Investigation of molluscan phylogeny using large-subunit and small-subunit nuclear rRNA sequences

The Mollusca represent one of the most morphologically diverse animal phyla, prompting a variety of hypotheses on relationships between the major lineages within the phylum based upon morphological, developmental, and paleontological data. Analyses of small-ribosomal RNA (SSU rRNA) gene sequence have provided limited resolution of higher-level relationships within the Mollusca. Recent analyses suggest large-subunit (LSU) rRNA gene sequences are useful in resolving deep-level metazoan relationships, particularly when combined with SSU sequence. To this end, LSU (approximately 3.5 kb in length) and SSU (approximately 2 kb) sequences were collected for 33 taxa representing the major lineages within the Mollusca to improve resolution of intraphyletic relationships. Although the LSU and combined LSU+SSU datasets appear to hold potential for resolving branching order within the recognized molluscan classes, low bootstrap support was found for relationships between the major lineages within the Mollusca. LSU+SSU sequences also showed significant levels of rate heterogeneity between molluscan lineages. The Polyplacophora, Gastropoda, and Cephalopoda were each recovered as monophyletic clades with the LSU+SSU dataset. While the Bivalvia were not recovered as monophyletic clade in analyses of the SSU, LSU, or LSU+SSU, the Shimodaira-Hasegawa test showed that likelihood scores for these results did not differ significantly from topologies where the Bivalvia were monophyletic. Analyses of LSU sequences strongly contradict the widely accepted Diasoma hypotheses that bivalves and scaphopods are closely related to one another. The data are consistent with recent morphological and SSU analyses suggesting scaphopods are more closely related to gastropods and cephalopods than to bivalves. The dataset also presents the first published DNA sequences from a neomeniomorph aplacophoran, a group considered critical to our understanding of the origin and early radiation of the Mollusca.     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data

We investigated evolutionary relationships among deuterostome subgroups by obtaining nearly complete large-subunit ribosomal RNA (LSU rRNA)-gene sequences for 14 deuterostomes and 3 protostomes and complete small-subunit (SSU) rRNA-gene sequences for five of these animals. With the addition of previously published sequences, we compared 28 taxa using three different data sets (LSU only, SSU only, and combined LSU + SSU) under minimum evolution (with LogDet distances), maximum likelihood, and maximum parsimony optimality criteria. Additionally, we analyzed the combined LSU + SSU sequences with spectral analysis of LogDet distances, a technique that measures the amount of support and conflict within the data for every possible grouping of taxa. Overall, we found that (1) the LSU genes produced a tree very similar to the SSU gene tree, (2) adding LSU to SSU sequences strengthened the bootstrap support for many groups above the SSU-only values (e.g., hemichordates plus echinoderms as Ambulacraria; lancelets as the sister group to vertebrates), (3) LSU sequences did not support SSU-based hypotheses of pterobranchs evolving from enteropneusts and thaliaceans evolving from ascidians, and (4) the combined LSU + SSU data are ambiguous about the monophyly of chordates. No tree-building algorithm united urochordates conclusively with other chordates, although spectral analysis did so, providing our only evidence for chordate monophyly. With spectral analysis, we also evaluated several major hypotheses of deuterostome phylogeny that were constructed from morphological, embryological, and paleontological evidence. Our rRNA-gene analysis refutes most of these hypotheses and thus advocates a rethinking of chordate and vertebrate origins.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Interrelationships and evolution of the tapeworms (Platyhelminthes: Cestoda)

