which play an important ecological role in the benthos of modern sunlit oceans and likely played a crucial part in the evolution of early animals by structuring benthic habitats and providing novel niches. By applying a phylogenomic approach, we resolve deep relationships of the core Chlorophyta (Ulvophyceae or green seaweeds, and freshwater or terrestrial Chlorophyceae and Trebouxiophyceae) and unveil a rapid radiation of Chlorophyceae and the principal lineages of the Ulvophyceae late in the Neoproterozoic Era. Our time-calibrated tree points to an origin and early diversification of green seaweeds in the late Tonian and Cryogenian periods, an interval marked by two global glaciations with strong consequent changes in the amount of available marine benthic habitat. We hypothesize that unicellular and simple multicellular ancestors of green seaweeds survived these extreme climate events in isolated refugia, and diversified in benthic environments that became increasingly available as ice retreated. An increased supply of nutrients and biotic interactions, such as grazing pressure, likely triggered the independent evolution of macroscopic growth via different strategies, including true multicellularity, and multiple types of giant-celled forms.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

Microbial Diversity in the Eukaryotic SAR Clade: Illuminating the Darkness Between Morphology and Molecular Data.

Despite their diversity and ecological importance, many areas of the SAR-Stramenopila, Alveolata, and Rhizaria-clade are poorly understood as the majority (90%) of SAR species lack molecular data and only 5% of species are from well-sampled families. Here, we review and summarize the state of knowledge about the three major clades of SAR, describing the diversity within each clade and identifying synapomorphies when possible. We also assess the "dark area" of SAR: the morphologically described species that are missing molecular data. The majority of molecular data for SAR lineages are characterized from marine samples and vertebrate hosts, highlighting the need for additional research effort in areas such as freshwater and terrestrial habitats and "non-vertebrate" hosts. We also describe the paucity of data on the biogeography of SAR species, and point to opportunities to illuminate diversity in this major eukaryotic clade. See also the video abstract here: https://youtu.be/_VUXqaX19Rw.

Land Plant Model Systems Branch Out.

The genome of the liverwort Marchantia polymorpha is an important step toward development of a new plant model system (Bowman et al., 2017). Liverworts may be the sister taxon to all other land plants, and the genome shows features that illuminate the ancestor of all land plants and give insights into how plant systems function and evolved.

Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics.

Dinoflagellates are key species in marine environments, but they remain poorly understood in part because of their large, complex genomes, unique molecular biology, and unresolved in-group relationships. We created a taxonomically representative dataset of dinoflagellate transcriptomes and used this to infer a strongly supported phylogeny to map major morphological and molecular transitions in dinoflagellate evolution. Our results show an early-branching position of Noctiluca, monophyly of thecate (plate-bearing) dinoflagellates, and paraphyly of athecate ones. This represents unambiguous phylogenetic evidence for a single origin of the group's cellulosic theca, which we show coincided with a radiation of cellulases implicated in cell division. By integrating dinoflagellate molecular, fossil, and biogeochemical evidence, we propose a revised model for the evolution of thecal tabulations and suggest that the late acquisition of dinosterol in the group is inconsistent with dinoflagellates being the source of this biomarker in pre-Mesozoic strata. Three distantly related, fundamentally nonphotosynthetic dinoflagellates, Noctiluca, Oxyrrhis, and Dinophysis, contain cryptic plastidial metabolisms and lack alternative cytosolic pathways, suggesting that all free-living dinoflagellates are metabolically dependent on plastids. This finding led us to propose general mechanisms of dependency on plastid organelles in eukaryotes that have lost photosynthesis; it also suggests that the evolutionary origin of bioluminescence in nonphotosynthetic dinoflagellates may be linked to plastidic tetrapyrrole biosynthesis. Finally, we use our phylogenetic framework to show that dinoflagellate nuclei have recruited DNA-binding proteins in three distinct evolutionary waves, which included two independent acquisitions of bacterial histone-like proteins.

Transcriptome Profiling of the Green Alga Spirogyra pratensis (Charophyta) Suggests an Ancestral Role for Ethylene in Cell Wall Metabolism, Photosynthesis, and Abiotic Stress Responses.

It is well known that ethylene regulates a diverse set of developmental and stress-related processes in angiosperms, yet its roles in early-diverging embryophytes and algae are poorly understood. Recently, it was shown that ethylene functions as a hormone in the charophyte green alga Spirogyra pratensis Since land plants evolved from charophytes, this implies conservation of ethylene as a hormone in green plants for at least 450 million years. However, the physiological role of ethylene in charophyte algae has remained unknown. To gain insight into ethylene responses in Spirogyra, we used mRNA sequencing to measure changes in gene expression over time in Spirogyra filaments in response to an ethylene treatment. Our analyses show that at the transcriptional level, ethylene predominantly regulates three processes in Spirogyra: (1) modification of the cell wall matrix by expansins and xyloglucan endotransglucosylases/hydrolases, (2) down-regulation of chlorophyll biosynthesis and photosynthesis, and (3) activation of abiotic stress responses. We confirmed that the photosynthetic capacity and chlorophyll content were reduced by an ethylene treatment and that several abiotic stress conditions could stimulate cell elongation in an ethylene-dependent manner. We also found that the Spirogyra transcriptome harbors only 10 ethylene-responsive transcription factor (ERF) homologs, several of which are regulated by ethylene. These results provide an initial understanding of the hormonal responses induced by ethylene in Spirogyra and help to reconstruct the role of ethylene in ancestral charophytes prior to the origin of land plants.

