Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

Dinoflagellate-targeted PCR reveals highly abundant and diverse communities of parasitic dinoflagellates in and near Zhubi Reef,South China Sea

While diversity of symbiodiniacean dinoflagellates has been a focus of coral reef ecological research, information on the diversity of planktonic dinoflagellates in reef ecosystems remains limited. We used dinoflagellate-targeted PCR to investigate dinoflagellate diversity for a coral reef ecosystem. In the summer of 2007, plankton samples were collected from a lagoon, atoll, and open sea area of Zhubi Reef in the Nansha Islands, South China Sea. Sequencing of dinoflagellate-specific SSU rDNA clone libraries from samples in each of these habitats revealed high diversity and numerous novel dinoflagellate lineages. Gymnodiniales were most abundantly represented in all three water areas. Lagoon assemblages were co-dominated by Syndiniales and Gonyaulacales, the atoll by Gonyaulacales and Peridiniales, and the open sea by Syndiniales and Prorocentrales taxa. Species in the Syndiniales (group II) genus Amoebophrya were represented by eight new sequences and 13 previously described clades and were dominated by species reported to infect Gymnodiniales, Gonyaulacales, Peridiniales, and Prorocentrales taxa. And Amoebophrya were particularly abundant and diverse in the lagoon. Our results suggest that Amoebophrya probably play an important role in regulating dynamics of dinoflagellate assemblages in the Zhubi Reef coral ecosystem. In contrast, the few symbiodiniacean taxa detected occurred only in the open sea, suggesting planktonic aposymbiotic Symbiodiniaceae rarely occur in the reef ecosystem. We demonstrate the usefulness of a dinoflagellate-specific molecular technique for profiling dinoflagellate communities, and uncover diversity and the potential importance of parasitic lineages in a coral reef ecosystem.

     

Inhibition of dinoflagellate growth by extracts of barley straw (Hordeum vulgare)

Growth of dinoflagellates representing three orders, the Gymnodiniales,Peridiniales, and the Prorocentrales was examined following treatment withbarley straw extract. Selected dinoflagellate taxa showed growth responsessimilar to those reported for freshwater algae including: inhibition(Gyrodinium galatheanum, Gymnodiniumsanguineum, Heterocapsa triquetra andH. pygmaea); stimulation (Gyrodinium instriatum,Prorocentrum minimum and P. micans); and no effect(Gyrodinium estuariale, G. uncatenum,Ceratium furca, Peridinium sp.).Although barley straw extracts do not appear to have value as a universalmanagement tool for dinoflagellates, they may have potential in management ofspecific taxa and possibly taxonomic groups.     

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.     

PHYLOGENY OF DINOFLAGELLATES BASED ON MITOCHONDRIAL CYTOCHROME B AND NUCLEAR SMALL SUBUNIT RDNA SEQUENCE COMPARISONS1

Despite their evolutionary and ecological importance, dinoflagellate phylogeny remains poorly resolved. Here we explored the utility of mitochondrial cytochrome b (cob) in inferring a dinoflagellate tree and focused on resolving the relationship between fucoxanthin‐and peridinin‐containing taxa. Trees were inferred using cob and small subunit rDNA alone or in combination as concatenated data and including members of the six major dinoflagellate orders. Many regions of the cob DNA or protein and rDNA trees were congruent with support for the monophyly of Symbiodinium spp. Freudenthal and of the Prorocentrales and the early divergence of Crypthecodinium cohnii Seligo in Grasse. However, these markers provided differing support for the monophyly of Pfiesteria spp. Steidinger et Burkholder (only supported strongly by rDNA) and of the fucoxanthin dinoflagellates with Akashiwo sp. (Hirasaka) Hansen et Moestrup (Gymnodiniales, only supported strongly by the cob data). The approximately unbiased (AU) test was used to assess these results using 13‐and 11‐taxon (excluding apicomplexans) backbone maximum likelihood trees inferred from the combined cob+rDNA data. The AU test suggested that our data were insufficient to resolve the phylogenetic position of Symbiodinium spp. and that the ancestral position of C. cohnii might have resulted from long‐branch attraction to the apicomplexan outgroup. We found significant support, however, for the association of fucoxanthin dinoflagellates with Akashiwo sp. The monophyly and relatively derived position of the Gymnodiniales in our cob DNA and protein trees and in the cob+rDNA tree is consistent with the tertiary endosymbiotic origin of the plastid in fucoxanthin dinoflagellates.     