Interrelationships of the tapeworms (Platyhelminthes: Cestoda) were examined by use of small (SSU) and large (LSU) subunit ribosomal DNA sequences and morphological characters. Fifty new complete SSU sequences were added to 21 sequences previously determined, and 71 new LSU (D1-D3) sequences were determined for the complementary set of taxa representing each of the major lineages of cestodes as currently understood. New sequences were determined for three amphilinidean taxa, but were removed from both alignments due to their excessively high degree of divergence from other cestode sequences. A morphological character matrix coded for supraspecific taxa was constructed by the modification of matrices from recently published studies. Maximum-parsimony (MP) analyses were performed on the LSU, SSU, LSU+SSU, and morphological data partitions, and minimum-evolution (ME) analyses utilizing a general time reversible model of nucleotide substitution including estimates of among-site rate heterogeneity were performed on the molecular data partitions. Resulting topologies were rooted at the node separating the Gyrocotylidea from the Eucestoda. The LSU data were found to be more informative than the SSU data and were more consistent with inferences from morphology, although nodal support was generally weak for most basal nodes. One class of transitions was found to be saturated for comparisons between the most distantly related taxa (gyrocotylideans vs cyclophyllideans and tetrabothriideans). Differences in the topologies resulting from MP and ME analyses were not statistically significant. Nonstrobilate orders formed the basal lineages of trees resulting from analysis of LSU data and morphology. Difossate orders were basal to tetrafossate orders, the latter of which formed a strongly supported clade. A clade including the orders Cyclophyllidea, Nippotaeniidea, and Tetrabothriidea was supported by all data partitions and methods of analysis. Paraphyly of the orders Pseudophyllidea, Tetraphyllidea, and Trypanorhyncha was consistent among the molecular data partitions. Inferences are made regarding a monozoic (nonsegmented) origin of the Eucestoda as represented by the Caryophyllidea and for the evolution of the strobilate and acetabulate/tetrafossate conditions having evolved in a stepwise pattern.     

Morphological convergence characterizes the evolution of Xanthophyceae (Heterokontophyta): evidence from nuclear SSU rDNA and plastidial rbcL genes

Xanthophyceae are a group of heterokontophyte algae. Few molecular studies have investigated the evolutionary history and phylogenetic relationships of this class. We sequenced the nuclear-encoded SSU rDNA and chloroplast-encoded rbcL genes of several xanthophycean species from different orders, families, and genera. Neither SSU rDNA nor rbcL genes show intraspecific sequence variation and are good diagnostic markers for characterization of problematic species. New sequences, combined with those previously available, were used to create different multiple alignments. Analyses included sequences from 26 species of Xanthophyceae plus three Phaeothamniophyceae and two Phaeophyceae taxa used as outgroups. Phylogenetic analyses were performed according to Bayesian inference, maximum likelihood, and maximum parsimony methods. We explored effects produced on the phylogenetic outcomes by both taxon sampling as well as selected genes. Congruent results were obtained from analyses performed on single gene multiple alignments as well as on a data set including both SSU rDNA and rbcL sequences. Trees obtained in this study show that several currently recognized xanthophycean taxa do not form monophyletic groups. The order Mischococcales is paraphyletic, while Tribonematales and Botrydiales are polyphyletic even if evidence for the second order is not conclusive. Botrydiales and Vaucheriales, both including siphonous taxa, do not form a clade. The families Botrydiopsidaceae, Botryochloridaceae, and Pleurochloridaceae as well as the genera Botrydiopsis and Chlorellidium are polyphyletic. The Centritractaceae and the genus Bumilleriopsis also appear to be polyphyletic but their monophyly cannot be completely rejected with current evidence. Our results support morphological convergence at any taxonomic rank in the evolution of the Xanthophyceae. Finally, our phylogenetic analyses exclude an origin of the Xanthophyceae from a Vaucheria-like ancestor and favor a single early origin of the coccoid cell form.     

Independent terrestrial origins of the Halosphaeriales (marine Ascomycota)

A phylogenetic study of marine ascomycetes was initiated to test and refine evolutionary hypotheses of marine-terrestrial transitions among ascomycetes. Taxon sampling focused on the Halosphaeriales, the largest order of marine ascomycetes. Approximately 1050 base pairs (bp) of the gene that codes for the nuclear small subunit (SSU) and 600 bp of the gene that codes for the nuclear large subunit (LSU) ribosomal RNAs (rDNA) were sequenced for 15 halosphaerialean taxa and integrated into a data set of homologous sequences from terrestrial ascomycetes. An initial set of phylogenetic analyses of the SSU rDNA from 38 taxa representing 15 major orders of the phylum Ascomycota confirmed a close phylogenetic relationship of the halosphaerialean species with several other orders of perithecial ascomycetes. A second set of analyses, which involved more intensive taxon sampling of perithecial ascomycetes, was performed using the SSU and LSU rDNA data in combined analyses. These second analyses included 15 halosphaerialean taxa, 26 terrestrial perithecial fungi from eight orders, and five outgroup taxa from the Pezizales. In these analyses the Halosphaeriales were polyphyletic and comprised two distinct lineages. One clade of Halosphaeriales comprised 12 taxa from 11 genera and was most closely related to terrestrial fungi of the Microascales. The second clade of halosphaerialean fungi comprised taxa from the genera Lulworthia and Lindra and was an isolated lineage among the perithecial fungi. Both the main clade of Halosphaeriales and the Lulworthia/Lindra clade are supported by the data as being independently derived from terrestrial ancestors.     

