J Plant Res (2010) 123:171–184
DOI 10.1007/s10265-010-0309-6
JPR SYMPOSIUM
Cytoplasmic inheritance in green algae: patterns, mechanisms
and relation to sex type
Shinichi Miyamura
Received: 14 October 2009 / Accepted: 21 December 2009 / Published online: 30 January 2010
Ó The Botanical Society of Japan and Springer 2010
Abstract Cytological and genetic investigations of two
major groups of green algae, chlorophyte and streptophyte
green algae, show a predominance of uniparental inheri-
tance of the plastid and mitochondrial genomes in most
species. However, in some crosses of isogamous species of
Ulva compressa, these genomes are transmitted from mt?,
mt-, and both parents. In species with uniparental orga-
nelle inheritance, various mechanisms can eliminate
organelles and their DNA during male gametogenesis or
after fertilization. Concerning plastid inheritance, two
major mechanisms are widespread in green algae: (1)
digestion of plastid DNA during male gametogenesis,
during fertilization, or after fertilization; and (2) disinte-
gration or fusion of the plastid in the zygote. The first
mechanism also eliminates the mitochondrial DNA in
anisogamous and oogamous species. These mechanisms
would ensure the predominantly uniparental inheritance of
organelle genomes in green algae. To trace the evolution-
ary history of cytoplasmic inheritance in green algae, the
relations between uniparental inheritance and sex type
were considered in isogamous, anisogamous, and ooga-
mous species using sex-specific features that might be
nearly universal among Chlorophyta.
Keywords Anisogamy
Cytoplasmic inheritance

Green algae
Isogamy
Oogamy
Sex
S. Miyamura (&)
Graduate School of Life and Environmental Sciences,
University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
e-mail: miyamura@sakura.cc.tsukuba.ac.jp
Cytoplasmic inheritance
Introduction
Cytoplasmic inheritance of plastid and mitochondrial
genomes is widespread in sexual eukaryotes such as algae,
land plants, and animals (Sears 1980; Whatley 1982; Birky
1995, 2008). Since the first findings of maternal inheritance
by Correns (1909) and biparental inheritance by Baur
(1909) a century ago, a wealth of genetic evidence related
to these phenomena has been accumulated. It has become
apparent that maternal inheritance of plastid and mito-
chondrial genomes is predominant in most land plants
except for coniferous gymnosperms, in which plastid
genomes are transmitted paternally to their progeny, while
mitochondrial genomes are inherited maternally or pater-
nally (Mogensen 1996).
The origin of maternal inheritance and its underlying
mechanisms in land plants can be traced back to their green
algal ancestor, because phylogenetic evidence indicates
that land plants originated from green algae (Graham 1993;
Lewis and McCourt 2004). For example, the preferential
digestion of plastid and mitochondrial DNA during sper-
matogenesis is a mechanism that explains the maternal
inheritance of organelle genomes in angiosperms
(Miyamura et al. 1987; Corriveau and Coleman 1988;
Nagata et al. 1999). A similar event also occurs in the
zygotes of the unicellular green alga Chlamydomonas
reinhardtii (Kuroiwa et al. 1982; Nishimura et al. 1999),
which exhibits predominantly uniparental inheritance of
plastid genomes (Sager 1954), and male gametes of mul-
ticellular and siphonous green algae, such as Bryopsis
maxima (Fig. 1; Kuroiwa and Hori 1986). Nevertheless, it
remains unknown as to whether these mechanisms have a
common origin or are independently acquired during
evolution, because Chlamydomonas, Bryopsis and angio-
sperms belong to distantly related green plant lineages.
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Fig. 1 Differential interference contrast and fluorescence images of
the male gamete and zygote of the green alga Bryopsis maxima after
staining with DAPI. Chloroplast and mitochondrial nucleoids disap-
pear in the male gamete, while those from female gamete remain in
the zygote. cp Chloroplast, cpN chloroplast nucleoid, N nucleus, mtN
mitochondrial nucleoid. Scale bar 5 lm
To answer this question, it is necessary to trace the evo-
lutional history of cytoplasmic inheritance in green algae.
This is not easy because the information related to cyto-
plasmic inheritance in green algae is scarce compared to
that of land plants, except for that related to C. reinhardtii
(Sears 1980; Whatley 1982; Kuroiwa 1991; Gillham 1994;
Birky 1995; Mogensen 1996). In most green algae, cyto-
plasmic inheritance has been examined using light, fluo-
rescence, and electron microscopy; it has not been studied
using genetic markers (Sears 1980; Whatley 1982; Ku-
roiwa 1991; Birky 1995). However, molecular methods
have allowed restriction fragment length polymorphisms
(RFLP) and PCR–RFLP to be used recently as molecular
markers. Such methods have stimulated investigations of
the patterns and mechanisms of cytoplasmic inheritance in
green algae.
Green algae are a highly diverse group of unicellular
and multicellular organisms that inhabit various
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environments in fresh water, marine water, and so on. They
are divided into two major groups: chlorophyte and strep-
tophyte green algae (Lewis and McCourt 2004; Becker and
Marin 2009). Streptophyte green algae, along with land
plants, form Streptophyta, a sister lineage to that of Chlo-
rophyta, which comprises the remainder of green algae to
which Chlamydomonas belongs. In contrast to land plants,
both groups of green algae have three types of sexual
reproduction, isogamy, anisogamy, and oogamy (Graham
and Wilcox 2000). In anisogamous and oogamous organ-
isms, the two sex types are readily distinguishable based on
differences in gamete size: males produce small gametes
while females produce large gametes. In contrast, gametes
of isogamous organisms such as C. reinhardtii have a
similar appearance; the two mating types have often been
designated arbitrarily as ‘‘plus’’ (mt?) and ‘‘minus’’ (mt-),
so that it is unclear how the sex types of anisogamous and
oogamous organisms are related to the mating types of
isogamous ancestors. Considering this ambiguity, the use
of objective methods to elucidate these relations is
important in order to understand the patterns and mecha-
nisms of cytoplasmic inheritance and to trace their evolu-
tionary history in green algae, because the patterns of
cytoplasmic inheritance are linked to sex type (Gillham
1994; Armbrust 1998).
