Symbiodinum_WPl+Appendix_v2.doc 1
Reasons to sequence the genome of the dinoflagellate Symbiodinium Thomas G. Doak, Princeton, USA.
Dinoflagellates are typically unicellular, photosynthetic, free-swimming, biflagellate organisms that constitute an important component of freshwater, brackish, and marine phytoplanktonic communities. While most are autotrophic (photosynthetic), some are heterotrophic, saprophytic, or symbiotic. Some are highly modified parasites. The autotrophic dinoflagellates are either free-living, or associated with a broad range of hosts as endosymbionts. The dominant modern coral group, the Scleractinia, contain dinoflagellates–almost exclusively of the genus Symbiodinium–as endosymbiotic “zooxanthellae.” Reef-building scleractinian corals have an obligatory requirement for intracellular zooxanthellae, to provide photosynthate, which serves to fuel the energetically expensive deposition of calcium carbonate (Trench 1987). Corals deprived of zooxanthellae deposit far less calcium carbonate and do not build the reefs that currently dominate coastlines in many parts of the world. These reefs function to house an estimated biodiversity of 1-9 million species (Bryant et al. 1998), directly support an estimated 100 million people worldwide, underpin major industries and national earnings, and protect coastlines and other coastal habitats (Hoegh-Guldberg 1999). 2. Dinoflagellate biology 2.1 Dinoflagellate groups. Dinoflagellates have been grouped by the types of pigments they produce. The two major groups are the peridinin and the fucoxanthin containing dinoflagellates. The peridinin-containing group is the most prevalent and includes Symbiodinium (Fig. 2) and various species that cause red tides. 2.2 The dinoflagellate nucleus. Dinoflagellates possess a unique nuclear structure, the dinokaryotic nucleus. Chromosomes are continuously condensed throughout mitosis and interphase (Fig. 3), although they do unwind for DNA replication. During prophase, metaphase, and anaphase, chromosomes are membrane attached: the nuclear membrane remains intact during mitosis and the mitotic spindle develops in channels that permeate the nucleus and coordinate the segregation of chromosomes via membrane attachments (Bhaud et al. 2000; Soyer-Gobillard et al. 1999). Dinoflagellates lack histones H-2, -3, -4, and so do not have nucleosomes, but rather the DNA is in 2.5 nm fibers, as in bacteria, not in 25 nm fibers as in most eukaryotes (Loeblich 1984). Dinoflagellates do have a possible homologue of histone H1 (Kasinsky et al. 2001), which is thought to perform a DNA compaction function. Genome compaction in dinoflagellates is further maintained by extensive chelation of the DNA phosphate backbone with divalent cations, including Mg+2, Ca+2, and various transition metals (Herzog & Soyer 1983; Rizzo 1987). Dinoflagellate genome sizes are frequently greater than those of other eukaryotes, up to 200 pg/cell (Rizzo 1987; Veldhuis et al. 1997). In the course of writing this proposal, we have had a number of dinoflagellate genome sizes measured by quantitative staining, both for classic (large-genome) genera and for a range of Symbiodinium isolates. Samples were provided by R. Anderson of the Provasoli-Guillard National Center for Culture Symbiodinum_WPl+Appendix_v2.doc 3 of Marine Phytoplankton (CCMP), Todd La Jeunesse, and the authors. Measurements by G. Lambert & D. Galbraith (pers. comm.) show that most Symbiodinium spp. (including those we propose to sequence) have a genome size averaging 2.2±0.3 pg/cell, and that as expected many dinoflagellate species have much larger genomes. None of the ~20 dinoflagellates with measured genome sizes has a genome smaller than Symbiodinium’s. [See Appendix 1 for a detailed discussion of dinoflagellate genome sizes.] Renaturation experiments (Allen et al. 1975; Tripplet et al. 1993; Davies et al. 1988; Hinnebusch et al. 1980; Kite et al. 1988) have shown that even in dinoflagellates with large genomes there are high percentages of unique sequence: e.g. Heterocapsa pygmaea (9.1 pg) 75%; Crypthecodinium cohnii (7.3 pg) 40-45%; Glenodinium foliaceum (75 pg) 56% (Allen et al. 1975; Tripplet et al. 1993; Davies et al. 1988; Hinnebusch et al. 1980; Kite et al. 1988), and these results do not support the hypothesis of extensive polyploidy in the dinoflagellates (Triplett et al. 1993). However, multigene families arranged as extensive tandem arrays have been characterized in dinoflagellates (e.g. Li & Hastings 1998; Reichman et al. 2003). Dinoflagellate DNA composition is also distinctive. While dinoflagellates have methyl C (mCpG), as do most eukaryotes, more than 60% of thymines in several species have been replaced by hydroxymethyluracil (hmdU, as hmUpA and hmUpC; Steele & Rae, 1980). In Symbiodinium spp., hmdU ranges from 5% to 12% (Blank et al. 1988). This modification destabilizes the double helix, but the adaptive value for dinoflagellates is not known. 2.3 Dinoflagellate life cycles. Most dinoflagellate life cycles include sexual and asexual, motile and non-motile stages. Asexual reproduction takes place by fission. In the sexual reproduction of a typical dinoflagellate, each haploid vegetative cell differentiates without division to form a gamete. Pairs of gametes fuse, to form a non-dividing diploid zygote, and after a short resting period or a long dormancy, the zygote undergoes meiosis to produce the next generation of haploid vegetative cells. 2.4 Dinoflagellate plastids. Chloroplasts (plastids) are accepted to have originated as a cyanobacterium that was endocytosed, but not digested, by a protist, becoming a symbiotic organelle (endosymbiosis). However, in dinoflagellates that contain the pigment peridinin, the plastid has a triple membrane (van den Hoek et al. 1995), suggesting that this plastid arose from a secondary endosymbiosis: the third and outermost membrane representing the dinoflagellate’s modified food vacuole. The plastid was probably derived from an endocytosed red alga (Yoon et al. 2002a). In addition, some dinoflagellate genera appear to have undergone plastid replacement, with ones derived from green alga or haptophytes (Palmer 2003; van den Hoek et al. 1995), these being discernible by the presence of four membranes around the plastid (Palmer 2003). The plastid genome of peridinin-possessing dinoflagellates is divided into 13 mini-circles (B. Green, pers. comm.), each of which normally carries a single gene, including the 16S and 26S genes (Zhang et al. 2002). This is an entirely unique situation among plastid genomes, both to have the genome consist of unigenic minicircles and to be so highly reduced in gene number. Dinoflagellates are also unique in that Rubisco, an essential enzyme for carbon fixation, is nuclear-encoded, and of the prokaryotic (Form II) type, rather than the eukaryotic (Form I) type (Whitney et al. 1995, Morse et al. 1995, Rowan et al. 1996). 2.5 Other organelles of dinoflagellates. Dinoflagellate mitochondria have the tubular cristae typical of alveolates. There are reports that the mitochondrial genome is fragmented, consisting of not one, but many DNA elements (M. Gray, pers. comm.). In the case of the dinoflagellate Crypthecodinium cohnii, the mitochondrial large subunit rDNA appears to be fragmented (ibid), analogously to the rDNA in apicomplexan mitochondrial genomes (Wilson & Williamson 1997) and the mitochondrial genome contains coding mRNAs for only three genes: cytb, coxI, & coxIII (Chaput et al. 2002; Lin et al. 2002, Norman & Gray 2001). These are the same three protein coding genes that remain in the highly reduced apicomplexan mitochondrial genomes. RNA editing of mitochondrial protein coding genes occurs in dinoflagellates (Lin et al. 2002), but has not been reported in the apicomplexans. Many photosynthetic dinoflagellates have eyespots, light-sensitive organelles composed of lipid droplets located in proximity to a photosensitive membrane. These are often located close to the Symbiodinum_WPl+Appendix_v2.doc 4 base of the flagella to allow the organism to detect the direction of a light source, and cause a signal to be relayed to the flagella. The eyespots contain light-excitable carotenoid compounds, such as retinal. A few species have a more complex structure, in which a crystalline lens focuses light onto a retinoid-lined membrane. Many dinoflagellates are bioluminescent. Bioluminescence in dinoflagellates has the function of predation-avoidance (Esias & Curl 1972). The magnitude of the response is dependent on scintillon (an organelle) migration from a perinuclear to a cytoplasmic location, and is regulated by a circadian clock mechanism (Okamoto & Hastings 2003). 2.6 Dinoflagellate toxins. Dinoflagellates can accumulate in high numbers, causing concentrated “red tides” or harmful algal blooms (HABs) that impact marine and freshwater flora and fauna. Moreover, some produce potent toxins that accumulate in the food chain and sicken human or animal consumers of finfish and shellfish. Many dinoflagellate toxins are complex polyether molecules derived from a polyketide backbone (Rein & Borrone 1999) or alkaloid-like neurotoxins. Diseases caused by dinoflagellates include: Neurotoxic Shellfish poisoning (NSP) which is caused by brevetoxins; Diarrhetic Shellfish poisoning (DSP) caused by okadaic acid, pectinotoxins, and yessotoxins; Ciguatera Fish Poisoning (CFP) caused by the ciguatoxins; and Paralytic Shellfish poisoning (PSP) caused by saxitoxins. These represent a significant economic and public health problem throughout the temperate and tropical marine waters of all continents, due to closure of fisheries, loss of tourism and human illness (Anderson 1998, 2003). A recent review of the incidence of ciguatera by Lehane & Lewis (2000) has highlighted the growing number of victims throughout the Pacific, and the possible link between global climate change, mass coral bleaching, and incidence of ciguatera. Currently we know very little about the biosynthetic pathways for any of these toxins (Rein & Borrone 1999). 2.7 Dinoflagellates as parasites. Some dinoflagellates are highly specialized internal parasites of great economic importance (Coats 1999). Dramatically, some bore through the shells of crustaceans, grow internally as undifferentiated amoeboid tissue, and then emerge to form a spore-forming body on the host’s surface. Dinoflagellate parasites cause diseases such as bitter and pink crab disease, and impact various fisheries, including blue crab and lobster fisheries. Dinoflagellate fish parasites greatly impact both farmed and wild fisheries. The dinoflagellate Pfiesteria has in recent years been notoriously linked to numerous fish kills in the estuaries of the mid-Atlantic coast. Pfiesteria functions like a predator: cells swarm around victim fish, seeming to detect their odor. It has been proposed that Pfiesteria does not kill via toxins, but by some other–unknown–mechanism (Berry et al. 2002). Heterotrophic dinoflagellates parasitize many protists, including the very dinoflagellates responsible for HABs (Coats 1999). 3. Evolutionary placement of dinoflagellate. The alveolates have been proposed to form a super-group, the chromalveolates, along with the chromists (Cavalier-Smith, 1999). The chromists (roughly equivalent to the previously defined Stramenopile, Heterokonta, and Chromobionta groups) include not only algae, but also diatoms, kelps, water molds and downy mildews. It has been proposed that the chromalveolates were ancestrally photosynthetic, by virtue of a secondary plastid that was acquired once in a common ancestor, derived from a red alga (reviewed in Archibald & Keeling, 2002). If this is true, many lineages, such as the water molds and ciliates, have secondarily lost their plastids, to become heterotrophic. It has been further proposed that in the dinoflagellate lineage, the ancestral chromalveolate plastid was lost, to be replaced by a tertiary plastid, derived from the endosymbiosis of a chromist haptophyte algae containing chlorophyll a/c1+c2 and fucoxanthin (Yoon et al. 2002b). The “fucoxanthin dinoflagellates” retained these pigments, while the “peridinin dinoflagellates” lost c1 and replaced fucoxanthin with peridinin. Previously it was believed that the peridinin dinoflagellates represented the ancestral state, and that there was a plastid replacement in the branch leading to the fucoxanthin dinoflagellates (Yoon et al. 2002b). This issue is still unsettled (Palmer 2003). Aside from ciliates, apicomplexans, and dinoflagellates, the alveolates include a number of minor groups of great evolutionary interest. The parasitic Perkinsids are a sister group to the Symbiodinum_WPl+Appendix_v2.doc 5 dinoflagellates, while the Syndiniales and Oxyrrhis are very early branches to the true dinoflagellates (Loeblich 1984; Saldarriaga et al. 2003). The gregarines and Colpodella are basal to the apicomplexans (Kuvardina et al. 2002, Leander et al. 2003). Presumably, genome projects for these linking taxa will be undertaken in the near future to complete the picture of alveolate evolution. 4. Important areas of research in dinoflagellates. 4.1 Evolutionary origins of symbiotic and parasitic alveolates. The close relationship between symbiotic dinoflagellates and cnidaria is thought to be an ancient association (e.g. Stanley and Swart 1995). The obligate nature of apicomplexan parasitism on metazoans is also an ancient character as evidenced by the fact that all the known apicomplexans are parasitic. Symbiotic dinoflagellates and parasitic apicomplexa have in common the ability to reside in host cells for long periods. Since dinoflagellates are a sister group to the exclusively parasitic apicomplexans, much might be learned by comparing the intracellular adaptations of apicomplexans with those of symbiotic dinoflagellates. The presence of the apicoplast in apicomplexans supports the hypothesis that common biochemical adaptations exist, as this relict plastid (Wilson 2002) remains essential to the viability of apicomplexans (Ralph et al 2001, Foth & McFadden 2003). Similarly, parasitic dinoflagellates often have reduced plastids just as apicomplexans have, though some have lost the plastid altogether (van den Hoek et al. 1995). 4.2 Dinoflagellates as models for apicomplexan infection The infection processes of parasitic dinoflagellates and apicomplexans are here postulated to share common mechanisms. Dinoflagellate alveoli, comprised of a membrane complex, are homologues of the inner membrane complex of apicomplexans (Leander & Keeling 2003), and the inner membrane complex is a vital part of the machinery that allows apicomplexan parasites to invade their host (Bannister et al. 1990). Additionally, the apical apparatus of some dinoflagellates contains structures homologous to the apical complex of apicomplexan parasites (Leander & Keeling 2003; Kuvardina et al. 2002), and dinoflagellate ejectisomes are likely homologous to the rhoptries and micronemes of apicomplexans (Leander et al. 2003). These latter structures are also part of the parasite invasion apparatus and harbor many antigens that are currently being trialed as malaria vaccines (Chauhan & Bhardwaj 2003). Host specificity is an as-yet unexplored aspect of both apicomplexan and dinoflagellate associations with animals. A related phenomenon, of association-induced cell adhesion, has begun to be studied in cnidarian-zooxanthellae associations, where the host surface molecule Sym-32 is necessary (Weis et al. 1999). Sym-32 is in the fasciclin-1 super family of cell adhesion molecules (Reynolds et al. 2000), which also have recurring roles in other symbioses: e.g. lichen- cyanobacterium and plant-rhizobium. Interestingly, Plasmodium cell surface proteins required for infection of the human host include an integrin homologue (Sultan 1999), and integrins are postulated as the partners of the fas-1/Sym-32 family (Kim et al. 2002). 4.3 Integration of organellar and nuclear genomes. The dinoflagellate plastid is an extreme example of genetic integration following endosymbiosis. Peridinin-possessing dinoflagellates have a chloroplast genome consisting of multiple diverse mini-circles encoding only a few genes (~13); the rest of the algal derived genes will be found in the nucleus. The structure and function of dinoflagellate minicircles may be linked to a history of nuclear integration events. The mini-circles can contain inverted repeats, which, together with other non- coding conserved sequences, are thought to be recombinogenic motifs (Zhang et al. 2002; Barbrook et al. 2001; Moore et al. 2003). It has been proposed by Howe et al. (2002) that mini-circle inverted repeats may be related to bacterial integrons and that they may be vectors for transfer of organellar genetic material to the nuclear genome over evolutionary time. The nuclear genome could provide a record of such integration events. This is being addressed by looking for mini-circle-like sequences in the nuclear genome of various dinoflagellates (Maier & Zauner, e.g. see Genbank record gid:29468209). Paralleling the migration of genes inside dinoflagellate cells, the apicomplexan apicoplast is one of the few organellar genomes to have revealed that a component of its DNA was derived from another organelle, in this case from the mitochondrion (Obornik et al. 2002). Also a wide array of Symbiodinum_WPl+Appendix_v2.doc 6 evidence indicates that multiple endosymbionts have donated genes to apicomplexan nuclear genomes (Striepen et al. 2002; Dzierszinski et al. 2001; J. Kissinger, manuscript in preparation). These findings support the hypothesis that inter-compartmental DNA traffic has occurred in the alveolate lineage. It is not known whether genome fragmentation has any relation to organellar genome reduction, but the fragmentation of both the mitochondrial and chloroplast genomes of dinoflagellates makes these organisms good models in which to ask the question. Clearly in all eukaryotic organisms genes do migrate from organelles to nuclei over time, but few testable hypotheses have been put forward for the physical mechanism of transfer. The recent finding that Symbiodinium plastid minicircles share putative integron-like or transposon-like sequences with elements of fungal mitochondrial genomes (Moore et al. 2003), potentially alludes to a general integron transfer process in organellar evolution (Howe et al 2003). 