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J Plant Res (2014) 127:599615

DOI 10.1007/s10265-014-0645-z

REGULAR PAPER

Embryology of Biebersteinia (Biebersteiniaceae, Sapindales):


characteristics and comparisons with related families
Takenori Yamamoto Dionyssios D. Vassiliades
Hiroshi Tobe

Received: 4 March 2014 / Accepted: 12 May 2014 / Published online: 3 July 2014
The Botanical Society of Japan and Springer Japan 2014

Abstract Biebersteinia, a perennial herb of five species


distributed from Greece to Central Asia, was long considered to be placed in, or near Geraniaceae. Recent molecular
analyses, however, have shown that the genus is the sole
member of the family Biebersteiniaceae in Sapindales (not
including Geraniaceae). Here, we report the embryological
features of Biebersteinia and provide embryological corroboration for the molecular sapindalean affinities of the
genus. We compared its embryology with those of eight
other families of Sapindales, as well as with those of the
related orders Huerteales, Malvales, and Brassicales.
Overall comparisons showed that Biebersteinia fits in Sapindales because of the presence of anther tapetal cells with
polyploid nuclear mass and non-fibrous exotegmen. Further, the genus is characterized by three-celled pollen
grains, tetrasporic 16-nucleate Penaea-type female gametophyte, unitegmic ovules, pseudoporogamy, and the chalaza shifting its position near the concave side in the postfertilization stage. A considerable number of autapomorphies, combined with the lack of synapomorphies with
other sapindalean families, supports placing Biebersteinia
in its own family. Biebersteiniaceae appear to represent an
early divergent lineage of Sapindales. Previous

Electronic supplementary material The online version of this


article (doi:10.1007/s10265-014-0645-z) contains supplementary
material, which is available to authorized users.
T. Yamamoto  H. Tobe (&)
Department of Botany, Graduate School of Science,
Kyoto University, Kyoto 606-8502, Japan
e-mail: tobe@sys.bot.kyoto-u.ac.jp
D. D. Vassiliades
24 Issiodou St, 10674 Athens, Greece

descriptions of seed coats, which were considered to have


developed from bitegmic ovules, were revised.
Keywords Biebersteinia  Biebersteiniaceae 
Embryology  Ovule  Seed  Sapindales

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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Fig. 1 Phylogenetic tree of Sapindales and related orders, showing


the position of Biebersteinia. Asterisks indicate a weakly supported
clade. Modified from Muellner et al. (2007), Worberg et al. (2009)
and Wang et al. (2009)

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Kamelina and Konnova (1990) described the ovule of B.


multifida as unitegmic. Consequently, there is disagreement as to the number of integuments or seed coats, but as
we show later, the ovules of Biebersteinia have only a
single integument. Therefore, previous descriptions of the
seed coats, which were based on recognition of the testa
(developed outer integument) and tegmen (developed inner
integument), must be revised.
Of the embryological data we present here, some confirm data previously reported by Kamelina and Konnova
(1990); some revise the data previously described; and
others represent new data for the genus. On the basis of the
overall embryological data, we will compare Biebersteinia
with all other Sapindales to understand whether Biebersteinia fits in the order morphologically, and, if so, how the
genus is related to other Sapindales.

Materials and methods


Huang 1997), embryology (Kamelina and Konnova 1990),
and ovule and/or seed structure (Corner 1976; Boesewinkel
1997). In these studies, comparisons have been made with
Geraniaceae. Our interests are now in understanding how
Biebersteinia is characterized morphologically and related
to families within Sapindales.
Here, we present an embryological study of Biebersteinia. Embryological data provide more than 50 characters associated with the development of anthers, ovules,
and seeds and thus have contributed to a better understanding of the relationships within and between families
(Tobe 1989; for more recent examples, see Tobe and
Kadokawa 2010 for Araceae [Alismatales]; Tobe 2011 for
Leitneria Chapm. [Simaroubaceae]; Tobe and Raven 2011
for Irvingiaceae [Malpighiales]). Previously, Kamelina and
Konnova (1990) reported some embryological features of
Biebersteinia on the basis of B. multifida DC., but they did
not provide any illustrative evidence, except for diagrams
showing the tetrasporic Penaea-type female gametophyte.
The Penaea-type female gametophyte is rare in angiosperms, occurring in Penaeaceae (Myrtales) (Stephens
1909; Tobe and Raven 1984a) and some Euphorbiaceae
and Malpighiaceae (Malpighiales) (supplementary information in Tobe and Raven 2011; see also Endress et al.
2013). The occurrence of the Penaea-type female gametophyte in Biebersteinia needs confirmation. Corner (1976,
p. 148) gave descriptions of ovules and mature seeds of B.
multifida along with drawings, but he mistook fruits and
fruit walls as seeds and seed coats, as Boesewinkel (1997)
pointed out. Boesewinkel (1997) reported the structure of
mature seeds of B. multifida and B. orphanidis Boiss., but
did not describe their development. He also observed the
mature ovules of B. emodii Jaub. and Spach, and described
the ovules of Biebersteinia to be bitegmic. However,