This review first presents a summary of our current
understanding of the patterns and mechanisms of cyto-
plasmic inheritance in green algae. An examination of the
relations between cytoplasmic inheritance and sex type in
isogamous, anisogamous, and oogamous species in green
algae is then described.
Patterns and mechanisms of cytoplasmic inheritance
in green algae
This section presents a survey of the patterns and mecha-
nisms of cytoplasmic inheritance in chlorophyte and
streptophyte green algae. Generally, the pattern of cyto-
plasmic inheritance is studied by genetic analyses such as
crossing experiments using genetic markers. However, in
most green algae, cytoplasmic inheritance has never been
studied using genetic markers. Consequently, in addition to
the genetic evidence, the cytological evidence of plastid
(Table 1) and mitochondrial inheritance (Table 2) is
included in this review.
Chlorophyta
Chlorophyta includes diverse groups of green algae. Sexual
reproduction is known to occur in Ulvophyceae, Tre-
bouxiophyceae, Chlorophyceae (UTC clade), and
 Table 1 Inheritance patterns and the fates of plastids and their DNA in green algae
Taxon Species Types of Mode of Types of Fate of plastid Fate of plastidsd Methods of Sourcesg
sexual inheritancea evidenceb DNAc analysise
reproduction Sex/stage/fate Sex/stage/fate

Chlorophyta
Chlorophyceae
Volvocales Chlamydomonas reinhardtii Isogamy U (mt?) G mt-/zygote/– mt?, mt-/zygote/fusion GM/PCR–RFLP/FM/TEM 1, 2, 3
C. eugametos 9 C. moewusii Isogamy U (mt?) G ND ND RFLP 4
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C. moewusii Isogamy U C ND/zygote/– ND FM 5

Gonium pectorale Isogamy U (mt?) G ND mt?, mt-/zygote/fusion PCR–RFLP/LM 6, 7
Volvox carteri Oogamy M G Male/fertilization/– ND RFLP/FM 8, 9, 10
Ulvophyceae
Ulotrichales Monostroma latissimum Isogamy U C ND/zygote/– ND/zygote/swelling FM/TEM 11
Siphonocladales Dictyosphaeria cavernosa Isogamy U C ND/zygote/– ND FM 12
Dasycladales Acetabularia calyculus Isogamy U C ND/zygote/– ND FM 12
Ulvales Ulva compressa crosses 1, 2f Isogamy U (mt?) G ND ND PCR–RFLP 13, 14, 15
Ulva compressa crosses 3f Isogamy U (mt?)/U (mt-)/B G ND ND PCR–RFLP 15
U. mutabilis Isogamy U (mt-) G ND mt?/zygote/swelling UV/TEM/AR 16, 17, 18
Bryopsidales Bryopsis maxima Anisogamy M C, BC Male/gamete/– Male/zygote/disintegration FM/CsCl/TEM 19, 20, 21
B. plumosa Anisogamy M C Male/gamete/– ND FM 22
Derbesia tenuissima Anisogamy M C Male/gamete/– ND FM/IEM 23
Caulerpa brachypus Anisogamy M C Male/gamete/– ND FM 24
C. okamurae Anisogamy M C Male/gamete/– ND FM 24, 25
C. racemosa var. laetevirens Anisogamy M C Male/gamete/– ND FM 24
C. serrulata var. serrulata Anisogamy M C Male/gamete/– ND FM 24
f. lata
Trebouxiophyceae
Prasiolales Prasiola stipitata Oogamy M C ND Male/zygote/disintegration LM 26
Prasinophytes
Nephroselmis olivacea Isogamy U C ND ND/zygote/swelling TEM 27
Streptophyta
Charales Chara corallina Oogamy M C Male/gamete/– ND FM/IFM 28
173

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 Table 1 continued
174

Taxon Species Types of Mode of Types of Fate of plastid Fate of plastidsd Methods of Sourcesg
sexual inheritancea evidenceb DNAc analysise

123
reproduction Sex/stage/fate Sex/stage/fate

Zygnematales Spirogyra verruculosa Isogamy U C ND ND/zygote/disintegration TEM 29

S. neglecta Isogamy U (passive) C ND Active/zygote/disintegration LM 30
Zygnema stellinum Isogamy U (passive) C ND Active/zygote/disintegration LM 31
Temnogyra collinsii Anisogamy M C ND Male/gamete/disintegration LM 32
Cosmarium turpinii Isogamy U (mt?) C ND mt-/zygote/disintegration LM 33
Closterium ehrenbergii Isogamy U C ND ND/zygote/disintegration LM/TEM 34, 35
Cylindrocystis brebissonii Isogamy U (mt?)/U (mt-) C ND mt?, mt-/meiosis/ LM 36, 37
segregation
a
M, maternal inheritance; U, uniparental inheritance; U (mt?), uniparental inheritance from mt?; U (mt-), uniparental inheritance from mt-; U (passive), uniparental inheritance from passive
gamete; B, biparental inheritance
b
G, genetic evidence; C, cytological evidence; BC, biochemical evidence
c
ND, not determined; – disappearance of plastid DNA
d
fusion, fusion of two plastids; swelling, swelling of thylakoid membranes; disintegration, disintegration of plastid; active, active gamete
e
GM, genetic analysis by mutants; FM, fluorescence microscopy; UV, experiments with UV-irradiated gametes; TEM, transmission electron microscopy; LM, light microscopy; RFLP,
