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: hyxbe@263.net

Morphology and distribution of Pronoctiluca (Dinoflagellata, incertae sedis) in the Pacific Ocean
GÓMEZ 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

Abstract The diversity and distribution of Pronoctiluca, a marine dinoflagellate of enigmatic systematic position, was studied in the vicinity of the Kuroshio and Oyashio Currents, the Philippines, Celebes, Sulu and South China Seas, western and central equatorial and southeast Pacific Ocean. The abundance of Pronoctiluca was higher, with a wide vertical distribution, in eutrophic temperate regions, whereas it was nearly absent in surface waters of oligotrophic tropical regions. Most of the specimens corresponded to P . spinifera. Pronoctiluca pelagica, covered by hyaline layers and with no flagella, is considered as an encysted form. The bipartition was only observed in P . acuta-P . spinifera forms, that together with the occurrence of intermediate forms between P . spinifera and P . pelagica suggest that they may correspond to developmental stages of a single species. 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. Acta Oceanologica Sinica, 32(4): 71–76, doi: 10.1007/s13131-013-0300-x

1 Introduction Pronoctiluca 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 near the anterior ventral surface, one long and other short, are assumed to be the transverse and longitudinal flagella, respectively. 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 it lacked the dinokaryon. Pronoctiluca, placed in the family Protodiniferaceae Kof. & Swezy (=Pronoctilucaceae M. Lebour), comprised the type P . pelagica Fabre-Dom., P . acuta (Lohmann) J. Schiller, P . spinifera (Lohmann) J. Schiller (=P . tentaculata (Kof. & Swezy) Pavill.) and P . rostrata F.J.R. Taylor (Gómez, 2012). In the classical taxonomical schemes, Pronoctiluca has been placed in the dinoflagellate order Gymnodiniales (Kofoid and Swezy, 1921) or Noctilucales (Sournia, 1986; Taylor, 1987; Steidinger and Tangen, 1997). Oxyrrhis marina Dujard. has been placed in the same family as Protodiniferaceae or Pronoctilucaceae (Kofoid and Swezy, 1921; Dodge, 1982). The recent advances in molecular phylogeny are largely restricted to easily accessible species available in cultures or those that bloom in coastal waters close to the specialized laboratories. Molecular phylogeny revealed that the members of the order Noctilucales (Noctiluca scintillans (Macartney) Kof., Spatulodinium Cachon & Cachon-Enjumet) constitutes a basal

group of the dinoflagellate core (Gómez et al., 2010a). Oxyrrhis Dujard. has been intensively investigated from cultures, and placed as an early branch of the dinoflagellate lineage in the molecular phylogenies (Saldarriaga et al., 2003a; Lowe et al., 2011). Very little is known about the ecological distribution and morphological diversity of Pronoctiluca. The present study reports a first estimation of the abundance, vertical and spatial distributions in several contrasting regions in both hemispheres in the Pacific Ocean between sub-arctic to the equator. Although Pronoctiluca is easily recognizable at the genus level, it is more difficult to determine how many valid species comprise this genus. These new observations contribute to the knowledge of this unique marine protist, and to facilitate the records and recognition for the necessary molecular studies. 2 Materials and methods Samples were collected during 11 cruises in the Pacific Ocean. Sample collection and light microscopic methods and environmental conditions were described in the previous studies (Gómez and Furuya, 2007; Gómez et al., 2007). Water samples at each station collected using Niskin bottles were preserved with acidified Lugol’s solution and stored at 5◦ C. Samples of 500 ml were concentrated by sedimentation in glass cylinders. During a six-day settling period, the top 450 ml of the sample was progressively and slowly siphoned off with small-bore tubing. Fifty milliliters of the concentrate representing 500 ml whole water sample was settled in composite settling chambers. The entire chamber was scanned at 200× magnification under a Nikon or Olympus 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 the NW and Equatorial Pacific Ocean); the Project BIOSOPE of the LEFE-CYBER (studies with regard to the SE Pacific Ocean); the Ministerio Español de Cienciay Tecnología under contract No. JCI-2010-08492; Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico BJT under contract No. 370646/2013-14. ∗ Corresponding author, E-mail: fernando.gomez@fitoplancton.com