RpoA: a useful gene for phylogenetic analysis in diatoms

The aim of this study was to compare the usefulness of two chloroplast-encoded genes (rpoA and rbcL) and the nuclear-encoded small subunit (SSU) ribosomal RNA for reconstructing phylogenetic relationships among diatoms at lower taxonomic levels. To this end, the rpoA and rbcL genes for selected centric and pennate diatoms were sequenced. The new rpoA and rbcL sequences, and an existing nuclear-encoded SSU rRNA data set, were subjected to weighted/unweighted parsimony, maximum likelihood, minimum evolution, and Bayesian analyses. All of the tree-building methods employed showed, based on the support values, that the rpoA gene was the most useful, relative to the rbcL and SSU rRNA genes, in determining phylogenetic relationships among the sampled diatoms. The support values for the relationships among the pennate lineages were, in many instances, greater in the rpoA trees than in the SSU rRNA trees. These results suggest that rpoA might be of value in determining phylogenetic relationships among pennate lineages.     

A REEXAMINATION OF THE SYSTEMATICS OF THE ACROCHAETIALES (RHODOPHYTA) USING LARGE-SUBUNIT RDNA SEQUENCE DATA

The taxonomic history of the red algal order Acrochaetiales is chaotic. There is no consensus in the literature as to how many genera should be recognized or in the assignment of the over 400 species to these genera. Morphological and anatomical studies have provided a suite of possible characters to delineate genera within this order, but there have been major discrepancies in the assessment and use of these features. The phylogenetic placement of the Acrochaetiales has also been the focus of debate. Once thought to be the most ancestral florideophyte lineage, recent molecular systematic studies have illustrated that this order is a derived lineage closely related to the Nemaliales and Palmariales. Phylogenies using sequences of the small-subunit (SSU) rDNA have strongly supported two very divergent lineages within a possibly polyphyletic Acrochaetiales. The relationships between these two groups and among other closely related rhodophyte orders were not resolved. We have generated large-subunit (LSU) rDNA sequence data for representatives of the Acrochaetiales and related taxa. Distance and parsimony phylogenies based on LSU and combined SSU and LSU data will be presented. The increased phylogenetic signal afforded by this approach will shed light on previous conundrums in the systematics of this group.     

Testing the new animal phylogeny: first use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes

Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete LSU gene sequences were obtained, and combined LSU + SSU sequences were assembled, for 15 distantly related protostome taxa plus five deuterostome outgroups. When the aligned LSU + SSU sequences were analyzed by tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were indeed strongly supported (e.g., bootstrap values >90%), with higher support than from the SSU sequences alone. Furthermore, with the LogDet-based methods, the LSU + SSU sequences resolved some accepted subgroups within Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences grouped with the mollusc and lophophorates)-subgroups that SSU-based studies do not reveal. Also, the mollusc sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our LSU + SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at lower taxonomic levels, and its core region is easy to align across distant taxa-but its base frequencies tend to be nonstationary across such taxa. We conclude that molecular systematists should use combined LSU + SSU rRNA genes rather than SSU alone.     

