Sergio Seoane

Phytoplankton assemblages and their dominant pigments in the Nervion River estuary

In the Nervion River estuary surface samples were taken from March to September 2003 at six sites covering most of the salinity range with the aim to know the biomass and taxonomic composition of phytoplankton assemblages in the different segments. Nine groups of algae including cyanobacteria, diatoms, dinoflagellates, chlorophytes, prasinophytes, euglenophytes, chrysophytes, haptophytes, raphidophytes and cryptophytes were identified by means of a combination of pigment analysis by high-performance liquid chromatography (HPLC) and microscopic observations of live and preserved cells. Diatoms, chlorophytes and cryptophytes were the most abundant algae in terms of cells number, whereas fucoxanthin, peridinin, chlorophyll b (Chl b) and alloxanthin were the most abundant auxiliary pigments. Based on multiple regression analysis, in the outer estuary (stations 0, 1, 2 and 3) about 93% of the chlorophyll a (Chl a) could be explained by algae containing fucoxanthin and by algae containing Chl b, whereas in the rest of the estuary most of the Chl a (about 98%) was accounted for by fucoxanthin, Chl b and alloxanthin containing algae. The study period coincided with that of most active phytoplankton growth in the estuary and fucoxanthin was by far the dominant among those signature pigments. Several diatoms, chrysophytes, haptophytes and raphydophytes were responsible for fucoxanthin among identified species. Besides, dinoflagellates with a pigment pattern corresponding to chrysophytes and type 4 haptophytes were identified among fucoxanthin-bearing algae. Cryptophytes were the most abundant species among those containing alloxanthin. The maximum of Chl b registered at the seaward end in April coincided with a bloom of the prasinophytes Cymbomonas tetramitiformis, whereas the Chl b maxima in late spring and summer were accounted for by prasinophytes in the middle and outer estuary and by several species of chlorophytes in the middle and inner estuary. Other Chl b containing algae were euglenophytes and the dinoflagellate Peridinium chlorophorum. Dinoflagellates constituted generally a minor component of the phytoplankton.

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.

Chrysochromulina palpebralis sp. nov. (Prymnesiophyceae): a haptophyte, possessing two alternative morphologies

S. Seoane, W. Eikrem, R. Pienaar and B. Edvardsen. 2009. Chrysochromulina palpebralis sp. nov. (Prymnesiophyceae): a haptophyte, possessing two alternative morphologies. Phycologia 48: 165–176. DOI: 10.2216/08-63.1. A marine haptophyte flagellate, Chrysochromulina palpebralis sp. nov., is described from cultures originating from the Nervión River estuary in the Cantabrian Sea, northern Spain. In addition, cultures of similar organisms originating from Japanese and South African waters have been studied. Cells of C. palpebralis had an ovoid shape with an anterior shoulder and were 8–13 µm long and 6–9 µm wide. The cells possessed two equal to subequal flagella that measured 20–27 µm and a haptonema (7–15 µm long) that was flexible and shorter than the flagella. The life cycle included two motile cell forms indistinguishable at the light microscope level. At the electron microscope level, the two cell forms could be distinguished by having different types of body scales. Flow cytometric ploidy analysis showed that the nuclei of the two forms differ in DNA content by a factor of about four. One cell form, f. palpebralis, assumed to be tetraploid, was covered by three scale types, one with a central spiny projection and a peripheral rim with about 20 projections and two types of ovoid plate scales. The other form, f. bisquamata, assumed to be haploid, had two types of plate scales. The first cell form is known from the literature as ‘eyelash Chrysochromulina’ and has previously been recorded from coastal waters in Australia, Japan and South Africa. Phylogenetic analyses based on the nuclear encoded SSU and LSU ribosomal DNA sequences suggest that C. palpebralis is closely related to some nonsaddle-shaped Chrysochromulina species, clustering with C. polylepis and C. kappa as its closest relatives. Chrysochromulina palpebralis was nontoxic to nauplii of brine shrimp.

Phytoplankton pigments and epifluorescence microscopy as tools for ecological status assessment in coastal and estuarine waters, within the Water Framework Directive

Inverted microscopy is widespread employed for the analysis of phytoplankton composition within water quality monitoring networks. However, the analysis at the lowest taxonomical level is not always required for ecological status assessment. In addition, inverted microscopy can underestimate the small phytoplankton, and not always distinguish photoautotrophic from heterotrophic cells. In this study, as alternative tools, epifluorescence microscopy and High Performance Liquid Chromatography (HPLC) were employed to characterize phytoplankton communities within waters of different trophic condition. Epifluorescence microscopy confirmed its effectiveness to count the small phytoplankton. Furthermore, significant correlations between nutrients of anthropogenic origin and nanoplankton abundances were found. However, this technique resulted very time-consuming. HPLC together with the CHEMTAX program was more appropriate than inverted microscopy, in terms of cost-effectiveness. Also, the main variability patterns observed in the phytoplankton community structure by HPLC coincided with previous findings in the study area. Nevertheless, a rapid screening at the inverted microscope is recommended.

