ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : II. The Giant Ameba Pelomyxa carolinensis

Dividing nuclei from the giant ameba Pelomyxa carolinensis were fixed in osmium tetroxide solutions buffered with veronal acetate to pH 8.0. If divalent cations (0.002 M calcium, magnesium, or strontium as chlorides) were added to the fixation solution, fibrils that are 14 mµ in diameter and have a dense cortex are observed in the spindle. If the divalent ions were omitted, oriented particles of smaller size are present and fibrils are not obvious. The stages of mitosis were observed and spindle components compared. Fibrils fixed in the presence of calcium ions are not so well defined in early metaphase as later, but otherwise have the same diameter in the late metaphase, anaphase, and early telophase. Fibrils are surrounded by clouds of fine material except in early telophase, when they are formed into tight bundles lying in the cytoplasm unattached to nuclei. Metaphase and anaphase fibrils fixed without calcium ions are less well defined and are not observably different from each other. The observations are consistent with the concept that spindle fibrils are composed of polymerized, oriented protein molecules that are in equilibrium with and bathed in non-oriented molecules of the same protein. Partially formed spindle fibrils and ribosome-like particles were observed in the mixoplasm when the nuclear envelope had only small discontinuities. Remnants of the envelope are visible throughout division and are probably incorporated into the new envelope in the telophase. Ribosome-like particles are numerous in the metaphase and anaphase spindle but are not seen in the telophase nucleus, once the envelope is reestablished, or in the interphase nucleus.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

Mitosis in the protostelidPlanoprotostelium aurantium

Summary Mitosis in the protostelidPlanoprotostelium aurantium Olive andStoianovich is characterized by an open, centric spindle. The nuclear envelope breaks down prior to metaphase, begins to reform during late anaphase, and is complete by telophase. Centrioles are present at the poles throughout mitosis and are devoid of rootlet microtubules from metaphase to late anaphase. Chromosomes are small and numerous and are attached to single kinetochore microtubules during metaphase and early anaphase. Chromosome separation takes place by a presumed shortening of the chromosome to pole spindle followed by a lengthening of the interzonal spindle. Mitosis inP. aurantium is similar to that of certain other protostelid amoebae and to myxomycete amoebae, but it is considerably different from that of dictyostelid amoebae. The phylogenetic significance of this is discussed.This research represents part of a Ph.D. dissertation presented to the University of North Carolina.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

Pelomyxa palustris Greeff

Summary The Pelomyxa palustris amoebae used in this study were multinucleate, herbivorous protozoans. All nuclei within a single organism were similar, but several types of nuclei were seen in different amoeba. These nuclei might represent various stages of mitosis although metaphase and anaphase stages were never seen. Rod-shaped bacteria within vesicles characteristically surrounded the nuclei. Bacterial rods of this as well as another type also occurred within vesicles in the cytoplasm. The nuclear envelope contained annuli and it was covered externally by minute vesicles. Nucleoli and micronucleoli were most frequently located along the inner surface of the nuclear envelope. Clusters of electron-opaque spheroids were found within the nucleoli; sometimes, they existed free in the nucleoplasm. Intranuclear globules of lipidlike material were often seen.Mitochondria, Golgi bodies, contactile vacuoles, and crystal vacuoles were definitely absent in P. palustris. The cytoplasm contained many food vacuoles and clear vacuoles of various sizes. Vacuole-like aggregations, probably containing glycogen, were present.Work supported by the U. S. Atomic Energy Commission.     

