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Sexual reproduction in gymnosperms: an

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Functional Diversity of Plant Reproduction, 2009: 1-16 ISBN: 978-81-308-0360-9

Editors: Alicia Gamboa-deBuen, Alma Orozco-Segovia and Felipe Cruz-Garcia

1. Sexual reproduction in gymnosperms:

An overview
Alejandra Vázquez-Lobo
Instituto de Ecología, Universidad Nacional Autónoma de México, Apartado Postal 70-275
Ciudad Universitaria, Coyoacán, México DF, 04510, Mexico

Abstract. Extant gymnosperms comprise four distinct lineages of

seed plants with prevalent sexual reproduction. Characters and
processes involved in sexual reproduction of gymnosperms are
described in this chapter in a comparative approach. Phylogenetic
evidence suggests monophyly for this group and some characters
involved in sexual reproduction are shared between taxa, although
remarkable exceptions are always found. Breeding systems varies
between basal and derived taxa, prevailing dioecy in Ginkgoales
and Cycadales, monoecy in conifers and unfunctional gynodioecy
in Gnetales; in any case outcrossing mating systems are promoted.
These plants have long life spans and long reproductive cycles
characterized by a delayed gametogenesis. Male reproductive
structures are simple and morphological patterns are similar between
groups with exceptions in Gnetales and Taxaceae, whereas female
structures could be simple or compound showing an astounding
diversity. Gametogenesis is a slow process that usually ends until
pollination. Female gametogenesis consists of a coenocytical
megagametophyte development, followed by cellularization and
differentiation of two or more archegonia. Exceptions to this pattern

Correspondence/Reprint request: Dr. Alejandra Vázquez- Lobo, Universidad Nacional Autónoma de México
Instituto de Ecología, 3er circuito exterior, Ciudad Universitaria, Coyoacán, México DF, 04510, México
E-mail: eva@miranda.ecologia.unam.mx
2 Alejandra Vázquez-Lobo

are found in Gnetales and Cupressaceae, where unique processes inside seed plants
are found. Male gametogenesis involves meiosis to form tetrads of microspores and
distinct number of subsequent meiosis before sperm differentiation. Sperm could be
free nuclei or celled with or without mobility. Anemophilous pollination is common,
but pollen transportation by insects is present in two lineages. Pollination mechanisms
involve secretion of pollination drop and distinct pollen morphologies. Fertilization is
predominantly siphonogamous but in basal gymnosperms, pollen tube development is
haustorial. Embryogenesis usually begins with acellular stages and the number of
mitosis before cellularization varies between groups. Polyembryony is feasible to
occur by two distinct processes but only one embryo reaches maturity. Modest
knowledge has been achieved relative to genetic basis of processes related with sexual
reproduction and morphological diversification of reproductive structures in
gymnosperms, warranting future research in these areas.

Introduction
All extant non-flowering seed plants are comprised in a group without
taxonomic rank: the gymnosperms. This group includes four divergent
lineages –Ginkgoales, Cycadales, Gnetales and Coniferales orders- with
morphologies highly similar to the fossil record from Mesozoic and a
worldwide distribution. Sexual reproduction is characteristic for this group,
since a few cases of asexual reproduction have been documented. Opposite to
angiosperms, reproductive structures of gymnosperms are typically unisexual,
although occurrence of bisporangiated cones have been reported for gnetales
and some conifer species. Reproductive units are called cones due to its conic
shape or strobilla; the female is the ovulated (seed) cone or the megasporangiated
strobile, while the male is the pollen cone or microsporangiated strobile. Cones
are considered simple when the structures that bear gametes are directly attached
to the central axe of the cone, or compound when the gametes bearing structures
borne at the axil of a bract, making up clustered complexes.
The term gymnosperm –naked seed- was assigned to this group because
the seed is not covered by a fruit as in angiosperms, although in some
lineages the seed is not properly naked, instead is covered by distinct
structures with apparent homology. Different molecular phylogenies support
the monophyly of this group, although due to deep divergence times and
lineage extinctions, taxa included in gymnosperms are highly differentiated
morphologically and the phylogenetic relationships between the four orders
are unstable. In the past, misinterpretation of morphological characters lead
to relate Gnetales with angiosperms and recently some molecular data place
Gnetales as sister group of all seed plants, challenging the monophyly of
gymnosperms [1, 2]. Even though, the majority of molecular evidence
supports gymnosperms as a natural group [3, 4]. However, lack of agreement
Sexual reproduction in gymnosperms 3

