Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site characteristic plates.Differentvariations of which abuts the second precingular plate (2″ the presence ofonly three cingular plates, the ma
area, Peridiniales, Phytoplankton, Salinit Keywords: dinoflagellate cells. microscopy isveryuseful, anda species are mixed, theidentific those thatcan toleratebrackish water. In such might represent animportant source offoodforsome marine heterotrophic dinoflagellates, especially with dominant Because ofthe hypereutrophic cond spring, inan oligo-mesohaline range of thesalinity,whichisnot usualfor within thegenus are discussed. From anecological pointofview, arrangement is not exactly the same. The relationsh Protoperidinium bolmonense 2 1 http://dx.doi.org/10.2216/PH07-82.1 July 2008, Volume47,Issue 4,Pages392–403 Phycologia dinoflagellate with aplate formula of Po, X,4 France, the Bolmon lagoon (‘Étang de Bolmon’). Described is a newProtoperidinium Abstract: *: Corresponding author :N.Chomérat, emailaddress : Paris, France Paris, France France Cedex, Concarneau © 2008International Phycological Society Muséum National d'Histoire Naturelle, Département R.D. Département Naturelle, d'Histoire National Muséum F-29187 Kérose, ruede 13, Bretagne-Nord Finistère etRessources/ Laboratoire Environnement IFREMER, small dinoflagellatefromabrackish Protoperidinium bolmonense population, involvingthedivisionor ofpre-existing thefusion plates. Onlyone other Brackish water,Dinoflagellates, Dinophyc Planktothrix agardhii species, P. vorax Nicolas Chomérat ation of small dinoflagellates can be tedious. Scanning electron , isknownto possess only three ci simple methodis described to isolate, prepare and observe afew species from a brackishandhypereutrophic lagoon intheSouthof
(cyanoprokaryota) and other autotrophic species. So, the latter itions pertaining in thelagoon, the phytoplankton was abundant y, Scanningelectronmicroscopy, Taxonomy sp.nov.(Peridinia France) France) ′ ), themarked dorsoventralflattening and the shape of , 3a,7 the epithecal plate pattern were observed in environments, where lar 1, * rked extension of the second intercalary plate (2a) Protoperidinium bolmonense and AlainCouté ip with other congeneric species and the position D.M. – USM 505, 57, Rue Cuvier – Case 39, F-75005 F-75005 39, Case – Cuvier Rue 57, 505, D.M. –USM hypereutrophic lagoon (South of ″ , 3c,4(?)s,5 eae, Ecology, Coastal lagoon, Mediterranean [email protected] ‴ and 2″″.Itspeculiarfeatures are ngular plates, but the thecalplate
P. bolmonense les, Dinophyceae), a 2 Archive Institutionnelle del’Ifremer Archive Institutionnelle
ge amounts ofphytoplankton http://www.ifremer.fr/docelec/ Protoperidinium sp. nov. isa small
was present in Archimer species. P. 1
Introduction
Protoperidinium Bergh is a large genus of dinoflagellates (Dinophyceae), usually without chloroplasts and regarded as heterotrophic (Olseng et al., 2002). Because of its extreme diversity, this genus has had a long and complex taxonomic history (Gribble &
Anderson, 2006). Most of the species had been formerly described as Peridinium
Ehrenberg species, which included marine and freshwater organisms, and, originally, also species now placed in different genera. Bergh (1881) noticed that thecal plates always exhibit a certain basic pattern common within a genus and he introduced the genus Protoperidinium which included species with distinct sulcal lists, but leaving in
Peridinium those with antapical horns or without sulcal lists. The importance of the morphology in identifying dinoflagellates was emphasized by Schütt (1895) and the genus Peridinium was divided by several workers into subgroups based on the cell outline and girdle displacement. Kofoid (1909) proposed a detailed description of thecal plates, upon which the current and generally accepted classification is based.
Subsequently, Jörgensen (1912), emphasized the importance of thecal plate pattern in identifying species, and proposed new groups. Species with three intercalary plates were maintained in the genus Peridinium, whose subdivisions were based on the shape of the first apical 1′ plate, and the shape and position of the second intercalary 2a plate. The new genus Archaeperidinium was proposed for species with only two anterior intercalary plates. Some workers preferred to consider these genera as two sub-genera of Peridinium but used the subdivisions system of Jörgensen (Lebour, 1925) or adapted it (Paulsen, 1931). However, some investigators noticed that the number and shape of cingular and sulcal plates could be of a great interest in Perididium (sensu lato) taxonomy (cf. Taylor, 1976). Balech (1963) and Loeblich (1968) realized that marine
3 species of Peridinium were all found to possess three cingular plates plus a transitional plate, which contrasted with freshwater species that may have more cingular plates.
Bourrelly (1968) investigated the cingular plates of twelve freshwater species and found that there were five or six plates. Balech (1974) transferred 231 marine species of
Peridinium to the genus Protoperidinium. It included species with ortho, meta and para apical 1′ plate, intercalary plate with quadra, penta and hexa-type, four cingular plates, and six sulcal plates. Species with ortho-type of 1′ plate, five or six cingular plates and six sulcal plates were retained in Peridinium. Moreover, Balech (1974) found the subgenera useful to distinguish some abnormal plate numbers in the genus
Protoperidinium, e.g. two intercalary plates in Archaeperidinium, six precingular in
Minusculum and Protoperidinium for species with the typical plate formula Po, X, 4′,
3a, 7′′, 4c, 6(7)s, 5′′′, 2′′′′.
