Balechina Cucumeridinium

BALECHINA AND THE NEW GENUS CUCUMERIDINIUM GEN. NOV. (DINOPHYCEAE),UNARMORED DINOFLAGELLATES WITH THICK CELL COVERINGS1

Fernando G�omez2

Laboratory of Plankton Systems, Oceanographic Institute, University of S~ao Paulo, Prac�a do Oceanogr�afico 191, Cidade

Universit�aria, Butant~a, S~ao Paulo 05508-900, Brazil

Purificaci�on L�opez-Garc�ıa

Unit�e d’Ecologie, Syst�ematique et Evolution, CNRS UMR 8079, Universit�e Paris-Sud, Batiment 360, Orsay Cedex 91405, France

Haruyoshi Takayama

Hatami 5-20-13, Ondo-cho, Kure, Hiroshima 737-1207, Japan

and David Moreira

Unit�e d’Ecologie, Syst�ematique et Evolution, CNRS UMR 8079, Universit�e Paris-Sud, Batiment 360, Orsay Cedex 91405, France

The genus Balechina (=subgenus Pachydinium) wasestablished for heterotrophic gymnodinioiddinoflagellates with a thick cell covering. The typespecies, B. pachydermata (=Gymnodinium pachyderm-atum), showed numerous fine longitudinal striae,whereas B. coerulea (=G. coeruleum) showed ~24prominent longitudinal surface ridges or furrows and adistinctive blue pigmentation. We have investigated themorphology and molecular phylogeny of these taxaand the species Gymnodinium cucumis, G. lira andG. amphora from the western Mediterranean, Braziland Japan. Sudden contractions at the cingulum levelwere seen in B. pachydermata, which also showed ahigh morphological variability which includedmorphotypes that have been described as Amphidiniumvasculum, G. amphora, G. dogielii and G. gracile sensuKofoid and Swezy. Molecular phylogeny based onsmall subunit rRNA gene sequences revealed thatBalechina coerulea, G. cucumis and G. lira formed aclade distantly related to the clade of the type species,B. pachydermata, and G. amphora. We propose the newgenus Cucumeridinium for the species with longitudinalridges and a circular apical groove (Cucumeridiniumcoeruleum comb. nov., C. lira comb. nov. andC. cucumis comb. nov.), and Gymnodinium canus andG. costatum are considered synonyms of C. coeruleum.The genus Balechina remains for the species with adouble-layer cell covering, bossed surface with finestriae, and an elongated elliptical apical groove. Atpresent, the genus is monotypic containing onlyB. pachydermata.

Key index words: athecate Dinoflagellata; autotomy;blue pigmentation; Gymnodinium; intraspecific

variability; Mediterranean Sea; molecular phylogeny;new genus; North Pacific Ocean; South AtlanticOcean

Abbreviation: bp, base pairs

Two major groups of dinoflagellates can be distin-guished based on their cell coverings. Thecate (ar-mored) dinoflagellates with large amphiesmalvesicles filled with cellulosic material, and the athe-cate (unarmored or “naked”) dinoflagellates thatcontain hundreds of alveoli lacking cellulosic mate-rial (Morrill and Loeblich 1983). The nakeddinoflagellates are usually fragile and delicate, thecells easily lyse during the observation of live sam-ples, lyse due to the fixation, or the fixed cells aretoo distorted for proper identification. Particularly,gymnodinioid dinoflagellates are notoriously diffi-cult to preserve. The chemical fixatives produce mis-shapen cells, swollen membranes, and clumping ofspecimens (Kofoid and Swezy 1921). However, theseparation between armored and unarmored speciesis not clear cut and some gymnodinioid dinoflagel-lates are characterized by a thick cell covering. Thisis the case of the genus Balechina Loeblich et A.R.Loeblich, which has been placed either in the orderKolkwitziellales (Taylor 1987) or Ptychodiscales(Fensome et al. 1993).Kofoid and Swezy (1921) published a monograph

of the unarmored dinoflagellates known at thattime. They proposed the subgenus PachydiniumKofoid et Swezy for gymnodinioid species with athick cell covering. They included three new specieslacking surface longitudinal ridges (Gymnodiniumpachydermatum Kofoid et Swezy, G. dogielii Kofoid etSwezy, and G. amphora Kofoid et Swezy), and specieswith a surface covered by longitudinal ridges(Gymnodinium coeruleum Dogiel, and the new species

1Received 6 January 2015. Accepted 29 July 2015.2Author for correspondence: e-mail fernando.gomez@fito

plancton.com.Editorial Responsibility: S. Lin (Associate Editor)

J. Phycol. 51, 1088–1105 (2015)© 2015 Phycological Society of AmericaDOI: 10.1111/jpy.12346

1088

G. canus Kofoid et Swezy, G. costatum Kofoid etSwezy, and G. lira Kofoid et Swezy). Despite thelarge sizes and thick cell covering of these species,only G. coeruleum has sporadically been reported inthe literature, while the records of the other speciesare very rarely reported or only known from theoriginal descriptions (Dogiel 1906, Kofoid andSwezy 1921, Wood 1968). This may be due to thefact that G. coeruleum is characterized by a strikingblue or purple coloration, which is relatively rare innature, in particular in microbial species.

With no known observations, Loeblich and Loe-blich (1968) proposed that the subgenus Pachy-dinium should be raised at the genus rank. Theyproposed the new name Balechina because Pachy-dinium Kofoid et Swezy 1921 was a junior homonymof the thecate Pachydinium Pavillard 1915. This pro-posal was followed only by Taylor (1976), who trans-ferred G. coeruleum into Balechina, and proposed athird species Balechina marianiae F.J.R. Taylor (seeAppendix S1 in the Supporting Information for ataxonomic, nomenclatural and biogeographicalaccount). The genus Balechina was further used inthe literature (Lessard and Swift 1986, Taylor 1987,Fensome et al. 1993, Steidinger and Tangen 1997),while other authors considered Balechina as a syn-onym of Gymnodinium F. Stein (Sournia 1986,Balech 1988).

In the last 15 years our knowledge of the unar-mored dinoflagellates has increased with theadvances of molecular phylogeny, initially basedmostly on photosynthetic cultivable species (Daugb-jerg et al. 2000), abundant heterotrophic coastalspecies (Hansen and Daugbjerg 2004, Takano andHoriguchi 2004) and later, on other less abundantheterotrophic species (G�omez et al. 2009). How-ever, little is known about the unarmored dinoflag-ellates with a thick cell covering that reach largersizes (>200 lm long, i.e., Gymnodinium cucumis F.Sch€utt), or highly distinctive species with strikingblue or purple pigmentation (i.e., Balechina coerulea(Dogiel) F.J.R. Taylor). To the best of our knowl-edge, the type species of Balechina, B. pachydermata(Kofoid et Swezy) Loeblich et A.R. Loeblich,remains only known from the original description(Kofoid and Swezy 1921) and Wood (1968).