in relationships between the four basal branches difficulties to reconstruct

evolutionary history of morphological characters and processes involved in
sexual reproduction. Functionally unisexual reproductive structures,
unitegumented ovules, presence of three cell generations in seed, and
anemophilous pollination syndromes are common features for the group,
although exceptions to these patterns will be described in this chapter.

Life cycle

An overview of Pinus (Pinaceae) life cycle, illustrates the reproductive

process in gymnosperms, though variations to these pattern are found in
different species. Adult trees commonly bear both pollen and ovulates cones
(dioecius) and the age for first reproduction ranges from 6 to 20 years. Male
cones carry microsporophylls enclosing the microsporangium. This diploid
tissue originates pollen (microspores) through meiosis of diploid
microsporocytes (pollen mother cells). After a series of unequal cell divisions
the shed pollen contains four haploid cells (1n), including the generative cell
that in turn, originates sperm through mitosis. Whereas in female cones,
ovules develop adaxially to ovuliferous scales, each with only one
integument enclosing a megasporangium or nucellus (2n), which in turn
contains a megasporocyte or megaspore mother cell that undergoes meiosis
to produce four megaspores, of which three degenerate. The functional
megaspore divides into a multicellular megagametophyte (1n) containing two
or three archegonia each with one egg. Ovules produce a pollination drop
trough a small opening of integument (micropyle), where pollen arrives.
Pollen grains germinate developing a pollination tube that penetrates both
nucellus and archegonium to release its gametes in to the egg. Polyembryony
is feasible to occur due to embryo multiplication or multiple archegonia
fertilization, although, only one embryo will develop until maturity. Embryo
develops emerging from the seed as a seedling with multiple cotyledons [5].
Dioecious breeding systems without sexual dimorphism are common in
“basal” gymnosperms –Ginkgoales, Cycadales and Gnetales-, since male and
female structures borne in separate trees that are indistinguishable till they bear
such distinct reproductive structures. It seems that in Ginkgo (Ginkgoaceae) some
chromosomal differences are associated with sex determination, although
cultivated trees occasionally are monoecious [6]. In Gnetales abortive ovules
often are found in male cones therefore these plants are referred as gynodioecious
but, because ovules in hermaphroditic structures are unfunctional, also could be
referred to as dioecious plants [7]. Opposite, inside conifers monoecious systems
are common, with exception of Taxaceae family where exclusively dioecious
populations are found [8]. Inside Pinaceae, Araucariaceae and Podocarpace
4 Alejandra Vázquez-Lobo

families, some species have dioecious breeding systems. This phenomenon seems
to be related with ambiental factors more than with a genetic determination of
sex, because at different ages or ambiental conditions male or female trees might
develop both cone types.
Prevalence of monoecious breeding systems in basal gymnosperms and
anemophilous pollination syndromes promote outcrossing as mating system
in this group. In conifers, endogamy is avoided through different mechanisms
such spatial or phenological separation of female and male cones. In many
conifer species male cones are restricted to low branches whereas the females
growth only at highest branches (helping also to seed dispersal by air).
Phenological separation includes species where young trees produce only
pollen cones and older trees ovulated cones and species in which distinct
reproductive structures are mature at different seasons. Moreover there is
evidence of genetic post-fertilization systems to avoid the development of
autogamous embryos as in Pinus, where a genetic overdominant lethal
system stops the development of embryos produced by self-fertilization [9].
Reproductive cycle, from ovule differentiation to seed maturity, occurs
over a time span of one or two years. Exceptionally in Ephedra
(Ephedraceae) reproductive cycle is shorter than a year and some species of
Cycadales, Pinaceae and Cupressaceae delay until three years to complete the
reproductive cycle [6]. Trees can live many years, indeed the oldest living
trees are conifers, such as Pinus longaeva, Pinus aristata and Picea abies
(Pinaceae) that reach estimate ages of 8000 years old.

