Molecular phylogenetic study of the heterotrophic dinoflagellate genus Protoperidinium (Dinophyceae) inferred from small subunit rRNA gene sequences

In the present study, we investigated the intrageneric and intergeneric phylogenetic relationships of the heterotrophic marine dinoflagellate genus Protoperidinium. Using single‐cell polymerase chain reaction methods, we determined small subunit ribosomal RNA gene sequences for 10 Protoperidinium species belonging to four sections and two subgenera. Phylogenetic trees were constructed using maximum parsimony, neighbor joining and maximum likelihood methods. We found intraspecific variability of small subunit rDNA sequences in Protoperidinium conicum (Gran) Balech, Protoperidinium crassipes (Kofoid) Balech and Protoperidinium denticulatum (Gran et Braarud) Balech, but not in other species. The small subunit rDNA phylogeny revealed that the genus is monophyletic, but its phylogenetic position within the Dinophyceae could not be determined because of ambiguous basal topologies. Within the genus Protoperidinium, species of the subgenus Archaeperidinium with two anterior intercalary plates (2a) were shown to be monophyletic, but species of the subgenus Protoperidinium with three anterior intercalary plates (3a) were resolved as paraphyletic. The sections Avellana, Divergentia and Protoperidinium were shown to be monophyletic, while the section Conica was paraphyletic. Based on the trees obtained in the present study, most of the traditionally defined sections are supported by molecular phylogeny. It was also indicated that the section Avellana evolved from one of the Conica‐type dinoflagellates.     

Molecular phylogeny of onygenalean fungi based on small subunit ribosomal DNA (SSU rDNA) sequences

Phylogenetic analysis of nucleotide data from small subunit ribosomal DNA (SSU rDNA) sequences (ca. 1685 bp.) was performed on 19 taxa of the Onygenales and three related mitosporic fungi. Phylogenetic trees were constructed by the neighbor-joining method with the sequence data of related taxa obtained from DNA databases. The species in the Onygenales form two clusters and seven subclusters, and the tree topology reflects the traditional classification by Currah (1985) with some exceptions. The Myxotrichaceae is placed in the different lineage, separate from other plectomycetous taxa and among the Leotiales and the Erysiphales. Furthermore, two separate lineages in the Myxotrichaceae were found. Tree topology suggested the Onygenaceae is polyphyletic and composed of three subgroups; 1) most members of Onygenaceae, 2)Spiromastix warcupii, and 3) pathogenic dimorphic fungi classified inAjellomyces.     

Haramonas pauciplastida sp. nov. (Raphidophyceae, Heterokontophyta) and phylogenetic analyses of Haramonas species using small subunit ribosomal RNA gene sequences

A new species of the Raphidophyceae, Haramonas pauciplastida sp. nov. from Canada is described. The genus Haramonas has been described based on the type species Haramonas dimorpha and currently only two species are known. This new alga belongs to the genus because it possesses a tubular invagination at the posterior end of the cell, producing a large amount of mucilage and generating both motile and non-motile phases in its life cycle. The chloroplast color of H. pauciplastida is yellowish green, and is similar to that of Haramonas viridis Horiguchi et Hoppenrath . However, this alga differs from the other species of the genus in that it possesses fewer chloroplasts, which are rarely overlapping. The ultrastructual study shows differences between these two species in the number of thylakoids in the lamella, the presence of a scattered pyrenoid matrix, and the position of the plastoglobuli. The phylogenetic analyses of the small subunit ribosomal RNA gene from the Haramonas species reveal that three species can be distinguished genetically from each other and they form a robust clade in the Raphidophyceae. This result supports the notion that the characteristic features of Haramonas are synapomorphies. This is the first report of molecular data from the Haramonas species.     

New dinoflagellate species Protoperidinium haizhouense sp. nov. (Peridiniales,Dinophyceae), its cyst‐theca relationship and phylogenetic position within the Monovela group

The number of cingular plates has been used to differentiate Protoperidinium from Peridinium and related genera. Protoperidinium is characterized by the presence of three cingular plates plus a transitional plate (3C+t). However, many Protoperidinium species have been described that exhibit different cingular plate tabulations. How these species should be classified within the genus remains unclear. To address this question, the phylogenetic relationship of four Protoperidinium species, with three or four cingular plates and lacking a transitional plate, were examined in relationship to other Protoperidinium species. These four species were germinated from cysts deposited in surface sediments collected from the East China Sea, the Bohai Sea and the Yellow Sea. Three of the isolated species, P. tricingulatum, P. americanum and P. parthenopes, were described previously. The fourth is here described as P. haizhouense sp. nov. with the plate formula Po, X, 4′, 3a, 7′′, 3C, 6S, 5′′′, 2′′′′. Differences in the cyst stages of these four species, which can be taxonomically informative, were compared. Partial large subunit ribosomal DNA sequences were obtained by single‐cell polymerase chain reaction. Maximum‐likelihood and Bayesian inference showed that these four species, P. fukuyoi and Islandinium minutum form a monophyletic clade with maximal support. The genus as a whole, however, appeared polyphyletic. Our results suggest that the presence/absence of a transitional plate is significant in the phylogeny of Protoperidinium.     

