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.     

The Complete Sequence of Mitochondrial COII Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit II (COII) gene of Penaeinae shrimp Fenneropenaeus chinen- sis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COI and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COII gene has a faster evolutionary rate than that of the COI gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

The Complete Sequence of Mitochondrial COⅡ Gene of Fenneropenaeus chinensis and Its Applicability as a Marker for Phylogenetic Analysis

The complete mitochondrial cytochrome oxidase subunit Ⅱ （COⅡ） gene of Penaeinae shrimp Fenneropenaeus chinensis was cloned and sequenced. The gene is 688 bp in length and codes for 229 amino acids. It shows 83.2%, 87.0% and 83.8% sequence similarity to Marsupenaeus Japonicus, Penaeus monodon and Farfantepenaeus notialis, respectively. The A＋T content of the whole gene and that at the third position of codons are 64.7% and 78.2%, respectively. The phylogenetic relationship between F. chinensis and three other species representing genera Farfanatepenaeus, Marsupenaeus and Penaeus was analyzed. Results showed that the genetic distances among the four taxa ranged from 0.144 0 to 0.200 5, exceeding those estimated with COⅠ and partial 16S rRNA gene sequences among Marsupenaeus, Litopenaeus and Melicertus, and being therefore larger than the value among subgenera. It has been suggested that the COⅡ gene has a faster evolutionary rate than that of the COⅠ gene and partial 16S rRNA gene and could be used for phylogenetic analysis at genus or species level. The results of the present study indicated that Farfantepenaeus, Fenneropenaeus, Marsupenaeus and Penaeus are at a higher phylogenetic level than subgenus, which supports the opinion of the elevation of phylogenetic status of the four subgenera to genus level.     

Molecular identification based on ITS sequences for Kappaphycus and Eucheuma cultivated in China

The systematic classification of the Eucheumatoideae is difficult because of their variable morphology and interpretation of reproductive structures. Kappaphycus and Eucheuma specimens cultivated on the Hainan and Fujian coast of China were introduced from Vietnam, the Philippines and Indonesia. Combined with morphological characteristics, all Kappaphycus and Eucheuma cultivated strains were identified by internal transcribed spacer (ITS) sequences. The phylogenetic tree was constructed using neighbor-joining and maximum likelihood methods. The results indicate that different ITS sequence lengths occurred in the different genera and species. An obvious difference in morphology could be found in the protuberance shape between Kappaphycus and Eucheuma. The protuberance in Eucheuma was thorn-like and in Kappaphycus was wartlike or papillate. Their ITS sequence lengths differed significantly in nucleotide variation rates up to 58.55%-63.90%. All nucleotide variations occurred in the ITS1 and ITS2 regions except for five nucleotide transversions in the 5.8S rDNA region. In addition, the difference was at the branches among congeneric species. Kappaphycus sp. had branches with small buds, while K. alvarezii did not have such a feature. The nucleotide variation rates varied from 7.02% to 7.48% among species; within the same species of the clades it was <1.20%. Eucheumatoideae algae cultivated in China consisted of three clades, K. alvarezii, Kappaphycus sp., and E. denticulatum. The results indicate that ITS sequence analysis was an effective way for identification of interspecies and intraspecies phylogenetic relationships and might provide a clue for molecular identification of algal Eucheumatoideae.     

Identification of Microplankton in South China Sea with Image-Matching Individual PCR

In this study, marine microplankton were identified by combining standard light microscopy with Sanger 18S rRNA gene sequencing. The image-matching individual PCR technique was applied to identify the image collectable unicellular microplankton to genera. Instead of pure strain culture and morphological identification, microplankton individual cells were isolated and fixed with glutaraldehyde, frozen and stored for months. Finally, they were imaged under a microscope and molecularly identified via phylogenetic analysis of their 18S ribosomal RNA gene(18S rDNA). Microplankton cells were collected at 30 locations in South China Sea, and were assigned to 21 known and 4 unidentified genera(2 uncultured fungi and 2 uncultured stramenopiles) with phylogenetic analysis in parallel to the morphological identification.     

Genotype and Phylogenetic Diversity of Symbiodinium ITS2 Sequences Within Clade C in Three Typical Coral Species from Luhuitou Fringing Reef of the South China Sea

Dinoflagellates in the genus Symbiodinium, including nine clades(A–I), mainly form mutualistic symbioses with corals. More than 100 Symbiodinium molecular types have been identified by the ITS2-based genotype method within any given clade, and specifically within Symbiodinium clade C. However, the genotype identification method using the ITS2 sequence is likely to lead to high diversity estimates due to the intra-genomic variations in the ITS2 space; thus, further validation is essential for a correct identification. In this study, the molecular diversity of Symbiodinium ITS2 sequences cloned from two stone corals, Acropora sp. SY-01 and Pocillopora sp. SY-05, and one soft coral, Sarcophyton sp. SY-07, living in the northern part of South China Sea(SCS), were analyzed and compared using the ITS2-based genotype identification method, coupled with ITS2-based secondary structural and phylogenetic analyses. As the result, 12 Symbiodinium ITS2 genotypes were identified, while only six and three Symbiodinium ITS2 genotypes were supported by ITS2-based secondary structural and phylogenetic analyses, respectively. In addition, no shared Symbiodinium ITS2 genotypes were observed among the three coral species, suggesting coral species-dependent Symbiodinium genotypes were within clade C. In summary, the present study provides a theoretical basis for validating the molecular diversity of Symbiodinium ITS2 genotypes in corals.     

