Download - Revision of the genusTetraselmis (Class Prasinophyceae)

Bot. Mag. Tokyo 93: 317-339, 1980

Revision of the Genus Tetraselmis

(Class Prasinophyceae)

RICHARD E. ~ORRIS*, TERUMITSU HORI**

AND MITSUO CHIttARA**

* Department of Botany and Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington, U.S.A. 98250;

** Institute of Biological Sciences, The University of Tsukuba, Sakura.mura, lbaraki, 305

Information available on tile structure of species belonging to the genera Tetraselmis, Platymonas and Prasinoeladus has been reviewed. Detailed comparison of these data has convinced the authors that species in these genera all belong to the same genus. Stalk development by cast-off thecae, a characteristic used to define Prasinocladus, is variable within different species and is not reliable for separation of a genus. Similarly, the penetration of the pyrenoid by a lobe of the nucleus cannot be held reliable in separation of these genera because it occurs in varying degree in different species. Platymonas G.S. West (1916), Prasinocladus Kuckuck (1894) and Aalacochlamys Margalef (1946) are considered to be synonyms of Tetraselmis Stein (1878). Tetraselmis is redescribed using characters visible with light and electron microscopy as well as life-history characteristics. The description reviews information on many species of Tetraselmis that have been found in the western and eastern Pacific as well as species from Great Britain. It is determined that variations in life-histories may be explained by different environmental factors, whereas structure of vegetative cells, as viewed by electron microscopy, seems to be quite stable and characteristic for each species.

Key words: Aulacochlamys - - Platymonas - - Prasinocladws - - Prasinophyceae Taxonomy - - Tetraselmis.

Tetraselmis Stein (1878) is a genus of green flagellates (Prasinophyceae) t h a t

contains m a n y mar ine as well as a few freshwater species. Marine species often occur

in dense populat ions causing blooms in tide pools or bays, thereby being i mpor t a n t

to an unders tand ing of the dynamics of p lank ton growth in nerit ic habi tats . Tetraselmis

may also occur as a symbiont with marine animals (Provasoli et al., 1968).

Tetraselmis has been recognized as being very close, if not the same genus, to

Platymonas G.S. West (1916) (Parke and Green, 1976), bu t this unce r t a in ty was

recent ly decided by Melkonian (1979) who studied fine s t ructural characteristics of the

type species, T. cordiformis, and found tha t characterist ics of this species are the same as

those of the genus Platymonas. This impor t an t discovery corroborates the t r e a t me n t

of Butcher (1959) who t ransferred several species of Platymonas to Tetraselmis and

described several new species. Melkonian (1979) reviewed taxonomic problems tha t

318 R.E. NoRRIs et al.

have arisen because of the confusion between these two genera. Within Tetraselmis taxonomic decisions are difficult because reliable characteristics are not easily assessed with the light microscope, and also because there has been difficulty in recognizing limits in variability of cell characteristics.

In the following discussion, the authors present an information and collation of previously published analyses of Tetraselmis, Platymonas and Prasinecladus, adding their own unpublished information on these genera. In this way, the authors hope to establish the generic limits of Tetraselmis and to faciliate delimitation of species within the genus. The species of the genus, however, will be more completely described in future publications.

Materials and Methods

Most specimens used in this analysis of the genus Tetraselmis were collected in various parts of Japan, Washington State, U.S.A., and British Columbia, Canada. Several British specimens of Tetraselmis were supplied by the Culture Centre of Algae and Protozoa, Cambridge, England, and were studied. All of the cells figured in this publication are indexed by culture numbers; cultures of the Department of Botany, University of Washington, have three numbers separated by two dashes, those of the University of Tsukuba have numbers preceded by T, and those of the Culture Collec- tion of Algae and Protozoa, Cambridge, have two numbers separated by / .

Cultures of Tetraselmis were grown according to the methods and media described by Norris and Pearson (1975) and Tanimoto and Hori (1975). Preparation of specimens for electron microscopy was done following the techniques outlined by Norris and Pearson (1975).

Results and Di scuss ion

Living cells as observed with the light microscope Tetraselmis has at least three phases in its life-history (Provasoli et al., 1968;

Kobara and ttori, 1975; Tanoue and Aruga, 1975); a flagellate stage, a non-motile vegetative phase and a third stage in which the cells become converted into an aflagellate cyst with a thick, often ornamented wall (Fig. 35). Such cysts germinate by dividing into four cells. The term 'cyst' should be reserved for this thick-walled phase that divides into four cells, an uncommon phase compared with the occurrence of the other two phases in Tetraselmis' life-history. Species of Tetraselmis often remain in a vegetative, non-motile phase for very long period of time, and undoubtedly it is the dominant phase in the life-history of certain species under some environmental conditions. During this non-motile phase new walls develop, old walls being cast-off and accumulating as concentric rings around the cell or becoming densely arranged and polarized on one side of the cell forming a stalk. Prasinocladus has a similar non- motile phase, but in Prasinocladus cast-off walls accumulate to form long acellular stalks subtending single cells or small groups of cells. Kylin (1935) and Proskauer (1950) discussed wall formation and stalk development in Prasinocladus and compare this

Revision of Tetraselmis 319

development with similar wall formation in Tetraselmis (as Platymonas). Proskauer

(1950) outlines the following series of events in Prasinocladus ascus: 1) cells settle and throw off the flagella, 2) the protoplast retracts from the anterior part of the wall, 3)

the part of the wall that was in contact with the apical depression of the cell becomes everted, 4) if cells are in fresh media a new wall is secreted inside the original wall,

5) the new wall elongates and the outer wall is ruptured at its upper end, 6) the protoplast rounds off and lies at the upper end of the wall; as the wall grows in length a

tube is formed behind the protoplast, 7) the protoplast develops a new wall inside the tube, 8) the protoplast may divide, always longitudinally, 9) one daughter cell slides

past the other and the two cells lie obliquely in the apical end of the tube within their

mother celt wall, 10) one of the daughter cells may rotate 180 ~ within the mother

wall, 11) the upper cell may continue the formation of a tube, leaving the lower cell

behind, or 12) both daughter cells may repeat the above process, producing two tubes, each breaking through the original wall, or 13) each daughter cell may continue to

divide in situ. I f cells remain in nutrient depleted media, or are in some other way placed in an

unfavorable environment, they may behave in an entirely different way. In this situation the protoplast forms successive walls, each of which is broken in turn without appreciable elongation at each step. This type of development is more typical of the species of Tetraselmis than Prasinocladus, but Tetraselmis species may not have

polarized cast-off walls. Instead, the cast-off walls may form a concentric layer of wall material around the cell, the walls being broken in various positions, or, perhaps not

broken at all but becoming somewhat gelatinized.

