Chloroplast genome evolution and phylogeny of the early-diverging charophycean green algae with a focus on the Klebsormidiophyceae and Streptofilum.

The Klebsormidiophyceae are a class of green microalgae observed globally in both freshwater and terrestrial habitats. Morphology-based classification schemes of this class have been shown to be inadequate due to the simple morphology of these algae, the tendency of morphology to vary in culture versus field conditions, and rampant morphological homoplasy. Molecular studies revealing cryptic diversity have renewed interest in this group. We sequenced the complete chloroplast genomes of a broad series of taxa spanning the known taxonomic breadth of this class. We also sequenced the chloroplast genomes of three strains of Streptofilum, a recently discovered green algal lineage with close affinity to the Klebsormidiophyceae. Our results affirm the previously hypothesized polyphyly of the genus Klebsormidium as well as the polyphyly of the nominal species in this genus, K. flaccidum. Furthermore, plastome sequences strongly support the status of Streptofilum as a distinct, early-diverging lineage of charophytic algae sister to a clade comprising Klebsormidiophyceae plus Phragmoplastophyta. We also uncovered major structural alterations in the chloroplast genomes of species in Klebsormidium that have broad implications regarding the underlying mechanisms of chloroplast genome evolution.

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.

Dynamic plastid and mitochondrial genomes in Chaetopeltidales (Chlorophyceae) and characterization of a new chlorophyte taxon.

PREMISE: Chaetopeltidales is a poorly characterized order in the Chlorophyceae, with only two plastid and no mitochondrial genomes published. Here we describe a new taxon in Chaetopeltidales, Gormaniella terricola gen. et sp. nov. and characterize both of its organellar genomes. METHODS: Gormaniella terricola was inadvertently isolated from a surface-sterilized hornwort thallus. Light microscopy was used to characterize its vegetative morphology. Organellar genomes were assembled, annotated, and analyzed using a variety of software packages. RESULTS: The mitochondrial genome (66,927 bp) represents the first complete mitochondrial genome published for Chaetopeltidales. The chloroplast genome, measuring 428,981 bp, is one of the largest plastid genomes published to date and shares this large size and an incredible number of short, dispersed repeats with the other sequenced chloroplast genomes in Chaetopeltidales. Despite these shared features, the chloroplast genomes of Chaetopeltidales appear to be highly rearranged when compared to one another, with numerous inversions, translocations, and duplications, suggesting a particularly dynamic chloroplast genome. Both the chloroplast and mitochondrial genomes of G. terricola contain a number of mobile group I and group II introns, which appear to have invaded separately. Three of the introns within the mitochondrial genome encode homing endonucleases that are phylogenetically nested within those found in fungi, rather than algae, suggesting a possible case of horizontal gene transfer. CONCLUSIONS: These results help to shed light on a poorly understood group of algae and their unusual organellar genomes, raising additional questions about the unique patterns of genome evolution within Chaetopeltidales.

Induction of Conjugation and Zygospore Cell Wall Characteristics in the Alpine Spirogyra mirabilis (Zygnematophyceae, Charophyta): Advantage under Climate Change Scenarios?

Extreme environments, such as alpine habitats at high elevation, are increasingly exposed to man-made climate change. Zygnematophyceae thriving in these regions possess a special means of sexual reproduction, termed conjugation, leading to the formation of resistant zygospores. A field sample of Spirogyra with numerous conjugating stages was isolated and characterized by molecular phylogeny. We successfully induced sexual reproduction under laboratory conditions by a transfer to artificial pond water and increasing the light intensity to 184 µmol photons m-2 s-1. This, however was only possible in early spring, suggesting that the isolated cultures had an internal rhythm. The reproductive morphology was characterized by light- and transmission electron microscopy, and the latter allowed the detection of distinctly oriented microfibrils in the exo- and endospore, and an electron-dense mesospore. Glycan microarray profiling showed that Spirogyra cell walls are rich in major pectic and hemicellulosic polysaccharides, and immuno-fluorescence allowed the detection of arabinogalactan proteins (AGPs) and xyloglucan in the zygospore cell walls. Confocal RAMAN spectroscopy detected complex aromatic compounds, similar in their spectral signature to that of Lycopodium spores. These data support the idea that sexual reproduction in Zygnematophyceae, the sister lineage to land plants, might have played an important role in the process of terrestrialization.

