Marine Biology (2002) 140: 4ll-423 DOI 1 0. 1 007/s00221 01 00690

C. S. Lobban ' M. Schefter ' A. G. B. Simpson X. Pochon ' J. Pawlowski ' 'W'. Foissner Mart$entor dinoferus n. gen., n. sp., a giant heterotrich (Spirotrichea: Heterotrichidal with zooxanthellae, from coral reefs on Guam, Mariana lslands

Received: 5 January 2001 i Acceptecl: 3l July 2001 / Published online: l0 October 2001 @ Springer-Verlag 2001

Abstract Muristentor dino.ferus n. geo, n. sp., was dis- micronuclei and, on average, 101 somatic ciliary rows covered on coral reefs on Guam in 1996 and has since and 397 adoral membranelles. M. clhtof eran may be been for-rnd freqr-rently, at depths of 3-20 m. It forms closely related to limnetic spp., but differs in two black clusters, visible to the naked eye, especially on conspicLloLls features: (1) the cilia on the peristomial Padina spp. (Phaeophyta) and other light-colored bottom are scattered (orclered rows in Stentor spp.) and backgrounds. When fully extended, this sessile ciliate is (2) the paroral membrane is very short and opposite the trumpet-shaped, Llp to 1 mm tall and 300 pm wicle buccal portion of the adoral zone of membranelles (in across the cap. The ciliate is host to 500-800 symbiotic Stentor spp., it accompanies the entire membranellar algae. The anterior cap, or peristomial area, is divided zone). The cells appear dark due to stripes of cortical into two conspicLlolls lobes by a deep ventral indenta- granules; the granules are more concentrated in a "black tion. There is a single globular macronucleus, many band" below the cap. The cortical pigment(s) is red fluorescent with a broad absorption peak in the blue (ca. 420-480 nm), and sharp peaks in the yellow-green Communicated by J.P. Grassle, New Brunswick (ca. 550 nm) and red (600 nm). Illtrastructural and molecular data demonstrate that the symbiont is a di- Electronic sr-rpplementary merterial to this paper call be obtained by noflagellate of the genus Symbiodiniunt, the flrst Lrn- using the Springer Link server located zrt http://dx.doi.or-e/10.1007 I equivocal report of zooxanthellae in a ciliate. s002210 I 00690. Phylogenetic analysis of a portion of the large sr-rbr-rnit C.S. Lobban (X) ribosomal RNA gene (2BS rDNA) showed that the University of Guam, symbionts belong to Synnbiodiniunx sp. clade C, z lineage Division of Natural Sciences, Mangilao, that also inhabits many corals on Guam. The ciliate GIJ 96923, IJSA changes shape ut night, and the symbionts, which are E-mail: clobban@ uog.edu spread out in the eap during the day, are mostly with- Tel.: * I -67l-l352181 drawn into the stalk at night; these changes were Fax: + 1-671-1341299 apparently not simply a response to darkness. M. Schefter Box 5126, UOG Station, Mangilao, GU 96923, USA A.G.B. Simpson University of Sydney, lntroduction School of Biological Sciences, Sydney, NSW 2006, Australia The and flagellates of tropical reefs are a largely X. Pochon ' J. Pawlowski invisible and unexplored component of the benthos, IJniversity of Geneva, primarily in sediments (Patterson et al . l9B9; Larsen and I)epartment of Zoology and Animal Biology, Patterson 1990; Lynn et al . 1991); rarely do they become 1224 Chöne-Bougeries, Switzerland apparent to divers. Wichterman (1942) reported a new W. Foissner genus of ciliate, Paraeuplotes tortugensis, with zooxan- Universität Sal zburg, Institut für Zoologie, Hellbrunnerstrasse 34, 5020 Salzburg, Austria thellae from Tortugas, Florida. A macroscopic follicr"r- linid ciliate (" Hulofolliculinu corallasia" nomen nudem) Present address: A.G.B. Simpson has been conspicLrolrs aggre- Dalhousie University, recently reported to form Department of Biochemistry and Molecular Biology, gations on coral at several Indo-Pacific sites (Antonius Halifax, NS B3H 4H7, Canada 1999). The organism reportedly kills coral, and the dis- 4t2 ease has been termed "skeletal eroding band". Lobban and Schefter (1996) reported macroscopic aggregations Fig. la-j Maristentor dinoferus. Living specimens. a Underwater of an apparently novel Stentor-like ciliate on reef sub- photograph of undisturbed popr"rlation shows clusters of individ- uals on Padinasp. as well as on adjacent rock and crustose coralline strates in Guam. This organism does not appear cause to algae; b shows detail at approximately twice life size. c Group of harm and contains zooxanthellae reminiscent of those in three specimens in culture dish showing fully expanded posture corals, other invertebrates , and foraminifera on tropical (viewed lrom below with inverted microscope). Note subapical reefs (Rowan l99B; Carlos et al. 1999). In the present concentration of dark pigment granules and murcilage produced by cells (arrouls). Right lateral view fully expanded specimen, study we report on the morphology and behavior of the d of showing display of symbiotrc algzre in cap and stalk . Arrou,, marks Stentor-llke ciliate, for which we propose the new genus widely projecting left peristomial lobe. e Frontal view of fully and species names Maristentor dino.ferus) and \,ve exam- expanded cap (peristomial bottom). Note distribution of zooxan- ine the identity of its algal symbiont. thellae in cap and deep ventral indentation dividing peristomial bottom in twb conspicllous lobes of unequal size. f Cell in crawling posture, ofl microscope slide, showing macronucleLls, membranel- lar band, and zooxanthellae (differential interference contrast, Materials and methods flash). g Posterior portion of crawling cell in optical section, showing symbiotic algae; pyrenoid and nucleus visible in some Maristentor dinoferu^§ was first collected 20 m deep at "Pete's Reef' zooxanthellae (differential interference contrast, flash). h Ventral (Agat Bay) in July 1995. The principal study site was 3-5 m deep view of fully contracted specimen, showing nucle ar apparatus and on the reef at Scuba Beach, or the north shore of Apra Harbor, subapical pigment band (dark field illumination). i Cap of cell at Guam, Mariana Islands (I3'27'N; I4440'E), where we collected or night showing