Fine structure of division in ciliate protozoa. I. Micronuclear mitosis in Blepharisma

The mitotic, micronuclear division of the heterotrichous genus Blepharisma has been studied by electron microscopy. Dividing ciliates were selected from clone-derived mass cultures and fixed for electron microscopy by exposure to the vapor of 2% osmium tetroxide; individual Blepharisma were encapsulated and sectioned. Distinctive features of the mitosis are the presence of an intact nuclear envelope during the entire process and the absence of centrioles at the polar ends of the micronuclear figures. Spindle microtubules (SMT) first appear in advance of chromosome alignment, become more numerous and precisely aligned by metaphase, lengthen greatly in anaphase, and persist through telophase. Distinct chromosomal and continuous SMT are present. At telophase, daughter nuclei are separated by a spindle elongation of more than 40 µ, and a new nuclear envelope is formed in close apposition to the chromatin mass of each daughter nucleus and excludes the great amount of spindle material formed during division. The original nuclear envelope which has remained structurally intact then becomes discontinuous and releases the newly formed nucleus into the cytoplasm. The micronuclear envelope seems to lack the conspicuous pores that are typical of nuclear envelopes. The morphology, size, formation, and function of SMT and the nature of micronuclear division are discussed.     

MITOSIS IN THE ALGA VACUOLARIA VIRESCENS

Details of mitosis in the chloromonadophycean alga Vacuolaria virescens Cienk. have been studied with the light microscope. The chromosomes are relatively large (up to μ in length at metaphase) and so mitotic stages are readily distinguishable. Chromosomes can be recognized in interphase nuclei as fine strands of chromatin. Contraction of these chromosomes marks the beginning of mitosis and continues progressively until the transition from metaphase to anaphase. Disintegration of nucleoli is complete by late prophase and nucleolar reformation begins in telophase. Some chromosomes exhibit less densely stained regions; centromeres are also present as indicated by their differential staining and by the behavior of chromosomes at metaphase and anaphase. At anaphase progeny chromosomes move apart parallel to the division axis of the nucleus. As anaphase progresses the chromosomes fuse at the polar surface of the progeny chromosome groups. This process continues in telophase and the chromosome groups become more spherical. By the end of telophase nucleolar reformation has begun and the chromosomes have relaxed to their interphase condition.     

Studies on the Endoplasmic Reticulum : IV. Its Form and Distribution during Mitosis in Cells of Onion Root Tip

Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO₄. Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO₄-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell.     

Stratification within centrifuged amoeba nuclei

Summary Two species of large, fresh water amoebae were ultracentrifuged and studied with the electron microscope. Emphasis was placed on the stratification of the nucleoplasm, including nucleoli, within the confines of the nuclear envelope during interphase. Three major strata were found in the nuclei of both amoeba species, namely the centripetal nucleoplasm, the middle chromatin stratum, and the centrifugal nucleolar mass. In the highly radioresistant A. proteus, the nucleolar mass separated into a centripetal electron-opaque layer and a centrifugal electron-lucent layer. The latter layer appears to be missing from the radiosensitive P. illinoisensis. The nature of these nucleolar layers and their possible relationship to differences in radiosensitivity between the two species of amoebae is discussed. The contents of the heavier of the two nucleolar layers in A. proteus might be resistant to radiation damage and may possess radiorestorative capacity.Work supported by U.S. Atomic Energy Commission. A part of the work was reported at the 17th Meeting of the Society of Protozoologists, Boulder, Colorado in 1964.     

Cell division of binuclear cells induced by caffeine: Spindle organization and determination of division plane

Synchronously dividing binuclear cells were induced in root tips ofTriticum turgidum by caffeine treatment. Spindle and other microtubular configurations of such cells were studied using tubulin immunofluorescence and electron microscopy. The binuclear cells developed one, two or three preprophase microtubule bands longitudinally, transversely or rarely in a cross configuration. During the mitotic entry binuclear cells formed prophase spindles separately around each nucleus. When the nuclei were located fairly apart, their spindle structures developed independently throughout all mitotic phases. But when the nuclei were located closely together their metaphase and anaphase spindles shared a common polar region. However, the two spindles in such cells retained their functional autonomy. They display structurally independent minipoles in the common polar region. After anaphase the neighbouring nonsister chromosome groups of nuclei divided by a common polar region come to lie close together and in telophase, become enclosed by a common nuclear envelope. During cytokinesis of binuclear cells cell plates were formed only between sister nuclei. These cell plates may develop normally or may curve or branch giving rise to aberrant daughter cell walls. The peculiar mode of spindle and spindle polar region organization of binuclear cells and determination of the division plane in them are discussed.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum II. Metaphase I—Telophase I

