Sandra C. Craveiro

Diatoms and macroinvertebrates provide consistent and complementary information on environmental quality

Predictive models based on invertebrate assemblages are a powerful tool for assessing water quality. Yet, few attempts have been made to build predictive models with diatoms although they are widely used in freshwater assessment and are among the mandatory biological monitoring elements indicated in the European Water Framework Directive. We built two predictive models for central Portugal, one for macroinvertebrates and the other for diatoms, using data sampled simultaneously from 43 sites in the Spring of 2004 and 2005. Our objective was to test the performance of models based on the diatom community and compare the assessments made by the two indicator communities for the same sites. Each model was tested with 17 sites. Both models showed good performance in predicting reference sites to the correct group: 76 % for the macroinvertebrate model using the discriminant variables longitude, distance to source, alkalinity, depth, number of riffles and channel substrate diversity; 85 % for the diatoms model using latitude, runoff, mineralization, distance to source, slope, discharge and hardness. The water quality assessments of test sites produced by the two models were similar in 76 % of the cases. According to the predictive models, invertebrates were more sensitive to morphological alterations of the channel and habitat conditions (presence of dams and artificial walls, destruction of riparian corridor) while diatoms were more sensitive to changes in water chemistry. Moreover, in lowland streams, where the habitat and substrate heterogeneity are naturally low, assessment by diatoms was more useful than evaluations using invertebrates. We concluded that invertebrates and algae provide consistent and in some cases complementary information on environmental quality and recommend their simultaneous use for bioassessment.

Diversity and abundance of potentially toxic Pseudo-nitzschia peragallo in Aveiro coastal lagoon, Portugal and description of a new variety, P. pungens var. aveirensis var. nov.

The syndrome Amnesic Shellfish Poisoning (ASP) results from ingesting sea food contaminated by a neurotoxin, domoic acid. Diatoms of the genera Pseudo-nitzschia and Nitzschia are responsible for the production of this toxin. A total of eleven species of Pseudo-nitzschia were identified during two plankton surveys at the entrance and in the southern channels of Ria de Aveiro, the largest lagoon on the west coast of Portugal. During the first survey, conducted from February to October 2000, the following species were identified: P. australis, P. cuspidata, P. delicatissima, P. dolorosa, P. fraudulenta, P. pseudodelicatissima, P. subpacifica and a new variety of P. pungens. The second survey took place from September 2003 to April 2004, except for a break between December 2003-January 2004, along with one complementary sample taken in October 2004; it revealed three additional species, P. americana, P. calliantha and P. multistriata. In contrast, P. cuspidata, P. dolorosa and P. subpacifica were not found during the second survey. Five species: P. subpacifica, P. americana, P. calliantha, P. multistriata and P. cuspidata were reported for the first time in Portuguese waters. Overall, P. australis, P. pseudodelicatissima and a new variety of P. pungens were the most common species, the latter being present in all samples except October 2000. The highest concentrations of Pseudo-nitzschia cells registered were 90, 000 cells L–1 in August 2000 and 106, 000 cells L−1 in March 2004, with P. australis accounting for 65–75% of the cells. Cell concentrations were highest near the entrance of the lagoon and decreased along with salinity in the channels. Cultures of P. australis, P. americana, P. fraudulenta, P. multistriata and a new variety of P. pungens were established and tested for the production of domoic acid, but the results were negative. Morphological differences were found between our strains of P. pungens and two other previously described varieties of P. pungens regarding valve width, densities of striae, fibulae, poroids and band striae as well as differences in the morphology of the band striae. These differences were supported by phylogenetic analyses of ITS rDNA sequences, existence of a compensatory base change and mating studies indicate the presence of a separate taxon, which was described as a new variety, P. pungens var. aveirensis var. nov.

DESCRIPTION OF TYRANNODINIUM GEN. NOV., A FRESHWATER DINOFLAGELLATE CLOSELY RELATED TO THE MARINE PFIESTERIA-LIKE SPECIES 1

On the basis of morphological (light and electron microscopy) as well molecular data, we show that the widely distributed freshwater dinoflagellate presently known as Peridiniopsis berolinensis is a member of the family Pfiesteriaceae, an otherwise marine and estuarine family of dinoflagellates. P. berolinensis is a close relative of the marine species, which it resembles in morphology, mode of swimming, food‐uptake mechanism, and partial LSU rRNA sequences. It differs from all known genera of the family in plate tabulation. P. berolinensis is only distantly related to the type species of Peridiniopsis, P. borgei, and is therefore transferred to the new genus Tyrannodinium as T. berolinense comb. nov. T. berolinense is a very common freshwater flagellate that feeds vigorously on other protists and is able to consume injured metazoans much larger than itself. Production of toxins has not been reported.

