SAME BUT DIFFERENT: TWO NOVEL BICARINATE SPECIES OF EXTANT CALCAREOUS DINOPHYTES (THORACOSPHAERACEAE,PERIDINIALES) FROM THE MEDITERRANEAN SEA1

The diversity of extant calcareous dinophytes (Thoracosphaeraceae, Dinophyceae) is currently not sufficiently recorded. The majority of their coccoid stages are cryptotabulate or entirely atabulate, whereas relatively few forms exhibit at least some degree of tabulation more than the archeopyle. A survey of coastal surface sediment samples from the Mediterranean Sea resulted in the isolation and cultivation of several strains of calcareous dinophytes showing a prominent tabulation. We investigated the morphologies of the thecate and the coccoid cells and conducted phylogenetic analyses using Maximum Likelihood and Bayesian approaches. The coccoid cells showed a distinct reflection of the cingulum (and were thus cingulotabulate), whereas thecal morphology corresponded to the widely distributed and species‐rich Scrippsiella. As inferred from molecular sequence data (including 81 new GenBank entries), the strains belonged to the Scrippsiella sensu lato clade of the Thoracosphaeraceae and represented two distinct species. Morphological details likewise indicated two distinct species with previously unknown coccoid cells that we describe here as new, namely S. bicarinata spec. nov. and S. kirschiae spec. nov. Cingulotabulation results from the fusion of processes representing the pre‐ and postcingular plate series in S. bicarinata, whereas the ridges represent sutures between the cingulum and the pre‐ and postcingular series in S. kirschiae, respectively. Bicarinate cingulotabulation appears homoplasious among calcareous dinophytes, which is further supported by a comparison to similar, but only distantly related fossil forms.     

Who am I — and if so, how many? Species diversity of calcareous dinophytes (Thoracosphaeraceae, Peridiniales) in the Mediterranean Sea

The diversity of extant calcareous dinophytes (Thoracosphaeraceae, Dinophyceae) is not fully recorded at present. The establishment of algal strains collected at multiple localities is necessary for a rigorous study of taxonomy, morphology and evolution in these unicellular organisms. We collected sediment and water tow samples from more than 60 localities in coastal areas of the eastern Mediterranean Sea and documented 15 morphospecies of calcareous dinophytes. Internal transcribed spacer (ITS) barcoding identified numerous species of the Scrippsiella trochoidea species complex that were genetically distinct, but indistinguishable in gross morphology (i.e. with the same tabulation patterns of the motile theca and similar spiny coccoid stages). We assessed a possible minimal number of cryptic species using ITS ribotype networks that indicated the existence of at least 21 species within the Scrippsiella trochoidea species complex. Species diversity of calcareous dinophytes appears higher in the Mediterranean Sea than in other parts of the world??s oceans such as the North Sea. Our data underline the importance of field work to record the diversity of calcareous dinophytes and other unicellular life forms.     

Ultrastructure of Calcareous Dinophytes (Thoracosphaeraceae,Peridiniales) with a Focus on Vacuolar Crystal-Like Particles

Biomineralization in calcareous dinophytes (Thoracosphaeracaea, Peridiniales) takes place in coccoid cells and is presently poorly understood. Vacuolar crystal-like particles as well as collection sites within the prospective calcareous shell may play a crucial role during this process at the ultrastructural level. Using transmission electron microscopy, we investigated the ultrastructure of coccoid cells at an early developmental stage in fourteen calcareous dinophyte strains (corresponding to at least ten species of Calciodinellum, Calcigonellum, Leonella, Pernambugia, Scrippsiella, and Thoracosphaera). The shell of the coccoid cells consisted either of one (Leonella, Thoracosphaera) or two matrices (Scrippsiella and its relatives) of unknown element composition, whereas calcite is deposited in the only or the outer layer, respectively. We observed crystal-like particles in cytoplasmic vacuoles in cells of nine of the strains investigated and assume that they are widespread among calcareous dinophytes. However, similar structures are also found outside the Thoracosphaeraceae, and we postulate an evolutionarily old physiological pathway (possibly involved in detoxification) that later was specialized for calcification. We aim to contribute to a deeper knowledge of the biomineralization process in calcareous dinophytes.     

