Diatom Endosymbionts Research Articles

Diurnal transcriptional variation is reduced in a nitrogen-fixing diatom endosymbiont.

Many organisms have formed symbiotic relationships with nitrogen (N)-fixing bacteria to overcome N limitation. Diatoms in the family Rhopalodiaceae host unicellular, N-fixing cyanobacterial endosymbionts called spheroid bodies (SBs). Although this relationship is relatively young, SBs share many key features with older endosymbionts, including coordinated cell division and genome reduction. Unlike free-living relatives that fix N exclusively at night, SBs fix N largely during the day; however, how SB metabolism is regulated and coordinated with the host is not yet understood. We compared four SB genomes, including those from two new host species (Rhopalodia gibba and Epithemia adnata), to build a genome-wide phylogeny which provides a better understanding of SB evolutionary origins. Contrary to models of endosymbiotic genome reduction, the SB chromosome is unusually stable for an endosymbiont genome, likely due to the early loss of all mobile elements. Transcriptomic data for the R. gibba SB and host organelles addressed whether and how the allocation of transcriptional resources depends on light and nitrogen availability. Although allocation to the SB was high under all conditions, relative expression of chloroplast photosynthesis genes increased in the absence of nitrate, but this pattern was suppressed by nitrate addition. SB expression of catabolism genes was generally greater during daytime rather than at night, although the magnitude of diurnal changes in expression was modest compared to free-living Cyanobacteria. We conclude that SB daytime catabolism likely supports N-fixation by linking the process to host photosynthetic carbon fixation.

Prey preference in a kleptoplastic dinoflagellate is linked to photosynthetic performance

Dinoflagellates of the family Kryptoperidiniaceae, known as “dinotoms”, possess diatom-derived endosymbionts and contain individuals at three successive evolutionary stages: a transiently maintained kleptoplastic stage; a stage containing multiple permanently maintained diatom endosymbionts; and a further permanent stage containing a single diatom endosymbiont. Kleptoplastic dinotoms were discovered only recently, in Durinskia capensis; until now it has not been investigated kleptoplastic behavior and the metabolic and genetic integration of host and prey. Here, we show D. capensis is able to use various diatom species as kleptoplastids and exhibits different photosynthetic capacities depending on the diatom species. This is in contrast with the prey diatoms in their free-living stage, as there are no differences in their photosynthetic capacities. Complete photosynthesis including both the light reactions and the Calvin cycle remain active only when D. capensis feeds on its habitual associate, the “essential” diatom Nitzschia captiva. The organelles of another edible diatom, N. inconspicua, are preserved intact after ingestion by D. capensis and expresses the psbC gene of the photosynthetic light reaction, while RuBisCO gene expression is lost. Our results indicate that edible but non-essential, “supplemental” diatoms are used by D. capensis for producing ATP and NADPH, but not for carbon fixation. D. capensis has established a species-specifically designed metabolic system allowing carbon fixation to be performed only by its essential diatoms. The ability of D. capensis to ingest supplemental diatoms as kleptoplastids may be a flexible ecological strategy, to use these diatoms as “emergency supplies” while no essential diatoms are available.

Sterols of the ‘dinotom’ Durinskia baltica (Dinophyceae) are of dinoflagellate origin