Morphological patterns of reproductive female structures

Spite the low diversity of extant gymnosperms –in contrast with

angiosperms- significant differences in morphology and development of
female reproductive structures are observed between lineages. Female
structures could be simply a pair of naked ovules as in Ginkgo or complex
structures as the compound cones in conifers. Morphological differences had
been interpreted as adaptations to seed dispersal, seed protection and
pollination mechanisms. The extensive fossil record of gymnosperms suggest
that this morphologies appeared early during the evolution of this group and
just a few characters have a recent origin.
In Ginkgoales, paired ovules development initiates in terminal on short
stalks. Terminal ovules have just a three-layered integument, which becomes
fleshy at maturity. Whereas seed cones in Cycadales are simple since
megasporophylls -bearing a pair of ovules adaxially- grow directly from a
central axis with determined growing. Particularly in Cycas (Cycadaceae)
genus, as megasporophylls are not arranged in determined cones, after
Sexual reproduction in gymnosperms 5

reproductive development vegetative leaves could be differentiated again

from peripheral zone of the apical meristem. In this genus, megasporophylls
are petiolated and multiple ovules develop on both sides of the petiole [8].
Ovulated cones of Gnetales and conifers are considered compound, since
female reproductive axes are clustered, consisting of one or more ovules
associated to bracts. Though characters in Gnetales have been interpreted
similar to flowers, some features resemble female reproductive structures of
conifers. In Ephedraceae reproductive axes are clustered, each axis with six
or more sterile decussated bracts has two unitegumented ovules in axils of the
last two bracts. Female cones in Gnetaceae resemble a whorled spike; every
whorl form a circular structure called collar that bears fiver or more ovules
with three teguments. Welwitschiaceae, is a monotypic family with compact
seed cones similar to those observed in some conifers. Reproductive axes,
conformed by a pair of decussated bracts subtending only one ovule with two
teguments, develop decussated along a determined branch. The outer
tegument, supplied with two vascular bundles, develops to both sides shaping
two wings in the seed. In contrast with male structures where female infertile
ovules develop, female cones are always unisexual therefore many gnetalean
populations are gynodioecious, since some individuals are bisexual and some
individuals are exclusively female [7, 10].
In conifers, ovules develop on the adaxial surface of dorsoventrally
compressed structures called ovuliferous scales, which in turn develop on
bract axils bringing up a complex called bract-ovuliferous scale (bract-OS).
Multiple bract-OS complexes develop with helical phylotaxy along a
determined axis. Axilar position of ovuliferous scale respect to a bract along
to paleobotanical evidence, suggest that OS is homologous to a fertile dwarf
shoot with sterile and fertile appendages as those observed in Permian
conifers. Therefore the multiple vascular bundles observed in transversal
sections of OS are interpreted as sterile appendage remnants [11].
Seed cones pattern of Pinaceae usually is representative for all conifers,
although some differences are observed in lineages where conical shape of
cones is lost or where the bract is fussed to the OS or the OS is reduced or
lost, moreover in three of the seven families, outstanding anatomical
modifications are found. In Podocarpaceae number of complexes per cone is
reduced until two or one –except in Phyllocladus, where cones might develop
up to eight complexes. In this family, ovules develop associated to a structure
called epimatium that bends over covering the ovule then seeds are not
properly naked as in other gymnosperms. Epimatium also grows in the axil of
a bract and is considered homologous to ovuliferous scale, although, at
maturity acquires a fleshy appearance and often a showy color, as well in
some species of this family also bract is fleshy and colored [11, 12].
6 Alejandra Vázquez-Lobo

