Morphology,toxicity, and phylogeny of Alexandrium (Dinophyceae) species along the coast of China

The toxigenic genus Alexandrium includes ∼30 species, but information about its biogeography at a regional scale is limited. In this study, we explored the diversity of Alexandrium along the coast of China by incubating resting cysts collected from 7 sites. A total of 231 strains of Alexandrium belonging to 7 morphospecies were found. Among them, Alexandrium andersonii, Alexandrium fraterculum, Alexandrium leei, Alexandrium pseudogonyaulax, and Alexandrium tamutum were recorded from the China Sea for the first time. Partial large subunit (LSU) and/or internal transcribed spacer region (ITS1, ITS2, and 5.8S rDNA) sequences revealed two ribotypes of Alexandrium andersonii, Alexandrium leei, and Alexandrium tamarense: Atama complex Group I and IV. Atama complex Group I was exclusively distributed in the Yellow Sea and the Bohai Sea, whereas Group IV was restricted to the East China Sea and South China Sea. Atama complex Group I produced mainly N-sulfocarbamoyl toxins (C1/C2, 61–79% of total toxins) and gonyautoxins (GTX1/4, 17–37%). Alexandrium ostenfeldii strain ASBH01 produced NEO and STX exclusively (65% and 35%, respectively). Our results support the premise that Atama complex Group I is endemic to the Asian Pacific and includes cold water species, whereas Atama complex Group IV tends to inhabit warmer waters.     

DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species

Alexandrium species can be very difficult to identify, with A. catenella, A. tamarense, and A. fundyense that compose “Alexandrium tamarense species complex” (Atama complex) as a distinct example. DNA barcoding is promising to offer a solution but remains to be established. In this study, we examined the utility of ITS in resolving the Atama species complex, by analyzing previously studied strains plus unstudied Chinese strains within the LSU- and SSU-rDNA based group/clade frameworks recently established. We further investigated the presence of intragenomic polymorphism and its implications in species delimitation. Similar to the previous SSU and LSU results, our ITS-based phylogenies divided the complex to five clusters, but with longer and evener branch lengths between the clusters. Based on the ITS region, the inter-cluster genetic distances (p = 0.134–0.216) were consistently and substantially greater than intra-cluster genetic distances (p = 0.000–0.066), with an average inter-cluster (species) distance (p = 0.167) 7.6-fold of the average intraspecific difference (p = 0.022), qualifying the approximately 510–520 bp ITS as a DNA barcode for Atama complex. We detected varying levels of intragenomic polymorphism in ITS but found that this did not impact the taxon-resolving power of this gene. With this DNA barcode, the new East and South China Sea strains and one Antarctic strain were placed in Clade IIC/Group IV, even though there were 7–10 polymorphic sites in their ITS, in contrast to none in SSU. Furthermore, our results suggest that the five clusters are recognizable as distinct species according to the phylogenetic species concept. Based on the phylogenetic placements of the type-locality strains of the existing three morphospecies and the dominant localities of other strains, we propose that Group I/Clade I be designated as A. fundyense, Group III/Clade IIB as A. tamarense, Group IV/Clade IIC as A. catenella, Group II/Clade IIA as A. mediterranis, and Group V/Clade IID as A. australis.     

Molecular phylogeny and toxin profiles of Alexandrium tamarense (Lebour) Balech (Dinophyceae) from the west coast of Greenland

Detection of paralytic shellfish poisoning (PSP) toxins in scallops from the west coast of Greenland exceeding the 800 μg toxin/kg shellfish limit led to an investigation with the aim of finding the responsible organism(s). Three strains of Alexandrium Halim were established from single cell isolations. Morphological identification of the strains and determination of their position within the genus by LSU rDNA sequences was carried out. Light microscopy revealed that the three strains was of the Alexandrium tamarense morphotype, and bayesian and neighbor-joining analyses of the LSU rDNA sequences placed them within Group I of the A. tamarense species complex. The toxicity and toxin profiles of the strains were measured by liquid chromatography fluorescence detection (LC-FD) and their identity was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The three strains all turned out to be toxic and all produced large proportions (>60% total mol) of gonyautoxins 1 and 4 (GTX1/GTX4). This is the first record of saxitoxin producers from western Greenland. The toxin profiles were atypical for A. tamarense in their absence of N-sulfocarbanoyl C1/C2 or B1/B2 toxins. Rather the high molar percentage of GTX1/GTX4, the lesser amounts of only carbamoyl toxins and the absence of decarbamoyl derivatives are more characteristic features of A. minutum strains. This may indicate that the genetically determined toxin profiles in Alexandrium species are more complex than previously appreciated.     

