gomez 2013 pronoctiluca

Acta Oceanol. Sin., 2013, Vol. 32, No. 4, P. 71-76

DOI: 10.1007/s13131-013-0300-x

http://www.hyxb.org.cn

E-mail: [email protected]

Morphology and distribution of Pronoctiluca (Dinoflagellata,incertae sedis) in the Pacific OceanGÓMEZ Fernando1∗1 Department of Biological Oceanography, Institute of Oceanography, University of São Paulo, São Paulo,

SP 05508-120, Brazil

Received 23 December 2011; accepted 11 July 2012

©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2013

AbstractThe diversity and distribution of Pronoctiluca, a marine dinoflagellate of enigmatic systematic position, wasstudied in the vicinity of the Kuroshio and Oyashio Currents, the Philippines, Celebes, Sulu and South ChinaSeas, western and central equatorial and southeast Pacific Ocean. The abundance of Pronoctiluca was high-er, with a wide vertical distribution, in eutrophic temperate regions, whereas it was nearly absent in surfacewaters of oligotrophic tropical regions. Most of the specimens corresponded to P. spinifera. Pronoctilucapelagica, covered by hyaline layers and with no flagella, is considered as an encysted form. The bipartitionwas only observed in P. acuta-P. spinifera forms, that together with the occurrence of intermediate formsbetween P. spinifera and P. pelagica suggest that they may correspond to developmental stages of a singlespecies. Pronoctiluca is essential to understanding the evolutionary history of the alveolates.Key words: Alveolata, basal dinoflagellates, Dinophyceae, heterotrophic flagellate, Noctilucales, plankton

Citation: Gómez F. 2013. Morphology and distribution of Pronoctiluca (Dinoflagellata, incertae sedis) in the Pacific Ocean. ActaOceanologica Sinica, 32(4): 71–76, doi: 10.1007/s13131-013-0300-x

1 IntroductionPronoctiluca Fabre-Dom. is considered as an aberrant or

primitive dinoflagellate with an enigmatic systematic position.This heterotrophic protist possesses an antero-ventral tentacle,a short sulcus and no cingulum. Two flagella originating nearthe anterior ventral surface, one long and other short, are as-sumed to be the transverse and longitudinal flagella, respective-ly. The nucleus was large, with bead-like chromatin granules,as the typical dinokaryon of dinoflagellates (Kofoid and Swezy,1921; Takayama, 1998). In contrast, Fensome et al. (1993)reported that Pronoctiluca was not a dinoflagellate because itlacked the dinokaryon. Pronoctiluca, placed in the family Pro-todiniferaceae Kof. & Swezy (=Pronoctilucaceae M. Lebour),comprised the type P. pelagica Fabre-Dom., P. acuta (Lohman-n) J. Schiller, P. spinifera (Lohmann) J. Schiller (=P. tentacula-ta (Kof. & Swezy) Pavill.) and P. rostrata F.J.R. Taylor (Gómez,2012). In the classical taxonomical schemes, Pronoctiluca hasbeen placed in the dinoflagellate order Gymnodiniales (Kofoidand Swezy, 1921) or Noctilucales (Sournia, 1986; Taylor, 1987;Steidinger and Tangen, 1997). Oxyrrhis marina Dujard. hasbeen placed in the same family as Protodiniferaceae or Pronoc-tilucaceae (Kofoid and Swezy, 1921; Dodge, 1982).

The recent advances in molecular phylogeny are largelyrestricted to easily accessible species available in cultures orthose that bloom in coastal waters close to the specialized lab-oratories. Molecular phylogeny revealed that the members ofthe order Noctilucales (Noctiluca scintillans (Macartney) Kof.,Spatulodinium Cachon & Cachon-Enjumet) constitutes a basal

group of the dinoflagellate core (Gómez et al., 2010a). OxyrrhisDujard. has been intensively investigated from cultures, andplaced as an early branch of the dinoflagellate lineage in themolecular phylogenies (Saldarriaga et al., 2003a; Lowe et al.,2011). Very little is known about the ecological distribution andmorphological diversity of Pronoctiluca. The present study re-ports a first estimation of the abundance, vertical and spatialdistributions in several contrasting regions in both hemispheresin the Pacific Ocean between sub-arctic to the equator. Al-though Pronoctiluca is easily recognizable at the genus level, itis more difficult to determine how many valid species comprisethis genus. These new observations contribute to the knowl-edge of this unique marine protist, and to facilitate the recordsand recognition for the necessary molecular studies.