4.4 Genome structure and regulation. Dinoflagellate genomes are unique in a number of ways. Dinoflagellate chromosomes are condensed throughout the cell cycle (except in S-phase). However, loops of B-DNA have been detected protruding from chromosomes during the G1 phase of the cell cycle (Soyer-Gobillard et al. 1990), and it is expected that these are the regions being expressed. It is also possible that the apparently condensed regions of chromatin are expressing. How does a predominantly condensed genome allow gene expression? Further, how is the extrusion of B-DNA loops regulated? Dinoflagellate genomes are peculiar in possessing both a high level of mC and the unusual methylated base hydroxymethyluracil (Rizzo 1987). The nature of the gene sequences that are methylated is unknown, as is the mechanism of methylation. And more importantly, it is not known what function the unique hydroxymethyluracil serves. Methylation of eukaryotic nuclear DNA, in general, has the dual functions of switching off transposon activity (Matzke et al. 2000) and controlling developmental stages (Finnegan et al. 2000); DNA methylation in dinoflagellates might be related to either of these functions (ten Lohuis & Miller 1998a). 4.5 Dinoflagellates support coral reefs. Symbiotic dinoflagellates underpin an ecosystem that is rivaled only by rainforests in terms of biodiversity. Reef tourism and fishing earn billions of dollars each year for the United States alone (Hoegh-Guldberg 1999), and coral reefs form a vital resource for an estimated 100 million people worldwide (Wilkinson 1999; Hoegh-Guldberg 1999). Reefs are also critical to the integrity of most tropical coastlines, by forming a barrier to oceanic waves. Coral- zooxanthellae mass mortality is on the increase (Hughes et al. 2003), and is predicted to reach a point where whole reefs disappear worldwide: when this happens, human health will be directly compromised. Currently, there are several international initiatives aimed at rapidly filling the significant gaps in our knowledge of the causes of reef degradation (e.g. Global Environment Facility, http://www.gefcoral.org/). A complete understanding of the molecular biology of Symbiodinium would yield enormous insights into how reefs are likely to change and whether adaptation can keep pace with climate change. In addition, reef-dwelling organisms from diverse taxa are valuable sources of pharmaceuticals (Salm 1994; Fenical 1997; Luiten et al. 2003; Kohl et al. 2003) and the abundance of these taxa ultimately relies on foundations laid down by coral-Symbiodinium communities. Symbiodinium itself is currently the only culturable source of pseudopterosin (a terpene derivative), a valuable and unique pharmaceutical that inhibits phospholipase A2, and has anti-inflammatory activity (Kohl et al. 2003). 4.6 Biogenesis of dinoflagellate toxins. Many harmful algal bloom (HAB) dinoflagellates synthesize polyether toxins that are dangerous to human health (Rein & Borrone 1999; Anderson 1998, 2003), as do some symbiotic dinoflagellates. The biological function is not known for these toxins. It is thought that the toxin genes may have been horizontal gains from bacteria to the dinoflagellate nucleus over evolutionary time (Kodama 1990). 4.7 Circadian gene expression. Cues for the diel regulation of gene expression in diverse unicellular algae have a defined molecular signature (a 3’ UTR sequence motif), and this mechanism was originally discovered using dinoflagellates (Mittag 2003). Circadian rhythms play an important part in sleep research (Bunney & Bunney 2000), and are an area of interest in endocrinology, as Symbiodinum_WPl+Appendix_v2.doc 7 dinoflagellates and humans share the photoperiodic compound melatonin (Hardeland & Poeggeler 2003). Voluminous data has recently been gathered on circadian gene expression using EST microarrays of the dinoflagellate Pyrocystis lunula (Okamoto & Hastings 2003). A dinoflagellate genome sequence would greatly hasten the determination of the complete set of genes that are diely expressed in dinoflagellates. Applications of a model dinoflagellate to human biology and health Dinoflagellates have direct impacts on human biology and health, and will serve as workhorses to improve human health, in three main ways. First, as a sister group to the exclusively parasitic apicomplexans, dinoflagellates can serve to inform the search for antimalarial compounds, because their plastid functions may overlap with those of the apicoplast, and because the latter organelle is essential to survival in Plasmodium (Ralph et al. 2001; Waller et al. 2003; Foth & McFadden 2003). The genomic sequence comparison of dinoflagellates and apicomplexans is expected to yield insights into apicoplast evolution and alveolate vulnerabilities, and thus drug targeting. In a complementary fashion, dinoflagellates in culture can serve as first-screen organisms for the testing and development of anti-apicomplexan compounds (B. Baillie unpublished results), because they are easier to culture than apicomplexans. Additionally Baillie has suggested that Symbiodinium in symbiosis with a cnidarian host could function as an inexpensive screen for anti-apicomplexan compounds, because simultaneous harmlessness to the cnidarian animal host would indicate low toxicity to animals and by inference humans, pending required follow up trials in higher animals Second, this research can reduce the medical and economic impacts that dinoflagellates have on human food sources. Toxic and parasitic dinoflagellates decimate seafood supplies, killing the catch, or rendering it toxic when eaten (Durborow 1999). A deeper understanding of dinoflagellate biology, especially their sexual cycles, which largely govern their distributions (Anderson 1998, 2003), will help in the prediction of outbreaks. A host of different lifestyles are found within the harmful dinoflagellates, ranging from purely autotrophic to purely parasitic, and we argue that Symbiodinium represents aspects of all these lifestyles. By development of dinoflagellate transformation and gene knockout technology, knowledge about the biosynthesis of toxins will increase; the actual functions of dinoflagellate toxins in nature are currently unknown. Third, the prospects for exploiting coral reef ecosystems as a source of pharmaceuticals (e.g. Salm 1994; Fenical 1997; Luiten et al. 2003) will be enhanced, by understanding the factors contributing to reef degradation. Breakdown of the Symbiodinium-coral symbiosis impacts directly on reef biodiversity, and is an early indicator of a decline in marine biodiversity that is expected in the coming decades, due to global warming (Walther et al. 2002; Hughes et al. 2003). Understanding of the breakdown of this symbiosis may therefore lead to wider acceptance of the impact of global warming on the biosphere and through it, human health. Symbiodinium is the dinoflagellate of choice 1. Introduction. Symbiodinium is an emerging model for many aspects of dinoflagellate biology and by inference alveolate biology, and a complete genome sequence will greatly facilitate its continuing use. It is not yet a well-developed genetic system–nor is any dinoflagellate–and so a genome sequence offers the best and most immediate entry into the physiology of this group. Any one dinoflagellate genome will energize all dinoflagellate research, and we believe that Symbiodinium is the most usefully placed within the phylum. In this context, the small genome size and broad set of metabolic capabilities suggest that Symbiodinium will be a relevant model for all dinoflagellates. The fact that the vast majority of zooxanthellate marine invertebrates choose Symbiodinium indicates that Symbiodinium possesses pathways to take advantage of host resources, and we hypothesize it may share these pathways with the intracellular parasitic dinoflagellates and apicomplexans. Symbiodinium strains have direct cell adhesion with their hosts (Reynolds et al. 2000), and also display strong host-specific tendencies (Buddemeier et al. in press). As such, this genus may possess adaptations to infection route, host-specificity, and intracellular life that can provide insight on the similar lifestyles of apicomplexans. Few dinoflagellates other than Symbiodinium can model Symbiodinum_WPl+Appendix_v2.doc 8 intracellular lifestyle and simultaneously model the plastid biology that is relevant to apicoplast science. Symbiodinium are also periodically free-living (Trench 1987; Rowan 1998; La Jeunesse 2001; Baker 2003), making this genus a suitable model for free-living dinoflagellates. This and their placement in the peridinin-type plastid lineage means that Symbiodinium should represent and code for most of the basic cell biological pathways present in free-living HAB dinoflagellates. Symbiodinium, like many HAB dinoflagellates, produces polyether toxins (zooxanthellatoxins, Murata & Yasumoto 2000): one quarter of Symbiodinium cultures show genetic potential for toxin production (K. Rein & D. Mendola pers. comm.) through expression of the gene pks, which is central to toxin production (Rein & Borrone 1999). Isolation of more toxin biosynthetic genes, for reactions upstream and downstream of pks in Symbiodinium, is an avenue toward characterizing the toxin pathways and chemical abilities of a range of HAB dinoflagellates. 2. Symbiodinium as the Achilles heel of coral reefs. Symbiodinium is the dominant genus of algal symbiont in reef-building corals and in many other invertebrates in the world's equatorial, tropical, and temperate oceans. As such, Symbiodinium is not an ecologically incidental model. Symbiodinium forms the fundamental basis of coral reefs and therefore supports the millions of people that depend on coral reefs for their livelihoods. As a corollary to its role as a keystone genus, Symbiodinium is also a point of vulnerability to coral reefs. When corals are stressed by environmental perturbations such as declining water quality or climate change, the symbiotic relationship between host and dinoflagellate symbiont breaks down (Brown 1997; Hoegh-Guldberg 1999; Downs et al. 2002; Douglas 2003; Warner et al. 1999; Walther et al. 2002; Baker 2003; Hughes et al. 2003). The reasons for this symbiotic dysfunction, termed coral bleaching, are poorly understood. In the most extensive case of coral bleaching to date, an estimated 16% of corals died across the planet in 1998. In the Indian Ocean, in the same year, an estimated 46% of living coral was lost from a wide range of survey sites (Wilkinson 2000). It is now largely accepted that human-driven climate change has led to two decades of increasing coral bleaching (Hughes et al. 2003). An area of research is how coral-dinoflagellate symbioses will respond. Based on the blatant evidence of reef degradation it is apparent that the rate of reef-wide evolution towards more thermally robust symbioses is being exceeded by the rate of temperature change. Since corals’ reproductive rates are low, (normally spawning is annual; Harrison & Wallace 1990) any genetic adaptation will likely be among symbiotic dinoflagellates, in the form of recombination between existing ‘host-specificity’ traits and thermal tolerance traits. There is considerable debate around this issue, with the central issue being the major gaps in our knowledge of coral-Symbiodinium symbioses (e.g. Baker 2002 vs. Hoegh-Guldberg et al. 2002). Action has been taken in the USA to address how environmental changes will affect the world’s reef systems. A recent meeting of the US Coral Reef Task Force in Hawaii concluded that climate change was the largest threat to coral reef ecosystems. The National Oceanographic and Atmospheric Administration (NOAA) has launched an intensive effort to develop biomarkers of Symbiodinium and coral stress, for the monitoring and better understanding of bleaching events (Woodley et al. 2003; Downs et al. 2000, 2002). The Coral Disease and Health Consortium identified genome sequencing as a priority in their workshop proceedings “CDHC: A National Research Plan” (http://www.coral.noaa.gov/coral_disease/diag/). International efforts are also underway. Ove Hoegh-Guldberg is chair of the World Bank Targeted Working Group on Coral Bleaching and Climate Change. This group, part of a wider group of sixty scientists under the Global Environment Facility (http://www.gefcoral.org), is focused on understanding the changes currently effecting coral reefs. The research agenda includes molecular to ecological levels of investigation. 3. Mechanism of coral bleaching. Due to the intimate nature of the symbiosis between zooxanthellae and their hosts, it is not immediately apparent whether thermal stress first acts to disable the zooxanthellae or the coral host, and different host-symbiont pairings respond differently to this stressor. These differences suggest a potential basis for different tolerances by zooxanthellae- host symbioses, but neither the pathways nor the genetics of these differences have been mapped. Symbiodinum_WPl+Appendix_v2.doc 9
The ongoing EST microarrays will address many of the above issues in coral bleaching. Interactions specific for the symbiotic state can be identified, and the relative timing of events in symbiosis breakdown can be distinguished. Obviously, having the genome of Symbiodinium will greatly enhance these studies. 4. Symbiodinium’s relationship to other model dinoflagellates. Many of the dinoflagellates that are the subjects of EST projects might be reasonable genome models, if their genomes were small and they were more amenable to genetic manipulation, but they all have genomes larger than Symbiodinium, in some cases two to three orders of magnitude larger (Rizzo 1987; Veldhuis et al. 1997; G. Lambert & D. Galbraith pers. comm.) and few have been shown transformable (only Symbiodinium and Amphidinium so far; ten Lohuis & Miller 1998b). No dinoflagellate is a well-developed model for studies of genetic inheritance. The first and best- known experiments done in this respect were by Beam and Himes using Crypthecodinium cohnii (Beam & Himes 1974, 1984). The genome size of C. cohnii (~50 pg/cell; Rizzo 1987) precludes it being considered as a competitive model for dinoflagellate genome sequencing. Symbiodinium’s intracellular and free-living lifestyles, in combination with well developed microarray projects and a large scientific community, position it well as a general model for dinoflagellate cell biology and evolution, from the obligate parasitic forms, to the free-living organisms that cause HABs. 5. Suitability of Symbiodinium for experimentation. 5.1 Introduction. Despite being periodically sequestered in hosts in the wild, Symbiodinium live well in culture. We will exploit this to obtain DNA from axenic cell lines for the genome project. Zooxanthellae are also easily isolated directly from host organisms: wild-collected corals easily yield grams of dinoflagellates. Cells freshly extracted from a live host are healthy Symbiodinium cells that have divided recently and are arrested in G1(Smith & Muscatine 1999). Cultured Symbiodinium strains can be grown from single cells to colonies on agar (e.g. via simple dilution streaking), which can’t be done for most of the larger, more fragile, HAB species. Symbiodinium is thus more amenable to direct genetics–mutagenesis, transformation and antibiotic selection methods–than are the HAB species, and its small genome size reinforces this suitability. 5.2 Symbiodinium is a sexual organism. Population genetic studies have verified that Symbiodinium populations are sexual (Baillie et al. 1998, 2000; La Jeunesse 2001; Goulet & Coffroth 2003; Santos et al. 2003), although gametogenesis has not been reproducibly observed (Blank 1987). Given that Symbiodinium spp. populations are sexual, it is only a matter of time until signals for gametogenesis are identified. In free-living dinoflagellates, such as HAB-causing dinoflagellates, gametogenesis is brought on by nutrient limitation and/or temperature change. We suspect that for Symbiodinium, nutrient limitation occurs outside of the host (a richly nutritious environment) i.e. in seawater (tropical seas are very nutrient poor). Sex is probably not possible in the coral host, because there the symbionts are sequestered within a modified host endocytic vacuole. 