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Three species (Biebersteinia heterostemon Maxim., B.


multifida, and B. orphanidis) were investigated in this
study. Their respective collection data and developmental
stages available are listed in Table 1. Observations of the
development of anthers, ovules and seeds were based
principally on Biebersteinia orphanidis. Its flower buds and
fruits in various stages of development were fixed in FAA
(five parts stock formalin, five parts glacial acetic acid, 90
parts 50 % ethanol). We know that embryological
Table 1 Species studied of Biebersteinia, collection information, and
developmental stages
Taxon

Collection

Developmental
stage

Biebersteinia
heterostemon
Maxim.

China, Dagzi county


(29460 6.500 N 91530 31.900 E).
Yang Zhong s.n. no voucher

Young to
mature fruits

China, Xizang, Linzhi. D. E.


Boufford et al. 30182 (A)

Mature frutis

China, Xizang, Changdu Xian.


D. E. Boufford et al. 31189
(A)

Mature frutis

China, Sichuan, Ruoergai Xian.


D. E. Boufford et al. 40326
(A)

Mature frutis

Iraq, Kurdistan. Ertter BE8/


7(National Herbarium of
Kurdistan)

Mature fruits

Iraq, Sulaimani, Tawana


Mountain. S. A. Rahman s.n.
in 2011 (SUFA)

Mature fruits

Greece, Peloponnese, Mount


Killini. D. Vassiliades133
(ATHU)

Flower buds to
mature fruits

Biebersteinia
multifida DC.

Biebersteinia
orphanidis
Boiss.

J Plant Res (2014) 127:599615

characters other than seed characters are not variable


within a genus and even in a higher taxonomic category in
general (Tobe 1989; Tobe and Raven 1996). Therefore,
seed structures were observed in the three species B. heterostemon, B. multifida and B. orphanidis. Some of the
seeds were also fixed in FAA and others were dry. In the
latter case, they were soaked in 50 % ethanol prior to
anatomical observations.
For anatomical observations, some flower buds and seeds
were dehydrated through an ethanol series and then
embedded in Technovit 7100 (Kulzer, Wertheim, Germany)
for sectioning on a microtome. Serial resin sections cut at a
thickness of 45 lm were stained with Heidenhains
hematoxylin and mounted in Entellan (Merck, Darmstadt,
Germany). For observations of the number of cells in
mature pollen grains, some pollen grains collected from
liquid-preserved flowers were stained by 1 % acetocarmine
(Tobe and Raven 1984b). Their thick exine wall hindered
infiltration of the dye, but we could count the cell number of
pollen grains 24 h after staining. All microtome sections
and acetocarmine-stained pollen grains were observed with
an Olympus BX-51 microscope (Olympus, Tokyo, Japan).
For observations of the pollen-tube path for fertilization,
a few ovules at the time of fertilization were obtained in a
solution of 50 % ethanol by removing a fruit wall from
each pistil. They were cleared in 0.01 % sodium hypochlorite (NaClO) at room temperature overnight. After
rinsing two or three times in water, the ovules were macerated in 1 N NaOH at 60 C for 1 h. Pollen tubes within
the ovules were stained with 0.5 % aniline blue in 0.1 N
K3PO4 for 23 h and observations were made using fluorescence microscope Olympus BX-51.
For SEM-observations of the funicle of a mature ovule
which has an irregular shape during development, a few
specimens dehydrated through an ethanol series were
critical-point dried in CO2 and coated with platinum.
Observations were made using a Hitachi Miniscope TM1000.