Summary The three-dimensional structure of the spindle pole body (SPB) and meiotic spindle during early metaphase I through telophase I inPuccinia malvacearum is analyzed ultrastructurally from serial sections. During early metaphase I the spindle rotates from the perpendicular to a position oblique to the longitudinal axis and parallel to the sagittal plane of the cell. Tubular cisternae are present within the spindle at this stage. The half middle piece (MP) subtends a collateral disc (co-disc) which is inserted eccentrically within each SPB. The SPB, co-disc and half MP at opposite poles are in mirror image. During the transition from early metaphase I to full metaphase I, the spindle orients parallel to the lateral wall of the promycelium and the half MPs are lost. The co-discs partially detach from each discoid SPB and maintain this relation until the end of interphase I. Co-discs become further differentiated as they attach to the subtending sheath-like extension of the nuclear envelope previously occupied by the half MPs. Microvesicles within the nucleoplasm are specific to mid metaphase I. A metaphase plate is absent. The 14 bivalents, which are directly connected to each polar SPB by 2 to 3 kinetochore MTs, are spread over nearly the entire length of the central spindle. The first anaphasic movement involves asynchronous shortening of the kinetochore MTs while the second consists of extensive pole-to-pole elongation. Astral MTs first appear at early metaphase I and become most numerous at anaphase I. An intact nuclear envelope constricts against the central spindle at either end of the interzonal region. Concurrently, centripetal growth of the nuclear envelope under each SPB results in their gradual externalization by the end of telophase I. The sibling nuclei are cut off by constriction of the nuclear envelope at either end of the interzonal region. These meiotic stages inP. malvacearum are compared with those in other basidiomycetes and ascomycetes.     

ULTRASTRUCTURE AND TIME COURSE OF MITOSIS IN THE FUNGUS FUSARIUM OXYSPORUM

Mitosis in Fusarium oxysporum Schlect. was studied by light and electron microscopy. The average times required for the stages of mitosis, as determined from measurements made on living nuclei, were as follows: prophase, 70 sec; metaphase, 120 sec; anaphase, 13 sec; and telophase, 125 sec, for a total of 5.5 min. New postfixation procedures were developed specifically to preserve the fine-structure of the mitotic apparatus. Electron microscopy of mitotic nuclei revealed a fibrillo-granular, extranuclear Spindle Pole Body (SPB) at each pole of the intranuclear, microtubular spindles. Metaphase chromosomes were attached to spindle microtubules via kinetochores, which were found near the spindle poles at telophase. The still-intact, original nuclear envelope constricted around the incipient daughter nuclei during telophase.     

Mitotic Microtubule Development and Histone H3 Phosphorylation in the Holocentric Chromosomes of Rhynchospora Tenuis (Cyperaceae)

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.     

ELECTRON MICROSCOPIC STUDIES OF MITOSIS IN AMEBAE : I. Amoeba proteus

Individual organisms of Amoeba proteus have been fixed in buffered osmium tetroxide in either 0.9 per cent NaCl or 0.01 per cent CaCl₂, sectioned, and studied in the electron microscope in interphase and in several stages of mitosis. The helices typical of interphase nuclei do not coexist with condensed chromatin and thus either represent a DNA configuration unique to interphase or are not DNA at all. The membranes of the complex nuclear envelope are present in all stages observed but are discontinuous in metaphase. The inner, thick, honeycomb layer of the nuclear envelope disappears during prophase, reappearing after telophase when nuclear reconstruction is in progress. Nucleoli decrease in size and number during prophase and re-form during telophase in association with the chromatin network. In the early reconstruction nucleus, the nucleolar material forms into thin, sheet-like configurations which are closely associated with small amounts of chromatin and are closely applied to the inner, partially formed layer of the nuclear envelope. It is proposed that nucleolar material is implicated in the formation of the inner layer of the envelope and that there is a configuration of nucleolar material peculiar to this time. The plasmalemma is partially denuded of its fringe-like material during division.     