5.3 Transformation and selectable markers. In 1998, genetic transformation of dinoflagellates was achieved for both Symbiodinium and Amphidinium (ten Lohuis & Miller 1998b), showing the potential applicability of the method to a wider range of dinoflagellates. The selectable markers used were neomycin and hygromycin B phosphotransferases, driven by the Agrobacterium p1'2' and nos promoters. A recorder construct was also successfully used, coupling the b-glucoronidase gene with the p1'2' promoter, or the cauliflower mosaic virus 35S promoter. Strong native promoters for the nuclear encoded photosynthesis genes Rubisco (Rowan et al. 1996), and PCP (Reichman et al. 2003), are already available from Symbiodinium, as is a promoter for the plastid encoded gene psbA (Takishita et al. 2003, Moore et al. 2003). Other Symbiodinium promoters can be cloned based on probes that show regulated activity in the two Symbiodinium microarray projects. siRNA has not been tried in dinoflagellates to our knowledge. However, given that dinoflagellates can be transformed and that both ciliates and apicomplexans respond to siRNA, it is expected that Symbiodinum_WPl+Appendix_v2.doc 10 loss of function treatments can be developed in dinoflagellates and be a major way forward from the EST microarray and genome projects. Given that vegetative cells are haploid, siRNA, microarrays, and temperature sensitive mutants represent the best ways to develop a genetic system. It should be possible, as it is in all eukaryotes and prokaryotes, to produce insertional mutants, by transforming the nuclear DNA with a transposon or a cassette such as TDNA, which was shown by ten Lohuis and Miller (1998b) to be integrated in the nuclear genome. Cloning of nonessential genes by complementation will be possible, given transformation. It is unknown whether homologous recombination and targeted knockouts will be possible in dinoflagellates. 6. The Symbiodinium research community. The Symbiodinium community has grown exponentially since the naming of the genus in 1959, and is composed mainly of researchers who have been using Symbiodinium for fifteen years or less. Most of the interest is in molecular population genetics (biodiversity of the genus), as well as cell biology (see letters of support). A feature that unites the two camps is a desire to understand host-symbiont specificity, and to establish broad rules for why this occurs, and if there are obligate partnerships that are not adaptable (Baker 2001, 2002, 2003; Hoegh-Guldberg et al. 2002; Buddemeier et al. in press). This issue bears on the vulnerability of particular coral-zooxanthella partnerships to anthropogenic pressures. We expect that the Symbiodinium EST and microarray projects (section 7.2) will start to build an infrastructure for the management of the emerging genome sequence. The Hoegh-Guldberg lab consists of ten PhD students and 8 postdoctoral researchers, and is the largest Symbiodinium lab in the world and one of the largest dinoflagellate labs. The Medina microarray project (section 7.2) brings the genomics strengths of the JGI center to the community. Researchers working with other dinoflagellates or with apicomplexans will contribute to annotating the genome (see letters of support). The Coffroth, Medina, and Rizzo labs will be the main US bases for the genome project and will carry out the DNA purifications required for library creation and all algal culturing required for the genome project. Supplementary strains of Symbiodinium will be provided to the US labs by the Hoegh-Guldberg lab. In separate projects, the McFadden lab (alveolate evolution, Melbourne Australia) and the Carter lab (zooxanthellae speciation, Sydney Australia) intend to characterize the Symbiodinium genome using pulsed field gel electrophoresis. 7. The Symbiodinium genome and the status of its "pre-genomics" 7.1 The genome. While many dinoflagellates have huge genomes, as much as 20-200 pg per cell, Symbiodinium has a much smaller genome: average ~2.4 pg across a number of isolates from multiple sources (measured by DAPI staining and flow cytometry; G. Lambert & D. Galbraith pers. comm.). The GC content has been measured at 43-55% for Symbiodinium (Blank et al. 1988; Blank & Huss 1989). Preliminary renaturation analyses suggested that at least 20% of the genome was repetitive (Blank & Huss 1989). We are not aware of an estimate of dinoflagellate gene number, however, their nearest characterized free-living relatives, the ciliates, are estimated to have ~25,000 genes. Since vegetative Symbiodinium is haploid (Santos & Coffroth 2003), there will not be problems with sequencing and assembling two alleles. This is a huge advantage, since alleles are likely to be quite diverged, given the large population sizes of Symbiodinium, and would complicate genome assembly (e.g. Ciona savignyi; http://www-genome.wi.mit.edu/annotation/ciona/assembly.html). The lack of all polymorphism will make repetitive sequences particularly easy to deal with (Jaffe et al. 2003). There are established protocols to produce intact dinoflagellate nuclei (e.g. Rowan et al. 1996), and thus intact chromosomes, allowing production of the mega-base DNA needed to make BAC libraries. 7.2 ESTs/Microarrays. The Hoegh-Guldberg group is funded ($1 million over 2003-2006) to develop a microarray for Symbiodinium and its Indo-Pacific coral host (Acropora aspera), which will be used to identify key stress responses within the two organisms. The project (funded by the Australian Research Council, ARC) is embedded within the Global Environment Facility (GEF) group, which includes laboratories from the US Universities of Hawaii (R. Gates), New Hampshire (M. Lesser), Georgia (W. Fitt), and Florida Tech (Van Woesik). Concurrently, M. Medina (Joint Genome Symbiodinum_WPl+Appendix_v2.doc 11
Institute, University of California), who will also work with the Symbiodinium genome project, is directing a similar EST and microarray project at the Department of Energy’s JGI center, in collaboration with M. A. Coffroth (University at Buffalo) and A. Smzant (UNC-Wilmington). This latter project, funded through the NSF ($1 million over 2003-2007), will use Caribbean strains of Symbiodinium and the Caribbean coral Montastrea faveolata. As in the GEF microarray project, the major concern of the Caribbean microarray effort is the stress response of host and symbiont during coral bleaching. This duplication of research effort underlines the interest in Symbiodinium. As well, a mixed Symbiodinium/coral EST project is being conducted by NOAA, led by Cheryl Woodley. Proposal 1. Choice of Symbiodinium strains, and the proposed extent of sequencing. We propose that a finished genome be generated for a Symbiodinium isolate, plus lower coverage sequence of three other isolates, to allow a complete catalog of its genes to be generated, as has been achieved for budding yeasts and grasses (Kellis et al. 2003; Inada et al. 2003). The selection of four strains is aided by the existence of numerous closely related species in this genus, and by the large volume of taxonomic work that has been done on these. Symbiodinium, once thought to be a single pandemic species, is now known to include clades (designated A through G) that have been diverging for up to 100 million years (Rowan & Powers 1992; La Jeunesse 2001; Pochon et al. 2001; Santos et al. 2002; Rodriguez–Lanetty 2003). Molecularly, clades B and C are the most studied, due to their frequency in reef ecosystems. Clade C are the predominant coral zooxanthellae in the Pacific, including Hawaii, Guam, Samoa, Australia, East Africa, and SE Asia (La Jeunesse et al. 2003); in the Caribbean, clades B and C are found at comparable levels. The Australian EST/microarray project is using a clade C isolate, while the Medina/JGI project is planning to use a clade B isolate. We propose that, of these two, the primary strain for the genome project be the clade B isolate from Montastrea faveolata because 1) its genome size has been measured at 2.2±0.3 pg/cell (G. Lambert & D. Galbraith pers. comm.); 2) the lifecycle of clade B has been well characterized as haploid and sexual (Goulet & Coffroth 2003; Santos & Coffroth 2003, Santos et al 2003); 3) cultures are on hand; 4) this strain (from US waters)is the one that Medina will use for her EST project; 5) clade B are the workhorses of zooxanthellae research, because they are found in anemones (e.g. Aiptasia spp.) which are easily maintained and propagated in aquaria. In addition, we will choose two clade B isolates that are separated from the primary strain by 5- 20 million years for 5x sequencing, in order to allow the confident identification of ORFs, as well as of conserved non-coding sequences (phylogenetic shadowing: Kellis et al. 2003; Inada et al. 2003). Within each clade there are quite distinct genotypes, approximating species. Exactly which two of these will be used remains to be decided: while there are obvious candidates, nuclear sequences need to be characterized to assure that we pick strains with the optimum divergences (Kellis et al. 2003). We also propose that the clade C isolate from Acropora aspera, used in the Australian EST project, be sequenced to 5x. Clade B and C are estimated to be separated by ~65 million years, and clade C isolates have genomes of a similar size to B. The clade C sequence will begin to characterize the most widespread clade, it will compliment the Australian EST and microarray effort, and it will aid in ORF identification for all Symbiodinium spp. Importantly, pks mRNA is found in isolates of clades A, B and C (K. Rein & D. Mendola pers. comm.), so polyketide synthesis is widespread within the genus. Thus, we propose that the clade B strain from Montastrea faveolata be sequenced to closure, and two clade B isolates and a clade C isolate each be sequenced to 5x. 2. Genome sequencing, assembly, and annotation. The TIGR centre has indicted that they would enthusiastically take on the Symbiodinium genome project if permitted (C. Fraser pers. comm.). The finished genome will be primarily generated by assembling paired-end whole-genome shotgun reads, using short-insert plasmid libraries of various insert sizes, each of ~6-fold coverage, in addition to end sequences of a BAC library, to aid in long range assembly. A 15-fold coverage BAC library, average insert size 200 kbp, is being requested in a separate whitepaper to NHGRI, for the primary B strain from Montastrea faveolata, and a similar library for the clade C strain from Acropora aspera. The BAC library can also be used for the closure of major gaps. The sequences for the three low-passage genomes may be generated from “high-C0t” selected libraries (Peterson et al. Symbiodinum_WPl+Appendix_v2.doc 12
2002; Yuan et al. 2003); this will be decided in collaboration with the sequencing center. The two additional clade B sequence sets will be assembled using the first complete B sequence as a template. The clade C sequence may be too divergent to directly use the B genome as an assembly template, but we expect that there will be large regions of synteny where shared protein homologies can be used to guide assembly. All sequence traces will be deposited in the GenBank Trace Repository, and the sequencing center will provide public access to the sequence. The whole-genome shotgun sequence will be assembled using public-domain software packages such as ARACHNE (Jaffe et al. 2003), which was developed for assembly of mammalian genomes with levels of repeated sequence comparable to that of dinoflagellates (Triplett et al. 1993). The Generic Model Organism Database (GMOD; www.gmod.org) will serve as the database/bioinformatics core (and will soon have the Apollo gene annotation system added). Automated annotation will be performed using programs such as GlimmerM; the large number of Symbiodinium EST sequences (14,000) will allow a very good model for intron splice-sites to be generated, and dinoflagellate-specific ORF prediction will be augmented by EST data from other dinoflagellate projects. Where homologs are identified, gene files will be linked to Pfam, Prosite, and the Gene Ontology index. Bioinformatics and programming support have been offered to us by David Roos, within the apicomplexan genomes website (Bahl et al. 2003). There are now various programs to align genome-sized sequences against each other (e.g. Couronne et al. 3003, Delcher et al. 2002) and papers have recently been published that use “phylogenetic shadowing” to compare entire genomes in order to identify conserved non-coding sequences (CNS; Kellis et al. 2003; Inada et al. 2003; Thomas et al. 2003; The ENCODE Project: http://www.genome.gov/ENCODE/). Our planned data set will allow us to follow just this course of action. 3. Uses of the genome sequence. Perhaps the most immediate and notable effect of this project will be to allow all dinoflagellate researchers to go from EST sequences to full gene sequences, including the entire open reading frame and promoters. There are now many dinoflagellate EST projects underway. The major ones that we are aware of are: 1) Amphidinium carterae & Gonyaulax polyedra, 3200 & 1500 ESTs, Tsetso Bachvaroff & Charles Delwiche 2) Alexandrium ostenfeldii, 2500 ESTs, Linda Medlin, Allan Cembella, Uwe John & Klaus Valentin 3) Alexandrium sp., 10,000 ESTs, Debashish Bhattacarya 4) Heterocapsa triquetra & Kryptoperidinium foliacium 7,500 ESTs each, Mike Gray, Patrick Keeling, and others 5) Karenia brevis, 8000 ESTs, Fran Van Dolah 6) Perkinsus marinus, 2000 ESTs, Gerardo R. Vasta 7) Prorocentrum minimum, Karlodinium micrum, Pfiesteria piscicida, 900 genes, Senjie Lin 8) Pyrocystis lunula, 3,500 ESTs, J. Woodland Hastings 9) Scrippsiella nutricula, 2,000 ESTs, Rebecca Gast & David Caron 10) Symbiodinium sp., 4,000 ESTs, Ove Hoegh-Guldberg, David Yellowlees, Bill Leggatt, Sophie Dove, Bill Fitt, Ruth Gates & Todd La Jeunesse 11) Symbiodinium sp., 10,000 ESTs, Monica Medina
These EST projects address the issues of plastid and nuclear evolution, gene transfer, alveolate evolution, lifecycles and cell division, toxin chemistry, symbiotic integration, coral bleaching, stress markers and circadian rhythms. Proteomic approaches, that are particularly useful in characterizing organelles, will no longer be hindered in dinoflagellate research, because the genome project will provide genetic data to compare amino acid data against. Researchers may thus progress quickly from protein data to gene data. The existence of genome sequence will also enable Serial Analysis of Gene Expression (SAGE) studies. The phylogenetic shadowing strategy will uncover structural and regulatory regions in non-coding DNA, including genome-wide regulators of diel cycle, sexual differentiation, and nutrient toggling, which would not be possible to identify fully in a smaller project. Symbiodinum_WPl+Appendix_v2.doc 13
Insight into the evolutionary history of dinoflagellates and alveolates will be facilitated by automated annotation of the genome sequences of Symbiodinium. Automation will be augmented by a team of manual annotators (see letters of support). Particular attention will be paid to all photosynthetic gene hits, as well as to all genes with plastid import signals as done by Foth et al. (2003) for Plasmodium. The integration of the plastid and nuclear genomes will be of great interest to several dinoflagellate and apicomplexan labs that study this topic in an evolutionary and pharmaceutical context (Ralph et al. 2001; Waller et al. 2003; Foth & McFadden 2003). Intracellular gene transfers will be detected using software provided by Jessica Kissinger, and previously used to detect intracellular transfer between organellar genomes of apicomplexans. The complete gene list for Symbiodinium will be compared to those of apicomplexans and ciliates, to aid in reconstructing the common alveolate ancestor. Comparison of the whole Symbiodinium genome to that of apicomplexans may uncover similarities in genes used recurrently in the intracellular lifestyles of these two taxonomically related protists. Genome comparisons, together with Pfam and Prosite cross-referencing, will be used to obtain the sequences of any common cell adhesion proteins and outer-membrane transporters, that may be shared by Symbiodinium and the apicomplexa. Research on host infection and on membrane trafficking of host-derived nutrients will thereby be facilitated in both phyla. The Symbiodinium genome, expected to contain rhodopsins of unknown affinities, will be compared against genomes of animals, fungi, plants and algal-protists, to assess the affinities of visual-rhodopsin light receptor apparatus, present in animals but not in fungi (Idnurm & Howlett 2001; Brown et al. 2001), even though animals and fungi are each others closest relatives (Baldauf & Palmer 1993). This will address the question of whether animals gained visual rhodopsin secondarily (either autonomously or by intertaxonic combination with a protist) or fungi lost it. As well, the genes of Symbiodinium and Phytophthora (stramenopile) will be compared against others to assess the credibility of the chromalveolate grouping. Direct genetics will be developed for Symbiodinium based on the successful transformation protocol developed by ten Lohius & Miller (1998b). As well as using the Agrobacterium and plant vectors of ten Lohuis & Miller, alternative vectors such as dinoflagellate minicircle backbones, and alternative promoters, such as native dinoflagellate promoters, will be employed. Minicircles are particularly appealing as vectors because they are self-replicating and presumed to be recombination competent (Moore et al 2003; Barbrook et al. 2001; Zhang et al. 2002), and thus might be modified for integration into the nuclear genome as per their hypothesized evolutionary role (Howe et al. 2003). Direct genetics (gene knockouts) by any of these methods, beginning with the ten Lohuis and Miller method, will allow the dissection of pathways for the biosynthesis of toxins, pseudopterosins, UV-absorbing compounds (Shick et al.1999), and host-symbiont signalling compounds, leading to a more detailed understanding of these pathways. The mechanisms of coral bleaching will become understood by combining knowledge gained through the Symbiodinium genome project with knowledge gained from the GEF and JGI Symbiodinium and coral EST microarray projects, and subsequent SAGE studies. Technical developments in the creation and maintenance (in culture) of Symbiodinium mutants in biosynthetic pathways will subsequently serve as a precedent for the use of direct genetics to assess aspects of dinoflagellate cell biology, ontogeny, and bioenergetics. Symbiodinum_WPl+Appendix_v2.doc 14
