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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
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http://www.elsevier.com/copyright
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Protist, Vol. 161, 197–211, April 2010http://www.elsevier.de/protisPublished online date 30 December 2009
ORIGINAL PAPER
Phylogenetic Affiliations of MesopelagicAcantharia and Acantharian-like Environmental18S rRNA Genes off the Southern California Coast
Ilana C. Gilga,1,2, Linda A. Amaral-Zettlerb, Peter D. Countwaya, Stefanie Moorthic,Astrid Schnetzera, and David A. Carona
aDepartment of Biological Sciences, University of Southern California, 3616 Trousdale Pkwy AHF 301,Los Angeles, CA 90089-0371, USA
bThe Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine BiologicalLaboratory, 7 MBL Street, Woods Hole, MA 02543, USA
cCarl-von-Ossietzky Universitat Oldenburg, ICBM-Terramare, Schleusenstr. 1, D-26382 Wilhelmshaven,Germany
Submitted March 20, 2009; Accepted September 19, 2009Monitoring Editor: David Moreira
Incomplete knowledge of acantharian life cycles has hampered their study and limited ourunderstanding of their role in the vertical flux of carbon and strontium. Molecular tools can helpidentify enigmatic life stages and offer insights into aspects of acantharian biology and evolution. Weinferred the phylogenetic position of acantharian sequences from shallow water, as well asacantharian-like clone sequences from 500 and 880 m in the San Pedro Channel, California. Theanalyses included validated acantharian and polycystine sequences from public databases withenvironmental clone sequences related to acantharia and used Bayesian inference methods. Ouranalysis demonstrated strong support for two branches of unidentified organisms that are closelyrelated to, but possibly distinct from the Acantharea. We also found evidence of acanthariansequences from mesopelagic environments branching within the chaunacanthid clade, although themorphology of these organisms is presently unknown. HRP-conjugated probes were developed totarget Acantharea and phylotypes from Unidentified Clade 1 using Catalyzed Reporter DepositionFluorescence In Situ Hybridization (CARD-FISH) on samples collected at 500 m. Our CARD-FISHexperiments targeting phylotypes from an unidentified clade offer preliminary glimpses into themorphology of these protists, while a morphology for the aphotic acantharian lineages remainsunknown at this time.& 2009 Elsevier GmbH. All rights reserved.
Key words: 18S rRNA phylogeny; Acantharea; CARD-FISH; deep-sea protist; novel protistan lineage;sarcodine.
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1Corresponding author; fax +1 207 633 9661e-mail [email protected] (I.C. Gilg).2Current address: Bigelow Laboratory for Ocean Sciences, 180 McKown Point Rd., West Boothbay Harbor, ME 04575, USA.
& 2009 Elsevier GmbH. All rights reserved.doi:10.1016/j.protis.2009.09.002
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Introduction
Acantharia are amoeboid, mixotrophic protistsand common constituents of the microplanktoncommunity in euphotic oceanic ecosystems. Theyhave traditionally been distinguished from othersarcodine protists such as the polycystine andphaeodarian radiolaria and foraminifera by thecomposition of their exquisite skeletons, whichare comprised of SrSO4 in a Mullerian arrange-ment of 20 (in rare cases, 10) spicules (Fig. 1).Most acantharia possess tens to hundredsof photosynthetic eukaryotic symbionts percell (Michaels 1991; Michaels et al. 1995). Totalprimary production within the cytoplasmicnetwork of these and other sarcodines has beenmeasured at more than four orders of magnitudegreater than that of an equivalent volume ofsurrounding water (Caron et al. 1995). High ratesof primary productivity in these associationsreflect the fact that the microenvironmentof the sarcodine is highly enriched in algalbiomass relative to the surrounding oligotrophicenvironment, and to the observation thatsymbionts are typically at or close to theirmaximal gross growth rates within thesesymbiotic associations (Stabell et al. 2002).Together, these factors suggest that symbiontphotosynthesis can represent a significant portionof open ocean primary production when sarcodineabundances are high.
Acantharia often numerically dominate othersarcodines in open ocean ecosystems(Caron and Swanberg 1990). During ‘‘bloom-like’’conditions (Massera Bottazzi and Andreoli 1981,1982; Zas’ko and Vedernikov 2003), acantharianabundances have been reported at integrateddensities from 1.53 to 5.34�105 m�2. It isestimated that they comprise up to 41% of totalintegrated production during these events(Michaels 1988). These values represent signifi-cant amounts of carbon fixation in the oligotrophiceuphotic oceans and demonstrate the potentialfor acantharia to exert a substantial influence onmarine carbon budgets. Furthermore, acantharianblooms may contribute significantly to export flux(particularly Sr flux) from surface waters giventheir large cell size and dense skeletons (Michaelsand Silver 1993; Michaels et al. 1995).
Acantharia are thought to exert the mostsignificant biological influence on strontium bud-gets in the ocean due to their unique celestiteskeletons (Bernstein et al. 1987). Acantharia arethe only protists known to make their skeletonsentirely from SrSO4, although some radiolarian
swarmer cells produce individual crystals ofSrSO4 (Anderson 1983). Strontium was consid-ered a conservative element in marine environ-ments until slight variations in the Sr/Cl ratios ofthe upper 400 m were detected (Brass andTurekian 1974). Studies that followed implicatedthe crystallization and dissolution of acantharian
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Figure 1. (A) An example of Acanthometra sp. 1 inits vegetative state. (B) The same organism, afterreleasing swarmers. The swarmers are visible assmall dots around the skeleton. Note that the centralskeletal junction, an important feature for taxonomicidentification, is only visible after swarmers havebeen released. Scale bars=50 mm.
198 I.C. Gilg et al.
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celestite as the cause of this strontium flux(Bernstein et al. 1987; Bernstein and Byrne 2004;De Deckker 2004). Seawater is highly under-saturated with respect to strontium, so acanthariamust continuously expend energy to create theunique cellular structures that constitute theirskeletons. Sinking acantharian celestite from bothskeletons and cysts is thought to dissolve by900 m, where stabilization of the Sr/Cl ratios hasbeen observed (Bernstein et al. 1987). Theselective removal of Sr from surface waters, wherevegetative acantharia are most abundant, alsoaccounts for the variability of Sr/Ca ratios in theeuphotic zone (De Deckker 2004) and helpsinterpret anomalies in historical sea-surface datasets constructed from Sr/Ca ratios from corals.Acantharia have also been implicated in otherelemental cycling, most notably barium, whichcan comprise up to 0.4% of the skeleton. Theseconcentrations are ten times greater than that ofseawater (Bernstein et al. 1999) and may be thesource of mysteriously abundant strontium-richbarite particles in the deep ocean (Bernstein andByrne 2004).
