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Plant Systematics andEvolution ISSN 0378-2697Volume 292Combined 1-2 Plant Syst Evol (2010) 292:1-14DOI 10.1007/s00606-010-0388-9

Morphology, development and homologiesof the perianth and floral nectaries inCroton and Astraea (Euphorbiaceae-Malpighiales)

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ORIGINAL ARTICLE

Morphology, development and homologies of the perianthand floral nectaries in Croton and Astraea(Euphorbiaceae-Malpighiales)

Orlando Cavalari De-Paula • Maria das Gracas Sajo •

Gerhard Prenner • Ines Cordeiro • Paula J. Rudall

Received: 6 June 2010 / Accepted: 31 October 2010 / Published online: 26 November 2010

� Springer-Verlag 2010

Abstract New observations are presented on the ontogeny,

vasculature and morphology of both staminate and pistillate

flowers of Croton and Astraea. These data support earlier

hypotheses that the filamentous structures in pistillate flowers

represent reduced and transformed petals. Staminate flowers

of both genera possess five free nectaries, which are vascu-

larised by divergences of the sepal traces in Croton and

unvascularised in Astraea. In pistillate flowers, there are five

separate non-vascularised nectaries in Astraea, but in Croton

there is a single nectariferous disk that is vascularised by

divergences of the sepal traces. The nectaries are initiated late

in floral development, but their location indicates that they

could represent the outer stamen whorl transformed into

secretory staminodes. Other glandular structures occur in

pistillate flowers of most Croton species, resulting in flowers

with two secretory organ whorls. In these cases, the inner

whorl is formed by modified staminodes. Our observations

support the recent segregation of Astraea species from the

larger genus Croton. Despite strong similarities between the

two genera, there are clear structural differences, including

the presence of colleters in Astraea (absent in Croton),

moniliform trichomes on petals (rather than simple trichomes

in Croton), non-vascularised nectaries (vascularised in Cro-

ton) and reduced, non-secretory filamentous structures (well

developed and secretory in Croton).

Keywords Crotoneae � Euphorbiaceae � Flowers �Inaperturate crotonoids � Nectary evolution �Obdiplostemony � Petals � Staminodes

Introduction

As currently circumscribed, the family Euphorbiaceae

sensu stricto (order Malpighiales) is subdivided into four

subfamilies based on morphological and molecular studies:

Acalyphoideae, Cheilosoideae, Crotonoideae and Euphor-

bioideae (Wurdack et al. 2005; Tokuoka and Tobe 2006;

Tokuoka 2007; Davis et al. 2007; Wurdack and Davis

2009). The subfamily Crotonoideae is polyphyletic, and

some of its putative synapomorphies (such as crotonoid

pollen) are unsupported (Wurdack et al. 2005). Crotonoi-

deae includes 12 tribes and 74 genera from tropical and

temperate regions, distributed in 4 informal groups: Ade-

noclineae s.l., Gelonieae, the articulated crotonoids and the

inaperturate crotonoids (Webster 1994; Radcliffe-Smith

2001; Wurdack et al. 2005; Tokuoka and Tobe 2006;

Tokuoka 2007).

Croton and Astraea belong to the inaperturate croto-

noids, characterised by inaperturate pollen, ovules with

vascularised inner integument and petals present in at least

one of the floral types (i.e. pistillate or staminate flowers)

(Wurdack et al. 2005). Species of the large genus Croton

(ca. 1,200 tropical and subtropical species: Webster 1993;

Govaerts et al. 2000) are easily recognised by their star-

shaped or scaly trichomes, long or condensed monoclinous

inflorescences, frequent petiolar glands, and senescent

leaves that turn orange before abscising (Berry et al. 2005).

