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496
Evolution of multicellularity in the volvocine algae
David L Kirk
Recent studies reveal that relationships among the volvocine
algae are more complex than was previously believed.
Nevertheless, this group still appears to provide an unrivaled
opportunity to analyze an evolutionary pathway leading from
unicellularity (Chlamydomonas) to multicellularity with division
of labor (Volvox). Significant progress in this regard was made
in the past year when two genes playing key roles in Volvox
cellular differentiation were cloned, and clues were uncovered
regarding their mechanisms of action.
Figure 1
Addresses
Department of Biology, Washington University, Campus Box 1229,
St. Louis, MO 63130, USA; e-mail: kirk@biology.wustl.edu
Current Opinion in Plant Biology 1999, 2:496–501
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
ECM
extracellular matrix
gls
gonidialess
lag
late gonidia
rbcL
rubisco large subunit
regA
somatic regenerator
Introduction
Multicellularity has evolved many times on this planet. In
most cases, however, the pathway leading from a unicellular ancestor to a multicellular organism with differentiated
cell types has been obscured by the passage of time.
Although it is assumed that each of the major groups of
conspicuous modern eukaryotes (Figure 1) evolved from a
different unicellular ancestor more than a billion years ago
[1–3], we can do little more than speculate about what the
last unicellular ancestor of each of these groups may have
been like, or how the transition to multicellularity may
have occurred.
In contrast, within the volvocine algae multicellular organisms with differentiated cell types have evolved far more
recently — and more than once. Therefore, this group of
green flagellates, which includes members of the family
Volvocaceae and their close unicellular relative
Chlamydomonas reinhardtii, appears to provide an unrivaled
opportunity for exploring a pathway leading from unicellularity to multicellularity [4•].
The volvocine algae as an evolutionary model
The volvocine algae range in size and complexity from
unicellular Chlamydomonas, to Volvox, with its thousands of
mortal somatic cells and a few immortal germ cells. It has
long been commonplace for textbooks to arrange these
algae in a conceptual series such as that in Figure 2, in
which there is a regular progression in size, cell number,
abundance of extracellular matrix (ECM), and tendency to
produce sterile somatic cells. It is usually suggested that
Branching order of selected major eukaryotic lineages, based on data
in [2,3], and the family Volvocaceae, based on data in [5]. Note that
the Volvocaceae, represented by the bifurcation at the tip of the green
algal line, have been diversifying less than 10% as long as any of the
major multicellular groups.
this might represent the sequence by which Volvox
evolved. Although such a proposition contains a germ of
truth, it clearly is overly simplistic.
Molecular data indicate that the volvocine algae constitute
a surprisingly youthful group of close relatives: Volvox carteri and C. reinhardtii apparently last shared a common
ancestor 50–75 million years ago [5], and the germ-soma
division of labor that defines the genus Volvox has evolved
at least twice and possibly several times in this relatively
brief interval [4•,6]. This suggests that the transition from
unicellularity to the Volvox type of multicellularity must
have a relatively simple genetic basis, and may actually be
simple enough to be amenable to detailed analysis.
Such optimism is dampened only slightly by the accumulating molecular evidence that the volvocacean family tree
is far more complicated than was suspected a generation
ago. Volvox is not the only volvocine taxon that is polyphyletic. For example, the phylogenetic reconstruction
shown in Figure 3, which is based on rubisco large subunit
(rbcL) sequences, supports the long-standing assumption
that Eudorina, Pleodorina and Volvox are all closely related
but at the same time it reveals the striking degree to which
relationships within this section of the family Volvocaceae
may deviate from the simple ones suggested by Figure 2.
The data summarized in Figure 3 indicate clearly, for
example, that the genus Eudorina is polyphyletic, and that
its type species, E. elegans, is paraphyletic. Such deviations
from monophyly had already been suspected when only a
few Eudorina isolates had been analyzed [6], but the
extent of these deviations from monophyletic expectations
Evolution of multicellularity in the volvocine algae Kirk
Figure 2
497
Figure 3
A molecular phylogenetic reconstruction of the Eudorina–Pleodorina–
Volvox section of the family Volvocaceae based on rbcL sequences.
Numbers are strain identifications, as given in [7], from which the present
figure is adapted. The two taxa marked by asterisks were identified as
Eudorina illinoisensis in [7], but here they are labeled Pleodorina
illinoisensis, as they are in major algal culture collections, in order to
highlight some of the taxonomic uncertainties in this family.
A conventional representation of ‘the volvocine lineage’, with organisms
drawn at progressively decreasing magnification from bottom to top.
The diameters of the biflagellate cells would be in the range of
5–10 µm in each organism. Dark gray shading represents the cell
bodies, light gray shading represents the ECM and thick black lines
represent the tripartite layer that is discussed below.
has become increasingly apparent as more and more putatively ‘congeneric’ and ‘conspecific’ isolates have been
analyzed [7]. Therefore, we may anticipate a plethora of
additional complications when Pleodorina and Volvox
species are sampled with similar intensity, and when the
12–14 species of Volvox that are currently missing from
Figure 3 are added.
Relationships at the opposite end of the volvocine complexity spectrum are no simpler. Chlamydomonas turns out
to be a highly paraphyletic genus comprising many distinct clades [8], and C. reinhardtii is found to be much
more closely related to the volvocaceans than it is to most
Chlamydomonas species [8–10], including those species
once thought to be its closest relatives [10,11]. Although
analysis of rbcL data provide little support for the idea that
all species previously known as Gonium should be moved
from the Volvocaceae to two new families [12], they do
indicate that these species define two entirely separate
clades [6]; moreover, phylogenetic analysis at a higher
level of resolution demonstrated that Gonium pectorale consists of six reproductively isolated subclades [13••]. But
this is modest compared to the extraordinary reproductive, chromosomal and molecular diversity that has been
found within the morphologically uniform species
Pandorina morum [14–16].
In short, it appears that many volvocacean taxonomic categories identify developmental and/or organizational grades,
rather than phylogenetic clades. However, reconstructions
such as the one in Figure 3 (and others not shown) indicate
that transitions among these grades — in both directions —
must be easily accomplished and thus must have a relatively
simple genetic basis. This notion is supported by mutational studies: a single mutation is adequate to convert Volvox
powersii into a mimic of Pleodorina californica [17], and two
mutations abolish germ-soma differentiation in V. carteri and
convert it into a mimic of Eudorina [18].
Thus, an image of a highly branched, spreading bush has
replaced the earlier image of an upright, relatively
498
Cell biology
Figure 4
A schematic representation of the way in which certain extracellular
materials (predominantly glycoproteins and sulfated polysaccharides)
are arranged in selected volvocine taxa. Not drawn to scale.