restriction fragment length polymorphisms; CsCl, CsCl density gradient centrifugation; AR, autoradiography; IEM, immunoelectron microscopy; IFM, immunofluorescence microscopy
f
Crosses 1, 2 and 3 indicate the results of MGEC1 9 MGEC6, MGEC2 9 MGEC5, and other strains, respectively
g
1, Sager (1954); 2, Cavalier-Smith (1970); 3, Kuroiwa et al. (1982); 4, Lee et al. (1990); 5, Coleman and Maguire (1983); 6, Hamaji et al. (2008); 7, Stein (1958); 8, Adams et al. (1990); 9,
Coleman and Maguire (1982); 10, Kuroiwa et al. (1993a); 11, Kuroiwa et al. (1993b); 12, Kuroiwa et al. (1985); 13, Kagami et al. (2008); 14, Kawano (2008); 15, Mogi et al. (2009); 16,
Bråten(1971); 17, Fjeld (1971); 18, Bråten (1973); 19, Kuroiwa and Hori (1986); 20, Kuroiwa and Hori (1990); 21, Kuroiwa et al. (1991); 22, Ogawa (1988); 23, Lee et al. (2002); 24,
Miyamura and Nagumo (2007); 25, Miyamura and Hori (1997); 26, Friedmann (1960); 27, Suda et al. (2004); 28, Sun et al. (1988); 29, Ogawa (1982); 30, Fritsch (1956); 31, Kurssanow
(1912); 32, Lewis (1925); 33, Korn (1969); 34, Lippert (1967); 35, Ogawa and Hirota (2000); 36, Smith (1950); 37, Biebel (1973)
J Plant Res (2010) 123:171–184
J Plant Res (2010) 123:171–184 175

Table 2 Inheritance patterns and the fates of mitochondria and their DNA in green algae
Taxon Species Types of Mode of Types of Fate of Fate of Methods of Sourcesg
sexual inheritancea evidenceb mitochondrial mitochondriad analysise
reproduction DNAc
Sex/stage/fate Sex/stage/fate

Chlorophyta
Chlorophyceae
Volvocales Chlamydomonas Isogamy U (mt-) G mt?/ mt?, mt-/ RFLP/PCR– 1, 2
reinhardtii meiosis/– meiosis/ RFLP/FM
fusion
C. eugametos9C. Isogamy U (mt?) G ND ND RFLP 3
moewusii
Gonium pectorale Isogamy U (mt-) G ND ND PCR–RFLP 4
Volvox carteri Oogamy M G ND ND RFLP 5
Ulvophyceae
Ulvales Ulva compressa Isogamy U (mt?)/U G ND ND PCR–RFLP 6, 7
cross 1f (mt-)/B
Ulva compressa Isogamy U (mt?) G ND ND PCR–RFLP 6, 7
cross 2f
Bryopsidales Bryopsis maxima Anisogamy M C Male/ ND/zygote/ FM/CsCl/TEM 8, 9, 10
gamete/– disintegration
Derbesia tenuissima Anisogamy M C Male/ ND FM/IEM 11
gamete/–
Streptophyta
Charales Chara corallina Oogamy M C Male/ ND FM/IFM 12
gamete/–
a
M, maternal inheritance; U (mt?), uniparental inheritance from mt?; U (mt-), uniparental inheritance from mt-; B, biparental inheritance
b
G, genetic evidence; C, cytological evidence
c
ND, not determined; – disappearance of mitochondrial DNA
d
fusion, fusion of mitochondria; disintegration, disintegration of mitochondria
e
FM, fluorescence microscopy; TEM, transmission electron microscopy; RFLP, restriction fragment length polymorphism; CsCl, CsCl density
gradient centrifugation; IEM, immunoelectron microscopy; IFM, immunofluorescence microscopy
f
Cross 1 indicates the results of MGEC1 9 MGEC6, and cross 2 indicates the results of MGEC2 9 MGEC5
g
1, Boynton et al. (1987); 2, Aoyama et al. (2006); 3, Lee et al. (1990); 4, Hamaji et al. (2008); 5, Adams et al. (1990); 6, Kawano (2008); 7,
Mogi et al. (2009); 8, Kuroiwa and Hori (1986); 9, Kuroiwa and Hori (1990); 10, Kuroiwa et al. (1991); 11, Lee et al. (2002); 12, Sun et al.
(1988)

prasinophytes. The UTC clade is thought to have arisen
from distinct types of unicellular flagellates related to the
modern prasinophytes (Graham and Wilcox 2000).
Chlorophyceae
Chlorophyceaens are unicellular and multicellular green
algae that primarily occupy freshwater habitats (Graham
and Wilcox 2000). Sexual reproduction is widespread in
this class; cytoplasmic inheritance has been studied in
Chlamydomonas and other members of Volvocales.
Because the cytoplasmic inheritance in C. reinhardtii is
reviewed in other articles in this issue, it is only described
briefly here. During the fertilization of C. reinhardtii,
which involves the fusion of two isogametes of opposite
mating types (mt?, mt-), the chloroplast DNA of the mt?
gamete is transmitted preferentially to the next generation
(Sager 1954), while that of the mt- gamete is actively
digested by Ca2?-dependent nuclease within 30–40 min
after mating (Kuroiwa et al. 1982; Nishimura et al. 1999;
Nishimura et al. 2002). Finally, the two chloroplasts con-
tributed by the two gametes fuse to form a single chloro-
plast (Cavalier-Smith 1970). In contrast, the mitochondrial
genome is inherited from mt- gametes (Boynton et al.
1987) as a result of the selective digestion of mitochondrial
DNA from the mt? gamete during meiosis (Aoyama et al.
