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 PROROCENTRUM CONSUTUM SP. NOV. (DINOPHYCEAE), A NEW BENTHIC DINOFLAGELLATE FROM SOUTH BRITTANY (NORTHWESTERN FRANCE)1

A new marine benthic Prorocentrum species from sandy habitats of South Brittany (northwestern France), P. consutum sp. nov., is described using LM and SEM and molecular phylogenetic analyses. Cells have a subcircular to broadly ovoid shape and are plainly flattened. They are 57–61 μm long and 52–55 μm wide. A central pyrenoid is present, and the kidney‐shaped nucleus is positioned in the posterior region. In right valve view, the periflagellar area is deeply excavated, and the left valve forms a prominent apical ridge. The periflagellar area consists of nine platelets, and a small narrow collar is present around the flagellar pore. The ornamentation of this new species is very peculiar and is characterized by a ring of round areolae located at the periphery of the valves, each areola containing three or four pores. Apart from this ring of areolae, the cell surface is smooth and with scattered pores. Pores are not present in the center of the right or left valve. The intercalary band is generally narrow and faintly striated horizontally. The molecular phylogenetic position of P. consutum sp. nov. was inferred using SSU and LSU rDNA. In both analyses, this species branched with high support in the clade comprising species with a symmetric shape and appeared to be a sister group to that formed by P. lima and other tropical benthic species, such as P. arenarium, P. belizeanum, P. hoffmannianum, and P. maculosum.     

PROROCENTRUM BIMACULATUM SP. NOV. (DINOPHYCEAE,PROROCENTRALES), A NEW BENTHIC DINOFLAGELLATE SPECIES FROM KUWAIT (ARABIAN GULF)1

A new benthic dinoflagellate species, Prorocentrum bimaculatum sp. nov., is studied from Kuwait’s marine sediments, based on detailed morphological and molecular data. Cells are large, oblong oval in shape. They are 49.9–55.3 μm long and 38.4–43.2 μm wide. The ornamentation of this new species is peculiar, and characterized by smooth valves with large pores (0.32–0.50 μm) scattered on their surface, except in two circular patches of ～15 μm in diameter, devoid of ornamentation and located on both sides of the valve centers. The periflagellar area is widely triangular, located in a moderate excavation of the right valve, and comprises nine platelets. The intercalary band of P. bimaculatum is smooth. The molecular phylogenetic position of this new taxon was inferred from SSU and LSU rDNA genes. In both phylogenetic analyses, P. bimaculatum branched with high support with Prorocentrum consutum and formed a clade sister to the one including P. lima and related species such as P. arenarium, P. belizeanum, P. hoffmannianum, and P. maculosum. From the phylogenetic study, since most species related to P. bimaculatum are known for their toxic effects and production of okadaic acid, this new species can be considered as a potential toxin producer, but this has to be analyzed.     

PHYLOGENETIC ANALYSIS OF NINE SPECIES OF PROROCENTRUM (DINOPHYCEAE) INFERRED FROM 18S RIBOSOMAL DNA SEQUENCES, MORPHOLOGICAL COMPARISONS, AND DESCRIPTION OF PROROCENTRUM PANAMENSIS, SP. NOV.