Despite their important biogeochemical influ-ences in the ocean, relatively little is known aboutacantharian biology or ecology. Study of theirbiology has been hindered by the fact that they,like most large planktonic sarcodine protists,cannot be maintained in culture through succes-sive generations or even maintained very longafter capture. Although acantharia have not beencultured in the laboratory, formation and release of‘swarmer’ cells from freshly-collected organismshas been observed. This process involves thereorganization of the multinucleated vegetativecytoplasm or cyst into tens of thousands offlagellated, mononuclear cells (Caron and Swan-berg 1990). It has been hypothesized that certainacantharia sink before swarmer production occursin their natural environment, either by formingcysts or contracting their ectoplasm throughmicrofibrillar myonemes (Febvre and Febvre-Che-valier 1978). However, the next stage in theacantharian life cycle is entirely unknown. Knowl-edge of the complete life cycle could be especiallyuseful for more accurate estimates of nutrient fluxmodels, as current models consider only theexport of carbon from sinking surface-dwellingacantharia out of the euphotic zone in the openocean (Michaels 1988; Michaels and Silver 1993).
Environmental clone libraries have revealed thepresence of acantharian-like 18S rRNA genesfrom depths where acantharia have rarely, if ever,been observed (Alexander et al. 2009; Edgcomb
et al. 2002; Lopez-Garcia et al. 2001; Lovejoy et al.2006; Not et al. 2007). Acantharian-like clonesfrom deep-sea and mesopelagic environmentalclone libraries have been observed in the Atlantic(Countway et al. 2007) and in the San PedroChannel off the coast of California (Countwayet al. submitted; Schnetzer et al. in preparation)respectively. Countway et al. (2007) reportedsignificant percentages (up to 17%) of the totalprotistan phylotypes from 2500 m in the SargassoSea belonging to acantharia or acantharian-likeorganisms. Acantharian-like sequences have alsobeen reported in clone libraries from the SargassoSea (Moon-van der Staay et al. 2001; Not et al.2007) from 2000 m at the Antarctic polar front(Lopez-Garcia et al. 2001), sediment collectedfrom the mid-Atlantic ridge (Lopez-Garcia et al.2003), sediment in the southern Guaymas ventfield (Edgcomb et al. 2002), aphotic Arctic waters(Lovejoy et al. 2006), the anoxic Caricao basin(Stoeck et al. 2003), and from an anoxic hypersa-line basin (Alexander et al. 2009). This is strikingbecause, to our knowledge, acantharia have notbeen observed at such depths based on micro-scopical analyses. Heavily skeletonized formssuch as reproductive cysts have been observedas deep as 900 m (Bernstein et al. 1987) butdissolution of the skeleton is presumed to occurbelow this depth and the fate of the cells isunknown. The abundance of acantharian-likesequences at depths greater than 900 m suggeststhat there may be uncharacterized mesopelagic ordeep-sea life cycle stages that are presentlyunrecognizable by morphology alone.
Molecular diversity studies that have identifiedpossible acantharia often have not presentedcomplete sequences of these phylotypes andsome have not performed the phylogeneticanalyses necessary for conclusive identifica-tion. There are also conflicting interpretationsof the phylogenetic relationships between theAcantharea and the Polycystinea (Amaral Zettleret al. 1997; Danelian and Moreira 2004;Kunitomo et al. 2006; Lopez-Garcia et al. 2001;Nikolaev et al. 2004; Oka et al. 2005; Pawlowskiand Burki 2009; Takahashi et al. 2004). Histori-cally, the classes Acantharea, Phaeodarea andPolycystinea were merged into the superclass‘‘Radiolaria’’ based on the presence of a centralcapsule (Haeckel 1887). The Acantharea werelater removed from Haeckel’s Radiolaria based ondifferences in central capsule morphology andskeletal composition (Schewiakoff 1926). Defini-tions of groups comprising the Radiolaria havevaried in the literature and at times have included
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199Discovery of Acantharian-like Protists
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the Polycystinea and Acantharea, Polycystineaand Phaeodarea, and more recently the Polycys-tinea alone. Our goal was to clarify phylogeneticrelationships among acantharia and acantharian-like phylotypes and to investigate the possibility ofdeep-sea stages in the life cycle of acantharia. Wereport here phylogenetic analyses of 18S rRNAgene sequences from representatives of thePolycystinea and Acantharea along with new full-length validated acantharian and acantharian-likesequences from the San Pedro Channel, Califor-nia, in order to reveal more accurate phylogeneticassociations among these species, and to exam-ine the identity of sequences obtained from deepwater in the basin.
Results
Bayesian analysis (BA) of 18S rRNA genes stronglysupported the monophyly of the Acantharea(Fig. 2). The analysis also supported the sharedancestry of solitary skeleton-bearing spumellaridPolycystinea and Taxopodida initially observed byNikolaev et al. (2004). However, unlike previouslyreported phylogenies, the relationship of theAcantharea to other groups of Radiolaria waspoorly resolved in our analysis. Our analysissupported the polyphyly of the Polycystinea withthe skeleton-bearing spumellarian and taxopodidlineages branching away from the Nassellarida andcolonial skeleton-bearing and non-skeletalpolycystines as previously reported (Kunitomoet al. 2006; Nikolaev et al. 2004).
Three San Pedro clones from 880 m (MO010.880.00150, MO010.880.00119 and MO010.880.00133) and four from 500 m (MO010.500.00049,MO010.500.00040, MO010.500.00038 and MO010.500.000116) fell within the core acantharian clade inour analyses (Fig. 2). All but MO010.880.00119 wereclosely related to Chaunacanthid 6200, a Stauracon-like acantharian isolated from the surface waters ofthe San Pedro Channel. Although we were unable tomake a definitive identification of this organism, itmost resembled the genus Stauracon and possessedthe grape seed-shaped base of radial spines that isthe defining characteristic for all Chaunacanthida.
