Tinocladia sanrikuensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

The new species Tinocladia sanrikuensis sp. nov. H.Kawai, K.Takeuchi & T.Hanyuda (Ectocarpales s.l., Phaeophyceae) is described from the Pacific coast of the Tohoku region, northern Japan based on morphology and DNA sequences. The species is a spring–summer annual growing on lower intertidal to upper subtidal rocks and cobbles on relatively protected sites. T. sanrikuensis has a slimy, cylindrical, multiaxial erect thallus, slightly hollow when fully developed, branching once to twice, and resembles T. crassa in gross morphology. The erect thalli are composed of a dense medullary layer, long subcortical filaments, and assimilatory filaments of 11–35 cells, up to 425 μm long and curved in the upper portion. Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most closely related to T. crassa and has the same basic thallus structures but differs in having thinner and longer assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes support the distinctness of this species.     

A molecular and morphological investigation of Batrachospermum arcuatum (Batrachospermales, Rhodophyta) in China

Batrachospermum arcuatum specimens were analysed from seven stream segments in North China. Morphological characteristics were observed and cluster analysis was used to evaluate the divergence among thalli from. Sequence data of the rbcL gene (chloroplast gene) and cox2-3 spacer region (mitochondrial gene) were also utilized to evaluate genetic variation in specimens among stream segments. The specimens from four of the streams were monoecious, while the individuals at the other three locations were dioecious. Cluster analysis showed that the monoecious specimens were not separated from the dioecious specimens, based on morphology, but rather the specimens were grouped by geographical closeness and habitat similarity. Likewise, the combined analyses of rbcL and the cox2-3 spacer data from provided more evidence that breeding system (monoecy vs. dioecy) is not a good morphological character to distinguish species.     

Nuclear genotype affects mitochondrial genome organization of CMS-S maize

Summary A WF9 strain of maize with the RD subtype of the S male-sterile cytoplasm (CMS-S) was converted to the inbred M825 nuclear background by recurrent backcrossing. The organization of the mitochondrial genomes of the F₁ and succeeding backcross progenies was analyzed and compared with the progenitor RD-WF9 using probes derived from the S1 and S2 mitochondrial episomes, and probes containing the genes for cytochrome c oxidase subunit I (coxI), cytochrome c oxidase subunit II (coxII) and apocytochrome b (cob). Changes in mitochondrial DNA (mtDNA) organization were observed for S1-, S2-, and coxI-homologous sequences that involve loss of homologous restriction enzyme fragments present in the RD-WF9 progenitor. With the coxI probe, the loss of certain fragments was accompanied by the appearance of a fragment not detectable in the progenitor. The changes observed indicate the effect of the nuclear genome on the differential replication of specific mitochondrial subgenomic entities.     

PHYLOGENY OF ZYGNEMOPHYCEAE BASED ON COXIII GENE SEQUENCE DATA

Molecular phylogenetic analysis of the conjugating green algae (Class Zygnemophyceae) using nuclear (SSU rDNA) and chloroplast (rbcL) gene sequences has resolved hypotheses of relationship at the class, order, and family levels, but several key questions will require data from additional genes. Based on SSU and rbcL sequences, the Zygnemophyceae and Desmidiales are monophyletic, and families of placoderm desmids are distinct clades (Desmidiaceae, Peniaceae, Closteriaceae, and Gonatozygaceae). In contrast, the Zygnemataceae and Mesotaeniaceae are paraphyletic, although whether these two traditional families constitute a clade is uncertain. In addition, relationships of genera within families have proven resistant to resolution with these two oft‐used genes. We have sequenced the coxIII gene from the mitochondrial genome to address some of these ambiguous portions of the phylogeny of conjugating green algae. The coxIII gene is more variable than rbcL or SSU rDNA and offers greater resolving power for relationships of genera. We present preliminary analyses of coxIII sequences from each of the traditional families of Zygnemophyceae and contrast the resulting topologies with those derived from nuclear and chloroplast genes.     