I t is important to note that cells at any of the stages discussed above may develop flagella and become motile. In such cases, therefore, the cast-off walls may be vacated

entirely, or only one of a pair of daughter cells may swim from the cast-off walls leaving

the other in a non-motile condition at the end of the stalk or inside the concentric walls.

I t is also very important to note that for the life-history of the type species of

Tetraselmis, T. cordiformis, the only stage that has been studied (Melkonian, 1979) is

the flagellate cell. At the present time the authors do not have information on any

non-motile cell in the life-history of that species.

Hori and Chihara (1974a) showed that Prasinocladus marinus, when grown under

favorable conditions, develops a stalk of cast-off walls by contraction of the cell and its

subsequent emergence through a pore that develops in the posterior part of the wall, the cell remaining attached to the cast-off wall, forming a new one and repeatedly contracting and casting off old walls. Under unfavorable conditions the same species

was shown by Hori and Chihara (1974a) to accumulate cast-off wails concentrically around the cell, presumably the main difference being that the cell did not emerge from the cast-off wall. The demonstration of these two systems of behavior in the same isolate of Prasinocladus, one being more typical of Tetraselmis, conclusively demon- strates that the stalked nature of Prasinocladus colonies cannot be used to separate

that genus from Tetraselmis.

320 R.E. NORRIS et al.

Figs. I-8. Electron micrographs of Tetrasdmis species, showing tim structure and distribution of cell organelles. 1: Longitudinal section of a cell (cult. 15-8-17). Note two massive rhizoplasts (rh) originating from the basal body apparatus. Distal end of left rhizoplast connects to the broad cell surface where there is no special structure. The nucleus (N) lies between the rhizoplasts and beneath it is a small pyrenoid (P) surrounded by distinctly shaped, pyrenoid starch grains (PS) different from the starch grains elsewhere in the chloroplast (S). Flagella (F) emerge through the thecal slit in the bottom of the apical depression. Numerous flageliar pit hairs are present, x 7000. 2, 3: Sections of flagella. Small arrows indicate rod-shaped scales of the outermost layer; arrow heads indicate square tQ pentagonal scales of the innermost layer. Large arrows in Fig. 3 indicate hair-scales. Note (hat the scales in the outer layer overlap the gap between scales of the inner layer. 2, (cult. 66/12, T. levis), • 74000; 3, (cult. 8/6, T. chuii), • 37000. 4: Scanning electron micrograph of a cell. Note four flagella emerging from the flagellar pit, and the blunt distal end of the flagella. (cult. 15-8-61), • 2100. 5: Section of a theca having a rough surface. C, chloroplast. (cult. 15-8-61), • 17000. 6: Section of a theca consisting of three layers indicated by the number of 1-3. C, chloroplast; S, starch grain. (cult. 10-8-54), • 31000. 7: Section of a cyst wall. Note the outer electron dense layer with spines and the inner electron translucent layer. C, chloroplast; S, starch grain. (cult. 10-8-8), • 7000. 8: Scanning electron micrograph of an ornamented cyst wall. (cult. (cult. 8/6, T. chuii), • 13000.


Butcher (1959) used the term 'cyst' for cells that are surrounded by cast-off walls in Tetraselmis. It is difficult to determine from Butcher's discussion whether or not he actually observed cysts as those described by Kobara and Hori (1975) and Tanoue and Aruga (1975). Only one of Butcher's figures (Plate XII, Fig. 2b) shows a wall thick enough to suggest that it is a cyst. The presence of a papilla on the outermost layer of wall material in Butcher's figure suggests that a cast-off wall is present around that cell.

Proskauer (]950) briefly discussed and illustrated thick walled cysts for Prasino-

cladus ascus. It is difficult to determine whether or not this structure is the same as the true cyst. Proskauer's illustrations show a layered wall surrounding the cyst and two or three cells inside germinating cysts. The figure showing three cells in a germinating cyst suggests that this may be the same cyst as has been found in Tetraselmis, but additional studies are needed to determine the presence of such cysts in this species.

Flagellate cells of Tetraselmis are structurally identical with those of the non- motile vegetative cells with the exception that they are surrounded by only one wall layer whereas many non-motile cells have accumulated several wall layers.

The flagellate stage is the one described as characteristic of the genus Tetraselmis,

the cells having four flagella of equal length that are attached at the bottom of an apical, trough-shaped cell depression. Many species of Tetraselmis have compressed motile cells, although other species may be ellipsoidal to cylindrical in shape. Some species have slightly to strongly ridged cells, particularly in middle to posterior regions. Twisting of the ridges occurs in the posterior parts of some species. Also, most species have two or four slight to distinct creases extending much of the length of the cell wall (Fig. 4), the wall thereby being divided into two or four longitudinal segments. The only pore or opening in the wall, however, is the slit occurring at the base of the flagellar pit through which the flagella emerge. When walls are cast-off, that part of the wall in the trough-shaped flagellar pit becomes everted and then appears as a very short papilla in some species.

