Amphidoma parvula (Amphidomataceae), a new planktonic dinophyte from the Argentine Sea

Amphidoma is an old though poorly studied thecate dinophyte that has attracted attention recently as a potential producer of azaspiracids (AZA), a group of lipophilic phycotoxins. A new species, Amphidoma parvula, sp. nov. is described from the South Atlantic shelf of Argentina. With a Kofoidean thecal plate pattern Po, cp, X, 6′, 6′′, 6C, 5S, 6′′′, 2′′′′, the cultivated strain H-1E9 (from which the type material of Am. parvula, sp. nov. was prepared) shared the characteristic plate arrangement of Amphidoma each with six apical, precingular and postcingular plates. Amphidoma parvula, sp. nov. differs from other species of Amphidoma by a characteristic combination of small size (10.7–13.6 µm in length), ovoid shape, high length ratio between epitheca and hypotheca, and small length ratio between apical and precingular plates. Other morphological details, such as the number and arrangement of sulcal plates and the fine structure of the apical pore complex support the close relationship between Amphidoma and the other known genus of Amphidomataceae, Azadinium. However, Am. parvula, sp. nov. lacks a ventral pore, a characteristically structured pore found in all contemporary electron microscopy studies of Amphidoma and Azadinium. As inferred from liquid chromatography coupled with tandem mass spectrometry, Am. parvula, sp. nov. did not produce AZA in measurable amounts. Molecular phylogenetics confirmed the systematic placement of Am. parvula, sp. nov. in Amphidoma (as sister species of Amphidoma languida) and the Amphidomataceae. The results of this study have improved the knowledge of Amphidomataceae biodiversity.     

Revisiting the 1991 algal bloom in shelf waters off Argentina: Azadinium luciferelloides sp. nov. (Amphidomataceae,Dinophyceae) as the causative species in a diverse community of other amphidomataceans

Species of the marine dinophycean genera Azadinium and Amphidoma (Amphidomataceae) mostly attract attention because of their production potential of the lipophilic polyether phycotoxin azaspiracid (AZA). The genus Azadinium probably has a very wide geographical distribution. Blooms of Azadinium from the continental shelf off Argentina have been observed back in the early 1990, but were just recently published, and the causative species, identified at that time as Azadinium cf. spinosum, could not unequivocally be determined. Here we retrospectively analyzed old archived samples of one of the South Atlantic Azadinium bloom from 1991 with electron microscopy. It turned out that the dominant nanoplanktonic dinophycean species in fact represent a new species which we describe here based on the morphology. Azadinium luciferelloides sp. nov. is a small (approximately 9–14 μm cell length) thecate dinoflagellate with the dominant plate pattern of the genus (Po, X, 4′, 3a, 6″, 6C, 5S, 6″′, 2″″), and with a small antapical spine. Azadinium luciferelloides differed from all other described species of Azadinium by the position of the ventral pore, which was located on the right ventral side in a notch of an otherwise symmetric pore plate. In addition, we recorded and documented the presence of other similar sized species of the Amphidomataceae in the samples. Our finding of Az. spinosum, Az. dalianense, Az. dexteroporum, and Amphidoma languida are the first record for the South Atlantic and thus describe an important range extension of these species. The diversity and importance of the Amphidomataceae for South Atlantic spring bloom plankton is now known and taxonomically documented, but cultures and/or analysis of AZA in field samples of the area are needed to clarify the AZA production potential of the local species and populations in order to finally evaluate the risk potential of AZA for AZA shellfish contamination in the Southwestern Atlantic region.     

A new potentially toxic Azadinium species (Dinophyceae) from the Mediterranean Sea,A. dexteroporum sp. nov.