This study was based on observations of live mate-rial from several locations of the Mediterranean Sea(Marseille, Banyuls sur Mer, Villefranche sur Mer,Valencia), the South Atlantic Ocean on the coast ofBrazil (S~ao Sebasti~ao Channel and off Ubatuba),and the North Pacific Ocean on the coast of Japan(Kure, Hiroshima Prefecture). We provided the firstmicrographs of the species B. pachydermata, G. cu-cumis, G. dogielii, G. amphora and Amphidinium vascu-lum, and scanning electron microscopy pictures ofthe species B. coerulea, B. pachydermata, and G. lira,including their apical grooves. We reported for thefirst time the phenomenon of the sudden contrac-tion of B. pachydermata, and its high intraspecific

variability, mainly in the shape of the episome. Wepropose the species A. vasculum Kofoid et Swezy,G. amphora, G. dogielii and G. gracile sensu Kofoidand Swezy as synonyms of B. pachydermata. We alsoillustrated the phenomenon of autotomy in an unar-mored dinoflagellate based on our observations ofB. coerulea. We propose the species G. canusand G. costatum as synonyms of B. coerulea. Weprovided the first molecular data (SSU rRNAgene sequences) of the species B. pachydermata,B. coerulea, Gymnodinium amphora, G. cucumis andG. lira. We propose an emended description of thegenus Balechina, a new genus for the speciesB. coerulea, G. cucumis, G. lira, and a tentative unde-scribed species with intermediate characteristicsbetween B. coerulea and G. lira.

MATERIALS AND METHODS

Sampling and isolation of material. Specimens were collectedfrom the Mediterranean Sea by slowly filtering surface seawa-ter taken from the pier of the Station Marine d’Endoume atMarseille (43°160 48.05″ N, 5°200 56.22″ E, bottom depth3 m) from October 2007 to September 2008. A strainer of20, 40, or 60-lm mesh size (Millipore Inc., St. Quentin-Yve-line, France) was used to collect planktonic organisms fromwater volumes ranging between 10 and 100 L, depending onparticle concentration. The plankton concentrate wasscanned in settling chambers at 9100 magnification with aninverted microscope (Nikon Eclipse TE200; Nikon Inc.,Tokyo, Japan). Cells were photographed alive at 9200 or9400 magnifications with a Nikon Coolpix E995 digital cam-era. Further specimens were collected using the same methodfrom October 2008 to August 2009 in the surface waters(depth of 2 m) of the port of Banyuls sur Mer, France(42°280 50″ N, 3°080 09″ E). The concentrated sample wasexamined in Uterm€ohl chambers with an inverted micro-scope (Olympus IX51; Olympus Inc., Tokyo, Japan) and pho-tographed with an Olympus DP71 digital camera. Samplingcontinued from September 2009 to February 2010 in the Bayof Villefranche sur Mer, Ligurian Sea. For this location, sam-pling was performed at the long-term monitoring site Point B(43°410 10″ N, 7°190 00″ E, water column depth ~80 m).Water column samples (0–80 m) were obtained using a phy-toplankton net (53 lm mesh size, 54 cm diameter, 280 cmlength). Samples were prepared according to the same proce-dure as described above and specimens were observed withan inverted microscope (Olympus IX51; Olympus Inc.) andphotographed with an Olympus DP71 digital camera. Sam-pling continued from May 2012 to February 2013 in the portof Valencia, Spain (39°270 38.13″ N, 0°190 21.29″ W, water col-umn depth of 4 m). Specimens were obtained using a phyto-plankton net (20 lm mesh size). Samples were preparedaccording to the same procedure as described above andspecimens were observed with an inverted microscope (NikonEclipse T2000; Nikon Inc.) and photographed with an Olym-pus DP71 digital camera.

In the South Atlantic Ocean, sampling continued afterMarch 2013 in S~ao Sebasti~ao Channel (23°500 4.05″ S, 45°24028.82″ W), and from December 2013 to December 2014 offUbatuba (23°320 20.15″ S, 45°50 58.94″ W). The Brazilianspecimens were obtained using a phytoplankton net (20 lmmesh size) in surface waters. The living concentrated sampleswere examined in Uterm€ohl chambers at magnification of9200 with inverted microscopes (Nikon Diaphot-300 at S~aoSebasti~ao, and Nikon Eclipse TS-100 at Ubatuba), and

BALECHINA AND CUCUMERIDINIUM GEN. NOV. 1089

photographed with a digital camera (Cyber-shot DSC-W300;Sony, Tokyo, Japan) mounted on the microscope’s eyepiece.

In the North Pacific Ocean, samples were collected with aplankton net (30 lm mesh size) from the coastal Inland Seaof Japan at Kure (34°100 30″ N, 132°330 21.6″ E) in December2014. The living concentrated samples were observed at mag-nification of 9400 and 91,000 with an upright microscope(Olympus BH2; Olympus Inc.), and photographed with a dig-ital camera (Canon EOS Kiss F.; Canon Inc., Tokyo, Japan).

In all cases, each specimen was photographed and thenmicropipetted individually with a fine capillary into a cleanchamber and washed several times in a series of drops of0.2 lm-filtered and sterilized seawater. Finally, the specimenwas placed in a 0.2 mL tubes (ABgene; Thermo Fisher Scien-tific Inc., Courtaboeuf, France) filled with several drops ofabsolute ethanol. The sample was kept at room temperatureand in darkness until the molecular analysis could be per-formed.

After obtaining samples for DNA analysis, the subsequentspecimens of B. coerulea and B. pachydermata from Brazil wereisolated with the aim to establish cultures. These specimenswere individually placed in 6 or 12-well tissue culture plateswith 0.2 lm-filtered seawater. To have controlled environmen-tal conditions, the plates were placed in an incubator used formicroalgae culturing, at 23°C, 100 lmol photons � m�2 � s�1

from cool-white tubes and photoperiod 12:12 L:D. To feedBalechina spp., we added aliquots of cultures of the crypto-phyte Rhodomonas sp., the haptophyte Isochrysis sp., thedinoflagellates Heterocapsa sp. and Prorocentrum sp., and severalcentric diatoms isolated from field samples (Chaetoceros spp.,Thalassiosira spp., etc.). None of the potential preys that wereavailable in our culture collection contained blue pigments.

Scanning electron microscopy. Seawater samples were col-lected with a bucket from the coastal areas of the Inland Seaof Japan along Hiroshima Prefecture in 1980–1985 asdescribed in Takayama (1998). For scanning electron micro-scopy, dinoflagellate cells were pipetted individually, rinsedthree times in filtered seawater and placed on poly-lysine-coated coverslips. They were fixed in 2% osmium tetroxide inseawater for 20 min. After washing in distilled water for30 min, cells were dehydrated in an ethanol series, 10 min ineach change of 30%, 50%, 70%, 90%, and 95%, followed bytwo 30 min changes in absolute ethanol, and finally trans-ferred to amyl acetate. The cells were critical-point-driedusing liquid carbon dioxide and ion sputter coated with gold.They were observed using a scanning electron microscope(Hitachi S-430; Hitachi Ltd, Tokyo, Japan) operated at 15 kV.The method is explained in detail in Takayama (1998). Pic-tures were scanned and presented on a black backgroundusing Adobe Photoshop CS3 (Adobe Systems Inc., San Jos�e,CA, USA).