Citations [Authors who have provided letters of support are indicated by underlining.] Allen JR, Roberts M, Loeblich AR 3rd & Klotz LC. 1975. Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization. Cell. 6:161-169. Anderson DM. 1998. Testimony before the Subcommittee on Oceans and Fisheries. U.S. Senate Hearing on the Harmful Algal Bloom Eradication and Control Act of 1998 Anderson DM. 2003. Testimony before the Committee on Science Subcommittee on Environment, Technology and Standards U.S. House of Representatives. Hearing on the “Harmful Algal Bloom and Hypoxia Research Amendments Act of 2003 Archibald JM & Keeling PJ. 2002. Recycled plastids: a 'green movement' in eukaryotic evolution. Trends Genet. 18:577- 84. Bahl A, Brunk B, Crabtree J, Fraunholz MJ, Gajria B, Grant GR, Ginsburg H, Gupta D, Kissinger JC, Labo P, Li L, Mailman MD, Milgram AJ, Pearson DS, Roos DS, Schug J, Stoeckert CJ Jr & Whetzel P. 2003. PlasmoDB: the Plasmodium genome resource. A database integrating experimental and computational data. Nucleic Acids Res. 31:212-215. Baillie BK, Belda-Baillie CA, Silvestre V, Sison M, Gomez AV, Gomez ED & Monje V. 2000a. Genetic variation in Symbiodinium isolates from giant clams based on random-amplified-polymorphic DNA (RAPD) patterns. Marine Biology 136:829-836. Baillie BK, Monje V, Silvestre V, Sison M, & Belda-Baillie CA. 1998. Allozyme electrophoresis as a tool for distinguishing different zooxanthellae symbiotic with giant clams. Proc. Royal. Soc. Lond. Ser B 265:1949-1956. Baker AC. 2003. Flexibility and Specificity in Coral-Algal Symbiosis: Diversity, Ecology, and Biogeography of Symbiodinium. Ann. Rev. Ecol. Evol. Syst. in press. Baker AC. 2001. Reef corals bleach to survive change. Nature 411:765-766 Baker AC (2002) Reply to Hoegh-Guldberg et al. (2002) Nature, 415: 601-602. Baldauf SL, Palmer JD. 1993. Animals and fungi are each other's closest relatives: congruent evidence from multiple proteins. Proc. Natl. Acad. Sci. U. S. A. 90:11558-11562. Banaszak AT & Trench RK. 2001. Ultraviolet sunscreens in dinoflagellates. Protist. 152:93-101. Bannister LH & Dluzewski AR 1990 Ultrastructure of malaria invasion: a review. Blood Cells 16:257-292 Barbrook AC & Howe CJ. 2000. Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum. Mol. Gen. Genet. 263:152-158. Barbrook AC, Symington H, Nisbet RER, Larkum A. & Howe C. J. 2001. Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum. Mol. Genet. Genomics 266:632-638. Beam CA, Himes M. 1974. Evidence for sexual fusion and recombination in the dinoflagellate Crypthecodinium (Gyrodinium) cohnii. Nature 250:435-436. Beam CA, Himes M. 1984. Dinoflagellate genetics. In Dinoflagellates, Spector, DL. (ed.). Academic Press, NY. Pp 263-299. Berry JP, Reece KS, Rein KS, Baden DG, Haas LW, Ribeiro WL, Shields JD, Snyder RV, Vogelbein WK & Gawley RE. 2002. Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae. Proc. Natl. Acad. Sci. U. S. A. 99:10970-10975. Bhaud Y, Guillebault D, Lennon JF, Defacque H, Soyer-Golbillard MO & Moreau H. 2000. Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis. J. Cell. Sci. 113:1231-1239 Blank RJ & Huss VAR 1989. DNA divergency and speciation in Symbiodinium (Dinophyceae). Plant Syst. Evol. 163:153-163 Blank RJ, Huss VAR & Kersten W. 1988. Base composition of DNA from symbiotic dinoflagellates: a tool for phylogenetic classification. Arch. Microbiol. 149:515-520 Blank RJ. 1987. Presumed gametes of Symbiodinium: feintings by a fungal parasite? Endocytobiosis Cell Res. 4:297-304 Brown BE. 1997. Coral bleaching: causes and consequences. Coral Reefs 16 (Suppl.), S129-S138. Brown LS, Dioumaev AK, Lanyi JK, Spudich EN, Spudich JL. 2001. Photochemical reaction cycle and proton transfers in Neurospora rhodopsin. J. Biol. Chem. 276:32495-32505. Bryant D., Burke L., McManus, J. & Spalding M. 1998. Reefs at Risk: A map-based indicator of threats to the world’s coral reefs. World Resources Institute, p. 56. Buddemeier RW, Baker AC, Fautin DG & Jacobs JR. 2003. The adaptive bleaching hypothesis of bleaching: in Coral Health and Disease, Eugene Rosenberg (ed) Springer Verlag. In Press. Bunney WE & Bunney BG. 2000. Molecular clock genes in man and lower animals: possible implications for circadian abnormalities in depression. Neuropsychopharmacology. 22:335-45. Cavalier-Smith T. 1999. Principles of Protein and Lipid Targeting in Secondary Symbiogenesis: Euglenoid, Dinoflagellate, and Sporozoan Plastid Origins and the Eukaryote Family Tree. J. Eukaryot. Microbio. 46:347-366. Chaput H, Wang Y& Morse D 2002. Polyadenylated transcripts containing random gene fragments are expressed in dinoflagellate mitochondria. Protist 153:111-22. Chauhan VS & Bhardwaj D. 2003. Current status of malaria vaccine development. Adv Biochem. Eng. Biotechnol. 84:143- 182 Coats DW. 1999. Parasitic life styles of marine dinoflagellates. J. Eukaryot. Microbio. 46:402-409. Coles, SL & Jokiel, P.L. 1977. Effects of temperature on photosynthesis and respiration in hermatypic corals. Mar. Biol. 43:209-216. Symbiodinum_WPl+Appendix_v2.doc 15
Couronne O, Poliakov A, Bray N, Ishkhanov T, Ryaboy D, Rubin E, Pachter L & Dubchak I. 2003. Strategies and tools for whole-genome alignments. Genome Res. 13:73-80. Davies W, Jakobsen KS & Nordby O. 1988. Characterization of DNA from the dinoflagellate Woloszynskia bostoniensis. J. Protozool. 35:418-422. Delcher AL, Phillippy A, Carlton J, Salzberg SL. 2002. Fast algorithms for large-scale genome alignment and comparison. Nucleic Acids Res. 30:2478-2483. Douglas AE. 2003. Coral bleaching--how and why? Mar. Pollut. Bull. 46:385-392. Downs CA, Fauth JE, Halas J, Dustan P, Bemiss J, & Woodley CM. 2002. Oxidative Stress and Seasonal Coral Bleaching. Free Rad. Biol. Med. 33:533-543. Downs CA, Mueller E, Phillips S, Fauth JE & Woodley CM. 2000. A Molecular Biomarker System for Assessing the Health of Coral (Montastraea faveolata) during Heat Stress, J. Mar. Biotechnol., 2:533-544. Downs, C, Fauth, J, Dustan, P, Bemiss, J & Woodley, C. 2002. Oxidative stress and seasonal coral bleaching. Free. Radic. Biol. Med. 33:533. Durborow RM. 1999. Health and safety concerns in fisheries and aquaculture. Occup. Med. 14:373-406. Dzierszinski F, Mortuaire M, Dendouga N, Popescu O& Tomavo S. 2001. Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii. J Mol Biol. 309:1017-1027. Esaias WE & Curl H. C. 1972. Effect of dinoflagellate bioluminescence on copepod ingestion rate. Limnol. Oceanogr. 17:901-906. Fast NM, Xue L, Bingham S, Keeling PJ. 2002. Re-examining alveolate evolution using multiple protein molecular phylogenies. J. Eukaryot .Microbiol. 49:30-37. Fenical W. 1997. New pharmaceuticals from marine organisms. Trends Biotechnol. 15:339-341. Finnegan EJ, Peacock WJ, Dennis ES. 2000. DNA methylation, a key regulator of plant development and other processes. Curr Opin Genet Dev. 10:217-223. Foth BJ, McFadden GI. 2003. The apicoplast: a plastid in Plasmodium falciparum and other Apicomplexan parasites. Int Rev Cytol. 224:57-110. Foth BJ, Ralph SA, Tonkin CJ, Struck NS, Fraunholz M, Roos DS, Cowman AF, McFadden GI. 2003. Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science. 299:705-708. Freudenthal HD 1962. Symbiodinium gen. nov. and Symbiodinium microadriaticum sp. nov., a zooxanthella: taxonomy, life cycle, and morphology. J. Protozool. 9: 45-52 Goulet TL & Coffroth MA 2003. Genetic composition of zooxanthellae between and within colonies of the octocoral Plexaura kuna based on small subunit rDNA and multilocus DNA fingerprinting Marine Biology 142:233-239 Hardeland R & Poeggeler B. 2003. Non-vertebrate melatonin. J Pineal Res. 34:233-241. Harrison, P.L. & Wallace, C.C. 1990. Reproduction, dispersal and recruitment of scleractinian corals. Chapter 7. In: Z. Dubinsky (Editor), Coral Reef Ecosystems, Ecosystems of the World Vol. 25. pp. 133-207. Elsevier Science Publishers, Amsterdam. Herzog M & Soyer MO. 1983. The native structure of dinoflagellate chromosomes and their stabilization by Ca2+ and Mg2+ cations. Eur. J. Cell Biol. 30:33–41 Hinnebusch AG, Klotz LC, Immergut E & Loeblich AR 3rd. 1980 Deoxyribonucleic acid sequence organization in the genome of the dinoflagellate Crypthecodinium cohnii. Biochemistry. 19:1744-1755. Hoegh-Guldberg O. 1999. Coral bleaching, climate change and the future of the world’s coral reefs. Mar. Freshwater Res. 50:839-866. Hoegh-Guldberg O, Jones, RJ, Ward S.& Loh WK 2002. Is coral bleaching really adaptive? Nature. 415:601-602. Howe CJ, Barbrook AC, Koumandou VL, Nisbet RE, Symington HA & Wightman TF. 2003. Evolution of the chloroplast genome. Philos. Trans. R. Soc. Lond. B Biol. Sci. 358:99-106; discussion 106-107. Hughes TP Baird AH, Bellwood DR Card M, Connolly SR, Folke C, Grosberg R, Hoegh-Guldberg O, Jackson JBC, Kleypas J, Lough JM, Marshall P, Nyström M, Palumbi SR, Pandolfi JM, Rosen B & Roughgarden J. 2003. Climate Change, Human Impacts, and the Resilience of Coral Reefs; Science 301:929-933. Inada DC, Bashir A, Lee C, Thomas BC, Ko C, Goff SA & Freeling M. 2003. Conserved noncoding sequences in the grasses. Genome Res. 13:2030-2041. Idnurm A, Howlett BJ. 2001. Characterization of an opsin gene from the ascomycete Leptosphaeria maculans. Genome. 44:167-171. Jaffe DB, Butler J, Gnerre S, Mauceli E, Lindblad-Toh K, Mesirov JP, Zody MC & Lander ES. 2003. Whole-genome sequence assembly for mammalian genomes: Arachne 2. Genome Res. 13:91-96. Kasinsky HE, Lewis JD, Dacks JB & Ausio J. 2001. Origin of H1 linker histones. FASEB J. 15:34-42. Kellis M, Patterson N, Endrizzi M, Birren B & Lander ES. 2003. Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423:241-254. Kevin MJ, Hall WT, McLaughlin JA& Zahl PA. 1969. Symbiodinium microadriaticum Freudenthal, a revised taxonomic description, ultrastructure. J Phycol 5:341-350. Kim JE, Jeong HW, Nam JO, Lee BH, Choi JY, Park RW, Park JY & Kim IS. 2002. Identification of motifs in the fasciclin domains of the transforming growth factor-beta-induced matrix protein beta IG-h3 that interact with the alpha V beta 5 integrin. J. Biol. Chem. 277:46159-46165. Kite GC, Rothschild LJ & Dodge JD. 1988. Nuclear and plastid DNAs from the binucleate dinoflagellates Glenodinium (Peridinium) foliaceum and Peridinium balticum. Biosystems. 21:151-163. Symbiodinum_WPl+Appendix_v2.doc 16
Kodama M. 1990. Possible links between bacteria and toxin production in algal blooms. In: Graneli E, SundstromB, Edler L & Anderson DM (eds) Toxic Marine phytoplankton. Elsevier, Amsterdam, pp 52-61. Kohl AC, Ata A & Kerr RG. 2003. Pseudopterosin biosynthesis-pathway elucidation, enzymology, and a proposed production method for anti-inflammatory metabolites from Pseudopterogorgia elisabethae. J. Ind. Microbiol. Biotechnol. Aug 14 [Epub ahead of print] Kuvardina ON, Leander BS, Aleshin VV, Myl'nikov AP, Keeling PJ & Simdyanov TG 2002. The phylogeny of colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the free-living sister group to apicomplexans. J. Eukaryot. Microbiol. 49:498-504. La Jeunesse TC. 2001. Investigating the biodiversity, ecology, and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a "species" level marker. J. Phycol. 37:866-880. La Jeunesse T, Loh WKW, van Woesik R, Hoegh-Guldberg O, Schmidt GW & Fitt WK. 2003. Low symbiont diversity in southern Great Barrier Reef corals, relative to those of the Carribean. Limnol. Oceanog. 48:2046-2054 Leander BS, Clopton RE, Keeling PJ. 2003. Phylogeny of gregarines (Apicomplexa) as inferred from small-subunit rDNA and beta-tubulin. Int. J. Syst. Evol. Microbiol. 53:345-354. Leander BS & Keeling PJ. 2003. Morphostasis in alveolate evolution. Trends Ecol. Evol. 18:395-402. Lee JJ, Morales J, Bacus S, Diamont A, Hallock P, Pawlowski J, Thorpe J. 1997. Progress in characterizing the endosymbiotic dinoflagellates of soritid formaninfera and related studies on some stages in the life cycle of Marginopora vartebralis. J. Foraminiferal Res. 27:254-263. Lehane, L. & Lewis, RJ. 2000. Ciguatera: recent advances but the risk remains. International Journal of Food Microbiology 61:91–125. Li L & Hastings JW. 1998. The structure and organization of the luciferase gene in the photosynthetic dinoflagellate Gonyaulax polyedra. Plant Mol Biol. 36:275-284. Lin S, Zhang H, Spencer DF, Norman JE & Gray MW. 2002. Widespread and extensive editing of mitochondrial mRNAS in dinoflagellates. J. Mol. Biol. 320:727-739. Loeblich III AR. 1984. Dinoflagellate Evolution. In: DL Spector (ed), Dinoflagellates. Academic Press, pp. 481-522. Lough JM. 2000. 1997-98: unprecedented thermal stress to coral reefs? Geophysical Research Letters 27:3901-3904. Luiten EE, Akkerman I, Koulman A, Kamermans P, Reith H, Barbosa MJ, Sipkema D & Wijffels RH. 2003. Realizing the promises of marine biotechnology. Biomol. Eng. 20:429-439. Matzke MA, Mette MF, Matzke AJ. 2000. Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol. 43:401-415. Mittag M. 2003. The function of circadian RNA-binding proteins and their cis-acting elements in microalgae. Chronobiol Int. 20:529-541. Moore, RB, Ferguson, KM, Loh, WKW, Hoegh-Guldberg, O, & Carter, DA. 2003. Highly organised structure in the non- coding region of the psbA minicircle from clade C Symbiodinium. Int. J. Syst. Evol. Microbiol. In Press. Morse D, Salois P, Markovic P & Hastings JW 1995. A nuclear-encoded form II RuBisCO in dinoflagellates. Science 268:1622-1624. Murata M, Yasumoto T. 2000. The structure elucidation and biological activities of high molecular weight algal toxins: maitotoxin, prymnesins and zooxanthellatoxins. Nat. Prod. Rep. 17:293-314. Norman JE & Gray MW 2001. A complex organization of the gene encoding cytochrome oxidase subunit 1 in the mitochondrial genome of the dinoflagellate, Crypthecodinium cohnii: homologous recombination generates two different cox1 open reading frames. J. Mol. Evol. 53:351-63. Obornik M, Van de Peer Y, Hypsa V, Frickey T, Slapeta JR, Meyer A & Lukes J. 2002. Phylogenetic analyses suggest lateral gene transfer from the mitochondrion to the apicoplast. Gene 285:109-18. Okamoto OK & Hastings JW. 2003. Novel dinoflagellate clock-related genes identified through microarray analysis. J. Phycol. 39:519-526 Palmer JD. 2003. The symbiotic birth and spread of plastids: How many times and whodunit? J. Phycol. 39:4-11 Peterson DG, Schulze SR, Sciara EB, Lee SA, Bowers JE, Nagel A, Jiang N, Tibbitts DC, Wessler SR, Paterson AH. 2002. Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery. Genome Res. 12:795-807. Pfiester LA 1984. Sexual Reproduction. Dinoflagellates. D.L. Spector (ed.), Academic Press, Inc, pp. 181-199. Pochon, X, Pawlowski, J, Zaninetti & Rowan, R. 2001. High genetic diversity and relative specificity among Symbiodinium-like endosymbiotic dinoflagellates in soritid foraminiferans. Mar. Biol. 139:1069-1078. Ralph SA, D'Ombrain MC & McFadden GI. 2001. The apicoplast as an antimalarial drug target. Drug Resist. Updat. 4:145- 51. Reichman JR, Wilcox TP & Vize PD. 2003. PCP gene family in Symbiodinium from Hippopus hippopus: low levels of concerted evolution, isoform diversity and spectral tuning of chromophores. Mol. Biol. Evol. Aug 29 [Epub ahead of print]. Rein KS & Borrone J. 1999. Polyketides from dinoflagellates: origins, pharmacology and biosynthesis. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 124:117-31. Reynolds WS, Schwarz JA & Weis VM. 2000. Symbiosis-enhanced gene expression in cnidarian-algal associations: cloning and characterization of a cDNA, sym32, encoding a possible cell adhesion protein. Comp. Biochem Physiol. A Mol. Integr. Physiol. 126:33-44. Rizzo PJ. 1987. Biochemistry of the dinoflagellate nucleus. The Biology of Dinoflagellates. F.J.R. Taylor (ed.), Blackwell Botanical Monographs, pp. 143-173. Symbiodinum_WPl+Appendix_v2.doc 17
