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.     

Recombination in Mitochondrial DNA of European Mussels <Emphasis Type="Italic">Mytilus</Emphasis>

Mitochondrial DNA was long believed to be purely clonal and free from recombination. Major phylogenetic studies still depend on such assumptions. The peculiar genetic system of marine mussels Mytilus in which two divergent mitochondrial genomes exist provides a unique opportunity to study mtDNA recombination. Previous reports showed the existence of a few haplotypes having very strong recombination signal in the control region of mtDNA. Those recombinant variants have been found in a Baltic Sea population of Mytilus trossulus as well as in Mytilus galloprovincialis from the Black Sea. In both cases the mosaic genomes switched their transmission route and have been inherited paternally. In the present study rearranged mtDNA genomes found in all three European Mytilus species are described. The structure of their control region is a result of intra- and intermolecular recombination between mitochondrial genomes. Together with the phylogenetic reconstruction and geographic distribution, this suggests that two interlineage recombination events have occurred in the control region of mtDNA of European mussels Mytilus. Contrary to earlier observations, some of the mosaic genomes do not show any gender bias, which has important implications regarding the transmission and evolution of blue mussel mitochondrial genomes.     

Nucleotide Content Gradients in Maternally and Paternally Inherited Mitochondrial Genomes of the Mussel <Emphasis Type="Italic">Mytilus</Emphasis>

Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site’s time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (O_H) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (O_L) suggested that the most probable site lies within the ND3 gene. By adopting this O_L position we calculated times of exposure for 0_FD (nondegenerate), 2_FD (twofold degenerate), and 4_FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4_FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2_FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0_FD sites with exposure time, which is explicable as the result of biased usage of 4_FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. David Pollock]     

Genome-wide analysis of the chalcone synthase superfamily genes of <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon–intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.     

Mitochondrial Genome of <Emphasis Type="Italic">Savalia savaglia</Emphasis> (Cnidaria,Hexacorallia) and Early Metazoan Phylogeny

Mitochondrial genomes have recently become widely used in animal phylogeny, mainly to infer the relationships between vertebrates and other bilaterians. However, only 11 of 723 complete mitochondrial genomes available in the public databases are of early metazoans, including cnidarians (Anthozoa, mainly Scleractinia) and sponges. Although some cnidarians (Medusozoa) are known to possess atypical linear mitochondrial DNA, the anthozoan mitochondrial genome is circular and its organization is similar to that of other metazoans. Because the phylogenetic relationships among Anthozoa as well as their relation to other early metazoans still need to be clarified, we tested whether sequencing the complete mitochondrial genome of Savalia savaglia, an anthozoan belonging to the order Zoantharia (=Zoanthidea), could be useful to infer such relationships. Compared to other anthozoans, S. savaglia’s genome is unusually long (20,766 bp) due to the presence of several noncoding intergenic regions (3691 bp). The genome contains all 13 protein coding genes commonly found in metazoans, but like other Anthozoa it lacks most of the tRNAs. Phylogenetic analyses of S. savaglia mitochondrial sequences show Zoantharia branching closely to other Hexacorallia, either as a sister group to Actiniaria or as a sister group to Actiniaria and Scleractinia. The close relationships suggested between Zoantharia and Actiniaria are reinforced by strong similarities in their gene order and the presence of similar introns in the COI and ND5 genes. Our study suggests that mitochondrial genomes can be a source of potentially valuable information on the phylogeny of Hexacorallia and may provide new insights into the evolution of early metazoans. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Axel Meyer]     

New haplotype of the complete mitochondrial genome of <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> and its species-specific DNA markers: distinguishing <Emphasis Type="Italic">C. siamensis</Emphasis> from <Emphasis Type="Italic">C. porosus</Emphasis> in Thailand

Based on molecular phylogeny of available complete mitochondrial DNA (mtDNA) genome sequences reveals that Crocodylus siamensis and C. porosus are closely related species. Yet, the sequence divergence of their mtDNA showed only a few values under conspecific level. In this study, a new haplotype (haplotype2, EF581859) of the complete mtDNA genome of Siamese crocodile (C. siamensis) was determined. The genome organization, which appeared to be highly similar to haplotype1 (DQ353946) mtDNA genome of C. siamensis, was 16,814 bp in length. However, the sequence divergence between the two genomes differed by around 7–10 and 0.7–2.1% for the haplotype1 between C. siamensis and C. porosus (AJ810453). These results were consistent with the phylogenetic relationship among the three genomes, suggesting that C. siamensis haplotype1 mtDNA genome might be the hybrid or the intraspecific variation of C. porosus. On the other hand, our specimen was found to be a true C. siamensis. Simultaneously, the seven species-specific DNA markers designed based on the distinctive site between haplotype2 mtDNA sequences of C. siamensis and haplotype1 mtDNA sequence of C. siamensis–C. porosus were successfully used to distinguish C. siamensis from C. porosus. These effective markers could be used primarily for rapid and accurate species identification in population, ecology and conservation studies.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> and <Emphasis Type="Italic">Ceratodon purpureus</Emphasis>, mosses as model organisms in photosynthesis studies