distribution of symbionts and cortical pigment; observed populations at approximately bimonthly intervals. Oc- compare with e. j Surface view showing stripes of cortical granules, casional collections were made from other reefs on Guam: 15 m which also surrouncl nuclear apparatus (differentierl interference deep at "Coral Gardens" (Agat Bay), July 1996; 20 m deep at contrast, flash). The yellow zooxarrthellae show in the background. Apuntua Pt. (south side of Orote Peninsula), March 2000; J m deep Scalebars:10 mm (a);200 prm (c); 100 pm (d, e, f, h, );2A pm (g, j) at Dadi Beach (south side of Orote Peninsula), April 2001; and 3- (AZM adoral zone of membranelles; BB black pigment band; MA 5 m deep at several sites in Apra Harbor at various times since macronucleus) September 1998. Populations of M. dinoferus were collected by placing blades of the seaweed Padina spp. with adherent clusters (see Fig. la) into cells were washed, then post-fixed for ca. 30 min in ice-cold plastic bags fllled with ambient water. Collections were maintained 0.4% w/v osmium tetroxide. Other cells were immersed in ice-cold for up to 1 month in culture dishes, small aquaria, or tissue culture 5oÄ vlv glutaraldehyde,200 mM cacoclylate buffer (pH 1.4), and plates. Containers were placed near a window out of direct sun, in a l0% w/v sucrose, followed 15 s later by an equal volume of ice- room without air conditioning. Room temperatures were 2J-30"C, cold 0.8% w lv osmium tetroxide in 200 mM cacodylate and l0% compared to 29-30'C measured for lreshly collected seawater from sucrose. These cells were left on ice for ca. 30 min. In both cases the principal study site. Once the ciliates had left Padino spp., the cells were then washed free of fixative and dehydrated through an seaweed was removed from the dishes. We have so far been unable ascending series of ethanols. After passing through 100% ethanol, to establish long-term cultures in controlled conditions. the cells were infiltrated with Spurrs resin, then embedded. Silver- . Live specimens were studied with conventional and inverted gold ultrathin sections were cut with a diamond knife using a microscopes using bright field, dark field, phase contrast, and in- Reichert (Leica) IJltracut S r-rltramicrotome. Sections were terference contrast illumination. The ciliary pattern was revealed mounted on pioloform-coated slot grids after the techniqure of with protargol and scanning electron microscopy on cells fixed with Rowley and Moran (1915), carbon coated, and finally stained with Stieve's or Bouin's fluid, see Foissner (1991) for a detailed de- Reynolds lead citrate (Reynolds 1 963) and saturate,C uranyl acetate scription of these methods. Preliminary observations of day-night in 50oÄ ethanol. Sections were examined using a Zeiss 902 electron changes were made both during the natural light:dark cycle and microscope. when cells were kept under lights at night and in darkness during For DNA extraction, amplification, cloning and sequencing, six the day. samples of cells were taken from a collection from the principal site. Measurements on extended specimens were made from photo- DNA was extracted in guanidine lysis buffer (Tkach and Paw- graphic records of live cells in various postures and orientations in lowski 1999), precipitated with isopropanol and dissolved in dis- tissue culture dishes; measurements on silver-prepared specimens tilled water. Polymerase chain reaction (PCR) amplifications were were made on permanent slides with oil immersion using an eye- performed in atotalvolume of 50 pl using 40 cycles of 30 s at94"C, piece micrometer. Drawings of live cells were based on micro- 30 s at 52"C, and 120 s atJ2oC, followed by 5 min atJ2oC for final graphs; those of impregnated specimens were made with a camera extension. All ampliflcations were done using one specific ancl lucida. Numbers of symbionts were counted from cells that had one universal primer. Specific primers were designed according burst and spread on a drying slide. to the Syrubiodiniunt-related ribosomal DNA (rDNA) sequences Ciliate terminology follows that of Corliss (1919) and Foissner available in GenBank. Specific primer sequences were: and WöIfl (1994). Classiflcation in Alveolata follows Patterson S-DINO (5'CGCTCCTACCGATTGAGTGA3') and ITS_DINO (1999). By convention, the cells are illustrated and left/right iden- (5'GTGAATTGCAGAACTCC3). The seqLrence ol the universal tified according to hor,v they appear in the compoLrnd microscope; eukaryotic primer L_0 is 5'GCTATCCTGAGRGAAACTTCG3'. this is the opposite of their appearance in the dissection microscope. The localization of primers is shown in the electronic supplemen- Preliminary examination of cortical pigment (and algal pig- tary material (Appendix l). The amplified PCR products were ments, not reported here) was carried out using methanol extrac- purified using a High Pure PCR purification kit (Roche Diagnos- tion followed by phase partitioning with salt ancl ether, as described tics) then ligated into the pGEM-T vector system (Promega) and in Chapman (1988). The aqLreous extract contained the cortical cloned in XL-2 ultracompetent cells (Stratagene). Sequences were pigment(s), and the ether layer contained the algal pigments. Ab- prepared with an ABI-PRISM Big Dye terminator cycle sequencing sorption spectra for the cortical pigment were made with a Hitachi kit and analyzed with an ABI-3ll DNA seqLrencer (Perkin-Elmer), Perkin-Elmer spectrophotometer. all accordinrg to the manufacturers' instructions. For transmission electron microscopy, individual cells were For sequence analysis, a fragment of the 28S rDNA, with 529 selected by flne pipette and relaxed rn 7oÄ wlv MgCl2. Some cells aligned sites, was use,C for phylogenetic analysis. We chose 2BS were fixed for ca. 30 min in a cocktail of 2.5% v/v glutaraldehyde, rather than IBS rDNA, because the sequence variation in this gene 200 mM cacodylate buffer (pH 1.4), and l0% wlv sucrose. These is more suitable for analysis within the Symbiocliniunt complex 4t3

