PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

裳卷蛾变形孢虫SSU rRNA核心序列的克隆与系统发育分析

[目的]利用分子生物学手段探索裳卷蛾变形孢虫的遗传发育地位.[方法]微孢子虫的SSUrRNA序列是构建系统发育进化树的重要工具.试验通过T-A克隆法对裳卷蛾变形孢虫(Vairimorphaceraces)SSU rRNA核心序列进行了克隆,并采用近邻法构建了系统发育进化树.[结果]克隆得到了长为1228bp的核苷酸序列(GenBank EU267796).系统发育分析结果表明:裳卷蛾变形孢虫与分离于小菜蛾(Plutella xylostella)的Vairimorpha sp.Germany(GenBank AF124331)和Vairimorphaimperyecta(GenBank AJ131645)相似性最高,它们在系统发育进化树中与寄主为鳞翅目昆虫的Nosema属聚为一类,与纳卡变形孢虫(Vairimorpha necatrix)为代表的Vairimorpha属为相邻集.[结论]结合其生物学特征,裳卷蛾变形孢虫确实为Vairimorph a属的成员,但根据系统发育分析归入Nosematidae科可能更为合适.     

ORIGINS AND AFFINITIES OF THE FILAMENTOUS GREEN ALGAL ORDERS CHAETOPHORALES AND OEDOGONIALES BASED ON 18S rRNA GENE SEQUENCES

The order Chaetophorales includes filamentous green algae whose taxonomic relationships to other chlorophycean orders is uncertain. Chaetophoralean taxa include filamentous species which are both branched and unbranched. Ultrastructural studies of zoospores have revealed similar flagellar apparatuses in a number of genera, including Uronema, Stigeoclonium, and Fritschiella, suggesting a close phylogenetic relationship among these taxa. The order Oedogoniales represents a second group of branched and unbranched filamentous green algae whose relationships to other chlorophycean orders also has been unclear. A possible close relationship between the Chaetophorales and Oedogoniales has been suggested. Using DNA sequences from the small-subunit ribosomal RNA gene (SSU rRNA) of several members of each order, we have examined the monophyly of the Chaetophorales and Oedogoniales, as well as the nature of their relationship to other chlorophycean orders. Our results show that chaetophoralean and oedogonialean taxa form separate monophyletic groups. Results also suggest that the two orders are not closely related to each other.     

Molecular Phylogeny and Taxonomy of Two Novel Brackish Water Hypotrich Ciliates,with the Establishment of a New Genus,Antiokeronopsis gen. n. (Ciliophora,Hypotrichia)

Two novel brackish water urostyloid ciliates, Anteholosticha paramanca sp. n. and Antiokeronopsis flava gen. n., sp. n., isolated from the Shenzhen Mangrove Nature Protection Area on the coast of the South China Sea, were investigated using live observation and protargol impregnation techniques. Anteholosticha paramanca sp. n. is characterized by its spherical yellowish cortical granules arranged in lines, shortened midventral complex and three transverse cirri. Morphogenesis is similar to that in Anteholosticha manca. The new genus Antiokeronopsis is diagnosed by having a continuous adoral zone of membranelles, frontal cirri arranged in a bicorona, midventral complex composed of midventral pairs only, one marginal cirral row on each side, the presence of frontoterminal and transverse cirri, and the lack of buccal and caudal cirri. The type species A. flava sp. n. is characterized by its elongated body shape, brown to yellowish body color and two types of cortical granules. Small subunit ribosomal RNA gene sequence data justify the classification of both species. Phylogenetic analyses indicate that A. paramanca clusters with Bakuella subtropica within a clade that includes two other Anteholosticha species, while Antiokeronopsis groups within the core urostylids and is most closely related to the well‐known genera Pseudokeronopsis and Uroleptopsis.     

Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov

Sequences of the nuclear encoded small subunit (SSU) rRNA were determined for Pirsonia diadema, P. guinardiae, P. punctigerae, P. verrucosa, P. mucosa and three newly isolated strains 99-1, 99-2, 99-S. Based on phylogenetic analysis all Pirsonia strains, except P. mucosa, clustered together in one clade, most closely related to Hyphochytrium catenoides within the group of stramenopiles. However, P. mucosa was most closely related to Cercomonas sp. SIC 7235 and Heteromita globosa and belongs to the heterogenic group of Cercozoa. In addition to the SSU rDNA sequences, P. mucosa differs from the stramenopile Pirsonia species in some characteristics and was therefore redescribed in this paper as Pseudopirsonia mucosa. The three newly isolated strains 99-1, 99-2, and 99-S differed by 28 bp in their SSU rDNA sequences from their closest neighbour P. diadema and only 1 to 3 bp among themselves. These base differences and a host range similar to P. formosa were sufficient to assign them as new strains of P. formosa.     