Large Phylogenomic Data sets Reveal Deep Relationships and Trait Evolution in Chlorophyte Green Algae.

The chlorophyte green algae (Chlorophyta) are species-rich ancient groups ubiquitous in various habitats with high cytological diversity, ranging from microscopic to macroscopic organisms. However, the deep phylogeny within core Chlorophyta remains unresolved, in part due to the relatively sparse taxon and gene sampling in previous studies. Here we contribute new transcriptomic data and reconstruct phylogenetic relationships of core Chlorophyta based on four large data sets up to 2,698 genes of 70 species, representing 80% of extant orders. The impacts of outgroup choice, missing data, bootstrap-support cutoffs, and model misspecification in phylogenetic inference of core Chlorophyta are examined. The species tree topologies of core Chlorophyta from different analyses are highly congruent, with strong supports at many relationships (e.g., the Bryopsidales and the Scotinosphaerales-Dasycladales clade). The monophyly of Chlorophyceae and of Trebouxiophyceae as well as the uncertain placement of Chlorodendrophyceae and Pedinophyceae corroborate results from previous studies. The reconstruction of ancestral scenarios illustrates the evolution of the freshwater-sea and microscopic-macroscopic transition in the Ulvophyceae, and the transformation of unicellular→colonial→multicellular in the chlorophyte green algae. In addition, we provided new evidence that serine is encoded by both canonical codons and noncanonical TAG code in Scotinosphaerales, and stop-to-sense codon reassignment in the Ulvophyceae has originated independently at least three times. Our robust phylogenetic framework of core Chlorophyta unveils the evolutionary history of phycoplast, cyto-morphology, and noncanonical genetic codes in chlorophyte green algae.

Climate and seasonality drive the richness and composition of tropical fungal endophytes at a landscape scale.

Understanding how species-rich communities persist is a foundational question in ecology. In tropical forests, tree diversity is structured by edaphic factors, climate, and biotic interactions, with seasonality playing an essential role at landscape scales: wetter and less seasonal forests typically harbor higher tree diversity than more seasonal forests. We posited that the abiotic factors shaping tree diversity extend to hyperdiverse symbionts in leaves-fungal endophytes-that influence plant health, function, and resilience to stress. Through surveys in forests across Panama that considered climate, seasonality, and covarying biotic factors, we demonstrate that endophyte richness varies negatively with temperature seasonality. Endophyte community structure and taxonomic composition reflect both temperature seasonality and climate (mean annual temperature and precipitation). Overall our findings highlight the vital role of climate-related factors in shaping the hyperdiversity of these important and little-known symbionts of the trees that, in turn, form the foundations of tropical forest biodiversity.

Terrestrial Green Algae Show Higher Tolerance to Dehydration than Do Their Aquatic Sister-Species.

Diverse algae possess the ability to recover from extreme desiccation without forming specialized resting structures. Green algal genera such as Tetradesmus (Sphaeropleales, Chlorophyceae) contain temperate terrestrial, desert, and aquatic species, providing an opportunity to compare physiological traits associated with the transition to land in closely related taxa. We subjected six species from distinct habitats to three dehydration treatments varying in relative humidity (RH 5%, 65%, 80%) followed by short- and long-term rehydration. We tested the capacity of the algae to recover from dehydration using the effective quantum yield of photosystem II as a proxy for physiological activity. The degree of recovery was dependent both on the habitat of origin and the dehydration scenario, with terrestrial, but not aquatic, species recovering from dehydration. Distinct strains of each species responded similarly to dehydration and rehydration, with the exception of one aquatic strain that recovered from the mildest dehydration treatment. Cell ultrastructure was uniformly maintained in both aquatic and desert species during dehydration and rehydration, but staining with an amphiphilic styryl dye indicated damage to the plasma membrane from osmotically induced water loss in the aquatic species. These analyses demonstrate that terrestrial Tetradesmus possess a vegetative desiccation tolerance phenotype, making these species ideal for comparative omics studies.

Order, please! Uncertainty in the ordinal-level classification of Chlorophyceae.