SUMMARY‘Dinotoms’ are a relatively small group of dinoflagellates with aberrant tertiary plastids of diatom origin, thus differing from the majority of photosynthetic dinoflagellates which possess the carotenoid pigment peridinin and have secondary plastids of red algal origin. As part of our laboratory's continuing efforts to examine such unusual dinoflagellates in the search for clues to the evolution of their lipid compositions, we have examined the sterol composition of the dinotom Durinskia baltica. As such, we here compared its sterols to those of the previously examined dinotom, Kryptoperidinium foliaceum, more broadly to other photosynthetic, peridinin‐containing dinoflagellates, and to the diatom genus Nitzschia, which is the presumed ancestor of the D. baltica dinotom plastid. Sterols are ringed lipids, common to eukaryotes, thought to reinforce phospholipid bilayers. Many peridinin‐containing dinoflagellates have sterol compositions which are enriched by the presence of cholesterol (cholest‐5‐en‐3β‐ol) and 4α‐methyl‐substituted sterols such as dinosterol (4α,23,24‐trimethyl‐5α‐cholest‐22E‐en‐3β‐ol); this has also been found to be true for K. foliaceum despite its aberrant plastid ancestry. Our objective was to determine if this is also true for D. baltica as only the second dinotom to have its sterols characterized in detail, and to determine if there is any indication of prominent sterols which are uncommon to dinoflagellates, possibly originating from the diatom endosymbiont, as has been demonstrated previously with K. foliaceum and D. baltica chloroplast‐associated galactolipids of clear diatom origin. Our results demonstrate that like K. foliaceum, the major sterols of D. baltica are cholesterol, dinosterol, and other 4α‐methyl‐substituted sterols common to dinoflagellates. Although there were a number of minor sterols, none were found with obvious origin from the diatom endosymbiont, indicating that most originated with the dinoflagellate host itself, most likely before acquisition of the diatom tertiary plastid.

Diversity and flexibility of algal symbiont community in globally distributed larger benthic foraminifera of the genus Amphistegina

BackgroundUnderstanding the specificity and flexibility of the algal symbiosis-host association is fundamental for predicting how species occupy a diverse range of habitats. Here we assessed the algal symbiosis diversity of three species of larger benthic foraminifera from the genus Amphistegina and investigated the role of habitat and species identity in shaping the associated algal community.ResultsWe used next-generation sequencing to identify the associated algal community, and DNA barcoding to identify the diatom endosymbionts associated with species of A. lobifera, A. lessonii, and A. radiata, collected from shallow habitats (< 15 m) in 16 sites, ranging from the Mediterranean Sea to French Polynesia. Next-generation sequencing results showed the consistent presence of Ochrophyta as the main algal phylum associated with all species and sites analysed. A significant proportion of phylotypes were classified as Chlorophyta and Myzozoa. We uncovered unprecedented diversity of algal phylotypes found in low abundance, especially of the class Bacillariophyta (i.e., diatoms). We found a significant influence of sites rather than host identity in shaping algal communities in all species. DNA barcoding revealed the consistent presence of phylotypes classified within the order Fragilariales as the diatoms associated with A. lobifera and A. lessonii, while A. radiata specimens host predominately diatoms of the order Triceratiales.ConclusionsWe show that local habitat is the main factor influencing the overall composition of the algal symbiont community. However, host identity and the phylogenetic relationship among hosts is relevant in shaping the specific endosymbiont diatom community, suggesting that the relationship between diatom endosymbiont and hosts plays a crucial role in the evolutionary history of the genus Amphistegina. The capacity of Amphistegina species to associate with a diverse array of diatoms, and possibly other algal groups, likely underpins the ecological success of these crucial calcifying organisms across their extensive geographic range.

Morpho-molecular and spectroscopic characterization of the freshwater dinoflagellate Unruhdinium penardii var. robustum (Kryptoperidiniaceae, Peridiniales), blooming in the Loir River, France

A freshwater dinoflagellate formed extensive red tides in the Loir River, France in September 2018. Morphological observations using light microscopy and field emission scanning electron microscopy identified the causative organism as the“dinotom” Unruhdinium penardii var. robustum, with a tabulation of Po, x, 4ʹ, 0a, 6ʹʹ, 5c (or t+4c), 5s, 5ʹʹʹ, 2ʹʹʹʹ. The thecal plate overlap of the variety is documented for the first time. Division cysts were also observed in the plankton assemblage. Sequences obtained from the vegetative cells of small subunit (SSU) and large subunit (LSU) ribosomal DNA (rDNA) are identical to previously published sequences from Japan. This is the first unambiguous identification from French waters of this species and its variety. A SSU rDNA sequence is also reported for the diatom endosymbiont, which is close to the diatom genus Discostella. Fourier transform infrared spectroscopic analysis showed that division cysts and thecae of U. penardii var. robustum are cellulose-like, but with a higher degree of cross-linking than in microcrystalline cellulose and with a more complex macromolecular buildup.

