Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri: phylogenetic origins and recent insertion of introns

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene.     

DNA barcoding of a new record of epi-endophytic green algae Ulvella leptochaete (Ulvellaceae,Chlorophyta) in India

Epi-endophytic green algae comprise one of the most diverse and phylogenetically primitive groups of green algae and are considered to be ubiquitous in the world’s oceans; however, no reports of these algae exist from India. Here we report the serendipitous discovery of Ulvella growing on intertidal green algae Cladophora glomerata and benthic red algae Laurencia obtusa collected from India. DNA barcodes at nuclear ribosomal DNA Internal Transcriber Spacer (nrDNA ITS) 1 and 2 regions for Indian isolates from the west and east coasts have been generated for the first time. Based on morphology and DNA barcoding, isolates were identified as Ulvella leptochaete. Phylogenetic reconstruction of concatenated dataset using Maximum Likelihood method differentiated Indian isolates from other accessions of this alga available in Genbank, albeit with low bootstrap support. Monophyly of Ulvella leptochaete was obvious in both of our phylogenetic analyses. With this first report of epi-endophytic algae from Indian territorial waters, the dire need to catalogue its cryptic diversity is highlighted and avenues of future research are discussed.     

MOLECULAR EVIDENCE FROM nrDNA ITS SEQUENCES THAT LAMINARIOCOLAX (PHAEOPHYCEAE, ECTOCARPALES SENSU LATO) IS A WORLDWIDE CLADE OF CLOSELY RELATED KELP ENDOPHYTES

Marine brown algae living as endophytes in macroalgae are morphologically simple and their taxonomy is particularly difficult. A molecular phylogeny for endophytic taxa isolated from kelps and red algae, and for putative epiphytic and free-living relatives, was inferred from partial small subunit and complete internal transcribed spacer nuclear ribosomal DNA sequences. It has revealed the following results. (1) Three species of endophytes isolated from members of the Laminariales are closely related. They form a clade together with the epi-endophytic species Laminariocolax tomentosoides (Farlow) Kylin. Members of the clade possess uniseriate plurilocular sporangia, and they may form erect filaments. Laminariocolax eckloniae sp. nov., occurring in the South African host Ecklonia maxima (Osbeck) Papenfuss, is described. The new combinations, Laminariocolax aecidioides (Rosenvinge) comb. nov. and L. macrocystis (Peters) comb. nov., are proposed for two taxa previously classified in Gononema and Streblonema , respectively. (2) The genus Laminariocolax occurs worldwide in temperate areas, and the phylogeny of the taxa studied is in agreement with biogeographic distribution. (3) Laminariocolax belongs to the Ectocarpales sensu lato. The genus is more closely related to Chordaria than to Dictyosiphon, Ectocarpus, or Scytosiphon . (4) Two brown endophytes ( Streblonema spp.), isolated from red algae, are closely related to each other and may form a sister clade to Laminariocolax .     

Specificity of assemblage,not fungal partner species,explains mycorrhizal partnerships of mycoheterotrophic Burmannia plants

Mycoheterotrophic plants (MHPs) growing on arbuscular mycorrhizal fungi (AMF) usually maintain specialized mycorrhizal associations. The level of specificity varies between MHPs, although it remains largely unknown whether interactions with mycorrhizal fungi differ by plant lineage, species, and/or by population. Here, we investigate the mycorrhizal interactions among Burmannia species (Burmanniaceae) with different trophic modes using high-throughput DNA sequencing. We characterized the inter- and intraspecific dynamics of the fungal communities by assessing the composition and diversity of fungi among sites. We found that fully mycoheterotrophic species are more specialized in their fungal associations than chlorophyllous species, and that this specialization possibly results from the gradual loss of some fungal groups. In particular, although many fungal species were shared by different Burmannia species, fully MHP species typically host species-specific fungal assemblages, suggesting that they have a preference for the selected fungi. Although no apparent cophylogenetic relationship was detected between fungi and plants, we observe that evolutionarily closely related plants tend to have a greater proportion of shared or closely related fungal partners. Our findings suggest a host preference and specialization toward fungal assemblages in Burmannia, improving understanding of interactions between MHPs and fungi.Subject terms: Fungi, Plant sciences, Evolution     

Pneumocystis carinii shows DNA homology with the ustomycetous red yeast fungi

Pneumocystis carinii causes life-threatening pneumonia in T-lymphocyte-immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.     