(a) Chlamydomonas reinhardtii. The concentric black–white–black
ellipses at the periphery represent the ‘trilaminar’ layer of the cell wall,
while the stippled ellipse represents the more amorphous-looking inner
wall, and the central clear space represents the cell body. (b) Gonium
pectorale. A wall resembling that of Chlamydomonas surrounds each
cell body, but specializations (black rectangles) attach the outer layers
of neighboring walls to one another at specific points. Such
attachments vary in a species-specific way in different species of
Gonium. (c) Pandorina morum (part of a colony in cross section). The
inner wall and the inner portion of the tripartite layer surround each cell
as in (a) and (b), but the outermost portion of the trilaminar layer is
continuous over the surface of the organism. (d) Volvox carteri (part of
a somatic-cell layer in cross section). In Eudorina, Pleodorina and
Volvox, the entire tripartite layer is continuous over the surface of the
spheroid and most of the spheroid is occupied by a species-specific
assortment of fibrous layers (heavy stippling) and relatively amorphous
‘mucilaginous’ regions (lighter stippling), all of which appear to have
been derived by evolutionary amplification and elaboration of the inner
wall of Chlamydomona.
unbranched tree as a metaphor for volvocacean family history. But although this clearly means that reconstructing
the complete family history of the Volvocaceae will be a
much more challenging task than was once imagined, it
has done little to diminish optimism that it will be easier to
elucidate the molecular genetic origins of multicellularity
and cellular differentiation in the volvocine algae than in
any other lineage yet identified.
From unicells to colonies: sharing extracellular
materials
The prime feature distinguishing the volvocaceans from
Chlamydomonas is that the glycoprotein based ‘cell walls’
of volvocaceans are linked together in some species-specific manner to form an extracellular matrix (ECM);
however, many components of Chlamydomonas walls and
volvocacean ECM are clearly homologous. The most conserved extracellular feature of the group is a layer that has
a characteristic crystalline appearance when viewed en face
[19], but is more commonly seen as a dark–light–dark profile in electronmicrograph thin sections, and is therefore
usually called the ‘tripartite’ layer. This layer has an
extremely similar organization [19–21], and contains very
similar proteins [22–24] in all volvocine algae that have
been carefully examined; however, the disposition of this
layer with respect to the cell bodies varies in a taxon-specific way (Figure 4).
In Gonium, as in Chlamydomonas, the tripartite layer surrounds each cell and is separated from the plasmalemma
by only a very thin, nondescript ‘inner wall’; however, in
Gonium the walls of neighboring cells are linked together
(Figure 4a,b), with the nature of the linkages differing in a
species-specific manner [25,26]. A transition occurs in
Pandorina, where the tripartite layer is partially split, such
that its outer leaflet is continuous over the surface of the
colony, although its inner leaflet is still wrapped around
each cell body (Figure 4c) [27]. This transition is taken a
step further in the rest of the volvocaceans, in which the
entire tripartite layer is always continuous over the surface
(Figure 4d). In the larger volvocaceans (Eudorina to Volvox)
the ECM accounts for 90–99% of the volume of the organism, and it exhibits a wide variety of interesting
taxon-specific internal specializations [28]. The vast majority of this ECM appears to be an extensive evolutionary
amplification and elaboration of what in Chlamydomonas is
the remarkably unimpressive ‘inner wall’ (Figure 4a,d).
It would be surprising, therefore, if the architecturally
complex ECM of the larger volvocaceans was not significantly more complex biochemically than the cell wall of
C. reinhardtii, which contains > 30 kinds of glycoproteins,
some of which are glycine-rich but most of which are
hydroxyproline-rich [23,24]. Thus, the ten ECM proteins
of V. carteri that have been characterized to date by cDNA
cloning and sequencing [29••] constitute an impressive
and important start — but only a start — toward a full molecular characterization of the Volvox ECM.
The most striking feature shared by the extracellular glycoproteins of C. reinhardtii and V. carteri is their modularity.
One of the most recurrent themes is a hydroxyproline-rich,
rod-like module of variable length attached at one or both
ends to various globular modules, which often results in
molecules capable of extensive self-assembly [24,29••]. It
has been postulated that the propensity of the genes
encoding ECM modules for rapid diversification has
resulted in their exploitation for entirely different, but no
less important evolutionary ends: to encode species-specific sex proteins — sexual agglutinins in the case of
Chlamydomonas [30] and species-specific sex-inducing
pheromones in the case of Volvox [29••].
The conversion of a Chlamydomonas cell wall to a
volvocine ECM undoubtedly was facilitated by the
unique pattern of cell division that the green flagellates
exhibit: instead of undergoing binary fission as most cells
do, green flagellates undergo ‘multiple fission’. That is,
after growing 2n–fold in volume, they divide rapidly n
times within the mother-cell wall, to produce 2n daughter
cells (where the maximum value of n is a species characteristic) [4•,31]. In unicellular green flagellates,
cytokinesis is complete, so sister cells are able to move
Evolution of multicellularity in the volvocine algae Kirk
relative to one another while they are still within the
mother-cell wall and are forming their own cell walls [31];
as a result they develop as untethered, free individuals.
In contrast, in all volvocaceans that have been carefully
examined, cytokinesis is incomplete, and cytoplasmic
bridges that are formed in each division furrow hold all sister cells in fixed relationships until after the deposition of
‘wall’ components has begun [4•]. With the cells held
together in this way while extracellular materials are being
deposited, ‘walls’ invariably become fused in species-specific ways to form the ECM that ties volvocacean cells into
a coherent unit.
Thus, the critical first step in evolution of volvocine multicellularity was probably the invention of a mechanism
for delaying separation of sister cells until wall materials
had been deposited around and between them. A recently discovered flagellate, Gonium dispersum, appears to be
trying to make up its mind right now whether or not it
will permanently adopt such a mechanism. G. dispersum
divides two ways: one way results in sister cells being
held together between divisions, and results in formation
of colonies similar to those of other Gonium species; the
other way permits cells to move about between divisions, and generates Chlamydomonas-like unicellular
progeny [32]. Further study of this interesting schizoid
flagellate is warranted.
From colonies to multicellular organisms:
dividing life’s labors
V. carteri has become the premier model for studies of
volvocine ontogeny and phylogeny because of its genetic
accessibility and the clarity with which it can be used to
pose two of the central unanswered questions of biology:
how is a program for the development of cells of entirely
different phenotypes programmed in a genome and how
does such a program evolve?
In distinction to colonial volvocaceans such as Pandorina
or Eudorina, V. carteri is a bona fide multicellular organism
with a complete division of labor between two interdependent cell types [4•,33]. The ~2,000 biflagellate
somatic cells of V. carteri are specialized for motility but are
terminally differentiated and programmed to die after
serving their purpose. The ~16 gonidia (asexual reproductive cells), in marked contrast, are completely non-motile,
programmed for division and reproduction, and potentially immortal. When mature, each gonidium divides
repeatedly to produce an embryo containing all of the
cells of both types that will be present in an adult of the
next generation. A key step in germ-soma differentiation
occurs in mid-embryogenesis, when visibly asymmetric
divisions set apart large ‘gonidial initials’ from small
‘somatic initials’. Many types of evidence indicate that it
is this difference in size, and not any difference in cytoplasmic quality, that determines which pathway of
differentiation each cell will follow [34].