Yamadazyma ubonensis f.a., sp. nov., a novel xylitol-producing yeast species isolated in Thailand

Three hundred and thirty-seven xylose-utilizing yeast strains were isolated from various natural samples. Among these, 68 strains produced xylitol in the range of 0.1–0.69 g xylitol/g xylose. Thirty-nine xylitol-producing strains were identified to be Candida tropicalis. Ten strains were found belonging to 14 known species in the genus Candida, Cyberlindnera, Meyerozyma, Pichia, Wickerhamomyces, Yamadazyma and Cryptococcus. Two strains were identified to be two Candida species and two strains (DMKU-XE142^T and DMKU-XE332) were found to be a novel species. Strain DMKU-XE142^T was isolated from tree bark and DMKU-XE332 was obtained from decaying plant leaf collected in Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 region of the large subunit rRNA gene (LSU) and the internal transcribed spacer (ITS) region, the two strains were determined to represent a novel Yamadazyma species although formation of ascospores was not observed. The sequences of the D1/D2 region of the LSU rRNA gene and the ITS region of the two strains were identical but differed from Yamadazyma phyllophila, the closest species in terms of pairwise sequence similarity of the D1/D2 region, by 1.7 % nucleotide substitutions and 3.5 % nucleotide substitutions in the ITS region. The name Yamadazyma ubonensis f.a., sp. nov. is proposed (type strain is DMKU-XE142^T = BCC 61020^T = CBS 12859^T).     

IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

Possible spreading of toxic Alexandrium tamarense blooms on the Chukchi Sea shelf with the inflow of Pacific summer water due to climatic warming

A high abundance of resting cysts of the toxic dinoflagellate Alexandrium tamarense was recently reported in the vast continental shelf of the Chukchi Sea in the Arctic Ocean, suggesting that the species is widespread in the shelf. Nevertheless, little is known about the occurrence of A. tamarense vegetative cells in the water column of the arctic. Sea ice reduction and the inflow of Pacific summer water (PSW) through the Bering Strait have recently increased owing to warming in the shelf. To determine the spatial and temporal distributions of A. tamarense in the Chukchi Sea shelf and their relationship to the inflow of PSW, field samplings were conducted in the Chukchi Sea and north Bering Sea shelves three times during the summer of 2013 from July to October. Vegetative cells of A. tamarense was detected in both shelves at all sampling periods with a maximum density of 3.55 × 10³ cells L⁻¹. This species was also observed at the station at 73°N, indicating the northernmost record of this species to date. The center of the A. tamarense distribution was between the north Bering and south Chukchi Sea shelf during the first collection period, and spread to the north Chukchi Sea shelf during the second and third collection periods. The species occurrences were mainly observed at stations affected by the PSW, especially Bering shelf water. Water structure of PSW was characterized by warmer surface and bottom water temperatures, and increased temperatures may have promoted the cell growth and cyst germination of A. tamarense. Therefore, it is suggested that an increase in the PSW inflow owing to warming promotes toxic A. tamarense occurrences on the Chukchi Sea shelf.     

Intraregional variation among Alexandrium catenella (Dinophyceae) strains from southern Chile: Morphological,toxicological and genetic diversity

Morphological, toxicological and phylogenetic analyses, using the partial LSU gene and internal spacer (ITS) regions of the rDNA gene, were combined to evaluate the intraregional diversity of Alexandrium catenella occurring along the southern coast of Chile. Twenty-two strains isolated from different localities along the wide area of distribution of the species (from 42°S to 55°S) were examined by these three approaches. Morphologically, although the strains showed diagnostic characters according to the species definition, variations in these traits within and between strains were also observed. The absence of an apical or posterior attachment pore, for instance, was observed mainly in old isolates. Indirect connection between the apical and 1′ plates, traits normally seen in other species of the same genus, was also noted in some strains. However, the lack of a ventral pore on the 1′ plate was one of the most distinctive characteristics present in all the Chilean strains. Toxicologically, the Chilean strains were characterized by the dominance of N-sulfocarbamate (C1,2) and gonyautoxins (GTX1–4), but also by the scarcity or absence of saxitoxin. Considering the dominance of these toxins in each strain, at least two distinctive toxin patterns were distinguished. Through rDNA sequence analysis, the Chilean strains were segregated as part of Clade I (North American) of the Alexandrium tamarense species complex. Nevertheless, significant genetic diversity was also observed among the Chilean strains, especially using ITS sequences. Through these three approaches, Chilean strains of A. catenella showed significant intraregional variability, which is appropriate for a native species. However, the distribution of its genetic diversity seems to be inconsistent with the apparent northward expansion observed along the west coast of South America.     