Amphidoma languida sp. nov. (Dinophyceae) reveals a close relationship between Amphidoma and Azadinium

Toxic algae such as Alexandrium and Azadinium have an important ecological impact and have originated several times independently within the dinophytes. Their closest relatives, however, are mostly unknown at present. A new dinophyte species, Amphidoma languida sp. nov., was isolated from Bantry Bay (Ireland) during a period of elevated azaspiracid toxicity in mussels. The new species was described in detail, and its phylogenetic position was analysed, by using a combination of light and electron microscopy, chemical detection methods, and sequence comparison of concatenated ribosomal RNA sequence data. Morphological similarities, such as cingular and hypothecal plates, the number and arrangement of sulcal plates, and the characteristic apical pore complex with a small X-plate centrally invading the first apical plate, indicated a close relationship between Amphidoma and Azadinium. However, no known azaspiracid analogues were detected in A. languida by liquid chromatography coupled with tandem mass-spectrometry. In a molecular phylogeny, the Amphidomataceae including Amphidoma and Azadinium were an independent lineage among other monophyletic major groups of the dinophytes such as the Suessiales, Prorocentrales, Gonyaulacales, and Peridiniales. Thus, the taxonomic affiliation of Azadinium is clarified, and our data may prove helpful in the development of specific and reliable molecular detection methods of toxic Azadinium.     

PHYLOGENETIC AFFINITIES OF CRYSONEPHELE PALUSTRIS (CHRYSOPHYCEAE) BASED ON INFERRED NUCLEAR SMALL-SUBUNIT RIBOSOMAL RNA SEQUENCE

The nuclear small-subunit ribosomal DNA sequence was determined from the colonial chromophytic alga Chrysonephele palustris Pipes, Tyler, et Leedale using polymerase chain reaction methods. The inferred ribosomal RNA sequence was included in a multiple alignment, containing heterokont chromophytes, and subjected to molecular systematic analyses in order to determine the phylogenetic relationships of this alga . Chrysonephele palustris grouped within the Chrysophyceae and not in an intermediate position between the Chrysophyceae and Eustigmatophyceae, suggesting that Chrysonephele is not a phylogenetic link between these algal classes .     

HALOSTYLODINIUM ARENARIUM, GEN ET SP. NOV. (DINOPHYCEAE), A COCCOID SAND-DWELLING DINOFLAGELLATE FROM SUBTROPICAL JAPAN

A new genus and species of marine coccoid dinoflagellate from subtropical Japan, Halostylodinium arenarium Horiguchi et Yoshizawa-Ebata, gen. et sp. nov., is described. The dominant stage of the dinoflagellate is a nonmotile ovoidal to spheroidal cell with a distinct stalk. The stalk consists of an upper thick tubule, a lower thin tubule, and a discoidal holdfast. The dinoflagellate possesses a yellowish-brown chloroplast with multiple lobes radiating from a central pyrenoid. It reproduces by the formation of two motile cells, which swim for a short period and then transform directly into the stalked nonmotile cell. The stalk is produced during transformation from the apical stalk complex present in the apex of the motile cell. The apical stalk complex consists of a double-folded apical pore plate and doughnut-shaped holdfast-building material. The ultrastructure of the apical stalk complex is compared with those of Bysmatrum arenicola and Stylodinium littorale. Halostylodinium arenarium possesses delicate thecal plates, and the thecal plate formula is Po, 5', 2a, 7", 7c, 6s, 5"', 1p, 2"". A phylogenetic study based on the 18S ribosomal RNA gene did not show any clear affinities between this organism and any species included in the analysis.     