Water quality monitoring in Basque coastal areas using local chlorophyll-a algorithm and MERIS images

Accurate estimation of chlorophyll- a (chl- a ), a proxy of the eutrophication risk, is necessary in coastal areas for the assessment of water quality in accordance with European Directives. Local parameterization of remote sensing algorithms is useful to cope with the variability and specificity of optically-active in-water constituents. Using the Bay of Biscay coastal waters, affected by Basque river runoffs, as a case study, the objectives of this investigation are to: 1. develop an empirical algorithm to estimate water surface chl- a for the optically-complex Basque coastal waters; 2. explore the influence of suspended matter, phytoplankton species, and pigment content on the algorithm developed for medium resolution imaging spectrometer instrument (MERIS) imagery; 3. compare the local algorithm to three ocean color algorithms (OC4v6, Gitelson’s algorithm, and the OC5); and 4. apply the local algorithm to the MERIS images. For this purpose, two surveys were undertaken within the study area, the Batel-1 survey in 2007, and the Batel-2, in 2009. The empirical algorithm was developed with remote sensing reflectances ( R rs ), undertaken with a TriOS field spectrometer, and chl- a measured in situ from the Batel-2 survey. The algorithm was not affected by different concentrations of suspended matter in surface waters, within the range from 0.0 to 6.6     g · m − 3 . There was no significant effect of 23 accessory pigments found in the area on the algorithm. Eighty-four R rs and chl- a measurements from the Batel-1 survey were used to validate the local algorithm and to compare it with output of the other algorithms. The local algorithm provided the lowest root-mean-square difference ( RMS = 1.7     mg · m − 3 ), the best correlation with the observed data ( R = 0.8 ), together with the best slope-intercept combination between predicted and observed chl- a ( slope = 0.5 , intercept = 0.6 ). The chl- a algorithm developed here for MERIS imagery can assist in the assessment of water ecological status in the southeastern part of the Bay of Biscay, in a cost-effective manner.

New insights into the morphology and phylogeny of Heterosigma akashiwo (Raphidophyceae), with the description of Heterosigma minor sp. nov.

Abstract: Heterosigma akashiwo is one of the smallest raphidophytes and is known for its ability to form dense, fish-killing blooms. In many parts of the world, H. akashiwo is a permanent resident of the algal community, and blooms have caused extensive financial losses for the aquaculture industry. Different aspects of its biology have been the focus of numerous investigations over the last two decades but, perhaps because of its small size and fragile nature, few morphological studies have been carried out. We conducted phylogenetic and morphological studies of 24 strains of Heterosigma from the coastal waters of Europe, North America, Australia and New Zealand. Complete 18S, ITS region and the D1–D2 region of 28S rDNA were sequenced and compared with all available raphidophyte sequences, revealing a novel species of Heterosigma, Heterosigma minor sp. nov. All strains of H. akashiwo were genetically very similar across the rDNA region (> 99.8% similarity) and morphologically very similar in light microscopy and scanning electron microscopy. Heterosigma minor shared only 98.2% similarity with H. akashiwo, and there were several morphological differences: it was smaller, rounder and had fewer mucocysts than H. akashiwo. The secondary structure of ITS2 was reconstructed, revealing three compensatory base changes between the two species, further indicating that H. minor is a sister to H. akashiwo. The phylogeny of Raphidophyceae was reconstructed to include all currently described raphidophytes.

Bloom forming and toxic phytoplankton in transitional and coastal waters of Cantabria region coast (Southeastern Bay of Biscay, Spain)

Phytoplankton monitoring has extended to practically all the regions of the European coast due to the implementation of the European Water Framework Directive. In this way, the study of phytoplankton taxonomic composition and dynamic is being performed in many areas poorly studied or not studied before. During the last years, a monitoring programme has been carried out at the coast of Cantabria region (SE Bay of Biscay); the presence of some potentially toxic and bloom forming species (>7.5 × 10⁵ cells per litre) has been observed. Diatoms and cryptophytes are the main blooming taxa in this region in the majority of the estuaries and in some of the coastal sites. All estuaries and coastal stations showed at least one potentially toxic species, being the dinoflagellates the group with the highest number of taxa observed. The potentially toxic species found in highest concentrations were the genera Pseudo-nitzschia and Chrysochromulina.

Haptophytes of the Nervion River estuary, northern Spain

The meso-macrotidal Nervion River estuary in northern Spain was sampled monthly from March to September 2002 and 2003 to study species composition and abun- dance of haptophytes in the phytoplankton. From natural samples and uni-algal cultures, 17 haptophyte species were identified using light and electron microscopy. The species were found across a wide range of temperatures and salinities. The most frequently occurring haptophytes in the estuary were species of the genus Chrysochro- mulina; however, Isochrysis galbana reached highest abundances. Chrysochromulina lanceolata and Phaeo- cystis globosa were occasionally present at high abun- dances. Another bloom-forming species, Prymnesium parvum, was recorded only once. Of the genera identi- fied, eight species belonged to Chrysochromulina, two to Prymnesium and the rest of the genera were represented by one species only. The majority of the species were recorded for the first time in the Cantabrian Sea and one of them for the first time in the Atlantic Ocean.

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.