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DOI 10.1007/s10265-014-0645-z
REGULAR PAPER
Received: 4 March 2014 / Accepted: 12 May 2014 / Published online: 3 July 2014
The Botanical Society of Japan and Springer Japan 2014
Introduction
Biebersteinia Steph. ex Fisch. is a perennial herb of five
species distributed in temperate mountainous regions from
Greece to Central Asia (Knuth 1912; Muellner 2011; The
Plant List 2013). Its affinities and familial position were
uncertain when B. odora Steph. ex. Fisch. was described
under the new genus (Stephan 1811). Thereafter, although
Endlicher (1841) placed Biebersteinia between Zygophyllaceae and Geraniaceae, most authors placed it in
Geraniaceae (e.g., Boissier 1867; Knuth 1912; Scholz
1964; Cronquist 1981, 1988; Goldberg 1986; Thorne
1992), or near Geraniaceae as the distinct family Biebersteiniaceae (Takhtajan 1986; Dahlgren 1989). However,
since molecular evidence was published at the end of the
20th century (Bakker et al. 1998), the Angiosperm Phylogeny Group (APG 1998; APGII 2003; APGIII 2009) has
consistently accepted the monogenetic family Biebersteiniaceae in Sapindales, while placing Geraniaceae in Geraniales. More recent molecular analyses using rbcL
sequences suggested that Biebersteiniaceae are possibly
sister to the remaining eight families of Sapindales,
although the monophyly of the latter has weak support
(Muellner et al. 2007) (Fig. 1).
Biebersteinia is little known morphologically (Stevens
2001 onwards), although several publications are available
for morphological characters: for instance, chromosome
numbers and morphology (Aryavand 1975; Shen and
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Collection
Developmental
stage
Biebersteinia
heterostemon
Maxim.
Young to
mature fruits
Mature frutis
Mature frutis
Mature frutis
Mature fruits
Mature fruits
Flower buds to
mature fruits
Biebersteinia
multifida DC.
Biebersteinia
orphanidis
Boiss.
Results
Anthers and microspores
The flowers are bisexual and borne on inflorescences that
appear to be racemose (Fig. 2a), each flower bearing five
sepals, five petals, ten stamens, and a five-carpellate pistil
with a superior ovary or ovaries (Fig. 2b, c). Each anther is
tetrasporangiate (Fig. 2c). Prior to maturation, its wall is
composed of four to six cell layers: an epidermis, an
endothecium, one to three (usually two) middle layers, and
a tapetum (Fig. 2d). The middle layers have a common
histogenetic origin with the endothecium (Fig. 2d).
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Therefore, wall formation conforms to the Dicotyledonous type. The tapetum is glandular (Fig. 2e). Its cells are
initially uninucleate and later become binucleate (Fig. 2f),
and they normally develop further up to six to seven nuclei
due to nuclear divisions (Fig. 2g) and may contain up to 12
nuclei. The nuclei, however, fuse to form a single large
polyploid mass (Fig. 2h). During maturation, the middle
layers degenerate and cells of both the epidermis and
endothecium become enlarged (Fig. 2i). By the time of
anther wall dehiscence, the cells of the endothecium have
developed fibrous thickenings (Fig. 2i, j). Although the
cells of the epidermis are unspecialized, they were persistent. Anther dehiscence takes place by longitudinal slits,
with each slit common to two microsporangia of the theca
(Fig. 2j).
Meiosis in a microspore mother cell is accompanied by
simultaneous cytokinesis (Fig. 2k) and the resultant
microspore tetrads are predominantly tetrahedral. Pollen
grains are three-celled when shed (Fig. 2l), as Kamelina
and Konnova (1990) described for Bieberstenia multifida.
Female gametophytes and nucellus
Each carpel has a single ovule pendant from the upper
adaxial side of the ovary (Fig. 3a). An ovule primordium first
grows downwards (Fig. 3a) and then turns its apex toward
the horizontal direction and eventually upward (Fig. 3b). An
ovule with a developing (e.g., four-nucleate) female gametophyte is positioned almost horizontally. At maturity an
ovule is anatropous and epitropous ventral, having the
nucellar apex above and the raphe on the ventral side
(Fig. 3b, i). The funicle is massive, long, and bent irregularly
(Fig. 3b). The ovular body itself is also slightly twisted.