BACKGROUND: Chlorophyceae is one of three most species-rich green algal classes and also the only class in core Chlorophyta whose monophyly remains uncontested as gene and taxon sampling improves. However, some key relationships within Chlorophyceae are less clear-cut and warrant further investigation. The present study combined genome-scale chloroplast data and rich sampling in an attempt to resolve the ordinal classification in Chlorophyceae. The traditional division into Sphaeropleales and Volvocales (SV), and a clade containing Oedogoniales, Chaetopeltidales, and Chaetophorales (OCC) was of particular interest with the addition of deeply branching members of these groups, as well as the placement of several incertae sedis taxa. METHODS: We sequenced 18 chloroplast genomes across Chlorophyceae to compile a data set of 58 protein-coding genes of a total of 68 chlorophycean taxa. We analyzed the concatenated nucleotide and amino acid datasets in the Bayesian and Maximum Likelihood frameworks, supplemented by analyses to examine potential discordant signal among genes. We also examined gene presence and absence data across Chlorophyceae. RESULTS: Concatenated analyses yielded at least two well-supported phylogenies: nucleotide data supported the traditional classification with the inclusion of the enigmatic Treubarinia into Sphaeropleales sensu lato. However, amino acid data yielded equally strong support for Sphaeropleaceae as sister to Volvocales, with the rest of the taxa traditionally classified in Sphaeropleales in a separate clade, and Treubarinia as sister to all of the above. Single-gene and other supplementary analyses indicated that the data have low phylogenetic signal at these critical nodes. Major clades were supported by genomic structural features such as gene losses and trans-spliced intron insertions in the plastome. DISCUSSION: While the sequence and gene order data support the deep split between the SV and OCC lineages, multiple phylogenetic hypotheses are possible for Sphaeropleales s.l. Given this uncertainty as well as the higher-taxonomic disorder seen in other algal groups, dwelling on well-defined, strongly supported Linnaean orders is not currently practical in Chlorophyceae and a less formal clade system may be more useful in the foreseeable future. For example, we identify two strongly and unequivocally supported clades: Treubarinia and Scenedesminia, as well as other smaller groups that could serve a practical purpose as named clades. This system does not preclude future establishment of new orders, or emendment of the current ordinal classification if new data support such conclusions.

Assessing Combinability of Phylogenomic Data Using Bayes Factors.

With the rapid reduction in sequencing costs of high-throughput genomic data, it has become commonplace to use hundreds of genes to infer phylogeny of any study system. While sampling a large number of genes has given us a tremendous opportunity to uncover previously unknown relationships and improve phylogenetic resolution, it also presents us with new challenges when the phylogenetic signal is confused by differences in the evolutionary histories of sampled genes. Given the incorporation of accurate marginal likelihood estimation methods into popular Bayesian software programs, it is natural to consider using the Bayes Factor (BF) to compare different partition models in which genes within any given partition subset share both tree topology and edge lengths. We explore using marginal likelihood to assess data subset combinability when data subsets have varying levels of phylogenetic discordance due to deep coalescence events among genes (simulated within a species tree), and compare the results with our recently described phylogenetic informational dissonance index (D) estimated for each data set. BF effectively detects phylogenetic incongruence and provides a way to assess the statistical significance of D values. We use BFs to assess data combinability using an empirical data set comprising 56 plastid genes from the green algal order Volvocales. We also discuss the potential need for calibrating BFs and demonstrate that BFs used in this study are correctly calibrated.

Contemporaneous radiations of fungi and plants linked to symbiosis.

Interactions between fungi and plants, including parasitism, mutualism, and saprotrophy, have been invoked as key to their respective macroevolutionary success. Here we evaluate the origins of plant-fungal symbioses and saprotrophy using a time-calibrated phylogenetic framework that reveals linked and drastic shifts in diversification rates of each kingdom. Fungal colonization of land was associated with at least two origins of terrestrial green algae and preceded embryophytes (as evidenced by losses of fungal flagellum, ca. 720 Ma), likely facilitating terrestriality through endomycorrhizal and possibly endophytic symbioses. The largest radiation of fungi (Leotiomyceta), the origin of arbuscular mycorrhizae, and the diversification of extant embryophytes occurred ca. 480 Ma. This was followed by the origin of extant lichens. Saprotrophic mushrooms diversified in the Late Paleozoic as forests of seed plants started to dominate the landscape. The subsequent diversification and explosive radiation of Agaricomycetes, and eventually of ectomycorrhizal mushrooms, were associated with the evolution of Pinaceae in the Mesozoic, and establishment of angiosperm-dominated biomes in the Cretaceous.