Bayesian inference indicated strong support forthe formation of two lineages basal to identifiedacantharia, characterized in our analysis as Uni-dentified Clade 1 (UC1) and Unidentified Clade 2(UC2) (Fig. 2, PP=0.89 and 1.00 respectively).These clades support the findings of Not et al.(2007) whose novel groups RAD-1 and RAD-IIroughly correspond to UC1 and UC2 respectively.Three clones from 500 m in the present study(MO010.500.00307, MO010.500.00043 andMO010.500.00014) along with 9 Sargasso Seaclones (see Table 1, Not et al. 2007) comprisedUC1. UC2 contained one 880 m clone from thepresent study (MO010.880.00323), a 500 mSargasso Sea clone (SSRPB51, Not et al. 2007),an Antarctic deep-sea environmental clone(DH145-EKD17, Lopez-Garcia et al. 2001), a75 m clone from the Equatorial Pacific Ocean(OLI11032, Moon-van der Staay et al. 2001) and aclone from a hypersaline, anoxic basin (UI13C08,Alexander et al. 2009).
Three more clones from 880 m in the presentstudy and clone AT4-94 (Lopez-Garcia et al. 2003)branched basal to known spumellarian polycys-tines. One 880 m sequence branched among aclade encompassing the only known member ofthe Taxopodida, Sticholonche zanclea, and aselect group of spumellarian radiolaria.
Cells from the January 2006 sample collected at500 m successfully hybridized to the UC1 CARD-FISH probe (Fig. 3). Despite our detection ofacantharian 18S rRNA gene sequences in clonelibraries, the acantharian-specific probe a497 didnot detect these phylotypes on filters collected at500 m from May 2005 through January 2006.
Discussion
Phylogeny of Haeckel’s Radiolaria
Hypotheses on the phylogeny of ‘‘Haeckel’sRadiolaria’’ have changed repeatedly in recentyears through the application of DNA sequenceinformation. Amaral Zettler et al. (1997) were thefirst to infer a partial molecular phylogeny ofrepresentatives of these species based on 18S
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Figure 2. Phylogenetic relationships among 134 eukaryotic 18S rRNA gene sequences inferred from aBayesian analysis with 5,000,000 generations. 1115 positions were included in the analysis. Posteriorprobabilities 40.5 are displayed at the nodes. Sequences from this study are presented in boldface, Kindicates environmental sequences from this study collected at 500 m, and m indicates sequences from thesame location collected at 880 m. nClades were collapsed into single groups for this figure when sequencesimilarity resulted in poor internal branch resolution. Specific sequences falling within these clades may beviewed in Table 1.
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MO010.880.00119 ▲NW614.49C3_E029
0.93
C3_E013Chaunacanthid 6200 (Stauracon-like)
MO010.500.00116 ●MO010.500.00040 ●
MO010.500.00049 ●MO010.880.00133 ▲
MO010.880.00150 ▲T41D11
SCM27C24SCM15C12
0.90
MO010.500.00038 ●
0.56
Amphiacon denticulatusSymphyacanthida sp. 211Chaunacanthid 217Chaunacanthid 218
0.70
Acantharian 6201
0.71
Acanthometra sp. 1 6203Acanthometra sp. 1 6202Dorataspis sp. 813Acanthometra sp. 1 7201
Acanthometra sp. 2 7202Acantharian 6204Acantharian 6205Acanthometra sp.Haliommatidium sp.
0.83
Arthracanthid 206OLI11015
OLI110160.99
Amphibelone anomala 602
0.74
Amphibelone anomala 728Hexaconus sp. 09BAcanthometra sp. 3 813Sargasso Sea Env. Clones I*MO010.500.00307 ●
MO010.500.00043 ●0.59
MO010.500.00014 ●
0.98
0.89
MO010.880.00323 ▲DH147_EKD17
OLI11032SSRPB51
UI13C08
0.99
SSRPB68
Colonial Radiolaria*Thalassicolla nucleata
Thalassicolla pellucida1.00
SSRPB73
0.50
Pterocorys zancleusPterocorys zancleus 8030
Pterocanium trilobumArtostrobus sp. 2014
NW617.13 18S
0.73
DH145_HA2Lithomelissa sp. 2003
Pseudocubus obeliscus
0.98
0.62
Eucyrtidium hexagonatumEucyrtidium acuminatum 2051
Eucyrtidium hexastichum
0.71
Sargasso Sea Env. Clones II*C1_E045
0.88
Dicranastrum furcatumTriastrum aurivilliiDictyocorne profundaDictyocoryne truncatum
0.97
Euchitonia elegansSpongaster tetras
0.97
Spongodiscus biconcavus 8016
0.99
Didymocyrtis tetrathalamus
0.97
Styptosphaera sp. 2022
0.73
Tetrapyle octacanthaTetrapyle sp. 2098
Spongopyle osculosaStylodictya sp. 8037
0.89
0.91
0.99MO010.880.00197 ▲
MO010.880.00290 ▲MO010.880.00003 ▲MO010.880.00263 ▲
0.75
AT4_94
0.56
CS_E043Larcopyle butschlii 7039Spumellarian Radiolarian 7017
SCM38C17OLI011-75m.45
0.56
DH145_KW16SSRPC18SSRPD81
0.84
SSRPB44SSRPC73SSRPB80
Q2E12N5OLI016-75m.46
NW414.03NW415.02
MO010.880.00083 ▲SSRPB27
0.89
0.69
Sticholonche zanclea
0.81
SSRPB82LC22_5EP_23
0.87
0.96
0.93
0.63
Aulosphaera trigonopaPaulinella chromatophora
Massisteria marina
0.55
Clathrulina elegans
0.52
Achlya bisexualisFucus distichus
Schizochytrium minutum
0.10
1.00
1.001.00
1.00
1.001.00
1.001.00
Arthracanthida AC
AN
THA
REA
Unidentified Clade 1
Unidentified Clade 2
POLY
CYS
TIN
EA
Nassellarida
Spongodiscidae
Pyloniidae
Spongodiscidae
Spumellarida
Spumellarida
Taxopodida
CoccodiscidaeEthmosphaeridae
Pterocorythidae
Theoperidae
Plagoniidae
Theoperidae
ChaunacanthidaSymphacanthida
Chaunacanthida
Chaunacanthida
ArthracanthidaSymphacanthida
Gigartacontidae
Sphaenacanthida
Sphaenacanthida
Sphaenacanthida
SymphacanthidaArthracanthida
Amphilithiidae
1.00
1.001.00
1.00
1.001.00
1.00
1.001.00
1.00
1.00
1.001.00
1.00 1.00
1.00
1.001.00
1.001.00
1.00
1.001.00 1.00
1.001.00
1.00
1.00
1.00
1.00
1.00
1.001.00
1.00
1.001.00
1.00
CERCOZOA
STRAMENOPILES
UC2a
UC2b
Spumellarida
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rRNA genes. They concluded that the Acanthareaand Polycystinea formed distinct lineages, and didnot comprise a monophyletic assemblage. Othermolecular phylogenies restored the monophyleticassociation of the Polycystinea with Acantharea(Danelian and Moreira 2004; Lopez-Garcia et al.2002). With the addition of the taxopodid Sticho-lonche zanclea, the monophyly of Acantharea andspumellarian Polycystinea was again supported(Nikolaev et al. 2004). Subsequent molecularphylogenetic studies (Takahashi et al. 2004)revealed that polycystines are comprised of atleast two paraphyletic lineages: 1) colonial poly-cystines with and without skeletons and solitarypolycystines lacking skeletons and 2) shell-bear-ing solitary polycystine spumellarians. With the
addition of spongodiscid spumellarians and nas-sellarian sequences (Kunitomo et al. 2006; Yuasaet al. 2005), the monophyletic relationshipbetween the Acantharea and collodarian Polycys-tinea reported by Lopez-Garcia et al. (2002) hasnot been supported.
