Paramecium (Oligohymenophorea, Ciliophora) diversity in Thailand sheds light on the genus biogeography and reveals new phylogenetic lineages.

Paramecium (Ciliophora, Oligohymenophorea) is a good model to study ciliate biogeography. Extensive sampling mainly in northern hemisphere has led to 16 valid morphological species description thus far. However, a majority of hard-to-reach regions, including South East Asia, are underinvestigated. Our study combined traditional morphological and molecular approaches to reveal the biodiversity of Paramecium in Thailand from more than 110 samples collected in 10 provinces. Representatives of seven morphological species were identified from our collection, including the rare species, such as P. gigas and P. jenningsi. Additionally, we detected five different sibling species of the P. aurelia complex, described a new cryptic species P. hiwatashii n. sp. phylogenetically related to P. caudatum, and discovered a potentially new genetic species of the P. bursaria species complex. We also documented a variety of bacterial cytoplasmic symbionts from at least nine monoclonal cultures of Paramecium.

Fusiforma themisticola n. gen., n. sp., a new genus and species of apostome ciliate infecting the hyperiid amphipod Themisto libellula in the Canadian Beaufort Sea (Arctic Ocean), and establishment of the Pseudocolliniidae (Ciliophora, Apostomatia).

A novel parasitic ciliate Fusiforma themisticola n. gen., n. sp. was discovered infecting 4.4% of the hyperiid amphipod Themisto libellula. Ciliates were isolated from a formaldehyde-fixed whole amphipod and the DNA was extracted for amplification of the small subunit (SSU) rRNA gene. Sequence and phylogenetic analyses showed unambiguously that this ciliate is an apostome and about 2% divergent from the krill-infesting apostome species assigned to the genus Pseudocollinia. Protargol silver impregnation showed a highly unusual infraciliature for an apostome. There are typically 8 (6-9) bipolar somatic kineties covering the banana-shaped body. The anterior end of the oral cavity begins about 1/3 of the body length from the anterior end and is composed of an inpocketing that is lined on its anterior and left wall with an oral field of densely packed ciliated kinetosomes. Stomatogenesis begins with some dedifferentiation of the parental oral field and elongation of its paroral and oral kineties. A new oral field develops midventrally and the paroral and oral kineties break to form the oral apparatus of the opisthe, which completes development by additional kinetosomal proliferation and migration of the paroral. This morphology is novel among apostomes and justifies the establishment of a new genus and species.

Evolutionary relationships among marine cercozoans as inferred from combined SSU and LSU rDNA sequences and polyubiquitin insertions.

An insertion of one or two amino acids at the monomer-monomer junctions of polyubiquitin is a distinct and highly conserved molecular character that is shared by two very diverse clades of microeukaryotes, the Cercozoa and the Foraminifera. It has been suggested that an insertion consisting of one amino acid, like that found in foraminiferans and some cercozoans, represents an ancestral state, and an insertion consisting of two amino acids represents a derived state. However, the limited number of cercozoan taxa examined so far limits inferences about the number and frequency of state changes associated with this character over deep evolutionary time. Cercozoa include a very diverse assemblage of mainly uncultivated amoeboflagellates, and their tenuous phylogenetic interrelationships have been based largely on small subunit (SSU) rDNA sequences. Because concatenated datasets consisting of both SSU and large subunit (LSU) rDNA sequences have been shown to more robustly recover the phylogenetic relationships of other major groups of eukaryotes, we employed a similar approach for the Cercozoa. In order to reconstruct the evolutionary history of this group, we amplified twelve LSU rDNAs, three SSU rDNAs, and seven polyubiquitin sequences from several different cercozoans, especially uncultured taxa isolated from marine benthic habitats. The distribution of single amino acid insertions and double amino acid insertions on the phylogenetic trees inferred from the concatenated dataset indicates that the gain and loss of amino acid residues between polyubiquitin monomers occurred several times independently. Nonetheless, all of the cercozoans we examined possessed at least one amino acid insertion between the polyubiquitin monomers, which reinforced the significance of this feature as a molecular signature for identifying members of the Cercozoa and the Foraminifera. Our study also showed that analyses combining both SSU and LSU rDNA sequences leads to improved phylogenetic resolution and statistical support for deeper branches within the Cercozoa.

Molecular systematics of marine gregarines (Apicomplexa) from North-eastern Pacific polychaetes and nemerteans, with descriptions of three novel species: Lecudina phyllochaetopteri sp. nov., Difficilina tubulani sp. nov. and Difficilina paranemertis sp. nov.