2006). It is particularly interesting that this inheritance
pattern is identical to that of the isogamous colonial species
Gonium pectorale, which is the closest relative of C. rein-
hardtii (Hamaji et al. 2008), but the pattern differs from
those of the distantly related species pair C. eugame-
tos 9 C. moewusii, in which both plastid and mitochon-
drial genome markers are transmitted uniparentally from
mt? parents to the zygotes (Lee et al. 1990). By contrast, in

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the oogamous colonial green alga Volvox carteri f. na-
gariensis, RFLP analysis of the progenies of crosses
between two geographically distant strains indicates that
both plastid and mitochondrial genomes are transmitted
maternally to the progeny (Adams et al. 1990). Coleman
and Maguire (1982) and Kuroiwa et al. (1993a) investi-
gated the fate of plastid DNA during spermatogenesis and
fertilization of V. carteri using fluorescence microscopy
with DAPI (40 ,6-diamidino-2-phenylindole) staining. Ku-
roiwa et al. (1993a) showed that during spermatogenesis of
V. carteri f. kawasakiensis, the amount of chloroplast DNA
in each chloroplast is reduced stepwise by each cell divi-
sion; eventually almost all sperm contain up to three plastid
nucleoids, while the mature egg cells in the female colonies
have many dispersed plastid nucleoids in a large chloro-
plast. After plasmogamy, sperm chloroplast nucleoids
cannot be recognized in the egg cytoplasm (Kuroiwa et al.
1993a).
Ulvophyceae
Ulvophyceaen algae primarily occupy marine waters. They
display unicellular, multicellular or coenocytic organiza-
tion during their life cycles (Graham and Wilcox 2000).
Biflagellate isogametes and various degrees of anisoga-
metes are widespread in this class. The inheritance patterns
of the plastid and mitochondrial genomes have been clar-
ified in the isogamous Ulva compressa (Ulvales) using
microsatellite markers (Kagami et al. 2008; Kawano 2008).
They used two genetic lines [MGEC-1 (mt?) and MGEC-2
(mt-), MGEC-5 (mt?) and MGEC-6 (mt-)] isolated from
different coasts of Japan for crossing experiments. Using
primers designed for the psbF-psbL and petD-accD inter-
genic regions of the plastid genome, PCR–RFLP analyses
of MGEC-1 9 MGEC-6 sporophytes and MGEC-
2 9 MGEC-5 sporophytes indicate that the plastid gene is
inherited only from the mt? parent in all crosses. It is
particularly interesting that PCR–RFLP analyses of the
mitochondrial genome using microsatellite markers
exhibited different inheritance patterns from those of
chloroplast genomes (Kawano 2008; Mogi et al. 2009). The
MGEC-2 (mt-) 9 MGEC-5 (mt?) sporophytes showed
uniparental inheritance only from mt? gametes, although
MGEC-1 (mt?) 9 MGEC-6 (mt-) sporophytes exhibited
three inheritance patterns of mitochondrial genomes: uni-
parental inheritance from mt? gametes, uniparental inher-
itance from mt- gametes, and biparental inheritance
(Kawano 2008; Mogi et al. 2009). This pattern is appar-
ently an exception to the tenets of strict uniparental
inheritance, but it is consistent with the mounting evidence
indicating the presence of occasional paternal and bipa-
rental inheritance (Birky 1995; Xu 2005; White et al.
2008). Furthermore, recently, the presence of uniparental
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inheritance from mt- and biparental inheritance of chlo-
roplast genomes has been revealed by 12 interline crosses
(Mogi et al. 2009), indicating that this phenomenon is not
restricted to mitochondrial genomes. Kagami et al. (2008)
examined the fate of chloroplast DNA in the zygotes of U.
compressa using fluorescence microscopy after staining
with DAPI. The disappearance of chloroplast DNA derived
from mt- gametes occurred in only 6% of the spherical
zygotes (MGEC-1 9 MGEC-2) 3–4 h after mating and 0%
in the zygote (MGEC-1 9 MGEC-6) 6 h after mating,
which differs from other isogamous species, although we
cannot exclude the possibility that the disappearance of
chloroplast DNA occurs until the first cell division in U.
compressa. In Acetabularia calyculus (Dasycladales),
Dictyosphaeria cavernosa (Siphonocladales) (Kuroiwa
et al. 1985), and Monostroma latissimum (Ulotrichales)
(Kuroiwa et al. 1993b), the chloroplast DNA from one
parent was preferentially destroyed in the zygote soon after
the fertilization of isogametes in the same way as that of C.
reinhardtii (Kuroiwa et al. 1982). However, the mating
type that eliminated its chloroplast DNA has not been
confirmed using genetic markers in these ulvophycean
algae. The mating type of the inherited chloroplast has
been clarified in an isogamous species, U. mutabilis, using
cross experiments of UV-irradiated gametes, transmission
electron microscopy, and autoradiography of thin sections
(Fjeld 1971; Bråten 1971, 1973). They indicated that the
chloroplast originating from the mt? gamete disintegrated
rapidly soon after fertilization, while the chloroplast from
the mt- gamete was retained in the zygote, suggesting the
uniparental inheritance of the chloroplast genome from the
mt- gamete in U. mutabilis.
In contrast, the preferential disappearance of organelle
DNA occurs during male gametogenesis in anisogamous
species belonging to Bryopsidales, such as the siphonous
green algae, Bryopsis maxima (Kuroiwa and Hori 1986;
Kuroiwa et al. 1991), B. plumosa (Ogawa 1988), Derbesia
tenuissima (Lee et al. 2002), and Caulerpa (Miyamura and
Hori 1997; Miyamura and Nagumo 2007). Kuroiwa et al.