Ovules in Taxaceae borne terminal and solitary, except in Cephalotaxus,

where three or four ovules primordia differentiate, but generally only one
develops until maturity. In this group ovules are fully or partially covered by
a fleshy structure called aril that develops from the base and usually acquires
a reddish coloration. As a sterile structure associate to the ovule, aril could be
considered homologous to the OS or to the bract, but anatomical differences
of this sterile appendage suggest an evolutionary novelty for this group
[12, 13].
In Cupressaceae -a widely diversified family that includes genera
formerly assigned to Taxodiaceae-, seed cones show modifications in
ovuliferous complexes; predominantly fusion between components or
reduction of structures. In Cryptomeria japonica, the OS is a lobated
structure, suggesting that ancestral appendages are not totally fussed as in
other OS, whereas in Cunnighamia the OS is lost, therefore ovules are
associated directly to the bract. In Juniperus, seed cones are reduced to a few
ovuliferous complexes partially to totally fused, conferring a berry-like
appearance [11, 12].
Evolutionary changes in female structures could be interpreted as
adaptations to seed dispersal, ovule/seed protection, and pollination
mechanisms. Fleshy appearance and showy color of female structures has
been related to animal dispersal, although these characters had appeared
before the emerging of animal lineages involved in seed dispersal today,
which difficulties test adaptive hypothesis within a phylogenetic framework.

Morphological patterns of pollen cones

Pollen cones are morphologically more similar among gymnosperms.

Commonly, male cones are simple, since microsporophylls with abaxial
microsporangia, develop directly from a dwarf-shot with helicoidal
distribution. Microsporophylls could be compact as in cycads and conifers, or
loose as in Ginkgo. Male dwarf shots develop solitary or aggregated along
vegetative branches.
Remarkable extant exceptions to this general pattern are found in
Gnetales and in Cephalotaxus, where cones show unique characters. In these
lineages, groups of microsporophylls develop associated to bracteoles or
bracts making up complexes that develop clustered along terminal branches,
therefore pollen cones are compound. Cephalotaxus is the only extant conifer
with male compound cones and developmental evidence indicates that male
cones in Taxus (Taxaceae) and Torreya (Taxaceae), could be derived from
compound cones, therefore this simple cones wouldn’t be homologous to the
rest of simple pollen cones in other conifers [14].
Sexual reproduction in gymnosperms 7

Microsporophylls of Gnetales are fussed at the base in a structure called

microsporangiophore, where infertile ovules are often found. This
unfunctional hermaphroditism frequently appears in natural populations,
although Gnetales are commonly interpreted as dioecious plants.
Hermaphroditism has involved this group in hypotheses related with the
origin of flowers in angiosperms, even though molecular phylogenies do not
support close relationships between both groups. In contrast with conifers, in
Gnetales the structure of the male cone is similar to female cone, therefore
male cones in Gnetum (Gnetaceae) are whorled with collar structures
supporting microsporangiophores, whereas in Welwitschia (Welwitschiaceae)
a pair of decussated bracts support this male structure [15].

Gametogenesis

Gametogenesis in gymnosperms is a slow process that generally finishes

until pollination; for example gametes are not present in pollen when is shed,
and often archegonia differentiate until nucellus contact pollen grains or
fertilization begins. It seems that female gametogenesis is similar between
groups, whereas male gametogenesis is variable.
Female gametogenesis (or megagametogenesis) usually initiates with
only one megaspore mother cell inside the nucellus (2n), which undergoes
meiosis to form four haploid cells called megaspores. As three megaspores
degenerate and only one continues the process, the megagametophyte is
considered monosporic. Megaspore divides mitotically without cell-wall
formation, making up a coenocytical megagametophyte. Later, cellularization
occurs and two or more archegonia initials (occasionally one) differentiate at
the ovule tip, close to micropyle end. Each initial cell divides through
asymmetrical mitosis to form a small neck initial cell and a large central cell.
Central cell newly divides asymmetrically to arise a small ventral canal cell
and a large egg cell with a polar nucleus, whereas neck cell divides twice to
arise a four-cell neck (in some species two-cell or eight-cell necks have been
documented). At maturity, the egg nucleus migrates to a central position and
cytoplasm increases volume [6].
Megasporogenesis in Gnetum shows some unique characters respect
other seed plants. As product of differentiation, each ovule contains multiple
megasporocytes that undergo free nuclear meiosis producing several
“tetrasporic coenomegaspores” [16]. This phenomena has been documented
also inside conifers in Cupressus sempervirens (Cupressaceae), nevertheless
in this case only one tetraspore without wall-cells develops into a free nuclear
megagametophyte which later reaches the cellular stage [17]. Instead in
Gnetum each tetraspore develops through free nuclear divisions as other
8 Alejandra Vázquez-Lobo