A Preliminary Phylogenetic Analysis ofLuidia （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 substan- tial 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 tyro- chrome 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 phylogenetie relationships. The molecular phy- logeny 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. ehangi 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 Miiller ＆ Troschel from Chinese waters. Our molecular phylogeny results support the morphological Quinaria-Group and Alter- nata-Group assigned by D6derlein. Seven nominal species we sampled do not exhibit genetic distances that are large enough to rec- ognize them as separate species. Cryptic species may exist in ＇Luidia quinaria＇ from the Yellow Sea and the Sea of Japan. Meaning- ful morphological characters need further investigation in Luidia.     

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes （Pleuronectiformes）

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on genetic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the relationship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P olivaceus clustered with K.bicoloratus, but P cinnamomeus did not cluster with t3. olivaceus, which is worth further studying.     

The Appearance of Ulva laetevirens（Ulvophyceae,Chlorophyta） in the Northeast Coast of the United States of America

Introduced species may outcompete or hybridize with native species, resulting in the loss of native biodiversity or even alteration of ecosystem processes. In this study, we reported an alien distromatic Ulva species, which was found in an embayment（Holly Pond） connected with Long Island Sound, USA. The morphological and anatomical observations in combination with molecular data were used for its identification to species. Anatomy of collected specimens showed that the cell shape in rhizoidal and basal regions was round and the marginal teeth along the basal and median region were not found. These characteristics were primarily identical to the diagnostic characteristics of Ulva laetevirens Areschoug（Chlorophyta）. The plastid-encoding tufA and nucleus-encoding ITS1 were used for its molecular identification. Phylogenetic analysis for the tufA gene placed the specimens from Holly Pond in a well-supported clade along with published sequences of U. laetevirens identified early without any sequence divergence. In ITS tree, the sample also formed well-supported clades with the sequences of U. laetevirens with an estimated sequence divergence among the taxa in these clades as low as 1%. These findings confirmed the morpho-anatomical conclusion. Native to Australia, this species was reported in several countries along the Mediterranean coast after the late of 1990 s. This is the first time that U. laetevirens is found in the northeast coast of United States and the second record for Atlantic North America.     

DNA Barcoding Assessment of Green Macroalgae inCoastal Zone Around Qingdao,China

An assessment with assistance of DNA barcoding was conducted on green macroalgae in coastal zone around Qingdao,China, during the period of April-December, 2011. Three markers were applied in molecular discrimination, including the plastidelongation factor tufA gene, the internal transcribed spacer （ITS） region of the ribosomal cistron and rubisco large subunit gene 3＇regions （rbcL-3P）. DNA barcoding discriminated 8 species, excluding species of genus Cladophora and Bryopsis due to failures inamplification. We ascertained and corrected 4 species identified by morphological methods for effectively assisting the classification.The gene tufA presented more advantages as an appropriate DNA marker with the strongest amplification success rate and speciesdiscrimination power than the other two genes. The poorest sequencing success largely handicapped the application of ITS. Samplesidentified by tufA and rbcL as Ulvaflexuosa were clustered into the clade of U. prolifera by ITS in the neighbor-joining tree. Confu-sion with discrimination of the complex of U. linza, U. procera and U. prolifera （as the LPP complex） still existed for the three DNAmarkers. Based on our results, rbcL is recommended as a preferred marker for assisting tufA to discriminate green macroalgae. Indistinguishing green-tide-forming Ulva species, the free-floating sample collected from the green tide in 2011 was proved to be iden-tical with U. prolifera in Yellow Sea for ITS and rbcL genes. This study presents a preliminary survey of green macroalgae distrib-uted in the coastal area around Qingdao, and proves that DNA barcoding is a powerful tool for taxonomy of green macroalgae.     