CRUCIFORM NUCLEAR DIVISION IN WORONINA PYTHII (PLASMODIOPHOROMYCETES)

Somatic nuclear divisions in sporangiogenous plasmodia of Woronina pythii Goldie-Smith were studied with transmission electron microscopy. During metaphase, each nucleus formed a cruciform configuration as chromatin became aligned at the equatorial plate perpendicular to the persistent nucleolus. Except for polar fenestrations, the original nuclear envelope remained intact throughout the mitotic division. Intranuclear membranous vesicles appeared to bleb off the inner membrane of the original nuclear envelope, adhered to the surfaces of the separating chromatin, and eventually formed new daughter nuclear envelope within the original nuclear envelope. During the first 24 hr of vegetative plasmodial growth, each telophase nucleus exhibited an obvious constriction of the original nuclear envelope in the interzonal region. Similar constrictions were not evident in telophase nuclei found in 24–36-hr-old plasmodia. This variation in the ultrastructural morphology of cruciform division appears to be related to the age and size of each sporangiogenous plasmodium, and is the first to be documented within this group of fungal pathogens.     

The ultrastructure of mitosis during sporangiogenesis inWoronina pythii (Plasmodiophoromycetes)

Summary Mitotic divisions during sporangiogenous plasmodial cleavage inWoronina pythii were studied with transmission electron microscopy. We conclude that these nuclear divisions (e.g., transitional nuclear division, and sporangial mitoses) share basic similarities with the cruciform nuclear divisions inW. pythii and other plasmo-diophoraceous taxa. The major distinction appeared to be the absence of nucleoli during sporangial mitosis and the presence of nucleoli during cruciform nuclear division. The similarities were especially evident with regard to nuclear envelope breakdown and reformation. The mitotic divisions during formation of sporangia were centric, and closed with polar fenestrae, and characterized by the formation of intranuclear membranous vesicles. During metaphase, anaphase, and telophase, these vesicles appeard to bleb from the inner membrane of the original nuclear envelope and appeared to coalesce on the surface of the separating chromatin masses. By late telophase, the formation of new daughter nuclear envelopes was complete, and original nuclear envelope was fragmented. New observation pertinent to the mechanisms of mitosis in thePlasmodiophoromycetes include a evidence for the incorporation of membrane fragments of the original nuclear envelope into new daughter nuclear envelopes, and b the change in orientation of paired centrioles during sporangial mitosis.     

Closed spindle nuclear division in the plasmodial phase of the acellular slime moldEchinostelium minutum

Summary Mitosis in the plasmodium ofEchinostelium minutum is intranuclear (closed spindle) and centrioles are not present at the spindle poles. The nuclear envelope remains essentially intact throughout mitosis with polar fenestrae appearing in anaphase and persisting through telophase. During anaphase there is a shortening in the distance of the chromosomes to the poles followed by a further separation of the poles. Zippering of microtubules may be the basis for these two anaphasic movements. During telophase the polar MTOCs are extruded into the cytoplasm through the polar fenestrae prior to reconstitution of the nuclear envelope. It is proposed that during sporulation such MTOCs are responsible for the differentiation of the centrioles which subsequently persist in the myxamoebal phase of this species.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Genetic Control of Mitosis: Mutation ff3Causes Premature Beginning of Telophase Chromatin Reorganization in Drosophila melanogaster