499
Mutational and Mendelian analyses have led to the proposal that three major types of gene program V. carteri
germ-soma differentiation [18]. First the gonidialess (gls)
genes act in the cleaving embryo to cause asymmetric division. Then a pair of negative regulators are activated
differentially: the somatic regenerator (regA) gene product
acts in small cells to repress reproductive development,
while the late gonidia (lag) gene products act in the large
cells to repress somatic development.
The simple logic underlying such a negative-regulatory
program becomes apparent when viewed in an evolutionary context. In volvocine algae from Chlamydomonas to
Eudorina that have a single cell type, every cell first
develops as a biflagellate (somatic-like) cell, and then
later loses its flagella, rounds up, and transforms into a
reproductive (gonidia-like) cell that initiates a series of
rapid cleavage divisions [4•,31]. Thus, the basic program
of volvocine development is a sequential one: first biflagellate, then reproductive. In V. carteri, however, this
sequential program has been converted into a dichotomous one by repressing the biflagellate phase in gonidia
(with the lag genes), and the reproductive phase in
somatic cells (with the regA gene). When one of the lag
genes is inactivated, presumptive gonidia revert to the
ancestral ‘first biflagellate, then reproductive’ program,
and when the regA gene is inactivated the somatic cells do
the same [35]. Thus, it seems clear that these genes once
played as important a role in the evolution of V. carteri as
they now play in its development.
Detailed analysis of such genes awaited development of
molecular-genetic tools of the sort now considered commonplace in other systems. The concerted efforts of the
three laboratories now actively engaged in Volvox research
have resulted in development of such a tool kit: this now
includes a pair of selectable markers [36,37•], a nuclear
transformation system [38], a transposon suitable for gene
tagging [39], and reporter and inducible-promoter constructs [40]. Transformation has been used to modify
carbohydrate metabolism with a foreign gene [41], and to
achieve gene replacement by homologous recombination
[42]. More recently, transposon tagging has been used to
clone two key genes controlling germ-soma differentiation:
glsA, a gene required for asymmetric division [43••] and
regA, the gene whose product represses reproductive
development in somatic cells [44••].
GlsA, the protein encoded by glsA, was found to contain a
‘J domain’ that is essential for its function, and, like the
homologous human J protein MPP11, GlsA is associated
with the mitotic spindle in dividing cells [43••]. The J
domain is the conserved motif by which chaperones in the
DnaJ/Hsp40 class bind to partner chaperones in the Hsp70
class in order to target Hsp70 class chaperones to specific
sites and/or substrates. It has now been shown that an
Hsp70 is also associated with the V. carteri spindle, and the
mechanism by which this Hsp70 and GlsA may participate
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Cell biology
in shifting the division apparatus to an off-center site in an
asymmetrically dividing cell is under investigation
(SM Miller, personal communication).
germ-soma differentiation similar to that of V. carteri? Or
have they discovered some entirely different way to solve
the same evolutionary challenge?
RegA, the protein encoded by regA, is a nuclear protein
with many features of a transcriptional repressor [44••].
Earlier studies had identified a set of 18 genes defined by
‘maturation-abundant gonidial cDNAs’ [45], which have
expression patterns suggesting that they are under either
direct or indirect negative regulation by regA: transcripts of
these 18 genes accumulate to high levels in both gonidia
and regA– somatic cells but are undetectable in wild-type
(regA+) somatic cells after regA expression begins [18,45,46].
It was initially a surprise to learn that the first three of these
genes to be sequenced represented nuclear genes encoding
key chloroplast proteins [47] but this observation has now
been extended substantially. All 16 of these 18 candidate
target genes that have recognizable polypeptides encode
known or putative chloroplast proteins [48•]. These include
components of both light-harvesting complexes, the oxygen-evolving complex, the electron transport chain, the
chloroplast ATP-synthase, and the Calvin cycle, as well as a
regulator of chloroplast translation.
References and recommended reading
These unanticipated observations regarding the putative
targets of RegA regulation have led to the following
working hypothesis: RegA represses reproductive development in somatic cells by repressing genes whose
products are required for chloroplast biogenesis, thereby
preventing these cells from growing enough to reproduce. V. carteri is an obligate photoautotroph whose
growth is photosynthesis limited. Each somatic cell
inherits ~0.04% of the maternal chloroplast during gonidial cleavage; but if unable to make additional
chloroplast, it cannot grow significantly. And if it cannot
grow, it cannot reproduce.
Of the many additional observations that are judged to be
consistent with this hypothesis [48•], the most compelling
is that a significant number of somatic cells that are morphologically indistinguishable from their neighbors lack
any detectable chloroplast DNA [49]. This seems to
demonstrate quite clearly that chloroplast biogenesis is not
required for normal somatic cell development.
Conclusions
The family Volvocaceae is phylogenetically far more complex than was once believed. Nevertheless, it still holds
great promise as a model for studying the evolution of
multicellularity and cellular differentiation. V. carteri,
which has been studied more intensely than any other
member of the family, has begun to reveal the details of its
genetic program for germ-soma differentiation. This raises two fascinating questions to be addressed in the future:
what are the evolutionary roots of the gls, regA and lag
genes that now control cellular differentiation in V. carteri
and do other Volvox species that arose by independent
phylogenetic pathways exhibit a genetic program for
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
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phylogeny of green algae. J Mol Evol 1990, 31:18-24.
2.
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origins of the Metazoa: an evolutionary link with fungi. Science
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3.
Sogin ML: Early evolution and the origin of eukaryotes. Curr Biol
1991, 1:457-463.
4.
•
Kirk DL: Volvox: Molecular-Genetic Origins of Multicellularity and
Cellular Differentiation, vol 381. Cambridge: Cambridge University
Press; 1998.
A comprehensive treatise on the ontogeny and phylogeny of Volvox, particularly Volvox carteri. Most of the topics that are touched on in the present
review are discussed in considerably more detail in this monograph.
5.
Rausch H, Larsen N, Schmitt R: Phylogenetic relationships of the
green alga Volvox carteri deduced from small-subunit ribosomal
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8.
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flagellates: a study of 18S and 26S rRNA sequence data.
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9.
Larson A, Kirk MM, Kirk DL: Molecular phylogeny of the volvocine
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10. Coleman AW, Mai JC: Ribosomal DNA ITS-1 and ITS-2 sequence
comparisons as a tool for predicting genetic relatedness.
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11. Ettl H: Die Gattung Chlamydomonas Ehrenberg.
Beih Nova Hedwigia 1976, 49:1-1122. [Title translation: The genus
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12. Nozaki H, Itoh M: Phylogenetic relationships within the colonial
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on morphological data. J Phycol 1994, 30:353-365.
13. Fabry S, Köhler A, Coleman AW: Intraspecies analysis: Comparison
•• of ITS sequence data and gene intron sequence data with
breeding data for a worldwide collection of Gonium pectorale.
J Mol Evol 1999, 48:94-101.
The authors use sequences of the rDNA internal transcribed spacers (ITS)
[10], and three spliceosomal introns to perform two parallel high-resolution
phylogenetic analyses of G. pectorale isolates from five continents. The trees
generated by these two approaches have extremely similar topologies and
sort these 25 isolates into the same two major clades and six subclades.
