STRUCTURE AND ASEXUAL REPRODUCTION OF THE ENIGMATIC CHAROPHYCEAN GREEN ALGA ENTRANSIA FIMBRIATA (KLEBSORMIDIALES,CHAROPHYCEAE)1

As the closest relatives of embryophytes, the charophycean green algae (sensu Mattox and Stewart) may reveal the evolutionary history of characters in this lineage. Recent molecular phylogenetic analysis indicates that the little‐known species Entransia fimbriata Hughes is a member of the charophycean order Klebsormidiales. In this study LM and EM were used to identify and describe additional structural characters of Entransia so that comparisons could be made with Klebsormidium and with other charophycean algae outside the order Klebsormidiales. Features that Entransia shares with various members of the genus Klebsormidium include cylindrical cells in unbranched filaments that may spiral, parietal chloroplasts that cover only part of the circumference of the cell, H‐shaped cross walls, and vegetative reproduction by both fragmentation and formation of zoospores or aplanospores. Among the characteristics that distinguish Entransia from Klebsormidium are a highly lobed chloroplast with multiple pyrenoids; a single large vacuole; short cells that die and collapse, apparently facilitating filament fragmentation; and germinating filaments with condensed adhesive at the base and a tapering spine at the tip. Although Entransia has sometimes been tentatively considered to be a member of the Zygnemataceae, the presence of a flagellate life history stage distinguishes Entransia from this group. The pyrenoids of Entransia are typical of those of charophycean algae in having traversing membranes and surrounding starch. Presence of multiple such pyrenoids in each chloroplast of Entransia supports the hypothesis that the common ancestor of charophycean algae and embryophytes had a single chloroplast with multiple pyrenoids.     

PHYLOGENY OF THE CONJUGATING GREEN ALGAE BASED ON CHLOROPLAST AND MITOCHONDRIAL NUCLEOTIDE SEQUENCE DATA1

The conjugating green algae represent a lineage of charophyte green algae known for their structural diversity and unusual mode of sexual reproduction, conjugation. These algae are ubiquitous in freshwater environments, where they are often important primary producers, but few studies have investigated evolutionary relationships in a molecular systematic context. A 109‐taxon data set consisting of three gene fragments (two from the chloroplast and one from the mitochondrial genome) was used to estimate the phylogeny of the genera of the conjugating green algae. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) were used to estimate relationships from the 4,047 alignable nucleotides. This study confirmed the polyphyly of the Zygnemataceae and Mesotaeniaceae with respect to one another. The Peniaceae were determined to be paraphyletic, and two genera traditionally classified among the Zygnematales appear to belong to the lineage that gave rise to the Desmidiales. Six genera, Euastrum, Cosmarium, Cylindrocystis, Mesotaenium, Spondylosium, and Staurodesmus, were polyphyletic in this analysis. These findings have important implications for the evolution of structural characteristics in the group and will require some taxonomic changes. More work will be required to delineate lineages of Zygnematales in particular and to identify structural synapomorphies for some of the newly identified clades.     

Habitat-specific composition of morphotypes with low genetic diversity in the green algal genus Klebsormidium (Streptophyta) isolated from biological soil crusts in Central European grasslands and forests

Terrestrial filamentous green algae of the widely distributed, cosmopolitan genus Klebsormidium (Klebsormidiophyceae, Streptophyta) are typical components of biological soil crusts (BSCs). These communities occur in all climatic zones and on all continents, where soil moisture is limited or where there has been disturbance. BSCs form water-stable aggregates that have important ecological roles in primary production, nitrogen fixation, nutrient cycling, water retention and stabilization of soils. Although available data on Klebsormidium are limited, its functional importance in BSCs is regarded as high. Therefore, in the present study Klebsormidium strains were isolated from BSCs sampled from various grassland and forest plots of different land use intensities in Central Europe, as provided by the Biodiversity Exploratories, and its intraspecific genetic diversity was evaluated. Previous phylogenetic analyses revealed a relationship between sequence similarity and habitat preference with a higher genetic diversity than expected from a morphological classification. We isolated and sequenced 75 Klebsormidium strains. The molecular phylogeny based on the ITS regions showed that all strains belong to either the previously described clade B/C or clade E. This classification was supported by morphological characteristics: strains assigned to clade B/C were identified as Klebsormidium cf. flaccidum or Klebsormidium cf. dissectum, and strains from clade E as K. nitens or Klebsormidium cf. subtile. Within one clade the strains showed low sequence divergences. These minor differences were independent of the sampling region and land use intensity. Interestingly, most of the strains assigned to clade E were isolated from forest sites, whereas strains from clade B/C occurred equally in grassland and forest sites. Therefore, it is reasonable to assume that habitat with its microenvironmental conditions, and not biogeography, controls genetic diversity in Klebsormidium.     

