A New Halophilic Heterolobosean Flagellate,Aurem hypersalina gen. n. et sp. n., Closely Related to the Pleurostomum‐Tulamoeba Clade: Implications for Adaptive Radiation of Halophilic Eukaryotes

Halophilic protozoa are independently scattered across the molecular phylogeny of eukaryotes; most of which are assigned to Heterolobosea. Here, we isolated a biflagellate from a hypersaline water of 342‰ salinity. This isolate shared several morphological features with typical halophilic heterolobosean flagellates. In addition, molecular phylogenetic trees of the 18S rRNA gene sequences clearly indicated flagellate is a heterolobosean species closely related to the halophilic Tulamoebidae. However, the flagellate was not accommodated to any described genus. Cells were ovoid‐shaped, and no amoebae were observed. The two unequal flagella beat heterodynamically. An ear‐like bulge at the margin of a cytostomal groove was observed. Flagellates could grow at 100–200‰ salinity, suggesting an obligately halophilic species. Currently, it appears that the new halophilic Aurem hypersalina forms a strong clade with Tulamoebidae, and is sister to the Tulamoebidae, indicating that this new clade is composed almost entirely of obligate halophilic taxa. Thus, A. hypersalina and the Tulamoebidae clade currently represent a unique adaptive radiation of halophilic eukaryotes.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Morphology and Molecular Phylogeny of Trimyema koreanum n. sp., a Ciliate from the Hypersaline Water of a Solar Saltern

ABSTRACT. A new ciliate, Trimyema koreanum n. sp., isolated from hypersaline water (salinity of 293‰) from a solar saltern in Korea, was investigated using live observation, protargol impregnation, and gene sequencing. Trimyema koreanum is about 30 × 13 μm in vivo, has usually 23 longitudinal ciliary rows forming two distinct ciliary girdles visible both in vivo and in protargol impregnation. A third indistinct ciliary girdle as well as a girdle of mucocysts is distinguishable only in impregnated cells. We suggest T. koreanum as a new species, differing from the most similar species, T. marinum, by the presence of two distinct ciliary girdles (T. marinum usually has six ciliary girdles clearly visible in living cells and three anterior spirals that encircle the cell completely). Although the number of known 18S rRNA sequences in the genus Trimyema was limited, the Trimyema group including T. koreanum forms a strong clade. The phylogenetic position confirms that the isolate belongs to the genus Trimyema and is different from previously sequenced species. Trimyema koreanum is able to consume both prokaryotes and small eukaryotes (specifically, the alga Dunaliella sp.).     

Molecular Phylogeny and Ontogeny of a New Ciliate Genus,Paracladotricha salina n. g., n. sp. (Ciliophora,Hypotrichia)

A hypotrichous ciliate, Paracladotricha salina n. g., n. sp., was discovered in hypersaline waters (salinity about 80‰) from Qingdao, China. Its morphology and some major ontogenetic stages were studied and the phylogenetic position was estimated using standard methods. Paracladotricha salina is characterized by a flexible, more or less slender body (size 50–120 × 20–35 μm), a gonostomatid oral apparatus, one short and two long frontoventral rows, four macronuclear nodules, almost completely reduced dorsal kineties 1–3, and a loss of several parts of the ciliature, namely, the slightly shortened ciliary row of the adoral membranelles, the paroral, and the buccal, the postoral and pretransverse ventral, the transverse, and the caudal cirri. The ontogenesis is rather simple: anlage II of both filial products and anlage III of the opisthe originate de novo, while anlagen IV and V are formed within the parental rows. This combination of features requires the establishment of a new genus, Paracladotricha, which is, according to the morphological data, closely related to Schmidingerothrix and Cladotricha. The small‐subunit rRNA gene was sequenced, indicating that P. salina is, as also demonstrated by the oral apparatus, a member of the gonostomatids. We provide a first, vague hypothesis about the phylogenetic relationships of the Gonostomatidae, Cladotrichidae, and Schmidingerotrichidae. However, since molecular data of the type species of these higher taxa are lacking, their validity and relationships remain obscure.     

