Aitor Laza-Martínez

Morphological and genetic characterization of benthic dinoflagellates of the genera Coolia, Ostreopsis and Prorocentrum from the south-eastern Bay of Biscay

Benthic dinoflagellates of the genera Coolia, Ostreopsis and Prorocentrum isolated from coastal waters of the south-eastern Bay of Biscay were identified morphologically by means of light microscopy (LM) including epifluorescence microscopy, and scanning electron microscopy (SEM). To identify the strains to species level, molecular phylogenetic analyses using the nuclear large subunit rDNA (LSU) were performed for 16 strains of the three genera. These morphological and phylogenetic analyses revealed the presence of the following species: Coolia canariensis S. Fraga, Coolia monotis Meunier, Ostreopsis cf. siamensis Schmidt, Prorocentrum emarginatun Fukuyo, P. lima (Ehrenberg) Dodge, P. rhathymum Loeblich III, Sherley & Schmidt, and two as yet unidentified species, which in the phylogenetic tree were grouped with different strains of Prorocentrum emarginatun and P. fukuyoi Murray et Nagahama from GenBank. A strain from Minorca (Balearic Islands, western Mediterranean Sea) analysed in this study for comparative purposes and fitting morphologically into the P. emarginatum/P. fukuyoi group also appeared in this cluster, which seems to include morphologically cryptic or semicryptic species. The most common taxa were Coolia monotis, Ostreopsis cf. siamensis and Prorocentrum lima, which appeared at most sampling sites. Only the strains corresponding to Ostreopsis cf. siamensis and Prorocentrum lima were toxic to Artemia franciscana.

NOVEL UNARMORED DINOFLAGELLATES FROM THE TOXIGENIC FAMILY KARENIACEAE (GYMNODINIALES): FIVE NEW SPECIES OF KARLODINIUM AND ONE NEW TAKAYAMA FROM THE AUSTRALIAN SECTOR OF THE SOUTHERN OCEAN1

Six new species of unarmored dinoflagellates in the family Kareniaceae were isolated from the Australian sector of the Southern Ocean in March 2006: Takayama tuberculata de Salas sp. nov, Karlodinium antarcticum de Salas sp. nov., Karl. ballantinum de Salas sp. nov., Karl. conicum de Salas sp. nov., Karl. corrugatum de Salas sp. nov., and Karl. decipiens de Salas et Laza‐Martínez sp. nov. These new taxa were characterized using light and electron microscopy and sequencing of the LSU rDNA and are well supported based either on their morphology or molecular phylogeny. Takayama tuberculata, isolated just north of the polar front (55°–57° S), is genetically close to T. tasmanica, but smaller, with a significantly reduced number of amphiesmal vesicles. Medium‐sized Karl. antarcticum, also isolated from near the polar front, is characterized by its long ovoid cell outline and very long apical groove. The small Karl. ballantinum has a very short apical groove. The large Karl. conicum has a distinct conical epicone and spherical posterior nucleus. The small Karl. corrugatum, from just south of the polar front, has distinctive parallel striations on the epicone surface and a distinctively shaped and placed ventral pore. The large and widespread Karl. decipiens, distributed through Southern Ocean waters from the polar front to Tasmanian coastal waters, and coastal Spain, has a helicoidal chloroplast arrangement and a large central nucleus. This study represents the first description of species in the potentially ichthyotoxic family Kareniaceae recorded from the Southern Ocean.

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.

DIVERSITY OF PSEUDO-NITZSCHIA IN THE SOUTHEASTERN BAY OF BISCAY

Ten species of the genus Pseudo-nitzschia were identified in the Nervion River estuary from preserved net tow samples and 31 strains isolated from the estuary. Species identification was performed by means of ultrastructural analysis of valve ornamentation and genetic analysis of selected strains of the P. delicatissima complex. Identified species include: P. australis, P. fraudulenta, P. pungens and P. subpacifica from the P. seriata complex, and P. arenysensis, P. galaxiae, P. multistriata, P. pseudodelicatissima and two uncertain P. pseudodelicatissima-like genotypes from the delicatissima complex. Most clonal strains corresponded to P. pseudodelicatissima-like taxa, P. fraudulenta and P. galaxiae. We were unable to identify the strains of the P. delicatissima-like complex solely on the basis of morphology. A comparison of sequences of the rDNA ribosomal Internal Transcribed Spacers (ITS), including the regions ITS1, 5.8S and ITS2, of four strains of the P. delicatissima complex plus 23 sequences of taxonomically related strains available in GenBank, revealed the presence of P. arenysensis among strains of the P. delicatissima-morphologies and three different ITS types among the three P. pseudodelicatissima-like morphologies analysed. One corresponded to P. pseudodelicatissima sensu stricto (strain Ner-D5) whereas the other two (Ner-D6 and Ner-D8) constituted genetically distinct entities, which appeared in the phylogenetic tree as sister taxa to either P. cuspidata, the former, or to P. calliantha and P. mannii, the latter. The genetic differences among these three strains of P. pseudodelicatissima-like morphologies were corroborated by analysing their ITS2 secondary structure and comparing them with the ITS2 secondary structure of phylogenetically related species from GenBank.

