New taxonomic studies on six Chlorococcum species
The generic name Chlorococcum appears frequently in reports of soil flora investigations, but there is a marked lack of agreement regarding the circumscription of organisms to which this genus name applies (Starr, 1955). Furthermore, there is confusion as to the limits of the species comprising this group. This disagreement may partly be explained by the fact that the greater number of the species of Chlorococcum were described before 1900 by investigators who studied algae only from mixed collections in nature. The intensive study of Starr (1955) on Chlorococcum and other spherical, zoospore—producing genera of the Chlorococcales laid a firm foundation for the taxonomy of this venus. The three most consistent characteristics which are in occurrence and tend to group the species in putative natural alliance are: the type of zoospore, the type of chromatophore in the vegetative cell and one presence or absence of a pyrenoid in the vegetative cell. The genus Chlorococcum is differentiated from other genera in the Chlorococcaceae by its possession of three constant attributes: (l) asexual reproduction by means of Chlamydomonas type zoospores, (2) vegetative cells with a parietal, hollow spherical chromatophore and (3) vegetative cells with at least one pyrenoid (Starr, 1955). The importance of each as a taxonomic criterion at the generic level is fully discussed by Starr (1955). The zoospores of all species of this genus constantly retain their shape when becoming quiescent; all possess two flagella of equal length. Recently in classifying the taxonomically difficult unicellular algae, phycologists have found it necessary to resort to morphological descriptions together with certain physiological characteristics, such as hydrogenase activity, gelatin liquefaction, secondary carotenoid synthesis and the growth responses to various carbon and nitrogen sources (McLean, 1968). Bold and co-workers (Brown and Bold, 1964); Chantanachat and Bold, 1962; Mattox and Bold, 1962; Smith and Bold, 1966) have employed nutritional requirements, utilization of various carbon and nitrogen sources, growth in light and dark, ability to reduce nitrite, antibiotic reactions and immunochemistry to supplement the attributes of species in several Chlorococcacean genera. McLean (1968) measured the total chlorophyll and carotenoid concentrations in relation to total pigment content for eighteen Chlorococcum isolates which were grouped according to the color of the culture incubated under defined conditions for 6-7 weeks. He suggested that pigmentation of old cultures be used as the first criterion for distinguishing species of Chlorococcum. Comparative ultrastructure studies have also been utilized to supplement the microscopic morphological attributes. Gibbs (1962) employed the ultrastructure of the pyrenoids in the taxonomy of green algae above the generic level. Brown and Bold (1964) were the first to separate species of Tetracystis by comparing the ultra-structure of chloroplast, pyrenoid, mitochondria, Golgi apparatus and cell wall. Recently, Brown and McLean (1969) on the basis of studying the fine structure of pyrenoids in eighteen Chlorococcum species proposed three differentiating categories: (l) pyrenoids with unfragmented perforate starch plates, (2) pyrenoids with many separated fragmented starch plates, and (3) pyrenoids with hemispherical unfragmented starch plates. Pyrenoids were further distinguishable on the basis oi the number of thylakoid disks or tubules which penetrate the ground substance. The objectives attempted in this study were three-fold: to compare the morphology of six different species of the genus Chlorococcum growing on six different media under defined culture conditions, to determine any diagnostic physiological characteristics helpful in the separation of these species, and to examine and study the cell wall ultras ultrastructure of each. It was intended that such data might provide additional information for the separation of the species under investigation, and hopefully, the principles might be applied to the classification of other microalgae.