EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL AND RED ALGAL SMALL SUBUNIT RRNA GENES1

A previous study of the North American biogeography of the red algal genus Hildenbrandia noted the presence of group I introns in the nuclear small subunit (SSU) rRNA gene of the marine species H. rubra (Sommerf.) Menegh. Group IC1 introns have been previously reported at positions 516 and 1506 in the nuclear SSU RNA genes in the Bangiales and Hildenbrandiales. However, the presence of an unclassified intron at position 989 in a collection of H. rubra from British Columbia was noted. This intron is a member of the IE subclass and is the first report of this intron type in the red algae. Phylogenetic analyses of the intron sequences revealed a close relationship between this IE intron inserted at position 989 and similar fungal IE introns in positions 989 and 1199. The 989 IE introns formed a moderately to well‐supported clade, whereas the 1199 IE introns are weakly supported. Unique structural helices in the P13 domain of the 989 and 1199 IE introns also point to a close relationship between these two clades and provide further evidence for the value of secondary structural characteristics in identifying homologous introns in evolutionarily divergent organisms. The absence of the 989 IE intron in all other red algal nuclear SSU rRNA genes suggests that it is unlikely that this intron was vertically inherited from the common ancestor of the red algal and fungal lineages but rather is the result of lateral transfer between fungal and red algal nuclear SSU rRNA genes.     

BIOGEOGRAPHY AND ECOLOGY OF THE KELP/RED ALGAL SYMBIOSIS

A kelp/red algal symbiosis is described from nature based on extensive collections from the San Juan Islands, Washington. Kelp gametophytes were found as endophytes in the cell walls of seventeen species of red algae in three different kelp communities. Host red algae were mostly filamentous (e.g., Pleonosporium vancouverianum) or polysiphonous (e.g. Polysiphonia paniculata). The kelp gametophytes completed vegetative and reproductive development in the hosts with gametangia formed at the host surface and with sporophytes up to several mm in height being produced while still attached to the host. To date, none of the kelp gametophytes from nature have been identified to genus or species, although the gametophyte of Nereocystis luetkeana is a potential candidate for the symbiosis. Preliminary observations from Nova Scotia and the Isle of Man have not found the association in the Atlantic Ocean. Laboratory studies in Korea successfully reconstructed the symbiosis in the red alga Aglaothamnion oosumiense using zoospores of Undaria pinnatifida but not Laminaria religiosa. Here we outline the development of the symbiosis and discuss the potential adaptive significance of the kelp/red algal interaction.     

ALGAL TRANSGENICS IN THE GENOMIC ERA1

The last few years have witnessed significant advances in the field of algal genomics. Complete genome sequences from the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana have been published, the genomes for two more algae (Chlamydomonas reinhardtii and Ostreococcus tauri) are nearing completion, and several others are in progress or at the planning stage. In addition, large‐scale cDNA sequencing projects are being carried out for numerous algal species. This wealth of genome data is serving as a powerful catalyst for the development and application of recombinant techniques for these species. The data provide a rich resource of DNA elements such as promoters that can be used for transgene expression as well as an inventory of genes that are possible targets for genetic engineering programs aimed at manipulating algal metabolism. It is not surprising therefore that significant progress in the genetic engineering of eukaryotic algae is being made. Nuclear transformation of various microalgal species is now routine, and progress is being made on the transformation of macroalgae. Chloroplast transformation has been achieved for green, red, and euglenoid algae, and further success in organelle transformation is likely as the number of sequenced plastid, mitochondrial, and nucleomorph genomes continues to grow. Importantly, the commercial application of algal transgenics is beginning to be realized, and algal biotechnology companies are being established. Recent work has shown that recombinant proteins of therapeutic value can be produced in microalgal species, and it is now realistic to envisage the genetic engineering of commercially important species to improve production of valuable algal products. In this article we review the recent progress in algal transgenics and consider possible future developments now that phycology has entered the genomic era.     

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.     

