gomez 2005 brachidinium crypto algol

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Morphology ofBrachidinium capitatum F.J.R. Taylor(Brachidiniales, Dinophyceae) collected from the western

Pacific Ocean

Fernando GMEZa*, Sadaaki YOSH IM ATSUb& Ken FURUYA c

aStation M arine de Wimereux, Universitdes Sciences et Technologies de L il le28 avenue Foch, BP 80, F-62930 Wimereux, France

bA kashiwo Research I nstitute of K agawa Prefecture 75-5,Yashimahigashi Takamatsu, K agawa 761-0111, Japan

(Received 16 M ay 2003, accepted 15 A pr il 2004)Abstract This is a first report of the genus BrachidiniumF.J.R. Taylor from t he Pa cifi cO cean. Of the 17 specimens of B. capitatumreported here, 12 were collected from the vicin-ity of the Kuroshio in Ma y (2 specimens) and July (10 specimens), 3 from the w estern equa-torial Pacific Ocean, one from the Sulu Sea, and one from Tanabe Bay, Japan. Thelast-mentioned specimen wa s observed live. For the first time, the flagella a nd the sulcus ofa member of the order B rachidiniales A.R . Loeblich II I ex Sournia a re depicted in pho-tomicrographs. The ventral view corresponds to that seen when the position of the nucleusis in the left side of the cell. In several specimens, DA PI-staining showed a secondarynucleus located in the opposite side of the dinokary on nucleus. In the live specimen, thesulcus was visible and the lateral extensions were observed to be moveable.

Brachidinium /Brachydinium/ binucleate dinoflagellate / Pacific Ocean / phytoplankton /taxonomy

Rsum Morphologie deBrachidinium capitatum F.J.R. Taylor (Brachidiniales, Dino-phyceae) rcolt louest de lOcan Pacifique. Cest la premire signalisation du genreBrachidiniumF.J.R . Tay lor da ns lOca n P acifi que. Parmi les 17 spcimens de B. capitatumrapports ici, 12 ont t trouvs pro ximit du Kuroshio en mai (2 spcimens) et juillet(10 spcimens), 3 dans lOcan P acifiq ue quatorial occidental et enfin, un seul spcimendans la mer de Sulu ainsi que dans la B aie de Tanabe, Japon. Le dernier spcimen a t

observ vivant. Pour la premire fois, les fla gelles et le sulcus dun membre de lordre desB rachidiniales A.R . Loeblich I II ex Sournia sont illustrs par des photomicrographies. Lavue ventra le correspond ce que lon voit q uand le noya u est du ct ga uche de la cellule.D ans plusieurs spcimens, lutilisation de la colora tion a u DA PI a montr un noyau secon-da ire situ du le ct oppos au noyau dinokaryo n. Lobservation du spcimen vivant amontr nettement le sulcus ainsi q ue la motilit des prolongements lat raux.

Brachidinium /Brachydinium / dinoflagell binucl / Ocan Pacifique / phytoplancton /taxonomie

Cryptogamie, A lgol., 2005, 26 (2): 165-175 2005 Ada c. Tous droits rservs

* C orrespondence and reprints: [email protected] + 33 3 2199 2926. Fax + 33 3 2199 2901.

D epartment of A quatic Biosciences, The University of Tokyoc

1-1-1 Yayoi , Bunkyo, Bunk yo 113-8657, Japan


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INTRODUCTION

BrachidiniumF.J.R . Tay lor is a genus of photosynthetic planktonicmarine unarmored dinoflagellates [usually misspelled as Brachydinium, seeG mez (2003a)]. Brachidiniumand AsterodiniumSournia constitute the familyB rachidiniaceae, placed in the order B rachidiniales A.R . Loeblich III exSournia(Loeblich III, 1982; Sournia, 1984), or Ptychodiscales Fensome, Taylor, Norris,Sarjeant, Wharton etWilliams (Fensome et al., 1993). According to Steidinger &Tangen (1997, p. 468) Brachidiniumhas flattened cells with four elongate exten-sions radia ting from t he hyposoma and an apical process on the episoma. The sul-cus has not been observed, but an incomplete cingulum and chloroplasts arepresent. A large, ovoid nucleus occupies most of the cell body.