Pairwise tests then demonstrate that only members of the same subclade
are able to mate and produce fertile progeny.
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species Pandorina morum Bory de St. Vincent (Volvocaceae).
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and syngens in volvocacean green algae (Chlorophyta). J Phycol
1994, 30:80-90.
17.
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differentiation in Volvox gigas and Volvox powersii.
Arch Protistenkd 1971, 113:195-219.
Evolution of multicellularity in the volvocine algae Kirk
18. Tam L-W, Kirk DL: The program for cellular differentiation in Volvox
carteri as revealed by molecular analysis of development in a
gonidialess/somatic regenerator mutant. Development 1991,
112:571-580.
501
36. Gruber H, Goetinck SD, Kirk DL, Schmitt R: The nitrate reductaseencoding gene of Volvox carteri: map location, sequence and
induction kinetics. Gene 1992, 120:75-83.
20. Goodenough UW, Heuser JE: Molecular organization of cell-wall
crystals from Chlamydomonas reinhardtii and Volvox carteri.
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Hallmann A, Rappel A: Genetic engineering of the multicellular
green alga Volvox: a modified and multiplied bacterial antibiotic
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17:99-109.
This paper is important both as a demonstration of features that can be engineered into foreign genes to enhance their expression in V. carteri, and as a
description of a much-needed second selectable marker for use in transformation studies of Volvox.
21. Adair WS, Steinmetz SA, Mattson DM, Goodenough UW, Heuser JE:
Nucleated assembly of Chlamydomonas and Volvox cell walls.
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38. Schiedlmeier B, Schmitt R, Müller W, Kirk MM, Gruber H, Mages W,
Kirk DL: Nuclear transformation of Volvox carteri.
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22. Adair WS, Appel H: Identification of a highly conserved
hydroxyproline-rich glycoprotein in the cell walls of
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39. Miller SM, Schmitt R, Kirk DL: Jordan, an active Volvox transposable
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structure, biochemistry, and molecular biology. In Organization and
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WS, Mecham RP. San Diego: Academic Press; 1990:15-84.
24. Woessner JP, Goodenough UW: Volvocine cell walls and their
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27.
Fulton AB: Colony development in Pandorina morum. II. Colony
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comparative study and proposed system of nomenclature.
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29. Sumper M, Hallmann A: Biochemistry of the extracellular matrix of
•• Volvox. Int Rev Cytol 1998, 180:51-85.
In a comprehensive and lucid review, the authors summarize all that is known
to date (principally as a result of contributions from their own laboratory)
regarding the organization, composition, assembly, and roles played in the
life of the organism by the V. carteri extracellular matrix (ECM). Of particular
interest are their observations regarding sequence relationships between a
family of ECM glycoproteins and the extraordinarily powerful pheromone that
triggers sexual reproduction in V. carteri.
30. Goodenough UW: Chlamydomonas mating interactions. In
Microbial Cell–Cell Interactions. Edited by Dworkin M.
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1994:71-112.
31. Harris EH: The Chlamydomonas Sourcebook. A Comprehensive
Guide to Biology and Laboratory Use. San Diego: Academic Press;
1989.
32. Batko A, Jakubiec H: Gonium dispersum sp. nov., a new species of
Gonium from Poland. Arch Hyrobiol Suppl 1989, 82:39-47.
33. Starr RC: Control of differentiation in Volvox. Dev Biol Suppl 1970,
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34. Kirk MM, Ransick A, McRae SE, Kirk DL: The relationship between
cell size and cell fate in Volvox carteri. J Cell Biol 1993,
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Reproduction and Photosynthesis. Edited by Wiessner W,
Robinson DG, Starr RC. Berlin: Springer-Verlag; 1990:81-94.
37.
•
40. Hallmann A, Sumper M: Reporter genes and highly regulated
promoters as tools for transformation experiments in Volvox
carteri. Proc Natl Acad Sci USA 1994, 91:11562-11566.
41. Hallmann A, Sumper M: The Chlorella hexose/H+ symporter is a
useful selectable marker and biochemical reagent when
expressed in Volvox. Proc Natl Acad Sci USA 1996, 93:669-673.
42. Hallmann A, Rappel A, Sumper M: Gene replacement by
homologous recombination in the multicellular green alga Volvox
carteri. Proc Natl Acad Sci USA 1997, 94:7469-7474.
43. Miller SM, Kirk DL: glsA, a Volvox gene required for asymmetric
•• division and germ cell specification, encodes a chaperone-like
protein. Development 1999, 126:649-658.
This paper and its companion [44••] report the first uses of an inducible
transposon (see [39]) to tag and recover a developmentally important Volvox
gene. The glsA gene, which plays an essential role in the asymmetric divisions that set apart germ and somatic cell lineages, is shown to encode a
protein that contains two protein-binding sites, and that associates with the
mitotic spindle in dividing cells.
44. Kirk MM, Stark K, Miller SM, Müller W, Taillon BE, Gruber H,
•• Schmitt R, Kirk DL: regA, a Volvox gene that plays a central role in
germ-soma differentiation, encodes a novel regulatory protein.
Development 1999, 126:639-647.
This paper and its companion [43••] report the first uses of an inducible
transposon (see [39]) to tag and recover a developmentally important Volvox
gene. The regA gene, which prevents reproductive development in somatic
cells, is shown to be a nuclear protein with features of an active transcriptional repressor that is present in somatic cells, but not gonidia, for most of
the life cycle.
45. Tam L-W, Kirk DL: Identification of cell-type-specific genes of
Volvox carteri and characterization of their expression during the
asexual life cycle. Dev Biol 1991, 145:51-66.
46. Tam L-W, Stamer KA, Kirk DL: Early and late gene expression
programs in developing somatic cells of Volvox carteri.
Dev Biol 1991, 145:67-76.
47.
Choi G, Przybylska M, Straus D: Three abundant germ line-specific
transcripts in Volvox carteri encode photosynthetic proteins.
Curr Genet 1996, 30:347-355.
48. Meissner MK, Stark K, Cresnar B, Kirk DL, Schmitt R: Volvox
•
germline-specific genes that are putative targets of RegA
repression encode chloroplast proteins. Curr Gent 1999, in press.
This paper extends an analysis begun in [47], and shows that all of the putative target genes of regA [44••] that have recognizable open reading frames
are nuclear genes encoding chloroplast proteins. This leads to the hypothesis that regA exerts its effect on germ-soma differentiation by repressing
chloroplast biogenesis in somatic cells.
49. Coleman AW, Maguire MJ: A microspectrophotometric analysis of
nuclear and chloroplast DNA in Volvox. Dev Biol 1982,
94:441-450.
Evolution of multicellularity in the volvocine algae
David L Kirk
Recent studies reveal that relationships among the volvocine
algae are more complex than was previously believed.
Nevertheless, this group still appears to provide an unrivaled
opportunity to analyze an evolutionary pathway leading from
unicellularity (Chlamydomonas) to multicellularity with division
of labor (Volvox). Significant progress in this regard was made
in the past year when two genes playing key roles in Volvox
cellular differentiation were cloned, and clues were uncovered
regarding their mechanisms of action.