First detection of the bloom forming Unruhdinium penardii (Dinophyceae) in a Mediterranean reservoir: insights on its ecology, morphology and genetics

The freshwater genus Unruhdinium includes dinoflagellates hosting a tertiary diatom endosymbiont. Some of the species belonging to this genus form high-biomass blooms. In this study, data on the ecology, morphology and molecular identity of Unruhdinium penardii were reported for the first time from a Mediterranean reservoir (Cedrino Lake, Sardinia, Italy). The ecology of the species and its bloom events were examined along a multiannual series of data (2010-2017). Cell morphology was investigated using field samples and six cultures established by cell isolation. A molecular identification of the six strains was performed. Wild and cultured cells shared the same morphology, showing a prominent apical pore complex and two/three more or less prominent hypothecal spines as distinctive characters in light microscopy. Molecularly, the six cultured strains corresponded to the same taxonomic entity with sequences only differing in a few polymorphic positions for the studied markers SSU rDNA, LSU rDNA, ITS and endosymbiont SSU rDNA. All markers showed 99.5%−100% similarity with the available U. penardii sequences. Seasonality of U. penardii revealed its preference for the colder semester (from December to June) with bloom events restricted to late winter/early spring months. Three blooms resulting in reddish water discolorations were observed along the study period (2011, 2012 and 2017). GLMs revealed a significant role of water depth, temperature, and reactive phosphorous in determining the highest cell densities (&gt;5 x 104 cells L-1). The results obtained contribute to the increase of field ecology knowledge on this species, demonstrating it is well established in the Mediterranean area, and being able to produce recurrent high biomass blooms in the studied reservoir.

Marker gene analysis reveals the spatial and seasonal variations in the eukaryotic phytoplankton community composition in the Yangtze River, Three Gorges Reservoir, China

ABSTRACTThe eukaryotic phytoplankton community in the Three Gorges Reservoir (TGR) of the Yangtze River in China was investigated, based on marker gene analysis of the 18S rRNA V4 region. Community composition was investigated in four seasons (to assess seasonal variation) and in the mainstream and tributary of the river (to assess spatial variation). Chlorophyll a concentration was used to estimate eukaryotic phytoplankton biomass. A significant negative correlation of biomass and evenness was found in the TGR; this relationship may be due to a few highly competitive species dominating the eukaryotic phytoplankton community, suggesting that eukaryotic phytoplankton bloom events tend to occur in such communities. Our results showed that eukaryotic phytoplankton is more likely to bloom in the tributary when compared with mainstream, and we identified two unreported algal blooms, Tetraselmis and Mychonastes. Phylogenetic analysis revealed a novel lineage in Cryptophyta and substantially more diverse species in Bacillariophyta, two high relative abundance operational taxonomic units in Bacillariophyta classified to Peridiniopsis diatom endosymbionts; their high relative abundance may result from a high relative abundance of the genus Peridiniopsis. We found that Cryptomonas was the most widely distributed genus and had the highest relative abundance, and species in TGR that have higher relative abundance tend to be distributed more widely.