THE USE OF PLASTID DNA RESTRICTION ENDONUCLEASE PATTERNS IN DELINEATING RED ALGAL SPECIES AND POPULATIONS1

Plastid DNA band patterns generated by electrophoresis of endonuclease digests demonstrate remarkable conservation of DNA sequences at the species and subspecies level in flowering plants. Generally, patterns are identical or near-identical from different populations belonging to the same species. This methodology has now been applied to red algae to ascertain its value in systematic studies. Plastid DNA from nine bangiophycean and florideophycean red algae was isolated and cut with restriction endonucleases that recognize different 6-base pair sequences. The patterns generated upon the electrophoretic separation of digestion fragments show that within a species patterns are identical, but not within higher taxa. The proper identification of one Gracilaria population of uncertain taxonomic affinity was clearly established by this method of plastid DNA analysis. Differences between species in plastid DNA sequences were confirmed by probing blots of restriction fragments with known gene sequences. A number of heterologous plastid DNA probes were found to be sufficiently homologous to be useful in studying red algal DNA. Unexpectedly, supercoiled circular plasmids ranging in size from ca. 1.5–8 kb were found in some red algal species but not in others. The position of these plasmids in agarose gels following electrophoresis is uniform within a species but differs between different species of the same genus, contributing further patterns for taxonomic analysis.     

THE EVOLUTION OF PARASITISM IN THE RED ALGAE: MOLECULAR COMPARISONS OF ADELPHOPARASITES AND THEIR HOSTS1

In several groups of parasites including insect, flowering plant, fungal, and red algal parasites, morphological similarities of the parasites and their specific hosts have led to hypotheses that these parasites evolved from their hosts. But these conclusions have been criticized because the morphological features shared by parasite and host may be the result of convergent evolution. In this study, we examine the hypothesis, originally put forth by Setchell, that adelphoparasitic red algae, that is, parasitic red algae that are morphologically very similar to their hosts, evolved from their specific red algal hosts. Rather than comparing morphological features of parasites and hosts, small-subunit 18S nuclear ribosomal DNA and the internal transcribed spacer regions (ITSs) of the nuclear ribosomal repeat are compared for five parasites, their hosts, and related nonhosts from four red algal orders. These comparisons reveal that each of these adelphoparasites has evolved either directly from the host on which it is currently found, or it evolved from some other taxon that is closely related to the modern host. The parasites Gardneriella tuberifera, Rhodymeniocolax botryoides, and probably Gracilariophila oryzoides evolved from their respective hosts Sarcodiotheca gaudichaudii, Rhodymenia pacifica, and Gracilariopsis lemaneiformis, respectively. The parasite Faucheocolax attenuata evolved from either Fauchea laciniata or Fauchea fryeana and subsequently radiated onto the other host species. Presently this parasite is found on both hosts. Lastly, some parasitic genera such as Plocamiocolax are polyphyletic in their origins. A species of Plocamiocolax from an Antarctic Plocamium cartilagineum appears to have evolved from its host whereas the common Plocamiocolax pulvinata that occurs along the west coast of North America likely evolved from Plocamium violaceum and radiated secondarily onto its present day host, Plocamium cartilagineum.     

THE SYMBIOTIC BIRTH AND SPREAD OF PLASTIDS: HOW MANY TIMES AND WHODUNIT?

I discuss the evidence for a single origin of primary plastids in the context of a paper in this issue challenging this view, and I review recent evidence concerning the number of secondary plastid endosymbioses and the controversy over whether the relic plastid of apicomplexans is of red or green algal origin. A broad consensus has developed that the plastids of green algae, red algae, and glaucophytes arose from the same primary, cyanobacterial endosymbiosis. Although the analyses in this issue by Stiller and colleagues firmly undermine one of many sources of data, gene content similarities among plastid genomes used to argue for a monophyletic origin of primary plastids, the overall evidence still clearly favors monophyly. Nonetheless, this issue should not be considered settled and new data should be sought from better sampling of cyanobacteria and glaucophytes, from sequenced nuclear genomes, and from careful analysis of such key features as the plastid import apparatus. With respect to the number of secondary plastid symbioses, it is completely unclear as to whether the secondary plastids of euglenophytes and chlorarachniophytes arose by the same or two different algal endosymbioses. Recent analyses of certain plastid and nuclear genes support the chromalveolate hypothesis of Cavalier-Smith, namely, that the plastids of heterokonts, haptophytes, cryptophytes, dinoflagellates, and apicomplexans all arose from a common endosymbiosis involving a red alga. However, another recent paper presents intriguing conflicting data on this score for one of these groups—apicomplexans—arguing instead that they acquired their plastids from green algae.     

