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Diatom Research (2002), Volume 17 (2), 327–335

A MORPHOLOGICAL STUDY OF TENUISSIMUS MEUNIER, A LITTLE-KNOWN PLANKTONIC , WITH A DISCUSSION OF THE SECTION SIMPLICIA, SUBGENUS HYALOCHAETE

Eugenia A. Sar*

Departamento Científico Ficología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n, 1900 La Plata, Argentina

David U. Hernández-Becerril

Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apdo. postal 70–305, México, D. F. 04510, México

Inés Sunesen

Departamento Científico Ficología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n, 1900 La Plata, Argentina

The morphology of Chaetoceros tenuissimus Meunier, a little-known , has been studied for the first time with electron . C. tenuissimus has been placed as a synomyn of C. simplex Ostenfeld since approximately 1930, and has been misidentified or neglected in routine counts. The cells are very small and solitary, square to rectangular in girdle view. The setae are narrow and straight, arising from the two poles of the valve at an angle of 45° to its apical axis. The valve face possesses an annulus, delimited by a hyaline rim. Costae and small areolae radiate from the annulus. A single rimoportula occurs within the annulus. The rimoportula opens externally in a flatened tube and internally by a simple slit. The setae exhibit spirals of minute pores and an intricate pattern of spiral and perpendicular costae; spines are regularly present. Similarities with closely related species in Section Simplicia are discussed.

INTRODUCTION

Several solitary planktonic species, usually small ones, of the genus Chaetoceros Ehrenberg are frequently encountered but have possibly been misidentified in marine coastal all over the world. Most of them belong to the Section Simplicia of the subgenus Hyalochaete. Within this Section is included a little known species, Chaetoceros tenuissimus. This diminutive species was described by Meunier (1913). The protologue included only very small illustrations. Chaetoceros tenuissimus has been considered a synonym of C. simplex Ostenfeld by Hustedt (1930) and many subsequent authors (e.g. VanLandingham 1968). This doubtlessly has contributed to our poor knowledge of this taxon regarding its distribution, morphology and . However there appear to be good reasons to

* author for correspondence; e-mail address = [email protected] 328 E. A. SAR, D. U. HERNÁNDEZ-BECERRIL AND I. SUNESEN

consider the two taxa to be distinct entities. Chaetoceros simplex has been shown to produce resting spores and its general appearence is than that of C. tenuissimus, especially with respect to the direction of the setae and the larger size of the resting spores (Ostenfeld 1901, 1912, Paulsen 1905). Recognition of C. tenuissimus as a species in its own right by Hasle & Syvertsen (1997) and its recent description by Bérard-Therriault et al. (1999) brought the name to our attention. Since Gran’s (1897) proposal to divide the genus Chaetoceros into two subgenera (Phaeoceros and Hyalochaete), there have been additional proposals for further subdivisions into sections (Ostenfeld 1903). Section Simplicia, of the subgenus Hyalochaete (characterized by solitary, small, delicate cells), was revised by Ostenfeld (1912). He dealt with only 6 species and did not mention C. tenuissimus as it was described one later. Rines & Hargraves (1988) have discussed in some detail the situation of various species usually assigned to Section Simplicia, including C. tenuissimus. Recent studies which discuss species of Section Simplicia are those of Johansen & Rushforth (1985) on C. muelleri Lemmermann, Rines & Hargraves (1986) on C. ceratosporus var. brachysetus Rines et Hargarves, Rogerson et al. (1986) on the growth of C. gracilis Schütt (most likely misidentified as the illustrations may correspond to C. tenuissimus), Rushforth & Johansen (1986) on C. muelleri, C. muelleri var. subsalsum (Lemmermann) Johansen et Rushforth and C. cf. simplex, Johansen et al. (1990) on C. muelleri, C. muelleri var. subsalsum and what was later named C. transisetus Johansen et Boyer, by Johansen & Boyer (1995) on C. muelleri, C. muelleri var. subsalsum and C. transisetus, Hernández-Becerril (1996) on the morphology of C. gracilis and C. vistulae Apstein, by Jensen & Moestrup (1998) on C. ceratosporus var. brachysetus, and finally by Bérard-Therriault et al. (1999) on C. ceratosporus var. brachysetus and C. simplex. This paper is focused on the morphology of Chaetoceros tenuissimus, a relatively little-known species, which may be more widespread and abundant than thought. The relationship of C. tenuissimus to other members of Section Simplicia is also discussed.

