Biebersteinia EMBRYOLOGY_Yamamoto 2014

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R E G U L A R P A P E R

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 consid-ered 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 cor-

roboration 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 Sa-

pindales because of the presence of anther tapetal cells with

polyploid nuclear mass and non-fibrous exotegmen. Fur-

ther, the genus is characterized by three-celled pollen

grains, tetrasporic 16-nucleate Penaea-type female game-

tophyte, unitegmic ovules, pseudoporogamy, and the cha-

laza shifting its position near the concave side in the post-

fertilization stage. A considerable number of autapomor-

phies, 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

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 Zygo-

phyllaceae 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 Bieber-

steiniaceae (Takhtajan 1986; Dahlgren 1989). However,

since molecular evidence was published at the end of the

20th century (Bakker et al. 1998), the Angiosperm Phy-

logeny Group (APG 1998; APGII 2003; APGIII 2009) has

consistently accepted the monogenetic family Bieberstei-

niaceae in Sapindales, while placing Geraniaceae in Ge-

raniales. 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

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10265-014-0645-z ) contains supplementarymaterial, 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: [email protected]

D. D. Vassiliades

24 Issiodou St, 10674 Athens, Greece

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J Plant Res (2014) 127:599–615

DOI 10.1007/s10265-014-0645-z

http://dx.doi.org/10.1007/s10265-014-0645-zhttp://dx.doi.org/10.1007/s10265-014-0645-z


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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 Bieber-

steinia. Embryological data provide more than 50 charac-

ters associated with the development of anthers, ovules,

and seeds and thus have contributed to a better under-

standing 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 angio-

sperms, occurring in Penaeaceae (Myrtales) (Stephens

1909; Tobe and Raven 1984a) and some Euphorbiaceae

and Malpighiaceae (Malpighiales) (supplementary infor-

mation in Tobe and Raven 2011; see also Endress et al.

2013). The occurrence of the Penaea-type female game-

tophyte 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,

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

multifida as unitegmic. Consequently, there is disagree-

ment 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 con-

firm 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 Bieber-

steinia fits in the order morphologically, and, if so, how the

genus is related to other Sapindales.

Materials and methods

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

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)

Table 1 Species studied of Biebersteinia, collection information, and

developmental stages

Taxon Collection Developmental

stage

Biebersteinia

heterostemon

Maxim.

China, Dagzi county

(294606.500N 9153031.900E).

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

Biebersteinia

multifida DC.

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

Biebersteinia

orphanidis

Boiss.

Greece, Peloponnese, Mount

Killini. D. Vassiliades133

(ATHU)

Flower buds to

mature fruits

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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. het-

erostemon, 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 4–5 lm were stained with Heidenhain’s

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 sectionsand 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 hypo-

chlorite (NaClO) at room temperature overnight. After

rinsing two or three times in water, the ovules were mac-

erated 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

K 3PO4 for 2–3 h and observations were made using fluo-

rescence 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 TM-

1000.

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).

Therefore, wall formation conforms to the ‘‘Dicotyledon-

ous’’ 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 andendothecium 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 persis-

tent. Anther dehiscence takes place by longitudinal slits,

with each slit common to two microsporangia of the theca

(Fig. 2 j).

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 theovary (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 game-

tophyte 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 stain-

ing cytoplasm around the nuclei. As the female gameto-

phyte 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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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 gameto-

phyte, the apical dermal cells of the nucellus may, or maynot 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 develop-

ing 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 hypos-

tase (see Figs. 31 and 32 in Boesewinkel 1997, p. 288).

Integuments

The ovule is unitegmic (Fig. 4c–f), as described by

Kamelina and Konnova 1990) for the ovule of Bieber-

steinia multifida. The integument is initially two cell layers

thick. Cells of the inner epidermis further divide pericli-

nally, 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 differen-

tiate 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 throughthe 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 fertil-

ized and subsequently degenerated (Fig. 5a–f). Degenera-

tion 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. Fig-

ure 5b–f shows serial longitudinal sections of a young seed

that has an eight-celled proembryo with eight free endo-

sperm 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 devel-

ops, 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 endo-

sperm 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

bFig. 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 Trans-

verse 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

f –h

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massive tissue (Fig. 6e, f, h, i). But in the case of B. het-

erostemon, cells of the endosperm do not appear to have

cytoplasm (Fig. 6i). Thus, the mature seeds of B. heteros-

temon are nearly exalbuminous.We did not examine embryogenesis in detail, but frag-

mentary data on early and later embryogenesis indicated

that it proceeds normally to form globular and dicotyle-

donous embryos (Fig. 6a–d). The proembryos that we

observed in three young seeds were all transversely ellip-

soid to ellipsoid with no conspicuous suspensor (Fig. 6b).