2 Materials and methodsSamples were collected during 11 cruises in the Pacific O-

cean. Sample collection and light microscopic methods and en-vironmental conditions were described in the previous studies(Gómez and Furuya, 2007; Gómez et al., 2007). Water samples ateach station collected using Niskin bottles were preserved withacidified Lugol’s solution and stored at 5◦C. Samples of 500 mlwere concentrated by sedimentation in glass cylinders. Duringa six-day settling period, the top 450 ml of the sample was pro-gressively and slowly siphoned off with small-bore tubing. Fiftymilliliters of the concentrate representing 500 ml whole watersample was settled in composite settling chambers. The entirechamber was scanned at 200×magnification under a Nikon orOlympus inverted microscope equipped with a digital camera.

Foundation item: A Grant-in-aid for Creative Basic Research from the MEXT, Japan under contract No.12NP0201, DOBIS (studies with regard to theNW and Equatorial Pacific Ocean); the Project BIOSOPE of the LEFE-CYBER (studies with regard to the SE Pacific Ocean); the Ministerio Español deCienciay Tecnología under contract No. JCI-2010-08492; Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico BJT under contractNo. 370646/2013-14.∗ Corresponding author, E-mail: [email protected]

1

72 GÓMEZ Fernando Acta Oceanol. Sin., 2013, Vol. 32, No. 4, P. 71-76

Fig.1. Map of the station locations in the Pacific Ocean (marked by circles).

3 Results

3.1 Diversity of PronoctilucaThe specimens have been grouped in four morphotypes

under the assumption that they may be morphotypes of a sin-gle species:

Pronoctiluca spinifera (Figs 2a–j)In the present study the morphology of more of 9/10 of the

records of Pronoctiluca corresponded to fusiform cells, lengthabout two or three times the diameter, with a short slender an-terior tentacle that can flex considerably. Two flagella originat-ed near the anterior ventral surface. The specimens showed aposterior-pointed projection, hereafter named posterior spine.The nucleus was large, 2–3 times greater in length than width,and located longitudinally in the anterior cell body. One large orseveral smaller accumulation bodies were often observed in theposterior portion of the cell. The specimen illustrated in Figs 2i–j was apparently in a stage prior to the ejection of an accumu-lation body. Some specimens show a “feeding veil” that can bealso found in other heterotrophic dinoflagellates (Fig. 2h). Thecell dimensions of these specimens were 30–60 µm long and 9–22µm wide. These specimens have been ascribed to P. spinifera.None of the specimens of P. spinifera appeared encysted or cov-ered by a hyaline layer.

Pronoctiluca rostrata (Figs 2k–o)Some large specimens, 60–140 µm in length, showed a

long tubular tentacle that culminated by a short and thin cylin-drical terminal extension (Figs 2l–m). The mid-body was broad-est in the centre, tapering equally at both ends. The cell bodyshowed a hyaline area that occupied about one half of the cel-l body, located laterally (Figs 2l–m) or occupying the posteriorhalf of the cell body (Fig. 2n). From a conical antapex emergeda long spine with a pointed tip (Figs 2l–n). The spine was direct-ed posteriorly (Figs 2l–m) or obliquely from the lateral posteriorcell body (Figs 2n–o). The specimens were in concordance withthe description of P. rostrata.

Intermediate forms P. spinifera-P. pelagica (Figs 2o–q)Pronoctiluca pelagica mainly differs from P. spinifera in

lacking the posterior spine. Several specimens show the pos-terior spine of P. spinifera, although the cell body was ovate orglobular, close to P. pelagica, instead of fusiform as usual in P.spinifera. The posterior accumulation body in some specimensshow concentric circles (Fig. 2r). All these specimens were con-

sidered to be intermediate stages between of P. spinifera and P.pelagica.

Pronoctiluca pelagica (Figs 2s–y)Other specimens with a pyriform cell body lacking the

posterior spine were identified as P. pelagica (Figs 2s–y). Thetransdiameter was 12–20 µm. The anterior tentacle was orient-ed in different angles. No flagella were observed. The posteri-or half of the cell body was occupied by a large accumulationbody. Its colour in these Lugol-fixed specimens varied fromdark brown (Figs 2l–u, w) to hyaline (Fig. 2y). The specimensshowed one or several hyaline layers covering the posterior endof the cell. This suggests that P. pelagica may constitute an en-cysted form originat after the shape change and retraction ofthe posterior spine of P. spinifera (Figs 2l–y).

