Pelagomonas Research Articles

Genomic adaptation of the picoeukaryote Pelagomonas calceolata to iron-poor oceans revealed by a chromosome-scale genome sequence

The smallest phytoplankton species are key actors in oceans biogeochemical cycling and their abundance and distribution are affected with global environmental changes. Among them, algae of the Pelagophyceae class encompass coastal species causative of harmful algal blooms while others are cosmopolitan and abundant. The lack of genomic reference in this lineage is a main limitation to study its ecological importance. Here, we analysed Pelagomonas calceolata relative abundance, ecological niche and potential for the adaptation in all oceans using a complete chromosome-scale assembled genome sequence. Our results show that P. calceolata is one of the most abundant eukaryotic species in the oceans with a relative abundance favoured by high temperature, low-light and iron-poor conditions. Climate change projections based on its relative abundance suggest an extension of the P. calceolata habitat toward the poles at the end of this century. Finally, we observed a specific gene repertoire and expression level variations potentially explaining its ecological success in low-iron and low-nitrate environments. Collectively, these findings reveal the ecological importance of P. calceolata and lay the foundation for a global scale analysis of the adaptation and acclimation strategies of this small phytoplankton in a changing environment.

Genomic differentiation of three pico-phytoplankton species in the Mediterranean Sea.

For more than a decade, high-throughput sequencing has transformed the study of marine planktonic communities and has highlighted the extent of protist diversity in these ecosystems. Nevertheless, little is known relative to their genomic diversity at the species-scale as well as their major speciation mechanisms. An increasing number of data obtained from global scale sampling campaigns is becoming publicly available, and we postulate that metagenomic data could contribute to deciphering the processes shaping protist genomic differentiation in the marine realm. As a proof of concept, we developed a findable, accessible, interoperable and reusable (FAIR) pipeline and focused on the Mediterranean Sea to study three a priori abundant protist species: Bathycoccus prasinos, Pelagomonas calceolata and Phaeocystis cordata. We compared the genomic differentiation of each species in light of geographic, environmental and oceanographic distances. We highlighted that isolation-by-environment shapes the genomic differentiation of B. prasinos, whereas P. cordata is impacted by geographic distance (i.e. isolation-by-distance). At present time, the use of metagenomics to accurately estimate the genomic differentiation of protists remains challenging since coverages are lower compared to traditional population surveys. However, our approach sheds light on ecological and evolutionary processes occurring within natural marine populations and paves the way for future protist population metagenomic studies.

Coverage and SNP datasets for Bathycoccus prasinos, Pelagomonas calceolata and Phaeocystis cordata in the Mediteranean Sea, studied with Tara Oceans metagenomic samples

Coverage and SNP datasets for Bathycoccus prasinos, Pelagomonas calceolata and Phaeocystis cordata in the Mediteranean Sea, studied with Tara Oceans metagenomic samples

Transcriptomic Responses of Four Pelagophytes to Nutrient (N, P) and Light Stress

Pelagophytes are abundant picophytoplankton within open ocean ecosystems and the causative algae of harmful brown tide blooms in estuaries. The physiological capabilities facilitating the ecological success of pelagophytes in these diverse ecosystems remains poorly understood. Here, we investigated the transcriptional response of two coastal pelagophytes (Aureococcus anophagefferens and Aureoumbra lagunensis) and two open ocean pelagophytes (Pelagococcus subviridis and Pelagomonas calceolata) to conditions commonly found within the marine ecosystems where they thrive: low concentrations of nitrogen (N), phosphorus (P), or light. OrthoMCL was used to generate a total of 62,653 orthologous groups (OGs) with only a small fraction of these OGs (2,776 or 4.4%) being shared among all four pelagophytes. Of the commonly shared OGs, 8% were significantly differentially abundant under low N, P, or light with the majority associated with energy and lipid metabolism. Distinct responses among pelagophytes included increased abundance of transcripts encoding phosphate transporters (Aureococcus) and transcripts encoding a pyrophosphatase (Aureococcus and Pelagomonas) under low P, the expression of a suite of organic nitrogen-degrading enzymes under low N (Aureoumbra and Pelagomonas), increased abundance of transcripts encoding flavodoxins relative to ferredoxins (Pelagomonas) and transcripts encoding lysophospholipase (Pelagococcus) under low light, and both increases and decreases in abundance of transcripts encoding selenoproteins in all pelagophytes except Pelagococcus. Collectively, this study provides new information on the expressed gene compliment of these poorly characterized taxa and demonstrates that these pelagophytes possess a combination of shared and unique physiological features that likely facilitate their adaptation to distinct environmental conditions.