MATERIAL AND METHODS

Material for this study was collected in the northern area of San Matías Gulf, Patagonia, Argentina, at several locations along the coast: Los Álamos, Piedras Coloradas and Bajo de Oliveira (Fig. 1) during February 2000. General conditions for this date were temperature 21° C and salinity 34 psu. Phytoplankton samples were taken from the upper (0–5 m) with a 30 µm mesh net. Inmediately following sample collection, species of the nanoplankton size fraction were isolated by micropipette under a Wild M 20 microscope. Individual cells were washed several times in filtered and when free of contaminants they were transferred into tissue culture wells containing 2 ml of f/2 medium (Guillard 1975). The culture wells were maintained at room temperature (22ºC) under continuous supplied by cool-white fluorescent tubes and regularly checked for algal growth. When colour was detected, usually after 2 weeks, cultures were scaled up to 20 ml tubes and later 250 ml flasks. Several unialgal cultures were established. Field samples and aliquots of cultures containing Chaetoceros tenuissimus were fixed with 4% formalin and deposited in the Colección de Diatomeas Argentinas, Departamento Científico Ficología, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, under numbers 4629, 4630, 4632, 4642 and 4644. Field samples were rinsed with distilled water to remove salt and preservatives and the cleaned material was mounted for LM and SEM observations according to the method of Ferrario et al. (1995). Permanent mounts were made with Hyrax. For TEM, small aliquots of culture were placed with a micropipette on to formvar-coated grids and then rinsed in distilled water three times. Observations and photomicrographs were made using a Zeiss Axiolab photomicroscope and a Nikon Microphot–FX microscope (both equipped with phase contrast), a JEOL JSMT 100 scanning , and a JEOL 1200 EX transmission electron microscope. Terminology followed is that recommended by Ross et al. (1979), Brunel (1966, 1972), Rines & Hargraves (1988), Hernández-Becerril (1996) and Jensen & Moestrup (1998). MORPHOLOGY OF CHAETOCEROS TENUISSIMUS 329

Fig. 1. Map indicating locations where samples were taken for this study. Isobaths represent 0 and 10 m, respectively.

OBSERVATIONS

Chaetoceros tenuissimus Meunier

References: Meunier 1913, p. 49, pl. 7, fig. 55; Rines & Hargraves 1988, p. 97; Hasle & Syvertsen 1997, p. 223, pl. 47; Bérard-Therriault et al. 1999, p. 53, pl. 41, fig. g.

The description that follows is based entirely on the cultured material. The cells are usually solitary, rarely in pairs (Fig. 2). They are delicate, very small, and nearly square or rectangular in girdle view, with the pervalvar axis longer than the apical axis (Figs 2–4). The valves are flat or slightly convex. The setae are straight and arise from the poles of the cells at an angle of 45° to the apical axis 330 E. A. SAR, D. U. HERNÁNDEZ-BECERRIL AND I. SUNESEN

(Figs 2–5). The setae are very delicate and of narrow diameter. There is a single , occupying the entire cell. Resting spores are unknown. Electron microscopy shows the to be extremely weakly silicified and the valves to be elliptical or subcircular in valve view (Figs 3, 4). The valve face has a well-defined annulus, and costae which radiate from it (Figs 3–7). The annulus may be slightly eccentric or very near the valve face margin (Figs 3, 7). The valve face is separated from the valve mantle by a hyaline rim (Figs 5–7). The valve mantle is relatively high and has very weak costae or none at all (Figs 5, 7). Each valve possesses a single rimoportula within the annulus (Figs 4, 6, 8, 9). The rimoportula has an external projection, consisting of a flattened tube open to the outside (Figs 4, 8). The internal opening of the rimoportula is a single slit; no labia are present (Fig. 9). The rimoportula and its external projection are not visible in LM. The girdle is composed of several copulae (5–6), some of which are perforated by small poroids (Figs 4–6). The setae are uniform in width throughout and appear circular in cross-section. Distal to the point of origin they develop minute spines arranged in spirals (Figs 4, 8–12). Their walls show an intricate pattern of perpendicular costae, as well as costae in spirals, with some scattered poroids (Figs 10, 11). The setae are consistently oriented at a 45° angle to the apical axis. Measurements: for cells in culture apical axis 3.5–4 µm, pervalvar axis 4–11 µm. Cells with a longer pervalvar axis may have been dividing.

