Morphology and phylogeny of Prorocentrum caipirignum sp. nov. (Dinophyceae), a new tropical toxic benthic dinoflagellate

A new species of toxic benthic dinoflagellate is described based on laboratory cultures isolated from two locations from Brazil, Rio de Janeiro and Bahia. The morphology was studied with SEM and LM. Cells are elliptical in right thecal view and flat. They are 37–44 μm long and 29–36 μm wide. The right thecal plate has a V shaped indentation where six platelets can be identified. The thecal surface of both thecal plates is smooth and has round or kidney shaped and uniformly distributed pores except in the central area of the cell, and a line of marginal pores. Some cells present an elongated depression on the central area of the apical part of the right thecal plate. Prorocentrum caipirignum is similar to Prorocentrum lima in its morphology, but can be differentiated by the general cell shape, being elliptical while P. lima is ovoid. In the phylogenetic trees based on ITS and LSU rDNA sequences, the P. caipirignum clade appears close to the clades of P. lima and Prorocentrum hoffmannianum. The Brazilian strains of P. caipirignum formed a clade with strains from Cuba, Hainan Island and Malaysia and it is therefore likely that this new species has a broad tropical distribution. Prorocentrum caipirignum is a toxic species that produces okadaic acid and the fast acting toxin prorocentrolide.     

Studies on Woloszynskioid Dinoflagellates X: Ultrastructure,Phylogeny and Colour Variation in Tovellia rubescens n. sp. (Dinophyceae)

The external morphology and internal cell fine structure of a new species of Tovelliaceae, Tovellia rubescens n. sp., is described. Phylogenetic analyses based on partial LSU rDNA sequences place the new species in a clade containing Tovellia species that accumulate red pigments and identify T. aveirensis as its closest known relative. Cells of T. rubescens n. sp. were mostly round and had the cingulum located near the middle, with its ends displaced about one cingular width. Small numbers of distinctly flat cells appeared in culture batches; their significance could not be determined. Cells of the new species in culture batches progressively changed from a yellowish‐green, mainly due to chloroplast colour, to a reddish‐brown colour that appeared associated with lipid bodies. The switch to a reddish colour happened earlier in batches grown in medium lacking sources of N or P. Pigment analyses by HPLC‐MS/MS revealed the presence of astaxanthin and astaxanthin‐related metabolites in the new species, but also in T. aveirensis, in which a reddish colour was never observed. The chloroplast arrangement of T. rubescens n. sp. resembled that of T. aveirensis, with lobes radiating from a central pyrenoid complex. The flagellar apparatus and pusular system fell within the general features described from other Tovelliaceae. A row of microtubules interpretable as a microtubular strand of the peduncle was present. Spiny resting cysts with red contents and an ITS sequence identical to that of cultured material of the new species were found in the original locality.     

Characterization of Prorocentrum elegans and Prorocentrum levis (Dinophyceae) from the southeastern Bay of Biscay by morphology and molecular phylogeny

Benthic Prorocentrum species can produce toxins that adversely affect animals and human health. They are known to co‐occur with other bloom‐forming, potentially toxic, benthic dinoflagellates of the genera Ostreopsis, Coolia, and Gambierdiscus. In this study, we report on the presence of P. elegans M.Faust and P. levis M.A.Faust, Kibler, Vandersea, P.A. Tester & Litaker from the southeastern Bay of Biscay. Sampling was carried out in the Summer‐Autumn 2010–2012 along the Atlantic coast of the Iberian Peninsula, but these two species were only found in the northeastern part of the Peninsula. Strains were isolated from macroalgae collected from rocky‐shore areas bordering accessible beaches. Morphological traits of isolated strains were analyzed by LM and SEM, whereas molecular analyses were performed using the LSU and internal transcribed spacer (ITS)1‐5.8S‐ITS2 regions of the rDNA. A bioassay with Artemia fransciscana and liquid chromatography–high‐resolution mass spectrometry analyses were used to check the toxicity of the species, whose results were negative. The strains mostly corresponded to their species original morphological characterization, which is supported by the phylogenetic analyses in the case of P. levis, whereas for P. elegans, this is the first known molecular characterization. This is also the second known report of P. elegans.     