Croton was considered monophyletic with the exclusion of

section Astraea (elevated to the category of genus) and the

inclusion of Crotonopsis, Cubacroton, Eremocarpus,

O. C. De-Paula (&) � M. das Gracas Sajo

Universidade Estadual Paulista (UNESP), Rio Claro,

Sao Paulo 13506-900, Brazil

e-mail: orlandocavalari@gmail.com

G. Prenner � P. J. Rudall

Royal Botanic Gardens, Kew,

Richmond, Surrey TW9 3DS, UK

I. Cordeiro

Instituto de Botanica, Sao Paulo 04301-012, Brazil

123

Plant Syst Evol (2011) 292:1–14

DOI 10.1007/s00606-010-0388-9

Author's personal copy

Julocroton and Moacroton, which were previously

assigned to a tribe Crotoneae (Berry et al. 2005; van Ee

et al. 2008; Riina et al. 2009). The American genus Astraea

(10 species) is sister to Croton and shares many morpho-

logical features (Berry et al. 2005). Astraea is characterised

by the presence of deeply lobate leaves, glabrous floral

receptacle, staminate flowers with imbricate perianth mem-

bers, petals densely pilose at the base, stamen filaments

narrow and reddish, and stigma densely ramified. Its seeds

are quadrangular, rugose and carunculate (Miller and Web-

ster 1966; Berry et al. 2005; Caruzo and Cordeiro 2007).

Flowers of both Croton and Astraea are unisexual and

grouped in terminal inflorescences, usually with the pistil-

late flowers at the base and staminate flowers at the apex

(Caruzo and Cordeiro 2007). Male flowers possess a com-

plete perianth with calyx and corolla (Baillon 1858; Nair and

Abraham 1962; Venkata-Rao and Ramalakshmi 1968),

whereas in pistillate flowers only filamentous structures are

found in the positions of petals. These filamentous structures

have been interpreted as reduced petals, even though they

are non-vascularised (Baillon 1858; Nair and Abraham

1962; Venkata-Rao and Ramalakshmi 1968; Webster 1993;

Radcliffe-Smith 2001; Caruzo and Cordeiro 2007). Flowers

of both Croton and Astraea possess five-lobed nectaries

located opposite the sepals that are in staminate flowers and

range from entire to variably lobed in pistillate flowers

(Caruzo and Cordeiro 2007). Although the precise homol-

ogies of these nectaries are unknown, they have been

interpreted either as staminodes (Michaelis 1924) or nectary

disks (Caruzo and Cordeiro 2007). However, since nectary

disks develop from the floral receptacle (Weberling 1989)

and ontogenetic studies of Croton flowers are not yet

available, this interpretation remains to be tested.

In this paper, we investigate the morphology and floral

development of the pistillate and staminate flowers of

Croton and Astraea in order to clarify the homologies of

the different floral parts. Floral homologies among other

Euphorbiaceae are notoriously controversial (Prenner and

Rudall 2007; Prenner et al. 2008). Some Euphorbiaceae

possess perfect flowers (e.g. Jatropha), whereas in the

cyathium of Euphorbia and its allies, each flower is widely

interpreted as a single stalked stamen or a single naked

stalked pistil. Few previous studies have focused on the

flowers of non-cyathial euphorbiaceous taxa, which possess

less specialised inflorescences (Nair and Abraham 1962;

Venkata-Rao and Ramalakshmi 1968).

Materials and methods

Plants were collected in their natural environment, and

specimens were prepared and incorporated into the

Rioclarense Herbarium (HRCB) and the Herbario do

Estado ‘‘Maria Eneyda P. Kaufmann Fidalgo’’ (SP), as

follows: Astraea lobata (L.) Klotzsch, De-Paula 10

(HRCB)—Botucatu, Sao Paulo, Brazil; A. praetervisa

(Mull. Arg.) P.E. Berry, Cordeiro 3016 (SP)—Salvador,

Bahia, Brazil; Croton floribundus Spreng., De-Paula 25

(HRCB)—Botucatu, Sao Paulo, Brazil; C. fuscescens

Spreng., De-Paula 29 (HRCB)—Sao Paulo, Sao Paulo,

Brazil; C. glandulosus Mull. Arg., De-Paula 31

(HRCB)—Botucatu, Sao Paulo, Brazil; C. lundianus

(Didr.) Mull. Arg., De-Paula 26 (HRCB)—Jundiaı, Sao

Paulo, Brazil; C. piptocalyx Mull. Arg., De-Paula 28

(HRCB)—Campinas, Sao Paulo, Brazil; C. urucurana

Baill., De-Paula 30 (HRCB)—Botucatu, Sao Paulo,

Brazil; C. triqueter Lam., 3016 (SP)—Feira de Santana,

Bahia, Brazil. The specimens to be microscopically

examined were fixed in FAA 50 (Johansen 1940) and

subsequently stored in 50% ethanol.

For anatomical studies, entire flowers were dehydrated

through an ethanol series and embedded in Leica methac-

rylate. The material was sectioned using a rotary microtome

at 8–10 lm thickness. Sections were stained with 0.05%

toluidine blue at pH 4.7 (O’Brien et al. 1964) and mounted

with Entellan. Relevant features were recorded using a DFC

290 digital camera attached to a Leica DMLB microscope.