DISCUSSION

The general morphology of Chaetoceros tenuissimus is very similar to that of most members of the subgenus Hyalochaete. This subgenus was defined by Gran (1897) and revised more recently by Evensen & Hasle (1975). It includes delicate forms (either chains or solitary cells) with thin appendages (setae and external projections of the rimoportulae) and only in the body of the cell (not in the setae). The number of possible synonyms associated with Chaetoceros tenuissimus results from our poor knowledge of the degree of variation and lack of studies on morphological characters of species currently included in Section Simplicia. In addition, the ecological habits of most species in this section are similar. Rines & Hargraves (1988) cited four names that appear very closely related to C. tenuissimus. Chaetoceros galvestonensis Collier et Murphy is most likely a synonym of C. tenuissimus; however, it was described as possessing two chloroplasts. Chaetoceros calcitrans (Paulsen) Takano (= C. simplex var. calcitrans Paulsen) was considered a synonym of C. tenuissimus by Hasle & Syvertsen (1997), but it appears to be larger in its dimensions (Paulsen 1905 gave an apical axis length of 5–16 µm). Finally, C. calcitrans f. pumilus Takano seems to be only a smaller form of C. calcitrans and very similar to C. tenuissimus. Rines & Hargraves (1988) discussed their preference for use of the name C. tenuissimus over C. galvestonensis and C. calcitrans f. pumilus, but additionally considered “the possible existence of cryptic taxa”. Another species, possibly closely related to C. tenuissimus, is C. minutissimus Makarova et Proshkina-Lavrenko, which is comparable in size (length of apical axis = 3.6–9 µm as cited by Proshkina-Lavrenko & Makarova 1968) and the morphology of the . This is especially true of the position of the setae, which are oriented at a 45° angle to the apical axis, although they are not elevated at the valve ends, which are rounded. Chaetoceros minutissimus has not been reported to form resting spores and it has been found only in low salinity waters (12.8 psu) by Proshkina-Lavrenko & Makarova (1968). A comparison utilizing material from the locality type of C. minutissimus is necesary to prove conspecificity with C. tenuissimus. Morphological differences exist between Chaetoceros tenuissimus and other members of Section Simplicia. Chaetoceros simplex, C. gracilis, C. ceratosporus (and its var. brachysetus), C. muelleri (and its var. subsalsum) and C. transisetus form resting spores, a characteristic not found in

MORPHOLOGY OF CHAETOCEROS TENUISSIMUS 331

Figs 2–7. Chaetoceros tenuissimus. Scale bars = 5 µm (Fig. 3), 2 µm (Figs 2, 4, 5), or 1 µm (Figs 6, 7). Fig. 2. Several frustules of the species, SEM. Fig. 3. Two valves showing an annulus (arrowed) and costae, TEM. Fig. 4. Rather long frustule showing external tubes of rimoportulae on valve faces, SEM. Fig. 5. Nearly square frustule with several cingular bands, SEM. Fig. 6. Portion of frustule showing valve face and proximal part of setae, TEM. Fig. 7. Top valve with an annulus and costae radiating from it; annulus of the bottom valve arrowed, TEM. 332 E. A. SAR, D. U. HERNÁNDEZ-BECERRIL AND I. SUNESEN

Figs 8–12. Chaetoceros tenuissimus. Scale bars = 1 µm (Figs 8–10) or 0.5 µm (Figs 11, 12). Fig. 8. External view of valve showing external tube of rimoportula and costae, SEM. Fig. 9. Internal view of valve showing slit of rimoportula, SEM. Fig. 10. Portion of a seta showing intricate pattern of costae, TEM. Fig. 11. Detail of a seta showing some spines (arrows), TEM. Fig. 12. Detail of seta base, SEM.