t

-tr'hc (Lenaers et al. l99i; Scholin et al. 1994; Zardoya et al . 1995; Baker bootstrapping (Felsensteir-r 1988). with I .000 resalllplings. et al. 1997; Wilcox 1998), ancl a large numtrer of correspondiug PHYLO_WIN pr"ograrn (Galtier ancl Cior-ry 1996) wils usecl lirr seq Lrences a re available in CienBank. Six secl Llences obta i ned ll'orn clistatrse computations, tree-builcling. aucl bootstrappiug. The six M . dinof crus were aligned with 32 seqLlences ol' the S1'nrbiodirtiunt- S.t,ntbiodtrri.run sp. seclLlences have been clepositecl in CicnBank (ac- courplex availnble in the EMBL/GenBarrk database (sec electronic cessiorr nr-unbers: AJ278598 8603). as fbllorvs . AJZ] 8 5c)8. sr-rpplernentary material (Appenclix 2) lor references). StsA-VIh,W steu_? l8S rt{NA gene,5.85 rRNA^J27 geuc. l8S lt{l{A geue. intcrnal soltware (Claltier- ancl Ciouy 1996) was usecl lor alignrnent. The transcribecl spaccrl (lTSl) aucl internal transcri['ret] spuccr'2' rDNA seqlrcncres we rc analyzlecl r-rsir-rg the neighbor-joinin-u (NJ) (ITS2), isolate steu_2, AJ278599, stcn_8 28S rlLNA gene ancl in- 'isolate rr-rcthocl (Saitoh ancl Nei 1987), applied to clisl-arlces correctecl l'or tertral transcribecl spacer 2' (fTS2), sten_8; A,J27 8(r00. r-r-rultiplc hits. and lbr nnequal transition and transversion rates, stetr_10 28S rl{NA gene ancl iutcrnal transc'ribecl spacer 2' (ITS2), using the I(imura two-parameter- rnoclel (I(irnura 1980). The isolate ster-r_I0; AJ27B60l, sten_9 2BS rl{NA serle ancl intcrnal reliability ol'internal branches in the N.I tree was assessecl by transcribecl spacer 2' (lTS2), isolate ste n_9; AJ278 602. sten_ l0b 414