Morphology and molecular phylogeny of the benthic dinoflagellates (Dinophyceae,Peridiniales) Amphidiniopsis crumena n. sp. and Amphidiniopsis nileribanjensis n. sp.

Amphidiniopsis is a benthic, heterotrophic and thecate dinoflagellate genus that has a smaller epitheca and larger hypotheca. The genus contains 24 described species, but is considered to be polyphyletic based on morphological characters and molecular phylogenetics. In this study, two new species were discovered from two distant sampling localities, Amphidiniopsis crumena sp. nov. from Japan, and Amphidiniopsis nileribanjensis sp. nov., from Australia. These species have a uniquely shaped, additional second postcingular plate. Both species are dorsoventrally flattened, an apical hook is present, and have six postcingular plates. The plate formula is: APC 4′ 3a 7″ ?C 4?S 6″′ 2″″. The cells of these species were examined with LM and SEM, and molecular phylogenic analyses were performed using 18S and 28S rDNA. These species are distinguished by the presence of spines on the hypotheca and touching of the sixth postcingular plate and the anterior sulcal plate. Their shape and disposition of several thecal plates also differ. Molecular phylogenetic analyses showed that the two new species formed a monophyletic clade and did not belong to any morphogroup proposed by previous studies. Considering the morphological features and the molecular phylogenetic results, a new morphogroup is proposed, Amphidiniopsis morphogroup VI (‘crumena group’).     

Fulvifomes hainanensis sp. nov. and F. indicus comb. nov. (Hymenochaetales,Basidiomycota) evidenced by a combination of morphology and phylogeny

Fulvifomes hainanensis sp. nov. is described from tropical China. The new species resembles Fulvifomes rimosus, but differs by uncracked pileal surface, the presence of a black cuticle between tomentum and lower context, and ellipsoid basidiospores. In nuclear large subunit rDNA (nLSU) and internal transcribed spacer (ITS) based phylogenies, F. hainanensis formed a distinct lineage from other sequenced taxa in the Fulvifomes clade. Aurificaria indica, the type of Aurificaria, has typical morphological characters for Fulvifomes, such as lack of setae and production of colored basidiospores. It was transferred to Fulvifomes based on additional evidence from molecular phylogeny, and F. indicus was redescribed according to Chinese collections. Aurificaria, posterior to Fulvifomes, was thus treated as a taxonomic synonym of Fulvifomes, and the concept of Fulvifomes was accordingly emended to include Aurificaria species. Inonotus luteoumbrinus and I. porrectus are similar to F. indicus in morphology, and they were also supported as lineages in the Fulvifomes clade. However, taxonomical adjustments for the two species were not proposed as the specimens yielding their nLSU and ITS sequences were not reexamined in morphology in this study.     

Molecular phylogeny,morphology and toxigenicity of Ostreopsis cf. siamensis (Dinophyceae) from temperate south‐east Australia

Ostreopsis is a genus of dinoflagellates that includes species producing palytoxin and structurally related compounds. The distribution of Ostreopsis species in Australia is largely unknown, but they have been reported from north Queensland (18^° S) to Tasmania (41–43^° S). Ostreopsis spp. have been recurrently reported from estuaries around New South Wales, with persistent occurrences in Merimbula Lake inlet throughout the year. We isolated and characterized a strain of Ostreopsis cf. siamensis using light and scanning electron microscopy as well as molecular sequences of small subunit (SSU), large subunit (LSU) and ITS regions of ribosomal DNA. The strain grew significantly faster in low nutrient concentrations. Palytoxin‐like compounds were produced by the strain, as determined by chemical analysis, and the LD ₅₀ of the cell extract by intraperitoneal injection in mice was 25.1 mg kg^?1. This is the first comprehensive molecular, morphological and toxicological study of an Ostreopsis species from Australian waters. Increasing reports of Ostreopsis from temperate waters suggest an empirical need to expand the knowledge of their diversity and distribution to aid aquaculture monitoring in Australian estuaries.     