Crypthecodinium and Tetrahymena: An exercise in comparative evolution

Summary Nucleotide sequences have been determined for the highly variable D2 region of the large rRNA molecule for over 60 strains of dinoflagellates. These strains were selected from a worldwide collection that represents all the known sibling species (compatibility groups, Mendelian species) in the sibling swarm referred to as Crypthecodinium cohnii. A phylogenetic tree has been constructed from an analysis of the variations in a length of about 180 bases, using PHYLOGEN string analysis programs. The Crypthecodinium tree is compared with the previously published but here augmented tree constructed upon the same rRNA region for the sibling species of a worldwide collection of ciliated protozoa related to the genus Tetrahymena. The first reported sequence of Lambornella clarki, the parasite of tree-hole mosquitoes, is included.The dinoflagellate species complex is much more homogeneous with respect to ribosomal variation. The mean number of differences among sequences from different Crypthecodinium species is about 7, in comparison with 22 differences among the ciliate species examined. Moreover, all the diversity in the dinoflagellates can be explained by base substitutions, whereas insertions and deletions are common in the ciliates. The dinoflagellates are also much more uniform with respect to nutritional and genetic economies.The two complexes differ also in the relationship between molecular variations and breeding compatibility. All tetrahymenine sibling species thus far examined are monomorphic in the D2 region, but several dinoflagellate species are polymorphic. Several different dinoflagellate species, moreover, have identical D2 regions. This kind of ribosomal identity of incompatible strains is found in these ciliates only in one tight cluster of species—Group C.The tetrahymenine swarm is apparently much older than the Crypthecodinium swarm, and the dinoflagellate species produce incompatible progeny species much more readily than do the ciliates, perhaps by the acquisition of mutations that potentiate incompatibility in sympatric populations.Offprint requests to: D. L. Nanney     

Development and Evaluation of a PCR Based Assay for Detection of the Toxic Dinoflagellate, <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis>(Graham) in Ballast Water and Environmental Samples

Gymnodinium catenatum is a bloom forming dinoflagellate that has been known to cause paralytic shellfish poisoning (PSP) in humans. It is being reported with increased frequency around the world, with ballast water transport implicated as a primary vector that may have contributed to its global spread. Major limitations to monitoring and management of its spread are the inability for early, rapid, and accurate detection of G. catenatum in plankton samples. This study explored the feasibility of developing a PCR-based method for specific detection of G. catenatumin cultures and heterogeneous ballast water and environmental samples. Sequence comparison of the large sub unit (LSU) ribosomal DNA locus of several strains and species of dinoflagellates allowed the design of G. catenatum specific PCR primers that are flanked by conserved regions. Assay specificity was validated through screening a range of dinoflagellate cultures, including the morphologically similar and taxonomically closely related species G. nolleri. Amplification of the diagnostic PCR product from all the strains of G. catenatum but not from other species of dinoflagellates tested imply the species specificity of the assay. Sensitivity of the assay to detect cysts in ballast water samples was established by simulated spiked experiments. The assay could detect G. catenatum in all ‘blank’ plankton samples that were spiked with five or more cysts. The assay was used to test environmental samples collected from the Derwent river estuary, Tasmania. Based on the results we conclude that the assay may be utilized in large scale screening of environmental and ballast water samples.     