With the discovery of targeted gene replacement, moss biology has been rapidly advancing over the last 10 years. This study demonstrates the usefulness of moss as a model organism for plant photosynthesis research. The two mosses examined in this study, Physcomitrella patens and Ceratodon purpureus, are easily cultured through vegetative propagation. Growth tests were conducted to determine carbon sources suitable for maintaining heterotrophic growth while photosynthesis was blocked. Photosynthetic parameters examined in these plants indicated that the photosynthetic activity of Ceratodon and Physcomitrella is more similar to vascular plants than cyanobacteria or green algae. Ceratodon plants grown heterotrophically appeared etiolated in that the plants were taller and plastids did not differentiate thylakoid membranes. After returning to the light, the plants developed green, photosynthetically active chloroplasts. Furthermore, UV-induced mutagenesis was used to show that photosynthesis-deficient mutant Ceratodon plants could be obtained. After screening approximately 1000 plants, we obtained a number of mutants, which could be arranged into the following categories: high fluorescence, low fluorescence, fast and slow fluorescence quenching, and fast and slow greening. Our results indicate that in vivo biophysical analysis of photosynthetic activity in the mosses can be carried out which makes both mosses useful for photosynthesis studies, and Ceratodon best sustains perturbations in photosynthetic activity.     

Genome-wide analysis of intronless genes in rice and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Intronless genes, a characteristic feature of prokaryotes, constitute a significant portion of the eukaryotic genomes. Our analysis revealed the presence of 11,109 (19.9%) and 5,846 (21.7%) intronless genes in rice and Arabidopsis genomes, respectively, belonging to different cellular role and gene ontology categories. The distribution and conservation of rice and Arabidopsis intronless genes among different taxonomic groups have been analyzed. A total of 301 and 296 intronless genes from rice and Arabidopsis, respectively, are conserved among organisms representing the three major domains of life, i.e., archaea, bacteria, and eukaryotes. These evolutionarily conserved proteins are predicted to be involved in housekeeping cellular functions. Interestingly, among the 68% of rice and 77% of Arabidopsis intronless genes present only in eukaryotic genomes, approximately 51% and 57% genes have orthologs only in plants, and thus may represent the plant-specific genes. Furthermore, 831 and 144 intronless genes of rice and Arabidopsis, respectively, referred to as ORFans, do not exhibit homology to any of the genes in the database and may perform species-specific functions. These data can serve as a resource for further comparative, evolutionary, and functional analysis of intronless genes in plants and other organisms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The complete mitochondrial genome of the hard clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

Veneridae is a diverse, commercially important, and cosmopolitan family. Here we present the complete mitochondrial genome of the hard clam Meretrix meretrix (Bivalvia: Veneridae). The entire mitochondrial genome (mitogenome) sequence of M. meretrix is 19,826 bp in length, and contains 37 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 23 tRNAs. All genes are encoded on the heavy strand. In contrast to the typical animal mitochondrial genome, it lacks the protein-coding gene ATP8, and has only one copy of the tRNA^Ser gene, but three duplications of the tRNA^Gln, which is the first report among the present molluscan mtDNAs. We observed that the gene arrangement between M. meretrix and M. petechialis is same except one more tRNAGln gene in M. meretrix., and the sequence similarity is as high as 99%, indicating that M. petechialis and M. meretrix could be treated as a junior synonym of M. meretrix. Maximum Likelihood and Bayeslan analysis of 12 concatenated protein-coding amino acid sequences place the Unionidae as a sister group to other bivalves, which reflects the general opinion that the Unionidae deverged very early in Bivalvia evolution.     

In Silico Identification and Evolutionary Analysis of Plant <Emphasis Type="Italic">MAPKK6s</Emphasis>

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. Linking upstream MAPK kinase kinase (MAPKKK) to downstream MAPK, MAPK kinase (MAPKK) plays a crucial role in MAPK cascade. MAPKK6 is one member of the MAPKK family. In this study, we have found that plant MAPKK6 genes are widely distributed in different plant species, including moss, seedless vascular plants, gymnosperms, and angiosperms. However, no MAPKK6 can be found in genomes of algae. Analysis of exon–intron organization and intron phase showed that plant MAPKK6s are highly conserved genes during plant evolution. In Physcomitrella patens, Selaginella moellendorffii, and Picea glauca, MAPKK6s exist as multicopy genes. In most high plants, however, MAPKK6s exist as single-copy. Phylogenetic analysis indicated that the occurrence of single-copy of MAPKK6s in high plants is likely because of genomic copy-number loss.     