(1991) studied details of this process in B. maxima, and
showed that the size of the chloroplast and the number of
nucleoids per chloroplast in male and female gametangia
decreased markedly during gametogenesis as a result of
continuous chloroplast division; then the chloroplast DNA
disappeared in the male gametangia. By contrast, the
mitochondrial DNA in almost all male gametes disap-
peared subsequent to the disappearance of chloroplast
DNA, but those in a few gametes remained, while several
mitochondrial nucleoids were observed in all the female
gametes. These observations were supported by biochem-
ical analysis using CsCl density gradient centrifugation of
DNA extracted from male and female gametophytes and
gametes (Kuroiwa et al. 1991). They suggested that the
J Plant Res (2010) 123:171–184
chloroplast and mitochondrial DNA is preferentially
digested during male gametogenesis while the mitochon-
drial DNA in some male gametes and in all of the female
gametes remains (Fig. 1). Similar phenomena have been
reported in Caulerpa, for which the frequency of male
gametes that have plastid nucleoids varies from 0.6 to
18.2% among different species, and for which both male
and female gametes always contain 1–8 mitochondrial
nucleoids (Miyamura and Hori 1997; Miyamura and
Nagumo 2007). Furthermore, Kuroiwa and Hori (1990)
clarified the fate of the male chloroplast and mitochondria
in the zygotes of B. maxima using transmission electron
microscopy and histochemical methods that detect acid
phosphatase activity as a lysosome marker. They found that
some mitochondria were fused with the lysosome 4 h after
fertilization and were digested by 9.5 h. The origin of
digested mitochondria has not been determined because it
is extremely difficult to distinguish male from female
mitochondria based on their ultrastructure. On the other
hand, the small male chloroplast was fused with lysosome
9.5 h after fertilization and digested until 24–48 h, whereas
the large female chloroplast remained intact. These results
suggest that the lysosome plays a critical role in the
selective digestion of organelles in the zygote, and the
mechanisms of uniparental inheritance probably differ
between plastid and mitochondria because the selective
elimination of these organelles and their DNA occur at
different periods.
Trebouxiophyceae
Trebouxiophyceaen algae are freshwater and terrestrial
algae. Friedmann (1960), using light microscopy, exam-
ined the fertilization of the leaf-like green alga, Prasiola
stipitata. This alga produces motile biflagellate male
gametes and nonmotile larger female gametes. The male
chloroplast, which is definitely smaller than the female one,
was selectively destroyed after gamete fusion, which took
about 2–3 days under culture conditions. The female
chloroplast, on the other hand, increased in size in the
zygote and was subsequently transmitted to the progeny.
Prasinophytes
Prasinophytes retain characteristics that are believed to
have been present in the last common ancestor of green
algae (Lewis and McCourt 2004). Sex has been reported in
only one species (Nephroselmis olivacea), individuals of
which produce biflagellate isogametes (Suda et al. 1989). A
sign of uniparental inheritance of chloroplasts was found in
transmission electron micrographs of a two-day-old zygote,
in which the swelling of the thylakoid membranes occurred
in one of the two chloroplasts brought into a zygote through
177
gamete fusion, suggesting the selective degeneration of
chloroplast (Fig. 22 in Suda et al. 2004). These observations
suggest the possibility that uniparental inheritance of the
chloroplast genome occurs in N. olivacea, although the fate
of the degenerating chloroplast has not been clarified.
Streptophyte green algae
Cytoplasmic inheritance has only been examined in Zyg-
nematales and Charales. Zygnemataleans are characterized
by conjugation as their method of sexual reproduction,
which usually involves the fusion of two non-swimming
isogametes that are brought together by a conjugating tube
that forms between two vegetative cells. Light microscopic
studies in the filamentous algae Spirogyra neglecta (Fritsch
1956) and Zygnema stellinum (Kurssanow 1912) and the
desmids Closterium ehrenbergii (Lippert 1967) and Cos-
marium turpinii (Korn 1969) have revealed that the chlo-
roplasts derived from the gamete of one mating type are
destroyed in the zygote shortly after gamete fusion, sug-
gesting the predominantly uniparental inheritance of plas-
tid genomes. Details of this process have been examined
using transmission electron microscopy in S. verruculosa
(Ogawa 1982) and C. ehrenbergii (Ogawa and Hirota
2000). In both species, half the number of chloroplasts
exhibit disorganization of thylakoid membranes and
become fragmented and engulfed in the vacuolar structure,
where they are eventually destroyed. Early light micro-
scopic studies of Spirogyra (Fritsch 1956) indicated that
the disintegrating chloroplasts originated from the gamete
(active gamete) that passes through the conjugation tube
and unites with a gamete of opposite mating type (passive
gamete), but this has not been confirmed using electron
microscopy. It is particularly interesting that the male
gamete of the anisogamous species Temnogyra collinsii has
these characteristics while its chloroplast is disintegrated
selectively before conjugation (Lewis 1925).
The isogamous unicellular alga Cylindrocystis brebis-
sonii displayed another mechanism of uniparental inheri-
tance of the chloroplast genome. The zygote contained four
chloroplasts: two chloroplasts from each mating type. The
four cell products of meiosis received one chloroplast from
only one mating type or the opposite mating type of gamete
(Smith 1950; Biebel 1973). This inheritance mode of
plastid closely resembles that of the brown alga Ectocarpus
siliculosus, in which male and female plastids persisted
after fertilization and segregated into the two halves of the
sporophyte during the first cell division of the zygote
(Peters et al. 2004).
Charales is characterized by multicellular organization
and oogamous sexual reproduction. Fertilization is achieved
by the entry of biflagellate sperm into the oogonium. The
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fate of plastid and mitochondrial nucleoids during sperm
formation has been examined in stonewort, Chara corallina
(Charales), using fluorescence microscopy with DAPI
staining and immunofluorescence microscopy using anti-
DNA antibody (Sun et al. 1988). The plastid and mito-
chondrial nucleoids, which were present in vegetative cells
and cells at the early spermatid stage, disappeared at the late
stage of spermatogenesis, suggesting that these organelle
DNA might be digested during sperm formation. Similar
events have been reported in land plants, angiosperms
(Miyamura et al. 1987; Corriveau and Coleman 1988;
Nagata et al. 1999), ferns (Kuroiwa et al. 1988), and
hornwort (Izumi and Ono 1999) using the same cytological
methods in combination with microfluorometry. This is an
interesting similarity because Charales is a candidate for a
sister to land plants (Becker and Marin 2009). Therefore, it
seems likely that the mechanisms of maternal inheritance
employed in Charales became the prototype for those used
in land plants.