Mitochondrial Genome Evolution in Pelagophyte Algae.

The Pelagophyceae are marine stramenopile algae that include Aureoumbra lagunensis and Aureococcus anophagefferens, two microbial species notorious for causing harmful algal blooms. Despite their ecological significance, relatively few genomic studies of pelagophytes have been carried out. To improve understanding of the biology and evolution of pelagophyte algae, we sequenced complete mitochondrial genomes for A. lagunensis (CCMP1510), Pelagomonas calceolata (CCMP1756), and five strains of Aureoc. anophagefferens (CCMP1707, CCMP1708, CCMP1850, CCMP1984, and CCMP3368) using Nanopore long-read sequencing. All pelagophyte mitochondrial genomes assembled into single, circular mapping contigs between 39,376 bp (P. calceolata) and 55,968 bp (A. lagunensis) in size. Mitochondrial genomes for the five Aureoc. anophagefferens strains varied slightly in length (42,401–42,621 bp) and were 99.4–100.0% identical. Gene content and order were highly conserved between the Aureoc. anophagefferens and P. calceolata genomes, with the only major difference being a unique region in Aureoc. anophagefferens containingDNA adenine and cytosine methyltransferase (dam/dcm) genes that appear to be the product of lateral gene transfer from a prokaryotic or viral donor. Although the A. lagunensis mitochondrial genome shares seven distinct syntenic blocks with the other pelagophyte genomes, it has a tandem repeat expansion comprising ∼40% of its length, and lacks identifiable rps19 and glycine tRNA genes. Laterally acquired self-splicing introns were also found in the 23S rRNA (rnl) gene of P. calceolata and the coxI gene of the five Aureoc. anophagefferens genomes. Overall, these data provide baseline knowledge about the genetic diversity of bloom-forming pelagophytes relative to nonbloom-forming species.

Seasonal and Geographical Transitions in Eukaryotic Phytoplankton Community Structure in the Atlantic and Pacific Oceans

Much is known about how broad eukaryotic phytoplankton groups vary according to nutrient availability in marine ecosystems. However, genus- and species-level dynamics are generally unknown, although important given that adaptation and acclimation processes differentiate at these levels. We examined phytoplankton communities across seasonal cycles in the North Atlantic (BATS) and under different trophic conditions in the eastern North Pacific (ENP), using phylogenetic classification of plastid-encoded 16S rRNA amplicon sequence variants (ASVs) and other methodologies, including flow cytometric cell sorting. Prasinophytes dominated eukaryotic phytoplankton amplicons during the nutrient-rich deep-mixing winter period at BATS. During stratification (‘summer’) uncultured dictyochophytes formed ∼35 ± 10% of all surface plastid amplicons and dominated those from stramenopile algae, whereas diatoms showed only minor, ephemeral contributions over the entire year. Uncultured dictyochophytes also comprised a major fraction of plastid amplicons in the oligotrophic ENP. Phylogenetic reconstructions of near-full length 16S rRNA sequences established 11 uncultured Dictyochophyte Environmental Clades (DEC). DEC-I and DEC-VI dominated surface dictyochophytes under stratification at BATS and in the ENP, and DEC-IV was also important in the latter. Additionally, although less common at BATS, Florenciella-related clades (FC) were prominent at depth in the ENP. In both ecosystems, pelagophytes contributed notably at depth, with PEC-VIII (Pelagophyte Environmental Clade) and (cultured) Pelagomonas calceolata being most important. Q-PCR confirmed the near absence of P. calceolata at the surface of the same oligotrophic sites where it reached ∼1,500 18S rRNA gene copies ml–1 at the DCM. To further characterize phytoplankton present in our samples, we performed staining and at-sea single-cell sorting experiments. Sequencing results from these indicated several uncultured dictyochophyte clades are comprised of predatory mixotrophs. From an evolutionary perspective, these cells showed both conserved and unique features in the chloroplast genome. In ENP metatranscriptomes we observed high expression of multiple chloroplast genes as well as expression of a selfish element (group II intron) in the psaA gene. Comparative analyses across the Pacific and Atlantic sites support the conclusion that predatory dictyochophytes thrive under low nutrient conditions. The observations that several uncultured dictyochophyte lineages are seemingly capable of photosynthesis and predation, raises questions about potential shifts in phytoplankton trophic roles associated with seasonality and long-term ocean change.