Organellar phylogenomics inform systematics in the green algal family Hydrodictyaceae (Chlorophyceae) and provide clues to the complex evolutionary history of plastid genomes in the green algal tree of life.

PREMISE OF THE STUDY: Phylogenomic analyses across the green algae are resolving relationships at the class, order, and family levels and highlighting dynamic patterns of evolution in organellar genomes. Here we present a within-family phylogenomic study to resolve genera and species relationships in the family Hydrodictyaceae (Chlorophyceae), for which poor resolution in previous phylogenetic studies, along with divergent morphological traits, have precluded taxonomic revisions. METHODS: Complete plastome sequences and mitochondrial protein-coding gene sequences were acquired from representatives of the Hydrodictyaceae using next-generation sequencing methods. Plastomes were characterized, and gene order and content were compared with plastomes spanning the Sphaeropleales. Single-gene and concatenated-gene phylogenetic analyses of plastid and mitochondrial genes were performed. KEY RESULTS: The Hydrodictyaceae contain the largest sphaeroplealean plastomes thus far fully sequenced. Conservation of plastome gene order within Hydrodictyaceae is striking compared with more dynamic patterns revealed across Sphaeropleales. Phylogenetic analyses resolve Hydrodictyon sister to a monophyletic Pediastrum, though the morphologically distinct P. angulosum and P. duplex continue to be polyphyletic. Analyses of plastid data supported the neochloridacean genus Chlorotetraëdron as sister to Hydrodictyaceae, while conflicting signal was found in the mitochondrial data. CONCLUSIONS: A phylogenomic approach resolved within-family relationships not obtainable with previous phylogenetic analyses. Denser taxon sampling across Sphaeropleales is necessary to capture patterns in plastome evolution, and further taxa and studies are needed to fully resolve the sister lineage to Hydrodictyaceae and polyphyly of Pediastrum angulosum and P. duplex.

The terrestrial green macroalga Prasiola calophylla (Trebouxiophyceae, Chlorophyta): ecophysiological performance under water-limiting conditions.

The phylogenetic placement of Prasiola calophylla, from an anthropogenic habitat previously shown to contain a novel UV sunscreen compound, was confirmed by analysis of its rbcL gene. This alga has the capacity to tolerate strong water-limiting conditions. The photosynthetic performance and ultrastructural changes under desiccation and osmotic stress were investigated. Freshly harvested thalli showed an effective quantum yield of PSII [Y(II)] of 0.52 ± 0.06 that decreased to ∼60% of the initial value at 3000 mM sorbitol, and 4000 mM sorbitol led to a complete loss of Y(II). The Y(II) of thalli exposed to controlled desiccating conditions at 60% relative humidity (RH) ceased within 240 min, whereas zero values were reached after 120 min at 20% RH. All investigated samples completely recovered Y(II) within ∼100 min after rehydration. Relative electron transport rates (rETR) were temperature dependent, increasing from 5, 10, to 25 °C but strongly declining at 45 °C. Transmission electron microscopy of samples desiccated for 2.5 h showed an electron dense appearance of the entire cytoplasm when compared to control samples. Thylakoid membranes were still visible in desiccated cells, corroborating the ability to recover. Control and desiccated cells contained numerous storage lipids and starch grains, providing reserves. Overall, P. calophylla showed a high capacity to cope with water-limiting conditions on a physiological and structural basis. A lipophilic outer layer of the cell walls might contribute to reduce water evaporation in this poikilohydric organism.

Hiding in plain sight: Koshicola spirodelophila gen. et sp. nov. (Chaetopeltidales, Chlorophyceae), a novel green alga associated with the aquatic angiosperm Spirodela polyrhiza.