Pseudozyma vetiver sp. nov., a novel anamorphic ustilaginomycetous yeast species isolated from the phylloplane in Thailand

Three strains representing one novel yeast species were isolated from the phylloplanes of the vetiver grasses (DMKU-LV90 and DMKU-LV99^T) and sugarcane (DMKU-SP260) collected in Thailand by leaf washing followed by a plating technique. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics and the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer region (ITS), the three strains were found to represent a single novel anamorphic ustilaginomycetous yeast species in the genus Pseudozyma. The name Pseudozyma vetiver sp. nov. is proposed for this novel species. The type strain is DMKU-LV99^T (BCC 61021 = CBS 12824). The novel species showed phylogenetic relationships to the other members of the genus Pseudozyma and to teleomorphic fungal genera, namely Ustilago, Sporisorium and Anomalomyces in Ustilaginaceae, Ustilaginales. The three strains showed identical sequences both in the D1/D2 and ITS regions. The Pseudozyma species closest to the novel species in terms of pairwise sequence similarity in the D1/D2 region was Pseudozyma pruni but with 2.3 % nucleotide substitutions (14 nucleotide substitutions and no gaps out of 606 nt). The novel species and P. pruni differed by 10.9 % nucleotide substitutions (75 nucleotide substitutions and 31 gaps out of 691 nt) in the ITS region. The phylogenetic analysis based on the combined sequences of the ITS region and the D1/D2 region of the LSU rRNA gene showed that the novel species was found to be most closely related to Pseudozyma fusiformata but with 2.9 % nucleotide substitutions in the D1/D2 region and 7.4 % nucleotide substitutions in the ITS region.     

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.     

Inter- and intrasporal nuclear ribosomal gene sequence variation within one isolate of arbuscular mycorrhizal fungus, Diversispora sp.

Most molecular ecological studies of arbuscular mycorrhizal fungi (AMF) have been based on the rRNA gene sequences. However, information about intraspecific nucleotide variation is still limited in these fungi. In this study, we calculated the inter- and intrasporal nucleotide variation of Diversispora sp. EE1 using 78 cloned sequences from four spores within a ca 4960 bp fragment of the nuclear ribosomal operon spanning the near full length small ribosomal subunit (SSU) rRNA gene, the full internal transcribed spacer (ITS: ITS1-5.8S-ITS2) and ca 2740 bp of the large ribosomal subunit (LSU) rRNA gene. Data for each marker region (SSU, ITS and LSU) originated from the very same spores. Sequence variation resulting from point mutations and small indels was recorded in all regions. Highest sequence variation was observed in the ITS region at both the inter- and intrasporal levels. The ITS1 component was more variable than ITS2, whilst the 5.8S gene was the least variable component of the ITS region. Evolutionary divergence of gene copies between spores was intermediate for the LSU and lowest for the SSU. The SSU and the LSU genes had relatively similar evolutionary divergence per spore. Sequence variant richness was not exhaustive for any of the marker regions, indicating that multiple sequences per spore from multiple spores are needed when characterizing a species. This study provides reference sequences for ecological studies, permitting identification of AMF using any of the ribosomal regions or primer systems.     

Abundance and distribution of toxic Alexandrium tamarense resting cysts in the sediments of the Chukchi Sea and the eastern Bering Sea

Abundance and distribution of the toxic dinoflagellate Alexandrium tamarense species complex resting cyst were investigated in the eastern Bering Sea and the Chukchi Sea for the first time. Sediment samples (top 0–3 cm depth) were collected from the continental shelf of the eastern Bering Sea (17 stations) and the Chukchi Sea (13 stations) together with a long core sample (top 0–21 cm depth) from one station in the Chukchi Sea during 2009–2012. The cysts were enumerated using the primuline staining method. Species identification of the cysts was carried out with multiplex PCR assay and the plate morphology of vegetative cells germinated from cysts in the both areas. Alexandrium cysts were widely detected in the both areas, ranging from not detected (<1 cysts cm⁻³) to 835 cysts cm⁻³ wet sediment in the eastern Bering Sea and from not detected (<1 cysts cm⁻³) to 10,600 cysts cm⁻³ in the Chukchi Sea, and all isolated cysts were genetically and morphologically identified as the North American clade A. tamarense. Their cysts were mainly distributed in the shallow continental shelf where the water depth was less than 100 m in both areas. The cysts were detected from the deep layer (18–21 cm depth of sediment core) of the long core sample. The present study confirmed the abundant existence of A. tamarense with wide range of distribution in these areas. This fact suggests that A. tamarense vegetative cells have appeared in the water column in the both areas. Furthermore, these abundant cyst depositions indicate that this species originally distributed in the Arctic and subarctic regions and well adapted to the environments in the marginal ice zone.     