URGORRI COMPLANATUS GEN. ET SP. NOV. (CRYPTOPHYCEAE), A RED-TIDE-FORMING SPECIES IN BRACKISH WATERS(1).

The morphology, ultrastructure, phylogeny, and ecology of a new red‐tide‐forming cryptomonad, Urgorri complanatus Laza‐Martínez gen. et sp. nov., is described. U. complanatus has been collected in southwestern European estuaries, blooming in the inner reaches of several of them. The estuarine character of the species is also supported by its in vitro salinity preferences, showing a maximum growth rate at 10 psu. U. complanatus is a distinctive species and can be easily distinguished by LM from other known brackish and marine species. Cells are dorsoventrally flattened. The plastid has two anterior lobes. One pyrenoid is located in each of the lobes, and a third one on the posterior part. Thylakoids are arranged in pairs and do not penetrate pyrenoids. The plastid is reddish due to the presence of the phycoerythrin Cr‐PE545. An orange discoidal eyespot lies beneath the nucleus, in the posterior ventral face of the plastid. A long furrow runs from the vestibulum, and a gullet is lacking. The periplast is composed of an inner sheet. The nuclear 18S rDNA based molecular analysis reveals U. complanatus is not related to any of the main cryptomonad lineages. Based on ultrastructural and pigment data, the most probable relatives are those merged under the family Geminigeraceae. Its lack of derived characters, together with the presence of characters proposed in previous studies to be primitive, suggests Urgorri could be considered representative of the cryptophycean ancestral character state.

Relationships between the presence of Ostreopsis (Dinophyceae) in the Atlantic coast of the Iberian Peninsula and sea-surface temperature

Abstract An extensive sampling program was performed in the Atlantic side of the Iberian Peninsula on 17 sites during 2010 and 2011 summers, in order to characterize the distributional pattern of potential toxic dinoflagellates of the genus Ostreopsis. The study area presents a discontinuity in the species distribution in a pattern that parallels that of summer water temperature. Ostreopsis was not found in a relatively wide fringe of the Northwest side of the Peninsula, as water temperature is markedly lower than that in the Northeast and Southwest extremes where Ostreopsis has been found. Comparing the observed distribution of Ostreopsis on the Atlantic coast of the Peninsula with different sea surface temperature (SST) percentiles, it is notable that neither minimum nor maximum temperatures observed in the range of the study area can explain the species distribution but presumably is the length of the warm period what limits the genus presence. Thus we hypothesize that for Ostreopsis to be present in a certain area three continuous months with SST above 19.5°C may be necessary.

Characterization of a Strain of Fukuyoa paulensis (Dinophyceae) from the Western Mediterranean Sea

A single cell of the dinoflagellate genus Fukuyoa was isolated from the island of Formentera (Balearic Islands, west Mediterranean Sea), cultured, and characterized by morphological and molecular methods and toxin analyses. This is the first report of the Gambierdiscus lineage (genera Fukuyoa and Gambierdiscus) from the western Mediterranean Sea, which is cooler than its eastern basin. Molecular analyses revealed that the Mediterranean strain belongs to F. paulensis and that it bears LSU rDNA sequences identical to New Zealand, Australian, and Brazilian strains. It also shared an identical sequence of the more variable ITS‐rDNA with the Brazilian strain. Toxin analyses showed the presence of maitotoxin, 54‐deoxyCTX1B, and gambieric acid A. This is the first observation of the two latter compounds in a Fukuyoa strain. Therefore, both Gambierdiscus and Fukuyoa should be considered when as contributing to ciguatera fish poisoning. Different strains of Fukuyoa form a complex of morphologically cryptic lineages where F. paulensis stands as the most distantly related nominal species. The comparison of the ITS2 secondary structures revealed the absence of CBCs among strains. The study of the morphological and molecular traits depicted an unresolved taxonomic scenario impacted by the low strains sampling.

