Phylogeny of Klebsormidium (Kelbsormidiales; Charophyta) Based on rbcL,atpB, coxIII and nad5 Sequence Data

The charophycean order Klebsormidiales consists of two unbranched filamentous lineages, the fresh water Entransia and the semiterrestrial Klebsormidium. Molecular data have supported the close relationship Klebsormidiales have with land plants. A monograph exists for the European species of Klebsormidium; this monograph provides a cladistic analysis of morphological characteristics for eight Klebsormidium species, but a species level molecular phylogenetic analysis has not yet been performed for the group. We have obtained 50 strains of Klebsormidium, representing 11 morphological species, both from nature and from culture collections. Phylogenetic analyses using rbcL, atpB, coxIII and nad5 gene sequence data from these strains and 40 outgroup sequences suggest that several traditional Klebsormidium species may not be monophyletic.     

PHYLOGENY OF THE BASAL LINEAGES OF STREPTOPHYTA BASED ON RBCL AND ATPB GENE SEQUENCE DATA

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh‐water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbcL (the genes encoding the large subunit of rubisco) and atpB (the beta‐subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus, the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

THE PINGUIOPHYCEAE CLASSIS NOVA, CHROMOPHYTE ALGAE PRODUCING LARGE AMOUNTS OF OMEGA-3 FATTY ACIDS

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh-water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbc L (the genes encoding the large subunit of rubisco) and atp B (the beta-subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus , the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

The complete chloroplast DNA sequences of the charophycean green algae <Emphasis Type="Italic">Staurastrum</Emphasis> and <Emphasis Type="Italic">Zygnema</Emphasis> reveal that the chloroplast genome underwent extensive changes during the evolution of the Zygnematales

Background  

The Streptophyta comprise all land plants and six monophyletic groups of charophycean green algae. Phylogenetic analyses of four genes from three cellular compartments support the following branching order for these algal lineages: Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales, with the last lineage being sister to land plants. Comparative analyses of the Mesostigma viride (Mesostigmatales) and land plant chloroplast genome sequences revealed that this genome experienced many gene losses, intron insertions and gene rearrangements during the evolution of charophyceans. On the other hand, the chloroplast genome of Chaetosphaeridium globosum (Coleochaetales) is highly similar to its land plant counterparts in terms of gene content, intron composition and gene order, indicating that most of the features characteristic of land plant chloroplast DNA (cpDNA) were acquired from charophycean green algae. To gain further insight into when the highly conservative pattern displayed by land plant cpDNAs originated in the Streptophyta, we have determined the cpDNA sequences of the distantly related zygnematalean algae Staurastrum punctulatum and Zygnema circumcarinatum.     

Amino Acid Compositional Shifts During Streptophyte Transitions to Terrestrial Habitats

Across the streptophyte lineage, which includes charophycean algae and embryophytic plants, there have been at least four independent transitions to the terrestrial habitat. One of these involved the evolution of embryophytes (bryophytes and tracheophytes) from a charophycean ancestor, while others involved the earliest branching lineages, containing the monotypic genera Mesostigma and Chlorokybus, and within the Klebsormidiales and Zygnematales lineages. To overcome heat, water stress, and increased exposure to ultraviolet radiation, which must have accompanied these transitions, adaptive mechanisms would have been required. During periods of dehydration and/or desiccation, proteomes struggle to maintain adequate cytoplasmic solute concentrations. The increased usage of charged amino acids (DEHKR) may be one way of maintaining protein hydration, while increased use of aromatic residues (FHWY) protects proteins and nucleic acids by absorbing damaging UV, with both groups of residues thought to be important for the stabilization of protein structures. To test these hypotheses we examined amino acid sequences of orthologous proteins representing both mitochondrion- and plastid-encoded proteomes across streptophytic lineages. We compared relative differences within categories of amino acid residues and found consistent patterns of amino acid compositional fluxuation in extra-membranous regions that correspond with episodes of terrestrialization: positive change in usage frequency for residues with charged side-chains, and aromatic residues of the light-capturing chloroplast proteomes. We also found a general decrease in the usage frequency of hydrophobic, aliphatic, and small residues. These results suggest that amino acid compositional shifts in extra-membrane regions of plastid and mitochondrial proteins may represent biochemical adaptations that allowed green plants to colonize the land.     