Phylogenetic analysis of the non-pathogenic genusSpiromastix (Onygenaceae) and related onygenalean taxa based on large subunit ribosomal DNA sequences

The phylogenetic positioning of the non-pathogenic genusSpiromastix in the Onygenales was studied based on large subunit rDNA (LSU rDNA) partial sequences (ca. 570 bp.). FourSpiromastix species and 28 representative taxa of the Onygenales were newly sequenced. Phylogenetic trees were constructed by the neighbor-joining (NJ) method and evaluated by the maximum parsimony (MP) method with the data of 13 taxa retrieved from DNA databases.Spiromastix and dimorphic systemic pathogens,Ajellomyces andParacoccidioides, appear to be a monophyletic group with 74% bootstrap probability (BP) in the NJ tree constructed with the representative taxa of the Onygenales. The tree topology was concordant with the NJ tree based on SSU rDNA sequences of our previous work and corresponded to the classification system of the Onygenales by Currah (1985) and its minor modification by Udagawa (1997) with the exception of the classification of the Onygenaceae. The Onygeneceae sensu Udagawa may still be polyphyletic, since three independent lineages were recognized. The taxa forming helicoid peridial appendages were localized to two clades on the tree. The topology of the NJ tree constructed withSpiromastix and its close relatives suggested that the helicoid peridial appendages were apomorphic and acquired independently in the two clades of the Onygenales.     

Discovery of a novel type of body scale in the marine dinoflagellate,Amphidinium cupulatisquama sp. nov. (Dinophyceae)

A new species of Amphidinium, A. cupulatisquama Tamura et Horiguchi, from sand samples from Ikei Island, Okinawa Prefecture in subtropical Japan, is described based on light, scanning and transmission electron microscopy and the partial sequencing of the large subunit rDNA gene. The species has a typical morphology for the genus, but is distinguished from previously described species by having a combination of the following characteristics: (i) a relatively large cell (over 30 µm in length); (ii) possessing an eyespot on the dorsal side of the cingulum; (iii) the longitudinal flagellum emerging from a point close to the cingulum; (iv) cell division taking place in the motile phase; and (v) possessing body scales. This is the third species of this genus to possess body scales. The body scales of A. cupulatisquama are uniform and cup‐shaped in side view and elliptical in face view. Their dimensions are 136.4 nm by 91.0 nm by 81.8 nm high. In side view, the scale is seen to have a thick lower half and a thin upper half. This scale type is very different from those of previously reported Amphidinium species (HG114 and HG115). The molecular tree indicated that A. cupulatisquama and the two other strains of body scale‐bearing Amphidinium are distantly related within the Amphidinium clade.     

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.     

Plagiodinium ballux sp. nov. (Dinophyceae), a deep (36 m) sand dwelling dinoflagellate from subtropical Japan

A new species of marine sand‐dwelling dinoflagellate, Plagiodinium ballux N. Yamada, Dawut, R. Terada & T. Horiguchi is described from a deep (36 m) seafloor off Takeshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The species is thecate and superficially resembles species of Prorocentrum, but possesses an extremely small epitheca. The cell varies from ovoid to a rounded square, and is small (15.0–22.5 μm in length) and laterally compressed. The thecal plates are smooth and the thecal plate arrangement (Po, 1′, 0a, 5″, 5C, 2S, 5?, 0p, 1″″) is similar to that of Plagiodinium belizeanum, the type species of the genus. Molecular phylogenetic analyses based on SSU rDNA and partial LSU rDNA reveal that the dinoflagellate is closely related to P. belizeanum, but it can be clearly distinguished by its size and cell shape. This suite of morphological and molecular differences leads to the conclusion that this deep benthic dinoflagellate represents a new species of the genus Plagiodinium.     