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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b Fig. 2 Inflorescence of Biebersteinia orphanidis and the develop-

ment of anthers and microspores. a Inflorescence with an open flower


(an arrow). b Longitudinal section of a young flower bud. c Transverse section (TS) of a young flower bud. d TS of a young anther.
e TS of an old anther. f Uninucleate and binucleate tapetal cells.
g Multinucleate tapetal cell. h Polyploid nuclear mass in tapetal cell.
i TS of an old anther. j TS of a mature anther. Arrowheads indicate
the positions of longitudinal slits. k TS of an anther showing the
simultaneous cytokinesis in meiosis of pollen mother cells. l Whole
mature pollen grains stained with acetocarmine. br bract, ent
endothecium, ep epidermis, ml middle layer, pe petal, pmc pollen
mother cell, ps pistil, s sperm cell, se sepal, st stamen, t tapetum, and
v nucleus of a vegetative cell. Scale bars are 1 cm in a, 400 lm in
b and c, 100 lm in j, 50 lm in e, 20 lm in d, i, k, and l, and 10 lm in
fh

Each of the four nuclei undergoes two successive nuclear


divisions to form a quartet in each of the areas of the female
gametophyte where the nuclei were located originally,
resulting successively in an eight-nucleate (Fig. 3h) and a
16-nucleate female gametophyte (Figs. 3i, 5a). Four nuclei,
one each from the four quartets, move towards the center of
the female gametophyte, becoming polar nuclei (Fig. 5a).
The remaining three nuclei in each of the four quartets
become cellular and assume the appearance of an egg
apparatus consisting of an egg cell and two synergids
(Figs. 3i, 5a). Thus, the mode of female gametophyte
development is of the tetrasporic Penaea-type, as Kamelina
and Konnova (1990) reported in Biebersteinia multifida.
The 16-nucleate organized female gametophyte is widely
ellipsoidal (Fig. 3i) and the densely staining cytoplasm is
positioned peripherally and appears to link the 12 nuclei to
one another. Antipodal cells are absent.
Throughout the development of the female gametophyte, the apical dermal cells of the nucellus may, or may
not divide periclinally to form a two-cell-layered nucellar
cap (Fig. 4a, b). By the time of fertilization, a few of the
apical dermal cells of the nucellus are enlarged (Fig. 4f).
They may play a role in attracting pollen-tubes, because no
micropyle is formed by the time of fertilization, as
described later. Early in development, the nucellus is small.
Indeed, the ovule with the megaspore mother cell has few
cells below it in the nucellus (see Fig. 3e). However, as the
ovule develops, cell divisions continue around a developing female gametophyte, so that a thick nucellar tissue is
formed, particularly on the chalazal side (Fig. 3i; see also
Fig. 6a). No hypostase is formed (see the chalazal region of
the mature ovule in Fig. 3i), but it is interesting to note that
ovules of Bibersteinia emodii have a conspicuous hypostase (see Figs. 31 and 32 in Boesewinkel 1997, p. 288).
Integuments
The ovule is unitegmic (Fig. 4cf), as described by
Kamelina and Konnova 1990) for the ovule of Biebersteinia multifida. The integument is initially two cell layers

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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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b Fig. 3 Development of ovules and female gametophytes in Bieber-

steinia orphanidis. a Longitudinal section (LS) of pistil, showing a


pendant ovule primordium. b Scanning electron micrograph of mature
ovule. Note that the ovule is anatropous and epitropous ventral, with a
characteristic massive funicle. ci LSs of ovules, showing development of nucelli and female gametophytes c Ovule with an archesporial cell. d Ovule with a primary parietal cell and a primary
sporogenous cell. e Ovule with a megaspore mother cell. f Twonucleate female gametophyte. g Four-nucleate female gametophyte.
h Eight-nucleate female gametophyte. i Ovule with mature female
gametophyte. Arrowheads indicate the junctions between the nucellus
and the integument. Squares in g and h indicate digital superposition
of the nuclei from adjacent microtome sections. arc archesporial cell,
fn funicle, mmc megaspore mother cell, it integument, nc nucellar
tissue, op ovule primordium, p parietal tissue, pp primary parietal cell,
ps primary sporogenous cell, and rp raphe. Scale bars are 100 lm in
a, b, and i, 50 lm in e, and 20 lm in c, d, and fh