Figure 1
Addresses
Department of Biology, Washington University, Campus Box 1229,
St. Louis, MO 63130, USA; e-mail: kirk@biology.wustl.edu
Current Opinion in Plant Biology 1999, 2:496–501
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
ECM
extracellular matrix
gls
gonidialess
lag
late gonidia
rbcL
rubisco large subunit
regA
somatic regenerator
Introduction
Multicellularity has evolved many times on this planet. In
most cases, however, the pathway leading from a unicellular ancestor to a multicellular organism with differentiated
cell types has been obscured by the passage of time.
Although it is assumed that each of the major groups of
conspicuous modern eukaryotes (Figure 1) evolved from a
different unicellular ancestor more than a billion years ago
[1–3], we can do little more than speculate about what the
last unicellular ancestor of each of these groups may have
been like, or how the transition to multicellularity may
have occurred.
In contrast, within the volvocine algae multicellular organisms with differentiated cell types have evolved far more
recently — and more than once. Therefore, this group of
green flagellates, which includes members of the family
Volvocaceae and their close unicellular relative
Chlamydomonas reinhardtii, appears to provide an unrivaled
opportunity for exploring a pathway leading from unicellularity to multicellularity [4•].
The volvocine algae as an evolutionary model
The volvocine algae range in size and complexity from
unicellular Chlamydomonas, to Volvox, with its thousands of
mortal somatic cells and a few immortal germ cells. It has
long been commonplace for textbooks to arrange these
algae in a conceptual series such as that in Figure 2, in
which there is a regular progression in size, cell number,
abundance of extracellular matrix (ECM), and tendency to
produce sterile somatic cells. It is usually suggested that
Branching order of selected major eukaryotic lineages, based on data
in [2,3], and the family Volvocaceae, based on data in [5]. Note that
the Volvocaceae, represented by the bifurcation at the tip of the green
algal line, have been diversifying less than 10% as long as any of the
major multicellular groups.
this might represent the sequence by which Volvox
evolved. Although such a proposition contains a germ of
truth, it clearly is overly simplistic.
Molecular data indicate that the volvocine algae constitute
a surprisingly youthful group of close relatives: Volvox carteri and C. reinhardtii apparently last shared a common
ancestor 50–75 million years ago [5], and the germ-soma
division of labor that defines the genus Volvox has evolved
at least twice and possibly several times in this relatively
brief interval [4•,6]. This suggests that the transition from
unicellularity to the Volvox type of multicellularity must
have a relatively simple genetic basis, and may actually be
simple enough to be amenable to detailed analysis.
Such optimism is dampened only slightly by the accumulating molecular evidence that the volvocacean family tree
is far more complicated than was suspected a generation
ago. Volvox is not the only volvocine taxon that is polyphyletic. For example, the phylogenetic reconstruction
shown in Figure 3, which is based on rubisco large subunit
(rbcL) sequences, supports the long-standing assumption
that Eudorina, Pleodorina and Volvox are all closely related
but at the same time it reveals the striking degree to which
relationships within this section of the family Volvocaceae
may deviate from the simple ones suggested by Figure 2.
The data summarized in Figure 3 indicate clearly, for
example, that the genus Eudorina is polyphyletic, and that
its type species, E. elegans, is paraphyletic. Such deviations
from monophyly had already been suspected when only a
few Eudorina isolates had been analyzed [6], but the
extent of these deviations from monophyletic expectations
Evolution of multicellularity in the volvocine algae Kirk
Figure 2
497
Figure 3
A molecular phylogenetic reconstruction of the Eudorina–Pleodorina–
Volvox section of the family Volvocaceae based on rbcL sequences.
Numbers are strain identifications, as given in [7], from which the present
figure is adapted. The two taxa marked by asterisks were identified as
Eudorina illinoisensis in [7], but here they are labeled Pleodorina
illinoisensis, as they are in major algal culture collections, in order to
highlight some of the taxonomic uncertainties in this family.
A conventional representation of ‘the volvocine lineage’, with organisms
drawn at progressively decreasing magnification from bottom to top.
The diameters of the biflagellate cells would be in the range of
5–10 µm in each organism. Dark gray shading represents the cell
bodies, light gray shading represents the ECM and thick black lines
represent the tripartite layer that is discussed below.
has become increasingly apparent as more and more putatively ‘congeneric’ and ‘conspecific’ isolates have been
analyzed [7]. Therefore, we may anticipate a plethora of
additional complications when Pleodorina and Volvox
species are sampled with similar intensity, and when the
12–14 species of Volvox that are currently missing from
Figure 3 are added.
Relationships at the opposite end of the volvocine complexity spectrum are no simpler. Chlamydomonas turns out
to be a highly paraphyletic genus comprising many distinct clades [8], and C. reinhardtii is found to be much
more closely related to the volvocaceans than it is to most
Chlamydomonas species [8–10], including those species
once thought to be its closest relatives [10,11]. Although
analysis of rbcL data provide little support for the idea that
all species previously known as Gonium should be moved
from the Volvocaceae to two new families [12], they do
indicate that these species define two entirely separate
clades [6]; moreover, phylogenetic analysis at a higher
level of resolution demonstrated that Gonium pectorale consists of six reproductively isolated subclades [13••]. But
this is modest compared to the extraordinary reproductive, chromosomal and molecular diversity that has been
found within the morphologically uniform species
Pandorina morum [14–16].
In short, it appears that many volvocacean taxonomic categories identify developmental and/or organizational grades,
rather than phylogenetic clades. However, reconstructions
such as the one in Figure 3 (and others not shown) indicate
that transitions among these grades — in both directions —
must be easily accomplished and thus must have a relatively
simple genetic basis. This notion is supported by mutational studies: a single mutation is adequate to convert Volvox
powersii into a mimic of Pleodorina californica [17], and two
mutations abolish germ-soma differentiation in V. carteri and
convert it into a mimic of Eudorina [18].
Thus, an image of a highly branched, spreading bush has
replaced the earlier image of an upright, relatively
498
Cell biology
Figure 4
A schematic representation of the way in which certain extracellular
materials (predominantly glycoproteins and sulfated polysaccharides)
are arranged in selected volvocine taxa. Not drawn to scale.
(a) Chlamydomonas reinhardtii. The concentric black–white–black
ellipses at the periphery represent the ‘trilaminar’ layer of the cell wall,
while the stippled ellipse represents the more amorphous-looking inner
wall, and the central clear space represents the cell body. (b) Gonium
pectorale. A wall resembling that of Chlamydomonas surrounds each
cell body, but specializations (black rectangles) attach the outer layers
of neighboring walls to one another at specific points. Such
attachments vary in a species-specific way in different species of
Gonium. (c) Pandorina morum (part of a colony in cross section). The
inner wall and the inner portion of the tripartite layer surround each cell
as in (a) and (b), but the outermost portion of the trilaminar layer is
continuous over the surface of the organism. (d) Volvox carteri (part of
a somatic-cell layer in cross section). In Eudorina, Pleodorina and
Volvox, the entire tripartite layer is continuous over the surface of the
spheroid and most of the spheroid is occupied by a species-specific
assortment of fibrous layers (heavy stippling) and relatively amorphous
‘mucilaginous’ regions (lighter stippling), all of which appear to have
been derived by evolutionary amplification and elaboration of the inner
wall of Chlamydomona.
unbranched tree as a metaphor for volvocacean family history. But although this clearly means that reconstructing
the complete family history of the Volvocaceae will be a
much more challenging task than was once imagined, it
has done little to diminish optimism that it will be easier to
elucidate the molecular genetic origins of multicellularity
and cellular differentiation in the volvocine algae than in
any other lineage yet identified.