PCR amplification of small subunit rRNA genes (SSU rDNAs)and sequencing. The specimens fixed in ethanol were cen-trifuged for 5 min at 504 g. Ethanol was then evaporated in a

vacuum desiccator, and single cells were resuspended directlyin 25 lL of Ex TaKaRa buffer (TaKaRa, distributed by Lonza,Levallois-Perret, France). PCRs were done in a volume of30–50 lL reaction mix containing 10–20 pmol of the eukary-otic-specific SSU rDNA primers EK-42F (50-CTCAARGAY-TAAGCCATGCA-30) and EK-1520R (50-CYGCAGGTTCACCT-AC-30) (L�opez-Garc�ıa et al. 2001). PCRs were performedunder the following conditions: 2 min denaturation at 94°C;10 cycles of “touch-down” PCR (denaturation at 94°C for15 s; a 30 s annealing step at decreasing temperature from65 down to 55°C, employing a 1°C decrease with each cycle,extension at 72°C for 2 min); 20 additional cycles at 55°Cannealing temperature; and a final elongation step of 7 minat 72°C. A nested PCR was then carried out using 2–5 lL ofthe first PCR products in a GoTaq (Promega, Lyon, France)polymerase reaction mix containing the eukaryotic-specificprimers EK-82F (50-GAAACTGCGAATGGCTC-30) and EK-1498R (50-CACCTACGGAAACCTTGTTA-30; L�opez-Garc�ıaet al. 2001) and similar PCR conditions as described above. Athird, semi-nested PCR was carried out using the dinoflagel-late specific primer DIN464F (50-TAACAATACAGGGCATCCAT-30; G�omez et al. 2009) and the reverse primer EK-1498R.Negative controls without template DNA were used at allamplification steps. Amplicons of the expected size(~1,200 bp [base pairs]) were then sequenced bidirectionallyusing primers DIN464F and EK-1498R using an automated96-capillary ABI PRISM 3730xl sequencer (BC Genomics,Takeley, UK).

Phylogenetic analyses. The new SSU rDNA sequences werealigned to a large multiple sequence alignment containing~1,500 publicly available complete or nearly complete(>1,300 bp) dinoflagellate sequences using the profile align-ment option of MUSCLE 3.7 (Edgar 2004). The resultingalignment was manually inspected using the program ED ofthe MUST package (Philippe 1993). Ambiguously alignedregions and gaps were excluded in phylogenetic analyses. Pre-liminary phylogenetic trees with all sequences were con-structed using the Neighbor-Joining method (Saitou and Nei1987) implemented in the MUST package (Philippe 1993).These trees allowed identification of the closest relatives ofour sequences together with a sample of other dinoflagellatespecies, which were selected to carry out more computation-ally intensive Bayesian Inference analyses. These were donewith the program MrBayes 3.2.3 (Ronquist et al. 2012) apply-ing a GTR + Γ4 model of nucleotide substitution, taking intoaccount a Γ-shaped distribution of substitution rates with fourrate categories. Our sequences were deposited in DDBJ/EMBL/GenBank under accession numbers #KR139785–KR139792 (Table 1).

RESULTS

Balechina coerulea. This species is highly distinc-tive due to its striking blue or more rarely purple

TABLE 1. List of new SSU rDNA sequences used for the phylogenetic analysis. Accession numbers, geographic origin, andcollection dates are provided.

Taxa GenBank no. Geographic origin (date) Figure

Balechina coerulea FG754 KR139785 Banyuls sur Mer (28 Jul 2009) Fig. 1, a and bBalechina coerulea FG764 KR139786 Banyuls sur Mer (2 Jul 2009) Fig. 2, i and jGymnodinium lira FG1601 KR139787 Villefranche sur Mer (7 Jan 2010) Fig. 4, a–cGymnodinium cucumis FG1602 KR139788 Villefranche sur Mer (21 Jan 2010) Fig. 5, a–cBalechina pachydermata FGB22 KR139789 Valencia (11 May 2012) Fig. 6rGymnodinium amphora FGB8 KR139790 S~ao Sebasti~ao Channel (5 Aug 2013) Fig. 7eGymnodinium amphora FGB9 KR139791 S~ao Sebasti~ao Channel (9 Aug 2013) Fig. 7fGymnodinium amphora FGB11 KR139792 S~ao Sebasti~ao Channel (21 Jun 2013) Fig. 7g

1090 FERNANDO G �OMEZ ET AL.

http://www.ncbi.nlm.nih.gov/nuccore/KR139785










pigmentation. In previous observations based onLugol’s solution fixed samples, the specimens wereeasily overlooked as Gyrodinium-like species. How-ever, since 2007 and due to the observation of freshlive samples, B. coerulea appeared in all the samplingareas examined in warm waters such as in theMediterranean Sea (Marseille, Banyuls sur Mer,Villefranche sur Mer, Valencia), the Caribbean Sea(La Parguera and Bah�ıa Fosforescente, PuertoRico), and the South Atlantic Ocean (S~ao Sebasti~aoChannel and off Ubatuba, S~ao Paulo State, Brazil).The coloration of the specimens ranged from navyblue (Fig. 1, a–c), cobalt blue (Figs. 1, d, e and 2,a–e), plum (Fig. 1, f–i), purple (Fig. 1, j–n), peri-winkle (Fig. 1p), blue–green (Fig. 1, o, q–s, u) tocolorless (Fig. 1, t–w). As a general trend, the speci-mens from the same sample showed the same col-oration (Fig. 1s). The specimens with the mostintense blue pigmentation were observed in Mar-seille and Banyuls sur Mer. However, this phe-nomenon was very likely related to the samplingstress and time lapse between the collection and themicroscopic observations rather than to true differ-ences between the populations of different geo-graphic areas. The specimens from Marseille andBanyuls sur Mer were collected in front of the labo-ratory using a bucket and a soft filtration using astrainer, and observed just a few minutes after col-lection. That procedure reduced the stress and spec-imens showed a more natural pigmentation. Inother sampling areas, the samples were collectedusing plankton nets and with a longer delaybetween collection and observation (at least 2 h asin Valencia). Under the microscope, the blue coloris easily bleached due to the stress of sampling andobservation (Fig. 2f). For example, the isolated cellFG764 (GenBank accession number #KR139786)showed an intense blue pigmentation when firstobserved, but the pigmentation disappeared when itwas transferred into a clean chamber for isolation(Fig. 2, i, j). In other stressed specimens, the bluesubstance was released around the cells and it isbleached (Fig. 2, l–n, see Video S1 in the Support-ing Information, https://youtu.be/eLK5FMGNtTI).

The cell division of B. coerulea was by oblique bin-ary fission (Fig. 2a). During cell division, the apexof the episome of one of the daughter cells waselongated (Fig. 2, b, c). After the cell division, bothdaughter cells remained joined (Fig. 2, d, e), andexceptionally they may appear forming two pairs ofdividing cells (Fig. 2g). One of the daughter cellsshowed an elongated episome with a blunt trun-cated apex (Fig. 2d). This morphology corre-sponded typically to the species described asG. canus.