The xanthophyll cycle and NPQ in diverse desert and aquatic green algae

It has long been suspected that photoprotective mechanisms in green algae are similar to those in seed plants. However, exceptions have recently surfaced among aquatic and marine green algae in several taxonomic classes. Green algae are highly diverse genetically, falling into 13 named classes, and they are diverse ecologically, with many lineages including members from freshwater, marine, and terrestrial habitats. Genetically similar species living in dramatically different environments are potentially a rich source of information about variations in photoprotective function. Using aquatic and desert-derived species from three classes of green algae, we examined the induction of photoprotection under high light, exploring the relationship between nonphotochemical quenching and the xanthophyll cycle. In liquid culture, behavior of aquatic Entransia fimbriata (Klebsormidiophyceae) generally matched patterns observed in seed plants. Nonphotochemical quenching was lowest after overnight dark adaptation, increased with light intensity, and the extent of nonphotochemical quenching correlated with the extent of deepoxidation of xanthophyll cycle pigments. In contrast, overnight dark adaptation did not minimize nonphotochemical quenching in the other species studied: desert Klebsormidium sp. (Klebsormidiophyceae), desert and aquatic Cylindrocystis sp. (Zygnematophyceae), and desert Stichococcus sp. (Trebouxiophyceae). Instead, exposure to low light reduced nonphotochemical quenching below dark-adapted levels. De-epoxidation of xanthophyll cycle pigments paralleled light-induced changes in nonphotochemical quenching for species within Klebsormidiophyceae and Trebouxiophyceae, but not Zygnematophyceae. Inhibition of violaxanthin–zeaxanthin conversion by dithiothreitol reduced high-light-associated nonphotochemical quenching in all species (Zygnematophyceae the least), indicating that zeaxanthin can contribute to photoprotection as in seed plants but to different extents depending on taxon or lineage.     

Analysis of mitochondrial and chloroplast genomes in two volvocine algae: Eudorina elegans and Eudorina cylindrica (Volvocaceae,Chlorophyta)

The colonial volvocine algae span the full range of organizational complexity, from four-celled species to multicellular species, and this group of algae is often used for the study of evolution. In recent years, many organelle genomes have been sequenced using the application of next generation sequencing technology; however, only a few organelle genomes have been reported in colonial volvocine algae. In this study, we determined the organelle genomes of Eudorina elegans and Eudorina cylindrica and analysed the organelle genome size, structure and gene content between these volvocine species. This provided useful information to help us understand the composition of colonial volvocine organelle genomes. Based on the chloroplast genome protein-coding genes, we conducted a phylogenomics analysis of the volvocine algae. The result revealed an unexpected phylogenetic relationship, namely, E. elegans is more closely related to Pleodorina starrii than to E. cylindrica. The substitution rate of volvocine algae was then calculated based on organelle genome protein-coding genes; our analysis suggested the possibility that the two Eudorina species may be under similar evolutionary pressure. Lastly, the synteny analysis of the mitochondrial genome showed that gene arrangements and contents are highly conserved in the family Volvocaceae, and the synteny analysis of the chloroplast genome indicated that the genus Eudorina may have experienced genomic changes.     

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.     

Generic and species concepts in Microglena (previously the Chlamydomonas monadina group) revised using an integrative approach

Traditionally the genus Microglena Ehrenberg has been used to contain species that belong to the Chrysophyceae; however, the type species of Microglena, M. monadina, represents a green alga, which was later transferred to the genus Chlamydomonas. The taxonomic status of the genus has therefore remained unclear. We investigated 15 strains previously assigned to C. monadina and two marine species (C. reginae and C. uva-maris) using an integrative approach. Phylogenetic analyses of SSU and ITS rDNA sequences revealed that all strains form a monophyletic lineage within the Chlorophyceae containing species from different habitats. The strains studied showed similar morphology with respect to cell shape and size, but showed differences in chloroplast and pyrenoid structures. Some representatives of this group have the same type of sexual reproduction (homothallic advanced anisogamy). Three different morphotypes could be recognized. Strains belonging to type I have a cup-shaped chloroplast with a massive basal part, in which a large, single, ellipsoidal pyrenoid is located. The members of type II also have a cup-shaped chloroplast, which is partly lobed and has a thinner basal part than type I; here the pyrenoid is half-ring or horseshoe-shaped and occupies different positions in the chloroplast depending on the strain. The strains of type III have multiple pyrenoids, which appear to have developed from the subdivision of a single ring-shaped pyrenoid into several parts. We compared the results of our morphological investigations with the literature and found that 15 strains could be identified with existing species. Two strains did not fit with any described species. As a result of our study, we transfer all strains to the genus Microglena, propose 11 new combinations, and describe two new species. Comparison of the ITS-1 and ITS-2 secondary structures confirmed the species delineations. All species have characteristic compensatory base changes in their ITS secondary structures and are supported by ITS-2 DNA barcodes.     