Complex Ancestries of Isoprenoid Synthesis in Dinoflagellates.

Isoprenoid metabolism occupies a central position in the anabolic metabolism of all living cells. In plastid-bearing organisms, two pathways may be present for de novo isoprenoid synthesis, the cytosolic mevalonate pathway (MVA) and nuclear-encoded, plastid-targeted nonmevalonate pathway (DOXP). Using transcriptomic data we find that dinoflagellates apparently make exclusive use of the DOXP pathway. Using phylogenetic analyses of all DOXP genes we inferred the evolutionary origins of DOXP genes in dinoflagellates. Plastid replacements led to a DOXP pathway of multiple evolutionary origins. Dinoflagellates commonly referred to as dinotoms due to their relatively recent acquisition of a diatom plastid, express two completely redundant DOXP pathways. Dinoflagellates with a tertiary plastid of haptophyte origin, by contrast, express a hybrid pathway of dual evolutionary origin. Here, changes in the targeting motif of signal/transit peptide likely allow for targeting the new plastid by the proteins of core isoprenoid metabolism proteins. Parasitic dinoflagellates of the Amoebophyra species complex appear to have lost the DOXP pathway, suggesting that they may rely on their host for sterol synthesis.

Evaluation of BLAST-based edge-weighting metrics used for homology inference with the Markov Clustering algorithm.

BACKGROUND: Clustering protein sequences according to inferred homology is a fundamental step in the analysis of many large data sets. Since the publication of the Markov Clustering (MCL) algorithm in 2002, it has been the centerpiece of several popular applications. Each of these approaches generates an undirected graph that represents sequences as nodes connected to each other by edges weighted with a BLAST-based metric. MCL is then used to infer clusters of homologous proteins by analyzing these graphs. The various approaches differ only by how they weight the edges, yet there has been very little direct examination of the relative performance of alternative edge-weighting metrics. This study compares the performance of four BLAST-based edge-weighting metrics: the bit score, bit score ratio (BSR), bit score over anchored length (BAL), and negative common log of the expectation value (NLE). Performance is tested using the Extended CEGMA KOGs (ECK) database, which we introduce here. RESULTS: All metrics performed similarly when analyzing full-length sequences, but dramatic differences emerged as progressively larger fractions of the test sequences were split into fragments. The BSR and BAL successfully rescued subsets of clusters by strengthening certain types of alignments between fragmented sequences, but also shifted the largest correct scores down near the range of scores generated from spurious alignments. This penalty outweighed the benefits in most test cases, and was greatly exacerbated by increasing the MCL inflation parameter, making these metrics less robust than the bit score or the more popular NLE. Notably, the bit score performed as well or better than the other three metrics in all scenarios. CONCLUSIONS: The results provide a strong case for use of the bit score, which appears to offer equivalent or superior performance to the more popular NLE. The insight that MCL-based clustering methods can be improved using a more tractable edge-weighting metric will greatly simplify future implementations. We demonstrate this with our own minimalist Python implementation: Porthos, which uses only standard libraries and can process a graph with 25 m + edges connecting the 60 k + KOG sequences in half a minute using less than half a gigabyte of memory.

Dinoflagellate Gene Structure and Intron Splice Sites in a Genomic Tandem Array.

Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced, dinoflagellate genomic, tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR-based contigs. The sequence contains an intron-rich set of five highly expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3' splice site are repeated by an identical two to nine base pairs around the 5' splice site. The 5' and 3' splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary sequence might be useful in gene modeling and annotation of genomes.

Phylogenomic analysis of Emiliania huxleyi provides evidence for haptophyte-stramenopile association and a chimeric haptophyte nuclear genome.

Emiliania huxleyi is a haptophyte alga of uncertain phylogenetic affinity containing a secondarily derived, chlorophyll c containing plastid. We sought to characterize its relationships with other taxa by quantifying the bipartitions in which it was included from a group of single protein phylogenetic trees in a way that allowed for variation in taxonomic content and accounted for paralogous sequences. The largest number of sequences supported a phylogenetic relationship of E. huxleyi with the stramenopiles, in particular Aureococcus anophagefferens. Far fewer nuclear sequences gave strong support to the placement of this coccolithophorid with the cryptophyte, Guillardia theta. The majority of the sequences that did support this relationship did not have plastid related functions. These results suggest that the haptophytes may be more closely allied with the heterokonts than with the cryptophytes. Another small set of genes associated E. huxleyi with the Viridiplantae with high support. While these genes could have been acquired with a plastid, the lack of plastid related functions among the proteins for which they code and the lack of other organisms with chlorophyll c containing plastids within these bipartitions suggests other explanations may be possible. This study also identified several genes that may have been transferred from the haptophyte lineage to the dinoflagellates Karenia brevis and Karlodinium veneficum as a result of their haptophyte derived plastid, including some with non-photosynthetic functions.