gymnosperms, but only one develops until maturity without cellularization,

whereas the rest degenerate. Surprisingly neither archegonia nor egg cells are
found inside the mature gametophyte, and any free nuclei near to the
micropylar end could be fertilized. This phenomenon reported also in
Welwitschia, may represent a heterochronic event, since in this genera the
megagametophyte is sexually mature in comparatively early stages of ovular
development.
Microgametogenesis begins within microsporangia where diploid
microsporocytes or pollen mother cells undergo meiosis to form tetrads of
haploid microspores; each microspore will be a pollen grain covered with two
layers; an outer thick layer referred to as exine and an inner thin called intine.
After meiosis, a variable number of asymmetric mitosis arise differentiated
cells, including antheridial, prothalial, tube and sperm cells. Number of cells
in shed pollen varies between lineages from one to five, but always gametes
are absent since its differentiation occurs after pollination [18].
Ginkgo and cycad pollen is shed at four-cell and three-cell stages
respectively, both with a tube cell, one or two prothalial cells and one
antheridial initial. When arrives to pollen chamber, antheridal cell divides to
form a stalk cell and a generative cell, which in turns divides to form two
sperm cells [6]. Surprisingly in Ginkgo and cycads sperm cells are motile due
to multiple flagella, a unique feature for seed plants, since zoidogamy is
characteristic of spore-bearing plants. Motility in gametes of gymnosperms is
not fully useful, since sperm cells are enclosed and transported by pollen
grains; probably retention of this character is correlated with haustorial
development of microgametophytes in these groups. Fossil record suggests
that some extinct conifers were zoidogamic and mapping of this character
onto phylogenies could indicate that non-motile sperms have appeared
independently in angiosperms and gymnosperms [19].
Conifers and gnetalean plants have similar patterns of
microgametogenesis, although in some conifers multiple prothalial cells arise
before sperm differentiation. In Gnetales pollen is shed at four or five-cell
stage with an antheridial cell, one or two prothalial cells and a tube cell. After
pollination, antheridial cell divides to give a binucleate cell. Whereas early
divergent conifers such as those subscribed to Pinaceae, Araucariaceae and
Podocarpaceae families release pollen at four or five-cell stage and sperms
differentiate after five mitotic divisions, producing multiple prothalial cells.
More nested groups such as Cupressaceae and Taxaceae shed pollen at one or
two-cell stage and spermatogenesis only takes one or two mitotic divisions
without differentiation of prothalial cells [20]. In side conifers only sperm of
Cupressaceae is celled, whereas in other families gametes are free nuclei.
Cupressaceae pollen grains are shed at two-cell stage with a tube cell and a
Sexual reproduction in gymnosperms 9

generative cell, which at time of pollination divides arising two sperm cells.
In the phylogenetic context, reduced number of mitotic divisions prior to
spermatogenesis and celled sperm are homoplasic characters between
Ginkgoales, Cycadales and Cupressaceae.

Pollination mechanisms and fertilization

Besides modifications in ovule bearing structures, pollination

mechanisms are diverse in gymnosperms. Characters associated with
pollination, such as ovule direction, production and retraction of a pollination
drop and pollen shape are variable even between species from the same
genus. Pollen grains in gymnosperms are bigger than in angiosperms, though
are light due to low water content therefore pollen grains could be dispersed
by air through long distances, in some cases assisted by wings or aerial sacs,
which are expansions of outer wall (exine) [20]. Wind pollination requires
elevated pollen production to ensure that some pollen grains reach female
cones. Moreover, probably modification of exine surface in pollen grains –
such as minute orbicles or spines- and ovule secretions are adaptations to
promote pollen capture. Also has been proposed that female cone shape in
Pinaceae is an adaptation to improve pollen landing in ovuliferous scales
[21]. From this point of view, the conical shape and the arrangement of
ovuliferous scales create an aerodynamic effect similar to a turbine.
Although, test to this hypothesis with experimental and simulation
approaches do not support this suggestion [22].
Exceptions for anemophilous pollination syndromes are documented in
Cycadales and Gnetales where participation of insects in pollen transportation
has been documented. In fact, the pollination drop secreted by sterile ovules
in Gnetales perform functions of insect attractor; even in male flowers
without ovules some structures such as collar or bracts, are capable to
produce “nectar” [7]. Moreover, pollen is not produced in abundance and
frequent insect visits have been reported, although there is no evidence of
pollinator specificity. Whereas in cycads, entomophilous pollination is result
of a tight interaction between weevils life cycle and pollen cones, since they
use male tissues as both food resource and shelter. Female cones bear
idioblasts that release neurotoxins when are ruptured therefore insects only
feed on male cones and visit female cones bearing pollen and leaving them
intact [23].
Saccate pollen is present only in Pinaceae and Podocarpaceae families,
and probably the primary function of aerial sacs is the flotation in water
rather than through the air. Since those lineages with saccate pollen also
produce a pollination drop, has been proposed that sacs are flotation devices
10 Alejandra Vázquez-Lobo