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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about 270300 lm thick in B. orphanidis, 220250 lm


thick in B. multifida, and 85110 lm thick in B. heterostemon. In contrast, the seed coat (i.e., the developed
integument) is very thin, about 1520 lm thick in all the
three species examined. Seeds are exarillate and reniform
and transverse sections appear triangular (Fig. 6e).
Early in development, the seed coat is four to five cell
layers thick, consisting of an outer epidermis (exotesta),
two or three middle layers (mesotesta), and an inner epidermis (endotesta) (Fig. 7d) (we applied the testal terminology to the unitegmic seeds of Biebersteinia following
Schmid [1986]). As the seed develops, cells of the exotesta
become enlarged and round in shape; those of the endotesta
are small and accumulate a tannin-like substance, while
cells of the mesotesta degenerate (Fig. 7e). At maturity,
while the exotesta collapses to remain as a remnant, the
endotesta develops as a mechanical layer (Fig. 7fi). Endotestal cells are small, but their inner and radial walls are
thickened (Fig. 7fi). Since the endotesta (more strictly,
the inner epidermis of the integument) is the best developed mechanical layer, the seed coat is endotestal (terminology following Schmid 1986). There was no clear
difference in seed coat structure among the three species
investigated.

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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Fig. 4 Development of nucelli and integuments in Biebersteinia


orphanidis. a Longitudinal section (LS) of a young ovule with a
megaspore mother cell. b LS of a young ovule with megaspores. c LS
of a young ovule showing an initiation of the integument. d LS of an
immature ovule showing a developing integument. e LS of a mature
ovule showing a four to five cell layer integument. f LS of a mature

ovule. Note that a micropyle is not formed yet. g Mature ovule


observed with a fluorescence microscope, showing pollen-tube path at
the time of fertilization. h LS of a young seed in a post-fertilization
stage. ep epidermis, it integument, pt pollen tube. Scale bars are
100 lm in f, 50 lm in g and h, and 20 lm in ae

microspore mother cell cytokinesis simultaneous; microspore tetrads predominantly tetrahedral*; pollen grains
three-celled when shed.

Ovule anatropous* and crassinucellate*, having a long


massive funicle which is bent irregularly*. Ovule archesporium one-celled, dividing a primary parietal cell and a

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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

indicate digital superposition of the nuclei from adjacent microtome


sections. bf Five serial LSs of the female gametophyte in a postfertilization stage with an eight-celled proembryo and eight free
endosperm nuclei. Arrows indicate three degenerating egg apparati.
e.g. egg cell, fe free endosperm nucleus, pe proembryo, po polar
nucleus, and sy synergid cell. Scale bars are 50 lm in af

primary sporogenous cell; a three to four cell layer parietal


tissue formed*; female gametophyte development resulting
in a 16-nucleate tetrasporic Penaea-type; shape of mature
female gametophyte widely ellipsoidal*; nucellar cap not
formed or two cells thick and a few apical dermal cells
enlarged at the time of fertilization*; a thick nucellar tissue
formed particularly on the chalazal side*; hypostase present (B. emodii) or absent (B. orphanidis).*
Ovule unitegmic; integument two-cell-layered initially*,
becoming four- to five-cell-layered at maturity*; no

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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J Plant Res (2014) 127:599615


b Fig. 6 Development of seeds in Biebersteinia. ag B. orphanidis.

h B. multifida. i B. heterostemon. a Longitudinal section (LS) of


young seed. b Magnified view of the upper portion of a, showing a
globular proembryo. c LS of a developing seed. d Magnified view of
the upper portion of c, showing a dicotyledonous embryo. e Transverse section of mature seed. f, h, and i LSs of mature seeds. g Lateral
view of mature embryo. Arrowheads indicate the junctions between
the nucellus and integument, showing the position of the chalaza. ch
chalaza, cot cotyledon, em embryo, en endosperm, fe free endosperm
nucleus, nc nucellar tissue, pe proembryo. Scale bars are 1 mm in
c and eh, 500 lm in a and i, 200 lm in d, and 100 lm in b

on concave (raphal) side*. Embryogenesis unknown;


embryo in mature seed dicotyledonous and curved (bent at
a nearly right angle in B. heterostemon).
Seed small (smallest in B. heterostemon), curved and
slightly amphitropous*. Chalaza shifting its position
toward the concave side*. Fruits schizocarp, consisting of
five mericarps. Mature mericarp more or less reniform in
shape, one-seeded. Seeds exarillate*. Young seed coat
composed of four or five cell layers with exotestal cells
enlarged*. Mature seed coat endotestal with a mechanical cell ayer developed from the inner epidermis*. Exotestal cells collapsed, remaining as a remnant on the
endotesta*; endotestal cells small with inner and radial
walls thickened*.