Rodriguez-Lanetty M. 2003. Evolving lineages of Symbiodinium-like dinoflagellates based on ITS1 rDNA. Mol Phylogenet Evol. 28:152-168. Rowan R, Whitney SM, Fowler A & Yellowlees D. 1996. Rubisco in marine symbiotic dinoflagellates: form II enzymes in eukaryotic oxygenic phototrophs encoded by a nuclear multigene family. Plant Cell. 8:539-553. Rowan R. & Powers DA. 1992. Ribosomal RNA sequences and the diversity of symbiotic dinoflagellates (zooxanthellae). Proc. Natl. Acad. Sci. U. S. A. 89:3639-3643. Rowan R. 1998. Diversity and ecology of zooxanthellae on coral reefs. J. Phycol. 34:407-417. Saldarriaga JF, McEwan ML, Fast NM, Taylor FJ & Keeling PJ. 2003. Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage. Int. J. Syst. Evol. Microbiol. 53:355- 365. Salm R. 1994. Coral's hidden riches. People Planet. 3:19-21. Santos S.R., Gutierrez-Rodriguez, Lasker, H.R. & Coffroth, M.A. 2003. Patterns of Symbiodinium associations in the Caribbean gorgonian Pseudopterorgia elisabethae: high levels of genetic variability and population structure in symbiotic dinoflagellates of the Bahamas Marine Biology 143:111-120. Santos SR & Coffroth MA. 2003. Molecular Genetic Evidence that Dinoflagellates Belonging to the Genus Symbiodinium Freudenthal Are Haploid. Biol. Bull. 204:10-20. Santos SR, Taylor DJ, Kinzie RA, Hidaka M, Sakai K & Coffroth MA. 2002. Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences. Mol. Phylogenet. Evol. 23:97- 111. Shick JM, Romaine-Lioud S, Ferrier-Pagès C, and Gattuso J-P. 1999. “Ultraviolet-B radiation stimulates shikimate pathway-dependent accumulation of mycosporine-like amino acids in the coral Stylopora pistillata despite decreases in its population of symbiotic dinoflagellates.” Limnol. Oceanogr. 44: 1667-1682. Smith GJ & Muscatine L. 1999. Cell cycle of symbiotic dinoflagellates: variation in G1 phase-duration with anemone nutritional status and macronutrient supply in the Aiptasia pulchella-Symbiodinium pulchrorum symbiosis. Mar. Biol. 134:405-418. Soyer-Gobillard MO, Geraud ML, Coulaud D, Barray M, Theveny B, Revet B & Delain E. 1990. Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate. J. Cell. Biol. 111:293-304. Soyer-Gobillard MO, Gillet B, Geraud ML & Bhaud Y. 1999. Dinoflagellate chromosome behavior during stages of replication. Int. Microbiol. 2:93-102. Spector D. 1984. Dinoflagellate nucleus. In Spector, David L., ed., "Dinoflagellates", Academic Press, Inc. Stanley GD Jr & Swart PK. 1995. Evolution of the coral-zooxanthellae symbiosis during the Triassic: a geochemical approach. Paleobiology 21:179-199. Steele RE & Rae PM. 1980. Ordered distribution of modified bases in the DNA of a dinoflagellate. Nuc. Acids Res. 8:4709- 4725. Striepen B, White MW, Li C, Guerini MN, Malik SB, Logsdon JM Jr, Liu C & Abrahamsen MS. 2002. Genetic complementation in apicomplexan parasites. Proc Natl Acad Sci U S A. 99:6304-6309. Sultan AA. 1999. Molecular mechanisms of malaria sporozoite motility and invasion of host cells. Int. Microbiol. 2:155- 160. Takishita K, Ishikura M, Kioke K & Maruyama T 2003. Comparison of phylogenies based on nuclear-encoded SSU rDNA and plastid-encoded psbA in the symbiotic dinoflagellate genus Symbiodinium. Phycologia 42, 285 ten Lohuis MR & Miller, DJ. 1998a. Light-regulated transcription of genes encoding peridinin chlorophyll a proteins and the major intrinsic light-harvesting complex proteins in the dinoflagellate Amphidinium carterae hulburt (Dinophycae): changes in cytosine methylation accompany photoadaptation. Plant Physiol. 117:189-96. ten Lohuis MR & Miller DJ. 1998b. Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J. 13:427-435. Thomas JW, et al. 2003. Comparative analyses of multi-species sequences from targeted genomic regions. Nature 424:788- 793. Trench RK & Blank RJ. 1987. Symbiodinium-microadriaticum Freudenthal Symbiodinium-goreauii new-species Symbiodinium-kawagutii new-species and Symbiodinium-pilosum new-species Gymnodinioid dinoflagellate symbionts of marine invertebrates. J. Phycol. 23:469-481. Trench RK. 1987. Dinoflagellates in non-parasitic symbioses. The Biology of Dinoflagellates. F.J.R. Taylor (ed.), Blackwell Botanical Monographs, pp. 530-570. Triplett EL, Govind NS, Roman SJ, Jovine RV & Prezelin BB. 1993. Characterization of the sequence organization of DNA from the dinoflagellate Heterocapsa pygmaea (Glenodinium sp.). Mol. Mar. Biol. Biotechnol. 2:239-245. Udy JW, Hinde R, Vesk M. 1993. Chromosomes and DNA in Symbiodinium from Australian hosts. J. Phycology 29.3:314- 320 van den Hoek C, Mann DG & Jahns HM. 1995. Algae: An introduction to phycology. Chapter 16 Dinophyta. Cambridge University Press ISBN 0 521 30419 9 Veldhuis, M.J.W., Cucci, T.L. and Sieracki, M.E. 1997. Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. J. Phycology, 33:527-541. Waller RF, Ralph SA, Reed MB, Su V, Douglas JD, Minnikin DE, Cowman AF, Besra GS & McFadden GI. 2003. A type II pathway for fatty acid biosynthesis presents drug targets in Plasmodium falciparum. Antimicrob Agents Chemother. 47:297-301. Symbiodinum_WPl+Appendix_v2.doc 18
Walther GR, Post E, Convey P, Menzel A, Parmesan C, Beebee TJ, Fromentin JM, Hoegh-Guldberg O & Bairlein F. 2002. Ecological responses to recent climate change. Nature 416:389-95. Warner ME, Fitt WK & Schmidt GW. 1999. Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching. Proc. Natl. Acad. Sci. U. S. A. 96:8007-8012. Weis VM & Reynolds WS. 1999 Carbonic anhydrase expression and synthesis in the sea anemone Anthopleura elegantissima are enhanced by the presence of dinoflagellate symbionts. Physiol. Biochem. Zool. May- 72:307-316. Whitney SM, Shaw DC, Yellowlees D. (1995) Evidence that some dinoflagellates contain a ribulose-1,5-bisphosphate carboxylase/oxygenase related to that of the alpha-proteobacteria. Proc R Soc Lond B Biol Sci. 1995 Mar 22;259(1356):271-5. Wilkinson C, Linden, O. Cesar H, Hodgson G, Rubens J. & Strong AE. 1999. Ecological and Socioeconomic Impacts of 1998 Coral Mortality in the Indian Ocean: An ENSO impact and a warning of future change? AMBIO 26:188-196. Wilkinson C. (ed) 2000. Status of Coral Reefs of the World: 2000, Global Coral Reef Monitoring Network, Australian Institute of Marine Science, Townsville. Wilson RJ & Williamson DH. 1997. Extrachromosomal DNA in the Apicomplexa. Microbiol. Mol. Biol. Rev. 61:1-16. Wilson RJ. 2002. Progress with parasite plastids. J. Mol. Biol. 319:257-274. Woodley CM., Bruckner AW, Galloway SB, McLaughlin SM, Downs CA, Fauth JE, Shotts EB & Lidie KL. 2003. Coral Disease and Health: A National Research Plan. National Oceanic and Atmospheric Administration, Silver Spring, MD. 66 pp. Yoon HS, Hackett JD & Bhattacharya D. 2002a. A single origin of the peridinin- and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis. Proc. Natl. Acad. Sci. U. S. A. 99:11724-11729. Yoon HS, Hackett JD, Pinto G & Bhattacharya D. 2002b. The single, ancient origin of chromist plastids. Proc. Natl. Acad. Sci. U. S. A. 99:15507-12. Yuan Y, SanMiguel PJ & Bennetzen JL. 2003. High-Cot sequence analysis of the maize genome. Plant J. 34:249-55. Zhang Z, Cavalier-Smith T & Green BR. 2002. Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins. Mol. Biol. Evol. 19:489-500. Symbiodinum_WPl+Appendix_v2.doc 19
Appendix 1: The size of the Symbiodinium genome
When we started, there were no careful measurements of the Symbiodinium genome size; Bland & Huss (1998) had determined–from preliminary C0t analyses–that the genome must be >2x108 bp. For dinoflagellates as a whole, Rizzo (1987) had collated a number of published dinoflagellate genome sizes, and Velduis et al. (1997) had measured a number, in a broad survey of phytoplankton genome sizes. We asked Galbraith & Lambert (using DAPI staining and flow cytometry; Galbraith 1990; Galbraith et al. 1999; Johnston et al. 1999) to measure the genome sizes for a number of dinoflagellate species, as well as those for a number of Symbiodinium isolates, provided by a number of researchers (please see the final section for the results of an alternative and independent determination). [Much of this work is now published: Todd C. LaJeunesse, Georgina Lambert, Robert A. Andersen, Mary Alice Coffroth, David W. Galbraith. SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES. Journal of Phycology. Online publication date: 28-Jul-2005] The range of dinoflagellate genome sizes Bob Anderson of the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) selected a number of relatively small dinoflagellate species (thus compatible with the Lambert/Galbraith flow cytometry apparatus) for genome determination. Table 1 shows their results (all values in pg); compared to those of Rizzo (1987) and Velduis et al. (1997) (multiple determinations are shown separated by a slash). Clearly there is poor agreement between the Species Rizzo 1987 Velduis et al. 1997 Lambert/Galbraith CCMP # variou Amphidinium carterae 3.2/2.7 13.3/9.8 5.7/6 1314 s sets Cachonina neii 10.0/7.6 of Crypthecodinium cohonii 6.9/6.9/7.3 25.6/22.5 results Gonyaulax polyedra 200 139.7/125.8 in Gymnodinium sp. 15.4 16.0/14.7 almost Gymnodinium breve 101.6/112.5 all Gymnodinium nelsoni 143.0 cases, Gyrodinium resplendens 66.0 but it Peridinium trochoideum 34.0 is Protocentrum micans 42.0 278.1/227.1 imposs ible to A. tamarense 103.5 1598 know G. simplex 9.1/12.7 419 if this H. triquetra 24.1 449 is due H. pygmeae 3.4/4.1 1322) to K. brevis 57.1 2229 metho P. minimum 37.2/45.5 6.7/7.3 1329 d, or Polarella sp. (Arctic) 6.6/7.3 2088 to the Alexandrium catanella 228.1/168.7 specifi Heterocapsa triquetra 26.7/19.3 c Heterocapsa neie 63.5/29.0 isolate used for each species in each case; thus the results do not serve to “validate” the methods of Galbraith & Lambert. Symbiodinum_WPl+Appendix_v2.doc 20
Note that A. carterae has a particularly small genome, and is also a facultative endosymbiont of marine invertabrates. H. pygmeae also has a notable small genome; Velduis et al. (1997) show that genome size is strongly correlated with cell size, for all groups of marine phytoplankton. Symbiodinium genome sizes The first set of Symbiodinium isolates Lambert& Galbraith examined were provided by both B Moore and MA Coffroth, and include two of the strains we propose to sequence. There is a range of values, but all of them are small, some very small for a dinoflagellate. The Pseudopterogorgia
Symbiodinium host species and/or isolation number) DNA (pg/cell) source comments
Acropora aspera, Heron Island FIZ, clade C3 1.2/1.2 Moore Australian microarray strain Leptastrea purpurea, One Tree Island 13 - CZ no data " Leptastrea purpurea, One Tree Island 14 - CZ 3.1/3.6 " Leptastrea purpurea, One Tree Island 15 – CZ, clade C1? 2.3/2.5 " Pocillopora damicornis, One Tree Island 4 – CZ, clade C2? 5.4 "
PPf 2.9/2.7 Coffroth Same ITS and cp-genotype as Pseudopterogorgia elisabethae, clade B 1.9/1.9/1.8 " Medina microarray strain Mv 3 " MF1.5 4.2 " 702 2.9/2.8 " 579 2.2/2.9/2.7 " 571 2.5/2.3 " elisabethae isolate is genetically similar to the isolate that will be used in the Median CLADE ITS type DNA EST/microarray study (Montastrea faveolata), (=subgenus) (=species) Sample I.D. (pg/cell) and the strain that we propose for high coverage sequencing (based on ITS sequences A A1 61 2.2 (LaJeunesse 2002) and variation in domain V of A1.1 80 2.3 A3 168 3.2 the chloroplast 23s rDNA (Santos et al 2003)). A4 379 2.7 Symbiodinium genome sizes and clade B B1 2 2.1 B1 Pe* 1.9 Todd Christopher LaJeunesse B2 Ppf* 2.8 [email protected] have been very B2.1 141 2.4 involved in collecting and characterizing the B3 385 2.4 genetic structure of Symbiodinium (see refs), and B19 579* 2.6 provided another set of isolates that he has B? 571* 2.4 characterized by clade and ITS number (internal B? 702* 2.9 transcribed spacer region). He also included the C C1 152 4.8 Coffroth strains for which he have characterized C2 203 2.9 the clade and ITS (“*”). Again, there are a range D D1a A001 4.1 of values, but they are universally small, relative Carlos "D" 401 2.1 to other dinoflagellates. There is not a clear E E1 383 3.3 correlation between clade and genome size. F F1 Mv/135* 3.0 Note that the Montastrea faveolata strain, that F2 133 2.6 we propose for high coverage sequencing, is a ‘B1” ITS type. Symbiodinum_WPl+Appendix_v2.doc 21
An alternative method for size determination Finally, and mysteriously, Ryan Gregor http://www.genomesize.com/ measured the genome sizes for two Symbiodinium isolates, provided by Coffroth before we started this proposal. Gregor uses Feulgen image densitometry analyses. He determined that Symbiodinium SSPe (from the gorgonian Pseudoterogorgia elisabethae, a ITS-type B1) has a genome of ~0.58 pg, and reports that slides of the Montastraea isolate–that we propose sequencing–had values very close to these, when he looked at them more casually. Frustratingly, Gregor has been on sabbatical and traveling, and we have not been able to resolve the difference between his value of ~0.6 pg, and the Lambert & Galbraith value of 1.9 pg. Neither group can see their way to an explanation for the difference. In our proposal we choose to err on the side of presenting the Lambert & Galbraith FACs results, as the latter lab is experienced with autotrophs, and has obviously done a much more through job. References Galbraith DW, Herzenberg LA, Anderson MT. 1999. Flow cytometric analysis of transgene expression in higher plants: green fluorescent protein. Methods Enzymol. 302:296-315. Galbraith DW. 1990. Flow cytometric analysis of plant genomes. Methods Cell Biol. 33:5495-62. Johnston JS, Bennett MD, Rayburn AL, Galbraith DW, Price HJ. 1999. Reference standards for determination of DNA content of plant nuclei. Am J Bot. 86:609. LaJeunesse TC & R. K. Trench. 2000. Biogeography of Two Species of Symbiodinium (Freudenthal) Inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt). Biol. Bull. 199:126–134. LaJeunesse, T. C. 2001. Investigating the biodiversity, ecology, and phylogeny of endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS region: in search of a “species” level marker. J. Phycology 37:866–880. LaJeunesse, T. C. 2002. Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs. Marine Biology 141:387–400. Rizzo PJ. 1987. Biochemistry of the dinoflagellate nucleus. The Biology of Dinoflagellates. F.J.R. Taylor (ed.), Blackwell Botanical Monographs, pp. 143-173. Santos, S.R., Gutierrez-Rodriguez, and Coffroth, M.A. (2003) Phylogenetic identification of symbiotic dinoflagellates via length heteroplasmy in domain V of chloroplast large subunit (cp23S)-rDNA sequences. Marine Biotechnology 5:130-140 Veldhuis, M.J.W., Cucci, T.L. and Sieracki, M.E. 1997. Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications. Journal of Phycology, 33: 527- 541. Symbiodinum_WPl+Appendix_v2.doc 22
Letters of Support During preparation of the accompanying whitepaper, eighteen US labs and twenty one international labs contacted the whitepaper authors to offer concrete support, and have all supplied letters of support. Eight of the letters are presented in full. The rest of the letters are presented as excerpts, indicating experiments that each lab would do if the Symbiodinium genome sequence became available. Research of many of these supporters appears in the citations list in the accompanying whitepaper, where names of letter writers are underlined. Section 1: Annotation teams 1.1 Annotation team USA 1.2 Annotation team international Section 2: Importance of the dinoflagellate genome to basic science 2.1 J. Palmer – Member of the National Academy of Sciences USA Section 3: Full letters illustrating the health significance of the Symbiodinium genome proposal 3.1 G. McFadden, Howard Hughes Scholar 3.2 J. Kissinger, Center for Tropical and Emerging Global Diseases 3.3 D. Anderson, National Office for Marine Biotoxins and Harmful Algae 3.4 K. Rein, Advanced Research Cooperation in Environmental Health 3.5 L. Medlin, A. Cembella, U. John & K. Valentin – Wegener Institute 3.6 F. Van Dolah, National Oceanographic and Atmospheric Administration 3.7 A. Douglas, University of York Section 4: Excerpts of letters that illustrate the impact of the Symbiodinium genome proposal on knowledge of dinoflagellate biology 4.1 J. W. Hastings – Member of the National Academy of Sciences USA 4.2 Excerpts of letters from labs planning to utilize Symbiodinium sequence, grouped under molecular biological categories 4.2.1 Organellar and nuclear integration, intracellular gene transfer, & horizontal gene transfer 4.2.2 Transformation, chromosome structure/condensation, DNA methylation, & viruses in the genome 4.2.3 Dinoflagellate cell division, & gene complementation in yeast 4.2.4 Symbiosis, cell adhesion, signal transduction, & intercellular trafficking, 4.2.5 Karyotyping, speciation, sex, & phylogeny 4.2.6 Drug development & toxin biology