Flagella usually are shorter than the length of the cell (Fig. 35, A1) and emerge from the cell in two pairs, each pair parallel to the longer flat sides of the cell in the root position (Fig. 36K). The partners of the flagellar pairs diverge from one another toward opposite ends of the cell as they emerge from the flagellar pit in most species. In some species the flagellar partners remain close to one another as they emerge from the pit, thereby lying on the middle part of the fiat sides of the cell. Most Tetraselmis

species, however, have the diverging partners forming an X-configuration formed with

the opposite pair of fagella (Fig. 36). In T. convolutae divergence of the flagella in each

pair is extreme, the flagella lying along the narrowed sides of the flattened cells rather

than on the edges of the long fiat sides (Parke and Manton, 1967). Salisbury and Floyd (1978) discuss the flagellar activity of Tetraselmis as a breast-

stroke beat, the power stroke "is an oarlike beat, followed by a bend-propagated recovery stroke that encounters little resistance to the return movement". Their

322 R.E . :NoRRIs et al.

discussion continues to explain that the flagella are attached at the base of a deep depression "which places some peculiar geometric constraints on flagellar activity." " In particular, the degree of bend possible at the proximal end of the flagellum is less than that for cells without sunken flagellar bases."

Cells rotate on their axes while swimming, most species are apparently swimming in a straight line for fairly long distances and then abruptly changing direction without stopping. The activity of T. convolutae is discussed by Parke and Manton (1967) as being somewhat different from most Tetraselmis species in having repeated changes in direction without swimming for a very long distance in one direction. Also, this species apparently is somewhat different from others in attaching to the microscope slide with splayed flagella, although this behavior has been observed in other species.

A single large chloroplast is present in each cell, the shape of the chloroplast usually closely following the shape of the cell lobing. In most species a single pyrenoid is present within the chloroplast, usually in a central to posterior position. The pyrenoid occupies a more or less central position inside the cell, and some species have consider- able open lobing of the chloroplast posterior to the pyrenoid. The pyrenoid is surrounded by a starch shell in most cells that have grown in a well lighted environment, and grains of stroma starch are usually found in other parts of the chloroplast. A single stigma is usually present in the chloroplast, located on one of the cell's flattened sides. The position of the stigma along the length of the cell may vary in different species from near the anterior end of the cell to near the middle of the flat side to a more posterior position. This characteristic has been used to aid in determining species of Tetraselmis (Butcher, 1959). Melkonian and Robenek (1979) recently discussed the possible role of the eyespot in Tetraselmis phototaxis as well as structural features that may indicate its importance to cell orientation toward light. Some species have two to several eyespots or reddish-pigmented regions that resemble eyespots, usually all located in posterior regions of the cell. A few species of Tetraselmis accumulate large

amounts of haematochrome in the cell, causing them to become generally reddish in

color. Several Tetraselmis speices have been described as not having eyespots.

Lobing of the cell apex, texture of the chloroplast and arrangement of granules within the cell are other characteristics that have been used to separate species of

Tetraselmis (Butcher, 1959), but these characters do not seem to be reliable and may

not be observed in all species. Each cell of Tetraselmis has a single nucleus positioned centrally in the cell, directly beneath the flagellar depression. Division of organelles

occurs synchronously and immediately prior to nuclear division. Cytokinesis occurs

soon after mitosis and follows a phycoplastic pattern (Stewart et al., 1974).

Tetraselmis species divide in the non-motile state, and in most species one of the daughter cells inverts within the parent theca so that the two cells lie in reversed positions, anterior ends lying next to posterior ends. A few species do not seem to invert in post division periods and remain lying next to one another as they were formed, anterior next to anterior and posterior next to posterior. Tetraselmis roscoffensis has cells that do not invert after division and Butcher (1959) shows cells similarly

Revision of Tetraselmi~ 323

arranged in the mother theca of T. rubens, although he gives no discussion of this figure. Margalef (1946) placed great importance on this characteristic and used it as one of the primary ones for the establishment of the new genus, Aulacochlamys. Parke and Manton (1967) pointed out that daughter cells of T. convolutae sometimes inverted within the mother theca, but other daughter cells might not invert. The present authors find this to be true in other species as well and, for this reason, the validity of a separate genus based on absence of inversion of one daughter cell after cytokinesis, should be rejected, and the authors consider Aulacochlamys to be a synonym of Tetra- selmis.

Cells observed with the electron microscope Using electron microscopy, flagella of Tetraselmis are shown to be thick, blunt-ended

(Fig. 4) and to be covered by loosely attached, cross-striated flagellar hairs (hair-scales) and two layers of scales covering the entire flagellar membrane (Fig. 3). Flagella are also covered by a layer of mucilaginous-like material that may obscure the scales in whole mount preparations (Manton and Parke, 1965). The hair-scales are attached between scales of the inner layer and penetrate the mucilage, being visible in whole- mounts. The outer scale layer on the flagella is composed of small, somewhat rod- shaped scales that have an irregular fibrillar pattern, sometimes having short marginal proliferations (Figs. 2 and 3). This layer of scales is readily disorganized by fixation and manipulation of the cells for electron microscopy, but they seem to form a regular layer in which individual scales overlap the space between the scales in the inner scale layer (Fig. 2). These rod-shaped scales are similar to those found on flagella of Pseudoscourfieldia (Manton, 1975; Norris, 1980). The scales forming the inner layer on the flagella are similar to scales found on various other genera of prasinophytes (Norris, 1980). They are square to pentagonal in face view (Figs. 2 and 3), and have a rim of electron dense fibrillar material and a central protuberance of similar composition. A stellate pattern of electron translucent material is present around the central protuberance. The rim is as high or slightly lower than the central protuberance. The inner scale layer is compact and completely covers the flagella. All of these scales produced in the Golgi apparatus are transported into the scale reservoir (Fig. 10) before releasing on the flagellar surface.