Morphology, toxin composition and LSU rDNA phylogeny of Alexandrium minutum (Dinophyceae) from Denmark, with some morphological observations on other European strains

The morphology of Alexandrium minutum Halim from Denmark was studied and compared to the morphology of material from Portugal, Spain, France and Ireland. Strains from Denmark and the French coast of the English Channel differed from the typical minutum morphotype by the absence of a ventral pore. Cells without a pore also dominated field material from Ireland but a small fraction (6%) did have a pore. Many cells had a heavily areolated theca. In the exponential growth phase, the PSP-toxin profile of the Danish strain of A. minutum was dominated by C1 and C2 (up to 70%), whereas GTX2 and 3 made up more than 17%, and STX almost 13%. Cells entering the stationary phase contained 30% STX with a concomitant decrease of the other toxins. Partial large subunit rDNA sequences (664 bp) confirmed that the Danish A. minutum strain clusters together with other European strains of this species, and a strain from Australia. However, sequencing of this part of the gene did not resolve intraspecific relationships and could not differentiate populations with or without pore and/ or different toxin signatures. A strain from New Zealand had a remarkably high sequence divergence (up to 6%) compared to the other strains of A. minutum and its identity should be further investigated. A distribution map of A. minutum has been compiled and it is suggested that A. minutum and A. angustitabulatum Taylor are conspecific.     

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological,molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.     

Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China

A new species of entomopathogenic nematode, herein described as Steinernema akhursti sp. n., was recovered from soil samples collected from Yunnan Province, the People's Republic of China. Both morphological and molecular data show congruently that S. akhursti sp. n. belongs to the Steinernema feltiae group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For the first generation male, the new species can be recognized by spicule length 90 +/- 4.6 microm, spicule tip blunt with an aperture on the ventral side, gubernaculum with a long and needle-shaped cuneus, and tail conoid with a prominent mucron on the tip and a concave on ventral side. For the infective juvenile, the combination of the following characters: body length 812 +/- 19 microm, distance from anterior end to excretory pore 59 +/- 1.5 microm, tail length 73 +/- 2.9 microm, E% 77 +/- 4.5, lateral field with six evenly distributed and identical ridges at the middle body portion, and tail with long and slightly constrict hyaline portion can be used to separate the new species from other nematodes. For the female, the new species is characterized by: tail conoid with a short mucron and slightly swelling anal portion and a symmetrical, slightly protruding vulva with conspicuous double-flapped epiptygma. The nematode can be separated from other described species of Steinernema by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA and from the closely related species S. feltiae and Steinernema oregonense by cross-breeding tests.     

Morphology and molecular phylogeny of two new soil ciliates,Hemiurosomoida warreni nov. spec. and Hemiurosoma clampi nov. spec. (Ciliophora,Hypotrichia) from Tibet

Two novel hypotrichous ciliates, Hemiurosomoida warreni nov. spec. and Hemiurosoma clampi nov. spec., isolated from soil in the Lhalu Wetland and Motuo Virgin Forest in Tibet, respectively, were investigated using live observation and protargol staining. Hemiurosomoida warreni nov. spec. strongly resembles the type species H. longa but can be distinguished by its body size in vivo (110–145 × 30–40 μm vs. 50–100 × 18–40 μm), number of adoral membranelles (25–38 vs. 15–22), and numbers of right (29–39 vs. 14–23) and left (26–35 vs. 13–23) marginal cirri, transverse cirri (3 vs. 4 or 5) and macronuclear nodules (4–8 vs. 2). Hemiurosoma clampi nov. spec. is characterized by its vermiform body shape, colourless cortical granules distributed in irregular rows, two macronuclear nodules, three frontal cirri, one buccal cirrus, four frontoventral cirri ranged in a line, two transverse cirri, lacking postoral ventral and pretransverse ventral cirri, and marginal rows that are not posteriorly confluent. Phylogenetic analyses based on SSU rDNA gene sequences suggest that Hemiurosomoida is not monophyletic. A close relationship is revealed between Hemiurosomoida warreni nov. spec., Parakahilella macrostoma, Hemiurosoma clampi nov. spec., and the type species Hemiurosoma terricola. As expected, all these species are classified within the “non-oxytrichid Dorsomarginalia”.     