Fatty acid synthesis by slices from developing leaves

Fatty acid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivium L.) leaves. The basal regions of the leaves had the lowest rates of fatty acid synthesis and accumulated small amounts of very long chain fatty acids. Fatty acid synthesis was highest in the middle leaf sections in all four plants. Linolenic acid synthesis from [1-¹⁴C]acetate was highest in the distal leaf sections of rye grass. The labelling of the fatty acids of individual lipids of rye grass was examined and it was found that [¹⁴C]linolenic acid was highest in the galactolipids. Synthesis of this acid in the galactolipids was most active in leaf segment C. Only traces of [¹⁴C]linolenic acid were ever found in phosphatidylcholine and it is concluded that this phospholipid cannot serve as a substrate for linoleic acid desaturation in rye grass. The synthesis of fatty acids was sensitive to arsenite, fluoride and the herbicide EPTC. The latter was only inhibitory towards those leaf segments which made very long chain fatty acids. Formation of fatty acids from [1-¹⁴C]acetate was also studied in chloroplasts prepared from successive leaf sections of rye grass. Chloroplasts isolated from the middle leaf sections had the highest activity. Palmitic and oleic acids were the main fatty acid products in all chloroplast preparations. Linolenic acid synthesis was highest in chlorplasts isolated from the distal leaf sections of rye grass.     

Molecular phylogeny of Old WorldTrifolium (Fabaceae), based on plastid and nuclear markers

Phylogenies of Old WorldTrifolium species were constructed using nucleotide sequence data of the internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast DNA restriction site data from PCR-amplified genes and genic regions (rbcL,trnK, andrpoC1–C2). Biogeography, morphological evolution, and the existing classification forTrifolium were examined. The genusTrifolium is strongly supported as monophyletic, however, only one small section (Chronosemium) is monophyletic, although the data are in conflict regarding its placement. The two largest sections of the genus, Sects.Lotoidea andTrifolium, are not supported as monophyletic, as currently circumscribed. Many members of Sect.Lotoidea are basal within the genus, supporting previously-proposed hypotheses concerning plesiomorphic morphological characters and a Mediterranean-Mideast biogeographic origin of the genus.     

Cladosiphon takenoensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

The new brown algal species Cladosiphon takenoensis H. Kawai (Chordariaceae, Ectocarpales s.l.) is described from Takeno, Hyogo, Japan based on morphology and DNA sequences. The species is a spring annual, growing on subtidal rocks at more or less exposed sites. It resembles C. umezakii in its gross morphology, and the two often grow together, but is distinguishable from C. umezakii in having a more hairy appearance. Cladosiphon takenoensis has a slimy, cylindrical, multiaxial and sympodial erect thallus, branching once to twice, and is provided with long assimilatory filaments (up to 1.8 mm long, composed of up to 100 cells). Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most related to C. umezakii and has the same basic thallus structures, but differs from C. umezakii and other Cladosiphon species in lacking phaeophycean hairs and plurilocular zoidangia of the assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes and the nuclear rDNA ITS2 region support the distinctness of the species. The genus Cladosiphon was paraphyletic in our analyses because the clades of C. okamuranus/C. zosterae and C. takenoensis/C. umezakii were split by Mesogloia vermiculata. However, since the genus‐level taxonomy of Chordariaceae needs considerable revision, we suspend the genus‐level taxonomy of the new species, and tentatively describe it as C. takenoensis.     

Reinstatement of Myelophycus caespitosus (Scytosiphonaceae,Phaeophyceae) from Japan

Reinstatement of Myelophycus caespitosus Kjellman (Ectocarpales s.l., Phaeophyceae) described from western Japan is proposed based on the comparisons of DNA sequences of Japanese Myelophycus specimens, the type specimen of Chordaria simplex and lectotypified specimen of Myelophycus caespitosus. In the genetic analyses using mitochondrial cox1 and cox3, chloroplast atpB, psbA and rbcL DNA sequences, the specimens morphologically referable to M. simplex formed two distinct clades (clade‐1 and clade‐2) supported by high statistical values. Clade‐1 was distributed on the western coast of Japan (Honshu, Shikoku and Kyushu) from the Kii Peninsula to Tsushima and the Pacific coast of Aomori, northeastern Honshu, and clade‐2 on the Pacific coast of central Honshu from Sanriku to the Kii Peninsula. Based on the cox3 DNA sequences and the location of the type locality at Shimoda, Izu Peninsula, clade‐2 was concluded to correspond to true M. simplex. There were no recognizable differences in the representative morphological features (height and diameter of sporophytes and gametophytes, number of cells comprising cortical, subcortical and medullary layers, thickness of plurilocular gametangia, or length and diameter of unilocular zoidangia) between the specimens included in the two taxa. Analyses of the specimens of the two taxa, including sympatric populations on the Kii Peninsula, using a nuclear genetic marker ocm3 did not suggest any genetic exchanges between the two taxa. On the other hand, cox3 gene sequence of the voucher specimen of Myelophycus caespitosus collected from Goto, Kyushu, western Japan housed in the UPS herbarium was included in clade‐1. In conclusion, we propose the reinstatement of M. caespitosus and to lectotypify the specimen in UPS. Korean Myelophycus specimens reported from Cheju Island and Wando were considered to belong to M. caespitosus based on the reported rbcL sequences.     