Systematics of the Pezizomycetes--the operculate discomycetes

The Pezizomycetes (order Pezizales) is an early diverging lineage within the Pezizomycotina. A shared derived character, the operculate ascus, supports the Pezizales as monophyletic, although functional opercula have been lost in certain taxa. Phylogenetic relationships within Pezizales were studied using parsimony and Bayesian analyses of partial SSU and LSU rDNA sequences from 100 taxa representing 82 genera and 13 of the 15 families currently recognized. Three primary lineages are identified that more or less correspond to the A, B and C lineages resolved in previous analyses using SSU rDNA: (A) Ascobolaceae and Pezizaceae; (B) Discinaceae-Morchellaceae and Helvellaceae-Tuberaceae; (C) Ascodesmidaceae, Glaziellaceae, Pyronemataceae, Sarcoscyphaceae and Sarcosomataceae. In contrast the monotypic Rhizinaceae and Caloscyphaceae are resolved as two independent lineages. Bayesian analyses support a relationship among Rhizina and two species of Psilopezia (Pyronemataceae). Only lineage C is highly supported. The B and C lineages form a strongly supported monophyletic group. None of these lineages corresponds to earlier proposed suborders. The A and B lineages are supported by certain morphological features (e.g. ascus bluing reaction in iodine, cytology of spores and paraphyses, septal pore structures and excipulum structure); these characters have been subject to homoplasy. Lineage C is the largest and most heterogeneous, and no unifying morphological features support its recognition. The Pyronemataceae, in which almost half of the species in the order are found, is not monophyletic because the Ascodesmidaceae and Glaziellaceae are nested within it. The relationships among all families in the C lineage remain uncertain. The origin of various forms of ascomata, including hypogeous forms (truffles and truffle-like), epigeous cleistothecia, simple reduced apothecia and highly elaborate, stipitate forms (helvelloid and morchelloid), are discussed.     

Characteristics and utility of nuclear-encoded large-subunit ribosomal gene sequences in phylogenetic studies of red algae

Primer sequences are described for amplifying and sequencing a large fragment (approximately 2500 b.p.) of the nuclear-encoded large-subunit ribosomal RNA gene (LSU) from red algae. In comparison to RuBisCo large-subunit gene (rbcL) and nuclear-encoded small-subunit ribosomal RNA gene (SSU) sequence data, LSU sequence data was intermediate in the number of phylogenetically informative positions and sequence divergence. Parsimony analysis of LSU sequences for 16 Gelidiales species resolved some nodes unresolved in rbcL and SSU parsimony trees. An analysis of LSU sequences from 13 species of red algae classified in 11 orders suggests that this gene may be useful in studies of higher-level relationships of red algae.     

Molecular data place Hydnoraceae with Aristolochiaceae

Utilization of molecular phylogenetic information over the past decade has resulted in clarification of the position of most angiosperms. In contrast, the position of the holoparasitic family Hydnoraceae has remained controversial. To address the question of phylogenetic position of Hydnoraceae among angiosperms, nuclear SSU and LSU rDNA and mitochondrial atp1 and matR sequences were obtained for Hydnora and Prosopanche. These sequences were used in combined analyses that included the above four genes as well as chloroplast rbcL and atpB (these plastid genes are missing in Hydnoraceae and were hence coded as missing). Three data sets were analyzed using maximum parsimony: (1) three genes with 461 taxa; (2) five genes with 77 taxa; and (3) six genes with 38 taxa. Analyses of separate and combined data partitions support the monophyly of Hydnoraceae and the association of that clade with Aristolochiaceae sensu lato (s.l.) (including Lactoridaceae). The latter clade is sister to Piperaceae and Saururaceae. Despite over 11 kilobases (kb) of sequence data, relationships within Aristolochiaceae s.l. remain unresolved, thus it cannot yet be determined whether Aristolochiaceae, Hydnoraceae, and Lactoridaceae should be classified as distinct families. In contrast to most traditional classifications, molecular phylogenetic analyses do not suggest a close relationship between Hydnoraceae and Rafflesiaceae. A number of morphological features is shared by Hydnoraceae and Aristolochiaceae; however, a more resolved phylogeny is required to determine whether these represent synapomorphies or independent acquisitions.     

