Phytoplankton aggregate formation: observations of patterns and mechanisms of cell sticking and the significance of exopolymeric material

Flocculation of ‘sticky’ phytoplankton cells intorapidly sinking aggregates has been invoked as a mechanism explainingmass sedimentation of phytoplankton blooms in the ocean. Phytoplanktonstickiness, defined as the probability of adhesion upon collision,is one key factor determining the potential for aggregate formation.In the laboratory, we examined variation in stickiness in fivespecies of diatoms and two species of flagellates grown in batchcultures. We also investigated the production of paniculatemucus by phytoplankton cells and its role in aggregate formation,and we studied the effects of solute exudates on cell stickiness.Four of the five diatoms investigated were significantly sticky,while one diatom and both of the flagellates were not sticky.Stickiness varied considerably within species. In the diatomSkeletonema costatum, the typical but not entirely consistentpattern was that stickiness decreased with age of the batchcultures. We were otherwise unable to establish consistent relationshipsbetween cell stickiness and the growth stage of the algae, environmentalconcentrations of inorganic nutrients, and abundances of suspendedand epiphytic bacteria. We showed that the diatom S.costatumat times excretes a solute substance that depresses flocculation.This may reduce cell losses from the euphotic zone during thegrowth phase due to flocculation and sedimentation. We demonstratedtwo different mechanisms of phytoplankton aggregate formation.In the diatom S.costatum, the cells are sticky in themselves,and coagulation depends on cell-cell sticking and does not involvemucus. Aggregates are composed solely of cells. Cells of thediatom Chaetoceros affinis, on the other hand, are not in themselvessticky. Transparent exopolymeric particles (TEP), produced bythe diatom, cause the cells to aggregate and coagulation dependson TEP-cell rather than cell-cell sticking. Aggregates are formedof a mixture of mucus and cells. We found several species ofdiatoms and one flagellate species to produce copious amountsof TEP. TEP from some species (e.g. Coscinodiscus sp.) is stickyand may cause other, non-sticky particles to coagulate. Thisemphasizes the potential importance of diatom-derived paniculatemucus for particle flocculation in the ocean.     

Diatom aggregation in the sea: mechanisms and ecological implications

Marine snow is a ubiquitous feature of the ocean and an important agent in the transport of energy and nutrients through marine ecosystems. Diatom aggregates, which form during blooms and, to a lesser extent, by the resuspension of benthic biofilms, are a primary source of marine snow. Genera commonly found in diatom aggregates are: Nitzschia, Chaetoceros, Rhizosolenia, Leptocylindricus, Skeletonema and Thalassionema. Most fieldwork has been restricted to a limited number of locations in the Northern Hemisphere. To quantify the global impact of diatom aggregation there is a need to conduct fieldwork in a wider range of areas, particularly in the Southern Hemisphere. Aggregates form when particles collide and stick together. Collisions in the water column are affected by turbulence, differential settlement and animal feeding, whereas diatom stickiness is affected by extracellular polymeric substances (EPS). Laboratory experiments have demonstrated that diatoms produce more EPS under nutrient limitation, although little is known about how limitation by different nutrients affects the quantity and composition of EPS and subsequent stickiness. EPS form three pools in the environment: cell coatings, soluble EPS and transparent exopolymeric particles (TEP). There is a need to investigate the dynamics of conversion between the pools of EPS by both abiotic and biological processes and how these conversions affect aggregate concentration and structure. Processes governing disaggregation have been largely overlooked, although they are as important as aggregation in determining the dynamics of aggregate concentrations in the water column. The biogeochemical significance of diatom aggregates as a means of transporting carbon and other nutrients from the euphotic zone to the seabed is well established. However, the internal biogeochemistry of aggregates is not well understood. Aggregates contain anaerobic microsites and further work is required to establish whether aggregates are significant sinks for nitrogen in the water column through anaerobic denitrification. Several hypotheses have been proposed to explain diatom aggregation in the field, but many of these are flawed because the mechanisms and adaptive explanations proposed require natural selection to operate at the level of populations rather than genes or individuals.     