Brackish waters are defined as a mixing of fresh water and sea water and exhibit biological communities with peculiar characteristics, which differentiate them from adjacent marine and continental biomes. As brackish waters, and especially coastal lagoons, are an area of colonization of freshwater and marine organisms, species well adapted to the conditions of low salinity and specific to these habitats cohabit with halotolerant species from the adjacent sea and freshwater tributaries (Remane &
Schlieper, 1971). Since the separation of Peridinium and Protoperidinium into two distinct genera, the former is now considered as a freshwater or brackish genus while the latter is regarded as a strictly marine and cosmopolitan genus (Sournia, 1986;
Steidinger & Tangen, 1996). However, coastal lagoons are generally highly productive systems (Barnes, 1980) and blooms of autotrophic species may be favourable to the presence of heterotrophic marine dinoflagellates (Sarno et al., 1993; Chomérat et al.,
4 2004). In such conditions, some euryhaline Protoperidinium species may be present in brackish plankton (Caroppo, 2000).
The identification of Protoperidinium and Peridinium species in light microscopy is mainly based upon the observation of the cell size and shape, the presence of horns or spines, the shape of apical plate 1′, intercalary plates, the apical pore complex (APC), ornamentation of thecal plates, the displacement of the cingulum, and the number of cingular and sulcal plates. In plankton from brackish areas, where both of these genera can occur mixed in abundant plankton, a correct identification can be tedious when cells are small, rounded and without a characteristic outline shape and antapical appendages. Hence, the use of scanning electron microscopy (SEM) can greatly help to ensure a correct identification. For better results, this requires an isolation of cells and several dehydration steps prior to the observation (Couté, 2002).
Unavoidably, some cells to be identified are lost during the preparation process, which can be detrimental when only a few specimens are available. To minimize losses inherent to the dehydration, we propose here a simple method to prepare a few cells to be observed under SEM, and consequently, particularly adapted for the preparation of rare or ‘precious’ taxa.
This paper illustrates the thecal plate morphology of a new Protoperidinium, P. bolmonense, collected in a brackish hypereutrophic lagoon, which is an unusual habitat for a Protoperidinium species. This small dinoflagellate has a plate pattern and morphological features that do not correspond to any previously described within the genus.
5 Material and methods
Samples collection. Material was collected from the Bolmon lagoon (43°25′ N, 5°11′ E) which is a brackish (oligo-mesohaline) and hypereutrophic lagoon located in the South of France, near Marseille. It measures about 5 km long, up to 1.5 km wide, and is 0.58 km2 in area. Its depth does not exceed 2.2 m. A further description of this ecosystem and of the ten stations sampled is detailed in Chomérat (2005) and Chomérat et al.
(2007). Samples were collected with a sampling bottle just under the water surface to avoid floating detritus and were fixed with buffered formaldehyde for subsequent identification and scanning electron microscopy (SEM). Analyses of environmental parameters were carried out according to Chomérat et al. (2007).
Light microscopy. Cells were first isolated from field samples with a micropipette under an inverted microscope, and then observed and dissected with a
Zeiss Universal compound microscope (Zeiss, Oberkochen, Germany) equipped with plan-apochromatic objectives, Nomarski differential interference contrast (DIC), a digital camera and a drawing tube. Pictures and drawings were realized with the objectives 40/0.75 W, 40/0.95 or 63/1.40. Phytoplankton counts were carried out according to Chomérat et al. (2007) and abundances used in this paper were a monthly average of abundances in the ten stations sampled.
Preparation for SEM. Cells were isolated from the field samples with a capillary pipette and rinsed in deionised water to remove salts and fixative traces. When too many particles were present, two steps of isolation were performed. The suspension of isolated cells was then filtered on a polycarbonate membrane filter (Millipore GTTP
Isopore, 0.22 µm pores) using a syringe and a Swinnex filter holder (Millipore). To prevent cell losses during the different steps of preparation, we used a special device
6 made as follows. After the filtration of the cell suspension, the membrane covered with cells was deposited face upwards on a silicone gasket of the same diameter (12.9 mm) placed on a brass ring (1.5 mm thick). A second similar membrane filter (free of cells) was then carefully put above the first one, with an intercalary silicone gasket preventing contact between the two membranes. Then, another gasket and a second brass ring were put over the membranes. The assembly was compressed using a clamp (19 mm paper- clamp), maintaining the cells trapped within the two membranes (Fig. 1). The whole assembly was transferred through a graded series of ethanol solutions (15°, 30°, 50°,
70°, 90°, 95° and absolute) for about 1 hour in each step, and then critical-point dried.
Since cells released from the first membrane could be on either of the two filters, both of them were fixed onto a stub using carbon adhesive, great care being taken to maintain their respective orientation. Then, they were coated with gold with a sputter coater and finally observed with either a JSM–840A (Jeol Ltd, Tokyo, Japan) or a Quanta 200
(FEI, Eindhoven, The Netherlands) scanning electron microscope. The stubs numbered
07–C1, 07–C1′, 07–C4 and 07–C4′ have been deposited at the National Museum of
Natural History (RDDM Department, USM 505).
The measurements of cell sizes (n = 30) were taken by means of a calibrated eyepiece micrometer in LM and using Image J analysis software (Rasband, 1997-2006) on SEM digital pictures. SEM images were presented on a uniform background using
Adobe Photoshop CS2 (version 9.0.2, Adobe Systems, San Jose, CA, USA).
The terminology used for thecal plates in this paper is the generally accepted scheme based on the Kofoid system as modified by Balech (1980).
7 Results
Taxonomy
Class Dinophyceae Fritsch in West & Fritsch, 1927
Order Peridiniales Haeckel, 1894
Family Peridiniaceae Ehrenberg, 1831
Genus Protoperidinium Bergh, emend. Balech, 1974
Protoperidinium bolmonense Chomérat et Couté sp. nova (Figs 2–28).