Virescentia guangxiensis(Batrachospermales,Rhodophyta):a new freshwater red algal species from South China

Virescentia guangxiensis,a new species of Virescentia from Guangxi,South China,is described and illustrated based on morphological observations and phylogenetic analysis.This species was distinguished morphologically from other species by the presence of special expansion cells with a variable shape,obovoid,spherical,pear-shaped,located in the penultimate cells of primary or secondary fascicles,rarely terminal on primary fascicles,as well as by small whorls(250-350-μm wide) and short primary fascicles(5-7 cell stories).Phylogenetic analysis of molecular data from the rbcL and COI-5 P loci supported the separation of the proposed new species from other species in the genus Virescentia.This is the first species of the order Batrachospermales reported in Guangxi and the second species of the genus Virescentia reported in China.This study expands the known species diversity and geographical distribution of fre shwater Rhodophyta in China.     

AreAcila 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 taxon-omy, the mitochondrial CO1 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 CO1 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. di-varicata and A. mirabilis are two distinct species. The differences in the morphology and distribution between the two species were briefly discussed.     

Muelleria pseudogibbula,a new species from a newly recorded genus (Bacillariophyceae) in China

The genus Muelleria is reported for the first time from China in this paper. The new diatom species M uelleria pseudogibbula sp. nov. was observed during a survey of diatoms from Zoigê Wetland, Sichuan Province, China. The morphological characters of this new species were documented by light and scanning electron microscope and compared with similar species. M. pseudogibbula is characterized by its lanceolate to linear-lanceolate valve outline with rounded apices, two longitudinal canals running parallel to each side of the raphe, proximal ends of the raphe deflected to the same directions and finishing before the first row of areolae, and curved distal raphe ends, forming two divergent branches.     

A new record of Sebastes koreanus from China based on morphological characters and DNA barcoding

A new record of Sebastes koreanus(Kim and Lee,1994) was documented based on morphological characters and DNA barcoding. Fifty-six S ebastes specimens were collected from the coastal waters of northern China. Samples were identified as S. koreanus based on morphological characters. The coloration and morphometric measurements were consistent with those described from specimens collected in South Korea. In this study,specimens had the following morphological characteristics:light brown body with dark stripes and tiny dark spots,4–5 wide indistinct vertical patterns on the side,2 radial stripes behind and below the eyes,1 large dark blotch on the opercle. Additionally,the following meristic characters were recorded:dorsal fin XIV-13,pectoral fin 16,anal fin III-6–7,pelvic fin I-5,lateral line scales 29–30,and vertebrae 26. The fragment of cytochrome oxidase subunit I(C OI) gene of mitochondrial DNA was sequenced for phylogenetic analysis. The mean genetic distance within the species was 0.3%. Net genetic distances between S. koreanus and other S ebastes species ranged from 3.1% to 7.6%,which was greater than the threshold for species delimitation. The phylogenetic analysis strongly supports the validity of S. koreanus in China at the genetic level. The origion,evolution,patterns of speciation and unique features in genome divergence among primate lineages of this species still need future directions of research.     

DNA barcode assessment of Ceramiales (Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales(Rhodophyta) were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species,14 genera,and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers,namely,partial large subunit r RNA gene(LSU),universal plastid amplicon(UPA),and partial mitochondrial cytochrome c oxidase subunit I gene(COI). No intraspecific divergence was found in our collection for these markers,except for a 1–3 bp divergence in the COI of Ceramium kondoi,Symphyocladia latiuscula,and Neosiphonia japonica. Because short DNA markers were used,the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in Gen Bank or BOLD(Barcode of Life Data) Systems. Three specimens were presumed as H eterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification,which was the first record in the investigated area,implying that it might be a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers,H eterosiphonia japonica converged with D asya spp. and was distant from the other Heterosiphonia spp.,implying that H. japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species,but the COI had a higher ability to discriminate between species.     

Sequence and phylogenetic analyses of the chloroplast 16S rRNA, tufA, and rbcL genes from Bryopsis hypnoides

Using shotgun sequencing data, the complete sequences of chloroplast 16S rRNA and tufA genes were acquired from native specimens of Bryopsis hypnoides (Qingdao, China). There are two group I introns in the 16S rRNA gene, which is structurally similar to that of Caulerpa sertularioides (Bryopsidales, Chlorophyta). The chloroplast-encoded tufA gene sequence is 1 230 bp long, very AT-rich (61.5%), and is similar to previously published 16S rRNA sequences of bryopsidinean algae. Phylogenetic analyses based on chloroplast 16S rRNA and tufA gene sequence data support previous hypotheses that the Bryopsidineae, Halimedineae, and Ostreobidineae are three distinct lineages. These results also confirmed the exclusion of Avrainvillea from the family Udoteaceae. Phylogenetic analyses inferred that the genus Bryopsis as sister to Derbesia; however, this clade lacked robust nodal support. Moreover, the phylogenetic tree inferred from rbcL GenBank sequences, combined with the geographical distributions of Bryopsis species, identified a strongly supportive clade for three differently distributed Asian Bryopsis species. The preliminary results suggesting that these organisms are of distinct regional endemism.