Specimens of cells in division (Figs 2z–ag)Numerous specimens were observed in different phases

of the cell division cycle. All these specimens corresponded toP. spinifera or P. acuta and the flagella remained during all thephases. During the cell division the anterior tentacle was ab-sent and it did not appear until the separation of the daugh-ter cells (Figs 2z–ab). The anterior end showed a conical shapetapering progressively to a point, instead of a spine. This out-line resembled P. acuta (Figs 2z–ab). These elongated heart-likeshape specimens began to bifurcate longitudinally in the ante-rior end (Figs 2ac–ad). The nucleus was divided into two longi-tudinal ellipsoidal nuclei that migrated into each daughter cell.The posterior portion of the cell body showed a marked accu-mulation body (Fig. 2ad). In the next step, the entire cell divid-ed longitudinally. One of the daughter cells received the accu-mulation body from the parent cell (Figs 2ae–ag). Consequentlythe posterior accumulation body was pigmented in one daugh-ter cell and hyaline in the other one (Figs 2ae–ag). The cell pairsshowed an ellipsoidal shape and they remained attached in thelongitudinal plane at the level of the middle part of the anteri-or cell body. The anterior tentacle reappeared in each daughtercell. The marginal end of the tentacle was curved and projectedin opposite directions in each daughter cell. The acute posteri-or end of each daughter cell was transformed into the posteriorspine (Figs 2ae–ag).

3.2 Distribution of PronoctilucaA total of 397 specimens of the genus Pronoctiluca were

GÓMEZ Fernando Acta Oceanol. Sin., 2013, Vol. 32, No. 4, P. 71-76 73

Fig.2. Photomicrographs, bright-field optics, of the morphotypes of Pronoctiluca. See localization of the records in Table 1. a–j.P. spinifera. The arrow in Fig. 2h indicates the “feeding veil”. k–n. P. rostrata. The inset in Fig. 2l shows the marginal part of thetentacle. o–r. Intermediate forms between P. spinifera and P. pelagica. s–y. P. pelagica. The arrow in Figs 2s and v indicates hyalinelayers around the cell body. z–ag. Specimens of P. acuta and P. spinifera under division. See the marked accumulation body in theposterior part in Fig. 2ad. The arrows in Figs 2ae–af indicate the variable degree of pigmentation of the posterior accumulation bodyin the cell pairs. Scale bars=20 µm.

encountered. A latitudinal transect in the vicinity of theKuroshio Current in the south of Japan (138◦E) was investigated in May and July 2002. In May, 103 individuals of Pronoc-tiluca were found from 131 samples analysed (Fig. 3a). During

the cruise in July, the stations were re-visited and 42 specimenswere found in 144 samples analyzed (Fig. 3b). During the cruisein the marginal seas of the western Pacific, nine specimens wereobserved from 81 samples (Fig. 3c). In the western and central


Fig.3. Section plots of the records of Pronoctiluca in the Pacific Ocean indicated by filled rhombuses (see also Fig. 1). a. Recordsalong the meridian 138◦E in May, b. records from the same location in July, c. records from the Celebes, Sulu and South China Seas,d. records from the western and central equatorial Pacific, and e. records from the southeast Pacific. Isotherms are shown.

Table 1. Depth (m) and geographic coordinates (latitude, longitude) of the morphotypes of Pronoctiluca illustrated in Figs a–ag