Survival and recovery of the foraminifer Amphistegina gibbosa and associated diatom endosymbionts following up to 20 months in aphotic conditions

Abstract Dormancy in the Foraminifera has been observed widely across the phylum in reaction to a variety of triggers including, in the diatom symbiont-bearing foraminifer Amphistegina gibbosa, extended periods of darkness. Resumption of activity in the host-symbiont holobiont was noted, but not fully documented, in specimens reintroduced to light following up to 12 months in darkness. Here, criteria for documenting recovery included resumption of reticulopodial activity in the host and return of pre-treatment golden-brown color characteristic of an active symbiotic diatom population. Reticulopodial activity resumed in nearly all treatment specimens (>95%) following 12 months in darkness, and in >70% of the specimens when reintroduced to light following 20 months in darkness. Image analysis using the percent of the foraminiferal surface area showing golden-brown color as a proxy for recovery of the endosymbionts showed return of such color within days for shorter treatments (7 and 12 months in darkness), but slower and less complete return in longer treatments (15 and 20 months), indicating increased susceptibility to photic damage of symbionts as the length of dormancy increased.

Microbial Associations of Four Species of Algal Symbiont-bearing Foraminifers from the Florida Reef Tract, Usa

Abstract While microbiome research is a rapidly expanding field of study, relatively little is known of the microbiomes associated with Foraminifera. This preliminary study investigated microbes associated with four species of Foraminifera, representing two taxonomic orders, which host three kinds of algal endosymbionts. A major objective was to explore potential influences on the microbiome composition, including phylogenetic relatedness among the host species, similarities in algal symbionts hosted, and environmental conditions from which the specimens were collected. Samples examined from two locations along the middle Florida Keys reef tract included 45 foraminiferal specimens and four environmental samples. Bacterial DNA extraction from individual specimens was followed by amplification and amplicon sequencing of the V4 variable region of the 16S rRNA gene; results were obtained from 21 specimens. The Order Miliolida, Family Soritidae, was represented by 5–8 specimens of each of three species: Archaias angulatus and Cyclorbiculina compressa, which both host chlorophyte symbionts, and Sorites orbiculus, which hosts dinoflagellate symbionts. Three Ar. angulatus specimens from which the microbiome was successfully sequenced shared 177 OTUs. Six C. compressa specimens successfully sequenced shared 58 OTUs, of which 31 were also shared by the three specimens of Ar. angulatus. Four successfully sequenced S. orbiculus specimens shared 717 unique OTUs. The 13 soritid specimens shared 26 OTUs, 23 of which represented Proteobacteria, predominantly of the bacterial family Rhodobacteraceae. The fourth foraminiferal species, Amphistegina gibbosa (Order Rotaliida) hosts diatom endosymbionts. Bacterial DNA extraction was attempted on 16 Am. gibbosa, including both normal-appearing and partly-bleached specimens. Only six OTUs, four of which represented Proteobacteria, were found in all eight specimens successfully sequenced. The partly bleached specimens shared nearly twice as many unique microbial OTUs (32) as the normal-appearing specimens (19). All Am. gibbosa specimens shared only four microbial OTUs with the soritid species, three of which may have been contaminants, indicating minimal commonality between the microbiomes of Am. gibbosa and the soritid taxa.

Diverse Internal Symbiont Community in the Endosymbiotic Foraminifera Pararotalia calcariformata: Implications for Symbiont Shuffling Under Thermal Stress.

Many shallow-water tropical and subtropical foraminifera engage in photosymbiosis with eukaryotic microalgae. Some of these foraminifera appear to harbor a diverse consortium of endosymbiotic algae within a single host. Such apparent ability to contain different symbionts could facilitate change in symbiont community composition (symbiont shuffling) and mediate the ecological success of the group in a changing environment. However, the discovery of the intra-individual symbiont diversity was thus far based on symbiont culturing, which provides strong constraints on the vitality of the identified algae but provides poor constraints on their initial abundance and thus functional relevance to the host. Here we analyze the algal symbiont diversity in Pararotalia calcariformata, a benthic foraminifera sampled at four stations, inside and outside of a thermal plume in the eastern Mediterranean coast of Israel. This species has recently invaded the Mediterranean, is unusually thermally tolerant and was described previously to host at least one different diatom symbiont than other symbiont-bearing foraminifera. Our results using genotyping and isolation of algae in culture medium, confirm multiple associations with different diatom species within the same individual. Both methods revealed spatially consistent symbiont associations and identified the most common symbiont as a pelagic diatom Minutocellus polymorphus. In one case, an alternative dominant symbiont, the diatom Navicula sp., was detected by genotyping. This diatom was the third most abundant species identified using standard algae culturing method. This method further revealed a spatially consistent pattern in symbiont diversity of a total of seventeen identified diatom species, across the studied localities. Collectively, these results indicate that P. calcariformata hosts a diverse consortium of diatom endosymbionts, where different members can become numerically dominant and thus functionally relevant in a changing environment.