Rapid ribosomal RNA sequencing and the phylogenetic analysis of protists

A newly described technique for rapidly obtaining the partial nucleotide sequence of ribosomal RNA is being applied to investigate phylogenetic relationships among living organisms. Alan Johnson and Peter Boverstock describe the importance of this method to parasitology in providing new information on the phylogenetic relationships of parasitic organisms previously placed in groups of convenience. The phylum Apicomplexo in particular, has been the object of much study using this technique, but the technology is likely to extend soon to the restructuring of the phylogenetic trees of many groups of parasites.     

Phylogeny of calcareous dinoflagellates as inferred from ITS and ribosomal sequence data

The phylogenetic relationships of calcareous dinoflagellates (i.e., Calciodinellaceae and Thoracosphaera) are investigated. Molecular data from the ribosomal 5.8S rRNA and highly conserved motifs of the ITS1 show Calciodinellaceae s.l. to be monophyletic when few non-calcareous taxa are included. They segregate into three monophyletic assemblages in a molecular analysis that considers the 5.8S rRNA and both the Internal Transcribed Spacer regions ITS1 and ITS2: a clade comprising species of Ensiculifera and Pentapharsodinium (E/P-clade), Scrippsiella s.l. (including fossil-based taxa such as Calciodinellum and Calcigonellum), and a heterogeneous group (T/P-clade) of calcareous (e.g., Thoracosphaera) and non-calcareous taxa (e.g., the highly toxic Pfiesteria). The potential to produce calcareous structures is considered as apomorphic within alveolates, and non-calcareous taxa nesting with calcareous dinoflagellates may have reduced calcification secondarily. Molecular results do not contradict general evolutionary scenarios provided by previous morphological (mainly paleontological) investigations.     

Detecting morphological convergence in true fungi, using 18S rRNA gene sequence data.

For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.     

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).     

Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China

Molecular phylogenetic analyses are mainly based on the small ribosomal RNA subunit (18S rRNA), internal transcribed spacer regions, and other molecular markers. We compared the phylogenetic relationships of Babesia spp. using large subunit ribosomal RNA, i.e., 28S rRNA, and the united 28S + 18S rRNA sequence fragments from 11 isolates of Babesia spp. collected in China. Due to sequence length and variability, the 28S rRNA gene contained more information than the 18S rRNA gene and could be used to elucidate the phlyogenetic relationships of B. motasi, B. major, and B. bovis. Thus, 28S rRNA is another candidate marker that can be used for the phylogenetic analysis of Babesia spp. However, the united fragment (28S + 18S) analysis provided better supported phylogenetic relationships than single genes for Babesia spp. in China.     

Phylogenetic signal and noise: predicting the power of a data set to resolve phylogeny

A principal objective for phylogenetic experimental design is to predict the power of a data set to resolve nodes in a phylogenetic tree. However, proactively assessing the potential for phylogenetic noise compared with signal in a candidate data set has been a formidable challenge. Understanding the impact of collection of additional sequence data to resolve recalcitrant internodes at diverse historical times will facilitate increasingly accurate and cost-effective phylogenetic research. Here, we derive theory based on the fundamental unit of the phylogenetic tree, the quartet, that applies estimates of the state space and the rates of evolution of characters in a data set to predict phylogenetic signal and phylogenetic noise and therefore to predict the power to resolve internodes. We develop and implement a Monte Carlo approach to estimating power to resolve as well as deriving a nearly equivalent faster deterministic calculation. These approaches are applied to describe the distribution of potential signal, polytomy, or noise for two example data sets, one recent (cytochrome c oxidase I and 28S ribosomal rRNA sequences from Diplazontinae parasitoid wasps) and one deep (eight nuclear genes and a phylogenomic sequence for diverse microbial eukaryotes including Stramenopiles, Alveolata, and Rhizaria). The predicted power of resolution for the loci analyzed is consistent with the historic use of the genes in phylogenetics.     