ASSESSING PHYLOGENETIC AFFINITIES AND SPECIES DELIMITATIONS IN KLEBSORMIDIALES (STREPTOPHYTA): NUCLEAR‐ENCODED rDNA PHYLOGENIES AND ITS SECONDARY STRUCTURE MODELS IN KLEBSORMIDIUM,HORMIDIELLA, AND ENTRANSIA1

Nuclear‐encoded SSU, group I intron, and internal transcribed spacer (ITS) rDNA sequences were obtained for 16 strains of green algae representing species of Klebsormidium, Hormidiella attenuata, and Entransia fimbriata (for taxonomic authorities, see Table S1 in the supplementary material). The SSU phylogeny resolved a well‐supported clade Klebsormidiales in the Streptophyta that comprised authentic Klebsormidium isolates described recently in a monograph by G. M. Lokhorst and various strains from culture collections. The H. attenuata and En. fimbriata pair was the sister group of Klebsormidium. Certain isolates from culture collections previously identified as “Klebsormidium” emerged as Trebouxiophyceae. Strains assigned to Koliella, Gloeotila, and Stichococcus previously allied with Klebsormidium because of shared morphological and ultrastructural characteristics also belonged to Trebouxiophyceae. Group I introns inserted at Escherichia coli position 516 were found in K. nitens and SAG strain 384‐1, and at position 1506 in H. attenuata and En. fimbriata. Introns were not observed in other Klebsormidiales. Unambiguous alignment of ITS regions of Klebsormidiales was only possible after thermodynamic folding had predicted eight conserved helical domains. The ITS phylogeny provided support for five of the morphospecies recognized by Lokhorst (K. flaccidum, K. elegans, K. bilatum, K. crenulatum, K. mucosum), but the sequences of K. dissectum, K. fluitans, and K. nitens formed an unresolved clade. The species with the earliest origin in the Klebsormidium phylogeny was K. flaccidum. The incongruence between Lokhorst’s morphology‐based cladograms and the ITS phylogenies demonstrated the need for a critical reappraisal of the taxonomy and the morphological and molecular species concept in Klebsormidium on the basis of a more extensive taxonomic and geographic sampling strategy.     

The charophycean green algae provide insights into the early origins of plant cell walls

Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.     

Origin of land plants: Do conjugating green algae hold the key?

Background  

The terrestrial habitat was colonized by the ancestors of modern land plants about 500 to 470 million years ago. Today it is widely accepted that land plants (embryophytes) evolved from streptophyte algae, also referred to as charophycean algae. The streptophyte algae are a paraphyletic group of green algae, ranging from unicellular flagellates to morphologically complex forms such as the stoneworts (Charales). For a better understanding of the evolution of land plants, it is of prime importance to identify the streptophyte algae that are the sister-group to the embryophytes. The Charales, the Coleochaetales or more recently the Zygnematales have been considered to be the sister group of the embryophytes However, despite many years of phylogenetic studies, this question has not been resolved and remains controversial.     

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.     

COLEOCHAETE AND THE ORIGIN OF LAND PLANTS

For a century the green alga Coleochaete has figured prominently in considerations of the origins of land plants (embryophytes). Certain of its advanced features contributed to Bower's (1908) theories on the origin of the land plant sporophyte by intercalation. Though Bower's ideas were disputed in later years, recent investigations of Coleochaete and other green algae have lent strong support to them. At present it appears that further study of Coleochaete and other charophycean algae may contribute much to our understanding of how a number of plant features, including reproductive ones, originated.     