that allow pollen grains to float upwards in the pollination drop inside
inverted ovules. Although, not all the groups with inverted ovules or where
pollination drop has been observed exhibit saccate pollen, which implies that
sacs are not required for pollination, moreover in Abies and Tsuga, genera
that belong to Pinaceae, the pollen is saccate and there is no ovule secretion
[20, 24].
Ovular secretion must be an ancestral condition for pollination since only
a few lineages inside gymnosperms lack pollination drop (Abies, Tsuga and
Araucariaceae), although secretion and reabsorption mechanisms and drop
contents are quite variable within different genera. Apparently pollination drop
is secreted by the nucellus, since the integuments are not fully vascularized
whereas the withdrawal can occur passively (by evaporation) or actively by as
yet undescribed mechanism that is activated by pollen landing [24].
Erect ovules are present in Ginkgoales, Gnetales, Taxaceae, Cupressaceae
(with few exceptions), and some genera of Podocarpaceae. All of these
lineages produce ovular secretions where non-saccate pollen arrives and sinks
or is conducted to the ovule through evaporation or retraction of pollination
drop. Inverted ovules, when the micropyle points toward the central axe of
the cone, are present in Cycadales, Pinaceae, Araucariaceae, Podocarpaceae,
Sciadopityaceae and some Cupressaceae. Since only Pinaceae and
Podocapaceae have saccate pollen that float upwards, in the other groups
pollen must reach the nucellus surface by means of reduction of pollination
drop [20].
In absence of ovular secretion different mechanisms allow to capture
pollen and stimulate germination. In some cases, the ambiental humidity
accumulated between micropyle and cone central axe works as pollen drop,
capturing pollen and allowing germination. In Abies, Welwitschia and some
species of Araucariaceae has been documented the growing of nucellus
beyond micropyle to reach pollen. In other cases a delayed secretion has been
observed [Pseudotsuga and Larix (Pinaceae)], where the secretion is more
related with fertilization mechanisms rather than with pollination. In these
genera, two flaps in the micropyle grow gradually enclosing the pollen grains
and then a post-pollination drop fills the micropylar canal a few weeks after
pollination. After pollination, fertilization begins with the pollen tube growing
through megagametophyte and reaching the archegonium [20, 24].

Fertilization and embryogenesis

A time gap exists between pollination and fertilization, ranging from one
to several weeks. Pollen landing into micropyle trigger slow processes on
ovules and pollen grains that can take one to several weeks before egg and
Sexual reproduction in gymnosperms 11