Effects of different ion compositions on growth of obligately halophilic protozoan Halocafeteria seosinensis

Substantial halophilic organisms have been found in 100–200‰ salinities. These ranges represent a highly specialized halophilic environment to which only a few halotolerant species have adapted. Recent studies have underlined the existence of diverse obligately halophilic protozoa in the salinity ranges of 100–200‰. The ranges of salinity under which these organisms can grow have been examined to some extent, but the balance of specific ions that will support growth has not been investigated. The heterotrophic nanoflagellate Halocafeteria, the type strain of which grows optimally at 150‰ salinity and 35°C, is a commonly encountered obligate halophile found in very hypersaline environments. These extreme environments can vary in their Mg:Ca ratios (i.e. weight ratios) and sulfate concentrations. To examine growth response of Halocafeteria to the different chemical compositions, densities of Halocafeteria seosinensis strain EHF34 were monitored in seven different ion composition media for 9 days at 1- to 2-day intervals (at 150‰ salinity and 35°C, with no prey limitation). Halocafeteria does not grow at Mg:Ca ratios of 35 and 100 and at high sulfate concentrations of 11.6 and 31.6 g l⁻¹. It grows well in 0.6 g l⁻¹ sulfate media at Mg:Ca ratios of 2, 10 or 35, but not 100. The present study demonstrates that the growth of the obligate halophile Halocafeteria can be affected by different ion compositions in hypersaline environments. Therefore, Halocafeteria may not be ubiquitous in hypersaline environments due to its ionic requirements.     

Characterization of Selenaion koniopes n. gen., n. sp., an Amoeba that Represents a New Major Lineage within Heterolobosea,Isolated from the Wieliczka Salt Mine

A new heterolobosean amoeba, Selenaion koniopes n. gen., n. sp., was isolated from 73‰ saline water in the Wieliczka salt mine, Poland. The amoeba had eruptive pseudopodia, a prominent uroid, and a nucleus without central nucleolus. Cysts had multiple crater‐like pore plugs. No flagellates were observed. Transmission electron microscopy revealed several typical heterolobosean features: flattened mitochondrial cristae, mitochondria associated with endoplasmic reticulum, and an absence of obvious Golgi dictyosomes. Two types of larger and smaller granules were sometimes abundant in the cytoplasm—these may be involved in cyst formation. Mature cysts had a fibrous endocyst that could be thick, plus an ectocyst that was covered with small granules. Pore plugs had a flattened dome shape, were bipartite, and penetrated only the endocyst. Phylogenies based on the 18S rRNA gene and the presence of 18S rRNA helix 17_1 strongly confirmed assignment to Heterolobosea. The organism was not closely related to any described genus, and instead formed the deepest branch within the Heterolobosea clade after Pharyngomonas, with support for this deep‐branching position being moderate (i.e. maximum likelihood bootstrap support—67%; posterior probability—0.98). Cells grew at 15–150‰ salinity. Thus, S. koniopes is a halotolerant, probably moderately halophilic heterolobosean, with a potentially pivotal evolutionary position within this large eukaryote group.     

Marinamoeba thermophila, a new marine heterolobosean amoeba growing at 50 °C

Two amoeba strains were isolated from marine sediment taken at the same place with 18 months interval from a region of the sea floor heated by extended submarine hot springs and fumaroles. These thermophilic amoebae grow at temperatures up to 50 °C. Sequences of the internal transcribed spacer demonstrated that the two strains belong to the same species and are different from any genus for which sequences are known. Phylogeny using small subunit ribosomal RNA places the amoeba in the Heterolobosea. Their closest relatives are the hypersaline flagellate Pleurostomum flabellatum and the hypersaline amoeba Tulamoeba peronaphora. The freshwater amoeboflagellate genera Naegleria and Willaertia belong to the same phylogenetic clade in the Vahlkampfiidae. The new marine species does not transform into flagellates. It forms cysts, which are round to ellipsoidal with few pores. Because of their unique place in the molecular phylogenetic tree, and because there is no morphologically identical species found in the literature, these isolates are considered to be a new species and a new genus, Marinamoeba thermophila.     

Characterization of a Deep‐Branching Heterolobosean,Pharyngomonas turkanaensis n. sp., Isolated from a Non‐Hypersaline Habitat,and Ultrastructural Comparison of Cysts and Amoebae Among Pharyngomonas Strains

An unusual heterolobosean amoeba, isolate LO, was isolated recently from a sample with a salinity of ~4‰, from Lake Turkana in East Africa. 18S rDNA phylogenies confirm that isolate LO branches among halophilic amoeboflagellates assigned to Pharyngomonas. We examined the ultrastructure of the amoeba and cyst stages of isolate LO, as well as the amoebae and cysts of Pharyngomonas kirbyi (isolates AS12B and SD1A). The amoebae of all three isolates lacked discrete dictyosomes and had discoidal/flattened mitochondrial cristae, but the mitochondria were not enrobed by rough endoplasmic reticulum. The cysts of all three isolates showed a thick, bipartite cyst wall, and lacked cyst pores. The cysts of isolate LO were distinct in that the ectocyst was very loose‐fitting, and could contain “crypts”. No flagellate form of isolate LO has been observed to date, and a salinity‐for‐growth experiment showed that isolate LO can grow at 15–100‰ salinity, indicating that it is halotolerant. By contrast, other studied Pharyngomonas isolates are amoeboflagellates and true halophiles. Therefore, we propose isolate LO as a new species, Pharyngomonas turkanaensis n. sp. It is possible that P. turkanaensis descended from halophilic ancestors, and represents a secondary reestablishment of a physiology adapted for moderate salinity.     