Comparison with other Sapindales and related orders


We reviewed embryological data on 58 characters relevant
to the anther, ovule, and seed using published data for the
eight other families of Sapindales (for references see
Appendix 1) to compare with the embryological features of
Biebersteinia. Although very few data were available for
Kirkiaceae, the remaining seven families were relatively
well characterized embryologically. All the data for the
individual families are presented in Table S1 (supplementary data with online version of this article). We also
compared the embryological features of Biebersteinia with
those of families of the Huerteales, Malvales, and Brassicales, which form a clade sister to Sapindales (Worberg
et al. 2009; Wang et al. 2009; Soltis et al. 2011) (Fig. 1).
Within the clade, Huerteales are sister to Brassicales and
Malvales. For comparison, we used data from two of the
three constituent families of Huerteales: Tapisciaceae and
Dipentodontaceae. Likewise, we used data from Neuradaceae, the basal-most family in the Malvales (Soltis et al.
2000), and those of Akaniaceae and Tropaeolaceae, which
form a basally divergent clade in Brassicales. Embryological data for Tapisciaceae and Dipentodontaceae were
obtained from Corner (1976) and unpublished data,
respectively; those for the Neuradaceae from Murbeck
(1916), Corner (1976), Huber (1993), and unpublished

609

data; and those for the Akaniaceae and Tropaeolaceae,


from Tobe and Raven (2008 and references cited therein).
Overall comparison showed that Biebersteinia is similar
to families of the Huertales, Malvales, and Brassicales, as
well as those of the Sapindales for the majority of characters, but it agreed only with Sapindales in having anther
tapetal cells with a polyploid nuclear mass and having a
non-fibrous exotegmen in mature seed coats. In Brassicales, Huerteales, and Malvales anther tapetal cells are
binucleate, as in many other angiosperm families. However, B. orphanidis usually has six to seven nuclei (up to 12
nuclei) which fuse into a single large polyploid mass.
Kamelina and Konnova (1990) reported B. multifida to
have a single nucleus in anther tapetal cells, but the single
nuclei they observed might be the polyploid mass formed
by the fusion of more than two nuclei. Our literature survey
showed that in Sapindaceae, Rutaceae, Simaroubaceae, and
Meliaceae of the Sapindales, anther tapetal cells have a
polyploid mass consisting of more than two nuclei. In
Nitrariaceae (Sapindales) anther tapetal cells are reported
to have two nuclei in Nitraria sibirica Pall. (Li and Tu
1990a) and Peganum harmala L. (Kapil and Ahluwalia
1963). However, in P. harmala L., tapetal nuclei frequently
divide and fuse to become polyploid (Kapil and Ahluwalia
1963). In Anacardiaceae (Sapindales) binucleate tapetal
cells are likely common and are reported in a few genera
such as Lannea A. Rich. and Pistacia L. However, Toxicodendron diversilobum (Torr. and A.Gray) Greene has
two or more nuclei in each tapetal cell (Copeland and
Doyel 1940), although it is uncertain whether the nuclei
fuse to form a polyploid mass or not. In Burseraceae (Sapindales) anther tapetal cells are binucleate at least in
Boswellia serrata Roxb. (Narayana 1959) and Garuga
pinnata Roxb. (Narayana 1960), but they remain uninucleate in Bursera delpechiana Poiss. (Srivastava 1968).
The latter needs confirmation because there might have
been a multinucleate state prior to a uninucleate state, as
we observed in B. orphanidis. Because little attention has
been paid to the number and behavior of nuclei in anther
tapetal cells of Sapindales and related orders, previous
reports on this character are fewer than for the other
characters and are sometimes even dubious, as discussed
above. Nevertheless, available information indicates that
anther tapetal cells with a polyploid nuclear mass are
prevalent in Sapindales, but not in Huerteales, Malvales
(Neuradaceae) and Brassicales (Akaniaceae/Tropaeolaceae). Within Sapindales the binucleate condition is common in Anacardiaceae and Burseraceae which form a
monophyletic clade (see Fig. 1). However, we need to
check this character in these families, because if binucleate
anther tapetal cells occur consistently in the two families, a
reversal may have occurred from the multinucleate to the
binucleate state in the Anacardiaceae-Burseraceae clade.