sperm fusion. Once pollen is near to the nucellus, grains begin to hydrate and
pollen tube emerges from a distal aperture of exine referred to as leptoma -
therefore pollen tube is covered only with intine-, at the same time ovules
enlarge and micropyle seals through elongation of cells that line the canal,
forming a micropylar collar. When pollen tube reaches the nucellus begins to
destroy adjacent tissues -through synthesis of enzymes that affect cell walls-
until reaches neck cells of archegonial chamber. Neck cells and pollen tube
wall degenerate and release sperms and other cytoplasmic contents into
archegonia where one sperm is fused with egg nuclei and the other one is
reabsorbed by female megagametophyte [25].
Conifers and Gnetales are predominantly siphonogamous, but in Taxaceae
branching of pollen tube has been documented [6]. Opposite, in Gingko and
cycads penetration of nucellus by microgametophyte is haustorial rather than
tubular. Particularly in Ginkgo, at the beginning the growth of
microgametophyte is diffuse, resulting later in an extensive intercellular system
[26]. Gametes swim through a fertilization fluid produced by the nucellar cells
and only one penetrates the neck and remains attached to it releasing the
nucleus and preventing the entry of extra sperms into the egg [6].
In Gnetales double fertilization occur in two different ways, since
archegonia could be present (Ephedraceae) or absent (Gnetaceae and
Welwitschiaceae). In Ephedra each pollen tube contains two sperm nuclei;
one fuses to the egg nucleus whereas the other fuses with the ventral canal
nucleus. In Gnetum both nuclei are released into the coenocytical
megagametophyte and eventually each nuclei fuse with an egg. Double
fertilization in gnetales has been compared with double fertilization in
angiosperms, although there are many distinct features such as the fate of the
second fertilization, which is an embryo-nourishing structure in angiosperms
(endosperm) whereas in Gnetales both fertilization events yield two identical
diploid zygote nuclei that undergo synchronous mitotic divisions establishing
two individual proembryos, referred to as “clonal zygotes” [16].
As result of fertilization, gymnosperm seed include three cellular
generations. Seed coat originates from maternal sporophyte generation, is a
diploid tissue that include the integument and the nucellus remnants.
Embryo-nourishing tissue (megagametophyte) is the haploid maternal
generation and embryo is the new diploid generation, since definitive
embryos arise from a single fertilization event. After fertilization, zygote
undergoes nuclear duplication without cytokinesis, entering to a free nuclear
phase proembryo, generally with nuclei set in two groups, an upper one with
nuclei aligned in one plane and a lower one with variable arrangements.
Exceptions to these patterns are found in Gnetales and Sequoia (a member of
Cupressaceae) where no free-nuclei stages are recognized [27].
12 Alejandra Vázquez-Lobo

In Ginkgo and cycads, zygote nucleus divides without wall formation to

arise multiple nuclei (c.a. 350) that are evenly distributed in the cytoplasm
before cellularization. Cell-wall formation is followed by elongation of cells
near to the micropyle that form a suspensor, whereas those cells at the
opposite side will form an embryo with a differentiated meristem. In
cycadales, only those cells that will form an embryo –at the chalazal region-
undergo cellularization, whereas the others remain as free nuclei. In conifers,
the coenocytical proembryo early enters cellularization to form two tiers of
four cells or an eight-celled proembryo. Each tier divides resulting in four
tiers; first and second continues cytokinesis to form the embryo, whereas
third and fourth tiers elongate to constitute an embryonic suspensor. Embryo
continues growing through cell division creating a corrosion cavity while the
suspensor lengthens pushes the embryo into the female gametophyte [28].
Number of free nuclei in proembryo before cellularization is variable
reaching a maximum in members of Araucariaceae family, where up to 64
free nuclei arise. During this process megagametophyte provides nutrients to
the embryo by the means of programmed cell death. In Podocarpaceae wall
formation occurs early after the fourth mitosis, arising a two-tiered
proembryo, although in this family, division of these tiers originates a third
binucleate tier -instead four separate tiers- that will arise secondary
suspensors or will be part of embryo [29].
In gymnosperms polyembryony is feasible to occur by two different
processes: “simple polyembryony” and “cleavage” or “monozygotic
polyembryony”. Simple polyembryony is a common process due to
fertilization of eggs within different archegonia by different sperm cells,
whereas cleavage polyembryony is result of clonal multiplication of
immature embryo. It seems that cleavage polyembryony is restricted to some
genera of conifers and might occur after simple polyembryony arising up to
16 embryos within a seed. During the first development stages all the
embryos in an ovule have similar growth rates and thus apparently have an
equal opportunity for dominance. At one point, one embryo becomes
dominant while the growth of remaining subordinate embryos is
progressively reduced until it stops completely when the dominant has
reached the cotyledonary stage. Finally, those subordinate embryos are
progressively eliminated displaying autolytic and autophagic mechanisms
while dominant enters to a dormancy period [30].
Similar to conifers polyembryony occurs in Gnetales, but in a different
way since each megagametophyte contains two or more archegonia where
double fertilization might occur, therefore resulting embryos come from
different sperms and different cells functioning as eggs. In similar way, only
one embryo survives until maturity, but mechanisms related to one-embryo
Sexual reproduction in gymnosperms 13

dominance and elimination of subordinated embryos remains unknown for

this group, but probably are similar to conifers, since both groups are closely
related.