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Fig.1. Map of the station locations in the Pacific Ocean (marked by circles). 3 Results 3.1 Diversity of Pronoctiluca The specimens have been grouped in four morphotypes under the assumption that they may be morphotypes of a single 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, length about two or three times the diameter, with a short slender anterior tentacle that can flex considerably. Two flagella originated near the anterior ventral surface. The specimens showed a posterior-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 or several smaller accumulation bodies were often observed in the posterior portion of the cell. The specimen illustrated in Figs 2i– j was apparently in a stage prior to the ejection of an accumulation body. Some specimens show a “feeding veil” that can be also found in other heterotrophic dinoflagellates (Fig. 2h). The cell dimensions of these specimens were 30–60 µm long and 9– 22 µm wide. These specimens have been ascribed to P . spinifera. None of the specimens of P . spinifera appeared encysted or covered 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 cylindrical terminal extension (Figs 2l–m). The mid-body was broadest in the centre, tapering equally at both ends. The cell body showed a hyaline area that occupied about one half of the cell body, located laterally (Figs 2l–m) or occupying the posterior half of the cell body (Fig. 2n). From a conical antapex emerged a long spine with a pointed tip (Figs 2l–n). The spine was directed posteriorly (Figs 2l–m) or obliquely from the lateral posterior cell body (Figs 2n–o). The specimens were in concordance with the 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 posterior spine of P . spinifera, although the cell body was ovate or globular, close to P . pelagica, instead of fusiform as usual in P . spinifera. The posterior accumulation body in some specimens show concentric circles (Fig. 2r). All these specimens were considered 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). The transdiameter was 12–20 µm. The anterior tentacle was oriented in different angles. No flagella were observed. The posterior half of the cell body was occupied by a large accumulation body. Its colour in these Lugol-fixed specimens varied from dark brown (Figs 2l–u, w) to hyaline (Fig. 2y). The specimens showed one or several hyaline layers covering the posterior end of the cell. This suggests that P . pelagica may constitute an encysted form originat after the shape change and retraction of the 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 to P . spinifera or P . acuta and the flagella remained during all the phases. During the cell division the anterior tentacle was absent and it did not appear until the separation of the daughter cells (Figs 2z–ab). The anterior end showed a conical shape tapering progressively to a point, instead of a spine. This outline resembled P . acuta (Figs 2z–ab). These elongated heart-like shape specimens began to bifurcate longitudinally in the anterior end (Figs 2ac–ad). The nucleus was divided into two longitudinal ellipsoidal nuclei that migrated into each daughter cell. The posterior portion of the cell body showed a marked accumulation body (Fig. 2ad). In the next step, the entire cell divided longitudinally. One of the daughter cells received the accumulation body from the parent cell (Figs 2ae–ag). Consequently the posterior accumulation body was pigmented in one daughter cell and hyaline in the other one (Figs 2ae–ag). The cell pairs showed an ellipsoidal shape and they remained attached in the longitudinal plane at the level of the middle part of the anterior cell body. The anterior tentacle reappeared in each daughter cell. The marginal end of the tentacle was curved and projected in opposite directions in each daughter cell. The acute posterior end of each daughter cell was transformed into the posterior spine (Figs 2ae–ag). 3.2 Distribution of Pronoctiluca A total of 397 specimens of the genus Pronoctiluca were

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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 the tentacle. o–r. Intermediate forms between P . spinifera and P . pelagica. s–y. P . pelagica. The arrow in Figs 2s and v indicates hyaline layers around the cell body. z–ag. Specimens of P . acuta and P . spinifera under division. See the marked accumulation body in the posterior part in Fig. 2ad. The arrows in Figs 2ae–af indicate the variable degree of pigmentation of the posterior accumulation body in the cell pairs. Scale bars=20 µm.

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

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

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along 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.