A new photosynthetic planktonic marine dinoflagellate, Azadinium dexteroporum sp. nov., is described from the Gulf of Naples (South Tyrrhenian Sea, Mediterranean Sea). The plate formula of the species, Po, cp, X, 4′, 3a, 6″, 6C, 5?S, 6? and 2″″, is typical for this recently described genus. Azadinium dexteroporum is the smallest rep‐resentative of the genus (8.5 μm average length, 6.2 μm average width) and shares the presence of a small antapical spine with the type species A. spinosum and with A. polongum. However, it differs from all other Azadinium species for the markedly asymmetrical Po plate and the position of the ventral pore, which is located at the right posterior end of the Po plate. Another peculiarity of A. dexteroporum is the pronounced concavity of the second intercalary plate (2a), which appears collapsed with respect to the other plates. Phylogenetic analyses based on the large subunit 28S rDNA (D1/D2) and the internal transcribed spacer (ITS rDNA) support the attribution of A. dexteroporum to the genus Azadinium and its separation from the other known species. LC/MS‐TOF analysis shows that Azadinium dex‐teroporum produces azaspiracids in low amounts. Some of them have the same molecular weight as known compounds such as azaspiracid‐3 and ‐7 and Compound 3 from Amphidoma languida, as well as similar fragmentation patterns in some cases. This is the first finding of a species producing azapiracids in the Mediterranean Sea.     

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.     

Ultrastructure and large subunit rDNA sequences of Lepidodinium viride reveal a close relationship to Lepidodinium chlorophorum comb. nov. (=Gymnodinium chlorophorum)

The ultrastructure of the green dinoflagellate Lepididodinium viride M. M. Watanabe, S. Suda, I. Inouye Sawaguchi et Chihara was studied in detail. The nuclear envelope possessed numerous chambers each furnished with a nuclear pore, a similar arrangement to that found in other gymnodinioids. The flagellar apparatus was essentially identical to Gymnodinium chlorophorum Elbrächter et Schnepf, a species also containing chloroplasts of chlorophyte origin. Of particular interest was the connection of the flagellar apparatus to the nuclear envelope by means of both a fiber and a microtubular extension of the R3 flagellar root. This feature has not been found in other dinoflagellates and suggests a close relationship between these two species. This was confirmed by phylogenetic analysis based on partial sequences of the large subunit (LSU) rDNA gene of L. viride, G. chlorophorum and 16 other unarmoured dinoflagellates, including both the ‘type’ culture and a new Tasmanian isolate of G. chlorophorum. These two isolates had identical sequences and differed from L. viride by only 3.75% of their partial LSU sequences, considerably less than the difference between other Gymnodinium species. Therefore, based on ultrastructure, pigments and partial LSU rDNA sequences, the genus Lepidodinium was emended to encompass L. chlorophorum comb. nov.     

Ultrastructure and molecular phylogenetic position of a new marine sand-dwelling dinoflagellate from British Columbia,Canada: Pseudadenoides polypyrenoides sp. nov. (Dinophyceae)

Two monospecific genera of marine benthic dinoflagellates, Adenoides and Pseudadenoides, have unusual thecal tabulation patterns (lack of cingular plates in the former; and no precingular plates and a complete posterior intercalary plate series in the latter) and are thus difficult to place within a phylogenetic framework. Although both genera share morphological similarities, they have not formed sister taxa in previous molecular phylogenetic analyses. We discovered and characterized a new species of Pseudadenoides, P. polypyrenoides sp. nov., at both the ultrastructural and molecular phylogenetic levels. Molecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated a close relationship between P. polypyrenoides sp. nov. and Pseudadenoides kofoidii, and Adenoides and Pseudadenoides formed sister taxa in phylogenetic trees inferred from LSU rDNA sequences. Comparisons of morphological traits, such as the apical pore complex (APC), demonstrated similarities between Adenoides, Pseudadenoides and several planktonic genera (e.g. Heterocapsa, Azadinium and Amphidoma). Molecular phylogenetic analyses of SSU and LSU rDNA sequences also demonstrated an undescribed species within Adenoides.     