The genetic diversity of Mesodinium and associated cryptophytes

Ciliates from the genus Mesodinium are globally distributed in marine and freshwater ecosystems and may possess either heterotrophic or mixotrophic nutritional modes. Members of the Mesodinium major/rubrum species complex photosynthesize by sequestering and maintaining organelles from cryptophyte prey, and under certain conditions form periodic or recurrent blooms (= red tides). Here, we present an analysis of the genetic diversity of Mesodinium and cryptophyte populations from 10 environmental samples (eight globally dispersed habitats including five Mesodinium blooms), using group-specific primers for Mesodinium partial 18S, ITS, and partial 28S rRNA genes as well as cryptophyte large subunit RuBisCO genes (rbcL). In addition, 22 new cryptophyte and four new M. rubrum cultures were used to extract DNA and sequence rbcL and 18S-ITS-28S genes, respectively, in order to provide a stronger phylogenetic context for our environmental sequences. Bloom samples were analyzed from coastal Brazil, Chile, two Northeastern locations in the United States, and the Pribilof Islands within the Bering Sea. Additionally, samples were also analyzed from the Baltic and Barents Seas and coastal California under non-bloom conditions. Most blooms were dominated by a single Mesodinium genotype, with coastal Brazil and Chile blooms composed of M. major and the Eastern USA blooms dominated by M. rubrum variant B. Sequences from all four blooms were dominated by Teleaulax amphioxeia-like cryptophytes. Non-bloom communities revealed more diverse assemblages of Mesodinium spp., including heterotrophic species and the mixotrophic Mesodinium chamaeleon. Similarly, cryptophyte diversity was also higher in non-bloom samples. Our results confirm that Mesodinium blooms may be caused by M. major, as well as multiple variants of M. rubrum, and further implicate T. amphioxeia as the key cryptophyte species linked to these phenomena in temperate and subtropical regions.

Broad distribution of Coolia monotis and restricted distribution of Coolia cf. canariensis (Dinophyceae) on the Atlantic coast of the Iberian Peninsula

Abstract: The genus Coolia, which contains potentially toxic epibenthic dinoflagellate species, was studied throughout 17 sampling sites on the Atlantic coast of the Iberian Peninsula during the summers of 2010–2012. Cells from natural samples were isolated into culture, and the strains were observed by light and scanning electron microscopy in order to study and measure the morphological traits. A detailed compilation of previously compiled Coolia measurements was used for comparisons. To gain insight into the taxonomy of the strains, two regions of the nuclear-encoded rDNA were used as markers to infer phylogenetic relationships: the large subunit (LSU) and the internal transcribed spacers ITS1 and ITS2 plus the 5.8S region of the gene (ITS). Two species were found within the study area: Coolia monotis, which was widely distributed throughout the entire area, and Coolia cf. canariensis, which was found at only one sampling site. The observed morphology of the clonal cultures was supported by their accepted taxonomical descriptions in concordance with the phylogenetic analysis of both LSU and ITS regions. This study provided evidence of the limited geographic distribution of Coolia cf. canariensis in the studied area.

Molecular and morphological analyses of solitary forms of brackish Thalassiosiroid diatoms (Coscinodiscophyceae), with emphasis on their phenotypic plasticity

Abstract Blooms of centric diatoms are a common feature in the Bilbao estuary during summer when river flow is at its lowest and water temperature is above 20ºC. To gain insight into the specific composition of these diatom blooms, net samples and cultures of estuarine isolates were analysed under the scanning electron microscope (SEM) and by molecular analyses of the Internal Transcribed Spacers 1 and 2 plus the coding region 5.8S (ITS region) and the 28S rRNA gene. Seven species of solitary centric diatoms belonging to four genera were found in the estuary including: Conticribra weissflogii, Cyclotella atomus var. atomus, Cyclotella cryptica, Cyclotella marina, Cyclotella meneghiniana, Discostella pseudostelligera and Thalassiosira pseudonana. Dominant species during blooms were C. meneghiniana and Co. weissflogii in the upper estuary and D. pseudostelligera and T. pseudonana in the middle estuary. The morphological traits used to differentiate between species pairs of similar morphology (C. meneghiniana/C. cryptica or D. pseudostelligera/D. woltereckii) were observed to vary with environmental conditions, denoting a great deal of phenotypic plasticity which would hinder accurate identification of the species when using morphological approaches alone.