Mitochondrial DNA diversity and male sterility in natural populations of Daucus carota ssp carota

Mitochondrial variability was investigated in natural populations of wild carrot (Daucus carota ssp carota) in different regions: South of France, Greece, and various sites in the Mediterranean Basin and Asia. Total DNA was digested with two restriction endonucleases (EcoRV and HindIII) and probed with three mitochondrial DMA-specific genes (coxI, atp6, and coxII). Twenty-five different mitochondrial types were found in 80 analyzed individuals. Thirteen mitotypes were found among the 7 French populations studied. On average, 4.4 different mitotypes were observed per population, and these mitotypes were well-distributed among the populations. All of the mitochondrial types were specific to a single region. However, the proportion of shared restriction fragments between 2 mitotypes from different regions was not particularly lower than that which occurred among mitotypes from a single region. On the basis of the sexual phenotype [male-sterile (MS) or hermaphrodite] of the plants studied in situ and that of their progeny, 2 mitotypes were found to be highly associated with male sterility. Eighty percent of the plants bearing these mitotypes were MS in situ, and all of these plants produced more than 30% MS plants in their progeny. This association with male sterility was consistent in several populations, suggesting an association with a cytoplasmic male-sterility system. Moreover, these two mitotypes had very similar mitochondrial DNA restriction patterns and were well-differentiated from the other mitotypes observed in wild plants and also from those observed in the two CMS types already known in the cultivated carrot. This suggests that they correspond to a third cytoplasmic sterility.     

Molecular evidence for the coexistence of two sibling species in Pylaiella littoralis (Ectocarpales,Phaeophyceae) along the Brittany coast

The great phenotypic variability and the lack of diagnostic characters in the genus Pylaiella render the systematic study of this genus problematic. In this study, we investigated the diversity of Pylaiella littoralis along the Brittany (France) coast using a DNA barcoding multilocus approach with mitochondrial (cox1, nad1, and atp9) and chloroplastic (rbcL and atpB) markers associated with a population genetics approach using 10 microsatellite markers. In addition, spatio‐temporal sampling was conducted along the Brittany coast. We sampled 140 individuals from four sites located between Saint‐Malo and Concarneau (380 km) from April to October. Mitochondrial sequence data revealed the occurrence of two sibling species, with a minimum of 2.4% divergence between them. Microsatellite genotypic data congruently revealed two well‐supported clusters matching the two mitochondrial clades of Pylaiella. Although gene flow is limited between species, occurrence of genetic admixtures in some populations suggested that reproductive isolation is not complete. Our study highlighted the complementarity of barcoding and population genetics approaches to shed light on the evolutionary processes that lead to speciation.     

Molecular phylogeny of two unusual brown algae,Phaeostrophion irregulare and Platysiphon glacialis,proposal of the Stschapoviales ord. nov. and Platysiphonaceae fam. nov., and a re‐examination of divergence times for brown algal orders