28S rRNA gene and internal transcribed spacer 2' (ITS2), isolate monitored over a period of a month in September 1998 sten_l0b; AJ2TB603, sten_l2 28S rRNA gene and internal tran- were always clumped when observed during the late scribed spacer 2' (ITS2), isolate sten_l2. afternoon or evening, but were dispersed on 8 of 14 mornings; usually, all dishes were similar at a given time. Results M. dinr1fbrus exhibited distinct day-night changes (see Fig. le vs.Fig. li; Table l). The description given under Occurrence, ecology, and behavior "posture one" below reflects the appearance during the day, when the stalk was fully extended: the algal sym- To d,ate, Maristentor r)ino.fbrus has been positively bionts in the cap were spread out in a monolayer, and identified only from Guam (as listed in "Materials and very little pigment was visible in this region or in the methods"). There was a population at Scuba Beach stalk. The great majority of the algae were exposed in continuously from July 1999 to July 2000, sparse except clear areas of the cap and stalk, but some were in the during July and Augus t 1999, when it was abund ant and anterior pigmented band. Within about an hour after extensive. In July 1998, we found one similar-looking nightfall, however, the stalk contracted somewhat, few patch on Padina spp. from a reef in the Somosomo Strait algae were visible in the czp, especially towards the off Taveuni, trUi. We collected the sample and examined margins, and stripes of cortical pigment granules were it carefully with a hand lens but could not check rni- clearly visible in the cap (see trig. 1i).The cap remained croscopically. We cannot exclude the possibility of its extended, however, and the membranellar band seemed being another species, but we are confident that it was in as active as during the day. In contrast to the contracted the genus Maristentor " stalk, the cap remained transparent, notwithstanding the M. dino.fbrus was most commonly seen on Padina spp" pigment stripes. The changes did not appear to be simple (trig. la), but also occurred on other surfaces (Fig . la), responses to darkness; they may result from a circadian including coralline algae, coral rubble, and dead shell. rhythm, but tests in stan dardrzed culture conditions are Patches ranged from a few clusters on single blades of needed to examine this further. Padina spp. up to populations covering 400 cm' of substrate (Fig. 1a shows an area ^,10 cm wide of alarger patch; we distinguished over 400 individuals within area Morphology of the inset, Fig. 1b). Individuals were seen gliding on the substrate between clusters, and additional observa- M. dino.f erus exhibited four postures (shapes) in the tions in culture (see below) suggest that the clusters were laboratory, and we have seen three of these in nature. dynamic. Patches were evident from several feet away, Furthermore, M . dino.fbrus showed pronounced day- and at close range individual ciliates could be distin- night changes (described above). Morphometric data guished with the naked eye. If a blade of Padina sp. with (Table 1) show the variation of cells under various cir- clusters of M. dino.ferus was tapped, the individuals cumstances (trigs. l, 2, 3, 4, 5, 6). immediately contracted. This obvious behavior was an Posture one was the sedent ?ty, extended state, where aid to identification in the field. We have not ascertained the organism was up to I mm long and slenderly whether portions of the population are also in the trumpet-shaped (Figs. lc, d, e, 2a). It showed a wide, sediments or water column. asymmetric peristomial bottom ("cap") an,C a gradually In captivity, M. dinofbrus quickly left the seaweed" narrowing postoral portion ("stalk") attached to the Within 30 min many had left, and within 3 h virtually all substrate by a heavily ciliated holdfast (Figs. 2c, 4f). The had. They collected near the water surface, either at- holdfast very likely secreted the mucilage skein that was taching to the wall of the container or hanging from the observed on the side of the dish where LI. dino,f erus had water meniscus. Freshly collected M. dinoferus in a settled. We also noted mucilage around a cluster of three daylit bag stayed on Padino spp. for over I h; whereas, individuals in the tissue culture chamber (trig. 1c). in a comparable collection covered with black cloth, However, we never observed a mucilaginous lorica as is they swam to the top and sides of the bag within 30 min. known from several Stentnr species (Foissner and WöIfl Those in the daylit bag also left the seaweed overnight tee|). and did not return to it in the morning. However, during In the extended state, the peristomial area was spread two night dives (2100, 0400 hours), we observed that M. out and divided into two conspicuous lobes by a deep dinoferus in situ remained on Padina spp. ventral indentation. The left lobe, which contained the Cells showed a strong preference to be at or near the buccal part of the adoral zone of membranelles, was water surface on the lighted side of a container, or even higher and larger than the right lobe, giving the cap an to hang from the surface meniscus. Fresh specimens L-shape when seen from the left (Fig. 1c). The organism showed positive phototaxis: in dim light they swam to was thick and dark in the region below the ezp, because the bottom of tubes illuminated from below. They most of the pigment granules were concentratecl there; formed dynamic clusters of some 15-30 individuals. At thus, the ciliate appeared black at low magnification times, most of the cells in the dishes were in clusters; at (100x) and to the naked eye (Fig. la, b). The buccal other times, they were more uniformly dispersed. Cells apparatus was also obscured. 415

Table I M uri.,\'/.r,rt/.or diltof erus. Morphometric data (in pm). Peristomial bottom width measured as the lzrteral axis and depth as the clorsoventral axis (lV in vivo; P plotargol - impregnated, mounted specimens from field; N number ol specimens investigated) Clharacteristic Method Mean SD Median Min. Max. 1/

Boc1y. length lully extencled (day) IV 825 0 I 16.5 800 600 1 000 8 Rody, length fully extencled (night) IV 4s7 .0 3t .t 450 400 500 10 Peristornia,l bottom, wiclth (day) IV 253.5 23.0 2s0 I90 290 20 Peristomial bottorl, clepth (day) IV 213.0 25.2 200 150 250 20 Stalk, wiclth near base (day) IV 32.5 4.6 30 30 40 B Macronuclei, unrlber P 1.0 00 l0 I I 2t Macronuclens, long axis P 35.2 3.6 3s0 28 42 2t M acronucleus, shor-t axis P 32.6 4.4 330 23 40 2t Micronuclei, diarleter P 2.0 00 2o 2 2 21 Pigment granules. diameter IV 1.1 05 20 I 2 20 Ciliary rows in rnid-body, number P t0l.l 7.6 102.0 80 110 2l Acloral mernbranelles. number P 396.8 38.4 390 0 350 460 1l Symbiolic algae, cliarneter ry 95 0.9 r0.0 B 1t 20 Symbiotic al-eae, long axis P 80 0.1 8.0 7 I 2l Symbiotic algae, short axis P 1.6 0.1 BO 6 9 2t