PREMISE OF THE STUDY: Discovery and morphological characterization of a novel epiphytic aquatic green alga increases our understanding of Chaetopeltidales, a poorly known order in Chlorophyceae. Chloroplast genomic data from this taxon reveals an unusual architecture previously unknown in green algae. METHODS: Using light and electron microscopy, we characterized the morphology and ultrastructure of a novel taxon of green algae. Bayesian phylogenetic analyses of nuclear and plastid genes were used to test the hypothesized membership of this taxon in order Chaetopeltidales. With next-generation sequence data, we assembled the plastid genome of this novel taxon and compared its gene content and architecture to that of related species to further investigate plastid genome traits. KEY RESULTS: The morphology and ultrastructure of this alga are consistent with placement in Chaetopeltidales (Chlorophyceae), but a distinct trait combination supports recognition of this alga as a new genus and species-Koshicola spirodelophila gen. et sp. nov. Its placement in the phylogeny as a descendant of a deep division in the Chaetopeltidales is supported by analysis of molecular data sets. The chloroplast genome is among the largest reported in green algae and the genes are distributed on three large (rather than a single) chromosome, in contrast to other studied green algae. CONCLUSIONS: The discovery of Koshicola spirodelophila gen. et sp. nov. highlights the importance of investigating even commonplace habitats to explore new microalgal diversity. This work expands our understanding of the morphological and chloroplast genomic features of green algae, and in particular those of the poorly studied Chaetopeltidales.

Estimating Bayesian Phylogenetic Information Content.

Measuring the phylogenetic information content of data has a long history in systematics. Here we explore a Bayesian approach to information content estimation. The entropy of the posterior distribution compared with the entropy of the prior distribution provides a natural way to measure information content. If the data have no information relevant to ranking tree topologies beyond the information supplied by the prior, the posterior and prior will be identical. Information in data discourages consideration of some hypotheses allowed by the prior, resulting in a posterior distribution that is more concentrated (has lower entropy) than the prior. We focus on measuring information about tree topology using marginal posterior distributions of tree topologies. We show that both the accuracy and the computational efficiency of topological information content estimation improve with use of the conditional clade distribution, which also allows topological information content to be partitioned by clade. We explore two important applications of our method: providing a compelling definition of saturation and detecting conflict among data partitions that can negatively affect analyses of concatenated data. [Bayesian; concatenation; conditional clade distribution; entropy; information; phylogenetics; saturation.].

Comparative analyses of chloroplast genome data representing nine green algae in Sphaeropleales (Chlorophyceae, Chlorophyta).

The chloroplast genomes of green algae are highly variable in their architecture. In this article we summarize gene content across newly obtained and published chloroplast genomes in Chlorophyceae, including new data from nine of species in Sphaeropleales (Chlorophyceae, Chlorophyta). We present genome architecture information, including genome synteny analysis across two groups of species. Also, we provide a phylogenetic tree obtained from analysis of gene order data for species in Chlorophyceae with fully sequenced chloroplast genomes. Further analyses and interpretation of the data can be found in "Chloroplast phylogenomic data from the green algal order Sphaeropleales (Chlorophyceae, Chlorophyta) reveal complex patterns of sequence evolution" (Fucíková et al., In review) [1].

Chloroplast phylogenomic data from the green algal order Sphaeropleales (Chlorophyceae, Chlorophyta) reveal complex patterns of sequence evolution.

Chloroplast sequence data are widely used to infer phylogenies of plants and algae. With the increasing availability of complete chloroplast genome sequences, the opportunity arises to resolve ancient divergences that were heretofore problematic. On the flip side, properly analyzing large multi-gene data sets can be a major challenge, as these data may be riddled with systematic biases and conflicting signals. Our study contributes new data from nine complete and four fragmentary chloroplast genome sequences across the green algal order Sphaeropleales. Our phylogenetic analyses of a 56-gene data set show that analyzing these data on a nucleotide level yields a well-supported phylogeny - yet one that is quite different from a corresponding amino acid analysis. We offer some possible explanations for this conflict through a range of analyses of modified data sets. In addition, we characterize the newly sequenced genomes in terms of their structure and content, thereby further contributing to the knowledge of chloroplast genome evolution.

Desiccation tolerance in the chlorophyte green alga Ulva compressa: does cell wall architecture contribute to ecological success?