Changes in the community structure of free-living heterotrophic bacteria in the open tropical Pacific Ocean in response to microalgal lysate-derived dissolved organic matter.

Dissolved organic matter derived from phytoplankton (DOMP) can affect the bacterial biomass and community structure in aquatic ecosystems. Here, we examined the community response of free-living heterotrophic bacteria, with respect to cellular nucleic acid levels, to the DOMP lysates derived from three phytoplankton strains in the open tropical Pacific. The free amino acid (FAA) composition of each DOMP lysate differed among the microalgal strains. Terminal restriction fragment-length polymorphism analyses with 16S rRNA genes revealed that the community shifts of high nucleic acid (HNA) and low nucleic acid (LNA) bacteria varied significantly with the different DOMP lysate treatments. Furthermore, the FAA composition in DOMP lysates significantly affected the bacterial community shifts in HNA and LNA. Similarity percentage analysis using 16S rRNA gene deep-sequencing revealed that the DOMP lysates from the pelagophyte Pelagomonas calceolata caused relatively large community shifts with Alcaligenes predominating in the HNA fraction. In contrast, the DOMP lysate from the diatom Thalassiosira oceanica induced a community shift in the LNA fraction with a predominance of uncultured Actinobacteria Thus, the data indicate that the DOMP lysates from different microalgae constitute a primary factor altering the dominant bacterial groups in the open ocean.

Temperature-dependent growth and photophysiology of prokaryotic and eukaryotic oceanic picophytoplankton

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 466:43-55 (2012) - DOI: https://doi.org/10.3354/meps09898 Temperature-dependent growth and photophysiology of prokaryotic and eukaryotic oceanic picophytoplankton Gemma Kulk1,*, Pablo de Vries1, Willem H. van de Poll2, Ronald J. W. Visser1, Anita G. J. Buma1 1Department of Ocean Ecosystems, Energy and Sustainability Research Institute Groningen, University of Groningen, 9747 AG Groningen, The Netherlands 2Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, 1790 AB Den Burg, The Netherlands *Email: g.kulk@rug.nl ABSTRACT: It is expected that climate change will expand the open oligotrophic oceans by enhanced thermal stratification. Because temperature defines the geographic distribution of picophytoplankton in open-ocean ecosystems and regulates photophysiological responses, it is important to understand how temperature affects picophytoplankton growth and photophysiology. Two prokaryotic and 2 eukaryotic picophytoplankton strains were acclimated to 3 different temperatures, ranging from 16 to 24°C. Temperature-dependent growth and photophysiology were assessed by measurements of specific growth rates, cell size, pigment composition, absorption and electron transport rates. Growth of Prochlorococcus marinus (eMED4), Prochlorococcus sp. (eMIT9313), Ostreococcus sp. (clade B) and Pelagomonas calceolata was positively related to temperature, especially in the prokaryotic strains. Changes in photophysiology included increased light harvesting, increased electron transport and reduced photoinhibition at elevated temperatures. However, the changes related to light harvesting and electron transport could not fully explain the observed difference in growth. This suggests that other processes, such as Calvin cycle activity, are likely to limit growth at sub-optimal temperatures in these picophytoplankton strains. The overall changes in photophysiology during temperature acclimation will possibly allow photosynthesis at higher irradiance intensities, but the genetically defined low temperature tolerances and photosynthetic characteristics of the different ecotypes will likely be more important in determining picophytoplankton (depth) distribution and community composition. KEY WORDS: Prochlorococcus · Eukaryotic picophytoplankton · Temperature · Growth · Pigment · Absorption · Electron transport rate Full text in pdf format PreviousNextCite this article as: Kulk G, de Vries P, van de Poll WH, Visser RJW, Buma AGJ (2012) Temperature-dependent growth and photophysiology of prokaryotic and eukaryotic oceanic picophytoplankton. Mar Ecol Prog Ser 466:43-55. https://doi.org/10.3354/meps09898 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 466. Online publication date: October 15, 2012 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2012 Inter-Research.

