Relationship between the dinoflagellate cyst Spiniferites pachydermus and Gonyaulax ellegaardiae sp. nov. from Izmir Bay,Turkey, and molecular characterization

Here, we established the cyst‐motile stage relation‐ship for Spiniferites pachydermus through incubation of cysts with a characteristically microreticulate/perforate surface isolated from Izmir Bay in the eastern Aegean Sea of the eastern Mediterranean. The morphology of the motile stage was similar to Gonyaulax spinifera but had a different size, overhang, displacement and reticulations. Based on the distinct morphology of the cyst and morphological differences in motile cells, we assigned S. pachydermus from Izmir Bay to the new species Gonyaulax ellegaardiae. We elucidate the phylogenetic relationship of G. ellegaardiae through large and small subunit ribosomal DNA and show that it forms a clade with other species that belong to the G. spinifera complex.     

Morphology,ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales,Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium‐like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop‐shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.     

CYST–THECA RELATIONSHIP,LIFE CYCLE,AND EFFECTS OF TEMPERATURE AND SALINITY ON THE CYST MORPHOLOGY OF GONYAULAX BALTICA SP. NOV. (DINOPHYCEAE) FROM THE BALTIC SEA AREA1

A new species of Gonyaulax, here named Gonyaulax baltica sp. nov., has been isolated from sediment samples from the southeastern Baltic. Culture strains were established from individually isolated cysts, and cyst formation was induced in a nitrogen‐depleted medium. Although G. baltica cysts are similar to some forms attributed to Spiniferites bulloideus and the motile stage of G. baltica has affinities with G. spinifera, the combination of features of cyst and motile stage of G. baltica is unique. The culture strains were able to grow at salinity levels from 5 to 55 psu and formed cysts from 10 to 50 psu. Cultures at each salinity level were grown at 12, 16, and 20° C. Temperature‐ and salinity‐controlled morphological variability was found in the resting cysts. Central body size varied with temperature and salinity, and process length varied with salinity. Cysts that formed at extreme salinity levels displayed lower average process length than cysts formed at intermediate salinity levels, and central body length and width were lowest at higher temperature and lower salinity. Models for the relationship between central body size and temperature/salinity and process length and salinity have been developed and may be used to determine relative paleosalinity and paleotemperature levels. Our results on salinity‐dependent process length confirm earlier reports on short‐spined cysts of this species found in low salinity environments, and the model makes it possible to attempt to quantify past salinity levels.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

Huia caspica gen. & comb. nov., a dinoflagellate species that recently crossed the marine‐freshwater boundary

The dinoflagellate subfamily Diplopsalidoideae encompasses 11 genera whose plate patterns show a large diversity. In a recently published molecular phylogeny (Liu et al. 2015) some of these genera (e.g. Diplopsalis, Diplopelta) are polyphyletic, suggesting that further subdivision of these genera is needed. Here we established the cyst‐theca relationship of Diplopsalis caspica by incubating cysts collected from the East China Sea. Cells of D. caspica display a plate formula of Po, X, 3′, 1a, 6″, 3c+t, ?4s, 5″′, 1″″, characterized by a small, parallelogrammic anterior intercalary plate (1a) located in the middle of the dorsal part of the epitheca. The cysts are spherical and smooth‐walled with a theropylic archeopyle. In addition, we obtained four large subunit ribosomal DNA (LSU rDNA) sequences from the germinated motile cells by single‐cell polymerase chain reaction. Strains of D. caspica from the marine environment of the East China Sea differ at 0–2 positions of LSU rDNA sequences from that of lacustrine strains from NE China. In the molecular phylogeny, D. caspica was close to Lebouraia pusilla but distant from D. lenticula, the type species of Diplopsalis. Our results support the systematic importance of plate 1a, and therefore D. caspica was transferred to a new genus, Huia. The conservative LSU rDNA sequences in H. caspica suggest that the marine‐freshwater transition occurred recently.     

Molecular phylogeny of the Cladophoraceae (Cladophorales,Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen,and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica.     

Phylogenetic Reappraisal of the Genus Monomorphina (Euglenophyceae) Based on Molecular and Morphological Data

A morphological and molecular examination of the genus Monomorphina was conducted on 46 strains isolated mainly from Korea. The strains were divided into two types based on morphological data: Monomorphina aenigmatica and M. pyrum ‐ like species. Phylogenetic analysis based on a combined data set of nuclear SSU and LSU and plastid SSU and LSU rDNA showed that the strains could be divided into eight clades: Clade A of M. aenigmatica, Clade B of the isolates (M. pyropsis) from Michigan, USA, Clade C of M. pseudopyrum, Clade D of the isolates (M. pyroria) from Bremen, Germany, Clade E of M. soropyrum, Clade F of M. pyriformis, Clade G of M. parapyrum, and Clade H of M. pyrum. Six of these clades came from strains that would be considered M. pyrum sensu Kosmala et Zakry?, one of which could be recognized as a traditional species (M. pyrum) and five were designated as new species; each species had unique molecular signatures at nr SSU rDNA helix 17 and 17′ and spacer E23_14′‐E23_15. The species of Monomorphina had a wide range of genetic diversity with interspecies sequence similarity of 85.6%–97.1% and intraspecies similarity of 96.4%–99.9%. Our results suggested that genetic diversity found in the M. pyrum complex justifies the recognition of a minimum of eight species within this genus, based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Classification of crucigenoid algae: phylogenetic position of the reinstated genus Lemmermannia,Tetrastrum spp. Crucigenia tetrapedia,and C. lauterbornii (Trebouxiophyceae,Chlorophyta)1

The subfamily Crucigenioideae was traditionally classified within the well‐characterized family Scenedesmaceae (Chlorophyceae). Several morpho‐logical revisions and questionable taxonomic changes hampered the correct classification of crucigenoid species resulting in a high number of synonymous genera. We used a molecular approach to determine the phylogenetic position of several Tetrastrum and Crucigenia species. The molecular results were correlated with morphological and ontogenetic characters. Phylogenetic analyses of the SSU rDNA gene resolved the position of Tetrastrum heteracanthum and T. staurogeniaeforme as a new lineage within the Oocystis clade of the Trebouxiophyceae. Crucigenia tetrapedia, T. triangulare, T. punctatum, and T. komarekii were shown to be closely related to Botryococcus (Trebouxiophyceae) and were transferred to Lemmer‐mannia. Crucigenia lauterbornii was not closely related to the other Crucigenia strains, but was recovered within the Chlorella clade of the Trebouxiophyceae.     

