Rupert De Wachter

Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity

Picoplankton—cells with a diameter of less than 3 µm—are the dominant contributors to both primary production and biomass in open oceanic regions. However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known. Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa. Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters. Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75 m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes. Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians. We also found a novel lineage, closely related to dinoflagellates and not previously described.

Database on the Structure of Large Ribosomal Subunit RNA

About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/

Compilation of small ribosomal subunit RNA structures

The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk.

TREECON: A software package for the construction and drawing of evolutionary trees

A package of programs (run by a management program called TREECON) was developed for the construction and drawing of evolutionary trees. The program MATRIX calculates dissimilarity values and can perform boostrap analysis on nucleic acid sequences. TREE implements different evolutionary tree constructing methods based on distance matrices. Because some of these methods produce unrooted evolutionary trees, a program ROOT places a root on the tree. Finally, the program DRAW draws the evolutionary tree, changes its size or topology, and produces drawings suitable for publication. Whereas, MATRIX is suited only for nucleic acids, the modules TREE, ROOT and DRAW are applicable to any kind of dissimilarity matrix. The programs run on IBM-compatible microcomputers using the DOS operating system.

Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites

Motivation: To improve the estimation of evolutionary distances between nucieotide sequences by considering the differences in substitution rates among sites. Results: TREECON for Windows (Van de Peer.Y. and De Wachter.R. Comput. Applic. Biosci., 9, 569-570, 1994) is a software package for the construction and drawing of phylogenetic trees based on distance data computed from nucleic acid and amino acid sequences. For nucleic acids, we here describe the implementation of a recently developed method for estimating evolutionary distances taking into account the substitution rate of individual sites in a sequence alignment. Availability: TREECON for Windows is available on request from the authors. A small fee is asked in order to support the work and to reinvest in new computer hard- and software. More information about the program and substitution rate calibration can be found at URL http://bioc-www.uia.ac.be/uJ yvdp/treeconw. html. Contact: E-mail: yvdp@uia.ua.ac.be

Structure of the 16 S ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF ('Mastigocladus laminosus HTF') strain PCC7518, and phylogenetic analysis.

The thermophilic cyanobacterial strain, PCC7518, originally identified as ‘Mastigocladus laminosus HTF’ does not show branchings or heterocysts. The absence of branchings supports the later assignment to the genus Chlorogloeopsis. The absence of heterocysts may be the result of a mutation because heterocysts were observed in the original isolate. Alternatively, contamination may have happened. To solve this problem, the 16 S rRNA sequence was determined and used to infer a secondary structure model and build distance trees. The trees showed that strain PCC7518 belongs to the cluster of heterocystous species and has most probably lost the ability to produce heterocysts by mutation. It is only distantly related to Chlorogloeopsis fritschii PCC6718.

The determination and comparison of the 16S rRNA gene sequences of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships.

Summary As a consolidated effort on the part of several laboratories, partial and nearly complete sequence determinations of 165 rRNA genes have been applied as one of several analytical methods in a polyphasic study of the pseudomonads. Nearly-complete sequences have been determined of the PCR-amplified 16S rRNA genes of 21 species of the genus Pseudomonas (sensu stricto), including multiple strains of most species. Phylogenetic branching orders and the natural intrageneric relationships among the species have been inferred through sequence comparisons and cluster analysis and have not shown any obvious recognizable correlation with results derived through standard phenotypic criteria commonly used to group the species. This paper also focuses on the ability of 16S rRNA gene sequences, particularly the hypervariable sequence regions, to be used as nested identification markers and as target sites for the development of 16S rRNA sequence-based strategies for the identification of species of the genus Pseudomonas .

DCSE, an interactive tool for sequence alignment and secondary structure research.