The b.e. and Q types of 1,4-α-d-glucan: 1,4-α-d-glucan-6-glucosyltransferase isozymes in algae

The branching isozymes of the red alga, Rhodymenia pertusa are of two types: Q, which can branch, via the synthesis of α-1,6-glucosyl linkages, linear amyloses to amylopectin; and b.e., which can further branch the amylopectin formed to the more highly-branched floridean starch. Using the technique of tandem crossed-immunoelectrophoresis, it is shown that the Q branching isozyme of the red alga is more closely related to the b.e. type of branching isozymes of Anacystis nidulans and Cyanidium caldarium than it is to the exclusively Q types of branching isozymes found in Chlorella pyrenoidosa and other chlorophytes. The possibility of a biphyletic evolution of the red and the green algae from blue-green ancestral forms is discussed.     

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri : phylogenetic origins and recent insertion of introns

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene. Received: 27 November 1998 / Accepted: 21 April 1999     

DNA is present in the nucleomorph of cryptomonads: Further evidence that the chloroplast evolved from a eukaryotic endosymbiont

Summary The nucleomorph is a unique self-replicating organelle which is invariably present in the periplastidal compartment of cryptomonads. The nucleomorph ofCryptomonas abbreviata is located in a groove on the inner face of the pyrenoid. When JB-4-embedded sections ofC. abbreviata are stained with 4-6-diamidino-2-phenylindole (DAPI), the nucleomorph exhibits a blue fluorescence characteristic of DNA-DAPI complexes. This fluorescence is removed by DNase digestion, but not by RNase. When cells are prepared for electron microscopy by the method of Ryter and Kellenberger (Schreil 1964), a network of fine DNA-like fibrils is observed in the nucleomorph matrix. It is estimated that the nucleomorph contains between 10⁸ and 10⁹ daltons of DNA. The presence of DNA in nucleomorphs strongly supports the hypothesis that the nucleomorph is the vestigial nucleus of a eukaryotic endosymbiont. It is postulated that this eukaryotic symbiont was an ancestral red alga or an organism closely related to red algae. The cryptomonad host cell, on the other hand, is not evolutionarily close to any other group of algae.     

IDENTIFICATION OF GREEN ALGAL ENDOPHYTES AS THE ALTERNATE PHASE OF ACROSIPHONIA (CODIOLALES, CHLOROPHYTA) USING ITS1 AND ITS2 RIBOSOMAL DNA SEQUENCE DATA

The internal transcribed spacer regions of the nuclear ribosomal DNA cistron of green algal endophytes, previously identified as Chlorochytrium inclusum Kjellman and Codiolum petrocelidis Kuckuck, were sequenced. Culture studies have implicated marine Chlorochytrium and Codiolum -like cells in the life histories of a number of macrophytic green algae. The identity of the endophytes was resolved by comparing DNA sequences from unicells endophytic in the foliose red alga Mazzaella splendens (Setchell et Gardner) Hommersand and in the crustose phase of Mastocarpus papillatus (J. Agardh) Kylin, with sequences from species of Acrosiphonia, Urospora, Ulothrix, and Monostroma. All endophyte isolates were more closely related to species of Acrosiphonia than to those of Urospora, Ulothrix, or Monostroma. The results support previous culture studies, and conclusively identify the endophytes obtained in the field as alternate life history phases of one or more Acrosiphonia species in southern British Columbia, Canada.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Application of flow cytometry for exploring the evolution of Geosmithia fungi living in association with bark beetles: the role of conidial DNA content

Geosmithia belongs among fungi living in symbiosis with phloem-feeding bark beetles. Several species have altered their ecology to that of obligatory symbiosis with ambrosia beetles, which has led to a shift in their phenotype and caused formation of large spherical conidia. In this study, we pose the following questions; (1) Is the conidial DNA content of Geosmithia correlated with conidial volume?; (2) Is the DNA content of Geosmithia related to the degree of mutual dependence between Geosmithia and their vector? There was a positive and strong correlation between conidial DNA content and conidial volume in Geosmithia. Also species more narrowly associated with the vector tend to have a larger conidial DNA content and volume than less narrowly associated species. Ambrosia fungi achieved the biggest conidial DNA content and volume compared to other species. We suppose that polyploidisation occurred during the evolution of ambrosia species in the genus Geosmithia.     

Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates

Endosymbiosis has spread photosynthesis to many branches of the eukaryotic tree; however, the history of photosynthetic organelle (plastid) gain and loss remains controversial. Fortuitously, endosymbiosis may leave a genomic footprint through the transfer of endosymbiont genes to the "host" nucleus (endosymbiotic gene transfer, EGT). EGT can be detected through comparison of host genomes to uncover the history of past plastid acquisitions. Here we focus on a lineage of chlorophyll c-containing algae and protists ("chromalveolates") that are postulated to share a common red algal secondary endosymbiont. This plastid is originally of cyanobacterial origin through primary endosymbiosis and is closely related among the Plantae (i.e., red, green, and glaucophyte algae). To test these ideas, an automated phylogenomics pipeline was used with a novel unigene data set of 5,081 expressed sequence tags (ESTs) from the haptophyte alga Emiliania huxleyi and genome or EST data from other chromalveolates, red algae, plants, animals, fungi, and bacteria. We focused on nuclear-encoded proteins that are targeted to the plastid to express their function because this group of genes is expected to have phylogenies that are relatively easy to interpret. A total of 708 genes were identified in E. huxleyi that had a significant Blast hit to at least one other taxon in our data set. Forty-six of the alignments that were derived from the 708 genes contained at least one other chromalveolate (i.e., besides E. huxleyi), red and/or green algae (or land plants), and one or more cyanobacteria, whereas 15 alignments contained E. huxleyi, one or more other chromalveolates, and only cyanobacteria. Detailed phylogenetic analyses of these data sets turned up 19 cases of EGT that did not contain significant paralogy and had strong bootstrap support at the internal nodes, allowing us to confidently identify the source of the plastid-targeted gene in E. huxleyi. A total of 17 genes originated from the red algal lineage, whereas 2 genes were of green algal origin. Our data demonstrate the existence of multiple red algal genes that are shared among different chromalveolates, suggesting that at least a subset of this group may share a common origin.     

No support for occurrence of free-living Cladonia mycobionts in dead wood

Lichenised fungi are traditionally assumed to form obligate symbioses with algae or cyanobacteria and to be confined to the surface of their growing substratum. However, in a recent 454 pyrosequencing study of fungal communities in Picea abies logs, lichen-forming fungi were detected at a depth of more than 6 cm in dead wood, implying the existence of free-living lichen mycobionts. To determine whether this was the case, we investigated whether Cladonia spp., the most frequently encountered mycobionts, occurred in wood without their photobionts. We detected green algae in all samples with records of Cladonia spp. Hence, we found no evidence for free-living Cladonia mycobionts in wood. We suggest that the detected Cladonia DNA in these logs originates from vegetative propagules or thallus fragments dispersed into the logs by animals or water. However, the occurrence of free-living stages of other lichen-forming fungal taxa in dead wood cannot be excluded.     

Modulation of C-to-T mutation by recombination-independent pairing of closely positioned DNA repeats

Repeat-induced point mutation is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. Repeat-induced point mutation detects duplications (irrespective of their origin, specific sequence, coding capacity, and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely positioned repeats can induce mutation of the adjoining nonrepetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.     

论长城系微化石群的真核生物意义

本文主要以燕山地区长城系页岩相微化石群为基础,通过与现代生物形态标志的对比,论证了在18亿年前真核藻类即已繁盛发育,其中既包括有原始的绿藻和甲藻,也有低级的真菌,还可能有原始类型的褐藻,至于真核生物出现的时间则可追溯到古元古代。根据已知资料的综合,按体制阶段的发展规律给出了绿藻、甲藻以及褐藻的可能的各自发展路线,可供探讨藻类起源与演化时参考。     

Two new subfamilies of DNA mismatch repair proteins (MutS) specifically abundant in the marine environment