Phylogenetic positioning of the Antarctic alga Prasiola crispa (Trebouxiophyceae) using organellar genomes and their structural analysis

Antarctica is one of the most difficult habitats for sustaining life on earth; organisms that live there have developed different strategies for survival. Among these organisms is the green alga Prasiola crispa, belonging to the class Trebouxiophyceae. The literature on P. crispa taxonomy is scarce, and many gaps in the evolutionary relationship with its closest relatives remain. The goal of this study was to analyze the evolutionary relationships between P. crispa and other green algae using plastid and mitochondrial genomes. In addition, we analyzed the synteny conservation of these genomes of P. crispa with those of closely related species. Based on the plastid genome, P. crispa grouped with Prasiolopsis sp. SAG 84.81, another Trebouxiophyceaen species from the Prasiola clade. Based on the mitochondrial genome analysis, P. crispa grouped with other Trebouxiophyceaen species but had a basal position. The structure of the P. crispa chloroplast genome had low synteny with Prasiolopsis sp. SAG 84.81, despite some conserved gene blocks. The same was observed in the mitochondrial genome compared with Coccomyxa subellipsoidea C‐169. We were able to establish the phylogenetic position of P. crispa with other species of Trebouxiophyceae using its genomes. In addition, we described the plasticity of these genomes using a structural analysis. The plastid and mitochondrial genomes of P. crispa will be useful for further genetic studies, phylogenetic analysis and resource protection of P. crispa as well as for further phylogenetic analysis of Trebouxiophyceaen green algae.     

Misleading morphologies and the importance of sequencing type specimens for resolving coralline taxonomy (Corallinales,Rhodophyta): Pachyarthron cretaceum is Corallina officinalis

Coralline red algae play a key role in the ecology of near shore marine ecosystems and are increasingly being used to study the effects of climate change in the marine environment. Corallines are very difficult to identify to species, and even to genus, using morpho‐anatomy, likely complicating studies of their ecology, physiology, and biodiversity. We sequenced a 296 base pair fragment of chloroplast DNA from a 187‐year‐old isolectotype specimen of Pachyarthron cretaceum, a morphologically distinct geniculate species, to demonstrate that coralline morphology is often misleading and that species names can only be applied unequivocally by comparing DNA sequences from type material with sequences from field‐collected specimens. Our results indicate that Pachyarthron cretaceum is synonymous with Corallina officinalis.     

Reassessment of suitable markers for taxonomy of Chaetophorales (Chlorophyceae,Chlorophyta) based on chloroplast genomes

Filamentous green algae Chaetophorales present numerous taxonomic problems as many other green algae. Phylogenetic analyses based on nuclear genes have limited solutions. Studies with appropriate chloroplast molecular markers may solve this problems; however, suitable molecular markers for the order Chaetophorales are still unknown. In this study, 50 chloroplast genomes of Chlorophyceae, including 15 of Chaetophorales, were subjected to single protein-coding gene phylogenetic analyses, and substitution rate and evolutionary rate assays, and PCR amplification verification was conducted to screen the suitable molecular markers. Phylogenetic analyses of three chloroplast representative genes (psaB, tufA, and rbcL) amplified from 124 strains of Chaetophorales showed that phylogenetic relationships were not improved by increasing the number of samples, implying that the genes themselves, rather than limited samples, were the reason for the unsupported Topology I. Seven genes (atpF, atpI, ccsA, cemA, chlB, psbB, and rpl2) with robust support were selected to be the most suitable molecular markers for phylogenetic analyses of Chaetophorales, and the concatenated seven genes could replace the time-consuming and labor-intensive phylogenetic analyses based on chloroplast genome to some extent. To further solve the taxonomic problems of Chaetophorales, suitable chloroplast markers combined with more taxon-rich approach could be helpful and efficient.     

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.     

The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands

Background  

The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. While the basal position of the Prasinophyceae is well established, the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC) remains uncertain. The five complete chloroplast DNA (cpDNA) sequences currently available for representatives of these classes display considerable variability in overall structure, gene content, gene density, intron content and gene order. Among these genomes, that of the chlorophycean green alga Chlamydomonas reinhardtii has retained the least ancestral features. The two single-copy regions, which are separated from one another by the large inverted repeat (IR), have similar sizes, rather than unequal sizes, and differ radically in both gene contents and gene organizations relative to the single-copy regions of prasinophyte and ulvophyte cpDNAs. To gain insights into the various changes that underwent the chloroplast genome during the evolution of chlorophycean green algae, we have sequenced the cpDNA of Scenedesmus obliquus, a member of a distinct chlorophycean lineage.     

Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta)

The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.     