Fig.3. Section plots of the records of Pronoctiluca in the Pacific Ocean indicated by filled rhombuses (see also Fig. 1). a. Records

Table 1. Depth (m) and geographic coordinates (latitude, longitude) of the morphotypes of Pronoctiluca illustrated in Figs a–ag
Taxon P . spinifera P . spinifera P . spinifera P . spinifera P . spinifera P . spinifera P . spinifera P . spinifera P . spinifera P . rostrata P . rostrata P . rostrata P . rostrata P . spinifera-P . pelagica P . spinifera-P . pelagica Depth 200 40 50 150 100 200 200 30 150 60 110 100 60 150 100 Latitude 30◦ N 0◦ 0◦ 5◦ N 30◦ N 33◦ 45 N 0◦ 32◦ 42 S 33◦ N 33◦ 21 S 0◦ ◦ 33 21 S 34◦ 20 N 30◦ N 41◦ 30 N Longitude 138◦ E 160◦ E 175◦ E 121◦ E 138◦ E 138◦ E 165◦ E 84◦ 04 W 138◦ E 78◦ 06 W 170◦ E 78◦ 06 W 138◦ E 138◦ E 145◦ 47 E Fig. 2 a b c d e f g h i–j k l–m n o p q Taxon P . spinifera-P . pelagica P . pelagica P . pelagica P . pelagica P . pelagica P . pelagica P . pelagica P . pelagica P . acuta (division) P . acuta (division) P . acuta (division) P . acuta (division) P . spinifera (pairs) P . spinifera (pairs) Depth 50 150 100 200 10 45 100 60 50 70 15 100 25 30 Latitude 0◦ 0◦ 33◦ N 30◦ N 32◦ 30 N 23◦ 32 S 41◦ 30 N 0◦ ◦ 32 42 S 32◦ 42 S 33◦ 21 S 34◦ 20 N 33◦ 54.7 S 5◦ 13 N Longitude 160◦ E 165◦ E 138◦ E 138◦ E 138◦ E 117◦ 52 W 145◦ 47 E 175◦ W 84◦ 04 W 84◦ 04 W 78◦ 06 W 138◦ E 73◦ 21.8 W 120◦ 46 E Fig. 2 r s t u v w x y z aa ab ac–ad ae af–ag

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

In the Kuroshio Current and adjacent waters in the south of Japan, the number of specimens was higher in spring than in summer (Figs 3a–b). The highest abundance was encountered near 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 seas were too scarce to discern a pattern in the distribution (Fig. 3c).

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As a general trend, the records occurred below 50 m-depth in the oligotrophic waters of the western and central equatorial Pacific, and in the South Pacific Gyre (Fig. 3d). The abundance was higher in more eutrophic regions, i.e., the vicinity of the Marquesas Islands and in the Perú-Chile Current, with values up 40 cells/L. The highest abundance, 120 cells/L, was encountered in the Chilean upwelling off Concepción (Fig. 3e). 4 Discussion 4.1 Geographical and ecological distribution The abundance of Pronoctiluca was higher in the more eutrophic areas. In contrast, Pronoctiluca was nearly absent in the surface waters of the western Equatorial Pacific sampled during the El Niño conditions and in the South Pacific Gyre, two of the most oligotrophic regions in the world oceans. For a same region, i.e., the vicinity of Kuroshio Current the abundance was higher in spring, a more productive period than summer. The highest abundance was encountered in the Chilean upwelling off Concepción. Takayama (1998) reported that P . spinifera reached an abundance of 3 000 cells/L in the southern coast of Japan. In the cold sub-arctic waters of the Oyashio Current off Hokkaido, despite of the high eutrophic conditions, only a few specimens were encountered. Pronoctiluca is a heterotrophic species which development seems to be favoured in areas 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 end that resembled the outline of P . acuta (Figs 2z–ab). It is hypothesised that P . acuta constitutes a morphotype of P . spinifera. The little known Pronoctiluca rostrata, the larger species, was described from a few formalin-preserved specimens collected by net sampling from the Indian Ocean (Taylor, 1976). This inappropriate fixation method for unarmoured dinoflagellates is responsible of the highly deformed outline of the specimens. Saldarriaga et al. (2003b) doubted on the validity of P . rostrata as a Pronoctiluca species and they related it to the armoured dinoflagellate Lessardia elongata J.F. Saldarriaga & F.J.R. Taylor. The latter taxon is a member of the peridinian family Podolampadaceae (Gómez et al., 2010b). The length of P . rostrata was four or five times as long as L. elongata. Pronoctiluca rostrata did not possess a cingulum, whereas L. elongata showed an equatorial cingulum. This suggests that P . rostrata is a truly member of Pronoctiluca. The cylindrical tentacle of P . rostrata is divided into two sections of different diameter (Fig. 2l). Takayama (1998) by scanning electron microscopy revealed the two sections, the proximal more reduced, in the tentacle of P . spinifera. Consequently doubts appear on the validity of P . rostrata as a separate species because it seems to be a large morphotype of P . spinifera (Figs 2k–m). While the records of the previous taxa are scarce, Pronoctiluca pelagica and P . spinifera are more commonly reported. The lack of cultures and molecular data render our knowledge on the intraspecific morphological variability incomplete, and the co-specificity of P . pelagica and P . spinifera remains unclear. Pavillard (1922) illustrated P . spinifera and P . pelagica as a single species. Kofoid and Swezy (1921) erected the genus Protodinifer Kof. & Swezy with the species P . tentaculatum (=Pronoctiluca spinifera) and P . marinum (=Pronoctiluca pelagica). According to these authors the difference between the species was a stout tentacle for body length of 54 µm for P . tentaculatum, whereas P .