The size of the specimens usually ranged from90–120 lm long and 45–65 lm wide, and we excep-tionally observed specimens that reached 150 lm.This coincided with the morphology of G. costatum(Fig. 1s). In fact, the cells of B. coerulea showed

different shapes. The shape was biconical in the lessstressed specimens (Fig. 1, a, b, d–i) or ellipsoidal(Fig. 1, c, j–n). Other specimens showed a conicalepisome and a wide antapex (Fig. 1r). Other speci-mens showed a dome-shaped episome, and a slightlybifurcated antapex in ventral or dorsal views (Fig. 1,q, t, u). Other specimens showed a conical episomeand a reduced episome (Fig. 1, v, w). When a cellwas stressed, the pigmentation disappeared (seeVideo S1, https://youtu.be/eLK5FMGNtTI). Theshape also changed, usually being rounder (Fig. 2i)and later ellipsoidal (Fig. 2j); the most extendedphenomenon being that the cell changed progres-sively from biconical to ellipsoidal (Fig. 2, l–n).Specimens also showed other responses during

the microscopic observations. At first, the blue pig-mentation disappeared and the apex becamerounder (Fig. 2o). A constriction encircled the mid-dle of the episome and hyposome (Fig. 2p) and thisconstriction progressed until the cell was separatedinto three parts, which did not lyse during the pro-cess (Fig. 2, q, r). The central section kept thenucleus and the transversal flagellum that remainedbeating (Fig. 2, r). The complete process required~5 min, and it was similar in the Mediterranean andBrazilian specimens (Fig. 2s). In the plankton sam-ples, cells with a complete hyposome and a reducedepisome were observed (Fig. 2, t, u). This suggeststhat after the autotomy, at least the part of the cellthat contained the nucleus was able to regenerate,and the hyposome was the first part of cell torecover the normal size and shape.Despite a context of high intraspecific variability

in color and shape, the specimens of B. coeruleashowed several common morphological characters.The cingulum was median and had a cingulumdescending ~4 times its width. In ventral view, thehyposome showed a shallow depression at the anta-pex. The sulcus was well marked and extended fromthe antapex to the base to the apex. The cell sur-face was covered with well-marked longitudinalequidistant ridges (Fig. 1n). The nucleus was spheri-cal and small (~20 lm in diameter), and situated inthe central part of the hyposome, more visible indorsal view. The nucleus showed a well-developedperinuclear membrane (Fig. 1, p, s, u). The cellsshowed prominent food vacuoles, mainly located inthe middle cell and in the episome. Food vacuoleswere colorless or exhibited a brownish color (Figs. 1and 2).Under SEM, the longitudinal ridges showed dif-

ferent lengths. There were more ridges on the hypo-some than the episome (Fig. 3a). However, not allthe ridges that emerged from the cingulum reachedthe antapex or the base of the apex. In the epi-some, there were 24 ridges that extended anteriorlyfrom the upper cingular groove, and about one halfextended anteriorly more than 2/3 of the length ofthe episome, and ending at the base of the apicalgroove. In the hyposome, there were 48 ridges that



https://youtu.be/eLK5FMGNtTI


FIG. 1. Light micrographs of Balechina coerulea. (a–e) Specimens from Banyuls sur Mer. (a and b) Isolated cell FG754 (GenBank acces-sion number #KR139785). (f–i, o) Specimens from Marseille. (j–n) Specimen from Valencia. (p–w) Specimens from S~ao Sebasti~ao Chan-nel. (p) Note the different of size between the specimens. n = nucleus; scale bars, 50 lm.



extended from the base of the cingulum, and onlyabout one half of them reached the antapex(Fig. 3a). The separation between the ridges was ~6and 3 lm in the junction of the sulcus and episomeand hyposome, respectively. The apex was free ofridges and showed an apical groove with a shapealmost circular that encircled the apex (Fig. 3b).The diameter of the apical groove was ~14 lm

(Fig. 3c). In dorsal view, the groove was not dis-sected by any ridge (Fig. 3c).We tried to culture B. coerulea by feeding it with

different microalgae under laboratory conditions.The specimens of B. coerulea showed the typicalbiconical shape of the non-stressed specimens andeven they divided during the first days. However,after the first day, the specimens became colorless

FIG. 2. Light micrographs of Balechina coerulea. (a–e, h–n) Specimens from Banyuls sur Mer. (f) Specimens from Marseille. (m–q) Spec-imens from Valencia. (g, s–u) Specimens from S~ao Sebasti~ao Channel. (a–f) Dividing cells by oblique binary fission. (b, c) Specimen withan elongate apex, similar to Gymnodinium canus. (e, f) Two daughter cells before separation. (f) Depigmentation. Note that the coloredspheres remained in the apex and antapex. (g) Two pairs of daughter cells. The arrow points the elongate episome attached to the epi-some of the daughter cell. (h) Blue color is bleached. (i, j) Change in coloration and shape of a single specimen. Isolated cell FG764(GenBank accession number #KR139786). (k–m) Serial micrographs of the depigmentation and shape change in a single specimen. (m)Note the dissolution of the pigment in the surrounding water. (m–r) Serial pictures of the autotomy of a single specimen. (o) The arrowpoints the place where the autotomy begins in the episome and hyposome. (q) The arrow points the transversal flagellum. (s) Specimenbeginning the autotomy. (t, u) Cells under regeneration after autotomy. n, nucleus; tf, transversal flagellum; scale bars, 50 lm.



and the large food masses were absent. We wereable to increase survival time, up to 1 week, whenB. coerulea was fed a mix of diatoms from the seawa-ter samples enriched with nutrients and other con-taminant smaller microalgae in the culture. Incontrast, B. coerulea did not survive more than 2 dwhen it was fed with clonal cultures of Rhodomonassp., Isochysis sp., dinoflagellates or diatoms.Gymnodinium lira. The cell shape of G. lira was

ellipsoidal (95 lm long, 60 lm wide, Fig. 4, a–e) orround (70 lm long, 65 lm wide, Fig. 4, f–i), with adome-shaped or hemispherical episome. The sulcusextended from the antapex to the base of the apex(Fig. 4, b and g). The surface was covered with lon-gitudinal equidistant ridges. In contrast to the otherspecies, the nucleus was located in the episome(Fig. 4c). Brownish food vacuoles were observed inthe hyposome (Fig. 4b). The cell was colorless andthe main distinctive character was the presence ofred or pink corpuscles in the periphery, especiallyat both sides of the cingulum. These corpuscles orbody inclusions were more and less globular, withheterogeneous sizes and shapes, and located in themiddle longitudinal ridges (Fig. 4i). These corpus-cles did not disappear when the cells were stressed.

Under SEM, the cell had 24 and 36 ridges in theepisome and hyposome, respectively (Fig. 3d). Theridges were separated 9 and 6 lm in the episomeand hyposome, respectively. The apex was free ofridges, except the sulcus that finished more anteri-orly than the ridges (Fig. 3e). The apical groove wasalmost circular with a diameter that ranged from 10to 13 lm. The apical groove was not visible just in

the point of junction with the anterior end of thesulcus (Fig. 3f).We have obtained the sequence of a specimen

illustrated in Figure 4, a–c (isolated cell FG1601,GenBank accession number #KR139787).Gymnodinium cf. lira. These specimens were

more slender than G. lira (95–110 lm long, 40–45 lm wide), the cell body was biconical with apointed apex (Fig. 4, j–n). The cells showed periph-eral red corpuscles and the nucleus was located inthe hyposome. Brownish food vacuoles wereobserved in the episome (Fig. 4n). During themicroscopic observations, Gymnodinium cf. lira wasoverlooked with G. lira. However, a more carefulobservation suggests significant differences withG. lira and that these specimens likely correspondedto an undescribed species with intermediate charac-teristics between B. coerulea and G. lira. Gymnodiniumcf. lira shared the cell shape, size and nucleus posi-tion with B. coerulea, and it shared the presence ofred or pink corpuscles and lack of blue pigmenta-tion with G. lira. Unfortunately, we did not isolatespecimens for single-cell PCR.Gymnodinium cucumis. The vertical hauls with a

53 lm mesh size plankton net taken at the Bay ofVillefranche sur Mer from 80 m depth to the sur-face were mainly dominated by large thecatedinoflagellates. The few exceptions were large spe-cies with a thick cell covering such as B. coerulea,G. lira, or G. cucumis that resisted to this sampletreatment that is drastic for many unarmoreddinoflagellates. G. cucumis is a distinctive speciesand it could not go unnoticed, when present in the

FIG. 3. Scanning electronmicroscopy pictures of Balechinacoerulea (a–c) and Gymnodiniumlira (d–f) from South Japan. (a)Dorsal view. (b) Detail of theepisome. (c) The arrow pointsthe apical groove. (d) Ventralview. (e) Apical view. (f) Detail ofthe apical groove; scale bars, 50lm, b, c, f; scale bars, 5 lm.



surface samples collected from other locationsexamined in this study. Consequently, the paucity ofrecords could be associated with a preferential deepwater distribution of this species.