Strengths and limitations of molecular sequence comparisons for inferring the phylogeny of the major groups of fishes

Although the phylogenetic relationships of the major groups of fishes have been extensively studied with morphological characters, not all have been convincingly resolved. Analyses of molecular sequences from these groups may provide additional insights into problematical relationships, but are only just beginning to appear. We compare our own results from analyses of 18s ribosomal RNA sequences with those of other studies using globins, parvalbumins, insulin, 28s ribosomal RNA, and portions of two mitochondria1 genes (12S ribosomal RNA and cytochrome b ). Our evaluation of these studies reveals some of the difficulties encountered in reconstructing ancient divergences within the fishes, including unequal rates of evolution (among regions of a molecule as well as among lineages), gene duplication, extinction of lineages, and a possible rapid radiation of gnathostome higher taxa. The importance of evaluating the robustness of particular phylogenetic hypotheses is stressed. Some molecules appear to be inappropriate for investigating higher level divergences within the fishes; others are more promising, but must be examined in more taxa to allow an adequate evaluation of their utility. Convincing support for particular hypotheses of relationship will ultimately require congruence of trees generated from independent molecular data sets.     

Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima

At northern latitudes the sea anemones Anthopleura elegantissima and its congener A. xanthogrammica contain unidentified green chlorophytes (zoochlorellae) in addition to dinophytes belonging to the genus Symbiodinium. This dual algal symbiosis, involving members of distinct algal phyla in one host, has been extensively studied from the perspective of the ecological and energetic consequences of hosting one symbiotic type over the other. However, the identity of the green algal symbiont has remained elusive. We determined the phylogenetic position of the marine zoochlorellae inhabiting A. elegantissima by comparing sequence data from two cellular compartments, the nuclear 18S ribosomal RNA gene region and the plastid-encoded rbcL gene. The results support the inclusion of these zoochlorellae in a clade of green algae that form symbioses with animal (Anthopleura elegantissima), fungal (the lichen genus Nephroma), and seed plant (Ginkgo) partners. This clade is distinct from the Chlorella symbionts of Hydra. The phylogenetic diversity of algal hosts observed in this clade indicates a predisposition for this group of algae to participate in symbioses. An integrative approach to the study of these algae, both within the host and in culture, should yield important clues about how algae become symbionts in other organisms.     

Phylogenetic analysis of four nuclear protein-encoding genes largely corroborates the traditional classification of Bivalvia (Mollusca)

Revived interest in molluscan phylogeny has resulted in a torrent of molecular sequence data from phylogenetic, mitogenomic, and phylogenomic studies. Despite recent progress, basal relationships of the class Bivalvia remain contentious, owing to conflicting morphological and molecular hypotheses. Marked incongruity of phylogenetic signal in datasets heavily represented by nuclear ribosomal genes versus mitochondrial genes has also impeded consensus on the type of molecular data best suited for investigating bivalve relationships. To arbitrate conflicting phylogenetic hypotheses, we evaluated the utility of four nuclear protein-encoding genes-ATP synthase β, elongation factor-1α, myosin heavy chain type II, and RNA polymerase II-for resolving the basal relationships of Bivalvia. We sampled all five major lineages of bivalves (Archiheterodonta, Euheterodonta [including Anomalodesmata], Palaeoheterodonta, Protobranchia, and Pteriomorphia) and inferred relationships using maximum likelihood and Bayesian approaches. To investigate the robustness of the phylogenetic signal embedded in the data, we implemented additional datasets wherein length variability and/or third codon positions were eliminated. Results obtained include (a) the clade (Nuculanida+Opponobranchia), i.e., the traditionally defined Protobranchia; (b) the monophyly of Pteriomorphia; (c) the clade (Archiheterodonta+Palaeoheterodonta); (d) the monophyly of the traditionally defined Euheterodonta (including Anomalodesmata); and (e) the monophyly of Heteroconchia, i.e., (Palaeoheterodonta+Archiheterodonta+Euheterodonta). The stability of the basal tree topology to dataset manipulation is indicative of signal robustness in these four genes. The inferred tree topology corresponds closely to those obtained by datasets dominated by nuclear ribosomal genes (18S rRNA and 28S rRNA), controverting recent taxonomic actions based solely upon mitochondrial gene phylogenies.     