The role of transparent exopolymer particles (TEP) in the increase in apparent particle stickiness ({alpha}) during the decline of a diatom bloom

The termination of diatom spring blooms in temperate watershas been connected with the formation and subsequent rapid sedimentationof aggregates. According to coagulation theory, the rate ofaggregate formation depends on the probability of particle collisionand on the efficiency with which two particles adhere once theyhave collided (stickiness). During this study, the variationin particle stickiness was determined over the decline of adiatom bloom using the Couette Chamber assay with low shear(G = 0.86 s^–1). A mixed diatom population, dominated bySkeletonema costatum, was sampled during the spring bloom inthe Baltic Sea and incubated in the laboratory for 18 days.Measurements of diatom species composition, transparent exopolymerparticles (TEP) and bulk particle abundance, as well as chemicaland biological variables, were conducted in order to revealthe determinants of coagulation efficiency. The investigationshowed that an increase in TEP concentration relative to conventionalparticles at the decline of the bloom significantly enhancedapparent coagulation efficiencies. High proportions of TEP ledto apparent values of stickiness >1, which indicates thatcollision rates can be substantially underestimated when thestickiness parameter     

Some lower food web organisms in the nutrition of marine harpacticoid copepods: an experimental study

Some lower food web organisms from the marine littoral environment were studied as food for harpacticoid copepods. In laboratory experiments, it could be shown that, among the ciliates, the slow-movingUronema sp. was taken up while the fast-movingEuplotes sp. was not.Asterionella glacialis, a pennate diatom with spiny projections, was unsuitable as food. The centric diatomSkeletonema costatum was ingested by all harpacticoid species tested, includingTisbe holothuriae, Paramphiascella vararensis, Amphiascoides debilis andDactylopodia vulgaris. All are epibenthic and phytal species occurring in the shallow waters of Helgoland (North Sea). The amount of ciliate and algal carbon taken up was less than that provided by bacteria under laboratory conditions. However, some diatom food may be essential for the development ofD. vulgaris.     

Loss of waterborne brevetoxins from exposure to phytoplankton competitors

We tested whether interactions among phytoplankton competitors affect toxin dynamics involving the red tide dinoflagellate Karenia brevis, whose brevetoxins incapacitate and kill coastal wildlife. The addition of a live diatom, Skeletonema costatum, led to decreased concentrations of brevetoxin B (PbTx-2) associated with K. brevis cells in co-culturing experiments and with two of three natural bloom samples containing K. brevis. Similar decreases in PbTx-2 concentration, but not PbTx-3 concentration, occurred when a mixture of brevetoxins (without live K. brevis cells) was exposed to S. costatum, indicating that S. costatum metabolizes waterborne PbTx-2. Liquid chromatography–mass spectrometry (LC–MS) and ELISA analyses indicated that PbTx-2 is probably not transformed into other brevetoxins or into known brevetoxin metabolites, and instead is biotransformed by a previously unrecognized mechanism. Four different S. costatum strains from around the world caused similar loss of PbTx-2, suggesting that evolutionary experience with K. brevis is not a pre-requisite for the ability to metabolize PbTx-2. Additionally, phytoplankton-associated bacteria were found to play no role in the loss of PbTx-2, as bacteria-free S. costatum strains metabolized PbTx-2. Finally, loss of waterborne PbTx-2 caused by exposure to a dinoflagellate, a cryptophyte, and two additional diatom species indicates that this phenomenon is widespread among phytoplankton. Our results unexpectedly suggest that competing phytoplankton species present during K. brevis blooms, and possibly other red tides, could mediate bloom toxicity and therefore ecosystem-level consequences of red tides.     