The specimens of G. cucumis showed a slenderfusiform body of 190 lm long and 65 lm wide,slightly wider posteriorly, and tapering at both ends(Fig. 5, a–f). The episome was conical, slightlyasymmetrical with a narrow blunt apex. The hypo-some was broader with a narrow blunt antapex

(Fig. 5, a–f). The hyposome was slightly bifurcatedwith a shallow depression at the antapex (Fig. 5, cand e). The median cingulum had a descendingleft spiral course. The sulcus extended from theapex to the antapex (Fig. 5d). The cell surface wascovered with longitudinal equidistant ridges, ~18–20 of which crossed the ventral face of the episome(Fig. 5d). The specimens showed yellow-grayish pig-mentation. The nucleus was located in the hypo-some (Fig. 5a).

FIG. 4. Light micrographs of Gymnodinium lira (a–i) and Gymnodinium cf. lira (j–n). (a–c) Isolated cell FG1601 of Gymnodinium lira(GenBank accession number #KR139787) from Villefranche sur Mer. (b) The arrows point the brownish food vacuoles. (d, e) G. lira fromS~ao Sebasti~ao Channel. (f–i) G. lira from Villefranche sur Mer. (i) Note the different size of the red corpuscles. (j, k) Gymnodinium cf. lirafrom Marseille. (l, m) G. cf. lira from S~ao Sebasti~ao Channel. (n) G. cf. lira from Ubatuba. The arrow points the brownish food vacuole inthe episome. n, nucleus; scale bars, 50 lm.



A swollen cell shape was observed in moribundspecimens (Fig. 5g). In contrast to other gymnodin-ioid dinoflagellates, this species did not lyse when itwas fixed with ethanol. The cell contour was swol-len, with tentatively food masses and a well-markedrounder organelle in the middle of the hyposomethat corresponded to the nucleus (Fig. 5h). Weobtained the sequence of the specimen shown inFigure 5, a–c (isolated cell FG1602, GenBank acces-sion number #KR139788).Balechina pachydermata. In all the sampling

areas, we observed specimens with common charac-teristics: large size, hyposome larger than the epi-some, thick cell covering with a double-layerstructure, cingular displacement of ~4 cingularwidths, sulcus extended to the antapex and slightlyintruding onto the episome as an anterior sulcalnotch, nucleus placed in the hyposome, distinctiveochre pigmentation and prominent food vacuoles.It was problematic to assign these specimens toA. vasculum, G. amphora, G. dogielii, B. pachydermataor Gymnodinium gracile sensu Kofoid and Swezybecause of the occurrence of intermediate formsbetween these species, and the only available illus-trations are restricted to the original line drawings

(see Appendix S1). Our specimens showed a sud-den contraction at the cingulum level (Figs. 6, s–y;7, g–h and k–n; see Video S2 in the SupportingInformation, http://youtu.be/FDytvHEJsFg). Conse-quently, the specimens changed from the morphol-ogy of one described species to another in 1 s.Within this context, we assigned the specimens tothe five morphotypes described mainly based on theshape of the episome.The specimens with a reduced and almost low con-

ical episome and a conical hyposome were assignedto G. amphora (Figs. 6, a–j; 7, a, b, d–i, o). The iso-lated cells FGB8, FGB9, and FGH11 corresponded tothe Figure 7, e–g, respectively (GenBank accessionnumbers #KR139790, #KR139791, #KR139792). Spec-imens from the same sample had different sizes(Fig. 7g). When this species was under division, thedaughter cells showed the shape typical of G. am-phora (Figs. 6h and 7j). We assigned the specimenswith a triangular episome and a wide hyposome toA. vasculum (Fig. 7c), and we assigned the specimenswith a dome-shaped or hemispherical episome toB. pachydermata (Fig. 6, p–r), which included theisolated cell FGB22 (GenBank accession number#KR139789; Fig. 6r). The specimens with an

FIG. 5. Light micrographs ofGymnodinium cucumis from theBay of Villefranche sur Mer. (a–c) Live specimen used for single-cell PCR, isolated cell FG1602(GenBank accession number#KR139788). (d–f) Another livespecimen. (g) A moribundspecimen. (h) Specimen fixed inethanol. n, nucleus; scale bars,50 lm.



http://youtu.be/FDytvHEJsFg






elongate episome were assigned to Gymnodiniumdogielii (Fig. 6, l–n). Specimens with dome-shapedepisome specimens corresponded to G. gracile sensuKofoid and Swezy (Fig. 6k). Other specimens wereintermediate forms between Gymnodinium dogielii andG. amphora (Fig. 6o). The cell lengths varied fromthe smaller forms of B. pachydermata (110 lm long,Fig. 6r) to the larger morphotypes of G. dogielii,G. gracile, or G. amphora (180 lm long, Fig. 7g).

When B. pachydermata entered in contact withanother cell, it showed a sudden contraction, ~1 s,at the cingulum level, with a fast change toward around shape (Figs. 6, s–y; 7, h, i and k–n; seeVideo S2, http://youtu.be/FDytvHEJsFg).We examined at high magnification the morphol-

ogy of B. pachydermata (Fig. 8). This allowed observ-ing that the cell was covered of numerous thinlongitudinal striae (Fig. 8, c and h). The transverse

FIG. 6. Light microscopy pictures of Balechina spp. from the Mediterranean Sea. (a–g) Specimens from Banyuls sur Mer. (h) Specimensfrom Marseille. (i–y) Specimens from Valencia. (a–j) Gymnodinium amphora. (h) G. amphora under division. (k–n) Gymnodinium dogielii. (o)Gymnodinium amphora. (p–r) Balechina pachydermata. (r) Isolated cell FGB22 (GenBank accession number #KR139789). (s–y) Serial micro-graphs of G. amphora during the cell contraction. n, nucleus; scale bars, 50 lm.




flagellum emerged from a cavity in the hyposome(Fig. 8h). The apex showed a discontinuity in thecontour that corresponded to the apical groove(Fig. 8, e and f). The hyposome showed a kind ofrod-shaped structures that were almost radially dis-tributed, named rodlets by Kofoid and Swezy(Fig. 8, j–k).