Lunachloris lukesovae gen. et sp. nov. (Trebouxiophyceae,Chlorophyta), a novel coccoid green alga isolated from soil in South Bohemia,Czech Republic

Culture collections of microorganisms can still hold undiscovered biodiversity; with molecular techniques, considerable progress has been made in characterizing microalgae which were isolated in the past and misidentified due to a lack of morphological features. However, many strains are still awaiting taxonomic reassessment. Here we analysed the phylogenetic position, morphology and ultrastructure of the strain CCALA 307 previously identified as Coccomyxa cf. gloeobotrydiformis Reysigl isolated in 1987 from field soil in South Bohemia, Czech Republic. Molecular phylogenetic analyses based on SSU rDNA and the plastid rbcL gene revealed that the strain CCALA 307 formed a distinct sister lineage to Neocystis and Prasiola clades within the Trebouxiophyceae. We describe this strain as a new genus and species, Lunachloris lukesovae. Multiple conserved nucleotide positions identified in the secondary structures of the highly variable ITS2 rDNA barcoding marker provide further evidence of the phylogenetic position of Lunachloris. Minute vegetative cells of this newly recognized species are spherical or ellipsoid, with a single parietal chloroplast without a pyrenoid. Asexually, it reproduces by the formation of 2–6 autospores. Since the majority of recent attention has been paid to algae from the tropics or extreme habitats, the biodiversity of terrestrial microalgae in temperate regions is still notably unexplored and even a ‘common’ habitat like agricultural soil can contain new, as yet unknown species. Moreover, this study emphasizes the importance of culture collections of microorganisms even in the era of culture-independent biodiversity research, because they may harbour novel and undescribed organisms as well as preserving strains for future studies.     

陕北黄土高原生物结皮中7种微藻的形态与分子生物学鉴定

陕北黄土高原实施退耕还林后,生物结皮成为其典型的地表覆盖类型,含有丰富的土生藻类,对固定土壤和促进养分循环具有极其重要的作用。该研究通过平板法与水滴稀释法对陕北黄土高原生物结皮土生藻类进行分离培养,采用光学显微镜观察结皮微藻的形态特征,并对单藻种进行分子鉴定,为黄土高原生物结皮藻类的研究奠定基础。结果显示:(1)共纯化获得7种结皮藻类,经光学显微镜初步确定,其中5株为绿藻、2株为蓝藻。(2)5株绿藻SM-2-1、DB-2-1、DB-2-2、SD-1和SD-2的序列长度分别为664 bp、663 bp、662 bp、589 bp和688 bp, GC含量分别为33.43%、49.47%、50.15%、50.76%和51.01%;2株蓝藻YJ-3、YJ-2的序列长度分别为570 bp和465 bp, GC含量分别为46.31%和49.03%。(3)序列比对并构建系统树分析发现,5株结皮绿藻可分为4个分支,分别为栅藻科(Scenedesmaceae)2株(DB-2-1和SM-2-1)、衣藻目(Chlamydomonadales)、环藻科(Sphaeropleaceae)、真眼点藻科(Eust...     

THE RPS12 GENE IN SPROGYRA MAXIMA (CHLOROPHYTA) AND ITS EVOLUTIONARY SINGNIFICANCE1

The str operon consists fo four genes in eubacteria. Portions of his operon are conserved in the chloroplast genomes of green algae and land plants. In land plant chloroplasts, the str operon comprises only two genes, rps12 and rps7, and is arranged in a trans-spliced state. Since no other previously studied chloroplast genome contains this arrangement, and because the charophyte lineage is the sister group of land plants, we chose to look for this arrangement in the Charophyceae. The two str genes, rps12 and rps7, present in the chloroplasts of Spirogyra maxima Hanssall, were identified by hybridization of a Southern blot and requenced. The results indicate that Spirogyra contains a str operon almost identical to that of land plant chloroplasts. Based upon the structure of the operon in other chloroplasts and eubacterial genomes, the trans-spliced state most likely evolved early within the charophyte lineage.     

A multi-locus time-calibrated phylogeny of the siphonous green algae

The siphonous green algae are an assemblage of seaweeds that consist of a single giant cell. They comprise two sister orders, the Bryopsidales and Dasycladales. We infer the phylogenetic relationships among the siphonous green algae based on a five-locus data matrix and analyze temporal aspects of their diversification using relaxed molecular clock methods calibrated with the fossil record. The multi-locus approach resolves much of the previous phylogenetic uncertainty, but the radiation of families belonging to the core Halimedineae remains unresolved. In the Bryopsidales, three main clades were inferred, two of which correspond to previously described suborders (Bryopsidineae and Halimedineae) and a third lineage that contains only the limestone-boring genus Ostreobium. Relaxed molecular clock models indicate a Neoproterozoic origin of the siphonous green algae and a Paleozoic diversification of the orders into their families. The inferred node ages are used to resolve conflicting hypotheses about species ages in the tropical marine alga Halimeda.