Transparent exopolymer particle production and aggregation by a marine planktonic diatom (Thalassiosira weissflogii) at different growth rates

Transparent exopolymer particles (TEP) play an important role in the ocean carbon cycle as they are sticky and affect particle aggregation and the biological carbon pump. We investigated the effect of growth rate on TEP production in nitrogen limited semi‐continuous cultures of the diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle. Steady‐state diatom concentrations and other indicators of biomass (chl a, and total carbohydrate) were inversely related to growth rate, while individual cell volume increased with growth rate. There was no change in total TEP area with growth rate; however, individual TEP were larger at high growth rates and the number of individual TEP particles was lower. TEP concentration per cell was higher at higher growth rates. SYTOX Green staining showed that <5% of the diatom population had permeable cell membranes, with the proportion increasing at low growth rates. However, TEP production rates were greater at high growth rates, refuting our hypothesis that TEP formation is dependent on dying cells with compromised cell membranes in a diatom population. Measurements of particle size distribution in the cultures using laser scattering showed that they were most aggregated at high growth rates. These results indicate a coupling between TEP production and growth rate in diatoms under N limitation, with fast growing T. weissflogii producing more TEP and aggregates.     

EFFECT OF ZINC AVAILABILITY ON GROWTH,MORPHOLOGY, AND NUTRIENT INCORPORATION IN A COASTAL AND AN OCEANIC DIATOM1

We investigated the effect of Zn availability on growth rate (μ), cell morphology, and elemental stoichiometry and incorporation rate in two marine diatoms. For the coastal diatom Skeletonema costatum (Grev.) Cleve, the half‐saturation constant (K_S) for growth was 4.1 pM Zn²⁺, and growth ceased at ≤ 2.6 pM Zn²⁺, whereas for the oceanic diatom Thalassiosira oceanica Hasle, K_S was 0.5 pM Zn²⁺, and μ remained at ～40%μ_max even at 0.3 pM Zn²⁺. Under Zn‐limiting (Zn‐L) conditions, S. costatum decreased cell size significantly, leading to an 80% increase in surface area to volume ratio (SA/V) at Zn²⁺ of 3.5 pM compared to Zn‐replete (Zn‐R) conditions (at Zn²⁺ of 13.2 pM), whereas T. oceanica’s morphology did not change appreciably. Cell quotas of C, N, P, Si, and chl a significantly decreased under Zn limitation in S. costatum (at Zn²⁺ of 3.5 pM), whereas Zn limitation in T. oceanica (at Zn²⁺ of 0.3 pM) had little effect on quotas. Elemental stoichiometry was ～85C:10N:9Si:1P and 81C:9N:5Si:1P for S. costatum, and 66C:5N:2Si:1P and 52C:6N:2Si:1P for T. oceanica, under Zn‐R and Zn‐L conditions, respectively. Incorporation rates of all elements were significantly reduced under Zn limitation for both diatoms, but particularly for Si in S. costatum, and for C in T. oceanica, despite its apparent tolerance of low Zn conditions. With [Zn²⁺] in some parts of the ocean being of the same order (～0.2 to 2 pM) as our low Zn conditions for T. oceanica, our results support the hypothesis that in situ growth and C acquisition may be limited by Zn in some oceanic species.     

Responses of Two Coastal Algae (Skeletonema costatum and Chlorella vulgaris) to Changes in Light and Iron Levels1

Iron (Fe) is essential for phytoplankton growth and photosynthesis, and is proposed to be an important factor regulating algal blooms under replete major nutrients in coastal environments. Here, Skeletonema costatum, a typical red-tide diatom species, and Chlorella vulgaris, a widely distributed Chlorella, were chosen to examine carbon fixation and Fe uptake by coastal algae under dark and light conditions with different Fe levels. The cellular carbon fixation and intracellular Fe uptake were measured via ¹⁴C and ⁵⁵Fe tracer assay, respectively. Cell growth, cell size, and chlorophyll-α concentration were measured to investigate the algal physiological variation in different treatments. Our results showed that cellular Fe uptake proceeds under dark and the uptake rates were comparable to or even higher than those in the light for both algal species. Fe requirements per unit carbon fixation were also higher in the dark resulting in higher Fe: C ratios. During the experimental period, high Fe addition significantly enhanced cellular carbon fixation and Fe uptake. Compared to C. vulgaris, S. costatum was the common dominant bloom species because of its lower Fe demand but higher Fe uptake rate. This study provides some of the first measurements of Fe quotas in coastal phytoplankton cells, and implies that light and Fe concentrations may influence the phytoplankton community succession when blooms occur in coastal ecosystems.     