Sequences of 18S rRNA genes were obtained from eight species of Prorocentrum Ehrenberg: P. minimum (Pavillard) Schiller, P. mexicanum Osorio Tafall, P. emarginatum Fukuyo, P. lima (Ehrenberg) Dodge, P. arenarium Faust, P. maculosum Faust, P. concavum Fukuyo, and P. panamensis, sp. nov. Prorocentrum panamensis is a new species of tropical dinoflagellate isolated from a benthic coral reef on the Pacific coast of Panama and described here using scanning electron microscopy. Cells are heart shaped, 46–52 μm long and 43–46 μm wide. The valve surfaces are areolate except in the central area. Pores of 0.15 μm in diameter are scattered in areolae, mainly around the periphery of the cell. The right valve has a specific ovoid depression with numerous appressed pores; we named this structure the sieve-like depression. The periflagellar area is nearly ovoid, located in a shallow depression, and almost equally set into both valves. It is unornamented (no apical expansion) but has numerous depressions in platelets. The flagellar and auxiliary pores are different in size and shape. The intercalary band is transversally striated. Phylogenetic relationships of gonyaulacoid, peridinioid, gymnodinioid, and prorocentroid dinoflagellates were inferred from complete 18S rDNA sequences. Two distinct phylogenetic analyses are presented for armored and unarmored Dinophyceae in an attempt to make the phylogenetic relationships between these different kinds of organisms clearer. The Prorocentrales appear to have a common origin, although two groups of Prorocentrum spp. are apparent. The first group includes benthic, symmetrical species (P. lima, P. arenarium, P. maculosum, and P. concavum). The second group contains planktonic and bentho-planktonic species (P. micans Ehrenberg, P. minimum, P. mexicanum, and P. panamensis sp. nov.). Genetic distances between species within these two groups were high; however, the divergence between the two groups seems to have occurred late in dinoflagellate evolution. In addition, the bentho-planktonic P. emarginatum appeared distantly related to both groups; however,its 18S rDNA sequence shares specific nucleotide substitutions with the two groups, suggesting an older origin of this species compared to the others. A morphological interpretation of this phylogenetic analysis is made on the basis of the specific structure of the periflagellar area. Finally, genetic data and morphological observations support the hypothesis that the genus Prorocentrum is rather heterogeneous; several species could be considered to constitute distinct genera.     

A New Heterotrophic Dinoflagellate from the North‐eastern Pacific,Protoperidinium fukuyoi: Cyst–Theca Relationship,Phylogeny, Distribution and Ecology

The cyst–theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown‐coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst‐defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North‐east Pacific, its distribution reflecting optimal summer sea‐surface temperatures of ~14–18 °C and salinities of 22–34 psu.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF A NEW MARINE SAND‐DWELLING PROROCENTRUM SPECIES,P. TSAWWASSENENSE (DINOPHYCEAE,PROROCENTRALES), FROM BRITISH COLUMBIA,CANADA1

A new marine benthic, sand‐dwelling Prorocentrum species from the temperate region of the Pacific coast of British Columbia, Canada, is described using LM and EM and molecular phylogenetic analyses. The cells have a broad oval shape, 40.0–55.0 μm long and 30.0–47.5 μm wide, and a wide U‐shaped periflagellar area on the right thecal plate. The left thecal plate consists of a straighter apical outline in the form of a raised ridge. Five to six delicate apical spines in the center of the periflagellar area are present. The nucleus is located in the posterior region of the cell, and a conspicuous pusule is located in the anterior region of the cell. The cells have golden‐brown chloroplasts with a compound, intrachloroplast pyrenoid that lacks a starch sheath. The thecal plates are smooth with round pores of two different sizes. The larger pores are arranged in a specific pattern of radial rows that are evenly spaced around the plate periphery and of irregular rows (or double rows) that form an incomplete “V” at the apical end of the plates. Large pores are absent in the center of the left and right thecal plates. The intercalary band is striated transversely and also has faint horizontal striations. Trichocysts and two types of mucocysts are present. The molecular phylogenetic position of Prorocentrum tsawwassenense sp. nov. was inferred using SSU rDNA sequences. This new species branched with high support in a Prorocentrum clade containing both benthic and planktonic species.     

Molecular phylogeny of the spiny-surfaced species of the dinoflagellate Prorocentrum with the description of P. thermophilum sp. nov. and P. criophilum sp. nov. (Prorocentrales,Dinophyceae)