Informative characteristics of 12 divergent domains in complete large subunit rDNA sequences from the harmful dinoflagellate genus, Alexandrium (Dinophyceae)

The genus Alexandrium includes organisms of interest, both for the study of dinoflagellate evolution and for their impacts as toxic algae affecting human health and fisheries. Only partial large subunit (LSU) rDNA sequences of Alexandrium and other dinoflagellates are available, although they contain much genetic information. Here, we report complete LSU rDNA sequences from 11 strains of Alexandrium, including Alexandrium affine, Alexandrium catenella, Alexandrium fundyense, Alexandrium minutum, and Alexandrium tamarense, and discuss their segmented domains and structure. Putative LSU rRNA coding regions were recorded to be around 3,400 bp long. Their GC content (about 43.7%) is among the lowest when compared with other organisms. Furthermore, no AT-rich regions were found in Alexandrium LSU rDNA, although a low GC content was recorded within the LSU rDNA. No intron-like sequences were found. The secondary structure of the LSU rDNA and parsimony analyses showed that most variation in LSU rDNA is found in the divergent (D) domains with the D2 region being the most informative. This high D domain variability can allow members of the diverse Alexandrium genus to be categorized at the species level. In addition, phylogenetic analysis of the alveolate group using the complete LSU sequences strongly supported previous findings that the dinoflagellates and apicomplexans form a clade.     

A comparison of 18s ribosomal RNA and rubisco large subunit sequences for studying angiosperm phylogeny

Summary Partial sequences of 18s rRNA were obtained for 2 gymnosperms and 12 angiosperms from a wide range of families and these were analyzed with 5 other published sequences to form a phylogenetic tree. Using 16 published sequences of the large subunit of rubisco (rbcL), also from a wide range of angiosperm families, another phylogenetic tree was derived and the two approaches were compared. Both phylogenetic trees gave good grouping within families but in neither case was there resolution of the branching order of major taxa. Superficially the long rbcL sequences (whose base composition was homogeneous among all species) seemed very promising, but analysis showed that a large proportion of the variation did not affect the amino acid sequence. Although silent substitution contained some phylogenetic information, at the level required to order major taxa, much of it was random and obfuscating. It was concluded that neither macromolecule alone was likely to yield a solution to the problem of angiosperm phylogeny and therefore that studies of both, at least, will be required. For this reason, a method wa described for obtaining both DNA and RNA of good quality from the same preparation and which had been used successfully with a wide range of species including many with pungent leaves.     

Molecular phylogenetic study of the basidiomycetous anamorphic yeast genusTrichosporon and related taxa based on small subunit ribosomal DNA sequences

Small subunit ribosomal DNA sequences of all species of the basidiomycetous anamorphic yeast genusTrichosporon were determined, and phylogenetic trees were constructed by the neighbor-joining and maximum likelihood methods. The sequence data showed that, with the exception ofT. pullulans, the genus is monophyletic, although its members have two different major ubiquinones, Q9 and Q10. The genus can be divided phylogenetically into three major clusters. Species with Q10 as the major ubiquinone constitute a single cluster, while those with Q9 form two clusters.Trichosporon pullulans was phylogenetically distinct from other taxa of the genus. It is located in a cluster containingCystofilobasidium capitatum, Mrakía frigida, Xanthophyllomyces dendrorhous and three species ofUdeniomyces. This result sugests thatT. pullulans does not belong to the genusTrichosporon.     

Phylogenetic relationships ofPelvetia andPelvetiopsis (Phaeophyceae) based on small subunit ribosomal DNA sequences

Nucleotide sequences of the small-subunit (SSU) ribosomal DNA were determined forPelvetia babingtonii, P. canaliculate, Pelvetiopsis limitata, andAscophyllum nodosum in the family Fucaceae. A total of 1755 positions were aligned for the whole sequence. The positional differences in the primary structure among the taxa ranged from 16 to 30 nucleotide changes in pairwise comparisons. There was a minimum divergence betweenPs. limitata andP. babingtonii while a maximum betweenPs. limitata andP. canaliculata. The SSU rDNA trees showed that the genusPelvetia was not monophyletic and the genusPelvetiopsis was not closely related toPelvetia. Our results suggest that the taxonomic revision of the genusPelvetia as well as the family Fucaceae is needed based on detailed morphological observations.     

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.     

Morphological and molecular characterization of the small armoured dinoflagellate Heterocapsa minima (Peridiniales,Dinophyceae)