C. tenuissimus. Chaetoceros gracilis and C. vistulae have setae which are distally polygonal (i.e. four- sided in cross-section) (Hernández-Becerril 1996), whereas in C. tenuissimus the setae are circular in cross-section throughout their entire length. Only Chaetoceros tenuissimus and C. minutissimus have their setae consistently oriented at an angle of 45° to the apical axis. The single chloroplast occupies the entire cell in both species. We believe that the specimens studied by Rogerson et al. (1986) do not correspond to Chaetoceros gracilis as identified by these authors, but rather belong to C. tenuissimus. Their photomicrographs (figs 1–6) show dividing cells with a setae orientation of 45° to the apical axis and a single chloroplast occupying the entire cell. In addition, their fig. 9 shows a seta with characteristics MORPHOLOGY OF CHAETOCEROS TENUISSIMUS 333 similar to those reported herein for C. tenuissimus. Setae orientation and structure in C. gracilis differs from C. tenuissimus. Furthermore, the former species produces resting spores and is larger than the latter. Section Simplicia consists of solitary (or in pairs as cell division takes place), small, delicate (weakly silicified) species, with 1 or 2 chloroplasts and very thin setae. Most produce resting spores and inhabit shallow, neritic waters or lagoons. A few species occur in freshwater environments (Ostenfeld 1912, Rushforth & Johansen 1986). Thus, according to Ostenfeld (1912), Hustedt (1930), Rines & Hargraves (1988), and our own observations, the following taxa may be included in Section Simplicia, although we are aware that many of these taxa are poorly known.

Genus Chaetoceros Ehrenberg Subgenus Hyalochaete Gran Section Simplicia Ostenfeld Chaetoceros ceratosporus Ostenfeld var. ceratosporus Chaetoceros ceratosporus var. brachysetus Rines et Hargarves Chaetoceros distinguendum Lemmermann Chaetoceros gracilis Schütt Chaetoceros minutissimus Makarova et Proshkina-Lavrenko Chaetoceros muelleri Lemmermann var. muelleri Chaetoceros muelleri var. subsalsum (Lemmermann) Johansen et Rushforth (basionym = Chaetoceros subsalsus Lemmermann; synonym = Chaetoceros borgei Lemmermann) Chaetoceros simplex Ostenfeld Chaetoceros tenuissimus Meunier Chaetoceros transisetus Johansen et Boyer Chaetoceros vistulae Apstein

One former member of the section Simplicia is Chaetoceros septentrionalis Oestrup, (synonym = Chaetoceros tortilisetus Mangin), which was transfered to the genus West as A. septentrionalis (Oestrup) Crawford (Crawford et al. 1994). At present, no other member of Section Simplicia appears to belong to another genus. Inconsistency in the present classification of Chaetoceros, especially as regards the sections, has been discussed by Rines & Hargraves (1988) and Rines et al. (2000). The present classification (3 subgenera and 20 sections) does not seem to reflect a “natural” classification, in which sections and species would be clades produced by cladistical analysis. The species currently placed in Section Simplicia have very few morphological and ecological characters that permit us to gather them in a group. At present we do not even know if it is actually a “natural” group. Many species, which presently belong to other sections of the subgenus Hyalochaete, also produce single, solitary cells, but this is to begin the process of regenerating a new . Therefore these cells should not be considered part of Section Simplicia. Recent studies of species in Section Simplicia (Johansen & Rushforth 1985, Rines & Hargraves 1986, Rogerson et al. 1986, Rushforth & Johansen 1986, Johansen et al. 1990, Johansen & Boyer 1995, Hernández-Becerril 1996, Jensen & Moestrup 1998, Bérard-Therriault et al. 1999) have shown some characters that seem to be common amongst members of the section. Nevertheless, more knowledge is needed regarding life cycles, formation of resting spores, and autecology of the species (e.g. Mann 1999). Species of Section Simplicia appear to be highly specialized forms: small and solitary cells, often very abundant in coastal waters, and with a great tolerance to variations in salinity 334 E. A. SAR, D. U. HERNÁNDEZ-BECERRIL AND I. SUNESEN

allowing some to even inhabit freshwater environs. It is possible that most forms within this section are recognized species and the section, as a whole, may be a “natural” group. However, at the moment we are not certain. There is some preliminary evidence that the genus Chaetoceros is paraphyletic (Medlin et al. 2000, Medlin pers. comm.). Further studies may show that different genera should be named, and that there are also “cryptic” species, based not only on morphology, but also on ecological, physiological and molecular studies.

ACKNOWLEDGEMENTS

We are grateful for the excellent technical support provided by Dr M. Pascual (BID 1201 OC–AR PICT 98 Nº O4221 from Agencia de Promoción Científica y Tecnológica) and partial financial support by a grant (PIP 525/98) from CONICET to EAS. Also thanks are due to the Instituto de Fisiología Celular, UNAM, for use of their facilities. Drs J. Rines and G. Fryxell sent relevant literature to DUH–B, and J. Johansen provided an excellent review of the manuscript.

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