A Three-Gene Dinoflagellate Phylogeny Suggests Monophyly of Prorocentrales and a Basal Position for <Emphasis Type="Italic">Amphidinium</Emphasis> and <Emphasis Type="Italic">Heterocapsa</Emphasis>

Many outstanding questions about dinoflagellate evolution can potentially be resolved by establishing a robust phylogeny. To do this, we generated a data set of mitochondrial cytochrome b (cob) and mitochondrial cytochrome c oxidase 1 (cox1) from a broad range of dinoflagellates. Maximum likelihood, maximum parsimony, and Bayesian methods were used to infer phylogenies from these genes separately and as a concatenated alignment with and without small subunit (SSU) rDNA sequences. These trees were largely congruent in topology with previously published phylogenies but revealed several unexpected results. Prorocentrum benthic and planktonic species previously placed in different clusters formed a monophyletic group in all trees, suggesting that the Prorocentrales is a monophyletic group. More strikingly, our analyses placed Amphidinium and Heterocapsa as early splits among dinoflagellates that diverged after the emergence of O. marina. This affiliation received strong bootstrap support, but these lineages exhibited relatively long branches. The approximately unbiased (AU-) test was used to assess this result using a three-gene (cob + cox1 + SSU rDNA) DNA data set and the inferred tree. This analysis showed that forcing Amphidinium or Heterocapsa to relatively more derived positions in the phylogeny resulted in significantly lower likelihood scores, consistent with the phylogenies. The position of these lineages needs to be further verified. Reviewing Editor: Dr. Martin Kreitman     

Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae)

The morphology of an unarmored chain-forming harmful dinoflagellate Cochlodinium polykrikoides and its similar species such as Cochlodinium catenatum, Cochlodinium fulvescens, and Cochlodinium convolutum was carefully observed, emphasizing the single cell stage for clarifying taxonomically important morphological features. To differentiate C. polykrikoides from C. convolutum, the shape and the position of the nucleus are useful characters. C. polykrikoides also differs from C. fulvescens in being smaller in size, possessing many rod-shaped chloroplasts and having the sulcus running just below the cingulum on the dorsal surface. Careful observation of the ichnotype of C. catenatum suggests that C. catenatum sensu Kofoid and Swezy collected from off La Jolla, CA, USA, is not identical to C. catenatum sensu Okamura and is probably a different species, in having no chloroplasts and a nucleus positioned at the center of the cell. In addition, C. polykrikoides has many morphological features in common with C. catenatum sensu Okamura except for slightly elongate cells and is probably a junior synonym of this species.     

广东沿海几种赤潮生物的分类学研究

对１９９７年秋￣１９９８年春广东沿海多次赤潮发生期间的几种优势赤潮藻类进行了形态学和分类学研究。１种为我国首次报道引发赤潮的定革命金藻类（Ｐｒｙｍｎｅｓｉｏｐｈｙｔｅｓ）－球状棕囊藻（Ｐｈａｅｏｃｙｓｔｉｓ ｃｆ．ｇｌｉｏｂｏｓａ）。另有甲藻类７种,其中裸甲藻目（Ｇｙｍｎｏｄｉｎｉａｌｅｓ）３种：米氏裸甲藻（Ｇｙｍｎｏｄｉｎｉｕｍ ｃｆ．ｍｉｋｉｍｏｔｏｉ）、环节环沟藻（Ｇｙｒｏｄｉｎｉｕｍ ｉｎ     

Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group

The geographic range and bloom frequency of the toxic dinoflagellate Alexandrium minutum and other members of the A. minutum group have been increasing over the past few decades. Some of these species are responsible for paralytic shellfish poisoning (PSP) outbreaks throughout the world. The origins of new toxic populations found in previously unaffected areas are typically not known due to a lack of reliable plankton records with sound species identifications and to the lack of a global genetic database. This paper provides the first comprehensive study of minutum-group morphology and phylogeny on a global scale, including 45 isolates from northern Europe, the Mediterranean, Asia, Australia and New Zealand.Neither the morphospecies Alexandrium lusitanicum nor A. angustitabulatum was recoverable morphologically, due to large variation within and among all minutum-group clonal strains in characters previously used to distinguish these species: the length:width of the anterior sulcal plate, shape of the 1′ plate, connection between the 1′ plate and the apical pore complex, and the presence of a ventral pore. DNA sequence data from the D1 to D2 region of the LSU rDNA also fail to recognize these species. Therefore, we recommend that all isolates previously designated as A. lusitanicum or A. angustitabulatum be redesignated as A. minutum. A. tamutum, A. insuetum, and A. andersonii are clearly different from A. minutum on the basis of both genetic and morphological data.A. minutum strains from Europe and Australia are closely related to one another, which may indicate an introduction from Europe to Australia given the long history of PSP in Europe and its recent occurrence in Australia. A minutum from New Zealand and Taiwan form a separate phylogenetic group. Most strains of A. minutum fit into one of these two groups, although there are a few outlying strains that merit further study and may represent new species. The results of this paper have greatly improved our ability to track the spread of A. minutum species and to understand the evolutionary relationships within the A. minutum group by correcting inaccurate taxonomy and providing a global genetic database.     

Mixotrophy among Dinoflagellates1

Mixotrophy, used herein for the combination of phototrophy and phagotrophy, is widespread among dinoflagellates. It occurs among most, perhaps all, of the extant orders, including the Prorocentrales, Dinophysiales. Gymnodiniales, Noctilucales, Gonyaulacales, Peridiniales, Blastodiniales. Phytodiniales, and Dinamoebales. Many cases of mixotrophy among dinoflagellates are probably undocumented. Primarily photosynthetic dinoflagellates with their “own” plastids can often supplement their nutrition by preying on other cells. Some primarily phagotrophic species are photosynthetic due to the presence of kleptochloroplasts or algal endosymbionts. Some parasitic dinoflagellates have plastids and are probably mixotrophic. For most mixotrophic dinoflagellates, the relative importance of photosynthesis, uptake of dissolved inorganic nutrients, and feeding are unknown. However, it is apparent that mixotrophy has different functions in different physiological types of dinoflagellates. Data on the simultaneous regulation of photosynthesis, assimilation of dissolved inorganic and organic nutrients, and phagotophy by environmental parameters (irradiance. availablity of dissolved nutrients, availability of prey) and by life history events are needed in order to understand the diverse roles of mixotrophy in dinoflagellates.     

Improving the analysis of dinoflagellate phylogeny based on rDNA

Phylogenetic studies of dinoflagellates are often conducted using rDNA sequences. In analyses to date, the monophyly of some of the major lineages of dinoflagellates remain to be demonstrated. There are several reasons for this uncertainty, one of which may be the use of models of evolution that may not closely fit the data. We constructed and examined alignments of SSU and partial LSU rRNA along with a concatenated alignment of the two molecules. The alignments showed several characteristics that may confound phylogeny reconstruction: paired helix (stem) regions that contain non-independently evolving sites, high levels of compositional heterogeneity among some of the sequences, high levels of incompatibility (homoplasy), and rate heterogeneity among sites. Taking into account these confounding factors, we analysed the data and found that the Gonyaulacales, a well-supported clade, may be the most recently diverged order. Other supported orders were, in the analysis based on SSU, the Suessiales and the Dinophysiales; however, the Gymnodiniales and Prorocentrales appeared to be polyphyletic. The Peridiniales without Heterocapsa species appeared as a monophyletic group in the analysis based on LSU; however, the support was low. The concatenated alignment did not provide a better phylogenetic resolution than the single gene alignments.     

Dinoflagellate phylogeny as inferred from heat shock protein 90 and ribosomal gene sequences

Background

Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages.

Methodology/Principal Findings

We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved.