ULTRASTRUCTURE AND LSU RDNA‐BASED PHYLOGENY OF ESOPTRODINIUM GEMMA (DINOPHYCEAE), WITH NOTES ON FEEDING BEHAVIOR AND THE DESCRIPTION OF THE FLAGELLAR BASE AREA OF A PLANOZYGOTE1

A small, freshwater dinoflagellate with an incomplete cingulum, identified as Esoptrodinium gemma Javornický (=Bernardinium bernardinense sensu auctt. non sensu Chodat), was maintained in mixed culture and examined using light and serial section TEM. Vegetative flagellate cells, large cells with two longitudinal flagella (planozygotes), and cysts were examined. The cells displayed a red eyespot near the base of the longitudinal flagellum, made of two or three layers of pigment globules not bounded by a membrane. Yellow‐green, band‐shaped chloroplasts, bounded by three membranes and containing lamella with three thylakoids, were present in both flagellate cells and cysts. Most cells had food vacuoles, containing phagotrophically ingested chlamydomonads or chlorelloid green algae; ingestion occurred through the ventral area, involving a thin pseudopod apparently driven by the peduncle. The pusule was tubular, with numerous diverticula in its distal portion, and opened into the longitudinal flagellar canal. Three roots were associated with each pair of flagellar bases, both in vegetative cells and in a planozygote. The longitudinal microtubular root bifurcated around the longitudinal basal body. The planozygote contained a single peduncle and associated structures, and a single transverse flagellar canal with the two converging transverse flagella. Using two ciliates as outgroup species, phylogenetic analyses based on maximum parsimony, neighbor‐joining and posterior probability (Bayesian analysis) supported a clade comprising Esoptrodinium, Tovellia, and Jadwigia.

ULTRASTRUCTURE AND LSU rDNA–BASED REVISION OF PERIDINIUM GROUP PALATINUM (DINOPHYCEAE) WITH THE DESCRIPTION OF PALATINUS GEN. NOV.1

The name Peridinium palatinum Lauterborn currently designates a freshwater peridinioid with 13 epithecal and six cingular plates, and no apical pore complex. Freshwater dinoflagellate floras classify it in Peridinium group palatinum together with P. pseudolaeve M. Lefèvre. General ultrastructure, flagellar apparatus, and pusular components of P. palatinum were examined by serial section TEM and compared to P. cinctum (O. F. Müll.) Ehrenb. and Peridiniopsis borgei Lemmerm., respectively, types of Peridinium and Peridiniopsis. Partial LSU rDNA sequences from P. palatinum, P. pseudolaeve and several peridinioids, woloszynskioids, gymnodinioids, and other dinoflagellates were used for a phylogenetic analysis. General morphology and tabulation of taxa in group palatinum were characterized by SEM. Differences in plate numbers, affecting both the epitheca and the cingulum, combine with differences in plate ornamentation and a suite of internal cell features to suggest a generic‐level distinction between Peridinium group palatinum and typical Peridinium. The branching pattern of the phylogenetic tree is compatible with this conclusion, although with low support from bootstrap values and posterior probabilities, as are sequence divergences estimated between species in group palatinum, and typical Peridinium and Peridiniopsis. Palatinus nov. gen. is proposed with the new combinations Palatinus apiculatus nov. comb. (type species; syn. Peridinium palatinum), P. apiculatus var. laevis nov. comb., and P. pseudolaevis nov. comb. Distinctive characters for Palatinus include a smooth or slightly granulate, but not areolate, plate surface, a large central pyrenoid penetrated by cytoplasmic channels and radiating into chloroplast lobes, and the presence of a peduncle‐homologous microtubular strand. Palatinus cells exit the theca through the antapical‐postcingular area.