Taxon Depth Latitude Longitude Fig. 2 Taxon Depth Latitude Longitude Fig. 2

P. spinifera 200 30◦N 138◦E a P. spinifera-P. pelagica 50 0◦ 160◦E r

P. spinifera 40 0◦ 160◦E b P. pelagica 150 0◦ 165◦E s

P. spinifera 50 0◦ 175◦E c P. pelagica 100 33◦N 138◦E t

P. spinifera 150 5◦N 121◦E d P. pelagica 200 30◦N 138◦E u

P. spinifera 100 30◦N 138◦E e P. pelagica 10 32◦30′N 138◦E v

P. spinifera 200 33◦45′N 138◦E f P. pelagica 45 23◦32′S 117◦52′W w

P. spinifera 200 0◦ 165◦E g P. pelagica 100 41◦30′N 145◦47′E x

P. spinifera 30 32◦42′S 84◦04′W h P. pelagica 60 0◦ 175◦W y

P. spinifera 150 33◦N 138◦E i–j P. acuta (division) 50 32◦42′S 84◦04′W z

P. rostrata 60 33◦21′S 78◦06′W k P. acuta (division) 70 32◦42′S 84◦04′W aa

P. rostrata 110 0◦ 170◦E l–m P. acuta (division) 15 33◦21′S 78◦06′W ab

P. rostrata 100 33◦21′S 78◦06′W n P. acuta (division) 100 34◦20′N 138◦E ac–ad

P. rostrata 60 34◦20′N 138◦E o P. spinifera (pairs) 25 33◦54.7′S 73◦21.8′W ae

P. spinifera-P. pelagica 150 30◦N 138◦E p P. spinifera (pairs) 30 5◦13′N 120◦46′E af–ag

P. spinifera-P. pelagica 100 41◦30′N 145◦47′E q

equatorial Pacific, 11 specimens were found from 124 samples(Fig. 3d). In the southeast Pacific, 230 specimens of Pronoc-tiluca were found in the 100 samples analyzed (Fig. 3c). Sam-ples from six cruises carried out off Hokkaido (north of Japan)were also analyzed. A very few specimens of P. pelagica wereobserved in these cold waters under the influence of the sub-arctic Oyashio Current (Figs 2q, x).

In the Kuroshio Current and adjacent waters in the southof Japan, the number of specimens was higher in spring than insummer (Figs 3a–b). The highest abundance was encounterednear the slope waters in May (20 cells/L) at 60 m-depth (34◦N,138◦E) and in July (15 cells/L) at 30 m-depth (34◦25N, 138◦E).The records of Pronoctiluca in the Southeast Asia marginal seaswere too scarce to discern a pattern in the distribution (Fig. 3c).

GÓMEZ Fernando Acta Oceanol. Sin., 2013, Vol. 32, No. 4, P. 71-76 75

As a general trend, the records occurred below 50 m-depth inthe oligotrophic waters of the western and central equatorialPacific, and in the South Pacific Gyre (Fig. 3d). The abundancewas higher in more eutrophic regions, i.e., the vicinity of theMarquesas Islands and in the Perú-Chile Current, with valuesup 40 cells/L. The highest abundance, 120 cells/L, was encoun-tered in the Chilean upwelling off Concepción (Fig. 3e).

4 Discussion

4.1 Geographical and ecological distributionThe abundance of Pronoctiluca was higher in the more eu-

trophic areas. In contrast, Pronoctiluca was nearly absent inthe surface waters of the western Equatorial Pacific sampledduring the El Niño conditions and in the South Pacific Gyre, t-wo of the most oligotrophic regions in the world oceans. Fora same region, i.e., the vicinity of Kuroshio Current the abun-dance was higher in spring, a more productive period than sum-mer. The highest abundance was encountered in the Chileanupwelling off Concepción. Takayama (1998) reported that P.spinifera reached an abundance of 3 000 cells/L in the south-ern coast of Japan. In the cold sub-arctic waters of the OyashioCurrent off Hokkaido, despite of the high eutrophic conditions,only a few specimens were encountered. Pronoctiluca is a het-erotrophic species which development seems to be favoured inareas of high prey availability.

4.2 How many species of Pronoctiluca?In the present study, the specimens of P. spinifera prior

to the cell division showed an elongated pointed posterior endthat resembled the outline of P. acuta (Figs 2z–ab). It is hy-pothesised that P. acuta constitutes a morphotype of P. spinifer-a. The little known Pronoctiluca rostrata, the larger species, wasdescribed from a few formalin-preserved specimens collectedby net sampling from the Indian Ocean (Taylor, 1976). This i-nappropriate fixation method for unarmoured dinoflagellatesis responsible of the highly deformed outline of the specimens.Saldarriaga et al. (2003b) doubted on the validity of P. rostra-ta as a Pronoctiluca species and they related it to the armoureddinoflagellate Lessardia elongata J.F. Saldarriaga & F.J.R. Taylor.The latter taxon is a member of the peridinian family Podolam-padaceae (Gómez et al., 2010b). The length of P. rostrata wasfour or five times as long as L. elongata. Pronoctiluca rostratadid not possess a cingulum, whereas L. elongata showed an e-quatorial cingulum. This suggests that P. rostrata is a truly mem-ber of Pronoctiluca. The cylindrical tentacle of P. rostrata is di-vided into two sections of different diameter (Fig. 2l). Takayama(1998) by scanning electron microscopy revealed the two sec-tions, the proximal more reduced, in the tentacle of P. spinifera.Consequently doubts appear on the validity of P. rostrata as aseparate species because it seems to be a large morphotype ofP. spinifera (Figs 2k–m).