We observed under SEM two specimens ofB. pachydermata from different samples (Fig. 9).These specimens showed a high effect of shrinkagecompared to other species. The cell surface wasbossed and the fine longitudinal striae were visibleat high magnification. The striae were separated 3–4 lm one each other (Fig. 9d). The cells showed ananterior sulcal notch (Fig. 9, c and d). In both spec-imens, the cells showed a shrunk apex and apicalgroove (Fig. 9, b and e) compared to the observa-tions of live specimens (Fig. 8, e and f). Taken intoaccount the shrinkage of the cells, the apical groove

had an elongate elliptical shape (Fig. 9, b and e).Under culture conditions with different microalgae,the cells of B. pachydermata did not survive morethan 2 d. We did not observe the mechanism ofprey capture.Molecular phylogeny. The SSU rDNA sequences of

the three isolated cells (FGB8, FGB9 and FGB11) ofG. amphora from the South Atlantic Ocean (Fig. 7,e–g) were identical among them and to thesequence of an isolated cell (FGB22) of B. pachyder-mata from the Mediterranean Sea (Fig. 6r). Thesequences of the two specimens of B. coerulea wereidentical. G. lira and G. cucumis sequences were 98%and 95% identical to that of B. coerulea, respectively.The sequences of the type species, B. pachydermata,and of B. coerulea were 92% identical.We examined the phylogenetic position of G. am-

phora, G. cucumis, G. lira, B. coerulea and B. pachyder-mata using a data set including a variety of

FIG. 7. Light microscopy pictures of Balechina spp. from the South Atlantic Ocean. (a–j) Specimens from S~ao Sebasti~ao Channel. (k–o)Specimens from Ubatuba. (a, b) Gymnodinium amphora. (c) Amphidinium vasculum. (d–i) Gymnodinium amphora. (d) Isolated cell FGB8(GenBank accession number #KR139790). (e) Isolated cell FG9 (GenBank accession number #KR139791). (f) Note the different size. Iso-lated cell FGB11 (GenBank accession number #KR139792). (g, h) Serial micrographs of a cell contraction. (j) Gymnodinium amphora underdivision. (k–n) Serial micrographs of a cell contraction. (o) Gymnodinium amphora. n, nucleus; scale bars, 50 lm.





dinoflagellate SSU rDNA sequences and rootedusing syndinean sequences as the outgroup. TheBayesian phylogenetic tree showed that B. coeruleaand B. pachydermata branched in two clades distantlyrelated; one with short branches for the identicalsequences of B. pachydermata and G. amphora, andanother with long branches for the sequences ofG. cucumis, G. lira, and B. coerulea (posterior proba-bility of 1). In this clade, G. lira and B. coerulea weresister lineages and G. cucumis occupied a basal posi-tion (Fig. 10). However, these two clades did notshow any well-supported close affiliation to otherdinoflagellate groups present in public sequencedatabases. In fact, the new sequences branchedwithin the large lineage comprising Gymnodiniales,Peridiniales, Dinophysales, and Prorocentrales butwith poor support, making it difficult to infer theaffinity with any of these orders. Nevertheless, themolecular phylogeny clearly supported that B. pachy-dermata and B. coerulea should not be placed in thesame genus or even in the same family (Fig. 10).The taxonomic affinity of these two genera remainsunclear at the moment.Taxonomic revisions. Our morphological and

molecular data strongly support the separation of thespecies B. pachydermata and B. coerulea into two

distinct genera that are not related even at the familylevel. As B. pachydermata is the type species, the genusBalechina remains for the species with the characteris-tics of the type. We provide an emended descriptionof the genus Balechina and we propose the new genusCucumeridinium to accommodate the species with thecharacteristics of G. cucumis, G. lira, and B. coerulea.Balechina Loeblich et A.R. Loeblich 1968 emend.

F. G�omez, P. L�opez-Garc�ıa, H. Takayama et D. Mor-eira.Original publication: Loeblich and Loeblich (1968,

p. 210).Diagnosis: Unarmored heterotrophic dinoflagel-

lates with a double-layer thick cell covering. The cin-gulum is descending ~4 times its width. The sulcusextends to the antapex and slightly intrudes ontothe episome as an anterior sulcal notch. The apicalgroove is elongated and elliptical. The cell surface isbossed with fine longitudinal striae, and lackedprominent ridges or furrows.Type species: B. pachydermata (Kofoid et Swezy

1921) Loeblich et A.R. Loeblich 1968.Basionym: G. pachydermatum Kofoid et Swezy (1921,

pp. 239–240, plate 3, fig. 32, text-figure AA 5).Synonyms: A. vasculum Kofoid et Swezy 1921,

G. amphora Kofoid et Swezy 1921, Gymnodinium

FIG. 8. Light microscopypictures of Balechina pachydermatafrom South Japan. (a, b) Dorsalview. Note the brownish foodvacuoles. (b, c) Ventral view. (e,f) Detail of the apex. (g) Detailof the cingular displacement. (h)Detail of the cavity of thetransversal flagellum. See arrow.(i) Detail of the nucleus in thehypotheca. (j, k) Detail of thetentative ejectile bodies in thehyposome. c, cingular groove; eb,ejectile body; fv, food vacuole; lf,longitudinal flagellum; n,nucleus; s, sulcal groove; scalebars, 50 lm (a–d), 10 lm (e–k).


dogielii Kofoid et Swezy 1921 and G. gracile sensuKofoid and Swezy. The species B. coerulea (Dogiel)F.J.R. Taylor and B. marianiae F.J.R. Taylor do notbelong to the genus Balechina.

Iconotype: Fig. 11, a, bCucumeridinium F. G�omez, P. L�opez-Garc�ıa, H.

Takayama et D. Moreira, gen. nov.Diagnosis: Unarmored heterotrophic dinoflagel-

lates with prominent longitudinal ridges or furrowsin the cell surface covering. The cingulum isdescending ~4–7 times its width, and the sulcusextends from the antapex to the base of the apex.The apex is free of ridges, and the apical groove isalmost circular.

Synonyms: Balechina sensu Taylor 1976, auct. mult.Non: Balechina Loeblich et A.R. Loeblich 1968.Etymology: cucumis, cucumeris; Latin: cucumber.

The surface furrows or ridges resemble the skin ofsome fruits of the plant family Cucurbitaceae. Thegender is neuter.

Type species: Cucumeridinium coeruleum (Dogiel1906) F. G�omez, P. L�opez-Garc�ıa, H. Takayama etD. Moreira, comb. nov., hic designatus.

Basionym: Gymnodinium coeruleum Dogiel 1906, p.35, figs. 46, 47.

Non: G. coeruleum N.L. Antipova 1955.Homotypic synonym: B. coerulea (Dogiel) F.J.R. Tay-

lor 1976Heterotypic synonyms: G. cucumis sensu Sch€utt 1895,

fig. 64.1,3,4; Balechina marianiae F.J.R. Taylor 1976;G. costatum Kofoid et Swezy 1921; G. canus Kofoid etSwezy 1921.

Iconotype: Fig. 11, c, dOther species of the genus:Cucumeridinium cucumis (F. Sch€utt 1895) F.

G�omez, P. L�opez-Garc�ıa, H. Takayama et D. Mor-eira, comb. nov.

Basionym: G. cucumis F. Sch€utt 1895, p. 116, pl. 21,fig. 64.2. Sch€utt, F. 1895. Die Peridineen der Plank-ton-Expedition. Ergebnisse der Plankton-Expedition

der Humboldt-Stiftung 4:1–170, Lipsius and Tei-cher, Kiel.Non: G. cucumis F. Sch€utt 1895, pl. 21, figs. 64.1,

64.3, 64.4.Heterotypic synonym: B. coerulea sensu Taylor 1976.Cucumeridinium lira (Kofoid et Swezy 1921) F.