Posture two was the crawling state. Cells migrated Pignrent granules occurred in both the cortical region across the substrate, crawling with the stalk almost fully erncl tlle grouncl cytoplasm" In the cortex, they were a1-- extended and flattened against the substr ate, but with ranged in conspicLroLls str-ipes between the ciliary rows. the eap withdrawn (seen in situ and in culture dishes). which stood ouf as clezrr lirres (Figs. lj, 2cl). The pigrnent This was also how they appeared when constrained on a gran ules wer e I 2 prrr r-rcross ancl clar-k bloocl recl to microscope slide (trig. 10. In the crawling state, r-ecldish purple when observed uncler high magnification M. dinffirus resembled Faurö-Fremiet's (1936) illustra- and transmitted light (trig lg, j); uncler low magnifica- tion of auriculatus, except that the more tion, they often appearecl bluish ol- greenish. The aqLre- prominent or sharply defined lobe was the left one, not oLrs phase of the pigment extrnct, containing tlre cor-tical the right. They seemed to move with the right lateral pigurent(s), appearecl red in burlk (e.g" in a t-rottle 50 r-nm surface to the substrate (left lobe uppermost), and not diitrneter") but bluish or gray in srnaller qLlill'rtities (e.g. a with the oral (ventral) side to the substrate as Tartar spectrophotometer- cuvette). Some ciliate pigment ap- (1961) reported for ,Srentor spp peared to transl'er to the ether phase alc-rng with the Posture three is the contracted state (Figs. th, 2b,5a). zooxlurthellar pigments ancl remainecl nt the origin in When disturbed, and also spontaneously, cells con- pr-eliminary thin-layer chroma togt'aphy (clatil nr:rt tracted abruptly, becoming broadly conical or globular. shown). The abscrr-ption spectrum o[ the aqLreous phase Most of their algal symbionts were then obscured by a showed a broad pea[< irr the blue (ca. 420 480 ntr), and broad, subapical, transverse band of pigment (Figs. th, sharp peaks in tl're yellow-green (ca. 550 nrn) ancl t'ecl -l-his 2b), corresponding to the dark area of the extended cell. (600 nm) spectrzr (clata not shown). e.xtract fluo- Posture four was the free-swimming state. Cells swam resced recl under white, blue, ancl "black" light (elec- when f,rst transferred to a culture dish, but under most tronic supplementary material Appenclix 3)" The other conditions moved by crawling. M. dinoferus ernission appeared to be in the l-ange 600 650 nn.l, but swimming could not be observed in situ. They swam we did not have the equiprnent to nlerrsLrre the fluores- rapidly, spiraling clockwise, with the stalk and peristo- cence emission or exciti-rtion spectra, nor to assess flr-ro- mial bottom contracted, becoming shorter by more than rescence in vivo. The color o['the granules ils seen uncler half, but not completely contracted. Swimming cells the micloscope, especially with flash photography ancl were broadly conrcal or bullet-shaped, tapering toward high magnihcation (trig. lg, j), may be par"tly due to the posterior. When they contacted a surface they did so fluorescence in vivo. with the frontal area first, subsequently lying down and M. clinofbrus was holotrichously (completely) ciliated. extending the stalk to take up the crawling posture. The cilia emergecl ft-om closely spacecl (0.5 I prr"n in The nuclear apparatus lay subapically below the contrzrctecl, protargo[-irnpregnatecl specimens) dik ineIicls buccal cavity and consisted of a single, globular mac- (pairs of basal boclies) that were cilia(ed on only one ronucleus and many minute micronuclei. In vivo, it was basal body, possibly the anteriot' (trigs " 2d,, 6a). T'he often difficult to see, hidden by pigment granules and ciliary rows extended longituclinally in ttre sarne pattern symbiotic algae (Fig. 1c, d, i). The macronucleus was as in Stenlor spp. (Tartar 1961, Foissner et al. 1992). at about 40 pm across (Figs. lf, 2c) and contained many least postor-zrlly (Fig. 2c).The width of spacir-rg o[' the minute, pale nucleoli. There were more than ten mi- ciliary rows gritclecl irnper"ceptibly arouncl the circum- cronuclei of *2 pm diameter near and attached to the ference of the cell f'rom the nrost wiclely spacecl r ows macronucleus (Fig. ad; the exact number could be not (sepzrrated by the br'«radest pigrneut stripes) on the left o[ determined because the macronucleus always hid some the br-rcczrl cavity, to the most closely spacecl ciliary r-ows micronuclei (separzrted by the narrowest pigmerrt stripes) near t he 416

Fig. Za-i Maristentor dinrfe- rars. Postures (shapes) and cytologisal details from life fr: (a, b) and after protargol im- rjia a. t' pregnation (c, middle part of d, 'o. a.o ll rr e-j). a Left lateral view of Rb extended specimen. The peris- #'lo. tomial bottom is divided into .t:* two conspicuoLls lobes by a deep ventral indentation. b trully AZnn contracted specimen. c Total PD ventral view showing main e organelles. Note stripe contrast zone in cell's midline . Arrou,- head marks minute paroral AZ M I ffi membrane. d Surface view d showing main structures from outside to inside: stripes of cortical granules between ciliary A z tvl rows; paired basal bodies which ,l ,fi, give rise to the postciliodes- lt mata; and symbiotic algae. e-g Structure of adoral mem- branelles in the regions marked in trig. 3. h Cross section of tubular structure under adoral zone of membranelles. i Part of paroral membrane. j Distal and proximal end of adoral zone. Scctle bars: 200 prm (a, b); 100 pm (c); 10 pm (d) (AzM adoral zone of membranelles; BB black pigment band; f' fibers; G cortical granule ,,ry stripes; HF holdfast; LL left peristomial lobe; MA macro- nucleus; PD postciliodesma; RL right peristomial lobe; S,4 symbiotic algae; 7 tubular structure underneath AZM)