The type species Brachidinium capitatumF.J.R . Tay lor (Tay lor, 1963) a ndone other species, Brachidinium catenatumF.J.R . Tay lor, have been described fro mthe southwest India n O cean (Taylor, 1967; see Figs 2-4). The author mentionedthat the latter species might be a small neritic or summer form of B. capitatum(Tay lor, 1967) (Fig. 4). Sournia (1972) a lso reported B. capitatum(see Figs 5-6) anddescribed two new taxa, Brachidinium taylorii Sournia (see Fig. 7) andBrachidinium brevipesSournia (see Fig. 8), also from the southwest Indian O cean.According to Sournia (1972, p. 153) B. taylori ishows a robust aspect with thickerarms tha n the type species and a shorter apical protuberance. The surface of thecell is covered w ith fine peaks fi nes ctes ou crtes . Brachidinium brevipesshows shorter arms, a very reduced central body a nd is also covered with finepeaks (Sournia , 1972, p. 153-4).

Subsequently, B. capitatumand Brachidiniumsp. were reported from thesouthern waters of the I ndian O cean and the Arabian Sea, respectively (Sourniaet al., 1979; Tarran et al., 1999). The type species was also reported f rom t he north-east At lantic O cean (Margalef, 1973) and the Mediterranean Sea (Lger, 1971;Abboud-Abi Saab, 1985; Vilicic, 1998). In the northwest Mediterranean Sea,Estrada & Salat (1989) reported Brachidiniumas a component of the deep phy-toplankton assemblages and Palau et al. (1991) reported Brachidiniumsp. from acave. Brachidinium tayloriiSournia was reported from the southeast AtlanticO cean (K ruger, 1979) and the Mediterranean Sea (Margalef, 1995). The latterauthor also listed Brachidinium transversum but with no illustrations or addi-tional informa tion (Ma rgalef, 1995).

The morphology of the species of Bachidiniumis poorly known d ue to alack of records, deta iled illustrat ions and informa tion on t he ultrastructure (i.e.,fla gellum/fla gella; sulcus; nuclei; etc). Taylor (1963) did not observe the fl agel-lum/flagella or cingulum and pla ced Brachidiniumin the order DinococcalesP ascher. H e proposed a tenta tive orienta tion fo r the genus (Figs 2-3). Tay lor (1980,p. 67) showed a species of Brachidinium, which has a poorly defi ned sulcus, a lon-gitudinal flagellum and the dinokaryon nucleus in the left side of the cell (Fig. 9).Fensome et al . (1993, p. 3) included a line drawing of B. capitatumwith two fla-gella (Fig. 10). They did not mention fla gella or sulcus in the family B rachi-diniaceae (p. 56), but illustrated dorsal and ventral views of B. capitatum, in whichthe ventral view corresponds to the position of the dinokaryotic nucleus in theright side o f t he cell (Figs 5-6). Fensome et al . (1993, p. 56) reported tha t the ori-entation wa s based on Sournia (1972, 1986). H owever, Sournia (1972, 1986)included no illustra tion o f fl agellum/flagella or sulcus of Brachidinium. Sournia

(1972) introduced a terminology describing the orientation of the order Brachi-diniales and considered that the dinokaryotic nucleus is displaced to one side and

166 G omez F., Yoshimatsu S. & Furuya K.


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Brachid ini um capitatumfrom Pacifi c Ocean 167

Fig. 1. Location of the sampling stations in the western Pacific Ocean. The inset shows theTana be B ay w here a live specimen wa s collected.

the extensions or a rms closer to the nucleus were the left extensions.U ncertainties remained, however, and Sournia (1986, p. 49) doubted whether thecells were dorso-ventrally or la terally fl att ened.

Sournia (1986, p. 50), based on the records by Lger (1971), reportedthat Brachidiniumpossesses at least one fl agellum, but that the point of inser-tion rema ined unknown. Lger (1971) collected 30 specimens named as B. capi-tatum. H owever his figure, reproduced here (Fig. 11) does not seem to representthe type species. H is specimen dra wing has tw o extensions radiat ing from thehyposoma, and two extensions and a central process from the episoma. It isintermediate between A sterodinium and Brachidinium. Sournia (1972) com-mented on the possibility of an optical illusion in the position of the cingulum.His specimen has the dinokaryotic nucleus in the left side and a hypotheticalsulcal flagellum arising from the rear and the nucleus in the right side of thecell (Fig. 11). Lger (1971) reported tha t he w as unable to locate the insertion ofthe flagellum.