A clade uniting the green algae Mesostigma viride and Chlorokybus atmophyticus represents the deepest branch of the Streptophyta in chloroplast genome-based phylogenies

Background  

The Viridiplantae comprise two major phyla: the Streptophyta, containing the charophycean green algae and all land plants, and the Chlorophyta, containing the remaining green algae. Despite recent progress in unravelling phylogenetic relationships among major green plant lineages, problematic nodes still remain in the green tree of life. One of the major issues concerns the scaly biflagellate Mesostigma viride, which is either regarded as representing the earliest divergence of the Streptophyta or a separate lineage that diverged before the Chlorophyta and Streptophyta. Phylogenies based on chloroplast and mitochondrial genomes support the latter view. Because some green plant lineages are not represented in these phylogenies, sparse taxon sampling has been suspected to yield misleading topologies. Here, we describe the complete chloroplast DNA (cpDNA) sequence of the early-diverging charophycean alga Chlorokybus atmophyticus and present chloroplast genome-based phylogenies with an expanded taxon sampling.     

ULTRASTRUCTURE OF MITOSIS AND CYTOKINESIS IN KLEBSORMIDIUM MUCOSUM NOV. COMB., FORMERLY ULOTHRIX VERRUCOSA (CHLOROPHYTA)1

A transmission electron microscopy study of dividing cells of Ulothrix verrucosa Lokhorst has provided clear evidence that this species differs in many respects from other Ulothrix Kützing species. These differences include the presence of a microtubular sheath around the prophase nucleus, the complete disintegration of the nuclear envelope coinciding with the proliferation of extranuclear microtubules into the prometaphase nucleus and the intrusion of vacuoles into the interzonal spindle region in between the widely separated telophase nuclei. This necessitates the transfer of Ulothrix verrucosa to the charophycean genus Klebsormidium Silva, Mattox and Blackwell. The new combination Klebsormidium mucosum is proposed. On account of its mitotic pattern, this species can be placed in the (charophycean) evolutionary line towards the higher plants. However, because of its cytokinesis (annular centripetal ingrowth of the plasmalemma) this species probably should be considered as a blind offshoot of this line. It is emphasized that furrowing green algae with a persistent interzonal spindle at telophase (including the presently studied alga) often show an ill-defined cytokinetic microtubular system.     

Re-examination of ultrastructures of the stellate chloroplast organization in brown algae: Structure and development of pyrenoids

Some taxa of brown algae have a so‐called ‘stellate’ chloroplast arrangement composed of multiple chloroplasts arranged in a stellate configuration, or else a single chloroplast with radiating lobes. The fine structures of chloroplasts and pyrenoids have been studied, but the details of their membrane configurations as well as pyrenoid ontogeny have not been well understood. The ultrastructure of the single stellate chloroplast in Splachnidium rugosum and Scytothamnus australis were re‐examined in the present study, as well as the stellate arrangement of chloroplasts in Asteronema ferruginea and Asterocladon interjectum, using freeze‐substitution fixation. It was confirmed that the chloroplast envelope invaginated into the pyrenoid in Splachnidium rugosum, Scytothamnus australis and Asteronema ferruginea, but chloroplast endoplasmic reticulum (CER) remained on the surface of the chloroplast. The space between the invaginated chloroplast envelope and CER was filled with electron‐dense material. In Asteronema ferruginea, CER surrounding each pyrenoid was closely appressed to the neighboring CER over the pyrenoids, so that the chloroplasts formed a stellate configuration; however, in the apical cells chloroplasts formed two or more loose groups, or were completely dispersed. The pyrenoids of Asterocladon interjectum did not have any invagination of the chloroplast envelope, but a unique membranous sac surrounded the pyrenoid complex and occasionally other organelles (e.g. mitochondria). Immunolocalization of β‐1,3‐glucans showed that the membranous sac in Asterocladon interjectum did not contain photosynthetic products such as chrysolaminaran. Observations in the dividing cells of Splachnidium rugosum and Scytothamnus australis indicated that the pyrenoid in the center of the chloroplast enlarged and divided into two before or during chloroplast division.     