The theea closely encloses the cell in many species, but there may be places where the theca and cell membrane are widely separated because of irregularities in the shape of the cell (Figs. 17 and 23). The wall usually has smooth contours except for regular creases that may separate the wall into two or four sections in the apical end (Fig. 4). The twisted posterior part of the cell that occurs in some species may also be reflected in the shape of the wall in these species. The only opening in the wall is at the base of the apical depression in the form of a slit through which the flagella protrude (Figs. 1, 13 and 16). Extensions of the wall border the flagellar slit, extending upward for a short distance along the flagellar bases (Figs. 1 and 13). In cells losing flagella, these wall extensions close the slit but do not fuse together (Figs. 11 and 33). Peculiar thick, often curly, hairs are present at the base of the flagellar pit in most Tetraselmis species,


Figs. 9-14. Electron micrographs of Tetraselmis species, showing the structure and distribution of cell organelles, especially small particles, scale and half-desmosome. 9: Two dictyosomes (G) adjacent to the flagellar pit which are actively producing many small particles (small arrows) some of which, when released onto the cell surface, form the theca, as can be observed in this figure. Square or pentagonal flageilar scales (arrow heads) are also present in the same dictyosome cisternae, b, basal body; C, chloroplast; er, endoplasmic reticulum; N, nucleus. (cult. T164), x 13000. 10: Scale reservoir containing two types of flagellar scales, hair-scales (large arrow) and pentagonal or square scales (arrow head). Rod shaped scales are r/or clearly seen in this figure. (cult. 15-8-38), • 15000. 11: Longitudinal section of cell ~showing two half-desmosomes (Hd) and one basal body (b). Two dictyosomes (G) and endoplasmic reticulum (er) lie above the nucleus (N) on either side of the basal body apparatus, h, flagellr pit hairs. (cult. 66~7, T. hazenii) , • 18000. 12: Cross section of flagellar pit base showing tangential sections of four half-desmosomes (arrow-heads). Zig-zag arrangement of flagella is evident, (cult. 66/lc, T. tetrathele), x 20000. 13: Longitudinal section of flagellum (F) and half-desmosome (Hd). Flagellr pit hairs (h), theca (t) and plasmalemma (arrow head) are also shown, (cult. 15-8-17), • 14: Enlargement of half-desmosome shown in Fig. 13. Anchoring material (arrow) is evident on the half-desmosome (Hd) between the theca (t) and plasmalemma (arrow heads), • 38000.


Figs. 15-19. Electron micrographs of Tetraselmis species, showing the structure and distr ibution of flagellar apparatus . 15: Longitudinal section of a basal body (b) showing cylindrical material (el) and basal plate (bp). A half-desmosome (Hd) lies to the r ight of the basal body. Arrow indicates the flagellar stub. (cult. 15-8-78), x 43000. 16: Longitudinal section of four flagella (F) and their basal bodies, showing the basal bodies terminat ing on a common electron dense floor (arrow head) above nucleus (N). (cult. 66/lc, T. tetrathele), X 20000. 17: Cross section of a cell. Two rhizoplasts (rh) extend from the basal bodies (b), each towards one broad side of the cell. Four anter ior chloroplast lobes (c) are clearly seen. N, nucleus; G, dictyosome. (cult. I5-8-16), • 7500. 18: Cross section of four basal bodies (b l -b4) t h a t are uni ted by two str ia ted synistosomes (syl and sy2). Par t s of the two rhizoplasts (rh) are present. (cult. 15-8-19), X 54000. 19: Cross section of four basal bodies (b) Shut are uni ted by binding fibrils (arrow heads). The two masses of electron dense material on either side of the basal body row may be oblique sections of half-desmosomes. (cult. 66/6, T. rubens), X 50000.


Figs. 20-26. Electron micrographs of Tetrazelmis species, showing the structure of flagellar apparatus, stigma, pyrenoid and nucleus. 20: Cross section of four basal bodies (b) to which proximal branches of the rhizoplasts (rh) are attached. (cult. 15-8-80), • 28000. 21 : Cross section of five mierotubules (arrows) which are part of a half-desmosome. (cult. 10-8-4), • 44000. 22: A half-desn~osome with four microtubules (arrows). (cult. 15-8~1), • 44000. 23: Longitudinal section of a cell having a pyrenoid (p) invaded from many directions by cytoplasmic canaliculi. The stigma (E) is located beside the pyrenoid. C, chloroplast; er, endoplasmic reticulum; G, dictyosome; m, mitochondrion; N, nucleus; Nu, nueleolus; S, starch grain. (cult. T090), • 24: Enlargement of stigma shown in Fig. 23. Note the thylakoid (arrow head) between the rows of electron dense globules. • 12000. 25: Pyrenoid (P) showing canaliculi and lens-shaped starch granules (S). (cult. 66/5, T. striata), • 14000. 26: Pyrenoid (P) having a cavity into which a lobe of the nucleus protrudes (N). S, starch grain. (cult. 66/7, T. hazenii), • 14000.


apparently attached to the outside of the wall (Figs. 11 and 13-15). These hairs are spirally cross striated (Fig. 14) in a way similar to the flagellar hair-scales, but they are much thicker and often longer than the hairs on the flagella. Similar hairs are also present in the fiagellar attachment region of Pseudoscourfieldia longifilis (Norris, 1980) and may indicate, along with the structure of the outer flagellar scales, that there is a close phylogenetic relationship between that genus and Tetraselmis. Such hairs are not known to occur in any other prasinophyte genera.

In most species of Tetraselmis thecae are smooth and one or two-layered, but species having more or less rough thecae (Fig. 5) and species having three layered one also occur (Fig. 6). Thecae are formed by deposition of small stellate-like scales, derived from the Golgi apparatus, on the cell membrane during and shortly after cytokinesis (Fig. 9). Only one type of scale is deposited in T. tetrathele and T. subcordiformis (Manton and Parke, 1965; Stewart et al., 1974), but two types of fibrilloid structures are deposited in formation of thecae in T. convolutae (Parke and Manton, 1967). Stewart et al. (1974) showed that these scale-like particles are apparently formed in a large dense mass inside the Golgi vesicles at prophase, and they suggested that these large particles may separate into smaller ones as they are deposited. Cohesion of the particles begins outside the cell membrane in the region adjacent to the pyrenoid in T. tetrathele (Manton and Parke, 1965) and gradually the fusion of scales extends forward. Because the pyrenoid is in a posterior position in most species of Tetraselmis, the last part of the theca to form around a cell is usually in the anterior

region (Fig. 9), probably at the base of the flagellar pit where the slit through which the flagella emerge is present.

Growth of fully formed walls, as occurs in development of stalked cells and colonies in Prasinccladus, has not been studied with the electron microscope. The dissolution of certain parts of walls that occurs in these species, thereby allowing the contained cell(s) to emerge, also needs study with the electron microscope.

Lewin (1958) and Gooday (1971) showed that the theca of Tetraselmis is composed

of a pectin-like material having galactose, galacturonic acid and arabinose as the

major components, and Manton et al. (1973) demonstrated that calcium is present in the theca of one species.