Digestion of organelle DNA and disintegration
of organelles as a major mechanism of uniparental
inheritance in green algae
Green algae exhibit various mechanisms of uniparental
inheritance of plastid and mitochondrial genomes: degra-
dation of organelle DNA during male gametogenesis or
after fertilization, disintegration of organelles, and segre-
gation of organelles into meiotic products (Tables 1, 2).
Fig. 2 Schematic drawing gametes
showing the mechanisms of
uniparental inheritance of a isogamy
plastid genomes in isogamous
(a) and anisogamous/oogamous
green algae (b). N nucleus N N
mt+ X mt-
or mt-
chloroplast
chloroplast nucleoid
b anisogamy/oogamy
N N
X
digestion of
chloroplast DNA
123
J Plant Res (2010) 123:171–184
Most of these mechanisms have been reported in other
eukaryotes (Sears 1980; Whatley 1982; Kuroiwa 1991;
Birky 1995, 2008; Mogensen 1996; Xu 2005). However,
concerning plastid inheritance, at least two major mecha-
nisms are apparently widespread in green algae: (1)
digestion of plastid DNA after fertilization (Fig. 2a), dur-
ing fertilization or male gametogenesis (Fig. 2b), and (2)
disintegration (Fig. 2a1, b1) or fusion (Fig. 2a2) of the
plastids in the zygote. The first mechanism also eliminates
the mitochondrial DNA in anisogamous and oogamous
species. The essence of these mechanisms has been pro-
posed by Kuroiwa (1991); the results of the survey pre-
sented in this study (Tables 1, 2) support his proposal. The
first mechanism is mainly deduced from the data using
fluorescence microscopy with DAPI staining, so that it is
difficult to deny the possibility that the dispersal of DNA
molecules, presumably caused by the digestion or relaxa-
tion of the proteins that form the nucleoids, engenders the
disappearance of DNA fluorescence. However, the accu-
racy of the data has been confirmed in C. reinhardtii
(Nishimura et al. 1999), Volvox carteri (Adams et al.
1990), B. maxima (Kuroiwa et al. 1991), and D. tenuissima
(Lee et al. 2002) using molecular or biochemical methods
or immunoelectron microscopy. Consequently, the disap-
pearance of DNA fluorescence is suggested to be a result of
the digestion of DNA molecules by nuclease. The nuclease
involved in this process has been characterized as Ca2?-
dependent nuclease in C. reinhardtii (Nishimura et al.
2002). However, the nature of the nucleases and the control
mechanisms remains to be clarified in most species,
zygotes
a1
N N
?
N N N N disintegration
of chloroplast
or mt+ a2
N N
digestion of
chloroplast DNA
fusion of
chloroplasts
flagellum
b1
N N N N
disintegration
of chloroplast
J Plant Res (2010) 123:171–184
although Ca2?-dependent nuclease activity has been iden-
tified in whole plants of a few green algae and land plants
(Nakamura et al. 1987; Sugiyama et al. 2000). In anisog-
amous and oogamous species, the disappearance of plastid
DNA is achieved by two steps: (1) a gradual decrease in the
number of plastid nucleoids or the DNA content per plastid
during male gametogenesis, and (2) the digestion of plastid
DNA during male gametogenesis or during fertilization
(Fig. 2b; Kuroiwa 1991). One interesting point is that the
timing of plastid DNA disappearance differs slightly
among species belonging to different lineages. For exam-
ple, digestion of plastid DNA occurs during gametogenesis
in Bryopsidales (Bryopsis, Derbesia, and Caulerpa) and
Charales (Chara), although it occurs during fertilization in
Volvocales (Volvox). Such differences may be the result of
the independent evolution of anisogamy and oogamy in
each lineage. Selective digestion of uniparental plastid and
mitochondrial DNA has also been reported in other
eukaryotes, including land plants (Miyamura et al. 1987;
Nagata et al. 1999), slime molds (Moriyama and Kawano
2003) and animals (Nishimura et al. 2006), and might be a
universal mechanism for uniparental inheritance of orga-
nelle genomes (Kuroiwa 1991; Nishimura et al. 2002).
In contrast to plastid fusion in the zygote (Fig. 2a2),
which was found only in isogamous species in the UTC
clade of Chlorophyta, such as C. reinhardtii, G. pectorale,
and Acetabularia (Whatley 1982), disintegrating plastids
and signs of disintegration were observed in a wide range
of green algae (Table 1), and always occurred after fertil-
ization irrespective of the type of sexual reproduction
(Fig. 2a1, b1). One notable point is that a sign of plastid
disintegration was found in the prasinophyte N. olivacea, in
addition to other members of Chlorophyta and Strepto-
phyta. Prasinophytes are believed to be ancestors of
Chlorophyta and Streptophyta, which suggests the possi-
bility that the selective disintegration of the plastid is a
fundamental mechanism of uniparental inheritance in green
algae. Another interesting point is that plasmid disinte-
gration was achieved in two steps in S. verruculosa,
C. ehrenbergii, and B. maxima: (1) disorganization or
swelling of thylakoid membranes; (2) subsequent digestion
of the plastid by a vacuolar structure (S. verruculosa,
C. ehrenbergii) or lysosomes (B. maxima). Although the
second step has been observed only in these species, it can
be presumed that this mechanism is prevalent in other
green algae because the swelling of thylakoid membranes
or disintegration of chloroplasts has been observed in many
green algae (Table 1). The selective disintegration of male
mitochondria has been reported in B. maxima (Table 2;
Kuroiwa and Hori 1990) and in other eukaryotes. For
example, sperm mitochondria are destroyed after fertil-
ization in ferns (Myles 1978) and mammals (Sutovsky
et al. 2000). In mammals, this process involves
179
ubiquitination of sperm mitochondria outer membranes,
with subsequent recognition and proteolysis (Sutovsky
et al. 1999, 2000). Similar sex-specific recognition and
subsequent digestion of organelles presumably takes place
in the zygote of green algae, but it remains unknown
whether ubiquitination and other mechanisms are involved
in the selective disintegration of plastids and mitochondria
in the zygote. Nevertheless, disintegration of the organelles
and their DNA would ensure the predominantly uniparental
inheritance of organelle genomes, although this system
might not be perfect and would probably allow paternal
leakage under some conditions.
