The closest living relatives of land plants.

The embryophytes (land plants) have long been thought to be related to the green algal group Charophyta, though the nature of this relationship and the origin of the land plants have remained unresolved. A four-gene phylogenetic analysis was conducted to investigate these relationships. This analysis supports the hypothesis that the land plants are placed phylogenetically within the Charophyta, identifies the Charales (stoneworts) as the closest living relatives of plants, and shows the Coleochaetales as sister to this Charales/land plant assemblage. The results also support the unicellular flagellate Mesostigma as the earliest branch of the charophyte lineage. These findings provide insight into the nature of the ancestor of plants, and have broad implications for understanding the transition from aquatic green algae to terrestrial plants.

Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools.

The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

Phylogenetic analyses indicate that the 19'Hexanoyloxy-fucoxanthin-containing dinoflagellates have tertiary plastids of haptophyte origin.

The three anomalously pigmented dinoflagellates Gymnodinium galatheanum, Gyrodinium aureolum, and Gymnodinium breve have plastids possessing 19'-hexanoyloxy-fucoxanthin as the major carotenoid rather than peridinin, which is characteristic of the majority of the dinoflagellates. Analyses of SSU rDNA from the plastid and the nuclear genome of these dinoflagellate species indicate that they have acquired their plastids via endosymbiosis of a haptophyte. The dinoflagellate plastid sequences appear to have undergone rapid sequence evolution, and there is considerable divergence between the three species. However, distance, parsimony, and maximum-likelihood phylogenetic analyses of plastid SSU rRNA gene sequences place the three species within the haptophyte clade. Pavlova gyrans is the most basal branching haptophyte and is the outgroup to a clade comprising the dinoflagellate sequences and those of other haptophytes. The haptophytes themselves are thought to have plastids of a secondary origin; hence, these dinoflagellates appear to have tertiary plastids. Both molecular and morphological data divide the plastids into two groups, where G. aureolum and G. breve have similar plastid morphology and G. galatheanum has plastids with distinctive features.

A plastid of probable green algal origin in Apicomplexan parasites.

Protozoan parasites of the phylum Apicomplexa contain three genetic elements: the nuclear and mitochondrial genomes characteristic of virtually all eukaryotic cells and a 35-kilobase circular extrachromosomal DNA. In situ hybridization techniques were used to localize the 35-kilobase DNA of Toxoplasma gondii to a discrete organelle surrounded by four membranes. Phylogenetic analysis of the tufA gene encoded by the 35-kilobase genomes of coccidians T. gondii and Eimeria tenella and the malaria parasite Plasmodium falciparum grouped this organellar genome with cyanobacteria and plastids, showing consistent clustering with green algal plastids. Taken together, these observations indicate that the Apicomplexa acquired a plastid by secondary endosymbiosis, probably from a green alga.

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids.

Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information. Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria. Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity. In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.

Phylogenetic perspective on microbial life in hydrothermal ecosystems, past and present.

Understanding hydrothermal ecosystems, both past and present, requires basic information on the types of organisms present. Traditional methods, which require cultivation of microorganisms, fail to detect many taxa. We have used phylogenetic analyses of small subunit rRNA sequences obtained from microorganisms of a hot spring in Yellowstone National Park to explore the archael (archaebacterial) diversity present. Analysis of these sequences reveals several novel groups of archaea, greatly expanding our conception of the diversity of high temperature microorganisms, and demonstrating that hydrothermal systems harbour a rich variety of life. Many of these groups diverged from the archael line of descent early during evolution, and an understanding of their common properties may assist in inference of the nature of the last common ancestor of all life. The data also show a specific relationship between low-temperature marine archaea and some hot spring archaea, consistent with a thermophilic origin of life. Future use of rRNA-sequence-based techniques in exploration of hydrothermal systems should greatly facilitate study of modern thermophiles and give us insight into the activities of extinct communities as well.

Phylogenetic analysis of tufA sequences indicates a cyanobacterial origin of all plastids.

DNA sequences of the gene tufA, encoding elongation factor Tu, were determined from five cyanobacteria and 21 plastids. Three were full-length (ca. 1230 bp) sequences from cloned DNA, and 23 were partial (ca. 740 bp) sequences from PCR fragments. These sequences were aligned with sequences available from the literature, creating a data set of 56 tufA sequences of eubacterial or plastid origin. Phylogenetic analysis was performed on inferred amino acid sequences with parsimony and neighbor joining techniques, and on first and second position nucleotide sequences with maximum likelihood, and bootstrapping was performed with each method. Trees determined by the three methods were highly congruent with respect to well supported nodes. All examined plastids, including those of green and red algae, chromophytes, and Cyanophora paradoxa, cluster strongly with the cyanobacteria in all analyses. A cyanobacterial origin of all plastids confirms phylogenetic analyses of 16S rRNA and atpB sequences, but conflicts with those of rbcL and rbcS sequences. This discrepancy may be attributable to an ancient gene transfer of the rubisco operon in an ancestor of red algae and chromophytes. Maximum likelihood analysis also provides some support for a monophyletic origin of all plastids, while neighbor joining and parsimony analyses showed cyanobacteria and red, brown, and green plastid lineages as an unresolved polytomy. These tufA analyses also provide a broad perspective on eubacterial evolution and, in conjunction with published rRNA trees, point to at least two major radiations within eubacteria and their descendants: one of many eubacterial phyla, a second of cyanobacteria, and possibly a third radiation early in plastid evolution.

Gene phylogenies and the endosymbiotic origin of plastids.

The endosymbiotic origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence. Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and/or eukaryotes. Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (including rhodophytes, chromophytes, a cryptophyte and a glaucophyte), green algae, euglenoids and land plants. Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae. One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbcS and tufA trees and with the non-green plastid lineage in SSU rRNA trees. Analysis of psbA, tufA, atpB and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages. In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyletic in origin, with plastids being derived independently from both purple bacteria and cyanobacteria. A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.

Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene.

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in (13)C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO(2). beta-Carotene, an isoprenoid plant constituent, was depleted in (13)C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in (13)C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less (13)C) than C(3) plant material but when plants are stressed the isotopic composition could vary.

Lignin-like compounds and sporopollenin coleochaete, an algal model for land plant ancestry.

Unusual cell wall structure and resistance to microbial degradation led to an investigation of resistant biopolymers in Coleochaete (Chlorophyta, class Charophyceae), a green alga on the evolutionary lineage that led to land plants. In Coleochaete that are undergoing sexual reproduction, vegetative cell walls contain material similar to lignin, a substance generally thought absent from green algae, and the zygote wall includes sporopollenin. Knowledge of chemically resistant compounds in Coleochaete may facilitate interpretation of the fossil record. Placental transfer cells in Coleochaete orbicularis and in the hornwort Anthoceros survive acetolysis and contain lignin-like compounds, implying a close relation between these taxa.