Organization of repetitive DNAs and the genomic regions carrying ribosomal RNA,<Emphasis Type="Italic"> cob</Emphasis>, and<Emphasis Type="Italic"> atp9</Emphasis> genes in the cucurbit mitochondrial genomes

Plants in the genus Cucumis (cucumber and melon) have the largest mitochondrial genomes known among all plants, due in part to the accumulation of repetitive DNAs of varying complexities. Recombination among these repetitive DNAs should produce highly rearranged mitochondrial genomes relative to the smaller mitochondrial genomes of related plants. We cloned and sequenced mitochondrial genomic regions near the rRNA, atp9 and cob genes from cucumber, melon, squash and watermelon (all members of the Cucurbitaceae family), and compared to the previously sequenced mitochondrial genomes of Arabidopsis thaliana and sugar beet to study the distribution and arrangement of coding and repetitive DNAs. Cucumber and melon had regions of concentrated repetitive DNAs spread throughout the sequenced regions; few repetitive DNAs were revealed in the mitochondrial genomes of A. thaliana, sugar beet, squash and watermelon. Recombination among these repetitive DNAs most likely produced unique arrangements of the rrn18 and rrn5 genes in the genus Cucumis. Cucumber mitochondrial DNA had more pockets of dispersed direct and inverted repeats than melon and the other plants, and we did not reveal repetitive sequences significantly contributing to mitochondrial genome expansion in both cucumber and melon.Disclaimer. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.Communicated by R. Hagemann     

The cellulose synthase (<Emphasis Type="Italic">CESA</Emphasis>) gene superfamily of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

The CESA gene superfamily of Arabidopsis and other seed plants comprises the CESA family, which encodes the catalytic subunits of cellulose synthase, and eight families of CESA-like (CSL) genes whose functions are largely unknown. The CSL genes have been proposed to encode processive β-glycosyl transferases that synthesize noncellulosic cell wall polysaccharides. BLAST searches of EST and shotgun genomic sequences from the moss Physcomitrella patens (Hedw.) B.S.G. were used to identify genes with high similarity to vascular plant CESAs, CSLAs, CSLCs, and CSLDs. However, searches using Arabidopsis CSLBs, CSLEs, and CSLGs or rice CSLFs or CSLHs as queries identified no additional CESA superfamily members in P. patens, indicating that this moss lacks representatives of these families. Intron insertion sites are highly conserved between Arabidopsis and P. patens in all four shared gene families. However, phylogenetic analysis strongly supports independent diversification of the shared families in mosses and vascular plants. The lack of orthologs of vascular plant CESAs in the P. patens genome indicates that the divergence of mosses and vascular plants predated divergence and specialization of CESAs for primary and secondary cell wall syntheses and for distinct roles within the rosette terminal complexes. In contrast to Arabidopsis, the CSLD family is highly represented among P. patens ESTs. This is consistent with the proposed function of CSLDs in tip growth and the central role of tip growth in the development of the moss protonema. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Accession numbers: DQ417756, DQ417757, DQ898284–6, DQ898147–54, DQ902545–51.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

<Emphasis Type="Italic">Hox</Emphasis> cluster duplication in the basal teleost <Emphasis Type="Italic">Hiodon alosoides</Emphasis> (Osteoglossomorpha)

Large-scale—even genome-wide—duplications have repeatedly been invoked as an explanation for major radiations. Teleosts, the most species-rich vertebrate clade, underwent a “fish-specific genome duplication” (FSGD) that is shared by most ray-finned fish lineages. We investigate here the Hox complement of the goldeye (Hiodon alosoides), a representative of Osteoglossomorpha, the most basal teleostean clade. An extensive PCR survey reveals that goldeye has at least eight Hox clusters, indicating a duplicated genome compared to basal actinopterygians. The possession of duplicated Hox clusters is uncoupled to species richness. The Hox system of the goldeye is substantially different from that of other teleost lineages, having retained several duplicates of Hox genes for which crown teleosts have lost at least one copy. A detailed analysis of the PCR fragments as well as full length sequences of two HoxA13 paralogs, and HoxA10 and HoxC4 genes places the duplication event close in time to the divergence of Osteoglossomorpha and crown teleosts. The data are consistent with—but do not conclusively prove—that Osteoglossomorpha shares the FSGD. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Resurrection of an ancestral gene: functional and evolutionary analyses of the Ng<Emphasis Type="Italic">rol</Emphasis> genes transferred from<Emphasis Type="Italic"> Agrobacterium</Emphasis> to<Emphasis Type="Italic"> Nicotiana</Emphasis>

The Ngrol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca. It is thought that bacterial infection resulted in the transfer of the Ngrol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not. Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes. The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species. This paper focuses on studies of the Ngrol genes in present-day plants and during the evolution of the genus Nicotiana. The functional sequences of several Ngrol genes may have been conserved after their ancient introduction from a bacterium to the plant. Resurrection of an ancestral function of one of the Ngrol genes, as examined by physiological and evolutionary analyses, is also described. The origin of the Ngrol genes is then considered, based on results of molecular phylogenetic analyses. The effects of the horizontal transfer of the Ngrol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.Seishiro Aoki is the recipient of the Botanical Society Award for Young Scientist, 2002.