Little is known about the diversity and biogeography of the toxigenic dinoflagellate genera Karlodinium and Karenia because their cells are unarmoured and often small. We isolated single cells from ...Chinese waters to establish five Karlodinium cultures, and carried out single-cell polymerase chain reactions of eight cells of Karlodinium and Karenia isolated directly from field samples. We also obtained large-subunit (LSU) ribosomal DNA and internal transcribed spacer (ITS) sequences from the same samples used for descriptions of Karenia digitata and Karenia longicanalis. Five cultures of Karlodinium from the South China Sea and Yellow Sea of China were established and subjected to light, scanning electron, and transmission electron microscopy. These strains had a sulcal intrusion, a short apical groove, and six chloroplasts, each with an internal lenticular pyrenoid, but lacked a ventral pore. Pigment analysis showed that fucoxanthin was the main accessory pigment. On the basis of unique morphology and corroboration by molecular phylogeny, these strains were described as Karlodinium zhouanum sp. nov. LSU or ITS sequences (or both) of another eight cells revealed the presence of Karlodinium australe, Karenia longicanalis, and K. papilionacea in the East China Sea for the first time. LSU sequences showed that Karenia digitata differed from Karlodinium decipiens at only one position (99.86% similarity), and Karenia longicanalis differed from K. umbella at only four positions (99.45% similarity). Maximum-likelihood and Bayesian inference analyses supported the classification of Karenia digitata within Karlodinium, and demonstrated that presence of a ventral pore was not a reliable generic character for Karlodinium. Our results also indicated that LSU sequences might be too conservative to differentiate species of Karenia and Karlodinium.
Many Artificial Reefs (ARs) have been used worldwide for marine habitat and coral reef restoration. However, the microbial community structure that colonize the ARs and their progressive development ...have been seldom investigated. In this study, the successive development of the microbial communities on environmentally friendly Artificial Biological Reef structures (ABRs)
made of special concrete supported with bioactive materials collected from marine algal sources were studied. Three seasons (spring, summer and autumn), three coral reef localities and control models (SCE) without bioactive material and (NCE) made of normal cement were compared. The structure of the microbial pattern exhibited successive shifts from the natural environment to the ABRs supported with bioactive materials (ABAM). Cyanobacteria, Proteobacteria, and Planctomycetota were shown to be the most three dominant phyla. Their relative abundances pointedly increased on ABAM and SCE models compared to the environment. Amplicon Sequence Variant (ASV) Richness and Shannon index were obviously higher on ABAM models and showed significant positive relationship with that of macrobenthos than those on the controls and the natural reef (XR). Our results offer successful establishment of healthy microbial films on the ABR surfaces enhanced the restoration of macrobenthic community in the damaged coral reefs which better understands the ecological role of the ABRs.
► We reported two distinct ribotypes of Azadinium poporum in China Sea. ► The European ribotype is different from the new ones reported here. ► Azaspiracid profiles of Chinese A. poporum turned out ...to be diverse. ► A novel Azaspiracid with a molecular mass of 871Da was detected.
Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.
•First record of Azadinium poporum ribotype C from the Mediterranean Sea.•Azadinium poporum ribotype A from Greece produce low level or no AZA.•Azadinium poporum ribotype C from Greece produce ...predominantly AZA-40.