Euchlorocystis gen. nov. and Densicystis gen. nov., Two New Genera of Oocystaceae Algae from High‐altitude Semi‐saline Habitat (Trebouxiophyceae,Chlorophyta)

The Oocystaceae family is generally considered to contain common freshwater eukaryotic microalgae, and few are reported living in semi‐saline habitats. Our latest ecological survey in Qinghai Lake and Angzicuo Lake, both large, closed, high‐altitude, semi‐saline lakes located on the Qinghai‐Tibet plateau in China, revealed Oocystaceae species as a dominant group among plankton. Since limited knowledge exists about semi‐saline species in the Oocystaceae family, a taxonomical study was carried out using morphological and phylogenetic methods. Using this approach, four new strains of Oocystaceae were identified and successfully cultured in the lab. Molecular results correlated with morphological characters and resolved these species into at least three genera. A new genus, Euchlorocystis, with type species Euchlorocystis subsalina, is described here as having the distinctive morphology of multiple pyrenoids per chloroplast among Oocystaceae, and an independent phylogenetic position at the base of the Oocystaceae. Similarly, the genus Densicystis, with type species Densicystis glomerata, is newly proposed here as having a unique colony morphology of dozens or hundreds of little cells tightly embedded in ellipsoid to round mucilage masses. Oocystis marina, originally described from the Baltic Sea, was also identified in Qinghai Lake and Angzicuo Lake and phylogenetically positioned in the semi‐saline clade of the Oocystaceae. The result that a marine species was detected in the closed inland lakes implies a further need to reevaluate the origins of these species.     

Effects of temperature and salinity on the larvae of two species of rhizocephalan (Crustacea: Cirripedia)

Summary

Responses of larvae of two rhizocephalan species to changes in seawater temperature and salinity were studied under laboratory conditions. Peltogasterella gracilis parasitizes the hermit crab Pagurus pectinatus, which occurs at stable salinity and gradually changing temperature in summer. Sacculina polygenea is a parasite of the crab Hemigrapsus sanguineus, which lives in the intertidal zone in summer where salinity and temperature can fluctuate during the day. The development of both species is comprised of five naupliar stages and the cyprid stage, and it was considered successful if more than 50% of the nauplii attained the cyprid stage. P. gracilis nauplii successfully developed at 12–20°C and 30–34‰, but at 22°C successful development occurred in a narrower salinity range (32–34‰). All nauplii died both at 25°C and in 26‰. S. polygenea nauplii successfully reached the cyprid stage at higher temperatures (18–25°C) and a wider salinity range (18–34‰) than P. gracilis nauplii, but at 12°C and 16‰ larval development of S. polygenea was suppressed. Under favorable conditions, naupliar development lasted 3.5 days in P. gracilis and 2–3 days in S. polygenea. The cyprids of both rhizocephalan species demonstrated a greater resistance to temperature and salinity changes than nauplii. However, P. gracilis cyprids were active in a narrower salinity range (16–34‰), as compared to S. polygenea cyprids (8–34‰). Under favorable conditions the cyprids of both species survived for 6 to 10 days.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

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

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

Characterization of Pharyngomonas kirbyi (= "Macropharyngomonas halophila" nomen nudum), a very deep-branching, obligately halophilic heterolobosean flagellate

The tetraflagellate Pharyngomonas is among the most commonly reported morphotypes of halophilic protozoa. We have established two cultures of Pharyngomonas kirbyi, SD1A and AS12B, from 300‰ and 210‰ salinity waters from the USA and Australia, respectively. 18S rRNA gene phylogenies confirm that Pharyngomonas is the same entity as 'Macropharyngomonas' (nomen nudum), and represents the deepest branch in the heterolobosean lineage. Pharyngomonas kirbyi (Strain SD1A) has flattened/discoidal cristae, and lacks conspicuous Golgi dictyosomes. It also has a heterolobosean 'double bikont' flagellar apparatus, with two right roots, each associated with an 'I' fibre and part of a rhizoplast-like complex. One right root splits shortly after its origin, and supplies most of the microtubules that support both the ventral groove, and the sub-anterior cytopharynx. Interestingly, Pharyngomonas has some potentially ancestral features not found in typical Heterolobosea, including elongated left roots associated with multilayered 'C' fibres, orthogonal basal bodies, and a spur structure that might represent a 'B' fibre homolog. Both isolates are obligate halophiles that grow best at 100-200‰ salinity and do not grow below 75‰ salinity. Pharyngomonas is therefore of considerable evolutionary importance, both as a deep-branching, plesiomorphic heterolobosean, and a borderline extreme halophile.     