Diagnosis : Cellulae cum lorica. Longitudo : 18–22 µm ; latitudo : 15–18 µm. Epitheca rotundata ; hypotheca plus minusve angularis et dorsiventraliter perspicue compressa.
Cingulum aequatorium profunde excavatum et leviter ascendens. Chloroplasti absentes.
Aliquot granula in cytoplasma. Laminarum tabulatio : Po, X, 4′, 3a, 7′′, 3c, 4(?)s, 5′′′,
4′′′′. Lamina 1′ ortho. Tres intercalares laminae leviter sinistorsum translatae.
Intercalaris 2a sexangularis, ampla et cum 2′′ lamina contacta. Intercalaris 3a regulatim hexagona. Cingulum cum tres laminis longitudine dissimilibus, C2 lamina in tota dorsale parte producta. Laminae laeves cum poris circum aperturam incrassatis extra laminas multiplicis apicalis pori (APC). Epithecae tabulationis mutationes continent 3′ laminae divisionem aut 4′′ et 5′′ laminarum conjunctionem.
Cells armoured. 18–22 µm long, and 15–18 µm wide. Shape rounded and markedly dorsoventrally flattened. The cingulum is excavated, located in the equatorial part of the cell, and slightly ascending. No chloroplast but several globular bodies are present in the cytoplasm. The plate tabulation: Po, X, 4′, 3a, 7′′, 3c, 4(?)s, 5′′′ and 2′′′′. Shape of 1′ is ortho. The three intercalary plates are shifted to the left part of the theca. The second
8 intercalary plate is hexagonal and markedly extended, and abuts the 2′′ plate. The third intercalary plate is hexagonal and symmetrical. The cingulum consists of three plates of different lengths. All thecal plates are smooth, and, except those of the APC, have sparse pores surrounded by circular rims. Variations of the plate pattern are observed on the epitheca, involving splitting of 3′ plate or fusion of 4′′ and 5′′ plates.
ETYMOLOGY: The specific epithet means from the Bolmon lagoon, where the species has been collected.
HOLOTYPUS: Fig. 8, collected by Chomérat in 2002.
ISOTYPUS: Figs 9–15
TYPE LOCALITY : Bolmon lagoon (43°25′ N, 5°11′ E; southeastern France)
This species was isolated in sample B09D10 collected from the Bolmon lagoon (May
2002), a hypereutrophic oligo-mesohaline in southeastern France. A sample fixed with formaldehyde is stored at the National Museum of Natural History (RDDM
Department, USM 505), Paris.
Protoperidinium bolmonense has a rounded, sometimes dome shaped, epitheca and a hypotheca more or less angular (Figs 2–4, 8–9, 16, 23–24). Epitheca and hypotheca are almost equal in size. Cells are 18–22 µm (mean 19.6 ± 1.6 µm) long and 15–18 µm
(mean 16.9 ± 1.4 µm) wide (transdiameter). In apical, antapical and lateral views, the cells are dorsoventrally compressed (Figs 6, 10–11, 13, 22, 25–26). In LM, the nucleus is median or in the right antapical part of the theca and a large accumulation body is observed in the episome of some cells (Fig. 3). Several globular bodies are observed randomly distributed in the cytoplasm of some cells (Fig. 7).
9 The apical pore complex is roughly in the centre of the epitheca apex (Figs 4, 8), and points to the right side of the theca (Fig. 10). Two platelets compose the APC, the pore plate (Po) that is almost oval in shape and the small and rectangular canal plate (X;
Fig 12). The apical pore lies in the centre of the Po plate and is surrounded by a comma- shaped rim (Fig. 12). The first apical plate (1′) is pentagonal and contacts APC and plates 1′′, 2′, 4′, 7′′ and S.a. (Figs 5, 8, 10, 13, 16, 22–23). The anterior right margin is the smallest while the posterior right is the longest; the anterior left margin is longer than the posterior left, leading to asymmetrical rhombic shape with a typical ‘ortho’ arrangement. Plate 2′ is regular pentagonal while 3′ and 4′ are hexagonal (Figs 10, 20,
25). Plate 3′ is not centrally located but displaced in the left side of the epitheca, its ventral margin being notched by the APC (Figs 10, 20, 25). The three intercalary plates are very differently shaped. Plate 1a and 2a are located on the left side of the epithecal:
1a is pentagonal and 2a roughly hexagonal, wider than high. The latter has a distinctive position, located below 1a and abutting the 2′′ plate (Figs 15, 25). In contrast, plate 3a is located dorsally on the right side of the theca and has the shape of a regular hexagon
(Fig. 9). The first precingular plate (1′′) is six-sided and has the shape of an irregular polygon with opposite sides almost parallels, its sutures with S.a. and 1a being the smallest (Fig 8). Plate 2′′ is pentagonal and has a very short contact with 3′′ that is rectangular and very narrow, laying below the long 2a plate (Figs 15, 25). Plates 4′′ and
6′′ are five-sided while 5′′ and 7′′ are quadrangular (Figs 13–14). In the precingular series, plates 7′′ and 6′′, and to a lesser extent 1′′, are higher than the other plates (Figs 8,
13–14, 25). The cingulum is wide (2.8–4.1 µm, mean 3.4 ± 0.2 µm), deeply excavated
(cavizone), displaced and slightly ascending about ¼ of its width, and consisted of three plates only. The first one (C1) is the smallest and has a rectangular trapezium shape
10 (Figs 18, 27). This plate is in contact with the sulcal plates S.a. and S.s. and with 1′′, 2′′,