From unicells to colonies: sharing extracellular
materials
The prime feature distinguishing the volvocaceans from
Chlamydomonas is that the glycoprotein based ‘cell walls’
of volvocaceans are linked together in some species-specific manner to form an extracellular matrix (ECM);
however, many components of Chlamydomonas walls and
volvocacean ECM are clearly homologous. The most conserved extracellular feature of the group is a layer that has
a characteristic crystalline appearance when viewed en face
[19], but is more commonly seen as a dark–light–dark profile in electronmicrograph thin sections, and is therefore
usually called the ‘tripartite’ layer. This layer has an
extremely similar organization [19–21], and contains very
similar proteins [22–24] in all volvocine algae that have
been carefully examined; however, the disposition of this
layer with respect to the cell bodies varies in a taxon-specific way (Figure 4).
In Gonium, as in Chlamydomonas, the tripartite layer surrounds each cell and is separated from the plasmalemma
by only a very thin, nondescript ‘inner wall’; however, in
Gonium the walls of neighboring cells are linked together
(Figure 4a,b), with the nature of the linkages differing in a
species-specific manner [25,26]. A transition occurs in
Pandorina, where the tripartite layer is partially split, such
that its outer leaflet is continuous over the surface of the
colony, although its inner leaflet is still wrapped around
each cell body (Figure 4c) [27]. This transition is taken a
step further in the rest of the volvocaceans, in which the
entire tripartite layer is always continuous over the surface
(Figure 4d). In the larger volvocaceans (Eudorina to Volvox)
the ECM accounts for 90–99% of the volume of the organism, and it exhibits a wide variety of interesting
taxon-specific internal specializations [28]. The vast majority of this ECM appears to be an extensive evolutionary
amplification and elaboration of what in Chlamydomonas is
the remarkably unimpressive ‘inner wall’ (Figure 4a,d).
It would be surprising, therefore, if the architecturally
complex ECM of the larger volvocaceans was not significantly more complex biochemically than the cell wall of
C. reinhardtii, which contains > 30 kinds of glycoproteins,
some of which are glycine-rich but most of which are
hydroxyproline-rich [23,24]. Thus, the ten ECM proteins
of V. carteri that have been characterized to date by cDNA
cloning and sequencing [29••] constitute an impressive
and important start — but only a start — toward a full molecular characterization of the Volvox ECM.
The most striking feature shared by the extracellular glycoproteins of C. reinhardtii and V. carteri is their modularity.
One of the most recurrent themes is a hydroxyproline-rich,
rod-like module of variable length attached at one or both
ends to various globular modules, which often results in
molecules capable of extensive self-assembly [24,29••]. It
has been postulated that the propensity of the genes
encoding ECM modules for rapid diversification has
resulted in their exploitation for entirely different, but no
less important evolutionary ends: to encode species-specific sex proteins — sexual agglutinins in the case of
Chlamydomonas [30] and species-specific sex-inducing
pheromones in the case of Volvox [29••].
The conversion of a Chlamydomonas cell wall to a
volvocine ECM undoubtedly was facilitated by the
unique pattern of cell division that the green flagellates
exhibit: instead of undergoing binary fission as most cells
do, green flagellates undergo ‘multiple fission’. That is,
after growing 2n–fold in volume, they divide rapidly n
times within the mother-cell wall, to produce 2n daughter
cells (where the maximum value of n is a species characteristic) [4•,31]. In unicellular green flagellates,
cytokinesis is complete, so sister cells are able to move
Evolution of multicellularity in the volvocine algae Kirk
relative to one another while they are still within the
mother-cell wall and are forming their own cell walls [31];
as a result they develop as untethered, free individuals.
In contrast, in all volvocaceans that have been carefully
examined, cytokinesis is incomplete, and cytoplasmic
bridges that are formed in each division furrow hold all sister cells in fixed relationships until after the deposition of
‘wall’ components has begun [4•]. With the cells held
together in this way while extracellular materials are being
deposited, ‘walls’ invariably become fused in species-specific ways to form the ECM that ties volvocacean cells into
a coherent unit.
Thus, the critical first step in evolution of volvocine multicellularity was probably the invention of a mechanism
for delaying separation of sister cells until wall materials
had been deposited around and between them. A recently discovered flagellate, Gonium dispersum, appears to be
trying to make up its mind right now whether or not it
will permanently adopt such a mechanism. G. dispersum
divides two ways: one way results in sister cells being
held together between divisions, and results in formation
of colonies similar to those of other Gonium species; the
other way permits cells to move about between divisions, and generates Chlamydomonas-like unicellular
progeny [32]. Further study of this interesting schizoid
flagellate is warranted.
From colonies to multicellular organisms:
dividing life’s labors
V. carteri has become the premier model for studies of
volvocine ontogeny and phylogeny because of its genetic
accessibility and the clarity with which it can be used to
pose two of the central unanswered questions of biology:
how is a program for the development of cells of entirely
different phenotypes programmed in a genome and how
does such a program evolve?
In distinction to colonial volvocaceans such as Pandorina
or Eudorina, V. carteri is a bona fide multicellular organism
with a complete division of labor between two interdependent cell types [4•,33]. The ~2,000 biflagellate
somatic cells of V. carteri are specialized for motility but are
terminally differentiated and programmed to die after
serving their purpose. The ~16 gonidia (asexual reproductive cells), in marked contrast, are completely non-motile,
programmed for division and reproduction, and potentially immortal. When mature, each gonidium divides
repeatedly to produce an embryo containing all of the
cells of both types that will be present in an adult of the
next generation. A key step in germ-soma differentiation
occurs in mid-embryogenesis, when visibly asymmetric
divisions set apart large ‘gonidial initials’ from small
‘somatic initials’. Many types of evidence indicate that it
is this difference in size, and not any difference in cytoplasmic quality, that determines which pathway of
differentiation each cell will follow [34].
499
Mutational and Mendelian analyses have led to the proposal that three major types of gene program V. carteri
germ-soma differentiation [18]. First the gonidialess (gls)
genes act in the cleaving embryo to cause asymmetric division. Then a pair of negative regulators are activated
differentially: the somatic regenerator (regA) gene product
acts in small cells to repress reproductive development,
while the late gonidia (lag) gene products act in the large
cells to repress somatic development.