Cyst walls usually have protuberances of varing structure that may be useful specific characteristics (Fig. 8). Although Tanoue and Aruga (1975, 1977) and Kobara and Itori (1975) studied environmental conditions that contribute to the formation of cysts in several species of Tetraselmis, the layering and formation of cyst walls have not been fully examined with the electron microscope (Fig. 7), and there is no information available at present on the composition of these walls.

Vegetative cells, whether flagellate or non-motile, remain fixed in their position inside the theca by means of four contact points (Figs. 12 and 37) consisting of microtubules (Figs. 21 and 22) and electron dense material (Figs. 11, 13 and 14). Fig. 12 shows a tangential section through the distal ends of these four contact points, while Figs. 13 and 14 are longitudinal sections of two or one of these four points. These occur at


Figs, 27-31. Electron micrographs of Tetraselmis species, showing the various types of pyrenoid and chloroplast, and distribution pattern of dictyosomes. 27: Pyrenoid (P) having a cavity that is completely filled with a nuclear lobe (N). A thylakoid (arrow heads) lies between the pyrenoid matri~ and starch grains (S). Chloroplast membrane (ce) and nuclear membrane (he) are present surrounding the nuclear lobe in the pyrenoid cavity. (cult. 66/23, T . verruco~a), • 22000. 28: Pyrenoid matrix (P) invaded from various directions by cytoplasmic channels that contain electron-dense material surrounded by a single membrane (arrow). ce, chloroplast envelope; S, starch grain. (cult. 15-8-51), • 12000. 29: Longitudinal section of a cell showing a chloroplast (C) that is not posteriorly lobed. G, dictyosome; N, nucleus; P, pyrenoid. (cult T095), • 8000. 30: Longitudinal section of the posterior end of a cell showing chloroplast lobes (C). P, pyrenoid; S, starch grain. (cult. 66/]3, T. gracilis), • 13000. 31: Cross section of the anterior part of a cell showing basal bodies (b), synistosome (sy), endoplasmic reticulum (er) and dictyosomes (G). (cult. 15-843), • 18000.


the base of the flagellar pit and have been termed half-desmosomes by Sehnepf and Maiwald (1970), and Norris (1980) considers them to be similar to nmltilayered systems of other green plants. Melkonian (1979) found these structures in T. cordiformis, referring to them as crueiate flagellar roots of a compound type. In that species two of the roots have four mierotubules and the other two, forming an

opposite pair, have two microtuhules. At the point where each of the roots reaches the plasmalemma, the plasmalemma has on its surface many proliferations that play a role fixing the plasmalemma with the theea (Fig. 14). According to Melkonian (1979) two of tile four contact points are oval ones, each of which has a four-mierotubular root,

whereas another two are smaller circular ones, each of which has a two-mierotubular root. The present authors' observations on a wide variety of Tetraselmis species show that the number of microtubules in the roots is variable (Figs. 21 and 22) and may be a characteristic of value in determination of species.

The mierotubules in these compound roots terminate in the half-desmosome pads (Fig. 37). Melkonian (1979) described the proximal regions of the compound

mierotubular roots. In T. cordiformis the two four-stranded roots end in the same plane proximally touching one another, but are not continuous. They end in the space between the two pairs of basal bodies. The two stranded roots end between the basal bodies in each pair, and are not in the same plane, being considerably displaced with one another.

As the present authors indicated above in their discussion of characteristics of Tetraselmis that are visible with the light microscope, the flagella emerge from the cell in two pairs. Electron microscopy shows that the two pairs are arranged in a slightly zig-zag row at an angle of 45 ~ from the median of the broad side of the cell (Figs. 17-20 and 31). Thin fibrils, shown in cross section to form two peripheral parallel strands and one diagonal strand (Fig. 19), unite the flagellar partners to one another, while the pairs are united by short fibrils lying parallel to one another at a point where the pairs are closest together.

The basal bodies penetrate the cell for approximately 0.6 #m (• #m) in depth,

the four basal bodies all terminating at the same level within the cell, lying parallel and fairly close together (Fig. 16). A narrow electron dense layer forms a common floor extending beneath all four basal bodies (Figs. 16 and 37). The central tubules of the

axoneme terminate at a point 0.25 #m (• tim) above the point where the flagella

enter the cell. The c-tubules of the triplet mierotubules composing the basal body terminate slightly beneath the level of the basal plate. A cylinder of electron dense

material occurs between the basal plate and the ends of the axoneme (Fig. 15). A larger region of little electron density subtends the axoneme tubules and there is no axosome present. The membrane collar of the flagella is located adjacent to this distal region of little density. The basal bodies of Tetraselmis resemble the Type II basal bodies illustrated by Pitelka (1974), but differ in not having an axosome and in the postition of the basal plate. The cylinder of electron dense material found in the region between the axoneme tubule ends and the basal plate is not present in Type II basal bodies.


Non-motile cells of Tetraselmis usually have flagellar stubs that do not emerge through the thecal slit (Fig. 15). Usually the stubs of the flagella protrude above the cell surface, the stubs terminating at the proximal ends of the axoneme tubules (Fig. 15). The stubs contain the electron dense cylinder as well as the basal plate that lies proximal to it.

There is no single synistosome am described for Pyramimonas parkeae by Norris and Pearson (1975), nor is there a distinct system of distal and proximal fibers as described for Carteria by Lembi (1975). Instead, in Tetraselmis there are two synistosomes that lie exterior to the basal bodies, one facing each broad side of the cell (Figs. 18, 31 and 37). Each synistosome lies opposite the rhizoplast, which attaches to three of four basal bodies. Synistosome one (syl in Fig. 18) abuts onto basal body one (bl in Fig. 18), extends past basal body two (b2 in Fig. 18) where it thickens and attaches to the middle of basal body three (b3 in Fig. 18), where it terminates (Fig. 18 and 37). Synistosome two (sy2 in Fig. 18) extends in a similar manner on the opposite side of the basal body complex from basal body four (b4 in Fig. 18) to basal body two (b2 in Fig. 18) (Fig. 37). Each synistosome is banded transversely and may be the same type of structure that Lembi (1975) called 'proximal fibers' in species of Carteria. In Tetraselmis the synistosomes seem to be uniting the partners in each of the pairs of basal bodies in addition to fusing the pairs of basal bodies into a single system (Fig. 37).