The effect of cell cycle mutation ff3 on chromosome segregation was studied on fixed cells of neural ganglia of Drosophila melanogasterlarvae. The cell distributions by diameter of interphase nuclei and by distance between sister chromatid sets were compared at anaphase and telophase. In the control wild-type strain Lausenne, the cell distribution by distance between sister chromatids in anaphase was similar to their distribution by nuclear size. The mean distance between segregating chromatids at anaphase (l _av) coincided with the mean diameter of interphase nuclei (d _av) and was 8.3 m. Cells passed to telophase when chromatids were at least 10 m apart. The mutant ff3 strain differed from the control strain Lausenne in cell distribution by interphase nuclear diameter and distance between sister chromatids in anaphase; the mean nuclear diameter and mean distance between segregating chromatids similarly increased to 9.3 m. A specific feature of mitosis in mutant strain ff3 was a premature beginning of telophase chromatin reorganization. This caused the occurrence of cells with abnormally short (less then the interphase nuclear diameter) distance between sister chromatid sets in telophase but not in anaphase, as if these cells had passed from anaphase to telophase prematurely, during the chromatid movement toward poles in anaphase A.     

Differential nuclear envelope assembly at the end of mitosis in suspension-cultured Apium graveolens cells

NMCP1 is a plant protein that has a long coiled-coil domain within the molecule. Newly identified NMCP2 of Daucus carota and Apium graveolens showed similar peripheral localization in the interphase nucleus, and the sequence spanning the coiled-coil domain exhibited significant similarity with the corresponding region of NMCP1. To better understand disassembly and assembly of the nuclear envelope (NE) during mitosis, subcellular distribution of NMCP1 and NMCP2 was examined using A. graveolens cells. AgNMCP1 (NMCP1 in Apium) disassembled at prometaphase, dispersed mainly within the spindle, and accumulated on segregating chromosomes, while AgNMCP2 (NMCP2 in Apium), following disassembly at prometaphase with timing similar to that of AgNMCP1, dispersed throughout the mitotic cytoplasm at metaphase and anaphase. The protein accumulated at the periphery of reforming nuclei at telophase. A probe for the endomembrane indicated that the nuclear membrane (NM) disappears at prometaphase and begins to reappear at early telophase. Growth of the NM continued after mitosis was completed. NMCP2 in the mitotic cytoplasm localized in vesicular structures that could be distinguished from the bulk endomembrane system. These results suggest that NMCP1 and NMCP2 are recruited for NE assembly in different pathways in mitosis and that NMCP2 associates with NM-derived vesicles in the mitotic cytoplasm.     

Ultrastructure of mitosis and cytokinesis inZygnema sp. (Zygnematales,Chlorophyta)

Summary Mitosis and cytokinesis have been studied in the green algaZygnema C. A. Agardh using interference-contrast light and transmission electron microscopy. At prophase, the nucleolus disintegrates and numerous extranuclear microtubules near the nuclear periphery penetrate into the nucleoplasm. When aligned in the equatorial plane of the open metaphase spindle the chromosomes are coated with persistent nucleolar fragments. At anaphase, vacuoles intrude into the interzonal spindle region and seemingly contribute to the anaphase movement of the chromosomes. At telophase, the spindle is persistent and the reforming nuclei are separated by cytoplasmic strands containing microtubules, interspersed with vacuoles. Extensive bundles of microtubules, dictyosomes and parallel, slightly inflated ER-profiles extend from the poles of the telophase nucleus along the longitudinal side of the chloroplast. Conceivably, these microtubules guide the nucleus during its post-mitotic migration towards its central interphase position between the two halves of the dividing chloroplast. Throughout the mitotic cycle, ubiquitous dictyosomes, positioned near the chloroplast core, seem very active. Arrays of microtubules run towards these dictyosomes and may conduct the dictyosome-vesicles to the cleavage plane. At metaphase, septum growth becomes visible as an annular ingrowth of the plasmalemma. At late telophase or at entering interphase, an extensive clump of vesicles, associated with longitudinal bundles of microtubules, appears between the leading edges of the advanced furrow. Apparent fusion of these vesicles with the head of the centripetally-growing furrow results in its completion. The pattern of mitosis and cytokinesis inZygnema is compared with that of closely related green algae.