The simple logic underlying such a negative-regulatory
program becomes apparent when viewed in an evolutionary context. In volvocine algae from Chlamydomonas to
Eudorina that have a single cell type, every cell first
develops as a biflagellate (somatic-like) cell, and then
later loses its flagella, rounds up, and transforms into a
reproductive (gonidia-like) cell that initiates a series of
rapid cleavage divisions [4•,31]. Thus, the basic program
of volvocine development is a sequential one: first biflagellate, then reproductive. In V. carteri, however, this
sequential program has been converted into a dichotomous one by repressing the biflagellate phase in gonidia
(with the lag genes), and the reproductive phase in
somatic cells (with the regA gene). When one of the lag
genes is inactivated, presumptive gonidia revert to the
ancestral ‘first biflagellate, then reproductive’ program,
and when the regA gene is inactivated the somatic cells do
the same [35]. Thus, it seems clear that these genes once
played as important a role in the evolution of V. carteri as
they now play in its development.
Detailed analysis of such genes awaited development of
molecular-genetic tools of the sort now considered commonplace in other systems. The concerted efforts of the
three laboratories now actively engaged in Volvox research
have resulted in development of such a tool kit: this now
includes a pair of selectable markers [36,37•], a nuclear
transformation system [38], a transposon suitable for gene
tagging [39], and reporter and inducible-promoter constructs [40]. Transformation has been used to modify
carbohydrate metabolism with a foreign gene [41], and to
achieve gene replacement by homologous recombination
[42]. More recently, transposon tagging has been used to
clone two key genes controlling germ-soma differentiation:
glsA, a gene required for asymmetric division [43••] and
regA, the gene whose product represses reproductive
development in somatic cells [44••].
GlsA, the protein encoded by glsA, was found to contain a
‘J domain’ that is essential for its function, and, like the
homologous human J protein MPP11, GlsA is associated
with the mitotic spindle in dividing cells [43••]. The J
domain is the conserved motif by which chaperones in the
DnaJ/Hsp40 class bind to partner chaperones in the Hsp70
class in order to target Hsp70 class chaperones to specific
sites and/or substrates. It has now been shown that an
Hsp70 is also associated with the V. carteri spindle, and the
mechanism by which this Hsp70 and GlsA may participate
500
Cell biology
in shifting the division apparatus to an off-center site in an
asymmetrically dividing cell is under investigation
(SM Miller, personal communication).
germ-soma differentiation similar to that of V. carteri? Or
have they discovered some entirely different way to solve
the same evolutionary challenge?
RegA, the protein encoded by regA, is a nuclear protein
with many features of a transcriptional repressor [44••].
Earlier studies had identified a set of 18 genes defined by
‘maturation-abundant gonidial cDNAs’ [45], which have
expression patterns suggesting that they are under either
direct or indirect negative regulation by regA: transcripts of
these 18 genes accumulate to high levels in both gonidia
and regA– somatic cells but are undetectable in wild-type
(regA+) somatic cells after regA expression begins [18,45,46].
It was initially a surprise to learn that the first three of these
genes to be sequenced represented nuclear genes encoding
key chloroplast proteins [47] but this observation has now
been extended substantially. All 16 of these 18 candidate
target genes that have recognizable polypeptides encode
known or putative chloroplast proteins [48•]. These include
components of both light-harvesting complexes, the oxygen-evolving complex, the electron transport chain, the
chloroplast ATP-synthase, and the Calvin cycle, as well as a
regulator of chloroplast translation.
References and recommended reading
These unanticipated observations regarding the putative
targets of RegA regulation have led to the following
working hypothesis: RegA represses reproductive development in somatic cells by repressing genes whose
products are required for chloroplast biogenesis, thereby
preventing these cells from growing enough to reproduce. V. carteri is an obligate photoautotroph whose
growth is photosynthesis limited. Each somatic cell
inherits ~0.04% of the maternal chloroplast during gonidial cleavage; but if unable to make additional
chloroplast, it cannot grow significantly. And if it cannot
grow, it cannot reproduce.
Of the many additional observations that are judged to be
consistent with this hypothesis [48•], the most compelling
is that a significant number of somatic cells that are morphologically indistinguishable from their neighbors lack
any detectable chloroplast DNA [49]. This seems to
demonstrate quite clearly that chloroplast biogenesis is not
required for normal somatic cell development.
Conclusions
The family Volvocaceae is phylogenetically far more complex than was once believed. Nevertheless, it still holds
great promise as a model for studying the evolution of
multicellularity and cellular differentiation. V. carteri,
which has been studied more intensely than any other
member of the family, has begun to reveal the details of its
genetic program for germ-soma differentiation. This raises two fascinating questions to be addressed in the future:
what are the evolutionary roots of the gls, regA and lag
genes that now control cellular differentiation in V. carteri
and do other Volvox species that arose by independent
phylogenetic pathways exhibit a genetic program for
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Devereux R, Loeblich AR III, Fox GE: Higher plant origins and the
phylogeny of green algae. J Mol Evol 1990, 31:18-24.
2.
Wainwright PO, Hinkle G, Sogin ML, Stickel SK: Monophyletic
origins of the Metazoa: an evolutionary link with fungi. Science
1993, 260:340-342.
3.
Sogin ML: Early evolution and the origin of eukaryotes. Curr Biol
1991, 1:457-463.
4.
•
Kirk DL: Volvox: Molecular-Genetic Origins of Multicellularity and
Cellular Differentiation, vol 381. Cambridge: Cambridge University
Press; 1998.
A comprehensive treatise on the ontogeny and phylogeny of Volvox, particularly Volvox carteri. Most of the topics that are touched on in the present
review are discussed in considerably more detail in this monograph.
5.
Rausch H, Larsen N, Schmitt R: Phylogenetic relationships of the
green alga Volvox carteri deduced from small-subunit ribosomal
RNA comparisons. J Mol Evol 1989, 29:255-265.
6.
Nozaki H, Itoh M, Sano R, Uchida H, Watanabe MM, Kuroiwa T:
Phylogenetic relationships within the colonial Volvocales
(Chlorophyta) inferred from rbcL gene sequence data. J Phycol
1995, 31:970-979.
7.
Nozaki H, Ito M, Uchida H, Watanabe MM, Kuroiwa T:
Phylogenetic analysis of Eudorina species (Volvocaceae,
Chlorophyta) based on rbcL gene sequences. J Phycol 1997,
33:859-863.
8.
Buchheim MA, Chapman RL: Phylogeny of the colonial green
flagellates: a study of 18S and 26S rRNA sequence data.
BioSystems 1991, 25:85-100.
9.
Larson A, Kirk MM, Kirk DL: Molecular phylogeny of the volvocine
flagellates. Mol Biol Evol 1992, 9:85-105.
10. Coleman AW, Mai JC: Ribosomal DNA ITS-1 and ITS-2 sequence
comparisons as a tool for predicting genetic relatedness.
J Mol Evol 1997, 45:168-177.
11. Ettl H: Die Gattung Chlamydomonas Ehrenberg.
Beih Nova Hedwigia 1976, 49:1-1122. [Title translation: The genus
Chlamydomonas Ehrenberg.]
12. Nozaki H, Itoh M: Phylogenetic relationships within the colonial
Volvocales (Chlorophyta) inferred from cladistic analysis based
on morphological data. J Phycol 1994, 30:353-365.