This study reports for the first time the flagella and the sulcus inBrachidinium, including photo micrographs on the moveab le extensions of a live

specimen, and the occurrence of a secondary nucleus (confirmed by DA P Istaining).


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Figs 2-11. Line dra wings of several species of Brachidiniumreported in the literature. Fig. 2.B. capitatumada pted fro m Taylor (1963). Fig. 3. Ventral view of B. capitatumaccord ing to Tay lor(1963). Fig. 4. Brachidi nium catenatumF.J.R . Tay lor a da pted from Tay lor (1967). Figs 5-6. B. capi-tatumad apt ed from So urnia (1972). These figures were also reproduced by Fensome et al. (1993),

who proposed that they represented ventral and dorsal views, respectively. Fig. 7. BrachidiniumtayloriiSournia ada pted from Sournia (1972). Fig. 8. Brachidi nium br evipesSournia adaptedfrom Sournia (1972). Fig. 9. A ventral view of a member of the genus Brachidiniumshowing thelongitudinal fla gellum ada pted from Taylor (1980, p. 67). Fig. 10. A ventral view of B. capitatumwith two flagella adapted from Fensome et al . (1993, p. 3) a pparently ba sed on Sournia (1972,1986). Fig. 11. B rachidinium capitatum ada pted from Lger (1971). Scale bars 20 m.

MATERIAL AND METHODS

Samples were collected during several cruises in the western PacificO cean: 1) Two cruises on boa rd R /V Soyo Maru(13-20 May and 3-10 July 2002)a long the merid ian 138 in the vicinity o f the Kuroshio Current. Nine sta tions weresampled from 30 30 N t o 34 15 N in M ay, and 10 stat ions were sampled fro m30 0 N to 34 20 N during the July cruise. At ea ch sta tion , 15 dept hs from 5-200m were sampled with Niskin bottles; 2) on boa rd R /V H akuho Maru(7 No-vember-18 D ecember 2002) in the C elebes, Sulu and South C hina Sea s. Sampleswere collected using Niskin bottles at 10 stations at six depths from 0 to 150 mdepth; 3) aboa rd R /V Mirai(15-28 January 2003) along the eq uator fro m 160 Eto 160 W (Fig. 1). Samples were collected w ith Niskin bot tles from 9 stat ions at14 depths betw een 0 to 200 m depth. D uring a ll the cruises, samples were pre-served with acidified L ugols solution (H asle and Syvertsen 1997, p. 334) and

stored at 5 C. Samples were pre-concentrat ed by settling in glass cylinders, andconcentrat es settled in sta ndard sedimentation chambers. Concentrates equivalent


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to 400 mL were observed with a Nikon inverted microscope equipped with aNikon digita l camera.

Several of the Lugol fixed specimens were isolated with a capillary fromthe chambers, transferred to a glass slide, and ob served with an Olympus micro-scope equipped w ith Nomarski D ifferential Interference Contra st (D.I.C.) system.

High magnification microphotographs (

600;

1000) were obtained with an Olym-pus digital camera. Several specimens were stained by a dding DA P I (4,6-diamidino-2-phenylindole). DA P I specifi cally binds to double stranded D NA, andwhen excited with U.V. light the DA P I-D NA complex fluoresces a bright blue(Porter & Feig, 1980). E pifluorescence microscopy wa s done with Olympus andZ eiss microscopes equipped with U V excitation fa cility.

In ad dition, one specimen collected from the coastal wa ters of Japa n wasobserved live. Seaw ater samples were monthly collected from a station in Tana beB ay (see inset in the Fig. 1) at 0,5, 10,15 m depths and one metre a bove the bot -tom (19.5 m depth). Samples (1 l) were fi ltered through an 8 mm pore sizeMillipore cellulose acetate filter at low pressure (


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located in the rea r focus of the cell. Thus, the a spect of the specimen in figures 12-14 corresponds to the dorsal view (nucleus in the right side o f the cell). This spec-imen was later successfully transferred to a glass slide and observed under amicroscope with D.I.C . The specimen appeared in the same view, also with thenucleus in the right side of the cell, but the TF w as now displaced f rom the grooveand a higher proportion o f the TF w as seen moving freely (Fig. 15).