///fN HF : midline slightly right of the buccal cavity. Along the The oral apparatus was composed of a prominent cell's midline the most closely spaced rows abutted the adoral zone of membranelles and a minute paroral most widely spaced rows at acute angles, forming a su- membrane (Figs. 3, 4c).The adoral zone, which gradu- ture, the so-called "stripe contrast" (trig. 4b). The per- ally narrowed toward both ends (trig. 2j), performed one istomial field, which was slightly elliptical (Table 1), spiral turn on the margin of the peristomial bottom and bore numerous dikinetids with single cilia. These cilia a second in the comparatively small buccal cavity left of were very scattered (trigs . 3, 5b) in contrast to Stentor the cell's midline (Figs . 2c, 4d, e). In its main portion, spp., where they form semicircular rows (Foissner et al. the individual membranelles consisted of two 8-12 pm 1992; Foissner and WöIfl 1994)" long ciliary rows and a single, oblique dikinetid proxi- Two fibrillar systems were recognizable after protar- mally (trig. 2e); in the buccal portion of the zone, the gol impregnation in the somatic cortex. The first com- membranelles had a short third row attached and lacked prised the postciliodesmata (trigs. 2d, 6a), which were the proximal dikinetid (Fig. 2f, g). Very fine fibers associated with the basal bodies of the cilia and extended originated from the membranelles and formed a con- close to the right of the ciliary rows; usually they im- spicuous tubular structure under the zone (trigs . 2h, 3). pregnated only faintly with protargol. The second, The paroral membrane extended from the proximal end which obviously represented the contractile (myoneme) of the adoral zone anteriorly on the opposite side of the system, also extended right of the ciliary rows and buccal cavity and ended near the buccal opening showed numerous bifurcations in the stripe contrast (Figs . 2c, 3). The paroral membrane was difficult to zone" The individual myonemes were l-2 pm wide recognize and composed of (ciliated?) dikinetids from bands of very fine fibers and were often impregnated which fibers (- l0 pm long) originated and extended into heavily with protargol (Fig. 4c). the buccal cytoplasm (Figs . 2i, 4c). 417

domains Cl-C1 of the 2BS rDNA (electronic supple- mentary material - Appendix 1). One clone was seqllenced for each DNA extract. Two sequences (sten_B and sten_10) were identical. Two oth- ers (sten_ I 2 and sten_ I 0b) differed by only two mutations (0.17 5% of sequence divergence). The mean divergence between all sequences obtained f r om M. dino.fbrarc was very low (0.42 + 0 .26%). This variation was due to three transitions, four transversions, and two insertions within the six analyzed sequences (data not shown). It colre- sponded to the mean seqlrence divergence (0.88 + 0 .99%) observed within Syrubiodinium sp. clade C, where oLrr seqüences branched according to phylogenetic analyses (see below). For comparison, the mean seqLrence diver- gence and other clades was, Fig. 3 Mari,;l.entor dinoferats. After protargol impregnertion. Fron- between clade C (4, B, D) tal view; note scattered baserl bodies on peristomial bottom and respectively, 22.1 1oÄ, 12.7 5%, and I 8.68%. short paroratl membrane. Scale bar. 50 pm (AZM erclorerl zone of The phylogenetic analysis of six seqLrences obtained membrernelles; PB peristomial bottom; PM paroral membrane; from M. dinqfbrus was cornpared with 32 underneath AZM; a, g denote regions shown 7 tubular structure .f, sequences (trig. sequences grouped flve in trig. 2e-g at higher magnification) l). All into clearly distinguished clades, each slrpported by 100% bootstrap value. One clade, composed of Gyrnnoc{iniunt M. dinot'brus contained about 500-800 zooxanthellae simplex and G. heii was chosen as the outgroLrp, fol- (trigs. 1g, 4a), mainly in the expanded czp, where they lowing previous analyses with more extensive samples of formed a single layer (trig. le), and in the stalk (trig. ld), and other (Wilcox 1 998). but also subapically, where they were almost invisible Three other clades correspond to clades A, B, and C due to the concentrated pigment granules. The individ- identified previously by several authors (Rowan and ual symbionts were B-10 pm across in vivo, with an Powers I99l; Rowan and Knowlton 1995; Baker et al. evident pyrenoid and nucleus (Fig. 1g). We observed 1991). The structure of the tree and branching order many dividing algae, showing that they were indeed between the clades is consistent with previons work symbionts and not merely ingested food. Transmission (Wilcox 1998). Two seqlrences, Atr 110148 and electron microscopy showed that the symbionts belong AF l10l49 (Baker 1999), form a fourth Sltrubiodirtiunt sp. to the Dinophyta and, probably, to Syrubiodiniunt sp. clade, here referred to as "D". They possessed the typical dinoflagellate nucleus with condensed chromosomes (F'ig. 6b), triple plastid lamel- lae (Fig. 6e) with no girdle lamellae, and cytoplasmic Diagnoses channels thror-rgh the dividing nucleus (trig. 6c, d). The pyrenoid was surrounded by the plastid membrane and o M aristentor n. gen. lacked invasive plastid thylakoids (Fig. 6b). Zooxan- . Diagnosis: Stentoridae Carus, 1863, having cilia thellae showed no significant thickening of the am- scattered (not in rows) on peristomial bottom and phiesmal vesicles (bV thecal plate material) but were paroral membrane restricted to proximal portion and enclosed in host vacuoles (Fig. 6b, c). We do not have opposite side of adoral zone of membranelles. any further information about the vacuoles; no con- . Type species: Maristentor dino.ferLts t't sp. nection with the ciliate cytoskeleton was evident, aI- o Etymology: Composite of names referring to locality though the zooxanthellae are moved within the cell, as (Mariana Islands) and body shape resemblance to the noted above. genus Stentor. . Maristentor dino./brus n sp. . Diagnosis: Length in vivo about 800 Ltffi, width of Molecular identification of the zooxanthellae: peristomial bottom about 250 pm. Distinctly trumpet- sequence data and phylogenetic analysis shaped when attached and fr-rlly extended, peristomial bottom slightly elliptical and dividecl into two con- For one sample, we obtained a 1,595-bp fragment which spicuous lobes by deep ventral indentation. Single, included the whole ITS rDNA region, starting at the globular macronucleus and many spherical micronu- 3' end of the 1BS rDNA and terminating in the con- clei. Cells dark at low magnification due to stripes of served region C4 of the 2BS rDNA. This fragment was dark blood red cortical granules and 500-800 dino- amplified with primers S_DINO and L_0 (electronic flagellate endosymbionts (zooxanthellae) in the genus supplementary material Appendix 1). For five other Symhiocliniunt. On average 101 somatic ciliary rows samples, a shorter fragment of abor-rt 1,060 bp was and 397 adoratr membranelles, forming a spiral on amplifled using a pair of primers ITS_DINO and L_0. both the margin of the peristomial bottom, and in the This fragment comprised only the ITS2 region and the buccal cavity. Gregarious.