The Transition From Algae to Embryophytes: Chloroplast Phylogenomic Evidence (II)

The transition of plant life from aquatic algae to land plants was one of the major events in the history of life. However, in hypothesizing the evolutionary path of the transition, limited shared phenotypic characters in aquatic algae and land plants (embryophytes) have been a major hinderance. Chloroplast genomes contain characters useful in tracing evolutionary histories. Embryophyte chloroplast genomes are distinguished from algal cpDNAs by the presence of over 20 group II introns and three ribosomal protein operons (rpl23, clpP and 3?rps12 operons). These phylogenomic features indicate a phylogenetic relationship of charophytes and embryophytes. In addition to these operons and introns, the evolution of rRNA and psbB operon evolution of streptophyte lineages will be incorporated with major biological phenotypic features to produce a phylogenetic tree. Basal embryophytes, the antithetic hypothesis, monophyly of embryophytes, and paraphyly of charophytes will be discussed. Strepotophytes are classified into three major groups (basal streptophytes, mid‐divergent streptophytes and late divergent charophytes‐embryophytes).     

DISTRIBUTION,MORPHOLOGY, AND PHYLOGENY OF KLEBSORMIDIUM (KLEBSORMIDIALES,CHAROPHYCEAE) IN URBAN ENVIRONMENTS IN EUROPE1

Klebsormidium is a cosmopolitan genus of green algae, widespread in terrestrial and freshwater habitats. The classification of Klebsormidium is entirely based on morphological characters, and very little is understood about its phylogeny at the species level. We investigated the diversity and phylogenetic relationships of Klebsormidium in urban habitats in Europe by a combination of approaches including examination of field‐collected material, culture experiments conducted in many different combinations of factors, and phylogenetic analyses of the rbcL gene. Klebsormidium in European cities mainly occurs at the base of old walls, where it may produce green belts up to several meters in extent. Specimens from different cities showed a great morphological uniformity, consisting of long filaments 6–9 μm in width, with thin‐walled cylindrical cells and smooth wall, devoid of false branches, H‐shaped pieces, and biseriate parts. Conversely, the rbcL phylogeny showed a higher genetic diversity than expected from morphology. The strains were separated in four different clades supported by high bootstrap values and posterior probabilities. In culture, these clades differed in several characters, such as production of a superficial hydro‐repellent layer, tendency to break into short fragments, and inducibility of zoosporulation. On the basis of the taxonomic information available in the literature, most strains could not be identified unambiguously at the species level. The rbcL phylogeny showed no correspondence with classification based on morphology and suggested that the identity of many species, in particular the type species K. flaccidum (kütz.) P.C. Silva, Mattox et W. H. Blackw., needs critical reassessment.     

Molecular phylogeny of Zeacarpa (Ralfsiales,Phaeophyceae) proposing a new family Zeacarpaceae and its transfer to Nemodermatales

Zeacarpa leiomorpha is a crustose brown alga endemic to South Africa. The species has been tentatively placed in Ralfsiaceae, but its ordinal assignment has been uncertain. The molecular phylogeny of brown algae based on concatenated DNA sequences of seven chloroplast and mitochondrial gene sequences (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1) of taxa covering most of the orders revealed the most related phylogenetic relationship of Z. leiomorpha to Nemoderma tingitanum (Nemodermatales) rather than Ralfsiaceae (Ralfsiales). Morphologically, Zeacarpa and Nemoderma share crustose thallus structure and multiple discoidal chloroplasts without pyrenoids in each cell, however, the formation of lateral unilocular zoidangia in tufts in loose upright filaments in Zeacarpa is distinctive in brown algae. Considering the relatively distant genetic divergence between the two taxa, comparable to that among families or orders in representative brown algae, in addition to the above‐mentioned unique morphological features, we propose the classification of Zeacarpa in a new family Zeacarpaceae in the order Nemodermatales.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