Challenges in using CellTracker Green on foraminifers that host algal endosymbionts.

The uses of fluorescent microscopy and fluorescent probes, such as the metabolically activated probe CellTracker™ Green CMFDA (CTG), have become common in studies of living Foraminifera. This metabolic requirement, as well as the relatively quick production of the fluorescent reaction products, makes CTG a prime candidate for determining mortality in bioassay and other laboratory experiments. Previous work with the foraminifer Amphistegina gibbosa, which hosts diatom endosymbionts, has shown that the species is capable of surviving both acute chemical exposure and extended periods of total darkness by entering a low-activity dormant state. This paper explores the use of CTG and fluorescent microscopy to determine mortality in such experiments, as well as to explore the physiology of dormant foraminifers. The application of CTG was found to be complicated by the autofluorescence of the diatom symbionts, which masks the signal of the CTG, as well as by interactions between CTG and propylene glycol, a chemical of interest known to cause dormancy. These complications necessitated adapting methods from earlier studies using CTG. Here we present observations on CTG fluorescence and autofluorescence in A. gibbosa following both chemical exposure and periods of total darkness. While CTG can indicate vital activity in dormant foraminifers, complications include underestimates of total survival and recovery, and falsely indicating dead individuals as live due to rapid microbial colonization. Nonetheless, the brightness of the CTG signal in dormant individuals exposed to propylene glycol supports previously published results of survival patterns in A. gibbosa. Observations of CTG fluorescence in individuals kept for extended periods in aphotic conditions indicate uptake of CTG may begin within 30 min of exposure to light, suggesting darkness-induced dormancy and subsequent recovery can occur on short time scales. These results suggest that CTG accurately reflects changes associated with dormancy, and can be useful in laboratory experiments utilizing symbiont-bearing foraminifers.

Two new generic names for dinophytes harbouring a diatom as an endosymbiont, Blixaea and Unruhdinium (Kryptoperidiniaceae, Peridiniales)

Kryptoperidiniaceae are a small group of dinophytes hosting a tertiary endosymbiont derived from a diatom (Tomas et al. 1973, Horiguchi &amp; Pienaar 1994). Those so called ‘dinotoms’ (Imanian et al. 2011) include Dinothrix, Durinskia, Galeidinium, Kryptoperidinium (= Phyllodinium) and some species currently assigned to “Peridiniopsis” and “Peridinium” (Tamura et al. 2005, Horiguchi &amp; Takano 2006, Hansen et al. 2007, Zhang et al. 2011). Besides ‘possessing a diatom endosymbiont’ as a highly derived trait, the monophyly of Kryptoperidiniaceae is also supported by a unique type of eyespot that has possibly derived from the original chloroplast (Moestrup &amp; Daugbjerg 2007). In molecular trees, Kryptoperidiniaceae constitute a well supported monophyletic group (Kretschmann et al. unpubl.), but it is not finally resolved at present, whether they are embedded in the Thoracosphaeraceae or constitute their sister group (Gottschling &amp; McLean 2013). Regarding habitat preference, molecular trees further indicate at least two independent marine→freshwater transitions in the Kryptoperidiniaceae during the late Paleogene at the latest (Žerdoner Čalasan et al. unpubl.).

Evolution of the Tetrapyrrole Biosynthetic Pathway in Secondary Algae: Conservation, Redundancy and Replacement.