marinum showed a slender tentacle and the body length 12–40 µm. Based on these diagnostic characters, P . tentaculatum and P . marinum are synonyms because the length constitute a poor diagnostic criterion, and the tentacle shape change along the cell development (Fig. 2). There also have been questions as to whether P . pelagica is the encysted form of P . spinifera (Figs 2s–y). All the specimens, observed and in division, corresponded to the form P . spinifera as reported in other studies (Herrera and Margalef, 1963) and there are no illustrations of P . pelagica dividing cells. Pronoctiluca pelagica lacking the flagella, is covered with hyaline layers, and intermediate forms of P . spiniferaP . pelagica are here illustrated (Figs 2o–r). This suggests that Pronoctiluca is considered as a monotypic genus. Although P . spinifera is the most common morphotype, the encysted form P . pelagica may have the priority. 4.3 Systematic position of Pronoctiluca The etymology of the name Pronoctiluca suggests affinity with Noctiluca scintillans (Macartney) Kofoid. Noctilucales are characterized by a locomotion based on rapid changes of the cell shape (Gómez et al., 2010a). The tentacle of Pronoctiluca that can be moved in all directions, the lack of cingulum and the numerous vacuoles are the main similarities with Noctiluca scintillans. Fensome et al. (1993) reported “Pronoctiluca has been included in this family by some previous authors (e.g., Loeblich, 1982) primarily because, like Noctiluca, it has a single flagellum”. This comment was unfortunate because Pronoctiluca possesses two flagella. Kofoid and Swezy (1921) observed live specimens and reported that the cell displacement differed from the typical dinoflagellate rotation and the swimming behaviour was similar to desmokont cells of Prorocentrum Ehrenberg. Oxyrrhis marina, considering the closer relative of Pronoctiluca, was placed in the family Pronoctilucaceae. Dodge (1982) placed it together to Pronoctiluca in the family Pronoctilucaceae of the Gymnodiniales. The flagella of Oxyrrhis are only slightly dissimilar and inserted posteriorly. A true undulate transverse flagellum is not present. Oxyrrhis has long been considered to be a dinoflagellate, but it was excluded by Fensome et al. (1993) because of its lack of most dinoflagellate features such as the chromosomes decondensation during interphase, which has an intranuclear spindle, and lacks a girdle and sulcus. Morphology and molecular phylogeny reveal that Oxyrrhis is an early branch in the dinoflagellate lineage (Saldarriaga et al., 2003a; Lowe et al., 2011). Oxyrrhis is divided by transverse binary fission, whereas the dinoflagellates tend to divide longitudinally or obliquely. In contrast, Pronoctiluca possesses a nucleus with chromatin strands, two anteriorly inserted dissimilar flagella, a short sulcus, and the cell division is longitudinal, being closer to the characteristics of the typical dinoflagellates. Molecular data of Pronoctiluca are an essential issue to understand the evolutionary history of alveolates. References
Dodge J D. 1982. Marine Dinoflagellates of the British Isles. London: Her Majesty’s Stationery Office Fensome R A, Taylor F J R, Norris G, et al. 1993. A Classification of Living and Fossil Dinoflagellates, American Museum of Natural History. Hanover: Sheridan Press Herrera J, Margalef R. 1963. Hidrografía y fitoplancton de la costa comprendida entre Castellón y la desembocadura del Ebro, de julio de 1960 a junio de 1961. Investigaciones Pesqueras (in Spanish), 24(1): 33–112 Loeblich III A R. 1982. Dinophyceae. In: Parker S P, ed. Synopsis and Classification of Living Organisms, I. New York: McGraw-Hill,