The addition of environmental sequences in thepresent study has destabilized the overall place-ment of the Acantharea relative to the otherRadiolaria. Unlike previously reported phylogenieswhich analyzed rRNA gene and actin sequences(Nikolaev et al. 2004), rRNA gene and polyubiqui-tin sequences (Pawlowski and Burki 2009) orrRNA genes alone (Kunitomo et al. 2006; Takaha-shi et al. 2004; Yuasa et al. 2005), we did notobserve a monophyletic relationship between the
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Table 1. Sequences within collapsed clades (n) in Figure 2.
Condensed Clade fromFigure 2
Clone Name Accession Number
Sargasso Sea Env. Clones I SSRPC49 EF172929SSRPC83 EF172911SSRPC07 EF172910SSRPC45 EF172909SSRPC15 EF172908SSRPB59 EF172842SSRPB70 EF172841SSRPB05 EF172840SSRPB06 EF172828
Colonial Radiolaria Collozoum pelagicum AF091146Collozoum inerme AY266295Collozoum serpentinum AF018162Collosphaera globularis-huxleyi AF018163Acrosphaera sp. CR6A AF091148Rhaphidozoum acuferum AF091147Sphaerozoum punctatum AF018161Siphonosphaera cyathina AF091145
Sargasso Sea Env. Clones II SSRPC01 EF172890SSRPC12 EF172891SSRPC17 EF172895SSRPC19 EF172905SSRPC22 EF172899SSRPC26 EF172901SSRPC34 EF172903SSRPC42 EF172902SSRPC52 EF172930SSRPC53 EF172914SSRPC59 EF172932SSRPC76 EF172900SSRPC78 EF172896SSRPC85 EF172915SSRPC87 EF172894SSRPD81 EF172984
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Acantharea, the spongodiscid Spumellarida andthe clade bearing Sticholonche zanclea. With theaddition of our new environmental sequencesalong with newly reported radiolarian-relatedsequences, our data may lack the phylogeneticsignal necessary for resolving higher-order taxo-nomic relationships.
Our findings supported the polyphyletic nature ofthe Spumellarida (Kunitomo et al. 2006; Yuasa et al.
2005) and the paraphyly of the Spongodiscidae(Kunitomo et al. 2006), with a spongodiscid Larcopylebutschlii and Spumellarian Radiolarian 7017 branch-ing among the larger clade that included S. zanclea.These results call into question the classification ofthis clade, which is often labeled ‘‘Taxopodida’’ in theliterature (Not et al. 2007; Pawlowski and Burki 2009),especially given that the majority of members in thisclade are unidentified.
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Figure 3. Photomicrographs of acantharia and relatives. CARD-FISH samples were hybridized to HRP-conjugated taxa-specific oligonucleotide probes and visualized with Alexa Fluors 488. All scale bars=10 mm.Each ‘‘a’’ image shows a hybridized cell and each ‘‘b’’ image shows the corresponding DAPI image. (1) Avegetative acantharian (Acanthometra sp. 1) hybridized with acantharian-specific probe a497. All but oneknown species of acantharian is polynucleated in the vegetative stage. Skeletal disassociation was observedbecause Sr was not added to the fixation medium. (2) An acantharian swarmer, targeted with a497. Note thesingle nucleus at this stage. (3) An example image from a positive control, targeted with the universaleukaryotic probe 1209R. This mid-water ciliate was imaged from an environmental sample taken at 500 m inJanuary, 2006. Positive controls revealed a mid-water community of dinoflagellates, ciliates, andnanoflagellates, as well as many other unidentifiable eukaryotes. (4-5) Cells targeted by UC1-899, a probespecific for Unidentified Clade 1. Both images were taken from an environmental sample collected in January2006 from 500 m. (6) Negative control from January 2006 sample.
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Phylogeny of Schewiakoff’s Acantharea
Our analysis indicated that the traditional classi-fication of the Acantharea is not completelyconsistent with their molecular phylogeny, but isapplicable in certain cases. For instance, our treelends support to the traditional classification ofthe Chaunacanthida, which remain a discretelineage in all analyses along with Symphacanthid211. The association of Symphacanthid 211 withthe Chaunacanthida has been reported elsewhere(Oka et al. 2005), so it is possible that Sympha-canthid 211 was originally misidentified. However,the classification of Arthracanthida sensu Sche-wiakoff is not supported by our data, a findingwhich also corroborates the analyses of Oka et al.(2005). The Acantharean orders Arthracanthidaand Symphacanthida appear to be polyphyleticin our analysis and probably require revision.Amphibelone anomala, Amphibelone cultellataand Haliommatidium sp., are traditionally classi-fied as Symphacanthida, an Order characterizedby the basal fusion of spines and a lack ofcapsular membrane (Schewiakoff 1926). In con-trast, Arthracanthida is characterized by discretebut tightly joined radial spines, the base of whichreassemble arrowheads (Schewiakoff 1926).Amaral Zettler and Caron (2000) reported the firstmolecular justification for the placement ofHaliommatidium among the Arthracanthida. Theynoted that Haliommatidium shares morphologicalfeatures with the Arthracanthida, including anoccasionally observed central capsule wall(Febvre et al. 1990) which is an unusual featurefor Symphacanthida sensu Schewiakoff, and theformation of a latticed shell by fusion of apo-physes. Curiously, there are few morphologicalcharacteristics shared by Amphibelone andArthracanthida. One potential synapomorphy isthe lack of cyst formation. Amphilithiidae is theonly family of Symphacanthida that does not formcysts (Cachon and Cachon 1982) and directswarmer formation without encysting is one ofthe defining characteristics of Arthracanthida.