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J Plant Res (2014) 127:599615


b Fig. 7 Fruits and seed coat development and structure in Bieberstei-

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

Further, it will be interesting to see whether Kirkiaceae (the


family sister to the Anacardiaceae-Burseraceae clade) have
binucleate or multinucleate anther tapetal cells.
With regard to the exotegmen (i.e., an outer epidermis of
the developed inner integument) in mature seed coats,
Biebersteinia orphanidis has no comparable layer in a strict
sense, because its ovules are unitegmic, not bitegmic as in
Sapindales. The only persistent mechanical cell layer in the
mature seed coat of B. orphanidis is the endotesta (inner
epidermis of the integument). The cells of the endotesta are
small with the inner and radial walls often thickened (B.
orphanidis and B. multifida), but they never become
fibrous. In the other sapindalean families (except Meliaceae
and Rutaceae), cells of the exotegmen are unspecialized or
crushed. Only in Meliaceae and Rutaceae are the cells of
the exotegmen very often fibrous (Corner 1976). Among
outgroups, both Dipentodontaceae/Tapisciaceae (Huerteales) and Neuradaceae (Malvales) have a fibrous exotegmen (Corner 1976). We need to know whether
Picramniales (Picramniaceae only), which are sister to a
clade of four related orders (Soltis et al. 2011), have a
fibrous exotegmen or not. At present, since data on seed
coat structure are not known yet for Picramniaceae, we
simply regard the lack of a fibrous exotegmen to be characteristic of Sapindales.
Thus, both the possession of anther tapetal cells with a
polyploid nuclear mass and the lack of the fibrous exotegmen, which are found in Biebersteinia, are very likely
synapomorphies of the Sapindales. Embryological evidence corroborates molecular evidence, supporting the
placement of Biebersteinia in this order.
Comparisons with other families of Sapindales
Within Sapindales, Biebersteinia agrees embryologically
with all the families in having some common features
(except for Kirkiaceae due to a paucity of data for this
family), rather than with a particular family or families.
They include the following: anther tetrasporangiate; anther
wall more than four cell layers thick; anther epidermis

611

persistent; endothecium fibrous; middle layers ephemeral;


tapetum glandular; cytokinesis in the microspore mother
cell simultaneous; microspore tetrads predominantly tetrahedral; ovule usually anatropous and crassinucellate;
endothelium absent; endosperm formation of the nuclear
type. However, most of these common embryological
features can be found in many other families from orders
other than Sapindales. Biebersteinia differs from the rest of
Sapindales because of the following six apomorphies (rare
or not known elsewhere in Sapindales): (1) three-celled
pollen grains, (2) a long, massive, irregularly bent funicle
(or, an ovule shifting its orientation from downward to
upward during its development), (3) tetrasporic 16-nucleate
Penaea-type female gametophyte, (4) unitegmic ovules, (5)
pseudoporogamy (pollen-tube(s) reaching the nucellar apex
for fertilization before the micropyle is formed), and (6) the
chalaza shifting its position near the concave side of the
seed.
The three-celled pollen grains are rare in Sapindales,
where two-celled pollen grains are prevalent. Except for
Biebersteinia, three-celled pollen grains occur only in
Azadirachta A. Juss. (Meliaceae) and Murraya Koenig ex
L. and Ruta L. in Rutaceae (Brewbaker 1967; see also Tobe
2011) (Table S1). Since no close relationship exists among
Biebersteinia, Meliaceae, and Rutaceae (see Fig. 1), it is
clear that evolution from two-celled to three-celled pollen
has occurred as a homoplasy in three separate lineages
within Sapindales.
The long, massive, irregularly bent funicle is pronounced in Biebersteinia orphanidis. Irregular bending of
the funicle occurs because an ovule shifts its growing
direction from downward to upward during its development. The long massive funicle of Biebersteinia recalls
those of Anacardiaceae, but Bachelier and Endress (2009)
summarized features of the funicles and ovules as follows:
ovules are apotropous, and the massive funicle is bent,
long, and forming a funicle-ovule complex, which has a
characteristic bridge (ponticulus) on the dorsal side of
the funicle that is connected with the lower end of the
pollen-tube transmitting tract in the styles at anthesis. Thus,
the funicles of Anacardiaceae are different from those of
Biebersteinia in their association with the ovule and style,
and all the other families in Sapindales have unspecialized
funicles.
The tetrasporic 16-nucleate Penaea-type female gametophyte was first reported by Kamelina and Konnova
(1990) in Biebersteinia multifida. We also observed the
Penaea-type female gametophyte in B. orphanidis and
documented in detail its development and structure. In
contrast, Nitrariaceae, Sapindaceae, Anacardiaceae, Burseraceae, Rutaceae, Simaroubaceae, and Meliaceae all show
female gametophyte development of the monosporic eight-