Morphology,phylogeny and toxin profiles of Gymnodinium inusitatum sp. nov., Gymnodinium catenatum and Gymnodinium microreticulatum (Dinophyceae) from the Yellow Sea,China

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.     

Yamadazyma siamensis sp. nov., Yamadazyma phyllophila sp. nov. and Yamadazyma paraphyllophila sp. nov., three novel yeast species isolated from phylloplane in Thailand and Taiwan

Four strains representing three novel anamorphic yeast species were isolated from the external surface of sugarcane leaves (DMKU-RK254^T), corn leaves (DMKU-RK548^T), bean leaves (K129) in Thailand and hengchun pencilwood leaves (TrB1-1^T) in Taiwan. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene, the internal transcribed spacer (ITS) region, the actin gene (ACT1) and the elongation factor 2 gene (EF2), the four strains were determined to represent novel Yamadazyma species although formation of ascospores was not observed. Strain DMKU-RK254^T was determined to be related to Candida diddensiae, Candida naeodendra and Candida kanchanaburiensis but with 1.8, 1.8 and 2.0 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, respectively. It was assigned to Yamadazyma siamensis sp. nov. (type strain DMKU-RK254^T = BCC 50730^T = NBRC 108901^T = CBS 12573^T). The sequences of the D1/D2 region of the LSU rRNA gene, the ITS region, ACT1 gene and EF2 gene of two strains (DMKU-RK548^T and K129) were identical but differed from that of strain TrB1-1^T by 0.6, 1.0, 3.3 and 5.9 % nucleotide substitutions, respectively. Therefore, the two strains (DMKU-RK548^T and K129) and strain TrB1-1^T were assigned to be two separate species. The closest species in terms of pairwise sequences similarity of the D1/D2 region to the two novel species was Yamadazyma philogaea but with 1.1–1.7 % nucleotide substitutions. The two strains (DMKU-RK548^T and K129) were assigned to Yamadazyma phyllophila sp. nov. (type strain DMKU-RK548^T = BCC 50736^T = NBRC 108906^T = CBS 12572^T) and the strain TrB1-1^T was named Yamadazyma paraphyllophila sp. nov. (type strain TrB1-1^T = BCRC 23030^T = CCTCC AY 204005^T = CBS 9928^T).     

Gymnodinium catenatum Graham isolated from the Portuguese coast: Toxin content and genetic characterization

The bloom forming marine dinoflagellate Gymnodinium catenatum Graham has been linked to paralytic shellfish poisoning (PSP) outbreaks in humans. Along the Portuguese coast (NE Atlantic), G. catenatum shows a complex bloom pattern, raising questions about the origin and affinities of each bloom population. In this work, the variability within six cultured strains of G. catenatum isolated from Portuguese coastal waters (S coast, W coast and NW coast), between 1999 and 2011, was investigated. The strains were analyzed for toxin profiling and intra-specific genetic diversity. Regarding the toxin profile, differences recorded between strains could not be assigned to the time of isolation or geographical origin. The parameter that most influenced the toxin profile was the life-cycle stage that originated the culture: vegetative cell versus hypnozygote (resting cyst). At the genetic level, all strains showed similar sequences for the D1–D2 region of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA) and shared complete identity with strains from Spain, Algeria, China and Australia. Conversely, we did not find a total identity match for the ITS-5.8S nuclear rDNA fragment. After sequence analysis, two guanine/adenine (R) single nucleotide polymorphisms (SNP 1 and 2) were detected for all strains, in the ITS1 region. This species has been reported to present very conservative LSU and ITS-5.8S rDNA regions, though with few SNP, including SNP1 of this study, already attributed to strains from certain locations. The SNP here described characterize G. catenatum populations from Portuguese waters and may represent valuable genetic markers for studies on the phylogeography of this species.     

Adding new pieces to the Azadinium (Dinophyceae) diversity and biogeography puzzle: Non-toxigenic Azadinium zhuanum sp. nov. from China,toxigenic A. poporum from the Mediterranean,and a non-toxigenic A. dalianense from the French Atlantic

The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44 fg cell⁻¹. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea.     

