The chloroplast genome sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) reveals unusual structural features and new insights into the branching order of chlorophyte lineages

One major lineage of green plants, the Chlorophyta, is represented by the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae. The Prasinophyceae occupies the most basal position in the Chlorophyta, but the branching order of the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae remains unresolved. The chloroplast genome sequences currently available for representatives of three chlorophyte classes have revealed that this genome is highly plastic, with Chlamydomonas (Chlorophyceae) and Chlorella (Trebouxiophyceae) showing fewer ancestral features than Nephroselmis (Prasinophyceae). We report the 195,867-bp chloroplast DNA (cpDNA) sequence of Pseudendoclonium akinetum (Ulvophyceae), a member of the class that has not been previously examined for detailed cpDNA analysis. This genome shares common evolutionary trends with its Chlorella and Chlamydomonas homologs. The gene content, number of ancestral gene clusters, and abundance of short dispersed repeats in Pseudendoclonium cpDNA are intermediate between those observed for Chlorella and Chlamydomonas cpDNAs. Although Pseudendoclonium cpDNA features a large inverted repeat, its quadripartite structure is unusual in displaying an rRNA operon transcribed toward the large single-copy (LSC) region and a small single-copy region containing 14 genes that are normally found in the LSC region. Twenty-seven group I introns lie in nine genes and fall within four subgroups (IA1, IA2, IA3, and IB); 19 encode putative homing endonucleases, and 7 have homologs at identical insertion sites in other chlorophyte or streptophyte organelle genomes. The high similarity observed among the 14 IA1 and 7 IA2 introns and their encoded endonucleases suggests that many introns arose from intragenomic proliferation of a few founding introns in the lineage leading to Pseudendoclonium. Interestingly, one intron (in atpA) and some of the dispersed repeats also reside in Pseudendoclonium mitochondria, providing strong evidence for interorganellar lateral transfer of these genetic elements. Phylogenetic analyses of 58 cpDNA-encoded proteins and genes support the hypothesis that the Ulvophyceae is sister to the Trebouxiophyceae but cannot eliminate the hypothesis that the Ulvophyceae is sister to the Chlorophyceae. We favor the latter hypothesis because it is strongly supported by phylogenetic analyses of gene order data and by independent structural evidence based on shared gene losses and rearrangement break points within ancestrally conserved gene clusters.     

The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

Background  

The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA) sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae), in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR) featuring an inverted rRNA operon and a small single-copy (SSC) region containing 14 genes normally found in the large single-copy (LSC) region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage.     

The flagellar apparatus structure inMicrospora (Chlorophyceae) confirms a close evolutionary relationship with unicellular green algae

The spatial configuration of the flagellar apparatus of the biflagellate zoospores of the green algal genusMicrospora is reconstructed by serial sectioning analysis using transmission electron microscopy. Along with the unequal length of the flagella, the most remarkable characteristics of the flagellar apparatus are: (1) the subapical emergence of the flagella (especially apparent with scanning electron microscopy); (2) the parallel orientation of the two basal bodies which are interconnected by a prominent one-piece distal connecting fiber; (3) the unique ultrastructure of the distal connecting fiber composed of a central tubular region which is bordered on both sides by a striated zone; (4) the different origin of the d-rootlets from their relative basal bodies; (5) the asymmetry of the papillar region which together with the subapical position of the basal bodies apparently cause the different paths of corresponding rootlets in the zoospore anterior; (6) the presence of single-membered d-rootlets and multi-membered s-rootlets resulting in a 7-1-7-1 cruciate microtubular root system which, through the different rootlet origin, does not exhibit a strict 180° rotational symmetry. It is speculated that the different basal body origin of the d-rootlets is correlated with the subapical implant of flagella. It is further hypothesized that in the course of evolution the ancestors ofMicrospora had a flagellar papilla that has migrated from a strictly apical position towards a subapical position. Simultaneously, ancestral shift of flagella along the apical cell body periphery has taken place as can be concluded from the presence of an upper flagellum overlying a lower flagellum in the flagellar apparatus ofMicrospora. The basic features of the flagellar apparatus of theMicrospora zoospore resemble those of the coccoid green algal generaDictyochloris andBracteacoccus and also those of the flagellate green algal genusHeterochlamydomonas. This strengthens the general supposition thatMicrospora is evolutionarily closely related to taxa which were formerly classified in the traditionalChlorococcales.     