Ultrastructure and Large Subunit rDNA-Based Phylogeny of Sphaerodinium cracoviense, an Unusual Freshwater Dinoflagellate with a Novel Type of Eyespot

ABSTRACT. Sphaerodinium cracoviense was collected near Cracow, Poland, and analysed by light microscopy, scanning electron microscopy, and serial‐section transmission electron microscopy. Thecae showed a peridinioid type of plate arrangement with unusual numbers in the anterior intercalary and postcingular plate series: 4 and 6, respectively. The apical pore of S. cracoviense differed from the typical arrangement seen in many thecate forms and included a furrow with knob‐like protuberances reminiscent of the apical area of some woloszynskioids. The flagellar apparatus included the three microtubular roots that extend to the left of the basal bodies and a striated root connective between the transverse striated root and the longitudinal microtubular root. Both the single‐stranded root that associates with the right side of the longitudinal basal body in peridinioids and gonyaulacoids, and the layered connective typical of peridinioids were absent. The eyespot was formed by a layer of vesicle‐contained crystal‐like units underlain by layers of variably fused globules not bounded by membranes, and represents a novel type. The pusular system included a long canal with a dilated inner portion with radiating tubules. Bayesian and maximum likelihood analyses based on large subunit rDNA placed Sphaerodinium as a sister taxon to a group of woloszynskioids and relatively far from Peridinium and its allies.

Studies on woloszynskioid dinoflagellates VI: description of Tovellia aveirensis sp. nov. (Dinophyceae), a new species of Tovelliaceae with spiny cysts

A new species of Tovellia, T. aveirensis, is described on the basis of light (LM) and scanning electron microscopy (SEM) of motile cells and resting cysts, complemented with transmission electron microscopy (TEM) of flagellate cells and phylogenetic analysis of partial sequences of the large subunit ribosomal rRNA gene. Both vegetative cells and several stages of a life cycle involving sexual reproduction and the production of resting cysts were examined in cultures established from a tank in the University of Aveiro campus. Vegetative cells were round and little compressed dorsoventrally; planozygotes were longer and had a proportionally larger epicone. Chloroplast lobes were shown by TEM to radiate from a central, branched pyrenoid, although this was difficult to ascertain in LM. The amphiesma of flagellate cells had mainly 5 or 6-sided vesicles with thin plates, arranged in 5–7 latitudinal series on the epicone, 3–5 on the hypocone. The cingulum had 2 rows of plates, the posterior row extending into the hypocone and crossed by a series of small projecting knobs along the lower edge of the cingulum. A line of narrow amphiesmal plates extended over the cell apex, from near the cingulum on the ventral side to the middle of the dorsal side of the epicone. Eight or 9 narrow amphiesmal plates lined each side of this apical line of plates (ALP). Resting cysts differed from any described before in having numerous long, tapering spines with branched tips distributed over most of the surface. Most mature cysts showed an equatorial constriction. Neither cysts nor motile cells were seen to accumulate red cytoplasmic bodies in any stage of the cultures. The phylogenetic analysis placed, with high statistical support, the new species within the genus Tovellia; it formed a clade, with moderate support, with T. sanguinea, a species notable for its reddening cells.

Ultrastructure and phylogeny of Theleodinium calcisporum gen. et sp. nov., a freshwater dinoflagellate that produces calcareous cysts

Craveiro S.C., Pandeirada M.S., Daugbjerg N., Moestrup Ø. and Calado A.J. 2013. Ultrastructure and phylogeny of Theleodinium calcisporum gen. et sp. nov., a freshwater dinoflagellate that produces calcareous cysts. Phycologia 52: 488–507. DOI: 10:2216/13–152.1 A freshwater photosynthetic dinoflagellate isolated from a shallow lake near Aveiro, Portugal, was examined by light microscopy, scanning electron microscopy (SEM) and serial-section transmission electron microscopy (TEM), and characterized genetically. Cells were small, spherical to slightly elongated, and had a projecting, nearly cylindrical, apical pore. The chloroplasts were yellowish-brown, arranged near the surface of the cell, and had up to four pyrenoids surrounded by starch sheaths. The cells had a peridinioid plate pattern with Kofoidian plate formula pp, cp, x, 3′ (seldom 4′), 2a, 7′′, 6c, 5s (6s?), 5′′′, 2′′′′. A small extruded peduncle was observed by SEM in cells with intact membranes. A microtubular basket, made of about 46 microtubules disposed in four rows, was seen in the ventral area in connection with the cytoplasmic extension that made the peduncle. The flagellar apparatus was typical of a peridinioid with two roots associated with each of the basal bodies and a layered connective linking the proximal ends of roots 1 and 4. In dense cultures, this organism produced a round resting cyst with a thick wall covered by irregular calcified elements. Energy dispersive X-ray analysis (EDS) analysis of the cysts showed calcium as the most abundant element. A total of 3048 nucleotides of the nuclear ribosomal operon were sequenced and used in a phylogenetic analysis that placed this organism as a sister group to a clade of Scrippsiella species and the parasitic Duboscquodinium collinii. Theleodinium calcisporum gen. et sp. nov. is described for the first freshwater dinoflagellate reported to produce calcareous cysts.