MAIN CONCLUSION: Desiccation leads to structural changes of the inner pectic cell wall layers in Ulva compressa. This contributes to protection against mechanical damage due to desiccation-rehydration cycles. Ulva compressa, characterized by rbcL phylogeny, is a common species in the Mediterranean Sea. Ulva as an intertidal species tolerates repeated desiccation-rehydration cycles in nature; the physiological and structural basis were investigated under experimental conditions here. Desiccation to 73% relative water content (RWC) led to a significant decrease of the maximum quantum yield of photosystem II (F v/F m) to about half of the initial value. A reduction to 48 or 27% RWC caused a more drastic effect and thalli were only able to recover fully from desiccation to 73% RWC. Relative electron transport rates were stimulated at 73% RWC, but decreased significantly at 48 and 27% RWC, respectively. Imaging-PAM analysis demonstrated a homogenous desiccation process within individual thallus discs. The different cell wall layers of U. compressa were characterized by standard staining procedures, i.e. calcofluor white and aniline blue for structural components (cellulose, callose), ruthenium red for pectins and toluidine blue for acidic polysaccharides. Already a reduction to 73% RWC caused severe changes of the cell walls. The inner pectin-rich layers followed the shrinkage process of the cytoplasm, while the outer denser fibrillar layers maintained their shape. In this way, the thalli were not plasmolyzed during water loss, and upon recovery not negatively influenced by any mechanical damage. Transmission electron microscopy corroborated the arrangement of the different layers clearly distinguishable by their texture and electron density. We suggest the flexibility of the pectin-rich cell wall layers as a major contribution to desiccation tolerance in Ulva.

Chodatodesmus australis sp. nov. (Scenedesmaceae, Chlorophyta) from Antarctica, with the emended description of the genus Chodatodesmus, and circumscription of Flechtneria rotunda gen. et sp. nov.

The family Scenedesmaceae is a taxonomically complicated group due to its simple morphology, high phenotypic plasticity, and the presence of cryptic taxa. Over the years several taxonomic revisions, based on molecular data, affected the family. Here, we describe a new scenedesmacean species from Antarctica, Chodatodesmus australis, based on phylogenetic analyses of data from nuclear (ITS2 spacer, 18S rDNA), and plastid (rbcL, tufA) markers. Morphological (LM and SEM) and ultrastructural (TEM) observations, carried out both on the holotype of C. australis and on the generitype of Chodatodesmus, allow us to emend the original generic description of this genus. Our molecular and phylogenetic data also reveal the existence of a new monotypic genus, Flechtneria, inside the family Scenedesmaceae and lead to the taxonomic reassignment of some microalgal strains available in International Culture Collections to new taxa. Of the considered genomic regions, the tufA gene was the easiest to amplify and sequence and it showed the highest phylogenetic signal, even if the number of sequences already available for this marker in the public databases was considerably lower than for the other chosen loci. The rbcL gene also provided good phylogenetic signal, but its amplification and sequencing were generally more problematic. The nuclear markers gave lower phylogenetic signals, but the 18S rDNA allowed distinction at the genus level and the ITS2 spacer had the advantage that secondary structures could be considered in the analyses. The use of more than one molecular locus is suggested to obtain reliable results in the characterization of scenedesmacean strains.

Gene arrangement convergence, diverse intron content, and genetic code modifications in mitochondrial genomes of sphaeropleales (chlorophyta).

The majority of our knowledge about mitochondrial genomes of Viridiplantae comes from land plants, but much less is known about their green algal relatives. In the green algal order Sphaeropleales (Chlorophyta), only one representative mitochondrial genome is currently available-that of Acutodesmus obliquus. Our study adds nine completely sequenced and three partially sequenced mitochondrial genomes spanning the phylogenetic diversity of Sphaeropleales. We show not only a size range of 25-53 kb and variation in intron content (0-11) and gene order but also conservation of 13 core respiratory genes and fragmented ribosomal RNA genes. We also report an unusual case of gene arrangement convergence in Neochloris aquatica, where the two rns fragments were secondarily placed in close proximity. Finally, we report the unprecedented usage of UCG as stop codon in Pseudomuriella schumacherensis. In addition, phylogenetic analyses of the mitochondrial protein-coding genes yield a fully resolved, well-supported phylogeny, showing promise for addressing systematic challenges in green algae.