G�omez, P. L�opez-Garc�ıa, H. Takayama et D. Mor-eira, comb. nov.Basionym: G. lira Kofoid et Swezy 1921, p. 227,

text-figure Z 11, pl. 3, fig. 30. Kofoid, C.A. & Swezy,O. 1921. The free-living unarmored Dinoflagellata.Memoirs of the University of California 5: 1–562.Non: G. lira Kofoid et Swezy 1921, p. 160, 162,

text-fig. W1, 2.An undescribed species of this genus could be

Gymnodinium cf. lira (Fig. 4, j–n) that may corre-spond to G. lira sensu Kofoid et Swezy 1921, p. 160,p. 162, text-fig. W1, 2 (non G. lira Kofoid et Swezy1921, p. 227, text-figure Z: 11, plate 3, fig. 30).Gymnodinium cf. lira differed from Cucumedinium lirain the larger and slender cell body, conical episomewith pointed apex and the nucleus placed in thehyposome. Gymnodinium cf. lira differed fromC. coeruleum in the lack of blue or purple pigmenta-tion, and the presence of red or pink peripheralcorpuscles. We refrain to describe this species untilthe molecular data will confirm the distinction fromC. coeruleum and C. lira.

DISCUSSION

The combination of microscopic observations oflive specimens from different locations and themolecular data was able to shed light on this groupof large species with a thick cell covering and anunusual resistance to sampling and fixation. Speciessuch as C. coeruleum showed a striking pigmentationthat could easily receive the attention of theobserver. Despite these features that could facilitatethe recognition of the species, the records in the

FIG. 9. Scanning electron microscopy pictures of two specimens of Balechina pachydermata from South Japan. (a) Ventral view. (b)Detail of the episome. The arrow points the apical groove. The inset shows the apical groove. (c) Another specimen in ventral view. (d)Inset of the cingular and sulcal grooves. The arrows point the fine longitudinal striae. (e) The arrow points the apical groove. The insetshows the apical groove; scale bars, 50 lm (a, c), 10 lm bars (b, d, e).


FIG. 10. Bayesian phylogenetic tree of dinoflagellate SSU rDNA sequences, based on 1,166 aligned positions. Names in bold representsequences obtained in this study. Numbers at nodes are posterior probabilities (values <0.50 are omitted). Accession numbers are pro-vided between brackets. The scale bar represents the number of substitutions for a unit branch length.

FIG. 11. Line drawings ofBalechina pachydermata (a, b) andCucumeridinium coeruleum gen. etcomb. nov. (c, d). (a, c) Ventralview. (b, d) Apical view.


literature have been scarce for most of the species.In some cases, the observations were restricted tothe original descriptions (see Appendix S1). Insome genera, the paucity of observations could beattributed to the deep ocean distributions (i.e.,Heterodinium Kofoid; G�omez et al. 2012). However,with the exception of C. cucumis, all the species ofBalechina and Cucumeridinium can also be found insurface waters in coastal areas. Cucumeridinium cucu-mis and C. coeruleum were described from the Bay ofNaples (Sch€utt 1895, Dogiel 1906), where up todate there is a high tradition of dinoflagellate stud-ies. Cucumeridinium lira, B. pachydermata and its syn-onyms were described from the pier and watersnear the Scripps Institute of Oceanography at SanDiego, California (Kofoid and Swezy 1921). B. pachy-dermata was still present at San Diego, the type local-ity, as revealed by recent environmental sequencingsurveys (GenBank accession number #KJ763266, Lieet al. 2014). All species can be found in the FrenchMarine Stations along the Mediterranean coasts.Consequently, we cannot attribute the lack of stud-ies to a remote distribution of these species far ofwell-equipped laboratories.

An alternative explanation may be that, in addi-tion to the general paucity of taxonomists interestedin non-toxic species, the high polymorphism andthe lack of micrographs make difficult the identifi-cation at the species level and many previous obser-vations have simply been pooled as Gymnodinium sp.The errors in the original descriptions of the spe-cies, often based on fixed specimens, or based onthe observation of a single or few specimens alsocontributed to the difficulties in the identification.We provided a brief summary of the problems inthe species descriptions (see details inAppendix S1). In the earlier publication of thesespecies based on preserved material, Sch€utt (1895)illustrated at least two species collected from anindeterminate place under the name Gymnodinumcucumis. The illustrations of smaller specimens cor-responded to G. coeruleum. In other cases, Sch€utt(1895) also described different taxa under the samespecies name (i.e., Dissodinium pseudolunula E.V.Swift and Pyrocystis lunula (F. Sch€utt) F. Sch€utt). Afew years later, Dogiel (1906) described G. coeruleumfrom the Bay of Naples, which is probably the sameplace where Sch€utt (1895) collected G. cucumis.Dogiel examined live specimens, and he was rightwhen he described G. coeruleum under two differentmorphologies: biconical specimens with blue pig-mentation and the stressed specimens that showedan ellipsoidal contour and lacked the pigmentation.

Kofoid showed a trend to be a splitter taxonomistand he often described new species based on theobservation of single specimens and, consequently,ignored the intraspecific variability. Kofoid andSwezy (1921) reported that they observed a highnumber of specimens of G. coeruleum. However,they did not illustrate any specimen, and only

reproduced the illustration of the ellipsoidal speci-men described by Dogiel (1906). This anomaly didnot allow us to know the intraspecific morphologicalvariability in G. coeruleum. Kofoid and Swezy (1921)described G. canus from a single specimen with blu-ish pigmentation. This species was considered to beone of the daughter cells of G. coeruleum (Fig. 2, b–d). They also described G. costatum that likely corre-sponds to a large specimen of G. coeruleum whichblue color is bleaching (Fig. 1s). These authorsdescribed G. lira with round apex and nucleus inthe episome. However, in other part of the text theyalso used the name G. lira for a specimen with apointed apex and the nucleus in the hyposome.They described five other species (A. vasculum,G. pachydermatum, G. amphora, G. dogielii, andG. gracile sensu Kofoid and Swezy) with several com-mon characters: large size, a distinct thick double-layer cell covering, low cingular displacement(descending ~4 times its width), surface lackingprominent longitudinal ridges, sulcus extended tothe antapex, and an anterior sulcal notch in the epi-some, radial rod-shaped structures, distinctive yellowor ochre pigmentation, nucleus in the hyposome,prominent food vacuoles, etc. The main differenceof these species was the shape of the episome. How-ever, Kofoid and Swezy (1921) did not observe thatthe specimens were able of a sudden contraction atthe cingulum level, so that the shape of the episomecan change from that of one species to another.Schiller (1933) created more confusion when he

illustrated G. pachydermatum with important differ-ences in the cell shape and nucleus position whencompared to the original description (seeAppendix S1). Loeblich and Loeblich (1968) didnot contribute to the taxonomy of these species,and they only proposed the new genus Balechina forthe type species of the subgenus Pachydinium. Theydid not transfer other species of the subgenus Pachy-dinium into Balechina. The genus was defined basedon the characteristics of G. pachydermatum, such asthe lack of prominent longitudinal ridges. Taylor(1976) examined net samples of formalin fixedspecimens. He proposed to class the speciesG. coeruleum, with prominent surface ridges, into thegenus Balechina, and he described a new species,B. marianiae. However, he proposed B. marianiaebased on specimens of G. coeruleum, and he con-fused specimens of G. cucumis with G. coeruleum.Balech (1988), to whom the genus Balechina wasdedicated, did not place G. coeruleum into Balechina.In his classification, Taylor (1987) placed the genusBalechina into the family Kolkwiziellaceae that con-tains thecate species such as Herdmania J.D. Dodgeand Kolkwitziella Er. Lindem. Later, Fensome et al.(1993) placed Balechina into the order Ptychodis-cales, a mixing bag of genera with no relationamong them. For example, Brachidinium F.J.R. Tay-lor is included in that order based on its thick cellcovering. However, Brachidinium and Karenia Gert


http://www.ncbi.nlm.nih.gov/nuccore/KJ763266

Hansen et Moestrup are closely related genera, ifnot synonyms (G�omez et al. 2005, Henrichs et al.2011) but up to date, no study has reported a thickcell covering for Karenia.