MutS proteins are ubiquitous in cellular organisms and have important roles in DNA mismatch repair or recombination. In the virus world, the amoeba-infecting Mimivirus, as well as the recently sequenced Cafeteria roenbergensis virus are known to encode a MutS related to the homologs found in octocorals and ɛ-proteobacteria. To explore the presence of MutS proteins in other viral genomes, we performed a genomic survey of four giant viruses (‘giruses'') (Pyramimonas orientalis virus (PoV), Phaeocystis pouchetii virus (PpV), Chrysochromulina ericina virus (CeV) and Heterocapsa circularisquama DNA virus (HcDNAV)) that infect unicellular marine algae. Our analysis revealed the presence of a close homolog of Mimivirus MutS in all the analyzed giruses. These viral homologs possess a specific domain structure, including a C-terminal HNH-endonuclease domain, defining the new MutS7 subfamily. We confirmed the presence of conserved mismatch recognition residues in all members of the MutS7 subfamily, suggesting their role in DNA mismatch repair rather than DNA recombination. PoV and PpV were found to contain an additional type of MutS, which we propose to call MutS8. The MutS8 proteins in PoV and PpV were found to be closely related to homologs from ‘Candidatus Amoebophilus asiaticus'', an obligate intracellular amoeba-symbiont belonging to the Bacteroidetes. Furthermore, our analysis revealed that MutS7 and MutS8 are abundant in marine microbial metagenomes and that a vast majority of these environmental sequences are likely of girus origin. Giruses thus seem to represent a major source of the underexplored diversity of the MutS family in the microbial world.     

DNA polymerase of a basidiomycete fungus, Coprinus cinereus.

DNA polymerase activity was studied in Coprinus cinereus, a basidiomycete fungus. Only one from of the enzyme could be demonstrated, whether by affinity or ion-exchange chromatography; this enzyme had a molecular weight of 185000 on Sephadex G-200, and was inhibited by mercaptoethanol. Coprinus, a representative of the most advanced type of the filamentous fungi, resembles other eukaryotic micro-organisms in its lack of a mammalian beta-type DNA polymerase. The properties of the polymerase are compared with those of two other fungi, and found to resemble most closely the yeast polymerase A in Mg2+ requirements and template preference.     

Differences and similarities in chromatin structure of Neurospora crassa and higher eucaryotes.

The subunit structure of Neurospora chromatin which contains a full histone complement (Goff, 1976) exhibits both differences and similarities to chromatin of higher eucaryotes. The size of the DNA per subunit is only 170 +/- 5 base pairs, as compared to 200 base pairs in higher eucaryotes. However, the internal structures of the subunits are closely related. They contain 140 base pairs of DNA that are more tightly associated with the histone core and similarly arranged on the outside of the subunit. Hence the difference in structure resides in a shorter linker region of adjacent subunits in Neurospora chromatin. This is supported by a reduced primary cutting site and a lower content of lysines in histone H1. The role of H1 and its relation to the linker region are discussed.     

Microbial Community Structure, Pigment Composition, and Nitrogen Source of Red Snow in Antarctica

“Red snow” refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly ¹⁵N-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.     

Hyaloraphidium curvatum is not a green alga, but a lower fungus; Amoebidium parasiticum is not a fungus, but a member of the DRIPs

The unicellular heterotrophic protist Hyaloraphidium is classified with a family of green algae, the Ankistrodesmaceae. The only species that exists in pure culture and that is available for taxonomic studies is H. curvatum. Comparative 18S ribosomal RNA sequence analyses showed that H. curvatum belongs to the fungi rather than to the algae. Within the fungi, H. curvatum preferentially clustered with Chytridiomycetes. Unlike Chytridiomycetes, H. curvatum propagates by autosporulation, and the presence of flagella has never been reported. Transmission electron microscopy indicated that H. curvatum in some respects resembles Chytridiomycetes, but no elements of a flagellar apparatus were detected. The habitus of H. curvatum is unlike that of other fungi except the trichomycete Amoebidium parasiticum. The cell wall sugar composition of H. curvatum was unique, but to some extent resembled that of A. parasiticum. However, H. curvatum and A. parasiticum are not closely related to each other according to 18S rRNA sequence data. Moreover, A. parasiticum clustered with protistan animals, the Mesomycetozoa (DRIPs). Combined molecular, ultrastructural and chemical data do not allow assignment of H. curvatum to any recognized clade of fungi. This suggests that H. curvatum may represent an independent evolutionary lineage within the fungi.