1′′′ and C2. Its suture with the C2 plate roughly faces the right margin of the second precingular (2′′) (Figs 8, 16, 18, 23). Plate C2 is the largest of the three cingular plates, and extends on the whole dorsal part of the theca (Figs 9, 14–15, 24). Its suture with the
C3 plate is located slightly after the suture of 5′′ and 6′′ on the epitheca and 3′′′ and 4′′′ on the hypotheca (Figs 9, 14). The C3 plate is rather long and occupies the right lateral part and half of the ventral part of the theca (Figs 13–14, 27). Four plates were observed on the sulcal area (Figs 8, 18, 28). The anterior sulcal plate (S.a.) is rather big, indents slightly the epitheca and contacts 1′ and 1′′ on the epitheca, C1 and C3 on the girdle, and 5′′′, S.d. and S.s. plates on the hypotheca (Figs 18, 28). The S.d. plate is rectangular in shape and its left margin bears a small right sulcal list (Figs 8, 18). The S.s. plate is the longest of sulcal plates, as long as 1′′′ plate, and with a very narrow list bordering its right margin (Figs 8, 18). It contacts both S.a. and C1 anteriorly and S.p. plate posteriorly. The posterior sulcal S.p. plate is rather wide and hexagonal, in contact with
5′′′, S.d., S.s., 1′′′ and both of the antapical plates 1′′′′ and 2′′′′ (Figs 8, 18). Plate 1′′′ is pentagonal and contacts shortly S.p. (Figs 8, 11). Plates 2′′′ and 4′′′ are both four-sided
(Fig. 11) while 3′′′ is pentagonal and 5′′′ hexagonal (Fig. 18). Antapical plates 1′′′′ and
2′′′′ are both five-sided and almost equal in size (Fig. 11).
The thecal surface is smooth with sparse pores surrounded by circular rims (0.26 µm in diameter) (Fig. 17). All thecal plates, including sulcal and cingular plates (Figs 17–18) but except those of the APC, have such pores preferentially located along the margins but also in their middle (Figs 8–11, 13–18). On the cingular plates, most of the pores appear located in the anterior half part (Fig. 9) while they seem absent in the posterior one. On most of observed cells, plate margins form ridges and sutures are large and
11 transversally striated (e.g. Figs 14, 17), characteristic of metacytic growth, but some cells were observed with thin sutures (Figs 18, 21, 23).
Variations of thecal plate pattern
Some cells are observed with a different epithecal plate pattern than previously described. On a cell, we observe only 6 precingular plates instead of 7 (Fig. 20). A detailed observation reveals that the major difference is the absence of the 5′′ plate described in the type of this new species (cf. Figs 9, 19). In this case, the 3a plate is penta (hexa in the type, Fig. 9) and the 4′′ plate extends farther on the dorsal part of the epitheca. Consequently, the following precingular plates were called 5′′ and 6′′ (Figs
19–20) and are analogous to, respectively, 6′′ and 7′′ in the type of P. bolmonense (Figs
9, 14).
Another type of variation of the thecal pattern, involving the presence of an extra-plate, is observed on a few other cells. A supernumerary plate located between 1a and 3′ plates is visible in the dorsal and apical views, reducing the size of 3′ (Figs 21–
22) while the other plates are similar to the previous description. An attentive observation shows that this plate may well result from a division of the 3′ plate into two smaller plates, called 3′ α and 3′β (Figs 21–22, 25), since considered jointly; these two plates have the same shape and occupy the same position as 3′ on the type of this new species (Figs 10, 25). Plate 3′ β is roughly triangular, in contact posteriorly with 3a
(sometimes shortly with 2a), and anteriorly with 4′, 2′ and 3′α plates, its ventral side being in contact with the APC. Plate 3′ α is four-sided and in contact with 2′, 1a, 2a and
3′ β.
12 Ecology
During the study in the Bolmon lagoon, P. bolmonense was present from February to
September 2002. This species was detected at very low levels in February and its abundance increased over 1 × 104 cell l-1 in March (Fig. 29). The abundance of this dinoflagellate varied among the different stations on one date, being absent in some stations and present in others (not shown). Consequently standard deviations are high
(Fig. 29), nevertheless, highest abundances of P. bolmonense (in average 35 × 104 cell l-
1) were observed in May in all stations (Fig. 29). At this date, a peak over 106 cell l-1 was observed in some stations of the lagoon. In June, the cell number fell drastically and stayed low (< 1 × 105 cell l-1) until September. Then, no other record of this species occurred in the end of 2002. In comparison, the abundance of another marine dinoflagellate found at the same period in the Bolmon lagoon, Oblea rotunda (Lebour)
Balech ex Sournia, increased earlier from January to March when it reached an average abundance in the same order of magnitude than P. bolmonense maximum (ca. 30 × 104 cell l-1, Fig. 29). Then O. rotunda decreased from April, while P. bolmonense was increasing.
P. bolmonense was observed in a salinity range varying from 2.2 to 11.1, with highest abundances between 5.4 and 8.1 (Fig. 30). Water temperature recorded when this dinoflagellate was present ranged from 9.5 to 26.0°C and the abundances over 106 cell l-1 were found when the water was around 20°C (Fig. 30).