MOLECULAR PHYLOGENY OF THE HETEROTROPHIC DINOFLAGELLATES,PROTOPERIDINIUM, DIPLOPSALIS AND PREPERIDINIUM (DINOPHYCEAE), INFERRED FROM LARGE SUBUNIT rDNA1

The genera Protoperidinium Bergh, Diplopsalis Bergh, and Preperidinium Mangin, comprised of species of marine, thecate, heterotrophic dinoflagellates in the family Protoperidinaceae Balech, have had a confused taxonomic history. To elucidate the validity of morphological groupings within the Protoperidinium and diplopsalids, and to determine the evolutionary relationships between these and other dinoflagellates, we undertook a study of molecular phylogeny using the D1–D3 domains of the large subunit (LSU) of the rDNA. Based on morphology, the 10 Protoperidinium species examined belonged to three subgenera and five morphological sections. Two diplopsalid species were also included. Single‐cell PCR, cloning, and sequencing revealed a high degree of intraindividual sequence variability in the LSU rDNA. The genus Protoperidinium appeared to be recently divergent in all phylogenetic analyses. In maximum parsimony and neighbor joining analyses, Protoperidinium formed a monophyletic group, evolving from diplopsalid dinoflagellates. In maximum likelihood and Bayesian analyses, however, Protoperidinium was polyphyletic, as the lenticular, diplopsalid heterotroph, Diplopsalis lenticula Bergh, was inserted within the Protoperidinium clade as basal to Protoperidinium excentricum (Paulsen) Balech, and Preperidinium meunieri (Pavillard) Elbrächter fell within a separate clade as a sister to the Oceanica and Protoperidinium steidingerae Balech. In all analyses, the Protoperidinium were divided into two major clades, with members in the Oceanica group and subgenus Testeria in one clade, and the Excentrica, Conica, Pellucida, Pyriforme and Divergens sections in the other clade. The LSU rDNA molecular phylogeny supported the historical morphologically determined sections, but not a simple morphology based model of evolution based on thecal plate shape.     

First report of the photosynthetic dinoflagellate genus Azadinium in the Pacific Ocean: morphology and molecular characterization of Azadinium cf. poporum

A strain of a dinoflagellate belonging to the genus Azadinium was obtained by the incubation of sediments collected from Shiwha Bay, Korea. This report of the genus Azadinium is the first outside of northern Europe and furthermore from the Pacific Ocean. The diagnostic morphological features of the isolate very closely resemble the recently described species Azadinium poporum isolated from the North Sea. However, the shape of the 3' apical plate and the occasional morphological variations unreported from A. poporum bring minor distinctions between strains from different locations. The DNA sequences of small subunit, ITS, and large subunit (LSU) rDNA differed by 0.2%, 2.6%, and 3.6%, respectively, from those of A. poporum, whereas the COI gene was identical to those found in all strains of Azadinium. Phylogenetic analyses of the ribosomal DNA regions generally positioned the Korean strain as a sister taxon of A. poporum. However, the Korean isolate tends to occupy a basal position within Azadinium species with ITS rDNA and LSU rDNA. Using liquid chromatography coupled with tandem mass spectrometry, no known azaspiracids were detected. The slight but discernible morphological differences, the distinct rDNA sequences, and the tendency of the Korean strain to diverge phylogenetically based on ITS rDNA and LSU rDNA from A. poporum do not enable us to clearly assign the isolate to A. poporum. However, these characteristics do not allow us to classify it as a distinct species, and it is therefore designated as Azadinium cf. poporum. The examination of more strains to find more diagnostic characteristics might enable the attribution of this material to a well-defined taxonomic position.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

POLYPHYLETIC ORIGIN OF THE DICTYOSPHAERIUM MORPHOTYPE WITHIN CHLORELLACEAE (TREBOUXIOPHYCEAE)1

The green algal Dictyosphaerium morphotype is characterized by spherical or oval cells connected by gelatinized strands to microscopic colonies, which are covered by prominent mucilaginous envelopes. Combined SSU and ITS rRNA gene sequence analyses revealed that this morphotype evolved independently both in the Chlorella and Parachlorella clades of the Chlorellaceae. It was shown that strains exhibiting the morphology of the type species Dictyosphaerium ehrenbergianum Nägeli established a sister lineage to Parachlorella. The strain D. ehrenbergianum CCAP 222/1A was designated as an authentic strain for establishing the epitype of the genus Dictyosphaerium. The comparison of this strain with the authentic strain of Parachlorella beijerinckii Krienitz, E. Hegewald, Hepperle, V. Huss, T. Rohr et M. Wolf (SAG 2046) showed considerable differences in the secondary structure of the ITS region. Within the whole ITS‐1 and ITS‐2 region, 27 compensatory base changes (CBCs) were recognized. In the conserved Helix III of the ITS‐2, five CBCs/HemiCBCs were detected. This is a conclusive argument for separation of these two species. The clear definition of Dictyosphaerium is intended to be the necessary starting point of taxonomic reevaluation of Dictyosphaerium‐like algae within different evolutionary lineages of the Chlorellaceae.     

PHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE,DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES1

Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis.     

Morphology and Phylogeny of Prorocentrum texanum sp. nov. (Dinophyceae): A New Toxic Dinoflagellate from the Gulf of Mexico Coastal Waters Exhibiting Two Distinct Morphologies

A new planktonic species of Prorocentrum is described from the Gulf of Mexico. First observed with the Imaging FlowCytobot, Prorocentrum texanum sp. nov. was characterized using LM, SEM, and TEM along with sequencing of the SSU, LSU, and ITS ribosomal regions and the mitochondrial cob and cox1 regions. P. texanum sp. nov. is a round to oval bivalvate dinoflagellate, with a prominent anterior, serrated solid flange on periflagellar a platelet and an opposing short, flat flange on the h platelet. The periflagellar area consists of 10 platelets. Both left and right valves have shallow round depressions and two‐sized valve pores. The anterior ejectosome pore pattern differs between the left and right valve in relation to the periflagellar area and margins. Ten to eleven rows of tangential ejectosome pores are present on each valve. P. texanum sp. nov. has two varieties which exhibit distinct morphotypes, one round to oval (var. texanum) and the other pointed (var. cuspidatum). P. texanum var. cuspidatum is morphologically similar to P. micans in surface markings, but is smaller, and has a serrated periflagellar flange, and is genetically distinct from P. micans. Cytologically, P. texanum has two parietal chlo‐roplasts, each with a compound, interlamellar pyrenoid, trichocysts, fibrous vesicles that resemble mucocysts, pusules, V‐ to U‐shaped posterior nucleus, golgi, and tubular mitochondria. No genetic difference was found between the two varieties in the five genes examined. Phylogenetic analysis of the SSU, LSU, and ITS ribosomal regions place P. texanum sp. nov. as a sister group to P. micans. One isolate of P. texanum var. texanum produces okadaic acid.     

THE MOLECULAR PHYLOGENETIC AND GEOMETRIC MORPHOMETRIC EVALUATION OF MICRASTERIAS CRUX‐MELITENSIS/M. RADIANS SPECIES COMPLEX1

We investigated nine strains of the Micrasterias crux‐melitensis (Ehrenb.) Hassall ex Ralfs and M. radians W. B. Turner species complex. A combination of molecular, morphological, and geometric morphometric data was used to reveal the patterns of their phenotypic and phylogenetic differentiation. The molecular data based on internal transcribed spacer (ITS) rDNA, glycine transfer RNA (trnG^uuc) intron, and SSU rDNA sequences revealed three phylogenetic lineages. One of them comprised the six European and North American strains that were morphologically identified as M. crux‐melitensis. Phenotypic data illustrated high morphological variability of strains within this genetically homogenous lineage that spanned several traditional infraspecific taxa, including strains corresponding to M. crux‐melitensis var. janeira (Racib.) Grönblad and M. crux‐melitensis var. superflua W. B. Turner, whose morphometric characteristics profoundly differed. Three strains of M. radians formed two separate phylogenetic lineages corresponding to traditional varieties M. radians var. evoluta (W. B. Turner) Willi Krieg. and M. radians var. bogoriensis (C. J. Bernard) G. S. West. The morphological types corresponding to the former variety have, so far, only been reported from Africa. Therefore, we cannot preclude that geographic isolation may play a role in species differentiation of relatively large freshwater protists, such as Micrasterias.     