The complete mitochondrial DNA sequence of Mesostigma viride identifies this green alga as the earliest green plant divergence and predicts a highly compact mitochondrial genome in the ancestor of all green plants.

To gain insights into the nature of the mitochondrial genome in the common ancestor of all green plants, we have completely sequenced the mitochondrial DNA (mtDNA) of Mesostigma viride. This green alga belongs to a morphologically heterogeneous class (Prasinophyceae) that includes descendants of the earliest diverging green plants. Recent phylogenetic analyses of ribosomal RNAs (rRNAs) and concatenated proteins encoded by the chloroplast genome identified Mesostigma as a basal branch relative to the Streptophyta and the Chlorophyta, the two phyla that were previously thought to contain all extant green plants. The circular mitochondrial genome of Mesostigma resembles the mtDNAs of green algae occupying a basal position within the Chlorophyta in displaying a small size (42,424 bp) and a high gene density (86.6% coding sequences). It contains 65 genes that are conserved in other mtDNAs. Although none of these genes represents a novel coding sequence among green plant mtDNAs, four of them (rps1, sdh3, sdh4, and trnL[caa]) have not been reported previously in chlorophyte mtDNAs, and two others (rpl14 and trnI[gau]) have not been identified in the streptophyte mtDNAs examined so far (land-plant mtDNAs). Phylogenetic analyses of 19 concatenated mtDNA-encoded proteins favor the hypothesis that Mesostigma represents the earliest branch of green plant evolution. Four group I introns (two in rnl and two in cox1) and three group II introns (two in nad3 and one in cox2), two of which are trans-spliced at the RNA level, reside in Mesostigma mtDNA. The insertion sites of the three group II introns are unique to this mtDNA, suggesting that trans-splicing arose independently in the Mesostigma lineage and in the Streptophyta. The few structural features that can be regarded as ancestral in Mesostigma mtDNA predict that the common ancestor of all green plants had a compact mtDNA containing a minimum of 75 genes and perhaps two group I introns. Considering that the mitochondrial genome is much larger in size in land plants than in Mesostigma, we infer that mtDNA size began to increase dramatically in the Streptophyta either during the evolution of charophyte green algae or during the transition from charophytes to land plants.     

Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.     

Extraction of pigments and fatty acids from the green alga Scenedesmus obliquus (Chlorophyceae)

In this paper, the efficiency of pigment and fatty acid extraction from resistant algae using Scenedesmus obliquus as an example was examined. We found that adding quartz sand and solvent to freeze-dried algal material and subsequent extraction in an ultrasound bath for 90min at –4°C resulted in excellent extraction of these compounds. This extraction method was compared with a method regularly used for extraction of fatty acids and pigments, i.e. addition of solvents to algal material with subsequent incubation. Our extraction using the ultrasound and sand method was about twice as efficient as this method for both pigments and fatty acids. The ultrasound method is simple, extracts over 90% of the different substances in one step and conserves the relationships of pigments and fatty acids. In addition, no alteration- or breakdown products were observed with the new method. Thus, this method allows accurate quantitative extraction of both pigments and fatty acids from Scenedesmus obliquus and other algae. The method was also been found to be as effective for Cryptomonas erosa (Cryptophyceae), Cyclotella meneghiniana (Bacillariophyceae), Microcystis aeruginosa (Cyanophyceae), and Staurastrum paradoxum (Chlorophyceae, Desmidiaceae) and is thus applicable to a wide spectrum of algae.     

The complete mitochondrial DNA sequence of the cestode Echinococcus multilocularis (Cyclophyllidea: Taeniidae)

The 13,738 bp mitochondrial DNA from the cestode Echinococcus multilocularis has been sequenced. It contains two major noncoding regions and 36 genes (12 for proteins involved in oxidative phosphorylation, two for rRNAs and 22 for tRNAs) but a gene for ATPase subunit 8 is missing. All genes are transcribed in the same direction. Putative secondary structures of tRNAs indicate that most of them are conventional clover leaves but the dihydrouridine arm is unpaired in tRNA(Ser(AGN)), tRNA(Ser(UCN)), tRNA(Arg) and tRNA(Cys). The base composition at the wobble positions of fourfold degenerate codon families is highly biased toward U and against C.     