When P. bolmonense was present, the phytoplankton community was dominated by cyanoprokaryota (cyanobacteria), mainly Planktothrix agardhii (Gom.) Anagn. &
Kom., with abundances in an order of magnitude of 109 cell l-1. The eukaryotic species were never dominant in this lagoon, but a spring phytoplankton bloom of the diatom
13 Cylindrotheca closterium (Ehrenberg) Reimann & Lewin and the small dinoflagellate
Heterocapsa rotundata (Lohmann) Hansen [synonym Katodinium rotundatum
(Lohmann) Loeblich], occurred in February (Chomérat et al., 2004). During the annual cycle, the other peridinioid dinoflagellates were very rare in the sample, some other
Protoperidinium spp. being sometimes collected in very few number. Only H. rotundata and prorocentroid species, mainly Prorocentrum minimum (Pavillard)
Schiller and Prorocentrum micans Ehrenberg were the most abundant among thecate dinoflagellates in the lagoon.
Discussion
Taxonomy
Since numerous species of Protoperidinium are described, several authors attempted to clarify and simplify the classification of these species with the use of subdivisions of the genus. Because of its simplicity and practical convenience (Abé, 1981), the subdivisions system proposed by Jörgensen (1912) has been adopted by majority of subsequent investigators (e.g. Barrows, 1918). Nevertheless, Paulsen (1931) emphasized the schematic and artificial nature of this system, based only upon the plate pattern and not taking into account the morphological features, and he proposed a modified version including new sections. Not initially taken into account, the sulcal plates were considerably emphasized by Abé (Abé, 1936, 1981), having found that they were remarkably conservative, and Balech (Balech, 1971, 1974, 1980, 1988) who considered them as good taxonomic criteria at the species level. Contrary to Balech who used only the details of sulcal and cingular plates for species identification, Abé (1981) proposed somewhat different infra generic groups for Peridinium (maintaining fresh and marine
14 water species in this single genus) based upon the combination of characters such as the shape of plates in the ventral area (both first apical plate and plates in the sulcal region, with major emphasis on the sulcal posterior plate) and the form of antapical appendages of the theca. However, it is very difficult to place P. bolmonense in one of these infra generic groups. According to the commonly accepted subdivisions of the genus proposed by Jörgensen, P. bolmonense can be attributed to subgenus Orthoperidinium and to section Conica (subgen. Orthoperidinium) owing to the ortho pattern of the 1′ plate, the presence of three intercalary plates and the six-sided 2a. According to Paulsen, some small ortho-hexa species would better be placed in the section Tabulata, that groups some freshwater and marine species typically ortho-penta (Paulsen, 1931). The pattern of intercalary plates asymmetrically arranged with respect to the APC and the extension of 2a observed in P. bolmonense is very atypical for the genus and is not found in any of the species of this section. The asymmetric pattern of intercalary plates, located on the left side of the theca is observed in P. asymmetricum (Abé) Balech, that is a ortho-penta species (Abé, 1927) and some species having only two intercalary plates, grouped in the subgenus Archaeperidinium. One species with two intercalary plates, P. fusiforme (Abé) Balech, has a reniform cingular section comparable to P. bolmonense but it can easily be differentiated by its bigger size, and the number of intercalary and cingular plates. Woloszyńska (1928) described another species from the
Baltic Sea, Protoperidinium grenlandicum (Woloszyńska) Balech, that is also morphologically and ecologically close to P. bolmonense. However, it is larger (30 – 40
µm) and possesses only two anterior intercalary plates, the dorsal one being very peculiar and 9-sided (Woloszyńska, 1928). From the illustrations of P. grenlandicum, it appears that the plate pattern is different between these two species (cf. figs 6–14, pl. 9
15 in Woloszyńska, 1928). Since P. grenlandicum stayed poorly described and because of the lack of information on cingular and sulcal series, its appurtenance to the genus
Protoperidinium is doubtful (Balech, 1974) and we could not make further comparisons with P. bolmonense. According to the subdivisions proposed Abé (1936; 1981) who did not use the number of intercalary plates to separate groups, P. bolmonense appear related to species of the Monovela group because of the shape of 1′ and S.p. plates and the outline of cells. A comparison of the morphological features of P. bolmonense and selected related species is listed in Table 1.
In comparison with these species, P. bolmonense can be distinguished by its smaller size. Species of the Monovela group such as P. asymmetricum, P. monovelum,
P. minutum, P. monospinum, P. mutsuense have a size generally greater than 30 µm while P. bolmonense was never observed over 22 µm (Table 1).
P. bolmonense differs from almost all other congeneric species based on two main morphological characters: (1) the shape and position of the second intercalary plate that extends below the 1a plate and abuts the second precingular plate; and (2) the presence of only three plates in the cingular series.
Almost all the Protoperidinium species have four cingular plates which is commonly considered as generic character (Sournia, 1986; Steidinger & Tangen, 1996).
Most of species have a first cingular plate reduced to a small transitional plate (t plate) between sulcal and cingular series but some others have a wide first cingular plate that can not be considered as a transitional plate, but better as a first cingular plate. For example, the first cingular plate in Protoperidinium americanum and P. parthenopes is large and extends beyond the suture of the 1′′ and 2′′ plates (Lewis & Dodge, 1987;
Zingone & Montresor, 1988), which is similar on P. bolmonense. In contrast with most
16 of the Protoperidinium species, where the suture between the third and fourth cingular plates (called C2 and C3, respectively, the first one being called t) is on the right ventral part of the theca (Balech, 1980), it is located dorsally in P. americanum and P. parthenopes and dorso-laterally in P. bolmonense. But unlike P. bolmonense that has only three cingular plates, P. americanum and P. parthenopes have four (Table 1). To our knowledge, only one species recently described, P. vorax Siano & Montresor, exhibits this feature and has a cingular plate pattern comparable to that of P. bolmonense (Siano & Montresor, 2005). In addition, these two species have a very comparable large second intercalary plate abutting the second precingular, which is distinctive in the genus Protoperidinium. For these reasons, they appear morphologically the most closely related (Table 1).