Tetrapyrroles such as chlorophyll and heme are indispensable for life because they are involved in energy fixation and consumption, i.e. photosynthesis and oxidative phosphorylation. In eukaryotes, the tetrapyrrole biosynthetic pathway is shaped by past endosymbioses. We investigated the origins and predicted locations of the enzymes of the heme pathway in the chlorarachniophyte Bigelowiella natans, the cryptophyte Guillardia theta, the “green” dinoflagellate Lepidodinium chlorophorum, and three dinoflagellates with diatom endosymbionts (“dinotoms”): Durinskia baltica, Glenodinium foliaceum and Kryptoperidinium foliaceum. Bigelowiella natans appears to contain two separate heme pathways analogous to those found in Euglena gracilis; one is predicted to be mitochondrial-cytosolic, while the second is predicted to be plastid-located. In the remaining algae, only plastid-type tetrapyrrole synthesis is present, with a single remnant of the mitochondrial-cytosolic pathway, a ferrochelatase of G. theta putatively located in the mitochondrion. The green dinoflagellate contains a single pathway composed of mostly rhodophyte-origin enzymes, and the dinotoms hold two heme pathways of apparently plastidal origin. We suggest that heme pathway enzymes in B. natans and L. chlorophorum share a predominantly rhodophytic origin. This implies the ancient presence of a rhodophyte-derived plastid in the chlorarachniophyte alga, analogous to the green dinoflagellate, or an exceptionally massive horizontal gene transfer.

Functional Relationship between a Dinoflagellate Host and Its Diatom Endosymbiont.

While we know much about the evolutionary patterns of endosymbiotic organelle origins, we know less about how the actual process unfolded within each system. This is partly due to the massive changes endosymbiosis appears to trigger, and partly because most organelles evolved in the distant past. The dinotoms are dinoflagellates with diatom endosymbionts, and they represent a relatively recent but nevertheless obligate endosymbiotic association. We have carried out deep sequencing of both the host and endosymbiont transcriptomes from two dinotoms, Durinskia baltica and Glenodinium foliaceum, to examine how the nucleocytosolic compartments have functionally integrated. This analysis showed little or no functional reduction in either the endosymbiont or host, and no evidence for genetic integration. Rather, host and endosymbiont seem to be bound to each other via metabolites, such as photosynthate exported from the endosymbiont to the host as indicated by the presence of plastidic phosphate translocators in the host transcriptome. The host is able to synthesize starch, using plant-specific starch synthases, as a way to store imported photosynthate.

Evidence for the retention of two evolutionary distinct plastids in dinoflagellates with diatom endosymbionts.

Dinoflagellates harboring diatom endosymbionts (termed “dinotoms”) have undergone a process often referred to as “tertiary endosymbiosis”—the uptake of algae containing secondary plastids and integration of those plastids into the new host. In contrast to other tertiary plastids, and most secondary plastids, the endosymbiont of dinotoms is distinctly less reduced, retaining a number of cellular features, such as their nucleus and mitochondria and others, in addition to their plastid. This has resulted in redundancy between host and endosymbiont, at least between some mitochondrial and cytosolic metabolism, where this has been investigated. The question of plastidial redundancy is particularly interesting as the fate of the host dinoflagellate plastid is unclear. The host cytosol possesses an eyespot that has been postulated to be a remnant of the ancestral peridinin plastid, but this has not been tested, nor has its possible retention of plastid functions. To investigate this possibility, we searched for plastid-associated pathways and functions in transcriptomic data sets from three dinotom species. We show that the dinoflagellate host has indeed retained genes for plastid-associated pathways and that these genes encode targeting peptides similar to those of other dinoflagellate plastid-targeted proteins. Moreover, we also identified one gene encoding an essential component of the dinoflagellate plastid protein import machinery, altogether suggesting the presence of a functioning plastid import system in the host, and by extension a relict plastid. The presence of the same plastid-associated pathways in the endosymbiont also extends the known functional redundancy in dinotoms, further confirming the unusual state of plastid integration in this group of dinoflagellates.