Molecular Identity of Holomastigotes (Spirotrichonymphea,Parabasalia) with Descriptions of Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp.

Holomastigotes is a protist genus (Parabasalia: Spirotrichonymphea) that resides in the hindguts of “lower” termites. It can be distinguished from other parabasalids by spiral flagellar bands that run along the entire length of the cell, an anterior nucleus, a reduced or absent axostyle, the presence of spherical vesicles inside the cells, and the absence of ingested wood particles. Eight species have been described based on their morphology so far, although no molecular data were available prior to this study. We determined the 18S rRNA gene sequences of Holomastigotes from the hindguts of Hodotermopsis sjostedti, Reticulitermes flavipes, Reticulitermes lucifugus, and Reticulitermes tibialis. Phylogenetic analyses placed all sequences in an exclusive and well‐supported clade with the type species, Holomastigotes elongatum from R. lucifugus. However, the phylogenetic position of Holomastigotes within the Spirotrichonymphea was not resolved. We describe two new species, Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp., inhabiting the hindguts of R. flavipes and R. tibialis, respectively.     

Vannella samoroda n. sp. (Amoebozoa) — First member of the genus from a continental saline habitat placed in a molecular tree

Vannella samoroda n. sp. (Amoebozoa, Vannellida) was isolated from the mouth of the Malaya Samoroda river flowing into Elton, the largest European hypersaline lake (Russia). Among all rivers of the area, it has the highest salt content (ca. 110‰). Amoebae maintained in seawater medium with ca. 77‰ salts concentration had a set of morphological characters typical of Vannella spp.: rounded, fan-shaped, or spatulate locomotive form, floating form with bent, blunt-ended hyaline pseudopodia, and a cell coat consisting of regularly packed palisade elements and scarce simple filaments. Phylogenetic analyses based on SSU rRNA and cytochrome C oxidase subunit 1 genes show that the amoeba is most closely related to Vannella ebro Smirnov, 2001, but represents a distinct species. The clade of V. ebro and V. samoroda branches among marine species of Vannella. The studied species is the first member of the genus Vannella from a continental saline habitat described using molecular data. Interestingly, it has a broad range of salinity tolerance: cells reproduce above 18‰, while survival of a few cells regularly occurs even in highly diluted Prescott and James medium. The normal culture restores itself when PJ medium is substituted with 77‰ seawater medium even after months of experimental incubation.     

Growth, maturation and photosynthesis of the brackish water alga Rhizoclonium sp. (Cladophoraceae, Chlorophyta) in relation to salinity

The effects of salinity on growth, maturation and photosynthesis were examined in the filamentous alga Rhizoclonium sp. (Cladophoraceae, Chlorophyta) growing in a brackish water habitat in a canal draining into Tokyo Bay, Japan. In this habitat Rhizoclonium sp. was exposed to a wide salinity range, both daily, 5–23‰ during November 1996, and hourly, 6–24‰ during the spring tide day. From the results of culture experiments, growth and maturation of Rhizoclonium sp. occurred in the wide salinity range of 10–40‰ at 20 μmol photons m‐²s‐¹ at 20°C, but did not occur at salinity of 0‰. Light saturation on the photosynthesis‐irradiance curve at 20°C at 20‰ was reached at 100 μmol photons m‐²s‐¹, which is characteristic for shade‐adapted algae. On the photosynthesis‐salinity curve at 20°C at saturated irradiance (160 μmol photons m‐²s‐¹), the net photosynthetic rate increased with increasing salinity up to 30‰ but decreased at 40‰. On the photosynthesis–salinity curve at 20°C at 20 μmol photons m‐²s‐¹ (at near in situ irradiance), the photosynthetic rates were almost the same in the salinity range from 0 to 40‰. Therefore, this species is able to grow, reproduce and photosynthesize with a relative efficiency in a wide salinity range, which shows that it is well adapted to a brackish water environment.     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.