The molecular diversity of freshwater picoeukaryotes from an oligotrophic lake reveals diverse, distinctive and globally dispersed lineages

The recent discovery of a diverse phylogenetic assemblage of picoeukaryotes from environments such as oceans, salt marshes and acidic habitats, has expanded the debates about the extent and origin of microbial eukaryotes. However, the diversity of these eukaryote microorganisms, that overlap bacteria in size, and their environmental and biogeographical ubiquity remains poorly understood. Here we survey picoeukaryotes (microbial eukaryotes of 0.2-5 microm in size) from an oligotrophic (nutrient deficient) freshwater habitat using ribosomal RNA gene sequences. Three taxonomic groups the Heterokonta, Cryptomonads and the Alveolata dominated the detected diversity. Most sequences represented previously unsampled species, with several being unassignable to known taxonomic groups and plausibly represent new or unsampled phyla. Many freshwater phylogenetic groups identified in this study appeared unrelated to picoeukaryotic sequences identified in marine ecosystems, suggesting that aspects of eukaryote microbial diversity are specific to certain aquatic environments. Conversely, at least five phylogenetic clusters comprised sequences from freshwater and globally dispersed and often contrasting environments, supporting the concept that a number of picoeukaryotic lineages are widely distributed.     

The phylogeny of the Hyphochytriomycota as deduced from ribosomal RNA sequences of Hyphochytrium catenoides

Based on biochemical and ultrastructural data, hyphochytriomycetes are believed to share an ancestor with oomycetes and heterokont algae. In order to study the phylogeny of the hyphochytriomycetes, we determined both the small- and large-subunit ribosomal RNA sequence of Hyphochytrium catenoides. Phylogenetic trees were constructed using the neighbor-joining and maximum-parsimony method and include representatives of Chlorobionta, Fungi, Metazoa, Alveolata, and all known Heterokonta. Our main conclusion is that the hyphochytriomycetes form a monophyletic group with the oomycetes and heterokont algae and that they are probably the closest relatives of the oomycetes. However, the order of divergence between the various heterokont algal phyla and the oomycete-hyphochytriomycete cluster remains uncertain.      

Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the protozoan family sarcocystidae

Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.     

The first complete mitochondrial genome from Bostrychus genus (Bostrychus sinensis) and partitioned Bayesian analysis of Eleotridae fish phylogeny

To understand the phylogenetic position of Bostrychus sinensis in Eleotridae and the phylogenetic relationships of the family, we determined the nucleotide sequence of the mitochondrial (mt) genome of Bostrychus sinensis. It is the first complete mitochondrial genome sequence of Bostrychus genus. The entire mtDNA sequence was 16508 bp in length with a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a noncoding control region. The mitochondrial genome of B. sinensis had common features with those of other bony fishes with respect to gene arrangement, base composition, and tRNA structures. Phylogenetic hypotheses within Eleotridae fish have been controversial at the genus level. We used the mitochondrial cytochrome b (cytb) gene sequence to examine phylogenetic relationships of Eleotridae by using partitioned Bayesian method. When the specific models and parameter estimates were presumed for partitioning the total data, the harmonic mean –lnL was improved. The phylogenetic analysis supported the monophyly of Hypseleotris and Gobiomorphs. In addition, the Bostrychus were most closely related to Ophiocara, and the Philypnodon is also the sister to Microphlypnus, based on the current datasets. Further, extensive taxonomic sampling and more molecular information are needed to confirm the phylogenetic relationships in Eleotridae.