The role of Azadinium spinosum (Dinophyceae) in the production of azaspiracid shellfish poisoning in mussels

Azaspiracids (AZAs) are a group of lipophilic polyether compounds first detected in Ireland which have been implicated in shellfish poisoning incidents around Europe. These toxins regularly effect shellfish mariculture operations including protracted closures of shellfish harvesting areas for human consumption. The armoured dinoflagellate Azadinium spinosum Elbrächter et Tillmann gen. et sp. nov. (Dinophyceae) has been described as the de novo azaspiracid toxin producer; nonetheless the link between this organism and AZA toxin accumulation in shellfish has not yet been established. In August 2009, shellfish samples of blue mussel (Mytilus edulis) from the Southwest of Ireland were analysed using liquid chromatography–tandem-mass spectrometry (LC–MS/MS) and were found to be above the regulatory limit (0.16 μg g⁻¹ AZA-equiv.) for AZAs. Water samples from this area were collected and one algal isolate was identified as A. spinosum and was shown to produce azaspiracid toxins. This is the first strain of A. spinosum isolated from Irish waters. The Irish A. spinosum is identical with the other two available A. spinosum strains from Scotland (3D9) and from Denmark (UTHE2) in its sequence of the D1–D2 regions of the LSU rDNA.A 24 h feeding trial of blue mussels (M. edulis) using an algal suspension of the Irish A. spinosum culture at different cell densities demonstrated that A. spinosum is filtered, consumed and digested directly by mussels. Also, LC–MS/MS analysis had shown that AZAs were accumulating in the shellfish hepatopancreas. The toxins AZA1 and -2 were detected in the shellfish together with the AZA analogues AZA3, AZA6, AZA17 and -19 suggesting that AZA1 and -2 are metabolised in the shellfish within the first 24 h after ingestion of the algae. The levels of AZA17 detected in the shellfish hepatopancreas (HP) were equivalent to the levels of AZA1 but in the remainder tissues the levels of AZA17 were four to five times higher than that of AZA1, only small quantities of AZA3 and -19 were present with negligible amounts of AZA6 detected after the 24 h period. This could have implications in the future monitoring of these toxins given that at present according to EU legislation only AZA1–AZA3 is regulated for. This is the first report of blue mussels’ (M. edulis) feeding on the azaspiracid producing algae A. spinosum from Irish waters.     

ON THE IDENTITY OF KARLODINIUM VENEFICUM AND DESCRIPTION OF KARLODINIUM ARMIGER SP. NOV. (DINOPHYCEAE), BASED ON LIGHT AND ELECTRON MICROSCOPY,NUCLEAR‐ENCODED LSU RDNA,AND PIGMENT COMPOSITION1

An undescribed species of the dinoflagellate genus Karlodinium J. Larsen (viz. K. armiger sp. nov.) is described from Alfacs Bay (Spain), using light and electron microscopy, pigment composition, and partial large subunit (LSU) rDNA sequence. The new species differs from the type species of Karlodinium (K. micrum (Leadbeater et Dodge) J. Larsen) by lacking rows of amphiesmal plugs, a feature presently considered to be a characteristic of Karlodinium. In K. armiger, an outer membrane is underlain by a complex system of cisternae and vacuoles. The pigment profile of K. armiger revealed the presence of chlorophylls a and c, with fucoxanthin as the major carotenoid. Phylogenetic analysis confirmed K. armiger to be related to other species of Karlodinium; thus forming a monophyletic genus, which, in the LSU tree, occupies a sister group position to Takayama de Salas, Bolch, Botes et Hallegraeff. The culture used by Ballantine to describe Gymnodinium veneficum Ballantine (Plymouth 103) was examined by light and electron microscopy and by partial LSU rDNA. Ultrastructurally, it proved identical to K. micrum (cultures Plymouth 207 and K. Tangen KT‐77D, the latter also known as K‐0522), and in LSU sequence, differed in only 0.3% of 1438 bp. We consider the two taxa to belong to the same species. This necessitates a change of name for the most widely found species, K. micrum, to K. veneficum. The three genera Karlodinium, Takayama, and Karenia constitute a separate evolutionary lineage, for which the new family Kareniaceae fam. nov. is suggested.     

Phylogenetic affiliation of the desert truffles <Emphasis Type="Italic">Picoa juniperi</Emphasis> and <Emphasis Type="Italic">Picoa lefebvrei</Emphasis>

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.