Although P. bolmonense and P. vorax have a very smiliar size and epithecal plate pattern, they can be differentiated by distinctive characters. In LM, the shape of cells is quite different, P. bolmonense being less spherical, more irregular in ventral view, and more compressed in apical view than P. vorax. Characteristic features on the cingular and sulcal plates also distinguish these two species. The presence of pores on sulcal and cingular plates is peculiar to P. bolmonense since they are devoid of pores in
P. vorax (Siano & Montresor, 2005). The position of the suture between C2 and C3 plates is slightly different between these species. In P. vorax, it faces the fifth precingular and the third postcingular plates (cf. Fig. 8 incorrectly numbered in Siano &
Montresor, 2005) while it faces the sixth precingular and the fourth postcingular plates in P. bolmonense. About sulcal plates, we can not conclude that their number differs between the two species (Table 1) as the small S.m. plate reported in P. vorax could have been unseen in P. bolmonense. Owing to the small size of cells, it may be present
17 and not observed in our dissections in LM and is probably hidden by other plates in
SEM. Its presence still remains to be verified.. The shape of the S.d. plate is also distinctive since its anterior end tapers in P. vorax, in contrast to P. bolmonense where this plate is rectangle-shaped. These two species can also be differentiated by the shape of the S.p. plate. In P. vorax, its posterior margin forms a small indentation in the suture between 1′′′′ and 2′′′′ (cf. Fig. 15 in Siano & Montresor, 2005), which is not observed in
P. bolmonense. Since cingular and sulcal plates are considered as very conservative at the species level and are good taxonomic criteria (Balech, 1980; Abé, 1981; Okolodkov,
2005), we conclude that these two species are unequivocally different.
Since the thecal formula of P. vorax and P. bolmonense differs from the type of the genus (P. pellucidum Bergh) for girdle plates, their appartenance to Protoperidinum can be questionable. As their epithecal and hypothecal plate arrangement is typical of the genus, we agree with Siano & Montresor (2005) that they are best maintained within
Protoperidinium until new molecular data, actually unavailable for both of these species, argue differently. It is established that some variations of the type formula occur in the genus Protoperidinium and subdivisions of the genus were proposed to identify groups. Species with only six precingular plates were for example grouped in the subgenus Minusculum by Balech (1974). More recently, Faust (2006) proposed the new subgenus Testeria for species with no APC, which is normally a distinctive feature of the genus typically described with an apical pore and a canal plate (Steidinger &
Tangen, 1996). However, recent molecular studies on the genus showed that it is highly possible that the subdivision system at a subgeneric rank, based on thecal plates, does not reflect phylogenetic affinities and thus, is probably not a good taxonomic character
(Yamaguchi et al., 2006). For example, a molecular analysis showed that P. bipes
18 (Paulsen) Balech, that is characterized by a very peculiar morphology, leading to an uncertain position in the genus, is unexpectedly included in the clade of the section
Protoperidinium of the genus (Yamaguchi et al., 2007). Consequently, since all our attempts to determine nuclear-encoded rDNA from isolated cells stayed unsuccessful, probably due to the formaldehyde-fixation of our samples, it is not possible to specify the affinities of P. bolmonense with a particular subgeneric division. On a morphological basis, we consider it within the subgenus Protoperidinium (Table 1).
Owing to the reduced number and disposition of cingular plates, P. bolmonense and P. vorax appear morphologically more closely related to marine species of the genus Protoperidinium, than to the freshwater genus Peridinium Ehrenberg. In comparison with the type-Protoperidinium (Balech, 1980), the relative position and shape of cingular plates are peculiar in P. vorax and P. bolmonense, C2 not extending to the right ventral part of the theca and C3 being larger. In contrast, in many freshwater
Peridinium species, the sutures of cingular plates are aligned with those of the postcingular series (Bourrelly, 1968).
Variations of the thecal plate pattern.
Variations of the thecal plate pattern of armoured dinoflagellates have been long recognized. Barrows (1918) found that dinophysoids were more constant than peridinioids, the hypothecal pattern being more stable than the epithecal pattern. Abé
(1981) noted, although one can find both fewer or more plates in each series, with the latter more common, the integrity of latitudinal series generally remains intact, which is true in P. bolmonense. As reported by Taylor (1987), examples of plate splitting (the first apical plate) and apparent sutural loss are rare events in Protoperidinium (Diwald,
19 1939 in Taylor, 1987). However, examples of epithecal variability can be found in some brackish species related to Peridinium. For example, Krytoperidinium foliaceum (Stein)
Lindemann [synonyms Peridinium foliaceum (Stein) Biecheler and Glenodinium foliaceum (Stein)?] and Peridinium balticum (Levander) Lemmermann exhibit some variations of their thecal plate pattern (Lebour, 1925; Chesnick & Cox, 1985), as observed in natural samples of P. bolmonense. Lefèvre (1932) considered plate variations as ‘modifications on a theme’ and proposed descriptive terms to the various forms that seemed to represent divisions or fusions of pre-existing plates, or to represent the movement of plate sutures. Division of a pre-existing plate to yield an additional plate in the series, as occurring for the 3′ plate of P. bolmonense, was referred to as a complexum modification. A similar split of the 3′ plate can be observed in P. balticum
(Chesnick & Cox, 1985) and following the terminology of Lefèvre (1932), the 3′α plate observed in P. bolmonense is a true apical (3′) while 3′β is called a preapical plate
(noted pr). Fusion of pre-existing plates to reduce the number of plates in a series was referred to as a simplex modification (Lefèvre, 1932). Such variation is not found in culture of P. balticum (Chesnick & Cox, 1985) but correspond quite well to the fusion of 4′′ and 5′′ observed in some cells of P. bolmonense, reducing the precingular series to six plates (Fig. 19).