Section 1: Annotation Team A team of 30 annotators has assembled for this genome project, showing the high level of interest that the dinoflagellate and apicomplexan communities have in the genetics of a model dinoflagellate. Each included their intentions to annotate either in a formal letter of support or in an email to the whitepaper authors. All of these annotators are specialists in genetics and, as such, are highly skilled in the gene alignments and discernment that would be required. 1.1 Annotation team USA: Jeff Palmer, Scott Santos, Rebecca Gast, J. Woodland Hastings, Todd La Jeunesse, William Fitt, Gregory Schmidt, Senjie Lin, Monica Medina, John Logsdon, Andrew Baker, Tom Doak. 1.2 Annotation team international: Brett Baillie, Christopher Howe, Anthony Larkum, Tadashi Maruyama, Geoff McFadden, David Miller, Robert Moore, Herve Moreau, David Morse, Xavier Pochon, Jan Pawlowski, David Yellowlees, Joseph Wong, Willie Wilson, Dan Franklin, John Berges, Bill Leggat, Ove Hoegh-Guldberg. Symbiodinum_WPl+Appendix_v2.doc 23
Section 2: Core letter on the importance of the dinoflagellate genome to basic science 2.1 Jeff Palmer, Member of the National Academy of Sciences USA It is with true enthusiasm, indeed great excitement that I write in support of the proposal to sequence the first dinoflagellate “genome,” from Symbiodinium. I say “genome,” because all nuclear genome sequencing projects inevitably also generate sequences for that organism’s organelle genome(s). Usually this hardly matters, because the organelle genome(s) from that organism has already been sequenced, or is trivially easy to isolate and sequence, and/or is not especially interesting. Dinoflagellates present exactly the opposite situation! We know just enough about both their plastid and mitochondrial genomes to realize that they are bizarre and fascinating in a number of important respects, indeed that their plastid genomes are by far the most extraordinary and interesting of any organism! Yet it is precisely their fascinating fragmentation that renders them extraordinarily difficult, if not impossible, to isolate and characterize to completion by purification of organellar DNAs. About 13 different plastid genes have been characterized so far as present on the unique unigene mini-chromosomes of dinoflagellate plastids, but only when all of the DNA in the dinoflagellate cell is sequenced (with the much higher copy number of the organellar genomes allowing truly complete censusing of their genome) will we be sure exactly how many and which plastid proteins are still encoded by plastid genes and which have been transferred to the nucleus. This is a compelling issue that will significantly stimulate follow-up experimental work on my lab’s part and I’m sure that of others. Why? Because it is already clear that dinoflagellates have transferred to the nucleus far more plastid genes than any other photosynthetic eukaryote. Yet we need a complete and accurate accounting of the distribution of genetic labor between the nucleus and plastid (and the same on the mitochondrial side, where a similar situation exists) in order to know exactly what specific genes and categories of genes that otherwise are always found in plastid genomes have successfully moved to the nucleus in dinoflagellates. Knowing this will provide the foundation for asking both how these genes have moved, and why. What factors, what selective forces or mechanistic constraints, have been altered to drive or facilitate greatly enhanced organelle- to-nucleus gene transfer in dinoflagellates? Addressing this question in dinoflagellates should considerably illuminate some of the central questions concerning organelle genomes: Why organelle genomes, i.e., why do these genomes persist in the face of entirely unidirectional gene transfer to the nucleus? Why are certain genes always (except in dinoflagellates in the case of the plastid) retained in the organelles in the face of this unidirectional transfer? I have been funded by NIH for over 10 years on a project titled “Transfer of Organelle Genes to the Nucleus.” Most of this work has focused on plants as a model group to study this fundamental process. Once the three genomes of a dinoflagellate are sequenced, a significant part of my lab’s research in this area will switch to dinoflagellates, for the reasons and to address the questions given above. My lab has also contributed significantly to our current understanding of plastid phylogeny, including the primary origin of plastid via cyanobacterial endosymbiosis and the multiple transfers of plastids among eukaryotes via secondary symbiosis. How exactly the dinoflagellate plastid arose is unclear and controversial, with conflicting and sometimes complex hypotheses involving perhaps multiple serial symbioses presented in the literature or in recent meeting talks. It will take a full accounting of the phylogenetic history of essentially all dinoflagellate plastid proteins to figure out exactly what the overall evolutionary history is of the dinoflagellate plastid, and of that portion of its nucleus that arose from plastid genes. To what extent has serial symbiosis with red algae (most likely), but possibly also green algae and haptophytes, contributed to the evolutionary complexity of dinoflagellate plastid proteins? Has horizontal transfer of individual genes also added to the brew of the whole-organism horizontal transfer represented by secondary symbiosis? My lab, and undoubtedly several others, plan to mine the Symbiodinium genome in what will be a challenging bioinformatic/phylogenomic effort to address these questions. In conclusion, I look forward with eager anticipation to joining the Symbiodinium “genome” project by serving as a full and active participant in its annotation and to the payoffs from this sequence for my lab’s future research. - JP Symbiodinum_WPl+Appendix_v2.doc 24
Section 3: Letters illustrating the health significance of the Symbiodinium genome proposal The properly annotated, full sequence of the Symbiodinium genome can have immense benefits to human health and environmental health, as shown in the following six major letters received. Each of the letter authors lays out their plans for experiments/bioinformatics that will utilize the sequences, and each specifically identifies medical or environmental health issues that will be effectively addressed using dinoflagellate genomic information. 3.1 Geoff McFadden, Howard Hughes Scholar, University of Melbourne, Australia I write in support of your proposal to sequence the genome of the dinoflagellate Symbiodinium. I’m enormously supportive of this venture. A dinoflagellate genome project will be a tremendous boost to a wide range of research areas including parasitology, algology and protistology, symbiosis and genomics generally. Sequencing a dinoflagellate is now a vital priority because this group is part of the supergroup of Alveolates. At this juncture genomes are complete or well underway for members of the two neighboring phyla (Ph. Apicomplexa & Ph. Ciliata) in this assemblage. Numerous genomes for parasites from the apicomplexans are complete or well advanced. Similarly, ciliate genome projects are also progressing rapidly. It is thus very timely to develop a dinoflagellate genome. Indeed, the absence of a dinoflagellate genome will soon become a severe limitation to our understanding of these organisms. There are already about 10,000 dinoflagellate ESTs ready to be released so a full genome project seems the next logical step. The Symbiodinium genome sequence has very real potential to aid research into new drugs against apicomplexan parasites. The genome will reveal plastid-targeted genes homologous to apicoplast genes from human parasites like malaria and Toxoplasma. The apicoplast has emerged as a prime target for anti-parasite drugs in recent years and a dinoflagellate genome will certainly be a significant boost to developing further leads in this area and hence spin-off into the development of anti-malarial pharmaceuticals. I believe the genome will also aid our understanding of how apicomplexan parasites invade their host cells. Dinoflagellates apparently have much of the same cellular (and presumably molecular) machinery as the parasites, so a genome will give us the tools to explore these processes. Since host cell invasion is a key facet of strategies to develop vaccines against these parasites, deeper understanding of these mechanisms will aid the rationale and optimal development of strategies targeting these apparatuses. The dinoflagellate genome program will almost certainly need to separate the chromosomes by PFGE to build the shotgun libraries successfully and to determine the genome size more accurately. My lab has some experience in PFGE and would be willing to help out if possible. Additionally, my group could lend a hand with identifying the genes for plastid targeted proteins as we did for the malaria genome (Gardner et al Nature 2002, Foth et al Science 2003). We could also assist with identifying mitochondrial proteins. I’d also very much like to develop a transfection system for dinoflagellates, and a genome project would motivate this effort a lot. - GM 3.2 Jessica Kissinger, The Center for Tropical and Emerging Global Diseases, University of Georgia I would like to offer my enthusiastic support for the generation of genome sequence for the dinoflagellate Symbiodinium. Dinoflagellates are the sister group to the Apicomplexa, a phylum containing approx 5,000 parasitic organisms including the agents of malaria, toxoplasmosis, cryptosporidiosis and numerous pathogens of veterinary significance. I am an evolutionary biologist working on apicomplexan genome evolution and my research suffers from the lack of a genome sequence which is an appropriate outgroup. Currently, there are 15 genome projects within the Apicomplexa and two within the Ciliates. A genome sequence from the third arm of the Alveolata (dinoflagellates) is desperately needed. I cannot determine the set of Symbiodinum_WPl+Appendix_v2.doc 25
“apicomplexan-specific” genes without such a reference. Obviously, such a set of genes would contain numerous potential therapeutic targets. As a part of my research, I have been involved in the generation of Eukaryotic protistan genome databases, PlasmoDB, ToxoDB, CryptoDB and TcruziDB. I can state emphatically that there are members of the community with the expertise and desire to utilize a dinoflagellate genome sequence to its maximum potential. - JK 3.3 Don Anderson, Director - U.S. National Office for Marine Biotoxins and Harmful Algae I’m writing to express my enthusiastic support for your proposal to sequence the genome of the dinoflagellate Symbiodinium. The time is clearly overdue for an effort of this type for the dinoflagellates, which are certainly one of the more important groups of unicellular organisms. As you well know, a major obstacle to the sequencing of a dinoflagellate genome is its size. Your choice of Symbiodinium is an appropriate one, in this regard, since it has a relatively small genome size compared to other dinoflagellates, yet still maintains many of the physiological and ecological characteristics that are shared by others within that class. As you note in your proposal, there are a number of EST and related projects that will benefit directly from your efforts. In my own laboratory, and in the laboratory of several colleagues, we are generating information on gene expression in dinoflagellates, but without a full genome sequence, we all face a difficult task in trying to ascertain the true nature and affinity of the sequences we have been obtaining. As you know, I work almost exclusively on toxic and harmful dinoflagellates, and this is a field that will benefit greatly from the widespread availability of the Symbiodinium genome. In particular, there are significant public health implications to the information you will generate. Symbiodinium produces polyether compounds similar to many of the neurotoxins produced by HAB dinoflagellates, which are responsible for human poisonings, as well as mass mortalities of fish, seabirds, and many marine mammals and animals. A major goal in current HAB research is the identification and characterization of genes involved in toxin production, and this important work will be greatly facilitated by your project. In the long run, one can envision better methods for detecting toxic organisms and their toxins as a result of the genetic information that will be provided. The public health benefits are thus significant. I am also excited about the possibility that other important physiological and ecological processes will be more amenable to study once the Symbiodinium genome is sequenced. For example, we are very interested in life history transformations within the dinoflagellates, such as the formation of gametes and the production of resting cysts. Here again, we should be able to make great strides towards identifying the relevant genes, using your database as the framework against which to interpret our own data. Thank you for taking on the responsibility of proposing this effort, which will provide benefits to all of those who study dinoflagellates and related organisms. I know that the community of those who study toxic and harmful dinoflagellates is supportive of your efforts, and wishes you success in this important activity. - DA 3.4 Kathleen Rein, Director - Adv. Res. Coop. in Environ. Health, Florida International University I am writing to express my enthusiasm and strongest support for your proposed project to sequence the Symbiodinium genome. I am interested in understanding the biosynthesis of polyketide toxins by dinoflagellates. These toxins can accumulate in fish and shellfish and are responsible for a number of human illnesses resulting from the consumption of tainted seafood. While for the most part polyketides derived from dinoflagellates are constructed like all polyketides, from smaller carboxylic acid units, there are some anomalies in their biogenesis that have been observed only in dinoflagellate-derived polyketides. While the zooxanthellatoxins from Symbiodinium spp. have not been associated with human poisoning events, the identification of any polyketide pathway from a dinoflagellate could provide tremendous insights into the construction of these unusual carbon skeletons. We have screened approximately 30 strains of Symbiodinium for polyketide synthase Symbiodinum_WPl+Appendix_v2.doc 26
(PKS) gene expression and have found expression of these genes in roughly on third of our strains. The identification of a toxin biosynthetic gene or pathway could open a number of avenues of investigation relating to HAB toxins including: (i) the harnessing of the unique biosynthetic capability of dinoflagellates for developing combinatorial biosynthetic technologies. (ii) The development of toxin gene expression assays which could result in more accurate prediction of when fisheries will be toxic (or unusable) or (iii) a better understanding the environmental factors that control toxin production. I frequently search the genetic databases for any new genes from dinoflagellates and, probably like many of my colleagues, am usually disappointed by the scarcity of dinoflagellate genes. The information that will be made available through this project, such as the identification of regulatory elements, and the techniques that will be developed, such as transformation protocols, will surely be the catalyst that drives research into dinoflagellate genetics forward. - KR 3.5 Linda Medlin, Allan Cembella, Uwe John & Klaus Valentin, Wegener Institute, Germany The Molecular Biology and Chemical Ecology units of the Alfred Wegener Institute for Polar and Marine Research strongly support the sequencing the Symbiodinium genome, the first dinoflagellate sequence to be done. Dinoflagellates are an enigmatic group within the alveolates with respect to their evolutionary origin, their life cycle, metabolism, and genomic organization. Among the dinoflagellates there are many species that can form Harmful Algal Blooms (HAB) and/or potent toxins. The genetic basis of neither bloom formation nor toxin production is well understood and for this reason we use an EST approach to identify genes involved in these processes. Our efforts to identify such genes in Alexandrium ostenfeldii and Alexandrium minutum, and to isolate full-length clones will strongly benefit from the planned genome project. Our preliminary data show that only a small proportion (9%) of dinoflagellate ESTs could be identified. In other organisms (plants, animals, fungi, diatoms) this percentage is in the range of 40-60%. Dinoflagellates therefore are a substantial reservoir of yet unknown genes with potentially interesting functions. Knowing a complete genome sequence will make available full-length sequences for many novel genes, comparison to EST data will ensure their functionality. - LM, AC, UJ & KV 3.6 Frances Van Dolah, National Oceanographic & Atmospheric Administration I am writing in support of the proposed sequencing of Symbiodinium as a model dinoflagellate. My research focuses on toxic dinoflagellates that have adverse effects on human health and the health of coastal ecosystems. As you point out in the white paper, many dinoflagellates elaborate potent neurotoxins that when present in high concentrations or “blooms” can result in extensive fish and shellfish kills, marine mammal deaths, and human illness and death associated with the consumption of toxin-contaminated seafood. The frequency of occurrence of such harmful algal blooms has increased on a global scale over the past quarter century. Thus, understanding the mechanisms that regulate their growth and toxicity is important to developing control and mitigation strategies. In the past decade we have gained significant insight into the ecology and oceanography of toxic dinoflagellates through the multi-agency sponsored ECOHAB Program (Ecology and Oceanography of Harmful Algal Blooms). However, our knowledge of these primitive eukaryotes at the cellular and molecular level remains rudimentary. My research into cellular level controls of bloom formation currently focuses on the Florida red tide, Karenia brevis. Because no molecular information was available on this organism, we developed a cDNA library from which we currently have ~8000 ESTs. This approach has facilitated the identification of cell cycle regulatory genes, stress genes, and signal transduction pathway components whose expression is currently under study. We are in the process of developing microarrays from this library with which to characterize global gene expression patterns in K. brevis. We anticipate that this functional genomic approach will yield significant insight into the coordinate regulation of genes and identification of cellular pathways responding to environmental cues that trigger the formation and demise of toxic blooms. However, the structure of the dinoflagellate Symbiodinum_WPl+Appendix_v2.doc 27 genome remains unexplored and thus understanding regulatory elements will remain incomplete. The sequencing of a dinoflagellate genome is beyond the capacity of an individual research laboratory because of their large sizes (K. brevis genome is ~1011 bp); it thus requires the investment of a larger genome program. Although I have no particular bias with regards to which model species is the best candidate, I do believe Symbiodinium is a good choice due to its comparatively small genome and its relevance to real environmental issues such as coral bleaching that need to be addressed immediately. I anticipate that the sequences and insight into the genome structure of Symbiodinium resulting from this project will be directly relevant to other dinoflagellates, and thus this project will be a valuable resource for a community of researchers that currently has no such resource. 