The impact of food type, temperature and starvation on larval development of Balanus amphitrite Darwin (Cirripedia: Thoracica)

The impact of diatom food species (Chaetoceros calcitrans and Skeletonema costatum), temperature and starvation on the larval development of Balanus amphitrite was evaluated. Starvation threshold levels for different ages of larvae (0- to 5-day-old) fed with C. calcitrans and S. costatum and then starved at 5, 15 and 25 °C temperature were estimated as ultimate recovery hour (URH; denoting the starvation point in hours at the end of which larvae can recover and continue development). Effect of temperature on starvation threshold varied significantly with larval age and food species. The URH declined with larval age at 5 °C, but not at 15 and 25 °C. The URH and grazing rates were high for early instars fed on C. calcitrans, and for advanced instars fed on S. costatum. Carbon gain through feeding was maximum for 2-day-old larvae when fed with C. calcitrans and decreased with larval age. However, when fed with S. costatum carbon gain increased with larval age. This confirms that with development the utility of food types changes. The differences in the carbon gain can be attributed to differences in grazing rate due to variations in the size of the diatom cells, larval intersetular distance, diatom sinking rate and the photo-taxic behavior of larvae. Molting was observed at times when larvae were undergoing starvation and this could be viewed as stress-induced molting, and it differed with the larval age and food organisms.     

Feeding by Phototrophic Red-Tide Dinoflagellates on the Ubiquitous Marine Diatom Skeletonema costatum

ABSTRACT We investigated feeding by phototrophic red‐tide dinoflagellates on the ubiquitous diatom Skeletonema costatum to explore whether dinoflagellates are able to feed on S. costatum, inside the protoplasm of target dinoflagellate cells observed under compound microscope, confocal microscope, epifluorescence microscope, and transmission electron microscope (TEM) after adding living and fluorescently labeled S. costatum (FLSc). To explore effects of dinoflagellate predator size on ingestion rates of S. costatum, we measured ingestion rates of seven dinoflagellates at a single prey concentration. In addition, we measured ingestion rates of the common phototrophic dinoflagellates Prorocentrum micans and Gonyaulax polygramma on S. costatum as a function of prey concentration. We calculated grazing coefficients by combining field data on abundances of P. micans and G. polygramma on co‐occurring S. costatum with laboratory data on ingestion rates obtained in the present study. All phototrophic dinoflagellate predators tested (i.e. Akashiwo sanguinea, Amphidinium carterae, Alexandrium catenella, Alexandrium tamarense, Cochlodinium polykrikoides, G. polygramma, Gymnodinium catenatum, Gymnodinium impudicum, Heterocapsa rotundata, Heterocapsa triquetra, Lingulodinium polyedrum, Prorocentrum donghaiense, P. micans, Prorocentrum minimum, Prorocentrum triestinum, and Scrippsiella trochoidea) were able to ingest S. costatum. When mean prey concentrations were 170–260 ng C/ml (i.e. 6,500–10,000 cells/ml), the ingestion rates of G. polygramma, H. rotundata, H. triquetra, L. polyedrum, P. donghaiense, P. micans, and P. triestinum on S. costatum (0.007–0.081 ng C/dinoflagellate/d [0.2–3.0 cells/dinoflagellate/d]) were positively correlated with predator size. With increasing mean prey concentration of ca 1–3,440 ng C/ml (40–132,200 cells/ml), the ingestion rates of P. micans and G. polygramma on S. costatum continuously increased. At the given prey concentrations, the maximum ingestion rates of P. micans and G. polygramma on S. costatum (0.344–0.345 ng C/grazer/d; 13 cells/grazer/d) were almost the same. The maximum clearance rates of P. micans and G. polygramma on S. costatum were 0.165 and 0.020 μl/grazer/h, respectively. The calculated grazing coefficients of P. micans and G. polygramma on co‐occurring S. costatum were up to 0.100 and 0.222 h, respectively (i.e. up to 10% and 20% of S. costatum populations were removed by P. micans and G. polygramma populations in 1 h, respectively). Our results suggest that P. micans and G. polygramma sometimes have a considerable grazing impact on populations of S. costatum.     