First record of Grammatodinium (Dinophyceae) for the American Eastern Pacific coast: Morphological,molecular and ecological confirmation

Grammatodinium Li & Shin is a monospecific genus described from the Tongyeong Bay area in Korea. In the current study, we describe its presence in the American Eastern Pacific coast for the first time, particularly in Acapulco Bay, Mexico, using morphological, molecular and environmental data. Sequences generated in this study with SSU and LSU formed a monophyletic group with other sequences from GenBank belonging to Gr. tongyeonginum, the only species known for the genus; however, genetic distance values between this species and our specimens (8.5% SSU; 2.8% LSU) were equivalent or even greater than those reported in other genera of dinoflagellates. Our phylogeny clearly showed the relationship of Grammatodinium with the families Pyrocystaceae and Gonyaulacaceae. In our specimens, cells appeared individually and in colonies of up to 16 cells, which were observed mainly during the dry season, so they could be confused with Gymnodinium catenatum, a common dinoflagellate in Acapulco with which they can coexist and share their general appearance, but they are clearly differentiated by the presence of longitudinal furrows throughout the body and a yellowish-green coloration, both absent in Gymnodinium catenatum. Although our evidence strongly suggests the presence of a new species for the region, more detailed morphological examinations are needed to confirm this statement.     

<Emphasis Type="Italic">Pythium apinafurcum</Emphasis>: its morphology,molecular phylogeny,and infectivity for plants

During a survey of Pythium species in soils of Japan, Pythium isolates growing at high temperatures were obtained from an uncultivated field soil in Wakayama Prefecture. All six isolates showed similar morphology to each other and had complexly branched secondary hyphae, globose nonproliferating sporangia, and smooth-surfaced oogonia that have one or two oospores per oogonium. The combination of these characteristics differentiated these isolates from other Pythium species reported. Phylogenetic analyses based on sequences of the ribosomal DNA ITS and D1/D2 region of the large subunit showed that all Pythium isolates were clustered in a single clade that was distantly related to other known clades of the genus. We described these isolates as a new Pythium species, Pythium apinafurcum, based on morphology and molecular phylogeny. The P. apinafurcum isolates nonsymptomatically infected the roots of seedlings of bermudagrass, cabbage, and cucumber in a pot inoculation test.     

Ultrastructure of Gyrodinium spirale, the type species of Gyrodinium (Dinophyceae), including a phylogeny of G. dominans, G. rubrum and G. spirale deduced from partial LSU rDNA sequences

A detailed ultrastructural analysis of the type species of Gyrodinium, G. spirale, was made based on cells collected from Skagerrak and southern Kattegat (Denmark). This material is considered very similar to the type material studied by Bergh from southern Kattegat. The analysis revealed many characters typical for dinoflagellates as well as a number of previously undescribed features. Here, emphasis was given to a three-dimensional configuration of the flagellar apparatus, the surface ridges, and the nuclear capsule. The latter had a rather complex ultrastructure consisting of two wall-like layers surrounded by membranes, with nuclear pores restricted to globular invaginations of these layers. To overcome difficulties with culturing of many auto- and heterotrophic dinoflagellates, we designed a specific reverse primer to amplify ca. 1800 base pairs of nuclear-encoded LSU rDNA. Using this approach, LSU rDNA sequences were determined from three heterotrophic species of Gyrodinium, including the type species. Using other alveolates (i.e. ciliates and Apicomplexa) as outgroup species, phylogenetic analyses based on Maximum Likelihood, Maximum Parsimony, and Neighbor-Joining supported Gyrodinium as a separate lineage. Unfortunately, the nearest sister group to Gyrodinium could not be established due to low bootstraps support for the deep branching pattern.     