3.7 Angela Douglas, University of York, UK Your proposal to sequence the Symbiodinium genome has my strongest support. The immediate basis for my support is as a research scientist investigating the basis of the symbiosis between Symbiodinium-coral symbiosis and the processes underlying the collapse of the symbiosis during coral bleaching events. At present, the research field is dominated by whole- organism physiological approaches, with extensive datasets spanning the last 30-40 years on the cellular organization of the symbiosis, patterns of inorganic carbon assimilation and nutrient flux, and coral calcification. With few exceptions, our understanding of these important issues lacks any molecular underpinning. The genome sequence of Symbiodinium is the one vital tool needed to resolve fundamental problems and to meet new challenges. This can be illustrated by an immediate example of a key upcoming challenge: to generate internationally-agreed protocols underpinned by an objective and rational scientific understanding to monitor and manage the impacts of global climate change on the Symbiodinium-coral symbiosis. Such understanding can only be achieved by post-genomic methods, i.e. it is predicated on access to the complete genome sequence of Symbiodinium. The significance of the Symbiodinium genome can be considered from a different perspective. The genomes of several symbiotic microorganisms (e.g. Buchnera, Rhizobium) have recently been sequenced, and research on these systems has been transformed. For Symbiodinium, the prize of the complete genome sequence is particularly great because of the global environmental importance of the Symbiodinium-based symbioses and the limited prior information on the molecular biology and genomics of dinoflagellates. The investment of various groups into constructing EST libraries is an indication of the importance the research community attaches to gaining large-scale sequence data– and of the research “hunger” of the community for the complete genome sequence. - AD Section 4: Letters illustrating the impact of the Symbiodinium genome on dinoflagellate biology Knowledge of basic biological processes, in particular the phylogeny and early events in evolution of eukaryotes, can benefit from the genomic study of dinoflagellates, as shown in the following letters received. Each of these letters contains plans for experiments that will utilize the Symbiodinium genomic sequence that the project will produce. Two full letters are presented followed by excerpts of the remaining letters, the latter grouped according to molecular biological category of experiment. 4.1 J. Woodland Hastings, Member, National Academy of Sciences USA My laboratory has been active in the cloning and identification of genes of bioluminescent dinoflagellates in relation to their luminescence but also with respect to their regulation by their circadian clock. In pursuing these studies we have prepared EST microarrays of about 3,500 genes from the dinoflagellate Pyrocystis lunula and used these to construct microarray chips for determination of circadian differences in transcript levels. We have also discovered a novel type of nucleotide conservation for which the basis remains to be elucidated. Your proposal to sequence the complete genome of Symbiodinium is ambitious, important and very reasonable. I endorse and strongly support the project; the data obtained should be of great value to me, as well as to others working on dinoflagellates. I will be prepared to be of assistance wherever possible and to participate in the annotation of the Symbiodinium genome. Symbiodinum_WPl+Appendix_v2.doc 28
4.2 Excerpts of letters from labs planning to utilize Symbiodinium sequence, grouped under molecular biological categories 4.2.1 Organellar and nuclear integration, intracellular gene transfer, horizontal gene transfer Debashish Bhattacharya, Carver Centre for Comparative Genomics, University of Iowa We study the phylogeny and evolution of dinoflagellates, in particular their plastids, and will benefit greatly from having access to the full genome sequence of a peridinin dinoflagellate. In this regard, we have a funded EST sequencing project (NSF Microbial Genome Sequencing Program) in which we are generating 10,000 unique ESTs for the toxic dinoflagellate Alexandrium tamarense. Presently we have a unigene set of 6,800 sequences. We have found evidence for rampant horizontal gene transfer of photosynthetic genes in this taxon. We plan to reapply to NSF to do limited sequencing of the Alexandrium genome to understand the process of foreign gene incorporation. However this genome is far too big for complete sequencing. Your proposed project should therefore be of fundamental importance for us to understand in more detail the dynamics of dinoflagellate genome evolution. Chris Howe, University of Cambridge, UK We have been working for some time on the dinoflagellate plastid genome, with its bizarre organization into minicircles carrying separate genes (or gene pairs or no genes at all) and lacking most of the conventional chloroplast gene complement. We need to know whether the rest of the genes are in the nucleus, and how they are organized. Are there blocks of plastid-derived genes together, for example? Is there any evidence of minicircle sequences being present in the nuclear genome as well as in the plastid? (That might give some indication of how genes are moved from one organelle to another and whether the similarity to bacterial integrons is significant.) The EST projects currently underway will not provide answers to these questions – genome sequence data are essential. Uwe Maier & Stefan Zauner, Philipps University Marburg, Germany We have started to characterize the plastid genome of dinoflagellates. The data set is incomplete and only two hands full of genes are known from dinoflagellates. As the genes seem to be genetically compartmentalized on different groups of molecules, your data set will be extremely useful for the characterization of the nucleus-encoded genes. Mike Gray, Dalhousie University, Nova Scotia, Canada The Genome Canada-funded Protist EST Program that I head as PI is currently sequencing a Heterocapsa triquetra cDNA library, so a complete dinoflagellate genome sequence will be invaluable in helping us to annotate these and other dinoflagellate ESTs that we obtain. Conversely, the annotated dinoflagellate ESTs that the Protist EST Program generates should substantially aid in the annotation of the Symbiodinium genome. Another reason for my enthusiasm for your project is that our studies to date of dinoflagellate mitochondrial DNA suggest that the dinoflagellate mitochondrial genome, like that of apicomplexa, encodes very few genes. Thus, nuclear genome sequence is going to be essential in order to identify those mitochondrial proteins that are encoded in mtDNA in other organisms, as well as other components of the dinoflagellate mitochondrial proteome. Thus, dinoflagellate nuclear genome sequences will supply key comparative data for addressing questions about the evolution of the mitochondrial proteome in different eukaryotic lineages. John Logsdon, Carver Centre for Comparative Genomics, University of Iowa With relatively few genome projects currently aimed at protists, the Symbiodinium genome sequence will deliver considerable insight into the evolution and function of eukaryotic genes and genomes. My lab is interested in using the Symbiodinium genome to look at the potential role of lateral gene transfer in eukaryotic genome evolution; there have been some previous suggestions of this process in dinoflagellates that need genome-based studies to be properly evaluated. 4.2.2 Transformation, chromosome structure/condensation, DNA methylation, viruses in the genome David Miller, Comparative Genomics Centre, James Cook University Qld, Australia Symbiodinum_WPl+Appendix_v2.doc 29
The Symbiodinium genomic sequence would greatly facilitate our work on coral/zooxanthellae interactions and dinoflagellate gene regulation. Because they lack true histones and their DNA is highly modified, dinoflagellates are of special significance to the chromatin scientific community; a genomic sequence for Symbiodinium can therefore potentially contribute a great deal to our understanding of many basic aspects of chromatin evolution. Of particular interest to us are genes that might play roles in chromatin organization, particularly those encoding proteins involved in epigenetic phenomena. David Morse, University of Montreal, Canada We are actively pursuing dinoflagellate transformation using modified versions of the ten Lohuis and Miller protocol, and would welcome the opportunity to collaborate by sharing technology and test constructs. Although our main emphasis will be to understand regulation of protein synthesis and protein targeting mechanisms in vivo, we would also be very interested in a collaborative effort to dissect the different functional regions in the promoters of circadian regulated genes that your project will produce sequence for. We apply a proteomic approach to the biochemistry of the cell, and in the course of our experiments have noticed that dinoflagellates often employ unusual variants of proteins found in universal biochemical pathways. At a practical level, the return to nucleic acid level in order to study the regulation of gene expression would be greatly facilitated by the availability of genomic sequence information. William Wilson, The Marine Biological Association, UK My own interest is in algal virus genome research and I would be intrigued to know if the Symbiodinium genome has virus sequences or even a complete virus genome within it. This in itself would lead to a whole new extremely important research strand that I would love to collaborate with you on. 4.2.3 Dinoflagellate cell division, and gene complementation in yeast Senjie Lin, University of Connecticut The life cycles of dinoflagellates are known to correlate to blooms in which we are extremely interested: the sexual phase being dormant, and the asexual stage being proliferative. We are concerned with characterizing the proliferative phase in detail, and we are excited that the Symbiodinium genome sequence will deliver the full set of cell division, photosynthesis, and metabolism regulatory genes. We have initiated an EST satellite project for three species representing different trophic modes, Pfiesteria piscicida - heterotroph, Karlodinium micrum– mixotroph, and Prorocentrum minimum–photoautotroph (~900 genes sequenced from these three combined), and we have separately characterized several cell division related genes (pcna, cyclins, mapk). Our analysis of some individual genes (and studies by W. Hastings) indicate a high copy number. These results strongly suggest the need to have a genome-wide genetic resource, from a model dinoflagellate with a small genome such as Symbiodinium. Joseph Wong, Hong Kong University of Science and Technology I have been studying the cell cycle control, signal transduction in cell growth & encystment and chromosomal proteins of the dinoflagellates, as well as developing molecular probe for the detection of harmful species. I am interested in cloning genes involved in the control of cell growth and in the regulation of chromosomal morphology of dinoflagellates. While we mostly use the heterotrophic species (Crypthecodinium cohnii for our own research, the sequence data of Symbiodinium spp. would be of great value to the isolation of corresponding homologues. Herve Moreau, Oceanological Observatory Banyuls sur mer, France I will be mainly interested to specifically annotate the Symbiodinium genes that regulate the cell cycle and gene expression. I also propose that in our group we could study the functionality of the consequently identified cell cycle genes by complementation in yeast, as we are currently doing with those of the green alga Ostreococcus tauri (Prasinophyceae). 4.2.4 Symbiosis, cell adhesion, signal transduction, intercellular trafficking Symbiodinum_WPl+Appendix_v2.doc 30
Cheryl Woodley, Nat. Oceano. & Atm. Adm., and Chair-Coral Disease & Health Consortium On behalf of the Coral Disease and Health Consortium (CDHC), I extend my strong support for your proposal to sequence the Symbiodinium genome. One of the recommendations identified by the CDHC in its National Research Plan is to sequence the genome of a symbiotic dinoflagellate. The Consortium, now representing more than 50 national and international partners, views this research as important in helping us understand areas, such as the biology of symbiotic relationships and mechanisms of coral bleaching. The availability of Symbiodinium sequences will also assist my own specific research needs. We have been studying the influence of several triazine compounds on different Symbiodinium clades associated with corals. Our preliminary results indicate that photosynthesis is differentially affected among clades. Access to these sequences will provide a means to probe molecular differences that can assist in developing mechanistic models for these toxicological responses. Our ultimate objectives are to improve technologies currently available to NOAA for coral health assessment and provide resource managers with new management options for coral reefs. Todd La Jeunesse, William Fitt & Gregory Schmidt, University of Georgia We are identifying the genes required for symbiosis of zooxanthellae with their cnidarian hosts. We anticipate that many of these symbiont-specific genes will have correlates with pathogenic systems, as Symbiodinium face the same obstacles of overcoming host defenses as do many intracellular parasites. This knowledge may ultimately reveal the molecular basis of coral bleaching, an often serious pathology akin to an immune response by the host against its mutualistic residents. Furthermore, similar investigations on the molecular cross-talk between host and symbiont should begin to explain host-symbiont specificity patterns we have observed and are attempting to explain with our current work. Virginia Weis & Mauricio Rodriguez-Lanetty, Oregon State University We are interested in the cellular and molecular interactions underlying the symbiosis between host corals and their dinoflagellate symbionts. Insight into these processes is critical to understanding the mechanisms underlying the symbiosis breakdown that causes coral bleaching and resultant reef degradation. Presently, we spend a significant amount of time and effort searching for genes of interest in the maintenance of the partnership, using techniques such as subtractive libraries and RT- PCR. Progress is indeed slow and few others have joined in