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612

nucleate Polygonum type. Obviously the Penaea-type


female gametophyte is restricted to Biebersteinia.
The ovule is unitegmic throughout its development in
Biebersteinia orphanidis. Kamelina and Konnova (1990)
also observed that the ovule is unitegmic in B. multifida, and
Corner (1976) and Boesewinkel (1997) described the ovules
of Biebersteinia (including B. multifida and B. orphanidis) as
bitegmic based on observations of seeds. In other families of
Sapindales bitegmic ovules are common, but unitegmic
ovules occur rarely in Anacardiaceae, Burseraceae, and
Rutaceae. In Anacardiaceae unitegmic ovules occur in a few
genera, i.e., Amphipterygium Schiede ex Standl., Anacardium L., Lithraea Miers ex Hooker and Arnott, Mangifera L.,
Pistacia, and Semecarpus L.f.; in Burseraceae they occur in
Canarium L., Commiphora Jacq., and Santiria Blume (for
review see Bachelier and Endress 2009); in Rutaceae they
occur in Glycosmis Correa (Boesewinkel and Bouman
1978). Looking at some of them in more detail, the ovules are
entirely unitegmic in Lithraea molleoides (Vell.) Engl.
(Carmello-Guerreiro and Paoli 2005), but basally unitegmic
and apically bitegmic in Anacardium occidentale L. (Copeland 1961), Pistacia spp. (Copeland 1955; Grundwag
1976), and Rhus mysurensis B. Heyne ex Wight and Arn.
(Kelkar 1958a). An apically bitegmic ovule also occurs in
Burseraceae: Commiphora sp. (Shukla 1954), Canarium
asperum Benth., and C. oleosum (Lam.) Engl. (Wiger 1935).
The occurrence of diverse ovules with respect to the development of integuments, as well as the sporadic distribution of
genera with entirely or partially unitegmic ovules in rather
derived clades of a family phylogeny, suggests that evolution
from bitegmy to unitegmy occurred independently not only
in Biebersteinia, but also in Anacardiaceae and Burseraceae
(for phylogenetic trees of Anacardiaceae, Burseraceae, and
Rutaceae see Pell 2004; Weeks et al. 2005; Groppo et al.
2008, respectively).
In Biebersteinia orphanidis fertilization is pseudoporogamous. This mode of fertilization was considered to play
a role in selecting from multiple pollen tubes that had
reached to the nucellar apex before the micropyle was
formed (Sogo and Tobe 2006), although we did not observe
more than one pollen tube reaching the nucellus in B. orphanidis. Kamelina and Konnova (1990) described fertilization as porogamous in B. multifida; however, for the
aforementioned reason, porogamy in B. multifida needs
confirmation. Pseudoporogamy is unknown elsewhere in
Sapindales, where porogamy is prevalent, except in Anacardiaceae, where chalazogamy is common (Bachelier and
Endress 2009).
Biebersteinia orphanidis demonstrated an interesting
developmental change of the position of the chalaza in
post-fertilization stages. As the seed develops from anatropous to slightly amphitropous, it brings the chalaza
toward the concave side. A similar developmental change

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J Plant Res (2014) 127:599615