Two rhizoplasts originate in the basal body complex, one directed toward each broad side of the cell (Figs. 1, 17 and 37). They are elongate, striated rootlets and approach the cell surface at their distal ends. The rhizoplast divides into several branches where it joins the basal body complex. As shown in Figs. 17, 20 and 37, these branches in each rhizoplast attach to the adjacent three basal bodies, and another ones continue with three of the half-desmosomes (Fig. 37). The plasmalemma often is indented at the points where the rhizoplasts are attached (Fig. 1). Melkonian (1979) mentioned that the rhizoplasts are linked to the nuclear envelope by small appendages, and that condensed chromatin is present in the nucleus adjacent to the rhizoplast. A few microtubules may lie alongside the rhizoplast, particularly in their middle to distal regions. The striation pattern of rhizoplasts changes along the length of the structure, and contraction of the rhizoplast, causing different striation patterns, is discussed by Salisbury and Floyd (1978).

Chloroplasts in species of Tetraselmis usually occur one in each cell and most often are massive, occupying much of the cell volume. The chloroplast has four anterior lobes that usually extend to the ends of the anterior cell lobes (Figs. 17 and 23). The posterior part of the chloroplast may or may not be lobed (Figs. 23, 29 and 30). Species having a more centrally located pyrenoid usually have a posteriorly lobed chloroplast, but the posterior lobes are shorter than the anterior ones. The posterior lobes are often irregularly shaped (Figs. 23 and 34) and difficult to see with the light microscope. Species having more posteriorly located pyrenoids usually have no posterior chloroplast lobes (Fig. 29). Some species of Tetraselmis, including the type species, have reticulate chloroplasts (Fig. 32). Thylakoids and lamellae are similar to those in other green algae, no definite number of thylakoids being found in the lamellae.

Revision of Tetra.~elmis 331

Figs. 32-34. Electron micrographs of Tetraselmis species, showing the structure of pyrenoid, chloroplast and nucleus. 32: Longitudinal section of the posterior part of a cell showing a reticulate chloroplast. Stigma (E) is posterior, and the pyrenoid (P) cavity contains cytoplasmic microtubules (mt). m, mitochondrion; N, nucleus; lp, lipid droplet. (cult. 10-8-8), • 10000. 33: Longitudinal section through an anterior part of a cell showing half-desmosomes (Hd), dictyosomes (G), cytoplasmic microtubules (mr), nucleus (N) and nucleolus (Nu). Large arrows show an anteriorally directed lobe of the nucleus. (cult. T164), • 11000. 34: Longitudinal section of the posterior part of the cell shown in Fig. 33. Pyrenoid matrix (P) is invaded by canaliculi from different directions. A microbody branch (mb) penetrates pyrenoid cavity, m, mitochondrion; mr, cytoplasmic microtubules; N, nucleus. (cult. T164), • 11000.


Grana have not been found in the species of Tetraselmis that have been studied at this time.

A pyrenoid is always present in species of Tetraselmis, although the matrix may be small. I t is located in a median to posterior position in the cell. The pyrenoid matrix is usually surrounded by specialized starch grains that are concave on the side adjacent to the matrix (Figs. 23, 26, 28-30, 32 and 34), but some species have starch grains around the pyrenoid matrix that are little different from the starch grains in other parts of the chloroplast, both types being convex on both sides (Fig. 25). Starch has been demonstrated to be of the same type as is found in other green algae and higher plants (Suzuki, 1974). Mannitol has been demonstrated to be present in Tetraselmis by Craigie et al. (1967) and by Suzuki (1974).

The pyrenoid matrix usually has a cavity on the side adjacent to the nucleus (Figs. 26, 29 and 32), but some species have no such cavity, the pyrenoid matrix instead being invaded by eanalieuli from several directions (Figs. 23 and 28). All of these cavities and eanalieuli are bounded by the extension of the chloroplast envelope. The eanalieuli usually contain cytoplasm and, sometimes, a lobe of the nucleus, a mito- ehondrion, or a lobe of the microbody. In some species including Tetraselmis cordiformis

and Prasinocladus marinus, one or both of the nuclear membranes may penetrate into some or all of the pyrenoid canaliculi (Fig. 27) (Parke and Manton, 1965; Provasoli et

al., 1968; Itori and Chihara, 1974a; Melkonian, 1979). In some other species including Platymonas convolutae and Prasinocladus ascus, the cytoplasm in the canaliculi is surrounded by a single membrane, which might not be the extension of the nuclear membrane (Fig. 28) (Parke and Manton, 1967; Hori and Chihara, 1974b). The structure of the pyrenoid is a useful taxonomic characteristic.

Prasinocladus marinus has a thylakoid extending around the pyrenoid matrix separating it from the enveloping starch grains (Fig. 27) (Manton and Parke, 1965; Provasoli et al., 1968; Hori and Chihara, 1974a), and this membrane also seems to be present in "culture 371" of Provasoli et al. (1968) but not in most other species of Tetraselmis that have been examined.

Most species of Tetraselmis have a conspicuous stigma in the chloroplast, usually located in a median to posterior position (Figs. 23 and 32), but in some species it may be in the anterior region. The stigma is always on one of the broad sides of the cell, sometimes marginal on that side, and is composed of two layers of lipid granules that lie adjacent to the chloroplast envelope. A thylakoid usually is present between the layers of lipid granules in the stigma (Fig. 24).

The nucleus is situated between the two rhizoplasts, resting between the anterior lobes of the chloroplast adjacent,to the pyrenoid. Anterior lobing of the nucleus may occur in some species (Fig. 33), particularly on the broad side of the cell. Posterior nuclear lobes occur in some species, invading the pyrenoid cavity (Fig. 26). The outer nuclear nlembrane forms rough endoplasmic reticulum that extends into the region of the forming face of the dictyosomes, and also extends into the other parts of the cytoplasm (Fig. 23).