The tetrapyrrole synthesis pathway as a model of horizontal gene transfer in euglenoids

The history of euglenoids may have begun as early as ~2 bya. These early phagotrophs ate cyanobacteria, archaea, and eubacteria, and the subsequent appearance of red algae and chromalveolates provided euglenoids with additional food sources. Following the appearance of green algae, euglenoids acquired a chloroplast via a secondary endosymbiotic event with a green algal ancestor. This endosymbiosis also involved a massive transfer of nuclear‐encoded genes from the symbiont nucleus to the host. Expecting these genes to have a green algal origin, this research has shown, through the use of DNA‐sequences and the analysis of phylogenetic relationships, that many housekeeping genes have a red algal/chromalveolate ancestry. This suggested that many other endosymbiotic/horizontal gene transfers, which brought genes from chromalveolates to euglenoids, may have been taking place long before the acquisition of the chloroplast. The investigation of the origin of the enzymes involved in the tetrapyrrole synthesis pathway provided insights into horizontal gene transfer in euglenoids and demonstrated that the euglenoid nuclear genome is a mosaic comprised of genes from the ancestral lineage plus genes transferred endosymbiotically/horizontally from green, red, and chromalveolates lineages.     

UTILITY OF THE MITOCHONDRIAL nad4L GENE FOR ALGAL AND PROTISTAN PHYLOGENETIC ANALYSIS1

Mitochondrial gene sequences, in contrast to their plastid counterparts, have been rarely exploited for use in determining algal phylogenetic relationships. Their utilization would prove advantageous particularly for the assessment of evolutionary relationships among protistan lineages that include photo synthetic species and their colorless heterotrophic dependents that contain only a reduced plastid genome as well as ancestral nonphotosynthetic relatives. Recent studies that have examined mitochondrial protein (coxl, cox2, cob, and nad5) rather than ribosomal RNA genes confirm that these algal sequences are sufficiently conserved to be used routinely in algal systematics. In this investigation, we sequenced the mitochondrial nad4L gene, encoding a small (ca. 99-amino acid) polypeptide subunit of the NADH dehydrogenase complex, from two chromophytic algae and an oomycete to determine its suitability for use in determining algal and protistan evolutionary relationships. Our analysis demonstrated that nad4L could clearly resolve these three organisms as a distinct clade, the stramenopiles, to the exclusion of terrestrial plants/chlorophytes and eufungi. However, the position of deeply rooted species within the stramenopiles proved tenuous and would best be examined in conjunction with the analysis of longer mitochondrial protein gene sequences.     

ALGAL MODEL SYSTEMS AND THE ELUCIDATION OF PHOTOSYNTHETIC METABOLISM

The main impact of algal investigations on a wider understanding of photosynthesis in O₂ evolvers has come about through studies on microalgae. Experiments with flashing light led to the concepts of photo- synthetic units and reaction centers. Work on photon yields and the Emerson enhancement effect with fluorescence and spectroscopy, as well as investigations with mutants, were crucial in elaborating and then testing the validity of the Z scheme for light reactions. The carbon reduction (or Calvin) cycle was discovered using ¹⁴C, chromatography, and Chlorella (plus Scenedesmus ). The availability of complete gene sequences for several cyanobacteria and the flowering plant Arabidopsis and very soon for the eukaryotic alga Chlamydomonas permit important advances in our understanding of photosynthesis in all these organisms. Eukaryotic microalgae and cyanobacteria remain advantageous in fundamental photosynthetic studies.     

The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria-containing eukaryotes and an alternative hypothesis on the origin of plastids

Abstract Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (α- and β-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1α, α-tubulin, and β-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various primary and secondary plastid-containing lineages (green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, and Heterolobosea. The red algae represented the sister lineage to Group B. Using 34 OTUs for which essentially the entire amino acid sequences of the four genes are known, MP, distance, quartet puzzling, and two types of maximum likelihood (ML) calculations all robustly resolved the monophyly of Group B, as well as the basal position of red algae within eukaryotic organisms. In addition, phylogenetic analyses of a concatenated 4639-amino-acid sequence for 12 nuclear genes (excluding the EF-2 gene) of 12 mitochondria-containing OTUs (including C. merolae) resolved a robust non-sister relationship between green plants and red algae within a robust monophyletic group composed of red algae and the eukaryotic organisms belonging to Group B. A new scenario for the origin and evolution of plastids is suggested, based on the basal phylogenetic position of the red algae within the large clade (Group B plus red algae). The primary plastid endosymbiosis likely occurred once in the common ancestor of this large clade, and the primary plastids were subsequently lost in the ancestor(s) of the Discicristata (euglenoids, Kinetoplastida, and Heterolobosea), Heterokontophyta, and Alveolata (apicomplexans and Ciliophora). In addition, a new concept of “Plantae” is proposed for phototrophic and nonphototrophic organisms belonging to Group B and red algae, on the basis of the common history of the primary plastid endosymbiosis. The Plantae include primary plastid-containing phototrophs and nonphototrophic eukaryotes that possibly contain genes of cyanobacterial origin acquired in the primary endosymbiosis.     