Most eugregarine apicomplexans infecting the intestines of marine invertebrates have been described within the family Lecudinidae and the type genus Lecudina. The diversity of these parasites is vast and poorly understood and only a tiny number of species has been characterized at the molecular phylogenetic level. DNA sequences coupled with high-resolution micrographs of trophozoites provide an efficient and precise approach for delimiting gregarine lineages from one another and also facilitate our overall understanding of gregarine biodiversity. In this study, phylogenetic analyses of small subunit (SSU) rDNA sequences from five (uncultivated) gregarines isolated from polychaetes and nemerteans in the North-eastern Pacific Ocean are presented. Lecudina phyllochaetopteri sp. nov. was isolated from the intestines of the parchment tubeworm Phyllochaetopterus prolifica (Polychaeta). Lecudina longissima and Lecudina polymorpha were both isolated from the intestines of Lumbrineris japonica (Polychaeta). Difficilina tubulani sp. nov. was isolated from the nemertean Tubulanus polymorpha and Difficilina paranemertis sp. nov. was isolated from the nemertean Paranemertes peregrina. This is the first report of molecular sequence data from gregarines that infect nemerteans. The two novel species of the genus Difficilina described in this study formed a strongly supported clade in the phylogenetic analyses. This Difficilina clade formed the sister group to a robust subclade of lecudinids consisting of Lecudina longissima, Lecudina phyllochaetopteri sp. nov. (which lacked epicytic folds), Lecudina tuzetae, species of the genus Lankesteria and several sequences derived from previous environmental DNA surveys of marine biodiversity.

An SSU rDNA barcoding approach to the diversity of marine interstitial cercozoans, including descriptions of four novel genera and nine novel species.

Environmental DNA surveys have revealed a great deal of hidden diversity within the Cercozoa. An investigation into the biodiversity of heterotrophic flagellates in marine benthic habitats of British Columbia, Canada, demonstrated the presence of several undescribed taxa with morphological features that resemble the cercozoan genera Cryothecomonas and Protaspis. Nine novel species of marine interstitial cercozoans are described that are distributed into five genera, four of which are new. Phylogenetic analyses of small subunit rDNA sequences derived from two uncultured isolates of Protaspis obliqua and nine novel cercozoan species (within four novel genera) provided organismal anchors that helped establish the cellular identities of several different environmental sequence clades. These data, however, also showed that the rarity of distinctive morphological features in cryomonads, and other groups of cercozoans, makes the identification and systematics of the group very difficult. Therefore, a DNA barcoding approach was applied as a diagnostic tool for species delimitation that used a 618 bp region at the 5' end of the SSU rDNA sequence. Nucleotide sequence analysis of this region showed high intergeneric sequence divergences of about 7% and very low intraspecific sequence divergences of 0-0.5%; phylogenetic analyses inferred from this barcoding region showed very similar tree topologies to those inferred from the full-length sequence of the gene. Overall, this study indicated that the 618 bp barcoding region of SSU rDNA sequences is a useful molecular signature for understanding the biodiversity and interrelationships of marine benthic cercozoans.

Ultrastructure, life cycle and molecular phylogenetic position of a novel marine sand-dwelling cercozoan: Clautriavia biflagellata n. sp.

Clautriavia is a genus of uncertain taxonomic affinity that was initially described as gliding cells with one prominent trailing flagellum and a mid-ventral groove. The genus has been classified either with euglenids on the basis of similar paramylon-like granules or with cercozoans, specifically Protaspis spp., on the basis of general similarities in cell morphology and behavior. We isolated and cultivated a novel species of Clautriavia, namely C. biflagellata n. sp., from marine sand samples collected from the west coast of Vancouver Island, Canada and characterized this isolate with high resolution microscopy (LM, SEM, and TEM) and small subunit (SSU) rDNA sequence. The gliding cells of C. biflagellata n. sp. were round to oval in outline (12-20 microm wide and 15-20 microm long), dorsoventrally flattened, and capable of engulfing other eukaryotic cells (e.g., diatoms). The cells possessed two recurrent flagella of unequal length that emerged from a subapical pit within a ventral depression: the longer prominent flagellum was about 2X the cell length; the shorter flagellum was inconspicuous and was confined to the ventral depression. Molecular phylogenetic analyses demonstrated that C. biflagellata n. sp. branched strongly within the Cercozoa, but was only distantly related to Protaspis spp. Instead, C. biflagellata n. sp. branched closely with the recently established Auranticordida clade, consisting of Auranticordis quadriverberis and Pseudopirsonia mucosa. This position was concordant with our ultrastructural data, which demonstrated several features shared by A. quadriverberis and C. biflagellata n. sp. that are not present in Protaspis spp.: (1) a dense distribution of pores on the cell surface; (2) a distinct layer of muciferous bodies immediately beneath the cell surface; (3) a robust microtubular root attached to the anterior end of the nucleus; (4) the absence of a thick cell covering; and (5) the absence of conspicuously condensed chromosomes.

Molecular phylogeny and description of the novel katablepharid Roombia truncata gen. et sp. nov., and establishment of the Hacrobia taxon nov.

BACKGROUND: Photosynthetic eukaryotes with a secondary plastid of red algal origin (cryptophytes, haptophytes, stramenopiles, dinoflagellates, and apicomplexans) are hypothesized to share a single origin of plastid acquisition according to Chromalveolate hypothesis. Recent phylogenomic analyses suggest that photosynthetic "chromalveolates" form a large clade with inclusion of several non-photosynthetic protist lineages. Katablepharids are one such non-photosynthetic lineage closely related to cryptophytes. Despite their evolutionary and ecological importance, katablepharids are poorly investigated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a newly discovered flagellate, Roombia truncata gen. et sp. nov., that is related to katablepharids, but is morphologically distinct from othermembers of the group in the following ways: (1) two flagella emerge from a papilla-like subapical protrusion, (2) conspicuous ejectisomes are aligned in multiple (5-11) rows, (3) each ejectisome increases in size towards the posterior end of the rows, and (4) upon feeding, a part of cytoplasm elastically stretch to engulf whole prey cell. Molecular phylogenies inferred from Hsp90, SSU rDNA, and LSU rDNA sequences consistently and strongly show R. truncata as the sister lineage to all other katablepharids, including lineages known only from environmental sequence surveys. A close association between katablepharids and cryptophytes was also recovered in most analyses. Katablepharids and cryptophytes are together part of a larger, more inclusive, group that also contains haptophytes, telonemids, centrohelids and perhaps biliphytes. The monophyly of this group is supported by several different molecular phylogenetic datasets and one shared lateral gene transfer; therefore, we formally establish this diverse clade as the "Hacrobia." CONCLUSIONS/SIGNIFICANCE: Our discovery of R. truncata not only expands our knowledge in the less studied flagellate group, but provide a better understanding of phylogenetic relationship and evolutionary view of plastid acquisition/losses of Hacrobia. Being an ancestral to all katablepharids, and readily cultivable, R. truncata is a good candidate for multiple gene analyses that will contribute to future phylogenetic studies of Hacrobia.

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes.

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three "classical" morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.

Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: Auranticordis quadriverberis n. gen. et sp. (Cercozoa).

BACKGROUND: Comparative morphological studies and environmental sequencing surveys indicate that marine benthic environments contain a diverse assortment of microorganisms that are just beginning to be explored and characterized. The most conspicuous predatory flagellates in these habitats range from about 20-150 mum in size and fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates, euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together in molecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studies have demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within the Cercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge of cercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrate important cellular innovations for understanding eukaryote evolution. RESULTS: A rare tetraflagellate, Auranticordis quadriverberis n. gen. et sp., was isolated from marine sand samples. Uncultured cells were in low abundance and were individually prepared for electron microscopy and DNA sequencing. These flagellates possessed several novel features, such as (1) gliding motility associated with four bundled recurrent flagella, (2) heart-shaped cells about 35-75 microm in diam., and (3) bright orange coloration caused by linear arrays of muciferous bodies. Each cell also possessed about 2-30 pale orange bodies (usually 4-5 microm in diam.) that were enveloped by two membranes and sac-like vesicles. The innermost membrane invaginated to form unstacked thylakoids that extended towards a central pyrenoid containing tailed viral particles. Although to our knowledge, these bodies have never been described in any other eukaryote, the ultrastructure was most consistent with photosynthetic endosymbionts of cyanobacterial origin. This combination of morphological features did not allow us to assign A. quadriverberis to any known eukaryotic supergroup. Thus, we sequenced the small subunit rDNA sequence from two different isolates and demonstrated that this lineage evolved from within the Cercozoa. CONCLUSION: Our discovery and characterization of A. quadriverberis underscores how poorly we understand the diversity of cercozoans and, potentially, represents one of the few independent cases of primary endosymbiosis within the Cercozoa and beyond.

Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena.

The mitochondrial cytochrome-c oxidase subunit 1 (cox1) gene has been proposed as a DNA barcode to identify animal species. To test the applicability of the cox1 gene in identifying ciliates, 75 isolates of the genus Tetrahymena and three non-Tetrahymena ciliates that are close relatives of Tetrahymena, Colpidium campylum, Colpidium colpoda and Glaucoma chattoni, were selected. All tetrahymenines of unproblematic species could be identified to the species level using 689 bp of the cox1 sequence, with about 11 % interspecific sequence divergence. Intraspecific isolates of Tetrahymena borealis, Tetrahymena lwoffi, Tetrahymena patula and Tetrahymena thermophila could be identified by their cox1 sequences, showing <0.65 % intraspecific sequence divergence. In addition, isolates of these species were clustered together on a cox1 neighbour-joining (NJ) tree. However, strains identified as Tetrahymena pyriformis and Tetrahymena tropicalis showed high intraspecific sequence divergence values of 5.01 and 9.07 %, respectively, and did not cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species. The mean interspecific sequence divergence of Tetrahymena was about 11 times greater than the mean intraspecific sequence divergence, and this increased to 58 times when all isolates of species with high intraspecific sequence divergence were excluded. This result is similar to DNA barcoding studies on animals, indicating that congeneric sequence divergences are an order of magnitude greater than conspecific sequence divergences. Our analysis also demonstrated low sequence divergences of <1.0 % between some isolates of T. pyriformis and Tetrahymena setosa on the one hand and some isolates of Tetrahymena furgasoni and T. lwoffi on the other, suggesting that the latter species in each pair is a junior synonym of the former. Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene as a taxonomic marker for 'barcoding' and identifying Tetrahymena species and some other ciliated protists.