Early in development, the ovule has a one-celled
archesporium differentiated beneath the apical dermal layer
of the nucellus (Fig. 3c). The archesporial cell divides
periclinally into the primary parietal cell upward and the
primary sporogenous cell downward (Fig. 3d). Thus, the
ovule is crassinucellate. The primary parietal cell further
divides periclinally and anticlinally, resulting in a three- to
four-cell-layered parietal tissue above the megaspore
mother cell, which develops directly from the primary
sporogenous cell (Fig. 3e). Meiosis in the megaspore
mother cell is not accompanied by cytokinesis, resulting
successively in a two-nucleate (Fig. 3f) and four-nucleate
female gametophyte (Fig. 3g). Both the two-nucleate and
four-nucleate female gametophytes contain densely staining cytoplasm around the nuclei. As the female gametophyte enlarges, the four nuclei move into peripheral
positions. Their arrangement is more or less crosswise,
with one nucleus on the micropylar end and one on the
chalazal end, and the remaining two opposite on the sides
(Fig. 3g).
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thick. Cells of the inner epidermis further divide periclinally, so that the integument becomes three cell layers
thick (Fig. 4d) and further becomes four to five cell layers
thick (Fig. 4e). The integument becomes thicker at the
apical part than at the lateral part. Some cells of the apical
part are enlarged (Fig. 4f). No vascular bundles differentiate in the integument. No obturator develops from the
integument.
Although the pollen-tube path is observed entering from
the nucellar apex (Fig. 4g), the micropyle is not formed by
the time of fertilization (Fig. 4f), or even shortly after
fertilization (Fig. 4h).
Endosperm and embryo
As documented above, one or more pollen tubes reach the
nucellar apex for fertilization before the micropyle is
formed. Therefore, fertilization is not porogamous. The
integument (or testa) is closed in post-fertilization stages,
so that it often appears as if the pollen tube passed through
the micropyle (this mode of fertilization was referred to as
pseudoporogamy in the study of Myrica [Fagales], Sogo
and Tobe 2006).
Of the four egg apparati positioned crosswise at the
periphery of the female gametophyte (Fig. 5a), only the
one nearest to the nucellar apex is fertilized. We examined
eight female gametophytes in the post-fertilization stages
and found that in all of them a proembryo was produced by
the egg apparatus on the apical side. The remaining three
positioned on the chalazal or lateral sides were not fertilized and subsequently degenerated (Fig. 5af). Degeneration of the egg apparatus may occur even before
fertilization (see an arrow on the chalazal side in Fig. 5a).
Endosperm formation is of the nuclear type. Free
endosperm nuclei, all being logically pentaploid because
they should be formed by the fusion of four polar nuclei
and a sperm nucleus, can be seen in young seeds. Figure 5bf shows serial longitudinal sections of a young seed
that has an eight-celled proembryo with eight free endosperm nuclei. In a developing seed, the free endosperm
nuclei are positioned at the periphery of the female
gametophyte and are connected via cytoplasm with one
another. Wall formation in free endosperm nuclei starts
from the apical side of the female gametophyte and extends
towards the chalazal side (Fig. 6b, c). As the seed develops, it curves as described later and the endosperm is
mostly digested. Generally in the mature seeds it is scanty
on the convex (antiraphal) side (Fig. 6e, f, h, i). The three
species examined clearly differ in an amount of the endosperm on the convex side. In Biebersteinia orphanidis it is
one-cell-layered, two- to four-cell-layered in B. multifida,
and completely lost in B. heterostemon. In contrast, on the
concave (raphal) side, the endosperm remains as a rather
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massive tissue (Fig. 6e, f, h, i). But in the case of B. heterostemon, cells of the endosperm do not appear to have
cytoplasm (Fig. 6i). Thus, the mature seeds of B. heterostemon are nearly exalbuminous.
We did not examine embryogenesis in detail, but fragmentary data on early and later embryogenesis indicated
that it proceeds normally to form globular and dicotyledonous embryos (Fig. 6ad). The proembryos that we
observed in three young seeds were all transversely ellipsoid to ellipsoid with no conspicuous suspensor (Fig. 6b).