The molecular phylogeny of brown algae was examined using concatenated DNA sequences of seven chloroplast and mitochondrial genes (atpB, psaA, psaB, psbA, psbC, rbcL, and cox1). The study was carried out mostly from unialgal cultures; we included Phaeostrophion irregulare and Platysiphon glacialis because their ordinal taxonomic positions were unclear. Overall, the molecular phylogeny agreed with previously published studies, however, Platysiphon clustered with Halosiphon and Stschapovia and was paraphyletic with the Tilopteridales. Platysiphon resembled Stschapovia in showing remarkable morphological changes between young and mature thalli. Platysiphon, Halosiphon and Stschapovia also shared parenchymatous, terete, erect thalli with assimilatory filaments in whorls or on the distal end. Based on these results, we proposed a new order Stschapoviales and a new family Platysiphonaceae. We proposed to include Phaeostrophion in the Sphacelariales, and we emended the order to include this foliose member. Finally, using basal taxa not included in earlier studies, the origin and divergence times for brown algae were re‐investigated. Results showed that the Phaeophyceae branched from Schizocladiophyceae ~260 Ma during the Permian Period. The early diverging brown algae had isomorphic life histories, whereas the derived taxa with heteromorphic life histories evolved 155–110 Ma when they branched from the basal taxa. Based on these results, we propose that the development of heteromorphic life histories and their success in the temperate and cold‐water regions was induced by the development of the remarkable seasonality caused by the breakup of Pangaea. Most brown algal orders had diverged by roughly 60 Ma, around the last mass extinction event during the Cretaceous Period, and therefore a drastic climate change might have triggered the divergence of brown algae.     

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.     

Organellar segregation,rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli

Summary Cauliflower protoplasts were fused to determine the effect of protoplast source and pretreatment on organellar segregation in fusion products. Mitochondrial and chloroplast type were determined for over 250 calli from eight fusions between iodoacetate-treated or -irradiated leaf or hypocotyl protoplasts with fertile or Ogura cytoplasms. Organelles in fusion-derived calli were identified with five mitochondrial probes and one chloroplast probe. Mitochondrial and chloroplast segregation were independent but biased. Most calli had B. oleracea chloroplasts, but more calli had Ogura mitochondria than B. oleracea ones. Neither protoplast source nor pretreatment alone affected organelle segregation. However, iodoacetate treatment of hypocotyl protoplasts reduced their mitochondrial contribution to the fusion products although it did not affect chloroplast segregation. Over half of the calli had mitochondrial genomes distinct from those of either fusion partner; many of these contained the complete mitochondrial genome of one partner along with some mitochondrial DNA from the other. Out of 258 calli, 83 showed evidence of mitochondrial recombination, most commonly by formation of a novel 11-kb PstI fragment near the atp9 region.     

RNA editing of 10 Didymium iridis mitochondrial genes and comparison with the homologous genes in Physarum polycephalum

Regions of the Didymium iridis mitochondrial genome were identified with similarity to typical mitochondrial genes; however, these regions contained numerous stop codons. We used RT-PCR and DNA sequencing to determine whether, through RNA editing, these regions were transcribed into mRNAs that could encode functional proteins. Ten putative gene regions were examined: atp1, atp6, atp8, atp9, cox1, cox2, cytb, nad4L, nad6, and nad7. The cDNA sequences of each gene could encode a functional mitochondrial protein that was highly conserved compared with homologous genes. The type of editing events and editing sequence features were very similar to those observed in the homologous genes of Physarum polycephalum, though the actual editing locations showed a variable degree of conservation. Edited sites were compared with encoded sites in D. iridis and P. polycephalum for all 10 genes. Edited sequence for a portion of the cox1 gene was available for six myxomycetes, which, when compared, showed a high degree of conservation at the protein level. Different types of editing events showed varying degrees of site conservation with C-to-U base changes being the least conserved. Several aspects of single C insertion editing events led to the preferential creation of hydrophobic amino acid codons that may help to minimize adverse effects on the resulting protein structure.     

绣球属(Hydrangea L.)植物分子系统学及系统发育分析

该研究基于对绣球属(Hydrangea L.)的大尺度取样,选取国内外61种绣球属和近缘属植物,分别基于核基因片段(ITS)和叶绿体基因片段(rbcL,trnL-F,atpB)重建了绣球属及其近缘种属的系统发育关系。结果表明:(1)核基因与叶绿体基因树之间在树形上没有明显的冲突,进而基于核基因和叶绿体基因联合数据重建了绣球属及其近缘种属的系统发育关系。(2)基于联合数据构建的系统树确认了2个大分支,并得到了果实顶端截平与否这一形态学证据的强力支持;每个大分枝又分为4个类群,共确定了8个类群。部分类群也得到了广义宏观形态性状的支持,如第1类群得到了叶形、花粉以及种子形态的支持。因此,该系统发育关系的重建对于全面理解绣球属及其近缘种属的演化关系具有重要的启发。