D.I.C photomicrographs of a second specimen with the nucleus in the leftside of the cell were t aken (Figs 16-21). Figures 16-17 show a simila r focus (bothright anta pical and the left latera l anta pical extension in focus), with the TFlocated a long the groove. The TF did not arise from the rear focus of the cell.Figure 18 shows, in a different focal plane, the left anta pical and the right lat eralanta pical extensions, with the TF visible in the right side o f the cell along t hegroove. The end of the TF was located in the centra l part of the cell (see alsofi g. 21). We were unable to focus on the a rea of the TF origin. The fla gellum didnot ar ise from the ba ck of the cell (Figs 16-17). This position, with the nucleus inthe left side of the cell, is the ventral view. Af ter these observations the specimenwa s shaken until the TF wa s partia lly separa ted f rom the cingulum. The TF turnedaround t he left side of the cingulum (Figs 19-20) (rear o f the cell, as in Fig. 16).At a different focal plane most of the TF wa s visible (Fig. 21). The aspect ofthis specimen, with the nucleus in the left side, corresponds to the ventral view(Figs 16-21).

A third specimen with the nucleus in the right side was also observedwith D.I.C. optics (Figs 22, 24) and inverted microscopy (Fig. 23). A part of a fla -gellum that could correspond t o t he longitudinal fl agellum was observed betw eenthe tw o a ntapical extensions (Fig. 22). The TF ra n a long the cingulum in a fronta l

focus (Fig. 24). This position, with t he nucleus in the right side, corresponds to t hedorsal view.

Table 1. Number of specimens recorded, date, stations, depth (m) in meters, geographiccoordinates (latitude, longitude) and fi gures of the records of Brachidiniumin the western PacificOcean.

# D ate Sta. (m) L at N L ong Figure

1 24/1/1995 20 19.5 33 42 135 21 E Figs 27-28

1 10/5/2002 C3 5 33 30 138 E

1 13/5/2002 C13 5 30 30 138 E

2 7/7/2002 C8 20 33 30 138 E Figs 16-21

1 7/7/2002 C7 50 33 138 E

1 7/7/2002 C6 5 32 30 138 E

1 6/7/2002 C5 40 32 138 E

1 6/7/2002 C2 60 31 30 138 E Fig. 26

1 4/7/2002 B 1 10 30 138 E Figs 22-25

3 4/7/2002 B 1 60 30 138 E1 3/12/2002 10 30 8 50 121 48E

1 15/1/2003 6 1 0 160 E

1 17/1/2003 7 90 0 165 E Figs 12-15

1 25/1/2003 13 80 0 165 W


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Figs 12-21. Pho tomicrographs of Brachidiniumshowing the tra nsverse fl agellum (TF). Figs 12-15.D orsal view of a specimen under inverted microscopy (Figs 12-14), and direct microscopy(Fig. 15). See the end o f the TF displaced f rom the cingulum and free moving in the rea r focus ofthe cell (Fig. 12). See the cingulum groove where the TF ran (Fig. 13). P lease note that TF a longthe cingulum does not a rise from the fronta l focus (Fig. 14). The specimens wa s transferred to aglass slide and observed w ith D.I.C . optic when TF a ppeared partially separa ted fro m the cingu-lum (Fig. 15). Figs 16-21. D.I .C. photomicrographs of the ventral view of other specimen. Figs 16-17 the TF ra n a ll along the cingulum in the rear focus of the cell. Fig. 18. the end of the TF w as ob-served in the centra l part o f t he cingulum in the fro nta l focus o f t he cell. Figs 19-21. The specimen

wa s shaken until the TF w as pa rtially d isplaced from the cingulum in the rear focus (Figs 19-20)and the frontal focus (Fig. 21). AG = Apical groove; N= dinokaryon nucleus; TF = transversalflagellum. Scale ba rs 20 m.