Algal Biogeography: Metagenomics Shows Distribution of a Picoplanktonic Pelagophyte

How can we determine the distribution of uncultured marine microorganisms? Targeted metagenomics has provided the complete chloroplast genome sequence, and the distribution, for a picoplanktonic pelagophyte alga.

Growth and photoregulation dynamics of the picoeukaryote Pelagomonas calceolata in fluctuating light

Growth, photosynthesis, and photoacclimation properties of batch cultures of Pelagomonas calceolata (Pelagophyceae) were compared for 1 week under three different fluctuating light regimes with the same total daily amount of light. Treatments consisted of a sinusoidal diurnal light cycle or a high‐frequency fluctuating light simulating two different regimes of vertical mixing (highly fluctuating light [HFL] or fluctuating light [FL]). Three to five samples were taken every day for analysis of pigments, absorption spectrum, variable fluorescence, nonphotochemical quenching (NPQ), electron transport rate vs. light curves, and cell concentration. Pelagomonas achieved the same growth rate during the exponential growth phase under all three light conditions, revealing a high degree of acclimation to light and also suggesting that the daily light dose is the main factor regulating growth and division. Photophysiological adjustments occurred in the cells in response to the three light regimes. Pelagomonas seems to adopt the n‐type photoacclimation in HFL, whereas the σ;‐type photoacclimation is applied in FL. The cells rapidly trigger photoprotective mechanisms such as the xanthophyll cycle and NPQ, even though these do not appear to be able to fully prevent photoinhibition. The enhanced costs for maintenance and repair associated with HFL may have limited the allocation of energy to growth, thus explaining the shorter duration of the exponential growth phase in this regime with respect to the two others.

Comparative Ecophysiology of the Xanthophyll Cycle in Six Marine Phytoplanktonic Species

The ecophysiology of the photoprotective xanthophyll cycle (XC) was compared in six chlorophyll c-containing pico- and nano-phytoplankton species. Different accessory pigment combinations and ecological properties characterize the six studied species, Bolidomonas mediterranea, Pelagomonas calceolata, Phaeocystis cordata, Phaeocystis sp. (strain RCC186), Mesopedinella arctica and Ochromonas sp. The first experimental set consisted in the study of the activity of the xanthophyll cycle and non-photochemical quenching (NPQ) during two gradual light increases, with photon flux density (PFD) ranging from 40 to 400 micromol photons m(-2)s(-1). Pigments, absorption spectra, flow cytometry measurements for cell counts and chlorophyll a autofluorescence and Electron Transport Rate (ETR) vs. light curves were determined at different times during the experiment. The second set of experiments consisted in using two inhibitors: carotenogenesis inhibitor (norflurazon) and de-epoxidation step (occurring in the xanthophyll cycle) inhibitor (dithiotreitol) during high-light shift, to compare the functioning of the xanthophyll cycle among the different species. Results highlighted a large diversity in the xanthophyll cycle functioning, possibly related to the ecological traits of the species. In view of the results, three groups of species have been identified as (i) high light-adapted, (ii) low light-adapted and (iii) variable light-adapted species.