The complete nucleotide sequence of Chinese hamster (Cricetulus griseus) mitochondrial DNA.

Mammalian mitochondria contain their own approximately 16.5 kb circular genome. Mitochondrial DNA (mtDNA) encodes for a subset of the proteins involved in the electron transport chain and depletion or mutation of the sequence is implicated in a number of human disease processes. The recent finding is that mitochondrial damage mediates genotoxicity after exposure to chemical carcinogens has focused attention on the role of mtDNA mutations in the development of cancer. Although the entire genome has been sequenced for a number of mammals, only a small fraction of the mtDNA sequence is available for hamsters. We have obtained here the entire 16,284 bp sequence of the Chinese hamster mitochondrial genome, which will enable detailed analysis of mtDNA mutations caused by exposure to mutagens in hamster-derived cell lines.     

Evidence for a facultative mutualist nutritional relationship between the green coccoid alga Bracteacoccus sp. (Chlorophyceae) and the zoosporic fungus Rhizidium phycophilum (Chytridiomycota)

Symbiotic interactions between fungi and photosynthetic partners are common among derived fungal lineages. The only fungal-phototroph interactions thus far reported from the early diverging zoosporic fungi are parasitic in nature. Rhizidium phycophilum is a terrestrial, saprotrophic chytrid, which appears to be able to enter a facultative mutualism with a coccoid green alga in the absence of refractory organic material, such as pollen and chitin. Liquid and solid culturing methods were used in a series of differential fitness experiments in conjunction with microscopic analyses to characterize the interaction between R. phycophilum and the alga. The alga in this partnership is identified as a member of the genus Bracteacoccus. Under certain culturing conditions, algal cells grown in coculture with R. phycophilum were shown to grow larger and more prolifically than when cultured axenically under the same conditions. Additionally, dialysis experiments demonstrate that R. phycophilum does not parasitize Bracteacoccus sp., and can be cultured in media infused with unknown algal exudates. Rhizidium phycophilum and Bracteacoccus sp. represent the first facultative positive interaction between a zoosporic fungus and a photoautotroph and may prove a tractable system for modelling interactions between early fungi and plants.     

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.     

DNA sequence,structure, and phylogenetic relationship of the mitochondrial small-subunit rRNA from the red alga Chondrus crispus (Gigartinales,Rhodophytes)

The entire nucleotide sequence containing the small-subunit ribosomal RNA gene (SSU rRNA) from the mitochondrial genome of Chondrus crispus was determined. To our knowledge, this is the first sequence of a mitochondrial 16S-like rRNA from a red alga. The length of this gene is 1,376 nucleotides. Its secondary structure was constructed and compared with other known secondary structures from eubacteria and from mitochondria of land plants, green and brown algae, and fungi. Phylogenetic trees were built upon SSU rRNA sequence alignment from mitochondria and eubacteria. The results show that rhodophytes and chromophytes provide additional links in the evolution of mitochondria between the green plant lineage and the nonplant lineages.Correspondence to: C. Boyen     

The complete mitochondrial DNA sequence of the harbor seal,Phoca vitulina

Summary The nucleotide sequence of the mitochondrial DNA (mtDNA) of the harbor seal, Phoca vitulina, was determined. The total length of the molecule was 16,826 bp. The organization of the coding regions of the molecule conforms with that of other mammals, but the control region is unusually long. A considerable portion of the control region is made up of short repeats with the motif GTACAC particularly frequent. The two rRNA genes and the 13 peptide-coding genes of the harbor seal, fin whale, cow, human, mouse, and rat were compared and the relationships between the different species assessed. At ordinal level the 12S rRNA gene and 7 out of the 13 peptide-coding genes yielded a congruent topological tree of the mtDNA relationship between the seal, cow, whale, human, and the rodents. In this tree the whale and the cow join first, and this clade is most closely related to the seal.Offprint requests to: Ú. Árnason     

Sensitivity of the surface coat of the halotolerant green alga Dunaliella parva (Volvocales, Chlorophyceae) to lysozyme

The surface coat of Dunaliella parva Lerche was investigated using several techniques. Degradation by several cell lytic enzymes and ultrastructural observation revealed that D. parva has a specialized cell surface structure containing a glycoprotein that is sensitive not only to proteinases but also to lysozyme. This sensitivity was also demonstrated by electrophoresis of the cells and measurement of released glycerol after enzyme treatment. Immunochemical labeling indicated that the surface glycoprotein of D. parva is analogous to pepti-doglycan.     