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.     

Cyst‐motile stage relationship,morphology, ultrastructure,and molecular phylogeny of the gymnodinioid dinoflagellate Barrufeta resplendens comb. nov., formerly known as Gyrodinium resplendens,isolated from the Gulf of Mexico

In the present study, we redescribed Gyrodinium resplendens through incubation of process bearing cysts extracted from sediment collected in the northern Gulf of Mexico. The morphology and ultrastructure of the motile stage and cyst stage were examined using light microscopy, scanning electron microscopy, and transmission electron microscopy and this revealed that the species should be transferred to the genus Barrufeta. This genus differs from other gymnodinioid genera in possessing a Smurf‐cap apical structure complex (ASC) and currently encompasses only one species, Barrufeta bravensis. B. resplendens shows a Smurf‐cap ASC that consists of three rows of elongated vesicles with small knobs in the middle one. B. resplendens is very similar to B. bravensis in cell morphology, but can be separated using the ultrastructure such as the shape and location of nucleus and pyrenoids, which highlights the importance of ultrastructure at inter‐specific level in the genus Barrufeta. The unique cysts of B. resplendens are brown and process bearing, and have a tremic archeopyle with a zigzag margin on the dorsal side of the epicyst, and not polar as in cysts of Polykrikos. The cysts do not survive the palynological treatment used here and probably have a wide distribution. Maximum‐likelihood and Bayesian inference were carried out based on partial large subunit ribosomal DNA (LSU rDNA) sequences. Molecular phylogeny supports that the genus Barrufeta is monophyletic, and that the genus Gymnodinium is polyphyletic. Our results suggest that details of the ASC together with ultrastructure are potential features to subdivide the genus Gymnodinium.     

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo‐nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo‐nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo‐nitzschia species containing only chlorophyll c₂ and c₃.     

Brandtodinium gen. nov. and B. nutricula comb. Nov. (Dinophyceae), a dinoflagellate commonly found in symbiosis with polycystine radiolarians

Symbiotic interactions between pelagic hosts and microalgae have received little attention, although they are widespread in the photic layer of the world ocean, where they play a fundamental role in the ecology of the planktonic ecosystem. Polycystine radiolarians (including the orders Spumellaria, Collodaria and Nassellaria) are planktonic heterotrophic protists that are widely distributed and often abundant in the ocean. Many polycystines host symbiotic microalgae within their cytoplasm, mostly thought to be the dinoflagellate Scrippsiella nutricula, a species originally described by Karl Brandt in the late nineteenth century as Zooxanthella nutricula. The free‐living stage of this dinoflagellate has never been characterized in terms of morphology and thecal plate tabulation. We examined morphological characters and sequenced conservative ribosomal markers of clonal cultures of the free‐living stage of symbiotic dinoflagellates isolated from radiolarian hosts from the three polycystine orders. In addition, we sequenced symbiont genes directly from several polycystine‐symbiont holobiont specimens from different oceanic regions. Thecal plate arrangement of the free‐living stage does not match that of Scrippsiella or related genera, and LSU and SSU rDNA‐based molecular phylogenies place these symbionts in a distinct clade within the Peridiniales. Both phylogenetic analyses and the comparison of morphological features of culture strains with those reported for other closely related species support the erection of a new genus that we name Brandtodinium gen. nov. and the recombination of S. nutricula as B. nutricula comb. nov.     

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.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Species concept and nomenclatural changes within the genera Elliptochloris and Pseudochlorella (Trebouxiophyceae) based on an integrative approach

The genera Elliptochloris and Pseudochlorella were erected for Chlorella‐like green algae producing two types of autospores and cell packages, respectively. Both genera are widely distributed in different soil habitats, either as free living or as photobionts of lichens. The species of these genera are often difficult to identify because of the high phenotypic plasticity and occasional lack of characteristic features. The taxonomic and nomenclatural status of these species, therefore, remains unclear. In this study, 34 strains were investigated using an integrative approach. Phylogenetic analyses demonstrated that the isolates belong to two independent lineages of the Trebouxiophyceae (Elliptochloris and Prasiola clades) and confirmed that the genera are not closely related. The comparison of morphology, molecular phylogeny, and analyses of secondary structures of SSU and ITS rDNA sequences revealed that all of the strains belong to three genera: Elliptochloris, Pseudochlorella, and Edaphochlorella. As a consequence of the taxonomic revisions, we propose two new combinations (Elliptochloris antarctica and Pseudochlorella signiensis) and validate Elliptochloris reniformis, which is invalidly described according to the International Code for Nomenclature (ICN), by designating a holotype. To reflect the high phenotypic plasticity of P. signiensis, two new varieties were described: P. signiensis var. magna and P. signiensis var. communis. Chlorella mirabilis was not closely related to any of these genera and was, therefore, transferred to the new genus Edaphochlorella. All of the taxonomic changes were highly supported by all phylogenetic analyses and were confirmed by the ITS‐2 Barcodes using the ITS‐2/CBC approach.