While the records of the previous taxa are scarce, Pronoc-tiluca pelagica and P. spinifera are more commonly reported.The lack of cultures and molecular data render our knowledgeon the intraspecific morphological variability incomplete, andthe co-specificity of P. pelagica and P. spinifera remains unclear.Pavillard (1922) illustrated P. spinifera and P. pelagica as a singlespecies. Kofoid and Swezy (1921) erected the genus ProtodiniferKof. & Swezy with the species P. tentaculatum (=Pronoctilucaspinifera) and P. marinum (=Pronoctiluca pelagica). Accordingto these authors the difference between the species was a stouttentacle for body length of 54 µm for P. tentaculatum, whereas P.

marinum showed a slender tentacle and the body length 12–40µm. Based on these diagnostic characters, P. tentaculatum andP. marinum are synonyms because the length constitute a poordiagnostic criterion, and the tentacle shape change along thecell development (Fig. 2). There also have been questions asto whether P. pelagica is the encysted form of P. spinifera (Figs2s–y). All the specimens, observed and in division, correspond-ed to the form P. spinifera as reported in other studies (Herreraand Margalef, 1963) and there are no illustrations of P. pelagicadividing cells. Pronoctiluca pelagica lacking the flagella, is cov-ered with hyaline layers, and intermediate forms of P. spinifera-P. pelagica are here illustrated (Figs 2o–r). This suggests thatPronoctiluca is considered as a monotypic genus. Although P.spinifera is the most common morphotype, the encysted formP. pelagica may have the priority.

4.3 Systematic position of PronoctilucaThe etymology of the name Pronoctiluca suggests affini-

ty with Noctiluca scintillans (Macartney) Kofoid. Noctilucalesare characterized by a locomotion based on rapid changes ofthe cell shape (Gómez et al., 2010a). The tentacle of Pronoc-tiluca that can be moved in all directions, the lack of cingulumand the numerous vacuoles are the main similarities with Noc-tiluca scintillans. Fensome et al. (1993) reported “Pronoctilu-ca has been included in this family by some previous authors(e.g., Loeblich, 1982) primarily because, like Noctiluca, it hasa single flagellum”. This comment was unfortunate becausePronoctiluca possesses two flagella. Kofoid and Swezy (1921)observed live specimens and reported that the cell displace-ment differed from the typical dinoflagellate rotation and theswimming behaviour was similar to desmokont cells of Proro-centrum Ehrenberg. Oxyrrhis marina, considering the closerrelative of Pronoctiluca, was placed in the family Pronoctilu-caceae. Dodge (1982) placed it together to Pronoctiluca in thefamily Pronoctilucaceae of the Gymnodiniales. The flagella ofOxyrrhis are only slightly dissimilar and inserted posteriorly. Atrue undulate transverse flagellum is not present. Oxyrrhis haslong been considered to be a dinoflagellate, but it was excludedby Fensome et al. (1993) because of its lack of most dinoflagel-late features such as the chromosomes decondensation duringinterphase, which has an intranuclear spindle, and lacks a girdleand sulcus. Morphology and molecular phylogeny reveal thatOxyrrhis is an early branch in the dinoflagellate lineage (Sal-darriaga et al., 2003a; Lowe et al., 2011). Oxyrrhis is dividedby transverse binary fission, whereas the dinoflagellates tendto divide longitudinally or obliquely. In contrast, Pronoctilucapossesses a nucleus with chromatin strands, two anteriorly in-serted dissimilar flagella, a short sulcus, and the cell divisionis longitudinal, being closer to the characteristics of the typicaldinoflagellates. Molecular data of Pronoctiluca are an essentialissue to understand the evolutionary history of alveolates.

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