Spiny-surfaced species of Prorocentrum form harmful algal blooms, and its taxonomic identity is obscure due to the size and shape variability. Molecular phylogenies reveal two major clades: one for P. cordatum with sequences mainly retrieved as P. minimum, and the other for P. shikokuense with sequences also retrieved as P. dentatum and P. donghaiense. Several closely related clades still need to be characterized. Here, we provide nuclear SSU and LSU rRNA genes, and nuclear ITS region (ITS1-5.8S gene-ITS2) sequences of the strain CCMP3122 isolated from Florida (initially named P. donghaiense) and strains Prorocentrum sp. RCC6871–2 from the Ross Sea, Antarctica. We describe Prorocentrum thermophilum sp. nov. based on the strain CCMP3122, a species also distributed in the open waters of the Gulf of Mexico, New Zealand, and the Arabian Gulf; and Prorocentrum criophilum sp. nov. based on the strain RCC6872, which is distributed in the Antarctic Ocean and Arctic Sea. Prorocentrum thermophilum is roundish (~14 μm long, ~12 μm wide), with an inconspicuous anterior spine-like prolongation under light microscopy, valves with tiny, short knobs (5–7 per μm²), and several (<7) large trichocyst pores (~0.3 μm) in the right valve, as well as smaller pores (~0.15 μm). Prorocentrum criophilum is round in valve view (~11 μm long, 10 μm wide) and asymmetrically roundish in lateral view, the periflagellar area was not discernible under light microscopy, valves with very tiny, short knobs (6–10 per μm²), and at least 12 large pores in the right valve. Other potentially undescribed species of spiny-surfaced Prorocentrum are discussed.     

Morphological and Molecular Characterization of a New Species of Stephanopogon,Stephanopogon pattersoni n. sp.

Stephanopogon is a taxon of multiciliated protists that is now known to belong to Heterolobosea. Small subunit ribosomal DNA (SSU rDNA) phylogenies indicate that Stephanopogon is closely related to or descended from Percolomonas, a small tetraflagellate with a different feeding structure, thus these morphologically dissimilar taxa are of ongoing evolutionary interest. A new strain of Stephanopogon, KM041, was cultured, then characterized by light microscopy, electron microscopy, and SSU rDNA sequencing. KM041 is 18–35 μm (mean 26.8 μm) long, with six main ventral ciliary rows, one ventro‐lateral ciliary row, and three anterior barbs. It closely resembles Stephanopogon minuta Lei et al. 1999 in morphology, and is very closely related to an extinct culture “S. aff. minuta”, yet is markedly dissimilar in SSU rDNA sequence from a different isolate identified as S. minuta. This confirms that there are at least two distinct lineages of S. minuta‐like cells, and we describe KM041 as a new species, Stephanopogon pattersoni n. sp. The ultrastructure of KM041 resembles that of previously studied Stephanopogon species, though it has a novel paraxonemal structure in a few cilia. We note that a sub‐basal‐body pad and bulbous axosome are unlikely to be apomorphies for the Stephanopogon–Percolomonas clade.     

Verrucophora farcimen gen. et sp. nov. (Dictyochophyceae,Heterokonta)—a bloom‐forming ichthyotoxic flagellate from the Skagerrak,Norway1

Since 1998, a heterokont flagellate initially named Chattonella aff. verruculosa has formed recurrent extensive blooms in the North Sea and the Skagerrak, causing fish mortalities. Cells were isolated from the 2001 bloom off the south coast of Norway, and monoalgal cultures were established and compared with the Chattonella verruculosa Y. Hara et Chihara reference strain NIES 670 from Japan. The cells in Norwegian cultured isolates were very variable in size and form, being large oblong (up to 34 μm long) to small rounded (5–9 μm in diameter) with two unequal flagella, numerous chloroplasts, and mucocysts. The SSU and partial LSU rDNA sequences of strains from Norway and Japan were compared and differed by 0.4% (SSU) and 1.3% (LSU), respectively. Five strains from Norway were identical in the LSU rDNA region. Phylogenetic analyses based on heterokont SSU and concatenated SSU + LSU rDNA sequences placed C. aff. verruculosa and the Japanese C. verruculosa within the clade of Dictyochophyceae, with the picoflagellate Florenciella parvula Eikrem as the closest relative. Ultrastructure, morphology, and pigment composition supported this affinity. We propose the name Verrucophora farcimen sp. et gen. nov. for this flagellate and systematically place it within the class Dictyochophyceae. Our studies also show that C. verruculosa from Japan is genetically and morphologically different but closely related to V. farcimen. The species is transferred from the class Raphidophyceae to the class Dictyochophyceae and renamed Verrucophora verruculosa. We propose a new order, Florenciellales, to accommodate V. farcimen, V. verruculosa, and F. parvula.     