An alternative method to elucidate the cell orientation is based on thelocat ion of t he sulcus (ventra l side). The sulcus wa s not visible in the Lugol-fi xedspecimens, but w as visible in the live specimen observed with a d irect microscope.The nucleus of this specimen was located in the right side of the cell (Figs 27-28).Figure 27 focusses on only one of the four extensions, corresponding to the frontof the specimen. Figure 28, showing three extensions in focus (closer to the glassslide), corresponds to the rea r of the cell, with t he sulcus being visible. Figures 27-

28, with the nucleus in the right side of the cell, correspond to the dorsal view.In these four specimens, ba sed on the transverse fl agellum or the sulcus,

the ventral view corresponds to the nucleus being on the left side of the cell (Figs29-30). The o ccurrence of a sulcus (Fig. 28) provides evidence of the existence o fthe longitudinal fla gellum in the genus Brachidinium (partially observed inFig. 22). This orienta tion is contra ry to Fensome et al . (1993, p. 3, 56). Taylor (1980,p. 67) reported a n illustrat ion of Brachidiniumwith a wea kly defi ned sulcus in theleft side of the cell (Fig. 9). From our observat ions, however, the sulcus is centrallylocated in Brachidinium(Fig. 28).

Binucleate specimens

The nucleus of Brachidiniumwa s relatively large, ovoid, and occupied asignificant proportion of t he body cell. It wa s clearly visible in Lugol-fi xed speci-


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Figs 22-28. Figs 22-25. D orsal view of a specimen. Figs 22, 24. D.I.C. photomicrographs.Fig. 22. See a part of a fla gellum, possibly the longitudinal flagellum. Fig. 23. Inverted microscopyphotomicrograph of the specimen. The arrow indicates the micronucleus (N) or seconda rynucleus. Fig. 24. Frontal focus showing the TF along the cingulum. Fig 25. E pifluorescence pho-tomicrograph of the same specimen stained with DA PI and illuminated w ith U.V. light. Thearrow indicates the secondary nucleus. Fig. 26. Inverted microscopy photomicrograph of other

specimen in do rsal view. The a rrow shows the secondary nucleus. Figs. 27-28. Pho tomicrographsof the d orsal view of a specimen observed live. Fig. 27. Frontal focus. Fig. 28. R ear focus. See thesulcus. An inset betw een the fi gures 27-28 shows a low ma gnification photomicrograph ta kenprior to the movement of the lateral extensions of the specimen; AG = Apical groove;N = dinokaryon nucleus; N = secondary nucleus; TF = transversal flagellum; LF = longitudinalfla gellum; S = sulcus. Scale bars 20 m.

mens and appears darker tha n the rest of the cell surface (e.g., Figs 14, 23, 26),whereas it was less visible in the cell observed live (Figs 27-28). The d inokaryonnucleus was confirmed by D AP I-staining (Fig. 25). In addition, several of theLugol-fixed specimens showed a secondary small nucleus in side of the cell oppo-

site the d inokaryon nucleus, with bo th nuclei staining the same da rk brown colour(Fig. 26). In a DA P I-stained specimen (Figs 22-24), the micronucleus fluoresced


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Figs 29-30. Line dra wings of the ventral (Fig. 29) a nd do rsal (Fig. 30) views in Brachidinium.

when excited w ith U.V. light and t he dinokaryon nucleus appeared brighter thanthe micronucleus (Fig. 25). Sournia (1972) did not fi nd chloroplasts inB. brevipes. Sournia (1972) and Lger (1972) reported one circular plast in theopposite side of the nucleus in their line drawings (Fig. 8 and Lger (1972, p. 29)).This could be interpreted as the first evidence of the occurrence of a secondary

nucleus in Brachidinium.The occurrence of binucleate dinofl agellates is very ra re. The freshwa ter

dinoflagellate K ryptoperidi nium foli aceum(Stein) Lindemann presented mononu-cleate and binucleate strains. K ryptoperidini um foli aceumcontained a fucoxan-thin-containing diatom as a cytoplasmic endo-symbiont (Kempton et al., 2002).The origin of the secondary nucleus in Brachidiniumwa s not clarifi ed in the pres-ent study.

Live cell and moveable extensions

The extensions in Brachidiniumare moveable, as reported by Lger(1971), ba sed on observations by Cachon on a live specimen collected from

Villefranche-sur-Mer (Ligurian Sea ). We include, for the fi rst time, photomicro-graphs of a live specimen of Brachidinium. D uring our microscopical observations,


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