Model for fluorescence quenching in light harvesting complex II in different aggregation states

Low-temperature (77 K) steady-state fluorescence emission spectroscopy and dynamic light scattering were applied to the main chlorophyll a/b protein light harvesting complex of photosystem II (LHC II) in different aggregation states to elucidate the mechanism of fluorescence quenching within LHC II oligomers. Evidences presented that LHC II oligomers are heterogeneous and consist of large and small particles with different fluorescence yield. At intermediate detergent concentrations the mean size of the small particles is similar to that of trimers, while the size of large particles is comparable to that of aggregated trimers without added detergent. It is suggested that in small particles and trimers the emitter is monomeric chlorophyll, whereas in large aggregates there is also another emitter, which is a poorly fluorescing chlorophyll associate. A model, describing populations of antenna chlorophyll molecules in small and large aggregates in their ground and first singlet excited states, is considered. The model enables us to obtain the ratio of the singlet excited-state lifetimes in small and large particles, the relative amount of chlorophyll molecules in large particles, and the amount of quenchers as a function of the degree of aggregation. These dependencies reveal that the quenching of the chl a fluorescence upon aggregation is due to the formation of large aggregates and the increasing of the amount of chlorophyll molecules forming these aggregates. As a consequence, the amount of quenchers, located in large aggregates, is increased, and their singlet excited-state lifetimes steeply decrease.     

EFFECT OF POLYCARBONATE CONTAINERS ON THE GROWTH OF TWO SPECIES OF MARINE DIATOMS1

Two species of marine diatoms [Skeletonema costatum (Greville) Cleve and Thalassiosira pseudonana (Hustedt) Hasle and Heimdal] were grown in glass and polyarbonate containers. S. costatum exhibited a signzJicantly lower exponential growth rate and maximal yield and a signajcantly longer lag phase when grown in polycarbonate. Exponential growth rate and maximal yield of T. pseudonana was significantly reduced (P < 0.05 in all cases). This study suggests that a difference in diatom growth between glass and polyarbonate containers might arise in certain cases. However, such a difference may not be detectable with all biomass measurement techniques or with low within-treatment replication.     

Larval development of Octomeris sulcata Nilsson-Cantell (Cirripedia: Thoracica: Chthamalidae) from Japan and Korea

Embryos obtained from gravid adults of the chthamalid barnacle Octomeris sulcata Nilsson-Cantell from Japan and Korea were cultured through six naupliar stages to the cyprid and juvenile barnacle stage in laboratory conditions, fed either the diatom Skeletonema costatum (Grev.) Cleve or the dinoflagellate Prorocentrum minimum (Pavillard) Schiller. The nauplii were planktotrophic and, depending on diet, reached the cyprid stage 9 or 17 days after hatching in individual cultures at 22 °C with 24 h illumination. The survival rate was higher and the duration of the naupliar stages was shorter when fed P. minimum rather than S. costatum. This is probably due to the presence of feathered setae on the antennae. Feathered or plumose setae in nauplii of different cirripede taxa are apparently linked to the type of phytoplankton in the seas when these taxa first evolved.The larval stages of O. sulcata are described, and morphological differences between larvae reared from Japanese andKorean adults are compared. The polygonal cephalic shield and unilobed labrum, a pair of posterior shield spines after naupliar stage IV, feathered setae and a hispid seta on the coxa of the antenna, a cuspidate seta on the mandible, and the gnathobase of the antenna are important in distinguishing the nauplii of this species from other species, including Chthamalidae.     