420

Fig.6a-e Mari,stentor dinoferus (a), Syntbiodinium sp. (b-e). genus Condylostonta. These two marine Stentor spp. are Transmission electron microscopy . a Transverse section of somatic much smaller (extended length: 350 pm) than M. din- cortex showing a ciliated basal body, which gives rise to a o.f (Kahl 1932). postciliary microtubule ribbon, several of which overlap forming erus and are colorless a conspiclrolrs postciliodesma. b Symbiont showing stalked Two other marine ciliates are very different from pyrenoid (arrow,points to stalk). c, d Cell with nucleus undergoing M . dino./brus. One is " Halo.f olliculin(I corallasia," a lor i- division, bundles of microtubules in fenestrae (enlarged in d). e cate heterotrich from the Indo-Pacific region, which Detail of plerstid showing triple lermellae characteristic of dinofla- appears as deep-purple to black bands on a wide variety gellate. Scale bars: 1 pm (a-c); 0.5 pm (d); 0.1 pm (e) (lf dinoflagellate nucleus; P pyrenoid; PD postciliodesma) of massive and branching corals (Antonius 1999). Sim- ilarly, M . dinofbrus is not related to Wichterman's (1942) Paraeuplotes trtrtugensis, which is a hypotrichous ciliate to the Stentoridae, which was previously monotypic (: Hypotrichia), for which Wichterman (Ttrffrau 1994). proposed a new family, Paraeuplotidae (not listed by Few Stentor species have been found in marine hab- S.mall and Lynn l9B5). itats (Foissner and WöIfl 1994). Of these, ,S. auricula IJltrastructu rally , the symbiont closely resembles (Kent 1880, lBBl, 1882) and ,S. auriculatus (Kahl 1932) spp. (Trench and Blank 1987), and mo- deserve special attention because Jankowski ( 1 978) lecular data conflrm the generic identity. Phylogenetic classified them in a new genLls , Condylostentor. Howev- analysis shows that all six sequences obtained from er, Condylostentor differs from Maristentor in having a M. dino.fbrus fall within clade C, as do symbionts iso- conspicuous paroral membrane, in lacking cilia on the lated from a variety of invertebrate hosts in Guam peristomial bottom, and in lacking pigmented cortical (Pochon et al., in press). Although this is the only grolrp granules (Kent 1880, l BB 1, 1BB2; Gn-rber 1884; Kahl of Syrubiocliniuru-hke symbionts found in this ciliate, 1932; Faurö-Fremiet 1936). As mentioned by Faurö- they do not seem to be host speciflc. The M. dino.fbrus Fremiet ( 1936), both S. auri,cula and S. auriculatus very symbionts do not form a single group, but are inter- likely belong to or are closely related to species in the spersed among other clade C sequences. It is difficult to 421

0 038 r------{ Metamorphosing corals of many species must re-es- tablish their symbiosis from the benthic pool of free-