There is little resolution within much of theArthracanthida clade in our analysis, although amore targeted analysis might reveal more insight.Dorataspis, several species of Acanthometra andHaliommatidium branch together (Fig. 2) andcomprise the core of this lineage. However, therewas strong support (PP=1) for a distinct cladecontaining Hexaconus and Acanthometra sp. 3(Fig. 2), indicating that the genus Acanthometra ispolyphyletic. Within the suborder Sphaena-canthina, Hexalaspitidae may need to be
expanded to include some Acanthometra species.At present, there is no molecular justification forthe families Acanthometridae and Dorataspitidaewithin Sphaenacanthida.
Phylogeny of Deep-Sea Acantharia andClose Relatives
We have uncovered full-length 18S rRNA genesequences from the deep-sea environment thatare presently best classified as members of theAcantharea. These clones, MO010.880.00150,MO010.880.00133, MO010.500.00040, MO010.500.00116, MO010.500.00038 and MO010.500.00049, may be chaunacanthids, based ontheir position in the tree (Fig. 2). Environmentalclones C3_E013, C3_E029, and NW614.49 haveoccasionally been identified as acantharia inprevious studies (Not et al. 2007), but the depthto which they branch away from Chaunacanthid6200 in our analysis casts uncertainty onto thatidentification. Not et al. (2007) reported otherclones (First Sargasso Sea group in Table 1) withinthe Acantharean clade among selected topolo-gies. Our data strongly support (PP=0.99) theplacement of these sequences in a clade basal tothe known acantharia which casts doubt on theiridentity.
Acantharia are common in the San PedroChannel year-round. Acanthometra and Doratas-pis species were frequently found among thesurface-dwelling acantharian population at oursampling site. It is therefore possible that ourchaunacanthid-like sequences from aphoticdepths came from sinking cellular debris, cystsless than 200 mm in diameter or swarmers, asChaunacanthids encyst before releasing swar-mers. However, if these organisms are vegetative,their occurrence at these depths would representa newly recognized ecosystem for this taxon. Thedominant species of acantharia at the surface ofthe channel from 2003-2005 (Acanthometra sp. 1,Dorataspis sp. 813 and Chaunacanthid 6200) werenot the same as those detected at depth in June2001. Unfortunately, we do not know the dominantspecies of acantharia in surface waters duringJune 2001, our environmental sampling period,because surface samples for microscopy were nottreated with excess strontium, a necessary addi-tive for the preservation of acantharian skeletons(Beers and Stewart 1970). It is therefore possiblethat our clone sequences were derived fromsinking acantharian cells or cysts. However, wedid not detect the putative chaunacanthid or
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MO010.880.00119 phylotypes among the surfaceclones in our 2001 libraries.
We present further evidence of and strongsupport for novel, undescribed phylogeneticlineages basal to the Acantharea (UnidentifiedClades 1 and 2), which corroborate the findings ofNot et al. (2007). Organisms in Unidentified Clade2 (UC2) are related to Acantharea, but given theirevolutionary distance from the core Acanthareaand depth of branching, it is more likely that theyrepresent a novel group. The members of thisgroup may even be endemic to mesopelagicmarine environments. With the exception ofOLI11032, all clones within this clade wereobtained from mesopelagic to abyssal depths.UC2 phylotypes were not detected among surfacesequences in clone libraries from our sample siteover a two-year period. However, these phylo-types were detected repeatedly in our clonelibraries at depth over the same sampling period.Although there are relatively few representativesfor UC2, our data provide preliminary support forthe formation of 2 subgroups within this clade(UC2a and UC2b) although more sequences ofrelated taxa will be needed to validate thisproposal. It appears that we are only just begin-ning to uncover the diversity of these organisms.
Unidentified Clade 1 (UC1) has a shorter evolu-tionary distance from the core Acantharea than UC2(Fig. 2). Our San Pedro clones branch deeper thanthe Sargasso Sea I clones (Table 1, Not et al. 2007)and may represent more divergent members ofUC1. Whether or not these organisms are acanthariaremains unresolved. One possibility is that this claderepresents the Holacanthida, a class of Acanthareathat currently lacks representation in public genedatabases. The placement of these sequences isconsistent with the current taxonomic classificationof Holacanthida, the most ancestral acantharianorder (Schewiakoff 1926). However, we did notobserve Holacanthida in our net tows during oursampling period. It is also possible that UC1 mayrepresent another group of arthracanthids, given itsposition basal to known Arthracanthida. There are30 described genera within Arthracanthida (Sche-wiakoff 1926), only four of which have representationfrom identified organisms in GenBank. While theidentity of phylotypes in UC1 remains unknown, wetentatively consider these phylotypes novel untilfurther information is available.
All environmental clones in this study wereobtained from 500 and 880 m at our sample sitein an environment that experiences persistenthypoxia. The dissolved oxygen concentrations at500 m at the San Pedro Ocean Time-series site
ranged from 0.14 to 0.78 ml l�1 (Countway et al.submitted; Schnetzer et al. in prep). Values at880 m typically do not exceed 0.22 ml l�1. Inter-estingly, other studies have detected acantharianand acantharian-like sequences in deep anoxicenvironments. T41D11 (Stoeck et al. 2006) andUI13C08 (Alexander et al. 2009) are clones fromtwo separate anoxic basins, while C3_E013 andC3_E029 were extracted from anoxic sediments(Edgcomb et al. 2002). It is unknown if thesesequences came from living organisms or simplyrepresent sinking debris or cysts. However, if theyrepresent active members of the microbial com-munity in these environments, it would suggestthat these species may have the ability to adapt tohypoxic or anoxic conditions.