Conclusions/Significance

The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

Description of a new dinoflagellate with a diatom endosymbiont, <Emphasis Type="Italic">Durinskia capensis</Emphasis> sp. nov. (Peridiniales,Dinophyceae) from South Africa

A new dinoflagellate Durinskia capensis Pienaar, Sakai et Horiguchi sp. nov. (Peridiniales, Dinophyceae), from tidal pools along the west coast of the Cape Peninsula, Republic of South Africa, is described. The dinoflagellate produces characteristic dense orange-red colored blooms in tidal pools. The organism is characterized by having a eukaryotic endosymbiotic alga. Ultrastructure study revealed the organism has a cellular construction similar to that of other diatom-harboring dinoflagellates. The cell is thecate and the plate formula is: Po, x, 4', 2a, 6', 5c, 4s, 5', 2', which is the same as that of Durinskia baltica, the type species of the genus Durinskia. D. capensis can, however, be distinguished from D. baltica by overall cell shape, the relative size of the 1a and 2a plates, the degree of cingular displacement, and the shape of the eyespot. Our molecular analysis based on SSU rDNA revealed that D. capensis is closely allied to D. baltica, thus supporting the assignment of this new species to this genus. This Durinskia clade takes a sister position to another diatom-harboring dinoflagellate clade, which includes Kryptoperidinium foliaceum and Galeidinium rugatum. Molecular analysis based on the rbcL gene sequence and ultrastructure study revealed that the endosymbiont of D. capensis is a diatom. The SSU rDNA gene trees indicated that four species with a diatom endosymbiont formed a clade, suggesting a single endosymbiotic origin.     

The application of a molecular clock based on molecular sequences and the fossil record to explain biogeographic distributions within the Alexandrium tamarense "species complex" (Dinophyceae)

The cosmopolitan dinoflagellate genus Alexandrium, and especially the A. tamarense species complex, contain both toxic and nontoxic strains. An understanding of their evolution and paleogeography is a necessary precursor to unraveling the development and spread of toxic forms. The inclusion of more strains into the existing phylogenetic trees of the Alexandrium tamarense species complex from large subunit rDNA sequences has confirmed that geographic distribution is consistent with the molecular clades but not with the three morphologically defined species that constitute the complex. In addition, a new clade has been discovered, representing Mediterranean nontoxic strains. The dinoflagellates fossil record was used to calibrate a molecular clock: key dates used in this calibration are the origins of the Peridiniales (estimated at 190 MYA), Gonyaulacaceae (180 MYA), and Ceratiaceae (145 MYA). Based on the data set analyzed, the origin of the genus Alexandrium was estimated to be around late Cretaceous (77 MYA), with its earliest possible origination in the mid Cretaceous (119 MYA). The A. tamarense species complex potentially diverged around the early Neogene (23 MYA), with a possible first appearance in the late Paleogene (45 MYA). A paleobiogeographic scenario for Alexandrium is based on (1) the calculated possible ages of origination for the genus and its constituent groups; (2) paleogeographic events determined by plate movements, changing ocean configurations and currents, as well as climatic fluctuations; and (3) the present geographic distribution of the various clades of the Alexandrium tamarense species complex.     

Morphological and molecular phylogenetic analysis of <Emphasis Type="Italic">Melampsora</Emphasis> species on poplars in China

Many species of Melampsora on Populus have been reported in China, based on morphological characteristics of both uredial and telial states, and on host species, but their morphology and taxonomy are still poorly defined. In this study, 196 specimens representing Melampsora species on poplars and collected from various areas of China were used for morphological observations. The morphological characteristics of urediniospores and teliospores were examined with light and scanning electron microscopy. The specimens could be classified into five groups based on their morphology. For the sequencing of the nuclear large subunit rDNA (D1/D2), 5.8S rDNA and their internal transcribed spacers, ITS1 and ITS2 region, 54 specimens were selected from the specimens used in morphological observations. These specimens were separated into six clades by phylogenetic analyses of the D1/D2 and ITS regions. Correlations among morphological groups and phylogenetic clades based on these results suggest a revision of these species. In particular, no evidence to discriminate specimens of M. acedioides, M. magnusiana, and M. rostrupii was found from either morphological characteristics or sequence analysis.Contribution no. 185 Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan