Insights into the phylogeny of sporadotrichid ciliates (Protozoa, Ciliophora: Hypotricha) based on genealogical analyses of multiple molecular markers

The sporadotrichid ciliates are an especially diverse group. A number of investigators have studied the morphological, morphogenetic, and molecular relationships among members of this group. Despite this, a consistent classification is still lacking and several important questions about the phylogenetic relationships within this group remain unsolved. To improve our understanding of these relationships, we constructed phylogenetic trees using the nucleotide sequences of the small-subunit rRNA (SSrRNA) gene and amino acid sequences of actin I and α-tubulin. Analyses of SSrRNA gene sequences indicated that: 1) the Sporadotrichida sensu Lynn (2008) and the Oxytrichidae are polyphyletic; 2) the Uroleptus species, which are classified to urostylids, formed a sister group with the oxytrichids; 3) Halteria grandinella, which is grouped morphologically with oligotrich species, clustered within the oxytrichids. These results are congruent with previous studies based on SSrRNA gene sequences. However, the amino acid sequences of actin I and α-tubulin yielded different topologies. The main results are: 1) in all phylogenetic trees, the genus Oxytricha was paraphyletic; 2) Uroleptus was sister to a subset of Urostyla and Holosticha, albeit with low supporting values; 3) Halteria grandinella was separated distantly from the Oxytrichidae in trees inferred from actin I amino acid sequences but clustered with oligotrichids in the α-tubulin analysis. The inconsistency among the trees inferred from these different molecular markers may be caused by rapidly accumulated genetic characterizations of ciliates. Further studies with additional molecular markers and sampling of more taxa are expected to better address the relationships among sporadotrichids.     

Taxonomic reassessment of Polysiphonia hainanensis(Rhodomelaceae,Rhodophyta) based on molecular and morphological analyses

Polysiphonia hainanensis was previously described from Hainan, China, but DNA sequence had never been provided and its phylogenetic position in Polysiphonieae has not been clear so far. The classification of Polysiphonieae has changed recently based on the molecular analyses and detail morphological anatomy, consequently, the generic assignment of some species must be reassessed. In this study, we analyzed phylogenetic relationships of P. hainanensis and the related taxa in Polysiphonieae and Streblocladieae based on rbcL sequences, with specimens collected from the type locality and the area around. Two genetic types were detected, and formed a clade with Bryocladia thyrsigera with strong bootstrap support, in which the divergence was less than 0.81%. Furthermore, based on morphological analyses, we found characteristics of Chinese specimens, number of pericentral cells, percurrent axis, determinate branchlets, branching pattern, spermatangial axes development, and arrangement of tetrasporangia, etc., were consistent with those of Bryocladia. Combined with morphological and molecular analyses, P. hainanensis was taken a synonymous of B. thyrsigera, and gametophytes of this species were found from China for the first time.     

Taxonomic Clarification of A Well-Known Pathogenic Scuticociliate,Miamiensis avidus Thompson & Moewus,1964(Ciliophora,Scuticociliatia)

Miamiensis avidus Thompson Moewus,1964,is a cosmopolitan and well-known marine pathogenic ciliated protist.However,the taxonomy of this species up to now has remained controversial,especially with respect to the validity of the morphologically similar species,Philasterides dicentrarchi,which was considered as a junior synonym of M.avidus.In this study,a population of M.avidus was collected from the skin of pharaoh cuttlefish(Sepia pharaonis) cultured near the East China Sea,Ningbo,China and its morphology and phylogeny were investigated in detail based on living characters,infraciliature,small subunit(SSU) r DNA and ITS1-5.8 S-ITS2 region sequences.In addition,the morphometrics of a previously reported free-living population,collected from the Bohai Sea,were rechecked and analyzed.We compared the present two isolates with all historic populations of M.avidus and P.dicentrarchi,and found that their morphological characters were either highly similar or exactly identical,indicating that they are the same morphospecies.However,the phylogenetic analyses based on SSU r DNA or ITS1-5.8 S-ITS2 region sequences revealed that most M.avidus and P.dicentrarchi populations formed one clade,and the two isolates of M.avidus from Weifang and American Type Culture Collection clustered in another clade,which indicated that there might be cryptic species in Miamiensis avidus.     

Monophyly or polyphyly? Possible conflict between morphological and molecular interpretations of the wellknown genus Zoothamnium(Ciliophora,Peritrichia)

In this paper,we explore possible conflict between morphological and molecular interpretations of phylogenetic relationships within the well-known peritrichous genus Zoothamnium. On the basis of morphological evidence,for a long time this genus has been believed to be a well-defined monophyletic taxon. Nonetheless,Z oothamnium exhibits higher genetic diversity than the gross morphology of its species. Here,we used all available genetic information for the small subunit of ribosomal RNA(SSU r RNA) and internal transcribed spacer region(ITS1-5.8S-ITS2) for this genus to reconstruct phylogenies for four datasets(SSU rRNA,ITS1,ITS2,and ITS1-5.8S-ITS2) and a combined dataset(SSU rRNA+ITS1-5.8SITS2) using different phylogenetic methods and with consideration of the secondary structure of the genes. Confidence in phylogenetic tree selection was assessed with the approximately unbiased test. The molecular results showed both that Zoothamnium is more likely to be polyphyletic,and morphologically similar genera Zoothamnopsis and Myoschiston were always nested among Zoothamnium species. Accordingly,as with some other groups of ciliates,to understand more fully the correct phylogeny of Zoothamnium there remains a need for additional data from both morphological and molecular studies,covering additional Zoothamnium spp. and members of closely related genera(e.g. Zoothamnopsis,Myoschiston,and Epistylis).     

A preliminary phylogenetic analysis of Luidia (Paxillosida: Luidiidae) from Chinese waters with cytochrome oxidase subunit I (COI) sequences

Luidia Forbes (Paxillosida: Luidiidae) are common soft bottom sea stars with 49 described species. Because of substantial morphological diversity, the taxonomy of the genus is complex and hasn’t been resolved definitely. In order to resolve general taxonomic issues, and determine species boundaries and phylogenetic relationships within the genus Luidia, the sequences of cytochrome oxidase subunit I (COI) gene from 24 specimens of Luidia, belonging to eight taxa in Chinese waters, were studied. Three sequences of two species in genus Luidia from GenBank were used to analyze the phylogenetic relationships. The molecular phylogeny exhibited three main clades, each with strong bootstrap support: Clade A including Luidia quinaria from the Sea of Japan; Clade B including seven nominal species (L. quinaria von Martens, L. yesoensis Goto, L. changi Liu, Liao and Li, L. orientalis Fisher, L. avicularia Fisher, L. longispina Sladen and L. hardwicki Gray) from Chinese waters; and Clade C including L. maculata Müller & Troschel from Chinese waters. Our molecular phylogeny results support the morphological Quinaria-Group and Alternata-Group assigned by Döderlein. Seven nominal species we sampled do not exhibit genetic distances that are large enough to recognize them as separate species. Cryptic species may exist in’ Luidia quinaria’ from the Yellow Sea and the Sea of Japan. Meaningful morphological characters need further investigation in Luidia.     

Relationships between two Pseudoalteromonas strains isolated from the Canada Basin and the Southern Ocean using a polyphasic approach

The evolutionary relationships and taxonomic position of two marine planktonic bacterial strains BSw20211 and BSw10014, isolated from the Canada Basin and from the Southern Ocean, respectively, were determined using a polyphasic taxonomic approach. There was a close phylogenetic relationship between the two strains and most phenotypic properties were shared. Nonetheless, they were found to belong to different species of the genus Pseudoalteromonas on the basis of genotypic analyses. Findings were consistent with the suggestion that gyrB gene sequence comparison and DNA-DNA relatedness might better define phylogenetic relationships of bacteria at the species level. However, a cut-off value of 90% gyrB gene sequence similarity was not reliable for the differentiation of species within the genus Pseudoalteromonas.     

Molecular phylogeny of oligotrich genera Omegastrombidium and Novistrombidium (Protozoa, Ciliophora) for the systematical relationships within Family Strombidiidae

The phylogeny of the oligotrich ciliates is currently a hot debate despite the availability of both morphological and molecular data. In the present paper, further small subunit rRNA (SS rRNA) genes were analyzed from the Genera Omegastrombidium and Novistrombidium, as well as from Strombidium, and combined with three new SS rRNA sequences from Strombidium basimorphum, S. sulcatum population QD-1, and Novistrombidium testaceum population GD. The phylogenetic positions of these organisms were inferred using Bayesian inference, Maximum Likelihood, and Maximum Parsimony methods. The main results are: (1) the SS rRNA gene sequence analyses match the recent findings about the molecular evolution of oligotrichs, indicating that the family Strombidiidae is paraphyletic; (2) the Genus Omegastrombidium is separated from the Genus Strombidium, as shown in recent cladistic analyses; (3) morphospecies in Genus Novistrombidium, based on similarity of somatic ciliature, are separated from each other in all topological trees, indicating that this genus could be a paraphyletic group; (4) the molecular data indicate a possibility of paraphyly for the genus Strombidium; and (5) the similarities of the SS rRNA gene of specimens identified as S. sulcatum and S. inclinatum are 99.8%–100%. However, present knowledge on the oligotrichs sensu stricto is still insufficient and further studies based on both molecular and other technologies are required.     

Integrating a DNA barcoding project with an ecological survey: a case study on temperate intertidal polychaete communities in Qingdao, China

In this study, we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes. Using 16S rDNA as a complementary marker and combining morphological and ecological characterization, some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status. We obtained 22 haplotype gene sequences of 13 taxa, including 10 CO1 sequences and 12 16S rDNA sequences. Based on intra- and inter-specific distances, we built phylogenetic trees using the neighbor-joining method. Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes, but other genes, such as 16S rDNA, could be used as a complementary genetic marker. For more accurate species identification and effective testing of species hypothesis, DNA barcoding should be incorporated with morphological, ecological, biogeographical, and phylogenetic information. The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.     

Molecular Phylogenetic Lineage of Plagiopogon and Askenasia (Protozoa,Ciliophora) Revealed by Their Gene Sequences

Prostomates and haptorians are two basal groups of ciliates with limited morphological characteristics available for taxonomy. Morphologically, the structures used to identify prostomates and haptorians are similar or even identical, which generate heavy taxonomic and phylogenetic confusion. In present work, phylogenetic positions lineage of two rare genera, Plagiopogon and Askenasia, were investigated. Three genes including small subunit ribosomal RNA gene(hereafter SSU r DNA), internal transcribed spacer region(ITS region), and large subunit ribosomal RNA gene(LSU r DNA) were analyzed, 10 new sequences five species each. Our findings included 1) class Prostomatea and order Haptorida are multiphyletic; 2) it may not be appropriate to place order Cyclotrichiida in subclass Haptoria, and the systematic lineage of order Cyclotrichiida needs to be verified further; 3) genus Plagiopogon branches consistently within a clade covering most prostomes and is basal of clade Colepidae, implying its close lineage to Prostomatea; and 4) Askenasia is phylogenetically distant from the subclass Haptoria but close to classes Prostomatea, Plagiopylea and Oligohymenophorea. We supposed that the toxicyst of Askenasia may be close to taxa of prostomes instead of haptorians, and the dorsal brush is a more typical morphological characteristics of haptorians than toxicysts.     

Sequences and phylogeny analysis of <Emphasis Type="Italic">rbcL</Emphasis> gene in marine chlorophyta

The rbcL gene of Ulva pertusa,Enteromorpha prolifera and Monostroma grevillei was amplified,sequenced and analyzed.By comparing the rbcL sequences with seven other Ulvales species retrieved from GenBank,the sequence divergences and the phyletic evolution were analyzed and the phylogenetic tree was constructed.From the phylogenetic tree,it can be found that U.pertusa,E.prolifera and U.californica group in one branch,while E.compressa,U.rigida and U.fenestrata cluster in another clade.Obviously,unlike the Enteomorpha species,the Ulva species do not gather in one branch.Therefore Ulva and Enteomorpha might be affiliates of one genus.E.compressa and E.intestinalis gathered together,which coincided with the morphological characters.However,the thallus of U.pertusa is thick and with many holes,which is different from E.prolifera in morphology.They cluster together in the phylogenetic tree with a genetic distance of 0.005.The results indicate that Ulva and Enteromorpha are not distinguished strictly.     

Are Acila divaricata and Acila mirabilis One Species or Two Distinct Species?Evidence from COI Mitochondrial DNA

Acila divaricata (Hinds, 1843) and A. mirabilis (Adams and Reeve, 1850) are common benthic bivalves in China. A number of researchers have proposed that the latter species is a junior synonym of the former species. Because of morphological similarities, it is difficult to distinguish these two species based on visual examination only. For better understanding of their taxonomy, the mitochondrial COI gene fragments of five individuals of A. divaricata from the East China Sea and six individuals of A. mirabilis from the Yellow Sea were sequenced in this study. The phylogenetic relationships of the obtained COI sequences, together with nineteen sequences of three species of the genus Nucula, were analyzed. The pairwise intra- and inter-specific distances for the COI sequences ranged from 0.002 to 0.017 and from 0.128 to 0.134, respectively, and no overlap was found. Phylogenetically, A. divaricata and A. mirabilis form distinct clades and cluster into a sister to all other Nucula species. The results indicated that A. divaricata and A. mirabilis are two distinct species. The differences in the morphology and distribution between the two species were briefly discussed.     

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of B angia(Bangiales,Rhodophyta) from China

Filamentous Bangia,which are distributed extensively throughout the world,have simple and similar morphological characteristics.Scientists can classify these organisms using molecular markers in combination with morphology.We successfully sequenced the complete nuclear ribosomal DNA.approximately 13 kb in length,from a marine Bangia population.We further analyzed the small subunit ribosomal DNA gene(nrSSU) and the internal transcribed spacer(ITS) sequence regions along with nine other marine,and two freshwater Bangia samples from China.Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences(0-0.003;0-0.006,respectively) from each other,but high divergences(0.123-0.126;0.198,respectively) from freshwater samples.An exception is the marine sample collected from Weihai,which shows high divergence from both other marine samples(0.063-0.065;0.129,respectively) and the freshwater samples(0.097;0.120,respectively).A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades,with the two marine sample clades containing Bangia spp.from North America,Europe,Asia,and Australia;and one freshwater clade,containing Bangia atropurpurea from North America and China.     

Establishment of Alloptilella splendida gen.et sp.nov.and resurrection of Scytalium veneris(Thomson & Henderson,1906),two sea pens(Cnidaria:Pennatulacea) from seamounts in the tropical Western Pacific

Sea pens are a highly specialized and morphologically distinct group of octocorallians,but many taxa were poorly described and their phylogenetic relationships are still poorly known.We describe two species of sea pens collected from the tropical Western Pacific:Alloptilella splendida gen.et sp.nov.and Scytalium veneris(Thomson Henderson,1906).Alloptilella splendida gen.et sp.nov.accords with the definition of the recently resurrected genus Ptilella Gray,1870 in most characters,but differs from the latter in the arrangement of autozooids and the location of mesozooids.The new species mostly resembles Pennatula naresi K(o|")lliker(1880),but differs by the location of me sozooids and the reddish color of sclerite s surrounding siphonozooids.The generic separation is also well supported by their genetic distances and the molecular phylogenetic trees constructed by the concatenated mtMutS-COI-28 S,where Alloptilella clustered with Scytalium Herklots,1858 and formed a clade with the cluster of Ptilella and Distichoptilum.Scytalium veneris,usually considered as a junior synonym of Scytalium sarsii Herklots,1858,is resurrected by recognizing their distinctive differences.This is the first record of Scytalium veneris outside its type locality,and the phylogenetic analysis indicates that Scytalium is a monophyletic group.Both the families Pennatulidae and Virgulariidae are polyphyletic,and more morphological and molecular data are needed to clarify the phylogenetic positions of pennatulacean families and genera.     

Polyphasic examination on Merismopedia tenuissima CHAB 7021 from Ganjiang River,China revealed the polyphyly of the genus Merismopedia(Cyanobacteria)

Species in the cyanobacterial genus Merismopedia are present in freshwaters at different trophic levels, with some species even as the components of cyanobacterial blooms. However, species diversity in this genus was not fully verified by molecular investigation and polyphasic taxonomic studies. In this study, Merismopedia-like strain tenuissima CHAB 7021 was isolated from Ganjiang River in Jiangxi Province, China, and polyphasic characterization of this strain was performed by morphological observation, ultrastructural examination, chemical detection of pigments and phylogenetic analysis based on 16 S rRNA gene sequences. Morphological identification of the strain was supported by the ultrastructural features, as the tiny species Merismopedia tenuissima Lemmermann. The phylogeny based on 16 S rRNA gene sequences revealed at least three clades formed by the strains of Merismopedia. The three M. tenuissima strains including M. tenuissima CHAB 7021 was gathered in clade III with distant relationship to the clade I formed by the six Merismopedia strains including the type species M. punctata, and such a genetic distance may propose Merismopedia tenuissima to separate from Merismopedia genus. However intermixture relationship in between strains of M. punctate and M. glauca in the phylogenetic tree still complicated the taxonomic status in the genus Merismopedia. The process for taxonomic revision in the Merismopedia genus still await for examination and further information on more strains of type species M. punctata.     

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of <Emphasis Type="Italic">Bangia</Emphasis> (Bangiales,Rhodophyta) from China

Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0–0.006, respectively) from each other, but high divergences (0.123–0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063–0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.