Fluorescence In Situ Hybridization
Two acantharian-specific regions of the 18S rRNAgene at base pair 497 (probe a497) and base pair899 (probe a899) were initially reported by AmaralZettler et al. (1997). These probe locations remainphylogenetically informative when compared tocurrent acantharian sequences in GenBank. ABLAST analysis at the time of this study revealedthat among the validated sequences possessingthe signature region of a497, all were acantharia.However, we detected occasional polymorphismsin a497 in some known acantharian sequences.One polymorphism was detected at the thirteenthbase of the probe region in Amphibelo-neAB178584, and at the fourth base in Hexaco-nusAB178587 and AmphiaconAB178585 (data notshown). Among all validated and putativeacantharian sequences from our dataset, all butone possessed the ‘a899’ marker. Only C3_E029exhibited a single mutation in a899 (data notshown). All the clones comprising UC2 alsopossessed the region a899 without polymorph-ism. BLAST analysis revealed that only the coreAcantharea and UC2 sequences possessed thea899 sequence. However, phylotypes from UC2demonstrated significant polymorphisms (6bases) of a497. This finding is echoed in thephylogenetic placement of these sequences,which lies between the core Acantharea and theirrelatives, the polycystines. Interestingly, membersof UC1 had two unique and phylogeneticallyinformative polymorphisms in a497 and 1-2unique polymorphisms in a899. There were nosequences in GenBank containing these uniquevariations in a497 and a899 at the time of ouranalysis. It therefore appeared that sequences ata497 and a899 were informative not only for most
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members of the Acantharea, but also for theirunknown relatives in UC1. Because UC2 shareda899 with known acantharia, only a497 was usedto search for known acantharia at depth.UC1-899, a derivative probe of a899 containingthe UC1-specific polymorphisms, was used tosearch for UC1 phylotypes. We did not design aprobe for UC2 phylotypes in this study.
We successfully hybridized phylotypes fromUC1 (Fig. 3) with CARD-FISH (Pernthaler et al.2002) using the unique UC1 probe UC1-899 tobind to SSU rRNA. It is unclear if the hybridizedUC1 phylotype detected in frame 4 of Figure 3represented a single cell or a cluster of cells. Wealso detected solitary cells (or perhaps a fragment)in the same sample (Fig. 3, frames 5a and b).Based on the size and shape of the solitaryhybridized objects and the fact that they havewhat appears to be a single nucleus, it is possiblethat they were swarmers.
It is not a forgone conclusion that the source ofthese phylotypes are metabolically active mem-bers of the community. It is possible that templateDNA for the San Pedro clones came from cysts,fecal pellets or cellular debris from the surface.However, since FISH probes target rRNA, it isunlikely that we would observe hybridization fromacantharian-derived material in fecal pellets ordecaying matter because of the rapid degradationof extracellular RNA. Positive CARD-FISH resultswere never observed with acantharian cysts inpreliminary tests. Theoretically, a positive hybridi-zation should occur only with metabolically activecells. It’s therefore likely that the organisms wedetected are active members of deep protistancommunities.
The acantharian-specific probe (a497) did nothybridize to cells in our slide preparations fromdeep samples, even with increased samplevolumes. It is possible that we missed theseorganisms because they are transient members ofthe protistan assemblage at depth or becausethey are rarely encountered. It is also possible thatthe putative acantharian clones from our librarycame from cysts, fecal pellets other aggregatedparticles such as marine snow containing nuclearremains of acantharia. Although it is possible forthese particles to contain acantharian DNA whichcould be cloned, they would most likely notcontain intact rRNA and would therefore nothybridize to our probe. The nature of theseorganisms at depth remains unresolved andawaits further investigation.
Estimates of total eukaryote abundances at500 m, based on cell counts of CARD-FISH
eukaryotic positive controls from May 2005–January 2006 ranged from 225 to 469 cells l�1.The sample in which UC1 organisms weredetected (January 2006) yielded an abundanceestimate of 8 cells l�1 of the novel phylotype, orapproximately 2-4% of the total eukaryote popu-lation. This was near the limit of detection bystandard methods, so cells in samples fromprevious months may have evaded detectionbecause abundances were below our limit ofdetection.
We conducted an analysis of all eukaryoticclone libraries collected at our San Pedro sam-pling location during 2000-2001 (Countway et al.submitted; Schnetzer et al. in prep). The UC1phylotype was detected among sequences in2001 at 150 m on 27 July and at 150 and 500 mon 29 October. These sequences comprisedapproximately 1% of the total eukaryotic phylo-types from each sample. Core acanthariansequences were also present in San Pedroeukaryotic clone libraries from mesopelagicdepths (150 and 500 m). These sequences aver-aged approximately 4% of the total eukaryoticsequences respectively, although Acantharianphylotypes in one sample (27 July 2001 500 m)comprised over 15% of total eukaryoticsequences. This finding demonstrates that theseorganisms, cysts or decaying cells were at times asignificant fraction of the mesopelagic eukaryoticclone libraries at our study site. It is thereforepossible that aphotic acantharian populations, ifthey are metabolically active members of theprotistan assemblage, undergo ‘boom and bust’cycles, dictated by currently undetermined cues.
Overall, our study indicated the presence of twoprotistan clades with ancestral affinities to theAcantharea, as well as core acanthariansequences, in mesopelagic environments. Thedepth and physical water parameters at whichthese phylotypes were collected indicate thatthere is still much to be discovered aboutacantharian biology and their ecological roles.
Methods
Acantharian sample collection in surface waters:Acantharia were collected in plankton nets (200 mm mesh)from the San Pedro Channel, between Los Angeles and SantaCatalina Island, California, on multiple cruises during 2003 and2004. Live vegetative acantharia were isolated and identifiedto family or genus based on the criteria of Schewiakoff (1926).Conclusive identification was difficult with live cells becausetaxonomically important features of the skeleton were oftenobscured by the cellular matrix (Fig. 1). Cells were characterized
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only as ‘acantharia’ when critical characteristics were notvisible. One organism bore a strong resemblance to the genusStauracon, although it did not quite meet all the criteria for thisdesignation. Therefore, it was labeled ‘Chaunacanthid 6200’,even though we strongly suspect it was a Stauracon. Uponisolation, each cell was rinsed 6 times in fresh 0.2 mm filteredseawater to dilute contaminants, then placed individually into6-well culture plates containing 0.2 mm filtered seawater.Acantharia were maintained at 16 1C in an incubator with a12 h light/dark cycle until they formed swarmers, or for amaximum of one week to allow sufficient time for digestion ofingested prey prior to molecular analyses. Swarmer cells werepreferred over vegetative cells for analysis because symbiontswere consumed prior to formation. Vegetative cells wereamplified without the concentration step described below ifswarmers did not form after 1 week.