Cryptococcus nanyangensis sp. nov., A New Basidiomycetous Yeast Isolated from the Gut of Wood-Boring Larvae

Two strains of a novel basidiomycetous yeast species were isolated from the gut of wood-boring larvae collected in the Baotianman Nature Reserve, the central China. Sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) regions showed that these yeasts belong to the Bulleromyces clade and formed a cluster together with eleven undescribed Cryptococcus species. The novel species differed from its closest known species, Cryptococcus rajasthanensis, by 3.3 % divergence (15 substitutions and 6 gaps over 630 bases) in the D1/D2 domains, and by 13.4 % divergence (41 substitutions and 27 gaps over 508 bases) in the ITS regions. Physiologically, the fermentation of glucose, galactose, sucrose, trehalose, and raffinose in Durham tubes was observed for the strains of this new yeast. Based on the phenotypical and molecular characteristics presented, the two strains are proposed as a new species, Cryptococcus nanyangensis sp. nov., with the type strain KCY-1^T (=CICC 1976^T = CBS 12474^T).     

Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov., two novel methylotrophic yeast species from phylloplane in Thailand

Three strains (KM03^T, KM13 ^T and KM15) representing two novel methylotrophic yeast species were isolated from the external surface of plant leaves, which were collected from Kanchanaburi province, Thailand, by three-consecutive enrichments in methanol broth. Strain KM03^T was isolated from phylloplane of a mango tree (Mangifera indica) and two strains, KM13^T and KM15, were obtained from phylloplane of different wine grapes (Vitis vinifera). The sequences of the D1/D2 region of the large subunit (LSU) rRNA gene of the two strains (KM13^T and KM15) were identical and differed markedly from that of strain KM03^T. In terms of pairwise sequence similarity of the D1/D2 region the closest species to the strains KM13^T and KM15 were Candida suzukii (CBS 9253^T) and Candida nitratophila (CBS 2027^T) but with 2.1 % nucleotide substitutions. Strain KM03^T differed from Ogataea wickerhamii (CBS 4307^T), its closest relative, by 2.3 % nucleotide substitutions. Phylogenetic analysis based on the D1/D2 sequences placed the three strains in the Ogataea clade. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analyses of the D1/D2 and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the three strains represent two novel Ogataea species although formation of ascospores was not observed. Ogataea kanchanaburiensis sp. nov. is proposed for strain KM03^T (=BCC 47626^T = NBRC 108603^T = CBS 12673^T). Two strains, KM13^T and KM15, were assigned to Ogataea wangdongensis sp. nov. (type strain KM13^T = BCC 42664^T = NBRC 107778^T = CBS 12674^T). GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 and the ITS regions of O. kanchanaburiensis KM03^T are AB734090 and AB734093, respectively, of O. wangdongensis KM13^T are AB734091 and AB734094, respectively, and of O. wangdongensis KM15 are AB734092 and AB734095, respectively.     

Yarrowia porcina sp. nov. and Yarrowia bubula f.a. sp. nov., two yeast species from meat and river sediment

Eleven yeast strains representing two hitherto undescribed species were isolated from different kinds of meat samples in Hungary and one from the sediment of a tropical freshwater river in Southeastern Brazil. The analysis of the sequences of their large subunit rRNA gene D1/D2 domain and the internal transcribed spacer (ITS) regions placed the two new species in the Yarrowia clade. Some of the seven strains representing the first new species can mate and give rise to asci and form ascospores embedded in capsular material, which qualifies it as the third teleomorph species of the Yarrowia clade. The name Yarrowia porcina sp. nov. (type strain: NCAIM Y.02100^T = CBS 12935^T = NRRL Y-63669^T, allotype strain UFMG-RD131^A = CBS 12932^A) is proposed for this new yeast species, which, based on physiological characters, is indistinguishable from Yarrowia lipolytica and some other species of the genus. Considerable intraspecific variability was detected among the sequences of the large subunit rRNA gene D1/D2 domains of the seven strains. The variability among the D1/D2 sequences exceeded the divergence observed among the ITS sequences and in some cases more than 1 % substitution among the D1/D2 sequences was detected. The conspecificity of these strains was supported by the low (0–3 substitutions) sequence divergence among their ITS sequences, the result of a parsimony network analysis utilizing the concatenated ITS and D1/D2 sequences and also by the fingerprint patterns generated by microsatellite primed PCR. No ascospore formation was observed in the group of the other five strains representing the second new species. These strains shared identical D1/D2 and ITS sequences. Yarrowia bubula f.a., sp. nov. (type strain: NCAIM Y.01998^T = CBS 12934^T = NRRL Y-63668^T) is proposed to accommodate these strains.