They become dicotyledonous later (Fig. 6di). The cotyledons are massive (Fig. 6ei), building nearly four fifth of
the whole embryo in mature seeds (Fig. 5eg). The
embryos in mature seeds curve to various degrees (Fig. 6f
i). In the case of mature seeds in Biebersteinia heterostemon, the embryo was bent at a nearly right angle (Fig. 6i).
Seeds and seed coat
The seed curves during its development, changing from
anatropous to slightly amphitropous, with its concave
region on the raphal or ventral side. Interestingly, the seed
shifts the position of the chalaza toward the concave side
(see arrowheads indicating the junctions between the
nucellus and the integument in Figs. 3i, 6a, c, f, showing
changes in the position of the chalaza). The nucellar tissue
remains in young seeds, but mostly disappears except in the
concave side in mature seeds (Fig. 6e, f).
The fruit is a schizocarp, consisting of five mericarps at
maturity (Fig. 7a). At maturity, the mericarp is more or less
reniform in shape and one-seeded with a thick, hard pericarp (Fig. 7b, c). The size of the mature mericarp is different in the three species investigated: about 6.06.5 mm
long and 4.55.2 mm wide (measured from side to side) in
Biebersteinia orphanidis, 5.35.8 mm long and
3.64.1 mm wide in B. multifida, and 2.32.8 mm long and
1.52.0 mm wide in B. heterostemon. The fruit wall is
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Discussion
Summary of the embryological features
of Biebersteinia
As reviewed in the introduction, there has been uncertainty with regard to the embryological characters of
Biebersteinia, because the previous study did not provide
illustrative figures to show the development of anthers,
ovules, and seeds (Kamelina and Konnova 1990) and
because seed coats were based on a misidentification of
fruit walls as seed coats (Corner 1976), or the incorrect
assumption that they developed from bitegmic rather than
unitegmic ovules (Boesewinkel 1997). Not only did the
results of the present study clarify most of these issues,
but it also enabled a substantial revision of the data for
seed coat characters. The overall information on the
embryological features of Biebersteinia can be summarized as follows (see also data on 58 characters in Table
S1). New or revised information is indicated by an
asterisk.
Anther tetrasporangiate; anther wall four to six cell
layers thick*, formation of the Dicotyledonous type; anther
epidermis persistent; endothecium fibrous; one to three
middle layers crushed; tapetum glandular, and its cells
multinucleate*; tapetal nuclei (increasing into six to seven
nuclei, up to 12 nuclei) fusing into a large polyploid mass*.
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microspore mother cell cytokinesis simultaneous; microspore tetrads predominantly tetrahedral*; pollen grains
three-celled when shed.
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Fig. 5 Development of female gametophytes of Biebersteinia orphanidis in pre- and post-fertilization stages. All figures are presented
with the apical side of the nucellus above. a Longitudinal section (LS)
of a 16-nucleate female gametophyte. Note that four egg apparati are
positioned crosswise, with one each on the micropylar and chalazal
ends, and the remaining two opposite on the sides. The one on the
chalazal side is degenerating as indicated by an arrow. Squares
vascular bundles differentiating in integument*; no obturator formed*; micropyle not formed by the time of fertilization, formed in post-fertilizations stages*.
Fertilization pseudoporogamous (occurring before the
micropyle is formed)*; of the four egg apparati positioned
crosswise, only one nearest to the nucellar apex being
fertilized; endosperm formation of the nuclear type; mature
seeds albuminous (B. multifida and B. orphanidis), or
nearly exalbuminous (B. heterostemon) *; in the former,
endosperm scanty on convex (antiraphal) side and massive
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nia. ag B. orphanidis. h B. multifida. i B. heterostemon. a Infructescence. b Lateral view of the whole mature mericarp. c Median
longitudinal hand section of the whole mature mericarp. d Longitudinal section (LS) of young seed (shown in Fig. 6a) showing seed
coat structure on the convex (antiraphal) side. e LS of young seed
(shown in Fig. 6c) showing seed coat structure on the convex side. f,
h, i LSs of mature seeds showing seed coat structure on the convex
side. g Transverse section of mature seed showing seed coat structure
on the convex side. em embryo, en endosperm, ents endotesta, exts
exotesta, fn funicle, mr mericarp, mts mesotesta, nc nucellar tissue,
and ts testa. Scale bars are 1 cm in a, 1 mm in b and c, and 20 lm in
di
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612
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