For scanning electron microscopy (SEM), flowers of

different developmental stages were carefully dissected,

dehydrated through an alcohol series and processed in an

Autosamdri-815B CPD CO2 critical point drier. The sam-

ples were mounted on aluminium stubs, coated with plat-

inum using an Emitech K550 sputter coater and examined

using a cold-field emission Hitachi SEM S-4700-II at the

Jodrell Laboratory, Royal Botanic Gardens, Kew.

Photographs were processed using Adobe Photoshop

software (Redwood City, CA). Illustrations were prepared

from digital photographs using Adobe Illustrator software

(Redwood City, CA).

Results

Ontogeny (Figs. 1, 2)

Flower primordia of both Astraea and Croton are sub-

tended by a single abaxial bract. Flowers are preceded by

two lateral bracteoles, which arise in a rapid sequence

(Fig. 1a–c). The sepal primordia develop in a spiral

sequence (Fig. 1c), except in C. fuscescens and C. triquet-

er, which have zygomorphic corollas and unidirectional

sepal initiation (Fig. 1d, e). Alternating with the sepals of

pistillate flowers, five primordia give rise to filamentous

structures (Fig. 1f–i). In C. triqueter, only one primor-

dium develops in an adaxial position (Fig. 1k, l), and in

C. fuscescens no such structure is formed (Fig. 1j).

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Fig. 1 Pistillate flower buds of Astraea and Croton (SEM).

a, b Croton fuscescens, floral primordium subtended by one abaxial

bract (a) and preceded by two lateral bracteoles (b). c C. glandulosus,

five sepal primordia initiated in a spiral. d Croton fuscescens,

unidirectional initiation of sepals starting on the adaxial side. e Crotonfuscescens, abaxial view on older bud. f A. lobata, five sepals initiated

in a spiral, five primordia of filamentous structures (arrow) and three

carpel primordia. Dotted line marks the symmetry plane. g C. pipto-calyx, fused carpels, filamentous structures alternating with the sepal

and nectary primordia (arrow). h Detail of (g) showing young

filamentous structure flanked by two nectary primordia. i Later stage

of development of the filamentous structure and nectaries. Nectary

primordia are fused. j Croton fuscescens, horseshoe-shaped nectary.

k C. triqueter, horseshoe-shaped nectary. Adaxial filamentous

structure (arrow). l C. triqueter, detail of adaxial filamentous

structures. m C. urucurana, syncarpous ovary and bifurcate stigmas

(arrows). n A. lobata, side view, two sepals removed to show

gynoecium, and basal filamentous structures alternating with nectar-

ies. o Detail of (n) showing the filamentous structures (arrows).

p C. urucurana, stamens are initiated instead of nectaries (arrow).

q Croton fuscescens, gynoecium at final stage of differentiation.

Coiled styles and the nectary at the base of the gynoecium.

b Subtending bract, bl bracteole, c carpel, f floral primordium, fsfilamentous structure, g gynoecium, n nectary, s sepal. Scale bars500 lm (i–k, n), 200 lm (f, g, l, m, p, q), 100 lm (c–e, h), 50 lm

(a–b, o)

Homologies of the perianth and floral nectaries in Croton and Astraea 3

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Alternating with the filamentous structures and opposite

the sepals, five pairs of primordia give rise to five nectaries

(Fig. 1g–i) that fuse at later stages to form a disk. In

C. fuscescens and C. triqueter, the nectary forms an arc

(Fig. 1j, k). In the pistillate flowers of Astraea, in addition

to the filamentous structures that alternate with the sepals,

there are other filamentous structures alternating with the

nectaries (Fig. 1n, o). In some pistillate flowers, the pri-

mordia that would normally be expected to give rise to

nectaries actually form stamens (Fig. 1p). The gynoecium

arises from three carpel primordia (Fig. 1f) that fuse into a

syncarpous ovary (Fig. 1g, m, p). The stigma is bifid

(Fig. 1m, n, p, q).