The complete mitochondrial genome sequence of the little egret (Egretta garzetta)

Many phylogenetic questions in the Ciconiiformes remain unresolved and complete mitogenome data are urgently needed for further molecular investigation. In this work, we determined the complete mitogenome sequence of the little egret (Egretta garzetta). The genome was 17,361 bp in length and the gene organization was typical of other avian mtDNA. In protein-coding genes (PCGs), a C insertion was found in ND3, and COIII and ND4 terminated with incomplete stop codons (T). tRNA-Val and tRNA-Ser (AGY) were unable to fold into canonical cloverleaf secondary structures because they had lost the DHU arms. Long repetitive sequences consisting of five types of tandem repeats were found at the 3′ end of Domain III in the control region. A phylogenetic analysis of 11 species of Ciconiiformes was done using complete mitogenome data and 12 PCGs. The tree topologies obtained with these two strategies were identical, which strongly confirmed the monophyly of Ardeidae, Threskiorothidae and Ciconiidae. The phylogenetic analysis also revealed that Egretta was more closely related to Ardea than to Nycticorax in the Ardeidae, and Platalea was more closely related to Threskiornis than to Nipponia in the Threskiornithidae. These findings contribute to our understanding of the phylogenetic relationships of Ciconiiformes based on complete mitogenome data.     

The complete mitochondrial DNA sequences of Nephroselmis olivacea and Pedinomonas minor. Two radically different evolutionary patterns within green algae.

Green plants appear to comprise two sister lineages, Chlorophyta (classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, and Prasinophyceae) and Streptophyta (Charophyceae and Embryophyta, or land plants). To gain insight into the nature of the ancestral green plant mitochondrial genome, we have sequenced the mitochondrial DNAs (mtDNAs) of Nephroselmis olivacea and Pedinomonas minor. These two green algae are presumptive members of the Prasinophyceae. This class is thought to include descendants of the earliest diverging green algae. We find that Nephroselmis and Pedinomonas mtDNAs differ markedly in size, gene content, and gene organization. Of the green algal mtDNAs sequenced so far, that of Nephroselmis (45,223 bp) is the most ancestral (minimally diverged) and occupies the phylogenetically most basal position within the Chlorophyta. Its repertoire of 69 genes closely resembles that in the mtDNA of Prototheca wickerhamii, a later diverging trebouxiophycean green alga. Three of the Nephroselmis genes (nad10, rpl14, and rnpB) have not been identified in previously sequenced mtDNAs of green algae and land plants. In contrast, the 25,137-bp Pedinomonas mtDNA contains only 22 genes and retains few recognizably ancestral features. In several respects, including gene content and rate of sequence divergence, Pedinomonas mtDNA resembles the reduced mtDNAs of chlamydomonad algae, with which it is robustly affiliated in phylogenetic analyses. Our results confirm the existence of two radically different patterns of mitochondrial genome evolution within the green algae.     

Complete sequence of the yak (Bos grunniens) mitochondrial genome and its evolutionary relationship with other ruminants

The yak (Bos grunniens) is the most important domesticated species in the Qinhai-Tibetan Plateau. In present study, the complete sequence of the yak mitochondrial genome was determined. Sequence analysis revealed that there are no differences with cattle in the yak mitochondrial genome organization. Interestingly, within the D-loop, the conserved sequence blocks are less conserved than surrounding regions. Neighbor-Joining (NJ) trees based on single genes, gene sets and concatenated genes of mitochondrial genome were constructed. The analysis identified the yak as a sister group of a cattle/zebu clade. Based on substitutions in 22 tRNA genes, 12S rRNA gene and 16S rRNA gene, the dating of divergence between yak and cattle/zebu, and yak and water buffalo, was proposed to have occurred 4.38-5.32 and 10.54-13.85 million years before present, respectively. This is consistent with the paleontologyical data. Yak and sheep/goat divergent dating predicts that their divergence occurred at 13.14-27.99 million years before the present day.     

Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.