Constancea 83, 2002
University and Jepson Herbaria
P.C. Silva Festschrift

The Ultrastructure and Taxonomic Placement of Diacanthos belenophorus Korš. (Chlorophyta, Trebouxiophyceae, Micractiniaceae)

Eberhard Hegewald
Institute of Chemistry and Dynamics of the Geosphere III (Phytosphere),
Research Centre Jülich, Jülich, Germany
Eberhard Schnepf
Lehrstuhl für Zellenlehre, Universität Heidelberg, Germany


Diacanthos belenophorus Korš. has two spine-like bristles which are in fine structure similar to those of Micractinium. As in that genus, Diacanthos also has a pyrenoid crossed by a thylakoid, a bipartite cell wall consisting of an inner electron-dense layer and an outer less electron-dense layer and mother cell wall fragments that remain attached to the autospore walls. Hindák's transfer of Diacanthos from Oocystaceae to Micractiniaceae is supported by this work.


Diacanthos belenophorus was described from a pond in Ukraine (Koršikov 1953) as a species with solitary cells, with very long “setae” on the opposite ends of the cells, with pyrenoid and reproduction by autospores, the autospores being released by splitting of the cell walls, and with spines formed towards the end of the cell separation (Koršikov 1987). The species was later recorded from Europe: France (Bourrelly 1966), Germany (Krienitz 1990 and our strain), Great Britain (Belcher and Swale 1962), Hungary (Hortobágyi 1966, Schmidt and Fehér 1998), Poland (Krzeckowska-Woloszyn et al. 1971), Russia (Sutov 1922 sub nom. Centritractus belenophorus), Slovakia (Hindák 1980), Africa: Namibia (Hegewald unpubl.), Asia :India (Hortobágyi 1966, 1969), and from New Zealand (Haughey 1968). More recently, it was reported from Asia: Japan (Yamagishi 1988), Korea (Chung and Kim 1994, Chung et al. 1994), and Taiwan (Yamagishi 1992). Koršikov (1953) placed Diacanthos in the Oocystaceae, subfamily Lagerheimioideae. He understood the family in a very wide sense, including all planktonic unicellular globular to ovate species, hence he excluded the Micractiniaceae, because these are colonial. Koršikov (1953) placed the genus in the subfamily Lagerheimioideae because here he placed all single-celled species with spines. This placement was accepted by subsequent workers except for Hindák (1980), who placed Diacanthos in the Micractiniaceae because he includes in that family "autosporiferous algae with spines on the surface of cells [having the character that when] the autospores release the mother cell wall does not extend but cracks or splits asunder into several portions". Hortobágyi (1966) described D. belenophorus var. monocanthos, characterized by having only one spine and D. belenophorus f. heterospinosus, characterized by having spines of different lengths. No other species have been assigned to the genus. The species has hitherto been studied only from nature and only by light microscopy. The present study was carried out on a strain isolated by Hegewald from Lake Lietzensee, Berlin, Germany (SAG 42.98 = Hegewald 1998-24). Our purpose was to elucidate the fine structural details of the cells in order to assess its taxonomic placement.


We verified the identity of strain SAG 42.98 by comparison with the original description. The strain was cultured in 20 ml tubes aerated with a 1% CO2-air mixture. The media and trace elements are as given in Hegewald et al. (1984), except that the stock solution of thiamin contains 250 mg/l. The cultures were grown at 16:8 h light:dark and 10 klux (corresponding to about 200 mol m-2s-1 if daylight had been used) at a temperature of 25C. Under these culture conditions the strain showed good growth.

For transmission electron microscopy the cells were fixed with phosphate-buffered (pH 7.4) glutaraldehyde (1%), post-fixed with OsO4 (1%) in the same buffer, dehydrated with acetone and embedded in Spurr resin. The thin sections were generally stained with uranyl acetate and lead citrate. Empty cell walls were negative-stained with phosphotungstic acid.