is known in Anacardiaceae. In Harpephyllum Bernh. ex


Krauss, Rhus, and Schinus L., seeds have the chalaza or
hypostase on the concave side (von Teichman and van
Wyk 1988; von Teichman 1991; Carmello-Guerreiro and
Paoli 2005). It is uncertain, however, whether such a
developmental change in the position of the chalaza has
any similar function in Biebersteinia and Anacardiaceae.
Thus, while there is no embryological synapomorphy
common to Biebersteinia and any particular family in Sapindales, many autapomorphies exist in Biebersteinia. This
supports placing Biebersteinia in its own family, Biebersteiniaceae, as does molecular evidence. With regard to
relationships within Sapindales, molecular evidence
weakly suggested a sister-group relationship between
Biebersteiniaceae and the eight other families (Muellner
et al. 2007). Many of the aforementioned autapomorphies
of embryological characters in Biebersteiniaceae, combined with the lack of a synapomorphy with any other
sapindalean family, imply that Biebersteiniaceae may
represent one of the early divergent lineages of the Sapindales. However, based on data currently available for
embryological characters, there are no synapomorphies
confined to all of the families other than Biebersteiniaceae,
though Stevens (2001 onwards) suggests that a papillate
stigma is a synapomorphy of the eight other families of
Sapindales. More extensive morphological studies
throughout the order, as well as molecular analyses using
more sequence data, are needed to determine the relationship of Biebersteiniaceae within the Sapindales.
The modern Biebersteiniaceae consist of only five species. Molecular clocks suggest that while the stem lineage
of Biebersteiniaceae dates back to the Late Paleocene, the
crown-group diversified in the Oligocene and Miocene,
extending its range from the east (Central Asia) westwards
to Greece (Muellner et al. 2007). Muellner et al. (2007)
showed that Biebersteinia multifida and B. orphanidis,
which occur in geographically adjacent western regions,
are sister to each other in the family and noted that they
share tuberous rhizomes, instead of the supposedly ancestral condition of scarcely thickened rhizomes. Our analyses
showed that, although there was no clear difference in seed
coat structure among the three species examined, both B.
multifida and B. orphanidis differed from B. heterostemon
in the morphology and structure of the mature seeds. B.
multifida and B. orphanidis have large, albuminous mature
seeds with a curved embryo, while B. heterostemon has
much smaller, nearly exalbuminous mature seeds with an
embryo bent at a nearly right angle. In the light of general
trends of character evolution, the seed features of B. heterostemon, rather than those of B. multifida and B. orphanidis, appear to represent apomorphies. How has the
seed morphology diversified within the genus and family?
We would prefer to leave this subject to future research,

J Plant Res (2014) 127:599615

because data on seeds of B. emodii and B. odora are not yet


available, and because phylogenetic relationships among
the five species are unknown.

Appendix 1. Selected sources of data on embryological


features for individual families of Sapindales
Anacardiaceae: Bachelier and Endress (2007, 2009),
Carmello-Guerreiro and Paoli (2005), Copeland (1955,
1959, 1961), Copeland and Doyel (1940), Corner (1976),
Grimm (1912), Grundwag (1976), Grundwag and Fahn
(1969) Kelkar (1958a, b, 1961), Martnez-Palle and Herrero (1995), Robbertse et al. (1986), Shuraki and Sedgley
(1997), Srinivasachar (1940), von Teichman (1988, 1991),
von Teichman and van Wyk (1988); Biebersteiniaeae:
Boesewinkel (1997), Corner (1976), Kamelina and Konnova (1990); Burseraceae: Bachelier and Endress (2009),
Corner (1976), Wiger (1935); Kirkiacee: Bachelier and
Endress (2008); Meliaceae: Boesewinkel (1981), Corner
(1976), Garudamma (1956, 1957), Ghosh (1966a, b), Nair
(1958, 1959a, b), Nair and Kanta (1961), Narayana (1958),
Prakash et al. (1977), Wiger (1935); Nitrariaceae:
Kamelina (1994), Kapil and Ahluwalia (1963), Li and Fang
(2011), Li and Tu (1990a, b, 1991a, b); Rutaceae: Bacchi
(1943), Banerji (1954), Boesewinkel (1977, 1978; 1984),
Boesewinkel and Bouman (1978), Corner (1976), Desai
(1962), Johri and Ahuja (1957), Mauritzon (1935, 1936),
Narayana (1963); Sapindaceae: Banerji and Chaudhuri
(1944), Corner (1976), David (1938), Guerin (1901),
Haskell and Postlethwait (1971), Khushalani (1963), List
and Steward (1965), Mauritzon (1936), Nair and Joseph
(1960), Netolitzky (1926), van der Pijl (1957), Tobe and
Peng (1990), Weckerle and Rutishauser (2003, 2005),
Zhou and Liu (2012); Simaroubaceae: Corner (1976),
Nair and Joseph (1957), Nair and Sukumaran (1960),
Narayana (1957), Pfeiffer (1912), Tobe (2011), Wiger
(1935).
We are grateful to Peter H. Raven, Yang Zhong, Hongya
Gu, Yang Zhong, Li-Jia Qu, David Boufford, Ihsan AlShehbaz, Christopher Davidson, and Kaka Saman for their
assistance in getting materials and information used for the
present study. The study was supported by a Grant-in-Aid
for Scientific Research from the Japan Society for the
Promotion of Science (No. 25440208).

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