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101–115 Lowe C D, Keeling P J, Martin L E, et al. 2011. Who is Oxyrrhis marina? Morphological and phylogenetic studies on an unusual dinoflagellate. Journal of Plankton Research, 33(4): 555–567 Gómez F. 2012. A checklist and classification of living dinoflagellates (Dinoflagellata, Alveolata). CICIMAR Oceánides, 27(1): 65–140 Gómez F, Claustre H, Raimbault P, et al. 2007. Two High-Nutrient LowChlorophyll phytoplankton assemblages: the tropical central Pacific and the offshore Peru-Chile Current. Biogeosciences, 4(6): 1101–1113 Gómez F, Furuya K. 2007. Kofoidinium, Spatulodinium and other kofoidiniaceans (Noctilucales, Dinophyceae) in the Pacific Ocean. European Journal of Protistology, 43(2): 115–124 Gómez F, Moreira D, López-García P. 2010a. Molecular phylogeny of noctilucoid dinoflagellates (Noctilucales, Dinophyta). Protist, 161(3): 466–478 Gómez F, Moreira D, López-García P. 2010b. Molecular phylogeny of the dinoflagellates Podolampas and Blepharocysta (Peridiniales, Dinophyceae). Phycologia, 49(3): 212–220 Kofoid C A, Swezy O. 1921. The Free-living Unarmoured Dinoflagellata. Berkeley: Univ California Press Pavillard J. 1922. Pronoctiluca et Noctiluca. Bulletin de la Société Botanique de France, 69: 365–370 Saldarriaga J F, McEwan M L, Fast N M, et al. 2003a. Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus

are early branches of the dinoflagellate lineage. International Journal of Systematic and Evolutionary Microbiology, 53(1): 355–365 Saldarriaga J F, Leander B S, Taylor F J R, et al. 2003b. Lessardia elongata gen. et sp. nov. (Dinoflagellata, Peridiniales, Podolampadaceae) and the taxonomic position of the genus Roscoffia. Journal of Phycology, 39(2): 368–378 Sournia A. 1986. Atlas du Phytoplancton Marin: I. Introduction, Cyanophycées, Dictyochophycées, Dinophyceés et Raphidophyceés (in French). Paris: Editions CNRS Steidinger K A, Tangen K. 1997. Dinoflagellates. In: Tomas C R, ed. Identifying Marine Phytoplankton. London: Academic Press, 387–584 Takayama H. 1998. Morphological and Taxonomical Studies of the Free-living Unarmored Dinoflagellates occurring in the Seto Inland Sea and Adjacent Waters [dissertation] (in Japanese). Tokyo: The University of Tokyo Taylor F J R. 1976. Dinoflagellates from the International Indian Ocean Expedition: a Report on Material Collected by the R.V. ’Anton Bruun’ 1963–1964. Stuttgart: E. Schweizerbart’sche Verlagsbuchhandlung Taylor F J R. 1987. Taxonomy and classification. In: Taylor F J R, ed. The Biology of Dinoflagellates. Botanical Monographs 21. Oxford: Blackwell, 723–731

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