Universal sex-specific features unravel the relations
between cytoplasmic inheritance and sex types in
isogamous, anisogamous, and oogamous green algae
Maternal uniparental inheritance is predominant in
anisogamous and oogamous green algae. It probably
evolved from uniparental inheritance in ancestral isoga-
mous species. To trace the evolutional history of unipa-
rental inheritance, it would be necessary to relate the sex
types of anisogamous and oogamous species to mating
types of isogamous ancestors. This task is now becoming
possible using sex-specific features (e.g., MID, opposite
positioning of cell fusion sites and GCS1) that are probably
universal in a wide range of green algae (Table 3; Ferris
and Goodenough 1994; Miyamura and Nagumo 2007;
Mori et al. 2006).
The MID gene is unique to the mating type minus locus
of C. reinhardtii (Ferris and Goodenough 1994; Ferris et al.
2002). Cells expressing an MID gene differentiate as mt-,
while deletion of MID prevents cells from differentiating as
mt- (Fig. 3; Goodenough et al. 2007). Recently, Nozaki
et al. (2006) and Hamaji et al. (2008) identified MID
orthologs in the volvocalean algae Pleodorina starii and G.
pectorale. The MID ortholog is located only in the mating
type locus of mt- in G. pectorale, and in males in P. starii.
Furthermore, Hamaji et al. (2008) described the presence
of MID orthologs in male strains of V. carteri in the
‘‘Discussion’’ section of their paper. They suggested that
males of oogamous species (P. starii and V. carteri)
evolved from mt- of isogamous species (C. reinhardtii and
G. pectorale) (Nozaki et al. 2006; Hamaji et al. 2008).
As a result of their findings (Nozaki et al. 2006; Hamaji
et al. 2008), it became apparent that the plastid genomes
are always inherited uniparentally from the same mating
types, mt? and female (MID-), by the progeny of these
volvocalean algae (Table 3). On the other hand, the mito-
chondrial genomes were transmitted from mt- (MID?) in
C. reinhardtii and G. pectorale, while they were inherited
from female gametes (MID-) in V. carteri (Table 3).
123
 180

123
Table 3 Relationship between the transmission patterns of organelle genomes and the sex-specific features in green plants
Taxon Species Sex-specific featuresa Mode of inheritanceb Origin of organelle genomes Sourcesd
Male/mt- Female/mt? Plastids Mitochondria Plastids Mitochondria

Chlorophyta
Chlorophyceae Chlamydomonas Type b/MID?/ Type a/MID-/ U (mt?) U (mt-) Type a/MID-/ Type b/MID?/GCS1? 1, 2, 3, 4, 5, 6
reinhardtii GCS1? GCS1- GCS1-
Gonium pectorale MID? MID- U (mt?) U (mt-) MID- MID? 7
Volvox carteri MID? MID- M M MID- MID- 7, 8
Ulvophyceae Ulva compressa cross1c Type a Type b U (mt?) U (mt?)/U (mt-)/ Type b Type a/Type b/Type a, b 9, 10, 11, 12
B
Ulva compressa cross2c Type a Type b U (mt?) U (mt?) Type b Type b 9, 10, 11, 12
Bryopsis maxima Type a Type b M M Type b Type b 13, 14
Caulerpa brachypus Type a Type b M ND Type b ND 15
C. okamurae Type a Type b M ND Type b ND 15, 16
C. racemosa var. Type a Type b M ND Type b ND 15
laetevirens
C. serrulata var. Type a Type b M ND Type b ND 15
serrulata f. lata
Streptophyta
Angiosperms Lilium longiflorum GCS1? GCS1- M M GCS1- GCS1- 17, 18
Arabidopsis thaliana GCS1? GCS1- M M GCS1- GCS1- 17, 18, 19
a
a and b types in opposite positioning of cell fusion sites, as shown in Fig. 3; MID?, presence of MID or MID homolog; MID-, absence of MID or MID homolog; GCS1?, expression of
GCS1 in the gametes; GCS1-, absence of expression of GCS1 in the gametes
b
U (mt?), uniparental inheritance from mt? parents; U (mt-), uniparental inheritance from mt- parents; M, maternal inheritance from female parents; B, biparental inheritance
c
Cross 1 indicates the results of MGEC1 9 MGEC6, and cross 2 indicates the results of MGEC2 9 MGEC5
d
1, Sager (1954); 2, Boynton et al. (1987); 3, Holmes and Dutcher (1989); 4, Ferris and Goodenough (1994); 5, Mori et al. (2006); 6, Liu et al. (2008); 7, Hamaji et al. (2008); 8, Adams et al.