MICROPHOTOMETRIC ASSESSMENT OF SPECTRAL ABSORPTION AND ITS POTENTIAL APPLICATION FOR CHARACTERIZATION OF HARMFUL ALGAL SPECIES

A comparison was made of microphotometric measurements and spectrophotometric measurements of particulate spectral absorption of four algal species, including the chlorophyte Dunaliella tertiolecta Butcher; a nontoxic dinoflagellate, Amphidinium carterae Hulburt; a diatom, Chaetoceros gracilis Schütt; and a toxic dinoflagellate, Gymnodinium breve Steidinger. Particulate spectral absorption of monospecies cultures was estimated as the product of the average absorption efficiency factor, Q _a, determined by microphotometry, the cellular cross-sectional area, and the cell number density. Estimates of particulate spectral absorption from microphotometric measurements were, in most cases, within one standard deviation of values determined from spectrophotometric measurements of algal suspensions. Estimates of Q_a(675) were shown to be consistent with values reported in previous studies for cells of similar size and pigmentation and were consistent with theoretical predictions. Absorption spectra of mixtures of C. gracilis and G. breve were numerically decomposed into contributions by absorption signatures of monospecies cultures using either spectrophotometric or microphotometric measurements as the basis for end members. Modeled contributions assigned to either species displayed trends consistent with the actual proportions contributed to the spectrum by each algal culture. However, the technique was sensitive to measurement variability, which reduced the level of agreement between modeled and actual contributions. The utility of this approach for identification of algal taxa will depend on the degree to which algal spectral absorption signatures differ and the capabilities for acquiring high-resolution data with low signal-to-noise ratios.     

PHYLOGENOMICS AND SECONDARY PLASTIDS: A LOOK BACK AND A LOOK AHEAD1

Despite their importance to evolution, ecology, and cell biology, eukaryotes that acquired plastids through secondary endosymbiosis remain poorly studied from a genomic standpoint. Chromalveolata, a eukaryotic supergroup proposed to have descended from a heterotrophic eukaryote that acquired a red algal plastid by secondary endosymbiosis, includes four major lineages (alveolates, cryptophytes, haptophytes, and heterokonts). The chromalveolates exhibit remarkable diversity of cellular organization, and the available data suggest that they exhibit equal diversity in their genome organization. One of the most obvious differences in cellular organization is the retention of a highly reduced red algal nucleus in cryptophytes (also known as cryptomonads), but there are other major differences among chromalveolate lineages, including the loss of photosynthesis in multiple lineages. Although the hypothesis of chromalveolate monophyly is appealing, there is limited support for the hypothesis from nuclear genes, and questions have even been raised about the monophyly of chromalveolate plastids. Evidence for the chromalveolate hypothesis from large‐scale nuclear data sets is reviewed, and alternative hypotheses are described. The potential for integrating information from chromalveolate genomics into functional genomics is described, emphasizing both the methodological challenges and the opportunities for future phylogenomic analyses of these groups.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

EVOLUTIONARY ANALYSES OF THE NUCLEAR‐ENCODED PHOTOSYNTHETIC GENE psbO FROM TERTIARY PLASTID‐CONTAINING ALGAE IN DINOPHYTA1

Although the dinophytes generally possess red‐algal‐derived secondary plastids, tertiary plastids originating from haptophyte and diatom ancestors are recognized in some lineages within the Dinophyta. However, little is known about the nuclear‐encoded genes of plastid‐targeted proteins from the dinophytes with diatom‐derived tertiary plastids. We analyzed the sequences of the nuclear psbO gene encoding oxygen‐evolving enhancer protein from various algae with red‐algal‐derived secondary and tertiary plastids. Based on our sequencing of 10 new genes and phylogenetic analysis of PsbO amino acid sequences from a wide taxon sampling of red algae and organisms with red‐algal‐derived plastids, dinophytes form three separate lineages: one composed of peridinin‐containing species with secondary plastids, and the other two having haptophyte‐ or diatom‐derived tertiary plastids and forming a robust monophyletic group with haptophytes and diatoms, respectively. Comparison of the N‐terminal sequences of PsbO proteins suggests that psbO genes from a dinophyte with diatom‐derived tertiary plastids (Kryptoperidinium) encode proteins that are targeted to the diatom plastid from the endosymbiotic diatom nucleus as in the secondary phototrophs, whereas the fucoxanthin‐containing dinophytes (Karenia and Karlodinium) have evolved an additional system of psbO genes for targeting the PsbO proteins to their haptophyte‐derived tertiary plastids from the host dinophyte nuclei.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

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.