Horizontal gene transfer and redundancy of tryptophan biosynthetic enzymes in dinotoms.

A tertiary endosymbiosis between a dinoflagellate host and diatom endosymbiont gave rise to “dinotoms,” cells with a unique nuclear and mitochondrial redundancy derived from two evolutionarily distinct eukaryotic lineages. To examine how this unique redundancy might have affected the evolution of metabolic systems, we investigated the transcription of genes involved in biosynthesis of the amino acid tryptophan in three species, Durinskia baltica, Kryptoperidinium foliaceum, and Glenodinium foliaceum. From transcriptome sequence data, we recovered two distinct sets of protein-coding transcripts covering the entire tryptophan biosynthetic pathway. Phylogenetic analyses suggest a diatom origin for one set of the proteins, which we infer to be expressed in the endosymbiont, and that the other arose from multiple horizontal gene transfer events to the dinoflagellate ancestor of the host lineage. This is the first indication that these cells retain redundant sets of transcripts and likely metabolic pathways for the biosynthesis of small molecules and extend their redundancy to their two distinct nuclear genomes.

Nitrogen-fixing cyanobacteria (free-living and diatom endosymbionts): their use in southern California stream bioassessment

A weight-of-evidence approach was used to examine how nutrient availability influences stream benthic algal community structure and to validate nutrient-response thresholds in assessing nutrient limitation. Data from 104 southern California streams spanning broad nutrient gradients revealed that relative abundance of N2-fixing heterocystous cyanobacteria (Nostoc, Calothrix), and diatoms (Epithemia, Rhopalodia)-containing cyanobacterial endosymbionts, decreased with increasing ambient inorganic N concentrations within the low end of the N gradient. Response thresholds for these N2 fixers were 0.075 mg l−1 NO3-N, 0.04 mg l−1 NH4-N, and an N:P ratio (by weight) of 15:1. The NO3-N threshold was independently validated by observing nitrogenase gene expression using real-time reverse transcriptase PCR. Morphometric analysis of cyanobacterial endosymbionts in Epithemia and Rhopalodia indicated that endosymbiont biovolume per diatom cell decreased with increasing NO3-N. Our findings indicate that abundance of heterocyst-containing cyanobacteria and endosymbiont-containing diatom cells are good indicators for rapid nutrient biomonitoring. Because heterocystous cyanobacteria and Epithemia/Rhopalodia were not always recorded together at N-limited sites, examining both assemblages jointly may provide a more comprehensive assessment of stream nutrient limitation than using either assemblage alone.

Tertiary endosymbiosis in two dinotoms has generated little change in the mitochondrial genomes of their dinoflagellate hosts and diatom endosymbionts.

BackgroundMitochondria or mitochondrion-derived organelles are found in all eukaryotes with the exception of secondary or tertiary plastid endosymbionts. In these highly reduced systems, the mitochondrion has been lost in all cases except the diatom endosymbionts found in a small group of dinoflagellates, called ‘dinotoms’, the only cells with two evolutionarily distinct mitochondria. To investigate the persistence of this redundancy and its consequences on the content and structure of the endosymbiont and host mitochondrial genomes, we report the sequences of these genomes from two dinotoms.Methodology/Principal FindingsThe endosymbiont mitochondrial genomes of Durinskia baltica and Kryptoperidinium foliaceum exhibit nearly identical gene content with other diatoms, and highly conserved gene order (nearly identical to that of the raphid pennate diatom Fragilariopsis cylindrus). These two genomes are differentiated from other diatoms' by the fission of nad11 and by an insertion within nad2, in-frame and unspliced from the mRNA. Durinskia baltica is further distinguished from K. foliaceum by two gene fusions and its lack of introns. The host mitochondrial genome in D. baltica encodes cox1 and cob plus several fragments of LSU rRNA gene in a hugely expanded genome that includes numerous pseudogenes, and a trans-spliced cox3 gene, like in other dinoflagellates. Over 100 distinct contigs were identified through 454 sequencing, but intact full-length genes for cox1, cob and the 5′ exon of cox3 were present as a single contig each, suggesting most of the genome is pseudogenes. The host mitochondrial genome of K. foliaceum was difficult to identify, but fragments of all the three protein-coding genes, corresponding transcripts, and transcripts of several LSU rRNA fragments were all recovered.Conclusions/SignificanceOverall, the endosymbiont and host mitochondrial genomes in the two dinotoms have changed surprisingly little from those of free-living diatoms and dinoflagellates, irrespective of their long coexistence side by side in dinotoms.