A New Freshwater Amoeba: Cochliopodium pentatrifurcatum n. sp. (Amoebozoa,Amorphea)

Cochliopodium pentatrifurcatum n. sp. (ATCC^© 30935^TM) is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium pentatrifurcatum n. sp. (length ~ 25 μm) is characterized by surface microscales (0.3 μm tall) containing a circular porous base (~ 0.6 μm diam.) with a thin peripheral rim. Five radially arranged feet, emanating from the base, support a short central column terminating apically as a funnel‐shaped collar (~ 0.5 μm diam.) composed of five radial, trifurcate rays extending from the center toward a thin peripheral rim. The central spine is 0.5–0.6 μm long. The comparative morphologies and combined molecular genetic evidence, SSU‐rDNA and COI, indicate that the new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

THREE NEW BENTHIC SPECIES OF PROROCENTRUM (DINOPHYCEAE) FROM BELIZE: P. NORRISIANUM SP. NOV., P. TROPICALIS SP. NOV., and P. RETICULATUM SP. NOV.1

Three new benthic, photosynthetic dinoflagellate species, Prorocentrum norrisianum, Prorocentrum tropicalis, and Prorocentrum reticulatum, from floating detritus and coral rubble of Central America are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, thecal plate ornamentation, and architecture of the periflagellar area and intercalary band. Cells of P. norrisianum are ovate with a cell size of 20–25 μm long and 13–16 μm wide. The theca is delicate, its surface smooth, pores species specific with 95 to 105 pores per valve. Pores are round with a diameter of about 0.1 μm. The periflagellar area is V-shaped, located on the right valve in a shallow depression. It has no ornamentation. The flagellar and auxiliary pores are unequal in size. The intercalary band is smooth. Prorocentrum tropicalis cells are ovoid, 50–55 μm long and 40–45 μm wide in valve view with maximum width behind the middle region, narrow at the anterior end. The periflagellar area, situated in the right valve, is a V-shaped wide triangle with a deeply indented depression; the left valve exhibits a flat ridge. The periflagellar area is unornamented, and the flagellar and auxiliary pores are unequal in size. The valve surface is rugose with evenly distributed valve poroids. Each poroid appears to have a small dome in the center. The intercalary band is rimlike around the cell margin, granulated, and horizontally striated. Prorocentrum reticulatum cells are oblong in valve view; cells are 55–60 μm long and 40–45 μm wide. Thecal surface is reticulated; it is composed of a labyrinth of ridges with alternating depressions that vary in size and shape. Each depression has a narrow, oblong-kidney-shaped opening about 0.6 μm long. The periflagellar area is a deep, V-shaped triangle. The right valve of P. reticulatum is excavated, and contains a large flagellar pore and a smaller auxiliary pore surrounded by a narrow apical collar. The left valve margin exhibits a curved flat ridge. The intercalary band is smooth.     

Are Prorocentrum hoffmannianum and Prorocentrum belizeanum (DINOPHYCEAE,PROROCENTRALES), the same species? An integration of morphological and molecular data

The taxonomic assignment of Prorocentrum species is based on morphological characteristics; however, morphological variability has been found for several taxa isolated from different geographical regions. In this study, we evaluated species boundaries of Prorocentrum hoffmannianum and Prorocentrum belizeanum based on morphological and molecular data. A detailed morphological analysis was done, concentrating on the periflagellar architecture. Molecular analyses were performed on partial Small Sub‐Unit (SSU) rDNA, partial Large Sub‐Unit (LSU) rDNA, complete Internal Transcribed Spacer Regions (ITS1‐5.8S‐ITS2), and partial cytochrome b (cob) sequences. We concatenated the SSU‐ITS‐LSU fragments and constructed a phylogenetic tree using Bayesian Inference (BI) and maximum likelihood (ML) methods. Morphological analyses indicated that the main characters, such as cell size and number of depressions per valve, normally used to distinguish P. hoffmannianum from P. belizeanum, overlapped. No clear differences were found in the periflagellar area architecture. Prorocentrum hoffmannianum and P. belizeanum were a highly supported monophyletic clade separated into three subclades, which broadly corresponded to the sample collection regions. Subtle morphological overlaps found in cell shape, size, and ornamentation lead us to conclude that P. hoffmanianum and P. belizeanum might be considered conspecific. The molecular data analyses did not separate P. hoffmannianum and P. belizeanum into two morphospecies, and thus, we considered them to be the P. hoffmannianum species complex because their clades are separated by their geographic origin. These geographic and genetically distinct clades could be referred to as ribotypes: (A) Belize, (B) Florida‐Cuba, (C1) India, and (C2) Australia.     