They become dicotyledonous later (Fig. 6d–i). The coty-

ledons are massive (Fig. 6e–i), building nearly four fifth of

the whole embryo in mature seeds (Fig. 5e–g). The

embryos in mature seeds curve to various degrees (Fig. 6f–

i). In the case of mature seeds in Biebersteinia heteroste-

mon, 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 peri-

carp (Fig. 7b, c). The size of the mature mericarp is dif-

ferent in the three species investigated: about 6.0–6.5 mm

long and 4.5–5.2 mm wide (measured from side to side) in

Biebersteinia orphanidis, 5.3–5.8 mm long and

3.6–4.1 mm wide in B. multifida, and 2.3–2.8 mm long and

1.5–2.0 mm wide in B. heterostemon. The fruit wall is

about 270–300 lm thick in B. orphanidis, 220–250 lm

thick in B. multifida, and 85–110 lm thick in B. heteros-

temon. In contrast, the seed coat (i.e., the developed

integument) is very thin, about 15–20 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 epi-

dermis (endotesta) (Fig. 7d) (we applied the testal termi-

nology 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. 7f–i). En-

dotestal cells are small, but their inner and radial walls are

thickened (Fig. 7f–i). Since the endotesta (more strictly,

the inner epidermis of the integument) is the best devel-oped mechanical layer, the seed coat is ‘‘endotestal’’ (ter-

minology 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 uncer-

tainty 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 summa-

rized 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*.

bFig. 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. c–i LSs of ovules, showing develop-

ment of nucelli and female gametophytes c Ovule with an archespo-

rial cell. d Ovule with a primary parietal cell and a primary

sporogenous cell. e Ovule with a megaspore mother cell. f Two-

nucleate 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 f –h

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microspore mother cell cytokinesis simultaneous; micro-

spore tetrads predominantly tetrahedral*; pollen grains

three-celled when shed.

Ovule anatropous* and crassinucellate*, having a long

massive funicle which is bent irregularly*. Ovule arche-

sporium one-celled, dividing a primary parietal cell and a

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 a–e

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primary sporogenous cell; a three to four cell layer parietaltissue 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 pres-

ent ( 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 obtu-rator formed*; micropyle not formed by the time of fer-

tilization, 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

Fig. 5 Development of female gametophytes of Biebersteinia or-

phanidis 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. b–f Five serial LSs of the female gametophyte in a post-

fertilization 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 a–f

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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 mechan-

ical cell ayer developed from the inner epidermis*. Exot-

estal 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 (supplemen-

tary data with online version of this article). We also

compared the embryological features of Biebersteinia with

those of families of the Huerteales, Malvales, and Brassi-

cales, 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 Neurada-

ceae, 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. Embryo-

logical 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

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 char-

acters, 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 Brassi-cales, Huerteales, and Malvales anther tapetal cells are

binucleate, as in many other angiosperm families. How-

ever, 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 reportedto 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, Toxi-

codendron 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 (Sa-

pindales) anther tapetal cells are binucleate at least in

Boswellia serrata Roxb. (Narayana 1959) and Garuga

pinnata Roxb. (Narayana 1960), but they remain uninu-

cleate 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/Tropaeola-

ceae). Within Sapindales the binucleate condition is com-

mon 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.

bFig. 6 Development of seeds in Biebersteinia. a–g 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 Trans-

verse 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 e–h, 500 lm in a and i , 200 lm in d, and 100 lm in b

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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 (Huerte-

ales) and Neuradaceae (Malvales) have a fibrous exoteg-

men (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 char-

acteristic of Sapindales.

Thus, both the possession of anther tapetal cells with a

polyploid nuclear mass and the lack of the fibrous exo-

tegmen, which are found in Biebersteinia, are very likely

synapomorphies of the Sapindales. Embryological evi-

dence 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

persistent; endothecium fibrous; middle layers ephemeral;

tapetum glandular; cytokinesis in the microspore mother

cell simultaneous; microspore tetrads predominantly tet-

rahedral; 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 pro-

nounced in Biebersteinia orphanidis. Irregular bending of

the funicle occurs because an ovule shifts its growing

direction from downward to upward during its develop-

ment. 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 game-

tophyte 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, Burs-

eraceae, Rutaceae, Simaroubaceae, and Meliaceae all show

female gametophyte development of the monosporic eight-

bFig. 7 Fruits and seed coat development and structure in Bieberstei-

nia. a–g B. orphanidis. h B. multifida. i B. heterostemon. a Infruct-

escence. b Lateral view of the whole mature mericarp. c Median

longitudinal hand section of the whole mature mericarp. d Longitu-

dinal 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

d–i

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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., Anacardi-

um 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 unitegmicand apically bitegmic in Anacardium occidentale L. (Co-

peland 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 devel-

opmentof integuments, as well as thesporadic 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 pseudopor-

ogamous. 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. or-

phanidis. Kamelina and Konnova (1990) described fertil-

ization 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 Ana-

cardiaceae, 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 anat-

ropous to slightly amphitropous, it brings the chalaza

toward the concave side. A similar developmental change

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 synapomorphycommon to Biebersteinia and any particular family in Sa-

pindales, many autapomorphies exist in Biebersteinia. This

supports placing Biebersteinia in its own family, Bieber-

steiniaceae, 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, com-

bined with the lack of a synapomorphy with any other

sapindalean family, imply that Biebersteiniaceae may

represent one of the early divergent lineages of the Sa-pindales. 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 relation-

ship of Biebersteiniaceae within the Sapindales.

The modern Biebersteiniaceae consist of only five spe-

cies. 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 ances-

tral 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. het-

erostemon, rather than those of B. multifida and B. or-

phanidis, 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,

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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), Martı́nez-Pallé and Her-

rero (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 Kon-

nova (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), Guérin (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 Al-

Shehbaz, 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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