The cells are ovate and in cross section round and have a bristle on each cell pole (Fig. 1). The cell is partly filled by a cup-shaped chloroplast with starch grains and with a pyrenoid. Usually, the pyrenoid is nearly completely surrounded by two cup-shaped starch grains and is crossed by a thylakoid which passes through between the starch cups (Fig. 2, 3). The nucleus is placed randomly and has an inconspicuous nucleolus (Fig. 3). A variable number of plastoglobuli is found in the chloroplasts (Fig. 2, 3) and a single dictyosome is situated close to and parallel with the nucleus, orientated with its cis-face toward the nucleus (Fig. 3, 4). Cell wall fragments of the mother cell wall can be seen attached to adult cells (Fig. 5). The cell wall consists of an inner electron dense layer of about 18 nm thick and a less electron dense outer layer, about 22 nm thick. On each cell pole is one bristle (Fig. 1, 6, 8) which is square and hollow in cross section (Fig. 7). Detached bristles are closely connected with a cap-like fragment of the cell wall (Fig. 8). Negative staining revealed the bristles to be composed of fine, slightly sinusoidal filaments, best seen at the tip of the bristle, where it frays out (Fig. 9). In the body of the bristle, they are regularly arranged. The “unit cells” (see Schnepf et al. 1980) seen in diagonal view appear in a rhombic pattern (Fig. 10), but in orthogonal view as parallel lines (Fig. 11). In Fig. 12, both aspects are seen.


The genus Micractinium was studied under the electron microscope by Hegewald & Schnepf (1984), Moestrup (1972, sub nom. Golenkinia minutissima), and Schnepf et al. (1980). They found a special spine structure at that time not known from any other alga, the spines being square and hollow and composed of wavy fibres. The pyrenoid was crossed by single thylakoids and the cell wall was described as consisting of two layers (Moestrup 1972). Similar structures are shown in Figs. 2, 3, 6, 7 for D. belenophorus. Additionally Micractinium has the same orientation of dictyosome (unpublished data) and a similar cell wall fragmentation as is well known from the literature (e.g., Koršikov 1953, 1987). The Oocystaceae have a cell wall with cellulosic fibers in various layers and with spines of different substructure (Hegewald et al. 1980), although these are bristles, as in D. belenophorus (see Hegewald & Schnepf 1984; the word “spines” is used as a general term for spikes and bristles). Oocystis is also different in that the trans-faces of the dictyosomes are orientated towards the nucleus (Schnepf et al. 1966: fig. 32). Therefore, D. belenophorus has to be transferred to the Micractiniaceae, as already recommended by Hindák (1980) and as recently verified by 18S rDNA measurements (Krienitz, Hegewald, Hepperle, Wolf, unpublished). Already Koršikov (1953) and Hindák (1980) mentioned the fragmentation of the mother cell wall, and Hindák (1980) also points to the attachment of the cell wall fragments, both typical for Micractiniaceae. The genera Micractinium and Diacanthos have been recently shown to be allied with the Chlorellaceae, which are assigned to Trebouxiophyceae rather than Chlorophyceae (Krienitz et al. 2002a, 2002b; Wolf et al. 2002).

Diacanthos belenophorus differs from all species of Micractinium, by having ovate cells with a single, but thick bristle on each cell pole in contrast to Micractinium with colonies and several spines, not arranged oppositely. Species of the genus Didymogenes, which was transferred to the Micractiniaceae by Schnepf and Hegewald (1993), are similar to Diacanthos belenophorus in the above-described pyrenoid structure, cell wall, and bristles (Schnepf and Hegewald 1993). Additionally, Diacanthos and Didymogenes have the mother cell wall fragments attached to the daughter cells (Schnepf & Hegewald 1993). The Didymogenes species, however, have a curvate cell shape, more or less flat coenobia and 1 or 2 bristles on each cell pole or lack bristles.

Under the light microscope, a morphologically similar species is Desmatractum indutum (Geitler) Pascher. This species, however, has a cell wall composed of cellulosic fibers, that loosely surrounds the cells, (Reymond 1981, 1989, Hegewald and Tsarenko 1998). As in D. belenophorus, the pyrenoid is crossed by a thylakoid and a golgi vesicle is placed in a depression of the nucleus (Reymond 1981, 1989). Although there seems to be no relationship to the Micractiniaceae, the similarity in cell organelles indicate that also Desmatractum belongs to the Trebouxiophyceae. So far as is known the pyrenoid crossed by thylakoids seems to be a diagnostic characteristic for distinguishing the classes.


We thank Prof. T. Friedl for the strain from the collection Göttingen (SAG) and Mrs. S. Gold, Heidelberg, for the TEM micrographs.


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