High-throughput pyrosequencing of the chloroplast genome of a highly neutral-lipid-producing marine pennate diatom, Fistulifera sp. strain JPCC DA0580

The chloroplast genome of the highly neutral-lipid-producing marine pennate diatom Fistulifera sp. strain JPCC DA0580 was fully sequenced using high-throughput pyrosequencing. The general features and gene content were compared with three other complete diatom chloroplast genomes. The chloroplast genome is 134,918 bp with an inverted repeat of 13,330 bp and is slightly larger than the other diatom chloroplast genomes due to several low gene-density regions lacking similarity to the other diatom chloroplast genomes. Protein-coding genes were nearly identical to those from Phaeodactylum tricornutum. On the other hand, we found unique sequence variations in genes of photosystem II which differ from the consensus in other diatom chloroplasts. Furthermore, five functional unknown ORFs and a putative serine recombinase gene, serC2, are located in the low gene-density regions. SerC2 was also identified in the plasmids of another pennate diatom, Cylindrotheca fusiformis, and in the plastid genome of the diatom endosymbiont of Kryptoperidinium foliaceum. Exogenous plasmids might have been incorporated into the chloroplast genome of Fistulifera sp. by lateral gene transfer. Chloroplast genome sequencing analysis of this novel diatom provides many important insights into diatom evolution.

ORGANIC AND INORGANIC CARBON PRODUCTION BY ALGAL SYMBIONT-BEARING FORAMINIFERA ON NORTHWEST PACIFIC CORAL-REEF FLATS

Algal symbiont-bearing benthic foraminifera are producers of both organic and inorganic carbon in coral-reef ecosystems. However, their contribution to the total budget of reef carbon production has not been adequately quantified. We used O 2 -respirometry and alkalinity-anomaly techniques to estimate the organic and inorganic carbon production rates of three species of symbiont-bearing large foraminifera common to coral reef flats in the northwest Pacific Ocean. The species studied were Baculogypsina sphaerulata and Calcarina gaudichaudii , which both harbor diatom endosymbionts, and Marginopora kudakajimensis , which harbors dinoflagellate endosymbionts. Live mature individuals were placed onto mesh in clear airtight containers filled with filtered seawater. The containers were incubated for several hours at a constant temperature and subjected to different levels of light intensity. Seawater was continuously mixed throughout the experiment. Dissolved oxygen, pH and total alkalinity were measured before and after incubation. Three species of larger benthic foraminifera were net organic and inorganic carbon producers. The P/R ratio exceeded 1 for all species. No photoinhibition was observed up to 1500 μmol photons m −2 s −1 , and calcification was enhanced by light. These findings suggest that the three species are net carbon producers adapted to high-light, reef-flat environments. In addition, we calculated that Baculogypsina sphaerulata and C. gaudichaudii together accounted for 2–7.5% of the gross primary production of a reef-crest community, whereas M. kudakajimensis supplied 1–11% of the gross primary production of a backreef community. The two calcarinids and M. kudakajimensis contributed ~1 and 10% of the inorganic carbon production of seaward reef flat and protected lagoon communities, respectively. Together with previous reports, these estimates suggest that algal symbiont-bearing foraminifera play a minor but essential role in the organic and inorganic carbon production of coral-reef ecosystems.