The marine dinoflagellate Azadinium poporum produce azaspiracids (AZA) and has been recorded widely in the world. However, information on its biogeography is still limited, especially in view of the fact that A. poporum comprises several genetically differentiated groups. A total of 18 strains of A. poporum were obtained from the Eastern Mediterranean area by incubating surface sediment collected from Ionian Sea of Greece. The morphology of these strains was examined with light microscopy and scanning electron microscopy. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from all cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences confirmed three ribotypes within A. poporum and revealed two subclades within ribotypes A and C. Greek strains of A. poporum ribotype A were nested within ribotype A2 together with strains from Western Mediterranean Sea and French Atlantic, and Greek strains of A. poporum ribotype C were nested within ribotype C2 together with a strain from the Gulf of Mexico. Growth experiments on four selected strains revealed that ribotypes A and C from Greece differed in their growth at higher temperatures, indicating that they are physiologically differentiated. Azaspiracid profiles were analyzed for 15 cultured A. poporum strains using LCMS/MS and demonstrate that the A. poporum ribotype A from Greece produce low level or no AZA and A. poporum ribotype C from Greece produces predominantly AZA-40 (9.6–30.2 fg cell−1) followed by AZA-2 (2.1–2.6 fg cell−1). The first record of AZA-40 producing A. poporum from the Mediterranean suggests that this species is a potential source for azaspiracid contaminations in shellfish from the Eastern Mediterranean Sea.
The genus Gymnodinium contains more than 230 extant species, approximately 30% of which have not been reported since their original description. Approximately eight Gymnodinium species have been ...reported or described in the coastal waters of China. This work reports the presence of Gymnodinium trapeziforme from Jiaozhou Bay, China, in 2020, and its morphological and phylogenetic characterization by using light and scanning electron microscopy and systematic analysis based on partial LSU rDNA sequences. We observed the typical diagnostic features of G. trapeziforme, including a small size, biconical to ovoid shape, and a sulcal extension intruded to the epicone and connected to the horseshoe-shaped apical structure complex (ASC). Additionally, we firstly observed that the ASC consisted of three parallel series of vesicles, with the central one possessing knobs, and having more than 10 amphiesmal vesicles within the ASC. The nucleus was cucurbit-shaped, and the amphiesmal vesicles covering the cell surface, which would be peeled off for the cells in stress. While our molecular phylogeny inferred with the maximum likelihood (ML) and Bayesian inference (BI) confirmed the conspecificity of our isolate with the holotype G. trapeziforme (accession No. EF192414), we found a difference of 14 bases in the D1–D6 domains of the LSU rDNA sequences between the two entities, which indicates a detectable speciation of the two populations. Our work provides a detailed morphological and molecular characterization of G. trapeziforme that was isolated from the coastal water of China, which also broadens the geographical distribution of this species.
•At least three ribotypes were identified within Protoceratium reticulatum.•One compensatory base change was present in ITS-2 secondary structure among ribotypes.•Ecophysiological and biogeographic ...differences were correlated with the ribotypes.•Yessotoxin (YTX) was produced by ribotypes A and B, but ribotype C produced homoYTX only.
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.
Around 30 epibenthic Prorocentrum species have been described, but information about their biogeography is limited. Some species are able to produce okadaic acid (OA) and its derivatives, which are ...responsible for diarrheic shellfish poisoning (DSP). In the present study, we examined the diversity of epibenthic Prorocentrum in the northern South China Sea by isolating single cells from sand, coral, and macroalgal samples collected from 2012 to 2015. Their morphology was examined using light microscopy and scanning electron microscopy. Among 47 Prorocentrum strains, seven morphospecies were identified as P. lima, P. rhathymum, P. concavum, P. cf. emarginatum, P. fukuyoi, P. cf. maculosum and P. panamense. The latter five species have not been previously reported in Chinese waters, and this is the first record of P. panamense outside its type locality. Partial large subunit (LSU) ribosomal DNA and internal transcribed spacer region sequences were obtained and molecular phylogenetic analysis was carried out using maximum likelihood and Bayesian inference. Chinese P. cf. maculosum strains share 99.5% similarity of LSU sequences with the strain from Cuba (close to the type locality), but Chinese P. lima strains share only 96.7% similarity of LSU sequences with the strain from the type locality. P. cf. emarginatum differs from P. fukuyoi mainly in the presence/absence of marginal pores and they form a well-resolved clade together with P. sculptile. OA was detected in all Chinese strains of P. lima and P. cf. maculosum based on liquid chromatography-mass spectrometry analysis, but dinophysistoxin was produced only by two P. lima strains. Chinese strains of P. concavum, P. rhathymum, and P. panamense do not produce detectable level of OA. Our results support the wide distribution of epibenthic Prorocentrum species and highlight the potential risk of DSP in the northern South China Sea.