AF r 70 t43 living Synobiodinium spp . M. dinq/brus might participate M ariste ntor din ofer u s en d osy mbionts AF l70lz17 ssll6348 | C' in this pool (suggested by the genetic similarity), or might maintain a separate stock through cell division. C We have not observed reproduction rn M . dinoferus, bvt AF I 70r 29 AFl70t50 presLrme that cell division would divide the algal poplt- AF 170138 lation between the dar-rghter cells. 4Hr 70137 4F170151 Although M. dino/brus was most commonly seen on 4F170145 aFt 70l33 blades of Padina spp., this may be largely due to this SSLJ63485 C alga being a very common substratum on Guam reefs l70t I I ^l; AIr06089l and providing a contrasting background against which ssl.r63484 Af',060892 clusters of the ciliate are more readily seen. We have AF I 70139 documented M . dino.f erarc on other surfaces, as noted AF 170140 AF t70 t44 above, patches backgrounds were -I ]B but on dark very dif- AFr70r49 , niri;;i*'J t) ficult to see. On the other hand, preliminary observa- l\til70128 -t - tions of other erect seaweeds adjacent to patches on At;170126 I SSUr,tasO n I Padina spp., indicated that M. dino.fbrus was not present AFlzorsa I on Halimeda sp. Tydencania expeditionis. More -AF r70 r30 I or work AI;060tt97 I A needs to be done to establish the substratum preferences AFo6oses I Tor 4r I (if any) of M. dinübrus. ^uArro6o8e6 I pigment are -AI:060895 ) The role of the cortical is unknown. We AF060qot\ intrigued that ciliate with endosymbiotic algae has - a AFo6oeoo ) extensive cortical pigment even at 20 m depth, and that Fig. 7 Syrubiocliniunt sp. Phylogenetic reconstrLlction of the com- the fluorescence of the pigment appears to be near the plex, inferred from partial LSU rDNA (28S) based on the red absorption peak for chlorophylls. Studies of Stentor including sequences from neighbor-joining method, six obtained spp. pigments (reviewed by Tartar 1961 , pp 47-48) offer Maristentor dinoferus. Analysis based on 529 aligned sites no insights. However, evidence from corals that some excluding all gaps . I'{untbers at nodes represent percentage bootstrap slrpport following 1,000 (neighbor joining) data resam- fluorescent host pigments may enhance zooxanthellar pling. simplex and G. beii were chosen as the photosynthesis (Schlichter et al. 1985, 1986, 1994; Sch- outgroup lichter and Fricke 1991) suggests a line of str"rdy on M . dinqfbrus. Schlichter and colleagues have found tur- quoise (430-500 nm) fluorescence in a variety of corals, determine if this is due to a very low sequence divergence especially the deepwater Leptoseris .fragilis. Gastroder- within this clade or to a lack of specificity in these par- mal chromatophores in this coral form a layer under the ticular symbionts. The analysis of ITS sequences, which zooxanthellae. Photosynthetic enhancement would be evolve faster than the 28S sequences (Lee and Taylor worth testing tn M. dino/erus. Perhaps in deep water, the 1992; Baldwin et al. 1995), did not show any unique pigmented cortical layer of M . dinqferus also serves as a pattern for M. dinof erus symbionts (data not shown). "light guide," making the cells optically black, as has M. dino.fbrus is the first ciliate with zooxanthellae that been found in deepwater seaweeds (Ramus 1981). That are unequivocally dinoflagellates, to the best of our is, we envision that the Lrpwardly oriented, funnel- knowledge. The only other tropical marine ciliate with shaped cells could retain almost all the light entering symbionts had yellowish-brown bodies that Wichterman them, either by direct absorption by the algal pigments, (1942) identified as Zooxanthella, but his description of or after fluorescence from the inner side of the pigment the zooxanthellae is derived entirely from an account of granules. In some shallow-water corals fluorescent pig- zooxanthellae from corals, and not from detailed ments may serve a protective role, lying above the algae observation of the symbionts. Green algae or cyano- rather than below (Salih et al. 2000) . M. dino.ferers offers bacteria occur as symbionts in other ciliates, including an interesting system in which to study the functional several species of Stentor (Taylor 1984; Foissner and role(s) of the cortical pigment(s), because of the different WöIfl 1994). The gymnostome ciliate Mesodiniut'n rLt- postures it can adopt and its ability to redistribute brum has a cryptophyte symbiont (Taylor 1982). While zooxanthellae and pigment granules. Green fluorescent dinoflagellates were previously unknown in ciliates, protein (GtrP) and other fluorescent proteins are widely many tropical reef invertebrates and foraminifera form used as in vivo markers of gene expression and protein symbioses with dinoflagellates, particularly Symbiodini- localization (Matz et al. 1999), but preliminary charac- um spp. (Rowan 1998; Carlos et al. 1999). The diurnal terization of M. dino.fbrus extracts indicated that this changes in morphology are also novel observations for fluorescent pigment is not a protein (M . Matz, personal ciliates, although a ttdal rhythm in the marine oligotrich communication). ciliate Strombidium oculatum was reported by Faurö- The relationship between this novel ciliate and the Fremiet (1948). Symbiodinium sp. is fascinating in itself, and in 422 comparison with other Syrnbiodinir.tm spp. symbioses on Informationsbericht 5192, Bayerische Landesamt ftir Wasser- coral reefs. Laboratory studies on the life history of wirtschaft, Munich, pp l-502 Galtier N, Gouy M (1996) SEAVIEW and PHYLO_WIN: two M. dino,f erus and its nutritional relationship with the graphic tools for sequence alignment and molecular phylogeny. symbionts, and field studies on its behavior and popu- Comput Appl Biosci 12:543-548 lation dynamics are needed. Gruber A (lSB4) Die Protozoen des Hafens von Genua. Nova Acta Leopold 46:473-539 Acknowledgements We wish to express our appreciation to several Jankowski AW (1978) Revision of the system of the class Polyhy- people who helped us achieve this description of the new species. menophora (Spirotricha) (in Russian). Dokl Akacl Nauk SSSR Dr. R. Rowan (UOG Marine Lab) offered advice and encourage- l9l8:39-40 ment based on his experience with zooxanthellae, provided equip- Kahl A (1932) [Jrtiere oder . I. Wimpertiere oder Ciliata ment in his laboratory, helped search for M. dinoferus, and (Infusoria). 3. Spirotrichzr. Tierwelt Dtl 25:399-65A provided a critical review of the manuscript. R. LeGrande located I(ent WS (1880) A manual of the infusoria: including zr description the major population of M. dincfbrars that made a real research of ;rll known flargellate, ciliate, and tenterculiferous protozoa effort possible, and assisted with monitoring populations. Dr. British anctr foreign, and an accollnt of the organization and D. Patterson (IJ. Sydney) opened his protozoology laboratory to affinities of the sponges, vol l. David Bogue, Lonclon C.L. and M.S. on a visit there, and provided advice based on living Kent WS (1881) A manual of the infusoria: including zr description material. In his laboratory we were also assisted by B.L. Dicks in of all known flagellate, ciliate, and tentaculiferous protozoa getting high magniflcation photos of living M. dinctferus. The British and foreign, and an account of the organization and Electron Microscopical [Jnit, U. 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