New considerations on the phylogeny of cyrtophorian ciliates (Protozoa,Ciliophora): expanded sampling to understand their evolutionary relationships

To rationalize the confusing relationships among the cyrtophorian ciliates, we expanded the taxon sampling by sequencing the small subunit ribosomal RNA (SSU rRNA) gene of representatives of 12 genera (20 species, 23 new sequences). The SSU rRNA sequences of Spirodysteria, Agnathodysteria, Brooklynella and Odontochlamys are reported for the first time. Phylogenetic trees were constructed, and secondary structures of variable region 4 (V4) of all genera for which SSU rRNA gene sequence data are available were predicted. The results indicate that (i) Brooklynella is likely an intermediate taxon between Dysteriidae and Hartmannulidae; (ii) the genus Dysteria is paraphyletic with Spirodysteria and Mirodysteria nested within it; (iii) the genus Agnathodysteria is well separated from Dysteria based on both molecular and morphological data; and (iv) Trithigmostoma is a basal genus of Chilodonellidae, based on both the morphological and molecular data.     

A phylogenetic reconsideration of suctorian ciliates (Protista,Ciliophora, Phyllopharyngea) based on small subunit rRNA gene sequences

Compared with other ciliated protozoa, molecular studies of phylogenetic relationships within the subclass Suctoria are rare. In this work, phylogenetic analyses focusing on this group were performed based on all data available. In addition, the small subunit ribosomal RNA (SSU rRNA) genes of three suctorian ciliates (Acineta compressa, Acineta tuberosa and Paracineta limbata) were newly sequenced. Furthermore, the putative secondary structures of the variable region 2 of the SSU rRNA gene were predicted and compared within the Suctoria. Our results show that (i) there is support for the monophyly of the subclass Suctoria, which is a sister clade to the cyrtophorids; (ii) based on combined morphologic and molecular features, we propose the following evolutionary routine within the Suctoria: Exogenina – Evaginogenina – Endogenina; (iii) the similarities of the secondary structures of the V2 region and the SSU rRNA gene sequences within the subclass Suctoria are consistent with the branching of the phylogenetic lineages.     

Molecular phylogeny of tintinnid ciliates (tintinnida, ciliophora)

We investigated the phylogeny of tintinnids (Ciliophora, Tintinnida) with 62 new SSU-rDNA sequences from single cells of 32 marine and freshwater species in 20 genera, including the first SSU-rDNA sequences for Amphorides, Climacocylis, Codonaria, Cyttarocylis, Parundella, Petalotricha, Undella and Xystonella, and 23 ITS sequences of 17 species in 15 genera. SSU-rDNA phylogenies suggested a basal position for Eutintinnus, distant to other Tintinnidae. We propose Eutintinnidae fam. nov. for this divergent genus, keeping the family Tintinnidae for Amphorellopsis, Amphorides and Steenstrupiella. Tintinnopsis species branched in at least two separate groups and, unexpectedly, Climacocylis branched among Tintinnopsis sensu stricto species. Tintinnopsis does not belong to the family Codonellidae, which is restricted to Codonella, Codonaria, and also Dictyocysta (formerly in the family Dictyocystidae). The oceanic genus Undella branched close to an undescribed freshwater species. Metacylis, Rhabdonella and Cyttarocylis formed a well supported clade with several Tintinnopsis species at a basal position. Petalotricha ampulla and Cyttarocylis cassis SSU-rDNA and ITS sequences were identical or almost identical. Therefore, we propose Cyttarocylis ampulla comb. nov. for them. Intensive use of single-cell isolation and sequencing revealed unexpected complexity in the evolutionary history of these relatively well-studied ciliates. Notably, the diversity of freshwater forms suggests multiple marine-freshwater invasions.     

Genetic relatedness among Filobasidiella species

The three accepted species of Filobasidiella, F. neoformans, F. depauperata, and F. lutea, are compared morphologically and by molecular analysis. Sequences of the internally transcribed spacer (ITS) and the small subunit (SSU) gene of the ribosomal RNA (rRNA) gene cluster were obtained, and analysed by Neighbor-joining and Maximum parsimony methods. The three species of Filobsidiella are shown to form a single monophyletic clade, rooted by Tremella mesenterica. F. lutea was recovered as a distinct, but closely related taxon with the Filobasidiella clade. This is the first report of DNA sequences from herbarium specimens of F. lutea.This revised version was published online in October 2005 with corrections to the Cover Date.     

Utility of mitochondrial‐encoded cytochrome c oxidase I gene for phylogenetic analysis and species identification of the planktonic diatom genus Skeletonema

Small subunit (SSU) and large subunit (LSU) rDNA sequences have been commonly used to delineate the taxonomy and biogeography of the planktonic diatom genus Skeletonema, but the genes occur as multiple copies and are therefore not suitable for barcoding purposes. Here, we analyzed phylogenetic relationships of Skeletonema using the mitochondrial‐encoded cytochrome c oxidase I gene (cox1), as well as partial LSU rDNA (D1–D3) and SSU rDNA, to identify the factors that define species and to evaluate the utility of these three markers for this taxon. Twelve Skeletonema species were divided into six clades, I–VI, each of which comprised the same species by the three markers: clades I (S. japonicum, S. grethae, S. pseudocostatum, and S. tropicum), II (S. menzelii), III (S. dohrnii and S. marinoi), IV (S. costatum, S. potamos, and S. subsalsum), V (S. grevillei), and VI (S. ardens). However, the branching order among these clades was incongruent among the markers. In clade III, six S. marinoi strains had identical cox1 sequences. These S. marinoi strains branched along with S. dohrnii, except for strains from the Gulf of Naples, with high support in cox1. Species delimitation between S. dohrnii and S. marinoi was therefore not supported. In clade IV, S. costatum and S. subsalsum were robustly clustered, with S. potamos as a sister clade in the cox1 tree, not in the LSU and SSU trees. In clade II, cox1 also confirmed that S. menzelii includes three subclades potentially distinguishable from each other by morphological features. Cox1 proved to be the most useful marker for the identification of Skeletonema species because it gave a tree with highly supported clades, has sufficient variation within and among species, encodes a protein in a single copy, and requires relatively few primers.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Morphological and Ribosomal DNA‐based Characterization of Six Antarctic Ciliate Morphospecies from the Amundsen Sea with Phylogenetic Analyses

We characterized six tintinnid ciliates from Antarctic waters using molecular markers and morphological traits: Amphorellopsis quinquealata, Codonellopsis gaussi, Cymatocylis convallaria, Cy. calyciformis, Cy. drygalskii, and Laackmanniella prolongata. The 100% similarity in SSU‐ITS1‐5.8S rDNA‐ITS2‐partial LSU rDNA sequences among Cy. convallaria, Cy. calyciformis, and Cy. drygalskii is supportive of synonymy. Codonellopsis gaussi and L. prolongata also showed high levels of similarity in SSU rDNA (99.83%) and the D2 domain of LSU rDNA (95.77%), suggesting that they are closely related. Phylogenetic analysis placed Cymatocylis in the Rhabdonellidae, Amphorellopsis in the Tintinnidae and L. prolongata/Co. gaussi within the Dictyocystidae.     

A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic species.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.     

Molecular phylogeny of the Cladophoraceae (Cladophorales,Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen,and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica.     

PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1

Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well‐known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.     

PHYLOGENY OF THE BASAL LINEAGES OF STREPTOPHYTA BASED ON RBCL AND ATPB GENE SEQUENCE DATA

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 rbcL (the genes encoding the large subunit of rubisco) and atpB (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.     

Snake evolution in Melanesia: origin of the Hydrophiinae (Serpentes,Elapidae), and the evolutionary history of the enigmatic New Guinean elapid Toxicocalamus

The venomous snake subfamily Hydrophiinae includes more than 40 genera and approximately 200 species. Most members of this clade inhabit Australia, and have been well studied. But, because of poor taxon sampling of Melanesian taxa, basal evolutionary relationships have remained poorly resolved. The Melanesian genera Ogmodon, Loveridgelaps, and Salomonelaps have not been included in recent phylogenetic studies, and the New Guinean endemic, Toxicocalamus, has been poorly sampled and sometimes recovered as polyphyletic. We generated a multilocus phylogeny for the subfamily using three mitochondrial and four nuclear loci so as to investigate relationships among the basal hydrophiine genera and to determine the status of Toxicocalamus. We sequenced these loci for eight of the 12 described species within Toxicocalamus, representing the largest molecular data set for this genus. We found that a system of offshore island arcs in Melanesia was the centre of origin for terrestrial species of Hydrophiinae, and we recovered Toxicocalamus as monophyletic. Toxicocalamus demonstrates high genetic and morphological diversity, but some of the molecular diversity is not accompanied by diagnostic morphological change. We document at least five undescribed species that all key morphologically to Toxicocalamus loriae (Boulenger, 1898), rendering this species polyphyletic. Continued work on Toxicocalamus is needed to document the diversity of this genus, and is likely to result in the discovery of additional species. Our increased taxon sampling allowed us to better understand the evolution and biogeography of Hydrophiinae; however, several unsampled lineages remain, the later study of which may be used to test our biogeographic hypothesis.     

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha‐proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early‐diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein‐coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.