The dinophycean genus Heterocapsa is of considerable interest as it contains a number of bloom-forming and/or harmful species. Fine structure of organic body scales is regarded as the most important morphological feature for species determination but currently is unknown for the species H. minima described by Pomroy 25 years ago. Availability of a culture of H. minima collected in the south-west of Ireland allowed us to provide important information for this species, including cell size, cell organelle location, thecal plate pattern, body scale fine structure and molecular phylogeny. Light microscopy revealed the presence of one reticulate chloroplast, an elongated centrally located nucleus, and the presence of one pyrenoid surrounded by a starch sheath. Scanning electron microscopy (SEM) of the thecal plate pattern indicated that Pomroy erroneously designated the narrow first cingular plate as a sulcal plate. In addition, SEM revealed as yet unreported details of the apical pore complex and uncommon ornamentations of hypothecal plates. Organic body scales of H. minima were about 400 nm in size, roundish, with a small central hole and one central, six peripheral and three radiating spines. They differ from other body scales described within this genus allowing for positive identification of H. minima. Heterocapsa minima shares gross cell morphological features (hyposome smaller than episome, elongated nucleus in the middle of the cell, one pyrenoid located in the episome on its left side) with H. arctica (both subspecies H. arctica subsp. arctica and H. arctica subsp. frigida), H. lanceolata and H. rotundata. These relationships are reflected in the phylogenetic trees based on LSU and ITS rDNA sequence data, which identified H. arctica (both subspecies), H. rotundata and H. lanceolata as close relatives of H. minima.     

The phylogenetic positions of three Basal-hexapod groups (protura, diplura, and collembola) based on ribosomal RNA gene sequences

This study combined complete 18S with partial 28S ribosomal RNA gene sequences ( approximately 2,000 nt in total) to investigate the relations of basal hexapods. Ten species of Protura, 12 of Diplura, and 10 of Collembola (representing all subgroups of these three clades) were sequenced, along with 5 true insects and 8 other arthropods, which served as out-groups. Trees were constructed with maximum parsimony, maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded strong support for a clade of Protura plus Diplura, here named Nonoculata, and for monophyly of the Diplura. Parametric-bootstrapping analysis showed our data to be inconsistent with previous hypotheses (P < 0.01) that joined Protura with Collembola (Ellipura), that said Diplura are sister to true insects or are diphyletic, and that said Collembola are not hexapods. That is, our data are consistent with hexapod monophyly and Collembola grouped weakly with "Protura + Diplura" under most analytical conditions. As a caveat to the above conclusions, the sequences showed nonstationarity of nucleotide frequencies across taxa, so the CG-rich sequences of the diplurans and proturans may have grouped together artifactually; however, the fact that the LogDet method supported this group lessens this possibility. Within the basal hexapod groups, where nucleotide frequencies were stationary, traditional taxonomic subgroups generally were recovered: i.e., within Protura, the Eosentomata and Acerentomata (but Sinentomata was not monophyletic); within Collembola, the Arthropleona, Poduromorpha, and Entomobryomorpha (but Symphypleona was polyphyletic); and in Diplura, the most complete data set (> 2,100 nt) showed monophyly of Campodeoidea and of Japygoidea, and most methods united Projapygoidea with Japygoidea.     

Phylogeny of the Stichotrichia (Ciliophora; Spirotrichea) reconstructed with nuclear small subunit rRNA gene sequences: discrepancies and accordances with morphological data

The Stichotrichia, known as an especially various and taxonomically difficult group, were intensely studied with morphological, morphogenetic, and molecular methods in the last years. Nevertheless, a consistent classification is lacking and several important questions about the phylogenetic relationships within this group remain unsolved. In order to gain deeper insights into these relationships, the nuclear small subunit rRNA genes of seven species of the Stichotrichia, representatives of the families Oxytrichidae, Amphisiellidae, and Pseudourostylidae, were phylogenetically analysed. Although our analyses resulted in a poor resolution of the phylogenetic relationships, some conclusions can be drawn. Firstly, following the current classification systems the Oxytrichidae as well as their subfamilies seem to be paraphyletic and the basic 18 FVT cirral pattern has been modified several times independently. Secondly, sequence analyses of several Oxytricha species resulted in a high molecular diversity, which does not support monophyly of this genus. Thirdly, several families of the order Urostylida (Urostylidae, Pseudokeronopsidae, and Pseudourostylidae) also do not form monophyletic groups.     

Evolutionary relationships among heterokont algae (the autotrophic stramenopiles) based on combined analyses of small and large subunit ribosomal RNA

In order to study the phylogenetic relationships within the stramenopiles, and particularly among the heterokont algae, we have determined complete or nearly complete large-subunit ribosomal RNA sequences for different species of raphidophytes, phaeophytes, xanthophytes, chrysophytes, synurophytes and pinguiophytes. With the small- and large-subunit ribosomal RNA sequences of representatives for nearly all known groups of heterokont algae, phylogenetic trees were constructed from a concatenated alignment of both ribosomal RNAs, including more than 5,000 positions. By using different tree construction methods, inferred phylogenies showed phaeophytes and xanthophytes as sister taxa, as well as the pelagophytes and dictyochophytes, and the chrysophytes/synurophytes and eustigmatophytes. All these relationships are highly supported by bootstrap analysis. However, apart from these sister group relationships, very few other internodes are well resolved and most groups of heterokont algae seem to have diverged within a relatively short time frame.