Viability of phytoplankton resting stages in the sediments of a coastal Swedish fjord

Viable diatom and dinoflagellate resting stages were recovered from sediments in Koljö Fjord on the west coast of Sweden. To determine the maximum survival time of buried resting stages, samples from sediment depths down to 50?cm were incubated at temperatures of 3, 10 and 18?°C. Sediment cores were dated by ²¹⁰Pb and the age of samples containing viable resting stages was determined using the constant rate of supply model. Dilution cultures of surface sediments allowed semiquantitative estimates of the potential seed bank. Dinoflagellate cysts from species such as Diplopsalis sp., Gymnodinium nolleri, Oblea rotunda and Protoceratium reticulatum were viable down to 15?cm depth, or 37 years old. Spores and resting cells of the diatoms Chaetoceros spp., Detonula confervacea and Skeletonema costatum were viable to over 40?cm depth, and may have been buried for many decades. The seed bank of living resting stages in surficial sediments was found to be rich (c. 57000 diatom resting stages g^?1 wet weight and c. 200 dinoflagellate cysts g^?1 wet weight), and the percentage of viable resting stages was higher for spore- and cyst-forming species. The oxygen-deficient sediments in Koljö Fjord appear to be a natural conservator of cell viability, a condition not easily simulated in laboratory studies. These results are ecologically important since spores and cysts are a repository of genetic material able to repopulate waters if resuspended and exposed to suitable light, temperature and nutrients.     

Planktonic ecosystems. The effects of dystrophic conditions on structure and function in the gulf of fos

In the areas studied, an unusual structure and dynamic behavior was exhibited by the plankton ecosystems, due for the most part to industrial and natural effluents from the Rhǒne and Durance. The ecosystem was kept at a low state of maturity, which is characterized by frequent periods of intense multiplication by small species with high metabolic rates, such as the diatom, Skeletonema costatum, and the dinoflagellates, Exuviaella and Prorocentrum. Brackish water seems congenial to that type of proliferation. The turnover rate of these populations decreases as they become older and cell size increases. A lack of competition by species of the same genus is a characteristic of the photo-autotrophic organisms in these environments. Secondary production follows the same cycle as the primary production by the dinoflagellate population. Zooplankton species of the genus Acartia have periods of intensive development in these areas.     

Growth and decline of a diatom spring bloom phytoplankton species composition, formation of marine snow and the role of heterotrophic dinoflagellates

During the spring of 1994, we determined the factors responsiblefor the decline of the seasonal diatom bloom in the Gullmarfjord, on the west coast of Sweden. Four species constituted>75% of the biomass—Detonula confervacea, Chaetocerosdiadema, Skeletonema costatum and Thalassiosira nordenskioeldii—reachingconcentrations of 4900, 350, 8200 and 270 cells ml^–1,respectively. Growth of phytoplankton was exponential (growthrate = 0.12 day^–1) from 3 to 21 March, after which a galewith winds >15 m s^–1 caused massive aggregation. Amaximum of 130 p.p.m. (v/v) of marine snow aggregates was observedby in situ video at the peak of the bloom. Critical concentrations(Jackson, Deep-Sea Res., 37, 1197–1211, 1990) were similarto observed showing that coagulation theory could explain thesudden decline of the bloom. The heterotrophic dinoflagellateGyrodinium cf. spirale increased exponentially after the peakof the bloom with maximum (temperature-adjusted) growth rates.After the rapid aggregation and sedimentation of the bloom,they were able to control any further growth of diatoms. Nitrateand silicate were never depleted, but phosphate may have beenlimiting by the end of the study period. We conclude that massaggregation during a gale marked the end of the bloom, and thatintense grazing by heterotrophic dinoflagellates prevented anysubsequent increase of diatoms.     