C. coeruleum is an amazing dinoflagellate, mainlydue to its striking blue or purple pigmentation andits capacity of autotomy. The blue corpuscles, cyano-phores, of similar size accumulated in the peripheryof cell. The blue color fades when the cell isstressed, and the blue substance is released aroundthe cell (see Video S1, https://youtu.be/eLK5FMGNtTI). The origin and function of theblue pigmentation is uncertain. Some dinoflagel-lates show a blue–green pigmentation due to thepresence of phycobilin from cryptophyte kleptoplas-tids (Hu et al. 1980, Takano et al. 2014). However,C. coeruleum has no plastids. In June 2008 at Mar-seille, we observed a high abundance of C. coeruleumwith specimens that exhibited an intense blue pig-mentation, also coinciding with blue nauplii (seeFig. S1 in the Supporting Information). The obser-vation of parallel non-concentrated samples revealedthe bottom of the Uterm€ohl chamber covered withthe massive presence of a non-motile small microal-gae with globular or ellipsoidal shapes (4–7 lmlong) and blue–green pigmentation. Unfortunately,the limitations of the microscope and camera didnot allow obtaining better quality images (seeFig. S1). We have not observed the phenomenonagain and we cannot demonstrate that the presenceof that microalga could be related to the increase inthe abundance and intense blue pigmentation ofC. coeruleum. In metazoans such as chameleons thecolor shift is due to guanine nanocrystals (Teyssieret al. 2015).

The closest relatives of C. coeruleum, i.e., C. lira,C. cucumis and Gymnodinium cf. lira, did not showthe blue or purple pigmentation. Cucumeridiniumlira and Gymnodinium cf. lira showed red or pinkcorpuscles of different size. Their color and bright-ness were similar to those of corpuscles found insome species of Kofoidinium Pavillard (G�omez andFuruya 2007). The accumulation of reddish corpus-cles is common in heterotrophic dinoflagellatessuch as Protoperidinium Bergh (Neveux and Soyer1976, Carreto 1985).

We observed prominent food vacuoles, most ofthem with a distinctive brownish color, in specimensof C. coeruleum from field samples. However, we didnot observed the feeding behavior and the nature ofthe preys. Taylor (1976) reported that the specimenobserved of C. cucumis (misidentified as C. coeruleum)had ingested diatoms. Our cultures of C. coeruleumonly survived for 1 week when fed with a mix of dia-toms, and the cells remained colorless since the firstday. We collected the specimens using a planktonnet (=>20 lm mesh pore size) and the samples wereconcentrated in settling samples. This proce-dure excluded the observation of the smaller sizefraction of organisms (i.e., blue–green cyanobacteria,

cryptophyte), so that we could not observe the fullrange of potential prey for C. coeruleum in the naturalsamples.Another amazing phenomenon in C. coeruleum was

the ability of autotomy, which to the best of ourknowledge was unknown in other unarmoreddinoflagellates. The autotomy was known in thecatedinoflagellates such as Tripos Bory, which is able to cuttheir antapical and apical horns (Kofoid 1908). How-ever, in this case the autotomy was only the voluntaryshedding of the horns (i.e., appendages or bodyextensions), and it did not affect to the main body ofthe cell. By contrast, C. coeruleum, that does not havebody extensions, was able to separate one part of thehyposome, and sometimes also one part of the epi-some, and the cell did not lyse during the process(Video S1, https://youtu.be/eLK5FMGNtTI). Theobservation of incomplete specimens in healthy con-ditions revealed that the cell is able to regenerate afterautotomy (Fig. 2, t, u). The autotomy seems to be amoderate response to stress. When the cells werehighly stressed, they lysed as the typical gymnodinoiddinoflagellates. However, in contrast to other unar-mored dinoflagellates, the lysis was slower and the cellmembrane seemed to be more resistant. In terrestrialanimal ecology, autotomy is considered an anti-preda-tor mechanism, which is literally left holding a part ofthe intended victim’s body. For example, somerodents, salamanders, and lizards autotomize theirtails, and the thrashing tail often distracts the preda-tor while the prey escapes (Fleming et al. 2007). How-ever, the possible advantage of this phenomenon ofautotomy in this unarmored dinoflagellate was uncer-tain. These unique characteristics, the combination ofthe blue or purple pigmentation and the autotomy,may be related to its ecological success becauseC. coeruleum is more ubiquitous and abundant than itscongeneric species.B. pachydermata showed a sudden contraction at

the cingulum level, with a fast change in cell shape(see Video S2, http://youtu.be/FDytvHEJsFg). Thecontraction of B. pachydermata seemed to be a defen-sive strategy in response to the approach of a poten-tial predator. Balechina pachydermatum ischaracterized by a thick double-layer cell coveringwith a rough surface and fine longitudinal striae.This ultrastructural feature may facilitate the suddencontraction without damage or lysis of the cell. Thiscontraction was common in some noctilucoiddinoflagellates such as the leptodiscaceans (i.e.,Scaphodinium Margalef, Abedinium Loeblich et A.R.Loeblich; G�omez and Furuya 2004, G�omez et al.2010). These noctilucoid dinoflagellates possessed anet of myofibrils that facilitated the change in shape(Cachon and Cachon 1967). Kofoid and Swezy(1921) illustrated A. vasculum, G. pachydermatum,G. dogielii, G. amphora, and G. gracile with a kind ofalmost concentric radial filaments that theydescribed as long slender rodlets or radial canals.Kofoid and Swezy did not refer to the nature of






these organelles. This organelle was visible inB. pachydermata under high magnification (Fig. 8, j,k). The shape and position, mainly around thenucleus, resembled those observed in Abedinium(G�omez et al. 2010). These structures in leptodis-caceans were ejectile bodies used for prey capture(Cachon and Cachon 1969). In B. pachydermata,these organelles were considered to be ejectile bod-ies used for the prey capture, more so than myofib-ril-like structures.

This study summarized the observations from sev-eral regions of the world’s oceans. Studies will con-tinue to obtain information of the ultrastructure,especially the thick cell covering, and ecological sig-nificance of the pigmentation, autotomy, contrac-tion, or ejectile bodies of these amazingdinoflagellates.

F.G. was supported by the Brazilian Conselho Nacional deDesenvolvimento Cient�ıfico e Tecnol�ogico (grant numberBJT 370646/2013-14). We acknowledge financial supportfrom the French CNRS, the European Research Councilunder the European Union’s Seventh Framework ProgramERC Grant Agreement 322669 ‘ProtistWorld’, and Ile deFrance (SESAME project 13016398 “Unicell”).

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Supporting Information

Additional Supporting Information may befound in the online version of this article at thepublisher’s web site:

Figure S1. Unidentified microalgae coincidingwith the proliferation of Gymnodinium coeruleum atMarseille in August 2008.

Appendix S1. Taxonomic, nomenclatural, andbiogegraphical account of Balechina spp. andrelated species.

Video S1. Cucumeridinium coeruleum, https://youtu.be/eLK5FMGNtTI.

Video S2. Balechina pachydermata, http://you-tu.be/FDytvHEJsFg.






Balechina Cucumeridinium

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