Picoplankton diversity in the South-East Pacific Ocean from cultures

Abstract. In late 2004, the BIOSOPE cruise sailed between the equatorial influenced waters off the Marquesas Islands and the nutrient enriched waters of the Chilean upwelling. Along the way, it explored the Southeast Pacific gyre centred around Easter Island, which is probably the most oligotrophic oceanic region on earth. During this cruise, we undertook a vigorous effort to isolate novel photosynthetic picoplanktonic eukaryotes. Two strategies were attempted on board: enrichment of filtered samples with culture medium and sorting of specific populations by flow cytometry based on size and chlorophyll fluorescence. Over 1900 pre-cultures were started and then further purified by flow cytometry, serial dilution or pipette isolation to yield a total of 212 strains. These strains were characterized morphologically and for more than 50% of them, genetically, through partial sequencing of the 18 S rRNA gene. Among the characterized strains, the largest number belongs to stramenopiles (Heterokontophyta) with a record of 38 strains belonging to the species Pelagomonas calceolata (Pelagophyceae). Strains from the recently described genera Bolidomonas and Florenciella have been re-isolated for the first time since their description. Two other abundant groups are the Chlorophyta, especially Prasinophyceae, and the Haptophyta, especially the genera Phaeocystis and Emiliania. A limited number of heterotrophic flagellates have also been isolated, all of them belonging to groups containing known species. Finally, over a dozen of unicellular cyanobacterial Synechococcus strains have been obtained, some forming unusual short chains. Overall our strategy was quite successful since it allowed us to isolate a large number of picoplankton strains. Still it failed in two respects. First, apparently very few novel taxa have been obtained. One set of strains is related to Prasinoderma coloniale (Prasinococcales, Prasinophyceae) but their sequences are sufficiently different from the latter to probably belong to a new genus or species. The sequences of two other strains, unfortunately later lost, were phylogenetically affiliated to stramenopile environmental sequences, probably corresponding to a new algal class. Second, very few strains have been obtained from the very oligotrophic central gyre itself. In order to be successful, future work in similar waters should probably combine flow cytometry sorting with culture media and cultivation approaches specifically developed for oligotrophic water species.

Hybridisation of picoeukaryotes by eubacterial probes is widespread in the marine environment

Most general and group-specific eubacterial probes hybridised picoeukaryotes in coastal waters (Brittany, France) and cultures of the dominant picoeukaryotes from this environment (Micromonas pusilla and Pelagomonas calceolata). This is either because they matched the 16S rRNA from organelles or because of the presence of symbiotic or antagonist intracellular bacteria. The general eubacterial probe (EUB338) hybridised 84% of the picoeukaryotes, while the group-specific probes hybridised 3, 16, 10 and 34% of the picoeukaryotes for cyanobacteria (CYA664), alphaproteobacteria (ALF968), gamma-proteobacteria (GAM42a) and Cytophaga-Flavo-Bacteria (CF319), respectively. The results show that the hybridisation of eukaryote 16S rRNA by prokaryote probes can lead to significant errors in prokaryote counts, in particular for less well-represented groups such as cyanobacteria, with errors of 17% in the studied sample. In addition, we revealed for the first time at this scale that up to 44% of the picoeukaryotes contained intracellular prokaryotes. This finding might have serious implications for understanding the functioning of the microbial loop. Finally, because SSU rRNA databases have significantly been extended in recent years, we showed that the probe PLA886, which targets Planctomycete 16S rRNA, labelled 87% of the picoeukaryotes by hybridising their 18S rRNA. Consequently, the design of this probe should be refined for future studies, and the presence of similar changes in probe specificity should be checked regularly when using hybridisation-based techniques.

Physiological responses of three species of marine pico-phytoplankton to ammonium, phosphate, iron and light limitation