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a “Well‐known” Euplotes Ciliate,Euplotes harpa Stein, 1859 (Ciliophora,Euplotida)

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and the previously well described Aspidisca lynceus (Müller, 1773 ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44–68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11–13 dorsal kineties with 17–25 dikinetids in the mid‐dorsal row, and dorsal silverline system of the double‐eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the “well‐known” Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this “well‐known” form in molecular databases requires further confirmation.     

Ultrastructure and Systematics of Two New Species of Dinoflagellate,Paragymnodinium Asymmetricum sp. nov. and Paragymnodinium Inerme sp. nov. (Gymnodiniales,Dinophyceae)1

The genus Paragymnodinium currently includes two species, P. shiwhaense and P. stigmaticum, that are characterized by mixotrophic nutrition and the possession of nematocysts. In this study, two new dinoflagellates belonging to this genus were described based on observations using LM, SEM, and TEM together with a molecular analysis. Cells of P. asymmetricum sp. nov., isolated from Nha Trang Beach, Vietnam, were 7.9–12.6 μm long and 4.7–9.0 μm wide. The species showed no evidence of feeding behavior and was able to sustain itself phototrophically. Paragymnodinium asymmetricum shared many features with P. shiwhaense, including presence of nematocysts, absence of an eyespot, and a planktonic lifestyle, but was clearly distinguished by the asymmetric shape of the hyposome, possession of a single chloroplast, and its nutritional mode. Cells of P. inerme sp. nov., isolated from Jogashima, Kanagawa Pref, Japan, were 15.3–23.7 μm long and 10.9–19.6 μm wide. This species also showed no evidence of feeding behavior. Paragymnodinium inerme was similar to cells of P. shiwhaense in shape and planktonic lifestyle, but its nutritional mode was different. The presence of incomplete nematocysts was also a unique feature. A phylogenetic analysis inferred from concatenated SSU and LSU rDNA sequences recovered the two dinoflagellates in a robust clade with Paragymnodinium spp., within the clade of Gymnodinium sensu stricto. This evidence, together with their morphological similarities, made it reasonable to conclude that these two dinoflagellates are new species of Paragymnodinium.     

Morphological and Molecular Identification of the New Species,Trichodina pseudoheterodentata sp. n. (Ciliophora,Mobilida, Trichodinidae) from the Channel Catfish,Ictalurus punctatus,in Chongqing China

It is difficult to differentiate similar trichodinids solely based on morphological examination, thus other identification methods, such as molecular identification, are necessary for identification. One mobilid ciliate named Trichodina pseudoheterodentata sp. n. was isolated from the gills of channel catfish, Ictalurus punctatus, in Chongqing, China. In the present study, its SSU rDNA was sequenced for the first time. Based on the results from both morphological identification and SSU rDNA sequencing, the new species was identified and compared with similar species. The morphological analysis revealed that T. pseudoheterodentata is a large Trichodina species (cell diameter 73.0–82.5 μm) and possesses robust denticles with broad blades and well‐developed blade connections. Characterization of its primary and secondary SSU rDNA structures indicated that T. pseudoheterodentata was distinctly different from congeneric species in H12, H15, E10_1, and V4 regions. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. pseudoheterodentata and its placement in the genus Trichodina.     

PHYLOGENETIC POSITION OF CRUSTOMASTIX STIGMATICA SP. NOV. AND DOLICHOMASTIX TENUILEPIS IN RELATION TO THE MAMIELLALES (PRASINOPHYCEAE,CHLOROPHYTA)1

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear‐encoded small subunit [SSU] rDNA and plastid‐encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zingone. Crustomastix stigmatica has a bean‐shaped cell body 3–5 μm long and 1.5–2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow‐green, cup‐shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid‐dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair‐like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonaxanthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.     