PHYLOGENY AND SYSTEMATICS OF THE GENUS MONOMORPHINA (EUGLENACEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA1

Morphological studies of 16 strains belonging to the genus Monomorphina revealed a single, parietal, orbicular chloroplast in their cells. The chloroplast has a tendency to be perforated and disintegrates in aging populations and thus may appear to be many chloroplasts under the light microscope. A single chloroplast in the cells of Cryptoglena skujae is also parietally located and highly perforated. It never forms a globular and closed structure, but is open from the side of the furrow, resembling the letter C. We have verified the Monomorphina pyrum group (M. pyrum–like) on the basis of phylogenetic analysis of SSU rDNA and morphological data. The strain CCAC 0093 (misidentified as M. reeuwykiana) diverges first on the SSU rDNA phylogenetic tree. The rest of the M. pyrum–like strains form a tight cluster, subdivided into several smaller ones. Because morphological differences between the M. pyrum–like strains (including the strain CCAC 0093) do not conform to the tree topology, we suggest that they all (except the strain CCAC 0093) belong to M. pyrum. We designate a new species, M. pseudopyrum, for the strain CCAC 0093, solely on the basis of molecular characters. We also suggest that M. reeuwykiana and similar species should stay in Phacus and Lepocinclis unless detailed molecular and morphological studies show otherwise. Emended diagnoses of the genera Monomorphina and Cryptoglena and the species M. aenigmatica are also proposed, as well as the delimitation of an epitype for M. pyrum, the type species for the genus Monomorphina.     

Combining Molecular Data with Classical Morphology for Uncultured Phagotrophic Euglenids (Excavata): A Single‐Cell Approach

Phagotrophic euglenids are one of the most diverse and important forms of heterotrophic flagellates in sediment systems, and are key to understanding the evolution of photosynthetic euglenids and ‘primary osmotrophs’, yet relatively little is known about their biodiversity and phylogenetic relationships. A wealth of light microscopy‐based information is available, but little progress has been made in associating this with molecular sequence data. We established a protocol to obtain light microscopy data and molecular data from single euglenid cells isolated from environmental samples. Individual cells from freshwater and marine benthic samples were isolated and rinsed by micropipetting, documented using high‐resolution photomicroscopy, then subjected to single‐cell nested PCR using taxon‐specific primers in combination with universal eukaryotic primers, generating > 75% or full‐length SSU rDNA sequences. As a proof‐of‐principle eight individuals were characterised and subjected to phylogenetic analyses. Many of these cells were identified as Anisonema or Dinema, and grouped with existing sequences assigned to these taxa, and with a ‘Peranema sp.’ sequence that we could now clearly demonstrate was misidentified or misannotated. Another cell is Heteronema c.f. exaratum, the first ‘skidding heteronemid’ for which sequence data are available. This is not closely related to Heteronema scaphurum, and intriguingly, branches as the sister group to primary osmotrophs. A cell similar to Ploeotia vitrea (the type of this genus), shows no particular phylogenetic affinity to Ploeotia costata, the best studied Ploeotia species. Our experimental protocol provides a useful starting point for future analyses on euglenid biodiversity (including environmental sequence surveys), and their evolution and systematics.     

REASSESSMENT OF THE LITTLE‐KNOWN CRUSTOSE RED ALGAL GENUS POLYSTRATA (GIGARTINALES), BASED ON MORPHOLOGY AND SSU rDNA SEQUENCES1

The taxonomic distinctiveness of the crustose red algal genus Polystrata Heydrich (Peyssonneliaceae) is confirmed on the basis of morphological and molecular data. The vegetative and reproductive morphology of the type species Polystrata dura Heydrich is newly described. Polystrata thalli are thick multi‐layered crusts, each crust of which is composed of a mesothallus, a superior perithallus, and an inferior perithallus. P. dura is characterized by a poorly developed inferior perithallus consisting of single‐celled perithallial filaments and each layer of multi‐layered crusts being closely adherent to the parental layer. This Polystrata species is identical to Peyssonnelia species, the type genus of the Peyssonneliaceae in the morphology of sexual reproductive organs: a carpogonial branch and an auxiliary cell branch are formed laterally on respective nemathecial filaments; the gonimoblasts are developed from connecting filaments and auxiliary cells; the spermatangia are produced in male and female nemathecia; and the spermatangial filament produces a series of one to four paired spermatangia that form a whorl surrounding each central cell (the Peyssonnelia dubyi‐type development). Polystrata fosliei (Weber‐van Bosse) Denizot is clearly distinguished from P. dura by an inferior perithallus as well‐developed as the superior perithallus, and each layer of multi‐layered crusts being loosely adherent to the parental layer. In our small subunit rDNA trees of the Peyssonneliaceae, these Polystrata species formed a clade with low to medium supports, although the phylogenetic position of Polystrata was unresolved in this family. Therefore, the thallus structure of Polystrata may be regarded as an important taxonomic character at the genus rank.