Experiments were conducted with three species of marine pico-phytoplankton: Synechococcus sp. (CCMP 839), Pelagomonas calceolata (CCMP 1756) and Prasinomonas capsulatus (CCMP 1617) in order to collect physiological parameters for pico-phytoplankton to be utilised in Ocean Biogeochemical Climate Models. The main parameters to follow the effects of ammonium, phosphate, iron and light limitation were cell growth rates (μ), half saturation constants for growth (K m), N, P and Fe quota (per cell or per mol C), and photochemical quantum efficiency (F v/F m). The nitrate and phosphate limitation experiments demonstrated that the small phytoplankton species could grow at low N and P concentrations. K m values were in the micro-molar (NH 4 +) and sub-micro-molar (PO 4 3−) range. N and P quota were in the femto-molar range per cell and varied from nutrient-deplete to nutrient-replete conditions. F v/F m values were only adversely affected at the lowest N and P concentrations in these experiments. In the Fe limitation experiments, it was shown that all three species were adversely affected only at extremely low Fe concentrations. Iron chelating agents had to be added to force the species in Fe limitation till ultimately growth stopped. K m values with respect to dissolved Fe were in the femto-molar range. Fe quota were in the low zepto-molar (10 −21 M) range per cell, and varied considerably from Fe limiting to Fe replete growth conditions. F v/F m values diminished only at the lowest iron concentrations. In the light limitation experiments, growth rates and photochemical quantum efficiencies were adversely affected only at irradiance levels below 10 μmol photons m −2 s −1. These results indicate that the pico-phytoplankton species will hardly ever be completely stopped in their growth by NH 4 +, PO 4 3−, Fe or light (separately) under natural conditions.

Isoprene production by Prochlorococcus, a marine cyanobacterium, and other phytoplankton

The oceans are a small source of light (C 2–C 6) non-methane hydrocarbons (NMHC), which influence the photo-oxidant chemistry of the remote marine atmosphere. Previous work has shown that water column sources of alkenes include photochemical processes, and that various phytoplankton species can produce isoprene. However, only a few phytoplankton species have been studied, and no assessment has been performed of the effects of other pelagic microorganisms on NMHC cycling. The dependence of phytoplanktonic isoprene production on light, temperature, and organism size has also not been investigated. In this work, laboratory cultures of five different marine phytoplankton species ( Prochlorococcus, Synechococcus, Micromonas pusilla, Pelagomonas calceolata, and Emiliania huxleyi) were examined for NMHC production capabilities. All species were found to produce isoprene at constant rates during the balanced exponential growth phase; rates ranged from 1×10 −21 to 4×10 −19 mol cell −1 day −1 over all cell species and growth conditions tested. No other NMHC was consistently produced or consumed by these cells. The presence of heterotrophic bacteria in phytoplankton cultures had no effect on isoprene production rates per phytoplankton cell. A positive allometric relationship was observed between isoprene production rate and cell volume; highest production rates were found for the largest cell, E. huxleyi, and lowest rates for Prochlorococcus, the smallest cell. Isoprene production was a function of light intensity and temperature in Prochlorococcus, with patterns that were similar to those between growth rate and these environmental variables. The maximum production with light intensity occurred in the photoinhibited regime, and the maximum with temperature was at the maximum of growth rate for this species, near 23 °C. Nanoflagellate grazing by Cafeteria roenbergensis on, and phage infection of, Prochlorococcus controlled total isoprene produced in the flask by controlling cell abundances. Phage infection also decreased the isoprene production rate per cell during latent period of infection as compared to healthy cells. With certain assumptions, combining the measured laboratory isoprene production rates with observed water column phytoplankton abundances resulted in a maximum estimated sea-to-air flux of isoprene that was on the same order of magnitude as previously reported values determined using in situ measured seawater and atmospheric measurements.