Dietyosomes are restricted to the region surrounding the basal body complex


(Figs. 9, 11, 23, 31 and 33). The number of dictyosomes in the Golgi apparatus is variable, but usually seems to be at least two or four, some species having as many as eight or more. I t seems to be a fairly reliable specific characteristic. The dictyosomes' forming face is usually on the side facing the chloroplast, the maturing face being directed toward the region of the basal bodies (Figs. 9, ]1 and 23). Flagellar scales and wall element scales are formed within dictyosome cisternae (Fig. 9) that often fuse with one another forming small reservoirs containing several scales (Fig. 10) that are eventually deposited on the cell surface (Fig. 9), usually during periods associated with cell division.

Mitochondria are located throughout the cytoplasmic cavity of the cell (Fig. 23). A microbody is present, usually in a position posterior to the nucleus between it and the chloroplast. The microbody often is branched and its lobes sometimes extend into pyrenoid matrix canaliculi (Fig. 34).

Microtubules radiate from the basal body complex and extend into posterior parts of the cell. Some microtubules extend along the inside of the chloroplast cup in a rather irregular arrangement (Fig. 34), but some of these may penetrate the perforations of the chloroplast or even canaliculi of the pyrenoid matrix, some terminating in the canaliculi (Fig. 32). A few microtubules extend forward from the basal body complex, one group anchoring in the basal body apparatus and being part of the compound root system described above, and the other microtubules not being so clearly anchored in the basal body apparatus and extending forward over the lobes of the chloroplast where they are directed posteriorly.

Other cytoplasmic inclusions that occur in Tetraselmis are lipid droplets that may be present within the central or posterior cell cytoplasm (Fig. 32). Vacuoles or lysosomes are also present in the interior cell cytoplasm in many species, and they usually contain various types of electron dense materials (Figs. 9, 17, 23 and 31-34).

Cells reproduce by dividing in a non-motile stage, most species dividing only once, producing two daughter cells. Prasinocladus ascus, may have one or two subsequent divisions and may produce either two, four or eight daughter cells. The daughter cells develop into flagellate ceils or remain non-motile according to the physiological and environmental conditions. Nuclear division is preceded by synchronous divisions of organelles within a relatively short period of time (Stewart et

al., 1975; Norris 1980), and cell division occurs by formation of a phyeoplast in the one species that has been studied (Stewart et al., 1975). The rhizoplast may disappear during mitosis, probably forming the spindle poles. The basal bodies remain in the plane of the metaphase chromosomes.

Taxonomy of Tetraselmis, Platymonas, Prasinocladus and Aulacochlamys As a result of the authors' studies on living cells as well as on the fine structure of

many species of these genera, they have concluded that all the species belong to the same genus. Retention of the genus Prasinocladus on the basis of stalked cells or pyrenoid structure, as has been maintained in the past, cannot be supported by the present studies. Pyrenoids penetrated by a nuclear lobe was a characteristic con-


Fig. 35. Diagram of the life history of species assigned to Tetraselmis. A1-A3, motile cell settling onto the substratum and losing flagella; B1-Bt, non-motile dividing stage with accumulated concentric cast-off walls, and release of motile cells ; C1-C5, cyst and its germination; TS~-TS~, settled cells, showing the formation of daughter cells, as found in Tetraselmis species without stalked cells; TP, TA~-TAs, settled cells, showing the formation of the colonial condition and daughter cells by extensive growth of the w~lls, as in Tetraselmis a~cus ~nd some other species with non-septate stalked cells; TP, TM~-TM2, settled cells showing the formation of the colonial condition and daughter cells, the walls not growing in length but forming a septate stalk by periodic release of the cell through a posterior pore, attaching of the cell to the old wall where it regenerates a new wall, as in Tetraselmis marina; TA t cells having concentric accumulated walls as in Tetraselmis azcu~s and other species with non- septate stalked cells grown under unfavorable conditions; TM3, non-motile stage grown under unfavorable conditions, having concentric accumulated layers of walls as in Tetraselmis marina. Cz,, C3,, and TSt, are basal polar views; Cx is a surface view of the cyst; the other figures are viewed laterally.

sidered to be possibly significant in separat ing Prasinodadus from Platymonas b y Parke

and Manton (1965), bu t Chihara and Hori (1972) pointed out tha t this, as well as other

characteristics, does not seem to be reliable for separat ion of these genera. Pyrenoids

penetra ted by a nuclear lobe occur in species t ha t have no characteristic stalk on their

cells as they should be in Pras~ocladus. Species of Tetraselmis and Platymonas m a y

produce stalked cells under certain conditions. The stalked na ture of the cells in species

assigned to Tetraselmis or Platymonas is not usual ly so extreme as in those species assign-

ed to Prasinocladus, bu t the lack of a clear way to define this characteristic and the

convincing evidence t h a t i t is s t rongly influenced by env i ronmenta l factors makes the

cell stalk unreliable as a characterist ic on which to base a genus. Also, to this t ime

there seem to be no basic differences in cytological details of cells in Tetraselmis,

Revision of Tetrazelmis 335

\

D E F

I J K

Fig. 36. Tetraselmis species showing cells at rest in polar view and the direction of extended flagella. A: T. contracta; B: T. apiculata; C: T. chuii; D: T. gracilis; E: T. inconspicua; F: T. rubens; G: T. tetrathele; H: T. verrucosa; I: T. convolutae; J: T. ascu~; K: Tetraselmis spp. (the authors consider this type to be most typical for Tetraselmis). A, after Carter (1937); B-H, after Butcher (1959); I, after Parke and Manton (1967); J, after Proskauer (1950); K, original. A-J, partially modified.

Fig. 37. A three dimensional, interpretive line drawing showing the structure of the basal body complex in the genus Tetraselmis. In order to simplify the drawing, the cell membrane and enclosed adjacent organelles are not shown. Flagellar microtubules and transitional zones are also omitted. The cartwheel structure is drawn only in the right-hand basal body. b, basal body; bf, binding fibrils; Cs, cartwheel structure; F, flagellum; Hd, half-desmosome; mt, microtubules connected to half-desmosomes ; N, nucleus; rh, rbizoplast; sy, synistosome.