Bacterial communities and toxin profiles of Ostreopsis (Dinophyceae) from the Pacific island of Okinawa,Japan

Variations in toxicity of the benthic dinoflagellate Ostreopsis Schmidt 1901 have been attributed to specific molecular clades, biogeography of isolated strains, and the associated bacterial community. Here, we attempted to better understand the biodiversity and the basic biology influencing toxin production of Ostreopsis. Nine clonal cultures were established from Okinawa, Japan, and identified using phylogenetic analysis of the ITS-5.8S rRNA and 28S rRNA genes. Morphological analysis suggests that the apical pore complex L/W ratio could be a feature for differentiating Ostreopsis sp. 2 from the O. ovata species complex. We analyzed the toxicity and bacterial communities using liquid chromatography-mass spectrometry, and PCR-free metagenomic sequencing. Ovatoxin was detected in three of the seven strains of O. cf. ovata extracts, highlighting intraspecies variation in toxin production. Additionally, two new potential analogs of ovatoxin-a and ostreocin-A were identified. Commonly associated bacteria clades of Ostreopsis were identified from the established cultures. While some of these bacteria groups may be common to Ostreopsis (Rhodobacterales, Flavobacteria-Sphingobacteria, and Enterobacterales), it was not clear from our analysis if any one or more of these plays a role in toxin biosynthesis. Further examination of biosynthetic pathways in metagenomic data and additional experiments isolating specific bacteria from Ostreopsis would aid these efforts.     

New record of Ascaridia nymphii (Secernentea: Ascaridiidae) from macaw parrot,Ara chloroptera,in China

Present study was performed to identify the species of ascarids from macaw parrot, Ara chloroptera, in China. Total 6 ascarids (3 males and 3 females) were collected in the feces of 3 macaws at Guangzhou Zoo in Guangdong Province, China. Their morphological characteristics with dimensions were observed under a light microscope, and their genetic characters were analyzed with the partial 18S rDNA, ITS rDNA and nad4 gene sequences, respectively. Results showed that all worms have no interlabia but male worms have two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and 11 pairs of papillae. The partial 18S rDNA, ITS rDNA and nad4 sequences were 831 bp, 1015 bp and 394 bp in length, respectively. They showed the highest similarity of 99.8% (18S rDNA) with Ascaridia nymphii, 93.8% identities (ITS rDNA) with A. columbae and 98.5% to 99.5% identities (nad4) with Ascaridia sp. from infected parrot. All Ascaridia nematodes from the macaws were clustered into one clade and formed monophyletic group of Ascaridia with A. columbae and A. galli in two phylogenetic trees. It is observed that the combining morphological and sequencing data from three loci, the present Ascaridia species was identified as Ascaridia nymphii, which is the first record of A. nymphii from macaw parrot in China.     

Cystotheca tjibodensis (Erysiphaceae,Ascomycota): rediscovery in Java after 90 years and first finding of anamorph

Cystotheca tjibodensis, formerly known as Lanomyces tjibodensis (Perisporiales), is a fungus found in 1920 in Indonesia. This species, hitherto only known from its type collection, is now regarded as belonging to the Erysiphales. However, molecular data are still required to verify the taxonomic affinity. In March 2011, we rediscovered this fungus at Cibodas Botanical Garden, Java. Detailed characterizations of this tropical powdery mildew are reported in this study based on morphological and molecular examinations. The anamorph of this species that was not found in the type specimen is also reported in this study.     

Morphology and molecular phylogeny of a new brackish pleurostomatid ciliate,Loxophyllum paludosum sp. n. (Ciliophora,Litostomatea, Haptoria), from a mangrove wetland in China

A new ciliate species of the genus Loxophyllum Dujardin, 1841, Loxophyllum paludosum sp. n., is described from a mangrove wetland near Daya Bay in Guangdong Province, southern China, based on morphological and molecular analyses. The new species can be distinguished from its congeners by a combination of the following characters: (1) 12–14 right kineties and 4–6 left kineties; (2) two macronuclear nodules and one micronucleus; (3) a single contractile vacuole located terminally; (4) extrusomes bar-shaped, evenly spaced along entire ventral margin, and clustered to form 5–7 warts along dorsal margin; and (5) presence of three ridges on the left side of cell. The new species is divergent from its congeners from 0.4% to 6.7% (5–104 nucleotide sites) based on the small subunit (SSU) rRNA gene sequence data. The validity of the new species is also supported by molecular phylogenetic trees inferred from SSU rRNA gene sequences.