DIEL PATTERNS OF GROWTH AND DIVISION IN MARINE PICOPLANKTON IN CULTURE

The effect of a 12:12‐h light:dark (LD) cycle on the phasing of several cell parameters was explored in a variety of marine picophytoplanktonic strains. These included the photosynthetic prokaryotes Prochlorococcus (strains MED 4, PCC 9511, and SS 120) and Synechococcus (strains ALMO 03, ROS 04, WH 7803, and WH 8103) and five picoeukaryotes (Bathycoccus prasinos Eikrem et Throndsen, Bolidomonas pacifica Guillou et Chrétiennot‐Dinet, Micromonas pusilla Manton et Parke, Pelagomonas calceolata Andersen et Saunders, and Pycnococcus provasolii Guillard et al.). Flow cytometric analysis was used to determine the relationship between cell light scatter, pigment fluorescence, DNA (when possible), and the LD cycle in these organisms. As expected, growth and division were tightly coupled to the LD cycle for all of these strains. For both Prochlorococcus and picoeukaryotes, chl and intracellular carbon increased throughout the light period as estimated by chl fluorescence and light scatter, respectively. In response to cell division, these parameters decreased regularly during the early part of the dark period, a decrease that either continued throughout the dark period or stopped for the second half of the dark period. For Synechococcus, the decrease of chl and scatter occurred earlier (in the middle of the light period), and for some strains these cellular parameters remained constant throughout the dark period. The timing of division was very similar for all picoeukaryotes and occurred just before the subjective dusk, whereas it was more variable between the different Prochlorococcus and Synechococcus strains. The burst of division for Prochlorococcus SS 120 and PCC 9511 was recorded at the subjective dusk, whereas the MED 4 strain divided later at night. Synechococcus ALMO 03, ROS 04, and WH 7803, which have a low phycourobilin to phycoerythrobilin (PUB:PEB) ratio, divided earlier, and their division was restricted to the light period. In contrast, the high PUB:PEB Synechococcus strain WH 8103 divided preferentially at night. There was a weak linear relationship between the FALSmax:FALSmin ratio and growth rate calculated from cell counts (r = 0.83, n = 11, P < 0.05). Because of the significance of picoplanktonic populations in marine systems, these results should help to interpret diel variations in oceanic optical properties in regions where picoplankton dominates.

Micro- and macrodiversity in rbcL sequences in ambient phytoplankton populations from the southeastern Gulf of Mexico

Ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO) large subunit genes (rbcL) were obtained by amplification and cloning of 554 or 614 bp sequences of indigenous phytoplankton populations at 2 stations in the southeastern Gulf of Mexico. One station (Stn 4) was located in a low salinity, high chlorophyll plume (the 'Green River') which has previously been shown to contain elevated levels of Form IA rbcL mRNA while the other (Stn 7) was in oligotrophic, oceanic water. A diversity of rbcL sequences was obtained, spanning 3 of the 4 evolutionary clades of Form I RuBisCOs. Six nucleotide sequences obtained from Stn 4 were closely related (92 to 96% similar) to the Form IA-containing Prochlorococcus GP2. Flow cytometry and pigment analysis indicated that Prochlorococcus was abundant at this site. Other sequences found included a Form IB rbcL closely related to prasinophytes, and Form ID sequences related to prymnesiophytes, diatoms, and pelagophytes. One sequences was nearly identical to the pelagophyte, Pelagomonas calceolata. At Stn 7, sequences were obtained that were more deeply rooted, and less similar to rbcLs in existing databases (77 to 83% similar), and no Form IA rbcLs were detected. HPLC pigment signatures and flow cytometry data were consistent with the forms obtained by cloning. The similarity of the 6 Prochlorococcus GP2-like sequences (93 to 98%) is consistent with the phenomenon of molecular microdiversity as found at other loci in marine (and other environmental) microorganisms.

THE FLAGELLAR DEVELOPMENT CYCLE OF THE UNIFLAGELLATE PELAGOMONAS CALCEOLATA (PELAGOPHYCEAE)1

ABSTRACT Vegetative cells of Pelagomonas calceolata Andersen & Saunders were confirmed to possess a reduced flagellar apparatus, consisting of a single basal body/flagellum that is not accompanied by either flagellar roots or a barren basal body. Just prior to division, the parental flagellum retracts (or is abscised) as two new basal bodies/flagella arise de novo. During cytokinesis the parental basal body segregates with a new basal body/flagellum, briefly producing a progeny cell typical of other known uniflagellates (i.e. containing a basal body/flagellum and accompanying barren basal body). The parental basal body then disintegrates or “transforms” out of existence, leaving both progeny cells with a single basal body/flagellum (i.e. neither progeny cell possesses any vestige of a mature flagellum/basal body). Pelagomonas calceolata belongs to a lineage of chromophyte algae characterized by having a reduced flagellar apparatus, but it is the only known species, not only in this lineage but among all eukaryotes, to have undergone the complete elimination of the mature flagellum /basal body.