Evolutionary history of the Corallinales (Corallinophycidae, Rhodophyta) inferred from nuclear, plastidial and mitochondrial genomes

Systematics of the red algal order Corallinales has a long and convoluted history. In the present study, molecular approaches were used to assess the phylogenetic relationships based on the analyses of two datasets: a large dataset of SSU sequences including mainly sequences from GenBank; and a combined dataset including four molecular markers (two nuclear: SSU, LSU; one plastidial: psbA; and one mitochondrial: COI). Phylogenetic analyses of both datasets re-affirmed the monophyly of the Corallinales as well as the two families (Corallinaceae and Hapalidiaceae) currently recognized within the order. Three of the four subfamilies of the Corallinaceae (Corallinoideae, Lithophylloideae, Metagoniolithoideae) were also resolved as a monophyletic lineage whereas members of the Mastophoroideae were resolved as four distinct lineages. We therefore propose to restrict the Mastophoroideae to the genera Mastophora, Metamastophora, and possibly Lithoporella in the aim of rendering this subfamily monophyletic. In addition, our phylogenies resolved the genus Hydrolithon in two unrelated lineages, one containing the generitype Hydrolithon reinboldii and the second containing Hydrolithon onkodes, which used to be the generitype of the now defunct genus Porolithon. We therefore propose to resurrect the genus Porolithon for the second lineage encompassing those species with primarily monomerous thalli, and trichocyte arrangements in large pustulate horizontal rows. Moreover, our phylogenetic analyses revealed the presence of cryptic diversity in several taxa, shedding light on the need for further studies to better circumscribe species frontiers within the diverse order Corallinales, especially in the genera Mesophyllum and Neogoniolithon.     

Clarification of the relationship beteen Apiaceae and Araliaceae based on matK and rbcL sequence data

Apiaceae and Araliaceae (Apiales) represent a particularly troublesome example of the difficulty in understanding evolutionary relationships between tropical-temperate family pairs. Previous studies based on rbcL sequence data provided insights at higher levels, but were unable to resolve fully the family-pair relationship. In this study, sequence data from a more rapidly evolving gene, matK, was employed to provide greater resolution. In Apiales, matK sequences evolve an average of about two times faster than rbcL sequences. Results of phylogenetic analysis of matK sequences were first compared to those obtained previously from rbcL data; the two data sets were then combined and analyzed together. Molecular analyses confirm the polyphyly of apiaceous subfamily Hydrocotyloideae and suggest that some members of this subfamily are more closely related to Araliaceae than to other Apiaceae. The remainder of Apiaceae forms a monophyletic group with well-defined subclades corresponding to subfamilies Apioideae and Saniculoideae. Both the matK and the combined rbcL-matK analyses suggest that most Araliaceae form a monophyletic group, including all araliads sampled except Delarbrea and Mackinlaya. The unusual combination of morphological characters found in these two genera and the distribution of matK and rbcL indels suggest that these taxa may be the remnants of an ancient group of pro-araliads that gave rise to both Apiaceae and Araliaceae. Molecular data indicate that the evolutionary history of the two families is more complex than simple derivation of Apiaceae from within Araliaceae. Rather, the present study suggests that there are two well-defined "families," both of which may have been derived from a lineage (or lineages) or pro-araliads that may still have extant taxa.     

Evolution of Filamentous Ascomycetes Inferred from LSU rDNA Sequence Data

Abstract: The nuclear LSU rRNA gene was examined in order to evaluate the current phylogeny of ascomycetes, which is mainly based on nuclear SSU rRNA data. Partial LSU rRNA gene sequences of 19 ascomycetes were determined and aligned with the corresponding sequences of 13 other ascomycetes retrieved from Genbank, including all classes traditionally distinguished and most of the recently accepted classes. The classification based on SSU rDNA data and morphological characters is supported, while the traditional classification and classifications based on the ascus type are rejected. Ascomycetes with perithecia and cleistothecia form monophyletic groups, while the discomycetes are a paraphyletic assemblage. The Pezizales are basal to all other filamentous ascomycetes. The monophyly of Loculoascomycetes is uncertain. The results of the LSU rDNA analysis agree with those of the SSU rDNA and RPB2 gene analyses, suggesting that most classes circumscribed in the filamentous ascomycetes are monophyletic. The branching order and relationships among these classes, however, cannot be elucidated with any of these data sets.     

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.     