Ecological data.
Since Balech (1974) split the old genus Peridinium, almost all the Protoperidinium species are typically marine and are not recorded in brackish waters, unlike a few
Peridinium species that may cope with a moderate presence of salt in water (Sournia,
1986; Taylor & Pollingher, 1987). Only a few species of Protoperidinium can be
20 observed in brackish environments, such as P. achromaticum (Trigueros et al., 2000). In contrast, species most frequently found in brackish environments comparable to the
Bolmon lagoon are Kryptoperidinium folicaceum and Peridinium quinquecorne Abé
[synonym Protoperidinium quinquecorne (Abé) Balech]. K. foliaceum is morphologically closely related to Peridinium and some authors considered it as belonging to this genus (Trigueros et al., 2000). It grows at salinities below 10 and can reach high abundances in brackish harbour and estuaries (Lebour, 1925; Nézan & Piclet,
1996). Surprisingly, this species has never been observed in the Bolmon lagoon where the salinity was varying from 5 to 10. P. quinquecorne is also common in coastal waters, and sometimes found in estuaries and lagoons (Shamsudin et al., 1996;
Trigueros et al., 2000). In the Berre lagoon, that is adjacent to the Bolmon lagoon, P. quinquecorne is more frequent than marine Protoperidinium species that are very rarely observed, probably always as a consequence of Mediterranean seawater inflows (Beker, pers. comm.). In a former study of the Berre lagoon, Kim & Travers (1984) reported only Peridinium balticum with no mention of any other species. P. balticum is a typical brackish species, dorso-ventrally flattened and with six cingular plates, resembling K. foliacaeum (Lebour, 1925) but has never been observed in the Bolmon lagoon.
Protoperidinium vorax, that is morphologically the closest species to P. bolmonense, was collected in the Gulf of Naples (Mediterranean Sea) but there is no mention of environmental data that can be compared. It is described as a heterotrophic species, grazing on unicellular (Thalassiosira sp.), and chain-forming (Skeletonema pseudocostatum, Leptocylindrus danicus) diatom species (Siano & Montresor, 2005). In the Bolmon lagoon, we found another marine heterotrophic species, Oblea rotunda and we supposed that the abundance of food in the hypereutrophic lagoon may explain its
21 presence in this unusual habitat (Chomérat et al., 2004). Since almost all the
Protoperidinium species are heterotrophic (Gribble, 2006), P. bolmonense may be abundant in the Bolmon lagoon for the same reasons than O. rotunda. Even if not observed, the heterotrophy of P. bolmonense is suggested by the presence of numerous globular bodies in the cytoplasm, also observed and identified as accumulation bodies equivalent to lysosomes in P. vorax (Siano & Montresor, 2005), but this has to be confirmed as no pallium-feeding cells have been observed. It is however interesting to notice that P. bolmonense appeared after O. rotunda and reached its maximum abundance in May, while O. rotunda was declining. This may be the consequence of a competition for prey but this still needs to be verified.
The geographic distribution of P. bolmonense is not yet known but it is probably also present in the Baltic Sea since Pertola et al. (2006) represented specimens of P. grenlandicum (cf. fig 5) that are not in the size range of this species and with a plate arrangement reminding that of P. bolmonense. Further studies are required for confirmation.
Acknowledgements
The authors are very grateful to P. Crassous who designed the assembly for SEM preparation and for his skills with SEM, R. Garnier who carried out major part of the sampling and environmental analyses, and P. Webber and E. Nézan for their numerous suggestions and improvements on the manuscript. We thank Dr. N. Daugbjerg and two anonym reviewers for their useful comments. This investigation was supported by grants from the Provence-Alpes-Côte-d’Azur Regional Council and the Public Interest
Group for the Berre lagoon Rehabilitation (GIPREB).
22
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31 Figure captions:
Figure 1: Assembly used for dehydration of cells prior to SEM examination. A:
Exploded view of the different elements (1, 7: brass rings; 2, 4, 6: silicone gaskets; 3, 5: polycarbonate membrane filters, dots on filter 3 figuring deposited cells). B: Side view of the clamped assembly (8: clamp).
Figures 2–7: Light photomicrographs of Protoperidinium bolmonense sp. nov.
Chomérat et Couté. Fig. 2: Cell in bright field. Fig. 3: Cell slightly compressed by the coverslip, with focus on the nucleus (n) and the sac pusule (pu). Fig. 4: Empty theca showing the outline shape and the presence of an apical pore (arrow). Fig. 5: Empty theca showing some of the ventral plates. Fig. 6: Cingular section of the same specimen as Figs 4 and 5, showing the dorsoventral flatenning. Fig. 7: Cell containing globular bodies in the cytoplasm (arrows). Figs 3–7 are taken with Nomarski diffential interference contrast (DIC). Scale bars represent 10 µm.
Figures 8–15: Scanning electron micrographs of P. bolmonense sp. nov. Chomérat et
Couté (from 07–C1 and 07–C1′). Fig. 8: Holotype. Ventral view. The epitheca is almost round while the hypotheca is more angular. Fig. 9: Dorsal view. Note the extension of
C2 on all the dorsal part of the theca. Fig. 10: Ventro-apical view. Note the asymmetrical – ortho shape of 1′ plate and the displacement of 3′ on the left side of the epitheca. Fig. 11: Antapical view. The cell is compressed dorso-ventrally and has a reniform section. Fig. 12: Detail of the apical pore complex (APC) showing the pore plate (Po) and the canal plate (X). Note the comma-shaped rim surrounding the pore.