Population growth patterns of Skeletonema costatum and nutrient levels in the lower East River,New York,U.S.A.

The population growth patterns of Skeletonema costatum and nutrient levels in the lower East River were examined through field measurements and laboratory experimentation. Maximum growth rates of this diatom (approximately 1.8 divisions per day) were obtained in water samples from the late winter-early spring months. Summer water samples supported little or no growth of this diatom. Measurements of NH₃-N, PO₄,-P, and Si in water samples from the lower East River indicated that nutrient saturated conditions exist year round in this area. The effect of toxic substances in the water column may be responsible for limiting S. costatum growth during the summer months.     

Cadmium and lead toxicity on tropical freshwater periphyton communities under laboratory-based mesocosm experiments

Periphyton constitutes an important community that is useful for assessment of ecological conditions in lotic systems. The objective of this study was to assess the effects of different mixtures of Cd and Pb on periphyton growth as well as Cd and Pb mixtures toxicity to diatom assemblages in laboratory mesocosm experiments. A natural periphyton community sampled from the Monjolinho River (South of Brazil) was inoculated into five experimental systems containing clean glass substrates for periphyton colonization. The communities were exposed to mixtures of dissolved Cd and Pb concentrations of 0.01 and 0.1 mg l⁻¹ Cd and 0.033 and 0.1 mg l⁻¹ Pb. Periphyton ash-free dry weight, growth rate, diatom cell density and diatom community composition were analyzed on samples collected after 1, 2 and 3 weeks of colonization. High Cd concentration (0.1 mg l⁻¹) has negative effects on periphyton growth while high concentration of Pb (0.1 mg l⁻¹) decreased the toxic effects of Cd on periphyton growth. Shifts in species composition (development of more resistant species like Achnanthidium minutissimum and reduction of sensitive ones like Cymbopleura naviculiformis, Fragilaria capucina, Navicula cryptocephala, Encyonema silesiacum, Eunotia bilunaris, and Gomphonema parvulum), decreases in species diversity of diatom communities with increasing Cd and Pb concentrations and exposure duration have been demonstrated in this study making diatom communities appropriate monitors of metal mixtures in aquatic systems.     

Utility of mitochondrial‐encoded cytochrome c oxidase I gene for phylogenetic analysis and species identification of the planktonic diatom genus Skeletonema

Small subunit (SSU) and large subunit (LSU) rDNA sequences have been commonly used to delineate the taxonomy and biogeography of the planktonic diatom genus Skeletonema, but the genes occur as multiple copies and are therefore not suitable for barcoding purposes. Here, we analyzed phylogenetic relationships of Skeletonema using the mitochondrial‐encoded cytochrome c oxidase I gene (cox1), as well as partial LSU rDNA (D1–D3) and SSU rDNA, to identify the factors that define species and to evaluate the utility of these three markers for this taxon. Twelve Skeletonema species were divided into six clades, I–VI, each of which comprised the same species by the three markers: clades I (S. japonicum, S. grethae, S. pseudocostatum, and S. tropicum), II (S. menzelii), III (S. dohrnii and S. marinoi), IV (S. costatum, S. potamos, and S. subsalsum), V (S. grevillei), and VI (S. ardens). However, the branching order among these clades was incongruent among the markers. In clade III, six S. marinoi strains had identical cox1 sequences. These S. marinoi strains branched along with S. dohrnii, except for strains from the Gulf of Naples, with high support in cox1. Species delimitation between S. dohrnii and S. marinoi was therefore not supported. In clade IV, S. costatum and S. subsalsum were robustly clustered, with S. potamos as a sister clade in the cox1 tree, not in the LSU and SSU trees. In clade II, cox1 also confirmed that S. menzelii includes three subclades potentially distinguishable from each other by morphological features. Cox1 proved to be the most useful marker for the identification of Skeletonema species because it gave a tree with highly supported clades, has sufficient variation within and among species, encodes a protein in a single copy, and requires relatively few primers.