these types of studies because of the hit- or-miss nature of the effort. Having the Symbiodinium genome in hand would profoundly shift our efforts to understanding the mechanism of candidate genes identified from the genome and would encourage other investigators to launch parallel studies. Such progress would profoundly change the pace of discovery in our field. Ruth Gates, University of Hawaii I am specifically interested in the exquisitely balanced symbiotic interactions between Symbiodinium populations and their scleractinian hosts. As part of this endeavor, my collaborators and I are constructing expression libraries for Symbiodinium to explore the biological impacts of environmental disturbance. The information from your sequencing efforts will provide a critically needed framework to interpret the results of our studies, and make available an alphabet with which to explore biological innovations that are associated with, and perhaps specific to, the symbiotic condition. Tony Larkum and Rosanne Quinnell, University of Sydney We have turned our attention to the phenomenon of coral bleaching and to the triggers that must exist to cause the symbiosome membrane to fuse with the plasma membrane of the coral host, which in effect causes the release of the zooxanthellae. While much is known in general about such signal transduction and membrane fusion systems, very little is known in the coral/zooxanthellae association. We are therefore trying to generate a proteomic map of the symbiosome membrane proteins and the effects of enhanced temperature. Specifically in our case, we would use the genome Symbiodinum_WPl+Appendix_v2.doc 31 to identify genes for carbon transport in photosynthesis and algal derived proteins and second messengers interacting with the symbiosome membrane. Rebecca Gast, Woods Hole Oceanographic Institute One of my research projects/interests is in the genetic communication between host and symbiotic alga in sarcodine symbioses, a system very similar to that of corals. We hypothesize that there are genes in common between all symbiotic algae that may result in symbiotic competence. To begin to identify these genes, we have been working to isolate and identify the genes expressed by the dinoflagellate alga in the symbiotic state using subtractive hybridization methods. Although we have been successful in recovering several symbiotically expressed gene fragments, it has been difficult to recover the full-length sequences due to the very limited amounts of RNA available from our organisms. The availability of the Symbiodinium genome would very useful for my work as it would allow the recovery of complete gene sequences that are held in common between Symbiodinium and the symbiotic dinoflagellates that I work with. This would represent a significant step forward for my research, and would more rapidly facilitate my projects that aim to study gene regulation and the timing of expression. David Yellowlees, James Cook University Qld, Australia It is only since molecular approaches became available that there has been significant progress in understanding the complexity of coral zooxanthellae and clam zooxanthellae symbioses. Our research is focussed on establishing the identity of candidate genes involved in inorganic carbon concentration; and determination of the proteins necessary for the establishment and maintenance of symbiosis in these organisms. The Symbiodinium gene sequence would be the most valuable resource in pursuing these aims. Monica Medina, DOE Joint Genome Institute I have recently been awarded an NSF biocomplexity grant to look at gene expression during the onset of coral-algal symbiosis. This project involves the development of microarrays from the Symbiodinium symbiont of the Caribbean coral Montastraea faveolata. Having access to an annotated Symbiodinium genome will greatly improve our identification and analysis of symbiosis genes. Bill Leggat, University of Queensland My research has focused on the uptake and fixation of inorganic carbon and bleaching caused by global warming. In all of these research areas we have found that it is algae that is driving, or having significant effects, on the response of the holosymbiont. Access to a complete genome database would provide a quantum leap in our ability to understand these processes in Symbiodinium, and therefore marine invertebrate/Symbiodinium symbioses. I would expect that, based on homology with other genes in the database, we would amplify (from the genome) genes involved in inorganic nitrogen fixation, inorganic carbon and stress responses and examine their responses to environmental stress. This work would allow us to make some predictive models on how these symbioses will respond to human driven anthropogenic stresses. It would also allow us to develop simple monitoring tools, based upon the suite of genes described above, that could be used in field based applications that could be used to evaluate the “health” of coral reefs worldwide. John Berges, Queens University, UK & Dan Franklin, University of Queensland, Australia We have worked on the cell death processes of Symbiodinium and its similarities with metazoan programmed cell death. We feel a number of valuable research directions could arise from the proposed sequencing work. We would like to be involved in mining the Symbiodinium genome and will subsequently undertake in-vivo studies to compare gene function with metazoans. This comparison could provide insight into the role of Symbiodinium cell death programs both under bleaching stress, and in regulating Symbiodinium numbers during functional symbiosis with corals. Gary Ostrander, Johns Hopkins University Symbiodinum_WPl+Appendix_v2.doc 32
Coral bleaching is a focus of my laboratory and along these lines we have developed long-term primary coral cell cultures. As these cultures have zooxanthellae present, it became apparent to us that we really needed to understand the requirements of the dinoflagellate in order to keep the coral cells alive. Understanding the molecular and cellular interactions that are critical to the functional integrity of symbiosis would advance our basic understanding of how coral reefs function and what causes this relationship to break down. Carolyn Smith, Australian Institute of Marine Science I would use Symbiodinium genome data to design algal specific PCR primers for a range of candidate gene loci that may be involved in determining Symbiodinium thermal tolerance. I would use these PCR primers to screen for genetic variation at these loci between natural populations of symbionts that differ in their thermal tolerance. Furthermore, I would use this data to design quantitative real time PCR assay’s to monitor changes in gene expression during time course heat stress experiments. Tadashi Maruyama, Japan Marine Science and Technology Centre I am very interested in genome data of Symbiodinium strains. We have been studying symbiotic interaction between marine invertebrates such as Tridacna clams and Symbiodinium, and are interested in characterizing genes which play important roles in the symbiotic interaction between the host and symbiont. Nancy Knowlton, Director, Centre for Marine Biodiversity and Conservation, Scripps Institute of Oceanography The availability of Symbiodinium genome sequences will stimulate entire categories of work (e.g. molecular signalling between corals and their symbionts) that today is dismissed as impractical. Stephen Hall, Director - Australian Institute of Marine Science The Australian Institute of Marine Science (AIMS) would like to offer their full support for the proposed sequencing of a dinoflagellate genome in the genus Symbiodinium. The risk and recovery research team has expressed that they would use sequence data from the Symbiodinium genome to develop high resolution genetic markers to clarify the population structure of algal symbionts.. The bioinnovation and water quality teams are currently developing sensitive bioindicator systems in corals and their symbionts, for detecting poor water quality on reefs. Access to genome sequence data for symbiotic dinoflagellate species would not only facilitate the achievement of the current AIMS research goals, but would enable AIMS to branch out into new areas of molecular research. Andrew Baker, Columbia University I am interested in understanding the molecular basis of variation in thermal sensitivity between members of the genus Symbiodinium and how this translates to mass coral reef bleaching following sustained seawater warming. The growing scientific community working on Symbiodinium recognizes the primitive nature of the techniques currently used to screen for diversity (principally partial nuclear and chloroplast rDNA sequences) and their weakness as indirect proxy measures for the physiological diversity we need to understand. A complete genome for any one of the principal taxa, together with partial genomes for others would be invaluable in this regard and provide a timely boost for field whose conservation implications have real urgency. Current extinction times for reef corals as a result of repetitive mass bleaching are estimated at 20-50 years in some areas. 4.2.5 Karyotyping, speciation, sex and phylogeny Chris Bolch, University of Tasmania, Australia Our understanding of the population genetics of Gymnodinium spp. and Alexandrium spp. is limited by the expense/time needed to developing suitably polymmorphic markers. Genome sequence will allow us to target and design potentially variable regions by direct database search and rapidly develop PCR methods for intergenic single nucleotide polymorphism markers (SNiPs) to examine population differentiation and gene flow. I plan to isolate and clone differentially expressed genes from different lifecycle stages of the dinoflagellate Gymnodinium catenatum. We hope to able to examine factors that initiate the sexual life cycle in both the laboratory and the field. This objective will be aided by Symbiodinum_WPl+Appendix_v2.doc 33 the annotated Symbiodinium genome sequence, which will provide a framework to rapidly assess position and function of unknown ORFs and the existence of up or downstream regulatory genes. Marina Montresor, Gabriele Procaccini, Wiebe Kooistra & Adriana Zingone, Zoological Station, Italy Here we mention a few aspects for which the dinoflagellate genome sequencing will be of interest for our future research activities: 1. Identification of new sequence stretches to be used as markers for species definition (this is a ‘hot’ issue for endosymbiontic Symbiodiniums, but also for other free-living closely related taxa). 2. Helping in the definition of the evolutionary relationships among dinoflagellates. Moreover, comparative genomic studies carried out with the genome of organisms belonging to other phylogenetic lineages will elucidate the evolutionary history of marine eukaryotes. 3. Allowing identification of genes involved in the transition among different life stages (free living cells, resting cysts etc.) in the dinoflagellate life cycle. Xavier Pochon and Jan Pawlowski, University of Geneva, Italy We are currently working and have been collaborating in studies involving phylogenetic analyses of various armoured and unarmoured dinoflagellates that cause Harmful Algal Blooms, and studies on the diversity and the specificity of the genus Symbiodinium amongst soritid foraminiferans. Molecular phylogeny of Symbiodinium spp. is presently almost exclusively based on the genes encoding ribosomal RNA. There is an urgent need for investigating other nuclear genes. Practically, the availability of Symbiodinium genome data would make our research much more efficient in terms of time and money, because we could easily find new molecular markers without having to screen libraries with degenerate primers. We will certainly be involved in annotating the genome and also in helping to run a common website (e.g. Roos’s) to allow full and easy community access to the data. Dee Carter, Katherine Ferguson, Michael Stat, University of Sydney, Australia Work in our lab centers on the population genetics of zooxanthellae on the Great Barrier Reef. We are interested in the issues of host specificity, adaptive bleaching, speciation and sexuality. We intend to develop PFGE for Symbiodinium and probe the electrophoretic karyotypes with single copy genes to examine chromosome number and karyotype stability. The Symbiodinium genome sequence will allow us to develop molecular markers from microsatellites and introns with far greater ease and less expense than is currently possible. Further, with the extension to partially sequence additional clades and subclades, we would gain a better understanding of the speciation process and would be able to develop intra and inter-clade specific markers. 4.2.6 Drug development and toxin biology Russell Kerr & Lory Santiago-Vázquez, Florida Atlantic University We have recently published data that strongly indicates that the biosynthetic machinery for the production of the pseudopterosins, strong anti-inflammatory compounds harvested from the soft coral Pseudopterogorgia elisabethae, is found in the symbiotic dinoflagellate Symbiodinium and not in the soft coral. Another exciting recent development was the isolation of the diterpene cyclase that is responsible for the first committed step of the pseudopterosin biosynthesis. We were able to get a partial amino acid sequence of this enzyme. Our immediate goals include the cloning of this enzyme into a bacterial host by using degenerate primers designed from the partial amino acid sequence. This project requires screening a large amount of genetic sequences to find those that have homology to other diterpene cyclases from plants and fungi. Our ultimate goal is to genetically engineer bacterial cells to produce pseudopterosins or intermediates of the pathway that are present in limited amounts. It has been established that the production of pseudopterosins can be greatly enhanced by incubating dinoflagellate cells with methyl jasmonate. A second related project uses differential display RT-PCR to look for differentially displayed genes between induced and non- induced populations of Symbiodinium. This project will pinpoint other genes that are potentially involved in the pseudopterosin biosynthetic pathway and also requires screening a large amount of genetic sequences. To have a complete database of the Symbiodinium genome available to our group Symbiodinum_WPl+Appendix_v2.doc 34 would enormously increase our ability to find genetic sequences related to natural product biosynthesis genes. Brett Baillie, Australian National University I am interested in the basis for host-specificity within marine invertebrate associations with Symbiodinium. Recently I have also become interested in the similarities between Symbiodinium and apicomplexans such as malaria: both are alveolates that form intracellular associations with animals. To this end we have performed preliminary tests of the sensitivity of Symbiodinium to different anti-malarial drugs. Our initial data (as-yet unpublished) indicates that the one Symbiodinium strain tested appears to be sensitive to the anti-malarials used in the study. Genomic information should form a basis for studies of the mechanisms by which anti-apicomplexan drugs inhibit specific pathways of Symbiodinium metabolism. Chris Bolch, (this author has plans his letter split into two categories, 4.2.6 and 4.2.5) I am currently examining the molecular interactions between ecto-symbiont bacteria that stimulate dinoflagellate growth and toxicity. We plan to isolate and clone genes involved with the establishment and maintenance of this important relationship. As Symbiodinium appears to produce compounds structurally related to many of the major phycotoxins, a genome sequencing project will assist in identifying the genes that synthesise toxins. Dominick Mendola, Principle, CalBioMarineTechnologies, USA We have a modest collection of rare marine symbiotic dinoflagellates in culture (including Symbiodinium species) which we have been investigating for pharmaco-activity. The project is based on previously published works showing that dinoflagellates (both free-living and symbiotic) produce a wide variety of novel and sometimes toxic polyketide compounds (e.g., amphidinioles and zooxanthellatoxins), and that polyketides as a structure in nature are intermediates in the biosynthesis of various antibiotic and antitumor drugs including rifampicin, erythromycin, tetracycline, epothilones and lovastatin, among others. Sequencing of the entire genome of the largely representative dinoflagellate Symbiodinium sp. will greatly aid–and speed CalBioMarine’s discovery and drug development work for new dinoflagellate-derived compounds, in that our present genomic work proceeds only slowly (and at considerable expense) due to its reliance on the use of degenerate primers, positional cloning and library screening CalBioMarine has gladly contributed to the accompanying whitepaper through permission to print its in-kind experimental findings (polyketide synthase RT-PCR data), which we have obtained for Symbiodinium investigated in our drug discovery project.