As in pistillate flowers, the staminate flowers of both

Astraea and Croton possess five sepal primordia that

develop in a spiral sequence. The petal primordia arise

simultaneously and alternate with the sepals (Fig. 2a). The

stamens of the outer whorl develop from primordia oppo-

site the sepals, and the inner stamen whorl alternates with

the outer one (Fig. 2b, c). Occasionally a single stamen is

observed in the centre of the flower (Fig. 2b). As in pis-

tillate flowers, nectaries develop late in ontogeny after the

development of the fertile structures (Fig. 2d).

Vasculature of pistillate flowers (Figs. 3, 4, 7)

In the pedicel of Astraea, there are ten vascular bundles

(Fig. 3b), which diverge at the receptacle into three rings of

bundles. The 15 bundles of the outer ring correspond with

the 5 sepal traces: 1 main and 2 synlateral per trace

(Fig. 3c). The five bundles of the intermediate ring lead to

five filamentous structures that are located inside and

alternating with the sepals (Fig. 3d). However, these bun-

dles do not extend into the interior of the filamentous

structures. Alternating with the five filamentous structures

are five non-vascularised nectaries positioned opposite the

sepals (Fig. 3d). The gynoecium is tricarpellate, and the

ovary is vascularised by three dorsal and six ventral bun-

dles (Fig. 3e, f). The styles are short, connate and vascu-

larised by the dorsal carpel bundles (Fig. 3g), which divide

and supply the ramified stigma (Fig. 3h).

In Croton species with actinomorphic flowers (C. flori-

bundus, C. lundianus, C. piptocalyx, C. urucurana), there

is a ring of ten vascular bundles in the pedicel (Fig. 4b). At

the base of the receptacle, five of these bundles diverge

towards the periphery (Fig. 4c) and ramify into three

bundles, each giving rise to the sepal traces (Fig. 4d). At

the same level of divergence, five alternating traces diverge

toward the filamentous structures, but do not extend into

them (Fig. 4d). Alternating with each of these traces, and

opposite the sepals, there are two traces that do not extend

to any flower part (Fig. 4d). The sepals, filamentous

structures and nectaries are united at the base of the flower

(Fig. 4e). The calyx is formed by five sepals, each one

supplied by five bundles (Fig. 4e). Alternating with the

sepals, there are five filamentous structures that are non-

vascularised (Fig. 4e), but each possess a vascular trace at

their base (Fig. 4d). All the filamentous structures appear

to be secretory (Fig. 4h–j). The nectary is five-lobed, with

the lobes opposite the sepals (Fig. 4e). The organisation

Fig. 2 Staminate flower buds

of Astraea and Croton (SEM).

a C. glandulosus with sepals

formed in a spiral,

simultaneously initiated petals

(asterisk) and stamens opposite

the sepals. b–c C. fuscescenswith petals (sepals removed)

and stamen primordia in three

whorls. d C. lundianus with

nectary primordium initiated

late, when stamens are already

well developed. a Stamen,

p petal, n nectary, s sepal. Scalebars 500 lm (c), 200 lm (a, d),

100 lm (b)

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Fig. 3 Vasculature of pistillate flower of Astraea lobata. a Flower in

longitudinal section indicating regions where transverse sections were

made. b Pedicel with ten vascular bundles. c Receptacle. Divergence

of sepal, petal and stamen traces. d Base of sepals with filamentous

structures (arrow) and nectaries (grey). e Median region of ovary.

f Uppermost part of ovary where ovule traces originate. g Style.

h Stigma. arrow Filamentous structures, at stamen traces, db dorsal

bundles of the carpel, dt dorsal traces of the carpel, ft traces of the

filamentous structures, n nectary, ov ovule, ot ovule trace, s sepal, sgstigma, sgb stigma bundle, sgt stigma trace, sl style, st sepal trace, vbventral bundles of the carpel, vt ventral carpel traces. Scale bars200 lm

Homologies of the perianth and floral nectaries in Croton and Astraea 5

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Fig. 4 Vasculature of pistillate flower of Croton. a–e Crotonfloribundus. a Flower in longitudinal section indicating regions where

transverse sections were made. b Pedicel with ten vascular bundles.

c Receptacle. Divergence of sepal traces. d Receptacle at base of

nectaries (grey). Divergence of petal and stamen traces. e Base of

sepals with filamentous structures (arrows) and nectaries (grey).