Acantharian DNA amplification, cloning and sequen-cing: Live swarmers were concentrated by centrifugation and1 ml concentrate was added to 4 ml Lyse-N-Gos PCR reagent(Pierce Biotechnology, Rockford IL). Samples were lysed in aniCycler or MyCycler automated thermocycler (BioRad, HerculesCA). Polymerase Chain Reaction (PCR) amplification, cloningand partial-length sequencing of 18S rRNA genes wereconducted according to protocols outlined in Countway (2005)and Countway et al. (2007). Briefly, full-length eukaryotic 18SrRNA genes were amplified by PCR with eukaryotic specificprimers EukA (50-AACCTGGTTGATCCTGCCAGT-30) and EukB(50-GATCCTTCTGCAGGTTCACCTAC-30) (Medlin et al. 1988).Pre-mixed PCR reagents (0.5 mM of each primer, 2.5 mM MgCl2,250 mM each dNTP, 300 ng ml�1 BSA, 1X Promega buffer B) and2.5 U of Taq DNA polymerase in 1X buffer B (Promega, MadisonWI) were added directly to each tube in the thermocycler.Amplification took place according to the following thermalprotocol: Initial denaturing at 95 1C for 2 min, followed by 35cycles of: denaturing at 95 1C for 30 sec, 60 1C annealing for30 sec and 72 1C extension for 2 min, with a final extension at72 1C for 10 min. PCR amplicons were purified from gels withZymocleanTM Gel DNA recovery kits (Zymo Research, OrangeCA). Ligation reactions were set up using the TOPO-TA Clonings
kit for sequencing with the PCRs 4-TOPOs cloning vector(Invitrogen, Carlsbad CA) using 4.6 ml of PCR product. Ligationreactions were run with undiluted Invitrogen salt solution (1.2 MNaCl, 0.06 M MgCl2) and purified prior to electroporationbecause this method resulted in substantially higher transforma-tion efficiencies than the manufacturer’s recommendation(Countway 2005). Ligations were purified with DNA Clean andConcentratorTM-5 spin columns (Zymo) and eluted in 12 ml ofsterile water before transformation into One Shot TOP10Electrocompetent E. coli (Invitrogen) with a Gene Pulser Xcell(BioRad). Aliquots of transformed cells were diluted 1:10 withfresh SOC medium and 15 ml of diluted transformants wereplated onto LB agar containing ampicillin (50 mg ml�1) forovernight growth at 37 1C. Colonies were picked the followingday into deep-well culture blocks containing 1.25 ml of TBmedium and ampicillin (50 mg ml�1), covered with AirPoreTM
tape (Qiagen, Valencia CA) and grown for 18-24 hours. PlasmidDNA was extracted using the Wizard SV96TM kit (Promega).Plasmid DNA for sequencing was eluted in 100 ml of sterilewater. Plasmids were stored at �20 1C until ready for DNAsequencing. Sequencing was performed on a CEQ 8000automated DNA sequencer (Beckman Coulter, Fullerton CA).All reads were manually trimmed and edited based on qualitybefore assembly in MacVectorTM v8.0. All sequences were runthrough Bellerophon (Huber et al. 2004) and Chimera-Check(Cole et al. 2003) servers to look for chimeric artifacts. Onechimeric sequence was identified and discarded.
Environmental sample collection and DNA extraction:Environmental samples were collected on 28 June 2001 fromthe San Pedro Channel (33 33 N, 118 24 W) at 880 m and500 m as part of the ongoing San Pedro Ocean Time-series(SPOT) conducted by the University of Southern CaliforniaWrigley Institute for Environmental Studies and an NSF-funded Microbial Observatory (http://www.usc.edu/dept/LAS/biosci/Caron_lab/MO/) led by Drs David Caron and JedFuhrman. Seawater was collected in 10 l Niskin samplingbottles on a CTD rosette (General Oceanics, Miami FL).Triplicate 2 l samples were fractionated successively through200 mm and 80 mm Nitex mesh housed in 47 mm inlinefilter-holders before final filtration onto GF/F filters. Thepre-filtration of samples through Nitex reduced the contribu-tion of metazoa to subsequent DNA extracts. Filters wereplaced into 15 ml falcon tubes pre-loaded with 2 ml lysisbuffer (40 mM EDTA, pH 8; 100 mM Tris, pH 8; 100 mM NaCland 1% SDS) and frozen in liquid N2. Samples were stored at�80 1C in the laboratory until ready for processing. Furtherdetails regarding sample collection, processing, and microbialdiversity at this site are described elsewhere (Countway 2005;Schnetzer et al. in prep).
For community DNA extraction, tubes were thawed at 70 1C,and vortexed with 0.5 mm silica/zirconium beads for 30 secondsto mechanically disrupt cells. This heating and bead-beatingcycle was repeated 3 times to maximize the release of genomicDNA. Lysates were filtered through a sterile 0.2 mm filter toremove the beads and debris into a clean tube to which a CTAB/NaCl solution was added (0.01% CTAB, 0.7 M NaCl, finalconcentration). Nucleic acids were extracted with three phenol:-chloroform:isoamyl alcohol (25:24:1) extractions, and a singlechloroform–isoamyl alcohol (24:1) extraction. The nucleic acidsin the final aqueous phase were precipitated overnight at�20 1Cby adding 1X volume 95% ethanol and 0.1X volume 10.5 Mammonium acetate. The following day, nucleic acids werepelleted by centrifugation (14,000�g) for 30 min at 4 1C,decanted and washed in 70% ethanol during an additional 15-min centrifugation, after which samples were decanted andinverted to dry. Dried DNA pellets were resuspended in 100 ml ofsterile TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) and storedfrozen at �20 1C until further analysis.