Fig. 13: Right ventro-lateral view showing the compression of the cell. Note the suture
32 between C2 and C3 plates (arrow). Fig. 14: Right lateral view of a cell. Fig. 15: Left dorsal view of a cell. 3′′ plate is very small, while 2a is large and exhibits a characterisc feature, located below 1a and abutting 2′′. Figs 9–15 are isotypes. Scale bars represent
10 µm.
Figures 16–18: Detailed morphology of P. bolmonense sp. nov. Chomérat et Couté
(SEM, from 07–C1 and 07–C1′). Fig. 16: Ventral view of a cell with a flagella (arrow) exiting from the sulcal furrow. Fig. 17: Detail of thecal surface morphology. Plates are smooth, with sparse pores enclosed in circular rims. Note the presence of pores in the cingular plates. Fig. 18. Detail of the ventral area and arrangement of sulcal plates. Note the presence of pores on sulcal plates and the narrow list on the right border of S.s.
(arrow). Scale bars represent 10 µm (Fig. 16) and 1 µm (Figs 17–18).
Figures 19–22: Variations of the epithecal plate pattern in P. bolmonense sp. nov.
Chomérat et Couté (from 07–C1 and 07–C1′). Fig. 19: Dorsal view of a cell with only six precingular plates. Note the five-sided 3a, the peculiar shape of plate 4′′ and the shape of plate 5′′ equivalent to 6′′ in the type species. Fig. 20: Apical view of the same cell with six precingular plates. Fig. 21: Dorsal view of specimen with thin sutures and a split 3′ plate. A supernumerary suture (arrow) divides the 3′ plate in two small plates
3′ α and 3′β . Fig. 22: Apical view of another cell with a split 3′ plate. Note the peculiar plate arrangement due to the supernumerary suture (arrow) and the difficulty to assign plate 3′ α in a series following the Kofoidean system. Scale bars represent 10 µm.
33 Figures 23–28: Line drawings of Protoperidinium bolmonense Chomérat et Couté. Fig.
23: Ventral view. Fig. 24: Dorsal view. Fig. 25: Apical view (note the supernumerary suture that sometimes divide plate 3′, dotted line). Fig. 26: Antapical view. Fig. 27:
Schematic drawing of the three cingular plates. Fig. 28: Detail of visible sulcal plates.
Scale bar for figs 23–26: 10 µm (others not to scale).
Fig. 29: Variations in the abundance of Protoperidinium bolmonense (filled boxes) and
Oblea rotunda (empty boxes) in the Bolmon lagoon from January to September 2002
(average of the ten stations sampled and standard deviation).
Fig. 30: Salinity and temperature in sampling stations where P. bolmonense was present. Open circles: abundances of P. bolmonense below 1 × 105 cell l-1; shaded circles: abundances between 1 × 105 and 1 × 106 cell l-1; filled circles: abundances over
1 × 106 cell l-1.
34 Table 1: Morphological features of P. bolmonense sp. nov. compared with Protoperidinium and Archaeperidinium spp. of similar size and shape.
Subgenera Protoperidinium Archaeperidinium ortho-hexa ortho-penta Species P. bolmonense P. vorax P. americanum P. parthenopes P. asymmetricum P. monovelum P. minutum P. monospinum P. mutsuense Cell shape Rounded and Spherical Spherical Spherical Spherical Rounded Spherical Spherical Spherical dorsoventrally pentagonal flattened Length (µm) 18–22 16–26 30–40 30–30.8 361 50–58 40–56 30–35 40–47 46–56 29–334 Width (µm) 15–18 16–25 28–38 26–35 34 51–54 20–25 36–43 APC Slightly Slightly Symmetric Slightly Asymmetric Asymmetric Symmetric Symmetric Asymmetric asymmetric asymmetric asymmetric Cingular plates 3c 3c t+3c 4 (t+3?)c t+3c t+3c t+3c t+3c ? Sulcal plates 4(?)s 5s 7s 6s 5s 5s 5s 5s 4s Shape of S.p. Triangular Triangular Triangular Triangular Rounded Triangular Triangular Rounded Triangular Plate Sparse pores Protuberances, Scattered pores Scattered pores Faint Scattered ornamentation bordered by pores and faint and points reticulation pores circular rims reticulation Pores along Present Present Present Present Absent Present Absent ? ? margins Pores on sulcal Present (S, C) Absent Present Present Absent Present (S) Present ? ? (S) and cingular (C) plates Cyst shape Unknown (not Unknown Spherical Unknown Unknown Unknown Spherical Unknown diameter observed) 35–52 µm 28–32 µm 41–43 µm wall Inner layer smooth, outer layer granular Spines 3–5 µm simple, Bifurcate and slender, curved acuminate processes 7–9 µm
References Present work Siano & Lewis & Dodge Zingone & Abé (1927) Abé (1936) Schiller(1933- Zonneveld & Abé (1936) Montresor (1987) Montresor 1937) Dale (1994) (2005) (1988) Abé (1936) Balech (1964) Balech (1974) Fukuyo et al. (1977)
Wall & Dale (1968) Abbreviations: APC = apical pore complex; S.p. = sulcal posterior plate
Fig. 1
Figs 2–7
Figs 8–15
Figs 16–18
Figs 19–22
Figs 23–28
Fig. 29 Fig. 30
90 12 80
) 10 -1 70 celll
4 60 8 50 .) u P.bolmonense . s 40 .
p 6 O. rotunda y ( y 30 t
20 salini 4 cell abundance (× 10 cell abundance 10 2 0 Jan Feb Mar Apr May Jun Jul Aug Sep 0 6 10 14 18 22 26 30 Temperature (°C)