Morphology and ultrastructure of Interfilum and Klebsormidium (Klebsormidiales,Streptophyta) with special reference to cell division and thallus formation

Representatives of the closely related genera, Interfilum and Klebsormidium, are characterized by unicells, dyads or packets in Interfilum and contrasting uniseriate filaments in Klebsormidium. According to the literature, these distinct thallus forms originate by different types of cell division, sporulation (cytogony) versus vegetative cell division (cytotomy), but investigations of their morphology and ultrastructure show a high degree of similarity. Cell walls of both genera are characterized by triangular spaces between cell walls of neighbouring cells and the parental wall or central space among the walls of a cell packet, exfoliations and projections of the parental wall and cap-like and H-like fragments of the cell wall. In both genera, each cell has its individual cell wall and it also has part of the common parental wall or its remnants. Therefore, vegetative cells of Interfilum and Klebsormidium probably divide by the same type of cell division (sporulation-like). Various strains representing different species of the two genera are characterized by differences in cell wall ultrastructure, particularly the level of preservation, rupture or gelatinization of the parental wall surrounding the daughter cells. The differing morphologies of representatives of various lineages result from features of the parental wall during cell separation and detachment. Cell division in three planes (usual in Interfilum and a rare event in Klebsormidium) takes place in spherical or short cylindrical cells, with the chloroplast positioned perpendicularly or obliquely to the filament (dyad) axis. The morphological differences are mainly a consequence of differing fates of the parental wall after cell division and detachment. The development of different morphologies within the two genera mostly depends on characters such as the shape of cells, texture of cell walls, mechanical interactions between cells and the influence of environmental conditions.     

The chloroplast genome sequence of Chara vulgaris sheds new light into the closest green algal relatives of land plants

The phylum Streptophyta comprises all land plants and six monophyletic groups of charophycean green algae (Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales, and Charales). Phylogenetic analyses of four genes encoded in three cellular compartments suggest that the Charales are sister to land plants and that charophycean green algae evolved progressively toward an increasing cellular complexity. To validate this phylogenetic hypothesis and to understand how and when the highly conservative pattern displayed by land plant chloroplast DNAs (cpDNAs) originated in the Streptophyta, we have determined the complete chloroplast genome sequence (184,933 bp) of a representative of the Charales, Chara vulgaris, and compared this genome to those of Mesostigma (Mesostigmatales), Chlorokybus (Chlorokybales), Staurastrum and Zygnema (Zygnematales), Chaetosphaeridium (Coleochaetales), and selected land plants. The phylogenies we inferred from 76 cpDNA-encoded proteins and genes using various methods favor the hypothesis that the Charales diverged before the Coleochaetales and Zygnematales. The Zygnematales were identified as sister to land plants in the best tree topology (T1), whereas Chaetosphaeridium (T2) or a clade uniting the Zygnematales and Chaetosphaeridium (T3) occupied this position in alternative topologies. Chara remained at the same basal position in trees including more land plant taxa and inferred from 56 proteins/genes. Phylogenetic inference from gene order data yielded two most parsimonious trees displaying the T1 and T3 topologies. Analyses of additional structural cpDNA features (gene order, gene content, intron content, and indels in coding regions) provided better support for T1 than for the topology of the above-mentioned four-gene tree. Our structural analyses also revealed that many of the features conserved in land plant cpDNAs were inherited from their green algal ancestors. The intron content data predicted that at least 15 of the 21 land plant group II introns were gained early during the evolution of streptophytes and that a single intron was acquired during the transition from charophycean green algae to land plants. Analyses of genome rearrangements based on inversions predicted no alteration in gene order during the transition from charophycean green algae to land plants.