DCSE provides a user-friendly package for the creation and editing of sequence alignments. The program runs on different platforms, including microcomputers and workstations. Apart from available hardware, the program is not limited in the size of the alignment it can handle. It deviates more from classical text editors than other available sequence editors because it uses a different approach towards editing. It shifts characters or entire blocks of aligned characters, rather than inserting or deleting gaps in the sequences. Alignment of a new sequence to an existing alignment is partly automated. Although DCSE can be used on protein sequence alignments, it is especially targeted at the examination of RNA. The secondary structure for every sequence can be incorporated easily in the alignment. DCSE also has extensive built-in support for finding and checking secondary structure elements. A sophisticated system of markers allows notation of special positions in an alignment. This system can be used to store information such as the position of hidden breaks, introns and tertiary structure interactions.

The European database on small subunit ribosomal RNA

The European database on SSU rRNA can be consulted via the World WideWeb at http://rrna.uia.ac.be/ssu/ and compiles all complete or nearly complete small subunit ribosomal RNA sequences. Sequences are provided in aligned format. The alignment takes into account the secondary structure information derived by comparative sequence analysis of thousands of sequences. Additional information such as literature references, taxonomy, secondary structure models and nucleotide variability maps, is also available.

Evolutionary Relationships Among the Eukaryotic Crown Taxa Taking into Account Site-to-Site Rate Variation in 18S rRNA

Abstract. In this study we constructed a bootstrapped distance tree of 500 small subunit ribosomal RNA sequences from organisms belonging to the so-called crown of eukaryote evolution. Taking into account the substitution rate of the individual nucleotides of the rRNA sequence alignment, our results suggest that (1) animals, true fungi, and choanoflagellates share a common origin: The branch joining these taxa is highly supported by bootstrap analysis (bootstrap support [BS] > 90%), (2) stramenopiles and alveolates are sister groups (BS = 75%), (3) within the alveolates, dinoflagellates and apicomplexans share a common ancestor BS > 95%), while in turn they both share a common origin with the ciliates (BS > 80%), and (4) within the stramenopiles, heterokont algae, hyphochytriomycetes, and oomycetes form a monophyletic grouping well supported by bootstrap analysis (BS > 85%), preceded by the well-supported successive divergence of labyrinthulomycetes and bicosoecids.On the other hand, many evolutionary relationships between crown taxa are still obscure on the basis of 18S rRNA. The branching order between the animal-fungal-choanoflagellates clade and the chlorobionts, the alveolates and stramenopiles, red algae, and several smaller groups of organisms remains largely unresolved.When among-site rate variation is not considered, the inferred tree topologies are inferior to those where the substitution rate spectrum for the 18S rRNA is taken into account. This is primarily indicated by the erroneous branching of fast-evolving sequences. Moreover, when different substitution rates among sites are not considered, the animals no longer appear as a monophyletic grouping in most distance trees.In this study we constructed a bootstrapped distance tree of 500 small subunit ribosomal RNA sequences from organisms belonging to the so-called crown of eukaryote evolution. Taking into account the substitution rate of the individual nucleotides of the rRNA sequence alignment, our results suggest that (1) animals, true fungi, and choanoflagellates share a common origin: The branch joining these taxa is highly supported by bootstrap analysis (bootstrap support [BS] > 90%), (2) stramenopiles and alveolates are sister groups (BS = 75%), (3) within the alveolates, dinoflagellates and apicomplexans share a common ancestor BS > 95%), while in turn they both share a common origin with the ciliates (BS > 80%), and (4) within the stramenopiles, heterokont algae, hyphochytriomycetes, and oomycetes form a monophyletic grouping well supported by bootstrap analysis (BS > 85%), preceded by the well-supported successive divergence of labyrinthulomycetes and bicosoecids. On the other hand, many evolutionary relationships between crown taxa are still obscure on the basis of 18S rRNA. The branching order between the animal-fungal-choanoflagellates clade and the chlorobionts, the alveolates and stramenopiles, red algae, and several smaller groups of organisms remains largely unresolved. When among-site rate variation is not considered, the inferred tree topologies are inferior to those where the substitution rate spectrum for the 18S rRNA is taken into account. This is primarily indicated by the erroneous branching of fast-evolving sequences. Moreover, when different substitution rates among sites are not considered, the animals no longer appear as a monophyletic grouping in most distance trees.