Studies on woloszynskioid dinoflagellates VII. Description of Borghiella andersenii sp. nov.: light and electron microscopy and phylogeny based on LSU rDNA

Freshwater woloszynskioid dinoflagellates were collected independently in Scotland and Portugal and found to belong to a previously unknown species of the genus Borghiella, here described as B. andersenii. The new species differs in morphology and nuclear-encoded LSU rDNA and ITS sequences from B. dodgei and B. tenuissima, the two species presently comprising the genus Borghiella. Unusual features of the new species were observed particularly during asexual reproduction, which took place in the motile stage – as in many other dinoflagellates – or in a so-called division cyst, recalling cell division in the family Tovelliaceae. Such diversity in cell division is rarely reported in dinoflagellates. Morphologically Borghiella andersenii differs from B. tenuissima in being only slightly compressed dorsoventrally whereas the latter species is flat. The slight compression is also visible in lateral view. Borghiella andersenii and B. dodgei are more challenging to discriminate but the apical structure complex is only half the length in B. andersenii compared with B. dodgei (3–4 vs 6 µm). This difference can only be accounted for in the scanning electron microscope. At the light microscopy level the epicone in B. andersenii is rounded whereas it is conical in B. dodgei. Sexual reproduction in Borghiella andersenii was homothallic by formation of planozygotes, followed by apparent resting cysts. Phylogenetic studies on woloszynskioids have recently shown that they comprise a polyphyletic assemblage, which has been divided into the three families Borghiellaceae, Tovelliaceae and Suessiaceae. New species of the three families are now being found rapidly in many parts of the world, proving that the techniques required to investigate these small, morphologically similar dinoflagellates are now in place and proving that such ‘gymnodinioids’ or ‘woloszynskioids’ comprise an often overlooked biological entity in both marine and freshwater biotopes. Based on LSU rDNA, B. andersenii is most closely related to B. tenuissima.

Studies on Peridinium aciculiferum and Peridinium malmogiense (=Scrippsiella hangoei): comparison with Chimonodinium lomnickii and description of Apocalathium gen. nov. (Dinophyceae)

Abstract: The fine structure of the freshwater Peridinium aciculiferum and the closely related Baltic Sea species currently known as Scrippsiella hangoei were examined in serial sections. The species name Peridinium malmogiense is shown to be an earlier synonym of Scrippsiella hangoei and is restored as the name of the species. Although both species have been included in the genus Peridinium, their phylogenetic positions are within the Thoracosphaeraceae, close to the specialized predators known as the pfiesteriaceans and the photosynthetic freshwater Chimonodinium lomnickii. The fine-structural features of the two species proved to be very similar, including the details of flagellar bases and roots, and the type of pyrenoid, which consisted of dilated areas of the chloroplast crossed by two-thylakoid lamellae and not associated with starch sheaths. Comparison with Chimonodinium revealed significant differences, in particular the absence of an eyespot and any trace of microtubules associated with a peduncle, which contrast with the multilayered eyespot and the distinct microtubular basket (MB) of C. lomnickii. The absence of a MB in P. aciculiferum and P. malmogiense is regarded as a character loss within a group of species hypothesized to be derived from a MB-containing ancestor. A phylogenetic analysis based on concatenation of nuclear-encoded small subunit rDNA, internal transcribed spacers 1 and 2 including 5.8S sequences agreed with published phylogenies based on genes of the ribosomal operon in closely grouping P. aciculiferum, P. malmogiense and two other species of peridinioids with a similar amphiesmal plate arrangement: P. euryceps and P. baicalense. The four species are regarded as members of the same genus. While one of the closest known relatives of these four species is C. lomnickii, the variable association of this species to several other groups of species in published phylogenies and the differences in fine-structure revealed in the present work advise against transferring the studied species to Chimonodinium. The new genus Apocalathium is described with P. aciculiferum as type species.