MORPHOLOGY AND MOLECULAR ANALYSES OF THREE TOXIC SPECIES OF GAMBIERDISCUS (DINOPHYCEAE): G. PACIFICUS, SP. NOV., G. AUSTRALES, SP. NOV., AND G. POLYNESIENSIS, SP. NOV.

Three new dinoflagellate species, Gambierdiscus polynesiensis, sp. nov., Gambierdiscus australes, sp. nov., and Gambierdiscus pacificus, sp. nov., are described from scanning electron micrographs. The morphology of the three new Gambierdiscus species is compared with the type species Gambierdiscus toxicus Adachi et Fukuyo 1979, and two other species: Gambierdiscus belizeanus Faust 1995 and Gambierdiscus yasumotoi Holmes 1998. The plate formula is: Po, 3′, 7", 6C, 8S, 5‴, 1p, 2". Culture extracts of these three new species displayed both ciguatoxin- and maitotoxin-like toxicities. The following morphological characteristics differentiated each species. 1) Cells of G. polynesiensis are 68–85 μm long and 64–75 μm wide, and the cell’s surface is smooth. They are identified by a large triangular apical pore plate (Po), a narrow fish-hook opening surrounded by 38 round pores, and a large, broad posterior intercalary plate (1p) wedged between narrow postcingular plates 2‴ and 4‴. Plate 1p occupies 60% of the width of the hypotheca. 2) Cells of G. australes also have a smooth surface and are 76–93 μm long and 65–85 μm wide in dorsoventral depth. They are identified by the broad ellipsoid apical pore plate (Po) surrounded by 31 round pores and a long and narrow 1p plate wedged between postcingular plates 2‴ and 4‴. Plate 1p occupies 30% of the width of the hypotheca. 3) Cells of G. pacificus are 67–77 μm long and 60–76 μm wide in dorsoventral depth, and its surface is smooth. They are identified by the four-sided apical pore plate (Po) surrounded by 30 round pores. A short narrow 1p plate is wedged between the wide postcingular plates 2‴ and 4‴. Plate 1p occupies 20% of the width of the hypotheca. These three newly described species were also characterized by isozyme electrophoresis and DNA sequencing of the D8–D10 region of their large subunit (LSU) rRNA genes. The consistency between species designations based on SEM microscopy and classification inferred from biochemical and genetic heterogeneities was examined among seven isolates of Gambierdiscus. Their classification into four morphospecies was not consistent with groupings inferred from isozyme patterns. Three molecular types could be distinguished based on the comparison of their LSU rDNA sequences. Although G. toxicus TUR was found to be more closely related to G. pacificus, sp. nov. than to other G. toxicus strains, the molecular classification was able to discriminate G. polynesiensis, sp. nov. and G. australes, sp. nov. from G. toxicus. These results suggest the usefulness of the D8–D10 portion of the Gambierdiscus LSU rDNA as a valuable taxonomic marker.     

The Taxonomy and Phylogeny of the Poorly known Heterotrich Ciliate Ampullofolliculina lageniformis Hadži, 1951 (Ciliophora: Heterotrichea)

Ampullofolliculina Had?i, 1951 is a monotypic genus, the only species being A. lageniformis which was first discovered in estuarine habitats in the U.S. and has never been redescribed. In the present study, we investigated the taxonomy and phylogeny of A. lageniformis Had?i, 1951 based on analyses of a population collected from a brackish wetland in Ningbo, China. The main characteristics of this species are as follows: trophont about 450–700 μm long in vivo with two short, rounded peristomial lobes of equal size; lorica flask‐shaped, transparent and smooth with a short, wide neck at the base of which two transparent valves are asymmetrically inserted; cortex with brownish to reddish cortical granules and greenish pigment granules; about 80 somatic kineties evenly arranged; moniliform macronucleus with 4–8 ellipsoidal nodules; swarmer dark green, vermiform in shape, about 200–350 μm long in vivo, with about 60 adoral membranelles and 85 somatic kineties, no mouth nor paroral membrane. Phylogenetic analyses inferred from SSU rDNA sequences show that A. lageniformis is closely related to Folliculina and Eufolliculina which nest within a large clade that comprises five families, i.e. Stentoridae, Blepharismidae, Fabreidae, Maristentoridae, and Folliculinidae.