(1990); 9, Miyamura (2003); 10, Kagami et al. (2008); 11, Mogi et al. (2008); 12, Kawano (2008); 13, Kuroiwa et al. (1991); 14, Miyamura et al. (2005); 15, Miyamura and Nagumo (2007); 16,
Miyamura and Hori (1997); 17, Mori et al. (2006); 18, Miyamura et al. (1987); 19, Kirk and Tilney-Bassett (1978)
J Plant Res (2010) 123:171–184
J Plant Res (2010) 123:171–184
mt+ /Type α/MID-/GCS1- mt- /Type β/MID+/GCS1+
mating structure
a no. 1 flagellum no. 2 flagellum no. 1 no. 2
eyespot eyespot
mating
structures
microtubular
b 1s 2d roots
1s 2d
no. 1 no. 2
no. 1 no. 2
2s 1d 2s
1d
eyespot mating structure eyespot
Fig. 3 Schematic diagram showing the relation between the opposite
positioning of mating structures/cell fusion sites, MID and GCS1 in
the gametes of Chlamydomonas reinhardtii. a Three-dimensional
illustration of mt? and mt- gametes showing the arrangement of
mating structures, eyespots, and flagella. b Spatial relation of mating
structures, eyespots and microtubular roots, as viewed from the cell
anterior. The mating structure of the mt- gamete is located at the cell
apex on the same side of the flagellar basal bodies as the eyespot (type
b), whereas the mt? structure is located in the opposite position (type
a) (Holmes and Dutcher 1989, 1992). The numbering of the flagella
(nos. 1 and 2) conforms to the general numbering system for green
algae (e.g., Melkonian 1989; Moestrup and Hori 1989).?, Presence of
MID or expression of GCS1; -, absence of MID or expression of
GCS1; 1d, 1d root; 1s, 1s root; 2d, 2d root; 2s, 2s root
Hamaji et al. (2008) suggested that the patterns of mito-
chondrial inheritance changed at some point during the
evolution of colonial Volvocales from uniparentally from
the MID-containing parent (MID?) to uniparentally from
the female (MID-).
Unfortunately, no MID ortholog has been identified and
characterized in other green algae, although probable MID
or Mid-like proteins have been found to be encoded in the
genomes of the unicellular green algae Micromonas and
Ostreococcus (prasinophyte) (Worden et al. 2009). In
Chlorophyta, instead of MID, opposite positioning of the
cell fusion site/mating structure (cell fusion apparatus) is
useful for the identification of sex types (Fig. 3; Miyamura
and Nagumo 2007). This feature is a kind of sexual
dimorphism that is probably universal among Chlorophyta
(Holmes and Dutcher 1989; Nakayama and Inouye 2000,
Suda et al. 2004; Miyamura and Nagumo 2007, among
others). In C. reinhardtii, the mt- gamete mating structure
is located at the cell apex on the same side of the flagellar
basal bodies as the eyespot, whereas the mt? structure is
located in the opposite position (Fig. 3a; Holmes and
Dutcher 1989). The microtubular roots that extend from the
181
basal bodies presumably define both positions (Fig. 3b;
Goodenough and Weiss 1978). The former type is desig-
nated the ‘‘b’’ type; the latter type is ‘‘a’’ (Miyamura and
Nagumo 2007). In Ulvophyceae, the mt- and mt? gametes
of U. compressa and the male and female gametes of
B. maxima and Caulerpa are classified, respectively, as
types a and b. As a result, it became apparent that the
plastid and mitochondrial genomes were usually inherited
from the same sex type, type b gametes, in these ulvo-
phycean algae (Table 3). However, the organelle genomes
were inherited from type a, type b, and both types in some
combinations of crosses in U. compressa (Kawano 2008;
Mogi et al. 2009), suggesting that the relation between
cytoplasmic inheritance and sex type is complicated.
In contrast to these ulvophycean algae, plastid and
mitochondrial genomes are transmitted from type a (mt?)
and type b (mt-) gametes to the next generation in
C. reinhardtii, respectively (Table 3). This pattern is not
consistent with that of ulvophycean algae. Why this
inconsistency occurs between these distantly related algal
species remains unknown. One way to address this ques-
tion is to find the MID ortholog in ulvophycean algae, to
analyze the arrangement of cell fusion sites/mating struc-
tures in volvocalean algae, or to use the other sex-specific
features universal in green algae. The sex-specific
expression of GCS1 (HAP2) is one candidate for such a
sex-specific feature. In fact, GCS1 is a key protein for
gamete fusion in fertilization that was first identified in
Lilium longiflorum pollen using differential display anal-
ysis. It plays an important role in cell fusion in angio-
sperms (Mori et al. 2006), C. reinhardtii (Liu et al. 2008),
and Plasmodium (Hirai et al. 2008; Liu et al. 2008). It is
particularly interesting that the expression of GCS1 is
specific to male gametes in angiosperms and Plasmodium
and mt- in C. reinhardtii (Mori et al. 2006; Hirai et al.
2008; Liu et al. 2008). These sex types have the common
feature of abandoning the attempt to transmit plastid
genomes to the next generation in the same way as MID?
in Volvocales and type a in ulvophycean algae (Kirk and
Tilney-Bassett 1978; Creasey et al. 1994; Sager 1954).
Genome surveys have revealed homologs in red alga,
slime molds, ciliates, choanoflagellates, and cnidarians,
suggesting that GCS1 is a highly conserved protein for
gamete fusion (Mori et al. 2006; Hirai et al. 2008; Liu
et al. 2008). Therefore, it is expected that GCS1 homologs
are present in ulvophyceans and other green algae, and
that they can be used as probes for the identification of sex
types.
At present, information related to these sex-specific
features in green algae remains limited. However, as this
information becomes available, it will be possible to better
understand the evolutionary history of cytoplasmic inheri-
tance and sex types in green algae.
123
182
Acknowledgments The author is greatly indebted to Prof. Tsu-
neyoshi Kuroiwa for the opportunity to write this article. I also thank
Prof. Shigeyuki Kawano and Yuko Mogi for information about
cytoplasmic inheritance in Ulva compressa. This work was partly
supported by a KAKENHI Grant-in-Aid for Scientific Research (B)
(No. 20370025).
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