Genetics of sexual reproduction

The most part of genes associated with reproductive structures

differentiation in gymnosperms belong to the MADS box gene family; some
undoubtedly homologous to previously reported gene families and others that
could conform new gene lineages unique to gymnosperms [31]. Though
gymnosperms and angiosperms diverged c. 330 million years ago, it seems
that key genes for development and organ differentiation of reproductive
structures are homologous between both groups, and evidence point toward
functional conservation. For example, in gymnosperm those genes
homologous to B-type genes are expressed in male cones, whereas in
angiosperms are involved in petal and stamen differentiation [32–34].
Similarly, C-type genes are expressed both in ovuliferous scales and male
cones, whereas in angiosperms are involved in stamen and carpel
differentiation [35–37]. Moreover, genes homologous to AGAMOUS-LIKE 6
(AGL6) and APETALA 2 (AP2), related with differentiation of floral organs
in angiosperms, seem to have an important role in development of
reproductive structures at least in conifers [38, 39]. Homologous sequences to
LEAFY (LFY) gene, which is involved in the switch between vegetative and
reproductive phases in Arabidopsis, have been found in the four lineages of
gymnosperms along with a paralog referred to as NEEDLY (NLY) [13, 40–
42]. Heterologous expression of these genes in wild-type and lfy lines of
Arabidopsis suggests certain degree of function conservation [41, 43],
although in contrast with LFY, NLY expression is not specifically coupled with
reproductive organ primordia in conifers [13]. Phylogenetic inferences on these
sequences imply that paralog duplication occurred before angiosperms-
gymnosperms split, and later NLY copy was lost in angiosperms [42].
In contrast to angiosperms, in gymnosperms little work in developmental
genetics has been done. Since gymnosperms have long generation times,
transgenic methodologies to test constitutive and heterologous gene
expressions are difficult to improve -but in pollen and pollen tube where
transformations are successful-, as consequence function of genes is
determined through comparative approaches and researches have been
focused on economically important trees as those subscribed to Pinaceae
family. Recently, plant genomic research and expressed sequence tag (EST)
libraries have allowed rapid identification of genes expressed in development
of embryo and other tissues, and have opened the door to comparative studies
14 Alejandra Vázquez-Lobo

to recognize genes exclusive to gymnosperms [28]. In the future sequencing

of complete genomes of distinct lineages of gymnosperms along with
microarrays and EST’s analysis will accelerate the achievement of
knowledge in genetics of sexual reproduction for this group of seed plants.

Conclusions
Gymnosperms are an ancient group of seed plants with a few shared
characters probably due to its deep divergence times. Although phylogenies
point toward monophyly and some features are common to all lineages, such
as outcrossing breeding systems, long generation and reproductive cycle
times, and presence of three cell generations in seed. Other characters are
common but exceptions are found at least in one species such as
unitegumented ovules, simple male cones, unisexual reproductive structures,
monosporic megagametophytes, pollination drop secretion and coenocytical
proembryos. Gnetales represent the most divergent lineage and is also the
most conflictive at the phylogenetic level, whereas Cycadales and Ginkgoales
have in common ancient characters of which the most notable is sperm
mobility. Conifers represent the most diversified lineage sharing characters
with the other three orders, and showing exclusive features for this group.
During the 20th century many efforts to document patterns and processes
in sexual reproduction of all gymnosperms were done, although in the last
decades research is focused in species of Pinaceae family because its
economic importance. Therefore morphological studies with modern techniques
along with genomic analysis will be fruitful to understand the evolutionary
history of gymnosperms and to understand the genetic basis of sexual
reproduction processes.

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