18S rDNA PCR amplification, cloning and sequencing wasconducted using the methods described for the acanthariansequences above. Partial sequences were initially obtained ona Beckman CEQ 8000 using the internal primer Euk-570F (50-GTAATTCCAGCTCCAATAGC-30) (Weekers et al. 1994) to readthrough one of the most variable regions of the gene. Thesereads were trimmed and subjected to a BLAST search againstsequences in GenBank in May 2005. Clones with top BLASThits matching acantharia in the database were selected forfull-length sequencing. Archived clones were re-grown, theplasmids were purified and full-length sequencing was carriedout using an ABI 3730XL (Applied Biosystems, Foster City,CA) at the Marine Biological Laboratory W. M. Keck Ecologicaland Evolutionary Genetics Facility. Individual sequence readswere assembled and edited using AlignIR (LI-COR Biotech,Lincoln, NE) software.
Phylogenetic analyses. We obtained sequences from publicdatabases to include representative rhizaria that encompassthe Polycystinea/Taxopodida/Acantharea, as well as certainenvironmental sequences of interest (Table 2). The corerhizarian alignment was obtained from Nikolaev et al. (2004).The Nikolaev alignment along with all acantharian andenvironmental samples were imported into ARB v. 05.05.26(Ludwig et al., 2004) and the alignment was adjusted using acombination of the Fast Aligner feature in ARB along with
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manual refinement. A sequence mask was applied to retainregions of unambiguous alignment and only those positionswere included in subsequent phylogenetic analyses. A dataset including just acantharian, polycystine sequences andselect outgroups (a total of 134 taxa and 1115 positions) wassubjected to Bayesian (BA) inference methods.
We conducted our Bayesian analysis using MrBayes,Version 3.0b4 (Ronquist and Huelsenbeck 2003) under theGTR model of substitution (Lanave et al. 1984; Rodriguez et al.1990; Tavare 1986), considering invariants and a gamma-shaped distribution of the rates of substitution among sites.The chain length for our analyses was 5,000,000 generationswith trees sampled every 100 generations using Markoff ChainMonte Carlo (MCMC) analysis. Chain parameters appeared tobe stationary after several thousand sampled trees; the first10,000 trees (1� 106 generations) were discarded as burn-infor the tree topology and posterior probability. Acantharianand environmental sequences were deposited in GenBankunder the Accession numbers GU246566-GU246591.
CARD-FISH for the detection of acantharian deep-seastages: Two rRNA-targeted 50HRP-conjugated probes weredeveloped to specifically target organisms of interest fromacantharian and unidentified clades. Probe A497, 50-TCATTC-CAATCAACTCAC-30 (Amaral Zettler et al. 1997) was used totarget acantharia and UC1-899, 50-TCATYATACAAAGGTCCA-30, was designed to target Unidentified Clade 1 phylotypes.The eukaryote-specific, 50HRP probe 1209R, 50-GGGCATCA-CAGACCTG-30 (Lim et al. 1993) served as the positive control.A HRP-conjugated sense probe of UC1-899 (50-TGGACCTTTGTATRACGA-30), served as the negative control.Before collecting samples, a series of stringency tests wereconducted on environmental samples enriched with vegeta-tive acantharia and acantharian cysts to empirically determineconditions necessary to minimize background and non-specific binding. Monthly o80 mm fractions of seawater from500 m were collected at the SPOT sampling site from May2005–January 2006. Cells were fixed at 4 1C for exactly 1 h in2% formaldehyde and gently filtered onto 25 mm white GTTPfilters (Millipore) and immobilized with 0.2% Metaphoragarose at 40 1C. Once dry, filters were dehydrated in 80%EtOH at room temperature for 1 minute, blotted and air-dried.Samples were then stored dry at �20 1C for several weeks.Our CARD-FISH protocol was adapted from Pernthaler et al.(2002) with some modifications. Filters were treated withproteinase K solution (2 ml proteinase K in 0.05 M EDTA and0.1 M Tris [pH 8]) at 37 1C for 1 h. Filters were washed withdH2O for 1 min, incubated in 0.01 M HCl for 20 min, rinsed 3times with fresh dH2O and air-dried. Filters were placed onclean glass slides and hybridized with 2 ml of 50 ng/ml probestock in 20 ml 40% formamide hybridization buffer (1.5 MNaCl; 33 mM Tris [pH 8]; 1 g dextran sulfate, 40% formamide;0.83% blocking reagent; 0.016% SDS). Negative controlsreceived the nonsense probe, blanks received only buffer.Filters were covered with glass cover slips and incubated inhumidified falcon tubes at 35 1C for 2-3 h. Filters weresubmerged in wash buffer (37 mM NaCl; 200 mM Tris [pH 8];10 mM EDTA [pH 7.5]; 0.01% SDS) at 37 1C for 10 min, thenblotted on blotting paper and incubated in 0.05% Triton X-100/PBS at room temperature for 15 min with mild agitation.Filters were blotted, but not allowed to dry, before tyramideamplification using a TSATM Kit #22 with HRP—streptavidinand Alexa Fluors 488 tyramide (Invitrogen). We preparedamplification buffer according to the first steps of the kitprotocol: 5 ml of tyramide working stock was combined with450 ml amplification buffer and 50 ml 10% blocking solutionand 100 ml of this tyramide solution was added to each filter
on a fresh slide. Filters were then covered with HybriSlipsTM
(Sigma) and incubated in the dark for 10 min at 37 1C. Filterswere washed with fresh 0.05% Triton X-100/PBS in the dark atroom temperature for 15 min, then washed with dH2O for1 min, 80% EtOH for 1 min and dried at 37 1C in the dark.Samples were mounted and stained with SlowFades Goldantifade reagent with DAPI (Invitrogen) and visualized on aLeica DM IBRE equipped with excitation and emission filtersets for FITC and DAPI, respectively.
Acknowledgements
We are grateful to Dr. Anthony Michaels for help withidentifications of live acantharia. Pratik Savai andSusie Theroux (MBL) provided technical supportand Dr. Timothy McLean offered advice regardingprotocols. The Wrigley Institute for EnvironmentalStudies provided important facilities and support forprocessing samples. We especially thank MichaelNeumann, Trevor Oudin and Reni and Gerry Smithof the Wrigley Institute for Environmental Studies,who helped with sample collection. Financial sup-port was received in part from the Wrigley Institutefor Environmental Studies, the International Censusof Marine Microbes (Amaral Zettler) funded by theSloan Foundation, NSF grant (MCB-0084231 andMCB-0703159), and the University of SouthernCalifornia Joint Initiative Program (Gilg).
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