13. Fabry S, Köhler A, Coleman AW: Intraspecies analysis: Comparison
•• of ITS sequence data and gene intron sequence data with
breeding data for a worldwide collection of Gonium pectorale.
J Mol Evol 1999, 48:94-101.
The authors use sequences of the rDNA internal transcribed spacers (ITS)
[10], and three spliceosomal introns to perform two parallel high-resolution
phylogenetic analyses of G. pectorale isolates from five continents. The trees
generated by these two approaches have extremely similar topologies and
sort these 25 isolates into the same two major clades and six subclades.
Pairwise tests then demonstrate that only members of the same subclade
are able to mate and produce fertile progeny.
14. Coleman AW: Sexual isolation in Pandorina morum. J Protozool
1959, 6:249-264.
15. Coleman AW, Zollner J: Cytogenetic polymorphism within the
species Pandorina morum Bory de St. Vincent (Volvocaceae).
Arch Protistenkd 1977, 119:224-232.
16. Coleman AW, Suarez A, Goff LJ: Molecular delineation of species
and syngens in volvocacean green algae (Chlorophyta). J Phycol
1994, 30:80-90.
17.
Vande Berg WJ, Starr RC: Structure, reproduction and
differentiation in Volvox gigas and Volvox powersii.
Arch Protistenkd 1971, 113:195-219.
Evolution of multicellularity in the volvocine algae Kirk
18. Tam L-W, Kirk DL: The program for cellular differentiation in Volvox
carteri as revealed by molecular analysis of development in a
gonidialess/somatic regenerator mutant. Development 1991,
112:571-580.
501
36. Gruber H, Goetinck SD, Kirk DL, Schmitt R: The nitrate reductaseencoding gene of Volvox carteri: map location, sequence and
induction kinetics. Gene 1992, 120:75-83.
20. Goodenough UW, Heuser JE: Molecular organization of cell-wall
crystals from Chlamydomonas reinhardtii and Volvox carteri.
J Cell Sci 1988, 90:717-733.
Hallmann A, Rappel A: Genetic engineering of the multicellular
green alga Volvox: a modified and multiplied bacterial antibiotic
resistance gene as a dominant selectable marker. Plant J 1999,
17:99-109.
This paper is important both as a demonstration of features that can be engineered into foreign genes to enhance their expression in V. carteri, and as a
description of a much-needed second selectable marker for use in transformation studies of Volvox.
21. Adair WS, Steinmetz SA, Mattson DM, Goodenough UW, Heuser JE:
Nucleated assembly of Chlamydomonas and Volvox cell walls.
J Cell Biol 1987, 105:2373-2382.
38. Schiedlmeier B, Schmitt R, Müller W, Kirk MM, Gruber H, Mages W,
Kirk DL: Nuclear transformation of Volvox carteri.
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22. Adair WS, Appel H: Identification of a highly conserved
hydroxyproline-rich glycoprotein in the cell walls of
Chlamydomonas reinhardtii and two other Volvocales. Planta
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39. Miller SM, Schmitt R, Kirk DL: Jordan, an active Volvox transposable
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5:1125-1138.
19. Roberts K: Crystalline glycoprotein cell walls of algae; their
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29. Sumper M, Hallmann A: Biochemistry of the extracellular matrix of
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In a comprehensive and lucid review, the authors summarize all that is known
to date (principally as a result of contributions from their own laboratory)
regarding the organization, composition, assembly, and roles played in the
life of the organism by the V. carteri extracellular matrix (ECM). Of particular
interest are their observations regarding sequence relationships between a
family of ECM glycoproteins and the extraordinarily powerful pheromone that
triggers sexual reproduction in V. carteri.
30. Goodenough UW: Chlamydomonas mating interactions. In
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31. Harris EH: The Chlamydomonas Sourcebook. A Comprehensive
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33. Starr RC: Control of differentiation in Volvox. Dev Biol Suppl 1970,
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34. Kirk MM, Ransick A, McRae SE, Kirk DL: The relationship between
cell size and cell fate in Volvox carteri. J Cell Biol 1993,
123:191-208.
35. Kirk DL: Genetic control of reproductive cell differentiation in
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•
40. Hallmann A, Sumper M: Reporter genes and highly regulated
promoters as tools for transformation experiments in Volvox
carteri. Proc Natl Acad Sci USA 1994, 91:11562-11566.
41. Hallmann A, Sumper M: The Chlorella hexose/H+ symporter is a
useful selectable marker and biochemical reagent when
expressed in Volvox. Proc Natl Acad Sci USA 1996, 93:669-673.
42. Hallmann A, Rappel A, Sumper M: Gene replacement by
homologous recombination in the multicellular green alga Volvox
carteri. Proc Natl Acad Sci USA 1997, 94:7469-7474.
43. Miller SM, Kirk DL: glsA, a Volvox gene required for asymmetric
•• division and germ cell specification, encodes a chaperone-like
protein. Development 1999, 126:649-658.
This paper and its companion [44••] report the first uses of an inducible
transposon (see [39]) to tag and recover a developmentally important Volvox
gene. The glsA gene, which plays an essential role in the asymmetric divisions that set apart germ and somatic cell lineages, is shown to encode a
protein that contains two protein-binding sites, and that associates with the
mitotic spindle in dividing cells.
44. Kirk MM, Stark K, Miller SM, Müller W, Taillon BE, Gruber H,
•• Schmitt R, Kirk DL: regA, a Volvox gene that plays a central role in
germ-soma differentiation, encodes a novel regulatory protein.
Development 1999, 126:639-647.
This paper and its companion [43••] report the first uses of an inducible
transposon (see [39]) to tag and recover a developmentally important Volvox
gene. The regA gene, which prevents reproductive development in somatic
cells, is shown to be a nuclear protein with features of an active transcriptional repressor that is present in somatic cells, but not gonidia, for most of
the life cycle.
45. Tam L-W, Kirk DL: Identification of cell-type-specific genes of
Volvox carteri and characterization of their expression during the
asexual life cycle. Dev Biol 1991, 145:51-66.
46. Tam L-W, Stamer KA, Kirk DL: Early and late gene expression
programs in developing somatic cells of Volvox carteri.
Dev Biol 1991, 145:67-76.
47.
Choi G, Przybylska M, Straus D: Three abundant germ line-specific
transcripts in Volvox carteri encode photosynthetic proteins.
Curr Genet 1996, 30:347-355.
48. Meissner MK, Stark K, Cresnar B, Kirk DL, Schmitt R: Volvox
•
germline-specific genes that are putative targets of RegA
repression encode chloroplast proteins. Curr Gent 1999, in press.
This paper extends an analysis begun in [47], and shows that all of the putative target genes of regA [44••] that have recognizable open reading frames
are nuclear genes encoding chloroplast proteins. This leads to the hypothesis that regA exerts its effect on germ-soma differentiation by repressing
chloroplast biogenesis in somatic cells.
49. Coleman AW, Maguire MJ: A microspectrophotometric analysis of
nuclear and chloroplast DNA in Volvox. Dev Biol 1982,
94:441-450.