•A new Gymnodinium species that produces microreticulate cysts was described.•Chinese Gymnodinium catenatum strains belong to the global population.•The profile of benzoate toxins was dominated by ...11-hydroxysulfate analogs.•A new ribotype of Gymnodinium microreticulatum was recovered.
Four Gymnodinium species have previously been reported to produce microreticulate cysts. Worldwide, Gymnodinium catenatum strains are conservative in terms of larger subunit (LSU) rDNA and internal transcribed spacer region (ITS) sequences, but only limited information on the molecular sequences of other species is available. In the present study, we explored the diversity of Gymnodinium by incubating microreticulate cysts collected from the Yellow Sea off China. A total of 18 strains of Gymnodinium, from three species, were established. Two of these were identified as Gymnodinium catenatum and Gymnodinium microreticulatum, and the third was described as a new species, Gymnodinium inusitatum. Motile cells of G. inusitatum are similar to those of Gymnodinium trapeziforme, but they only share 82.52% similarity in LSU sequences. Cysts of G. inusitatum are polygonal in shape, with its microreticulate wall composed of approximately 14 concave sections. G. microreticulatum strains differ from each other at 69 positions (88.00% similarity) in terms of ITS sequences, whereas all G. catenatum strains share identical ITS sequences and belonged to the global populations. Phylogenetic analyses, based on LSU sequences, revealed that Gymnodinium species that produce microreticulate cysts are monophyletic. Nevertheless, the genus as a whole appears to be polyphyletic. Paralytic shellfish toxins (PSTs) were found in all G. catenatum strains tested (dominated by 11-hydroxysulfate benzoate analogs and N-sulfocarmaboyl analogs) but not in any of the G. microreticulatum and G. inusitatum strains. Our results support the premise that cyst morphology is taxonomically informative and is a potential feature for subdividing the genus Gymnodinium.
The family Peridiniopsidaceae encompasses mainly freshwater species of the genera Peridiniopsis, Palatinus and Parvodinium. Only one benthic, marine species 'Scrippsiella' hexapraecingula has been ...attributed to this family. Here we established five strains by isolating single Parvodinium-like cells from the marine Gulf of Thailand, Hainan Island waters (China), off Manado (Indonesia) and from a freshwater reservoir in Fuzhou (China). All strains were examined with light, scanning and transmission electron microscopy, and their SSU, ITS-5.8S and partial LSU rRNA regions were sequenced. Four marine strains share a plate formula of Po, cp, X, 4′, 2a, 6′′, 6C, 4S, 5′′′, 2′′′′ and are herein attributed to a new genus Johsia as J. chumphonensis gen. et sp. nov. Its theca is characterized by an epitheca 1.5 times as long as the hypotheca in dorsal view and a 2a plate about half the size of 1a. A type B eyespot was observed in J. chumphonensis comprising two rows of lipid globules within a chloroplast with a single row of crystals overlying the eyespot. Production of spherical cysts was observed in culture. The freshwater strain shows a plate pattern of Po, cp, X, 4′, 2a, 7′′, 6C, 5S, 5′′′, 2′′′′, characterized by two unequal antapical plates and a lack of antapical spines, fitting the description of Peridinium parvulum, which was transferred to Parvodinium as P. parvulum comb. nov. In this species, a type A eyespot was observed comprising four rows of lipid globules within a chloroplast. A molecular phylogeny was inferred based on concatenated data from SSU, ITS-5.8S and partial LSU rRNA gene sequences using maximum likelihood and Bayesian inference. Our results show that Johsia is nested within the Peridiniopsidaceae and is a sister clade to Peridiniopsis borgei and the strain UTEX1948 identified as 'Scrippsiella' hexapraecingula with three anterior intercalary plates.
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Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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