THE PINGUIOPHYCEAE CLASSIS NOVA, CHROMOPHYTE ALGAE PRODUCING LARGE AMOUNTS OF OMEGA-3 FATTY ACIDS

The streptophytes comprise the Charophyceae sensu Mattox and Stewart (a morphologically diverse group of fresh-water green algae) and the embryophytes (land plants). Several charophycean groups are currently recognized. These include the Charales, Coleochaetales, Chlorokybales, Klebsormidiales and Zygnemophyceae (Desmidiales and Zygnematales). Recently, SSU rRNA gene sequence data allied Mesostigma viride (Prasinophyceae) with the Streptophyta. Complete chloroplast sequence data, however, placed Mesostigma sister to all green algae, not with the streptophytes. Several morphological, ultrastructural and biochemical features unite these lineages into a monophyletic group including embryophytes, but evolutionary relationships among the basal streptophytes remain ambiguous. To date, numerous studies using SSU rRNA gene sequences have yielded differing phylogenies with varying degrees of support dependent upon taxon sampling and choice of phylogenetic method. Like SSU data, chloroplast DNA sequence data have been used to examine relationships within the Charales, Coleochaetales, Zygnemophyceae and embryophytes. Representatives of all basal streptophyte lineages have not been examined using chloroplast data in a single analysis. Phylogenetic analyses were performed using DNA sequences of rbc L (the genes encoding the large subunit of rubisco) and atp B (the beta-subunit of ATPase) to examine relationships of basal streptophyte lineages. Preliminary analyses placed the branch leading to Mesostigma as the basal lineage in the Streptophyta with Chlorokybus , the sole representative of the Chlorokybales, branching next. Klebsormidiales and the enigmatic genus Entransia were sister taxa. Sister to these, the Charales, Coleochaetales, embryophytes and Zygnemophyceae formed a monophyletic group with Charales and Coleochaetales sister to each other and this clade sister to the embryophytes.     

Concatenated SSU and LSU rDNA data confirm the main evolutionary trends within myxosporeans (Myxozoa: Myxosporea) and provide an effective tool for their molecular phylogenetics

Views on myxosporean phylogeny and systematics have recently undergone substantial changes resulting from analyses of SSU rDNA. Here, we further investigate the evolutionary trends within myxosporean lineages by using 35 new sequences of the LSU rDNA. We show a good agreement between the two rRNA genes and confirm the main phylogenetic split between the freshwater and marine lineages. The informative superiority of the LSU data is shown by an increase of the resolution, nodal supports and tree indexes in the LSU rDNA and combined analyses. We determine the most suitable part of LSU for the myxosporean phylogeny by comparing informative content in various regions of the LSU sequences. Based on this comparison, we propose the D5–3′-end part of the LSU rRNA gene as the most informative region which provides in concatenation with the complete SSU a well resolved and robust tree. To allow for simple amplification of the marker, we design specific primer set for this part of LSU rDNA.     

Evolution according to large ribosomal subunit RNA

Evolutionary trees were constructed, by distance methods, from an alignment of 225 complete large subunit (LSU) rRNA sequences, representing Eucarya, Archaea, Bacteria, plastids, and mitochondria. A comparison was made with trees based on sets of small subunit (SSU) rRNA sequences. Trees constructed on the set of 172 species and organelles for which the sequences of both molecules are known had a very similar topology, at least with respect to the divergence order of large taxa such as the eukaryotic kingdoms and the bacterial divisions. However, since there are more than ten times as many SSU as LSU rRNA sequences, it is possible to select many SSU rRNA sequence sets of equivalent size but different species composition. The topologies of these trees showed considerable differences according to the particular species set selected.The effect of the dataset and of different distance correction methods on tree topology was tested for both LSU and SSU rRNA by repetitive random sampling of a single species from each large taxon. The impact of the species set on the topology of the resulting consensus trees is much lower using LSU than using SSU rRNA. This might imply that LSU rRNA is a better molecule for studying wide-range relationships. The mitochondria behave clearly as a monophyletic group, clustering with the Proteobacteria. Gram-positive bacteria appear as two distinct groups, which are found clustered together in very few cases. Archaea behave as if monophyletic in most cases, but with a low confidence.Abbreviations LSU rRNA large subunit ribosomal RNA - SSU rRNA small subunit ribosomal RNA - JC Jukes and Cantor - JN Jin and Nei Correspondence to: R. De Wachter