Platymonas, Prasinocladus or Aulacochlamys. I t is concluded, therefore, t ha t Prasino-

cladus, Platymonas, Aulacochlamys and Tetraselmis all belong to the same genus, and

the following nomencla tura l t r e a tmen t s are proposed:


T e t r a s e l m i s Stein, Org. Infusionsthiere 3(1): 143, pl. XVI. figs. 1-3. 1878. Type: Tetraselmis c~diformis (Carter) Stein. Synonyms: Prasinocladus Kuckuck, Wiss. Meeresunters. Abt. Helgoland N.F.

1: 261, fig. 28. 1894. Type species: Prasinocladus lubricus Kuc~mk. Platymonas G.S. West, J. Bot. London 54: 3, pl. V. fig. 14. 1916. Type species:

Platymonas tetrathele West. Aulacochlamys Margalef, Coll. Bot. 1: 105. 1946. Type species: Aulacochlamys

roscoffensis (P.A. Dangeard) Margalef. Tetraselmis adriatica nora. nov. Basionym: Platymonas mediterm.nea Roukhiyajnen, Nov. Sist. Nizsh. Rast. 9:

16, fig. 3. 1972. Non P. mediterranea (Lucksch) H. Ettl et O. Ettl, Arch. Protistenk. 104: 74. 1959.

Tetraselmis arnoldii (Proshkina-Lavrenko) comb. nov. Basionym: Carteria arnoldii Proshkina-Lavrenko, Nov. Sist. Nizsh. Rast. 5: 149,

pl. II. figs. 21-25. 1945. Synonym: Platymonas arnoldii (Proshkina-Lavrenko) Matvienko in Dedusenko-

Schegoleva, Matvienko et Shkorbatov, Opred. Presnovod. Vodor. SSSR 8: 165. 1959. Tetraselmis ascus (Proskauer) comb. nov. Basionym: Prasinocladus ascus Proskauer, Amer. J. Bot. 37: 65, figs. 1-40. 1950. Tetraselmis bichlora (H. Ettl et O. Ettl) comb. nov.

Platymonas bichlora H. Ettl et O. Ettl, Arch. Protistenk. 105: 281, Basionym: fig. 1. 1961.

Tetraselmis Basionym :

1. 1971. Tetraselmis Basionym: 65. 1944. Tetraselmis Basionym :

bilobata (Roukhiyajnen) comb. nov. Platymonas bilobata Roukhiyajnen, Nov. Sist. Nizsh. Rast. 7: 22, fig.

bolosiana (Margalef) comb. nov. Platymonas bolosiana Margalef, Publ. Inst. Bot. Barcelona 4 : 40 & 130,

convolutae (Parke et Manton) comb. nov. Platymonas convolutae Parke et Manton, J. mar. biol. Ass. U.K. 47:

461, fig. 1. 1967. Tetraselmis elliptica (G.M. Smith) comb. nov. Basionym: Platymonas elliptica G.M. Smith, Fresh-water Alg. U.S. 325, fig. 217.

1933. Tetraselmis fontiana (Margalef) comb. nov. Basionym: Platymonas fontiana Margalef, Coll. Bot. 1: 96, figs. a-h. 1946. Tetraselmis helgolandica (Kylin) Butcher var. tsingtaoensis (Tseng et T.J. Chang)

comb. nov. Basionym: Platymonas helgolandica Kylin var. tsingtaoensis Tseng et T.J. Chang

in Chang et al. Aeta Bot. Sin. 12: 110, figs. 1-24, 38-47. 1964. Tetraselmis impellucida (McLaehlan et Parke) comb. nov. Basionym: Platymonas impeUucida MeLachlan et Parke, J. mar. biol. Ass. U.K.

47: 730, pls. I - I I I . 1967.

Revision of Tetraselrais 337

Tetraselmis incisa (Nygaard) comb. nov.

Basionym: Platymonas incisa Nygaard, K. Danske Vid. Selsk. Biol. Skr. 7(1): 25, fig. 5. 1950.

Tetraselmis intermedia (Nasr) comb. nov. Basionym: Platymonas intermedia Nasr, Bull. Inst. l~gypte 26: 32, fig. 2. 1944.

Tetraselmis marina (Cienkowski) comb. nov.

Basionym: Chlorangium marinum Cienkowski, Trudy Sankt-Peterburg. Obshch.

Estestv. 12: 152, pl. I. figs. 7-11. 1881.

Synonym: Prasinocladus marinus (Cienkowski) Waern, Acta Pytogeogr. Suecica

30. 85, 1952.

Tetraselmis mediterranea (Lucksch) comb. nov.

Basionym: Carteria mediterranea Lucksch, Beih. Bot. Centralbl. 50 (1): 68, fig. 3.

1932.

Synonym : Platymonas mediterranea (Lucksch) H. Et t l et O. Ettl, Arch. Protistenk.

104: 74. 1959.

Tetraselmis roscoffensis (P.A. Dangeard) Butcher, Fish. Invest., Minist. Agricult., Fish. & Food, Set. IV, 1: 69, 1959.

Basionym: Platymonas roscoffensis P.A. Dangeard, Travaux cryptog~m, dedi~s L. Mangin 227, pl. 19. 1931.

Synonym: Aulacochlamys roscoffensis (P.A. Dangeard) Margalef, Coll. Bot. 1 : 105.

1946.

Tetraselmis viridis (Roukhiyajnen) comb. nov.

Basionym: Platymonas viridis Roukhiyajnen, Nov. Sist. Niszh. Rast. 2: 82, figs. 1-7. 1966.

This research was partially supported by grants from the Japan Society for the

Promotion of Sciences and the United State National Science Foundation (GA-27310

and GB-38232) as par t of the U.S.-Japan Cooperative Science Program (No. 5R052).

Special thanks are extended to Mrs. Barbara Reed for her assistance in the electron

microscope used in this research. The authors are also grateful to Dr. Paul C. Silva for

his generous aid in questions on the literature. This work constitutes a contribution from the Shimoda Marine Research Center,

The University of Tsukuba, No. 367.

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Received May 9, 1980