PHYLOGENY OF AUREOCOCCUS ANOPHAGEFFERENS AND A MORPHOLOGICALLY SIMILAR BLOOM‐FORMING ALGA FROM TEXAS AS DETERMINED BY 18S RIBOSOMAL RNA SEQUENCE ANALYSIS1

ABSTRACT The 18S ribosomal RNA genes from isolates of the east coast “brown tide” alga Aureococcus anophagefferens (strain Pt‐1) and the Texas “brown tide” alga (strain TBA‐2) were sequenced and compared to the gene sequences of Pelagomonas calceolata, a member of the Pelagophyceae, and 10 other organisms. The genes of A. anophagefferens and strain TBA‐2 consisted of 1814 and 2236 bases, respectively. The difference in length was largely due to a 423‐base insert occurring in the gene of strain TBA‐2. Excluding the insert, 93% of the bases of the aligned sequences were identical. Phylogenetic analyses were performed based on two different alignment refinement methods (eye refinement and Gatesy refinement). Trees inferred from the Jukes‐Cantor distance matrices using the neighbor‐joining method were similar for both alignment methods. In both trees, A. anophagefferens, strain TBA‐2, and P. calceolata formed a monophyletic group with A. anophagefferens and P. calceolata being sister taxa and strain TBA‐2 occurring on a deeper‐rooted branch. Bootstrap data sets for both alignment methods gave strong support for the Pelagophyceae group and the branches within that group. Parsimony analyses using the two alignments gave one tree for the eye‐refined alignment and two trees for the Gatesy method. All three trees had nearly the same topology as the trees inferred from the distance method. In addition, the Pelagophyceae group and the branches within that group were supported by bootstrap analyses and decay indices. Based on the available data, A. anophagefferens and strain TBA‐2 should be placed in the Pelagophyceae but not in the same order and family as P. calceolata.

Ribosomal RNA Evidence for Chloroplast Loss within Heterokonta: Pedinellid Relationships and a Revised Classification of Ochristan Algae

We have sequenced the 18S rRNA genes of the non-photosynthetic pedinellids Ciliophrys infusionum (syn. C. marina) and Pteridomonas danica. We have carried out molecular phylogenetic analysis by parsimony, maximum likelihood, and two distance methods (neighbor joining and Fitch). All four methods show that these ciliophryid pedinellids are most closely related to Pelagomonas calceolata, which supports the recent classification of the classes Pedinellea and Pelagophycea in the superclass Dictyochia. A very rare nucleotide substitution in a highly conserved region is shared by all three Dictyochia and no other Heterokonta. Our trees, based on 30 different heterokonts, show that these two ciliophryids evolved from a photosynthetic ancestor by the loss of chloroplasts. This is the first convincing molecular evidence for the total loss of plastids in any organism. We propose a new classification for the pedinellids in which the order Pedinellales is restricted to the genera with plastids, and a new order Ciliophryales is proposed for those that lack them. Each order is divided into two families according to whether or not a stalk is present: this requires the creation of a new family, Apedinellaceae, and the emendation of three existing families. We reduce the class Pedinellea to the rank of subclass (Pedinellidae), and create a new class, Actinochrysea, characterized by radiating microtubules attached basally to the nuclear envelope, to include pedinellids, silicoflagellates, and Rhizochromulinales. Rhizochromulinales are grouped with silicoflagellates in the subclass Dictyochidae, the putative sister group of Pedinellidae. The homogeneity of the Chrysophycea, already increased by the removal of pedinellids, bicoecids, and Rhizochromulina, is still further increased by removing Sarcinochrysidales and Chrysomeridales to form two new classes, Sarcinochrysea and Chrysomerophycea. We place Sarcinochrysea in the infraphylum Raphidoista, which includes the Dictyochia, and group Chrysomerophycea with Xanthophycea and Phaeophycea in a new superclass, Fucistia, of the infraphylum Chrysista. The remaining classes in the infraphylum Chrysista (Chrysophycea and Flavoretea) are grouped together in a new superclass Phagochrysia