Divergence of the dorsal carpel traces. f–i Croton glandulosus.

f Flower in longitudinal section, indicating the regions where the

transverse sections were made. g Base of receptacle. Divergence of

filament traces and dorsal and ventral carpel traces. h Basal region of

flower. i Median region of flower. The nectaries are fused to the sepals

and are vascularised filamentous structures (arrow). j–m Croton

triqueter. j Flower in longitudinal section, indicating regions where

transverse sections were made. k Base of receptacle. The nectariferous

tissues (grey) opposite the groups of sepal traces. l Base of sepals with

stamen traces in abaxial region, carpellary bundles in central region

and a single non-glandular filamentous structure in the adaxial region

(arrow). m Basal regions of the sepals and gynoecium, and median

region of the nectary (grey). Floral zygomorphy. arrow Filamentous

structures, at stamen trace, db dorsal bundle, dt dorsal trace, fb bundle

of the filamentous structures, ft trace of the filamentous structures,

g gynoecium, lo locule, n nectary, ov ovule, s sepal, sb sepal bundle, sgstigma, sl style, st sepal trace, vb ventral bundle, vt ventral carpel trace.

Scale bars 400 lm (a–e, g–h, j–m), 200 lm (f, i)

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and vasculature of the gynoecium resembles that of

Astraea.

In C. glandulosus, there are ten vascular bundles in the

pedicel, as in Astraea. At the base of the receptacle, five of

these bundles diverge to the sepals (Fig. 4g, h), and five

alternating traces diverge to supply the five filamentous

structures (Fig. 4g–i). The calyx, the filamentous struc-

tures, the nectaries and the gynoecium are fused at the

flower base (Fig. 4h). The calyx is formed by five sepals,

each with a single vascular bundle (Fig. 4i). The filamen-

tous structures alternate with the sepals; they are vascu-

larised (Fig. 4h, i) and apparently secretory (Fig. 4h–j).

The nectaries are fused to the sepals and are vascularised

by branches of the sepal traces (Fig. 4h, i). The tricarpel-

late ovary is vascularised by three dorsal and three ventral

vascular bundles (Fig. 4h, i). The style is short and vas-

cularised by dorsal carpellary bundles, which ramify and

supply the stigmas (Fig. 4f).

The filamentous structures of C. floribundus, C. lundi-

anus, C. piptocalyx, C. urucurana and C. glandulosus

appear secretory (Fig. 4h–j), so in these species the pistil-

late flowers possess two whorls of secretory structures.

In C. fuscescens and C. triqueter with zygomorphic

flowers, there are ten vascular bundles in the pedicel. At the

base of the receptacle, five groups of traces diverge to

supply each of the five sepals (Fig. 4k). In the same region,

four areas of nectariferous tissue alternate with the sepal

traces (Fig. 4k). The zygomorphic calyx is formed by two

free adaxial sepals and three larger abaxial ones that are

connate at their bases (Fig. 4l). Each of the larger sepals

possesses 5–7 vascular bundles and each of the smaller

sepals only one (Fig. 4m). In C. triqueter, a single non-

vascularised filamentous structure alternates with the

smaller sepals (Fig. 4l). In C. fuscescens there are no fila-

mentous structures. In these two species, the nectary is

abaxial and semicircular, and supplied by branches of the

sepal traces (Fig. 4l). Gynoecium vasculature resembles

that of C. glandulosus, though the ovary is zygomorphic in

both C. triqueter and C. fuscescens (Fig. 7i).

Vasculature of staminate flowers (Fig. 5)

The staminate flowers of Astraea are actinomorphic and

possess a horseshoe-shaped vascular bundle in the pedicel

(Fig. 5b), which divides at the base of the receptacle into

ten bundles: the five external bundles extend to the sepals,

and the five alternating inner bundles extend to the petals

(Fig. 5c–f). The calyx consists of five sepals united at their

bases, each with a single vascular bundle (Fig. 5e). The

corolla consists of five free petals alternating with the

sepals (Fig. 5e, f). Alternating with the petals there are five

free nectaries, adnate to the androphore (Fig. 5d, e). In the

centre of the flower there is an androphore with three

alternating whorls of five stamens (Fig. 5f). The outer-

whorl stamens are located opposite the petals. The stamens

are curved inwards in the flower bud, so that the filaments

appear external in transverse sections (Fig. 5f). Each anther

is supplied by a single bundle that extends to one-third of

its length (Fig. 5a).

The staminate flowers of Croton show the same pat-

tern of vasculature as those of Astraea, though the

pedicel in Croton possesses a ring of bundles and not an

arc (Fig. 5h). The nectaries are vascularised (Fig. 5k), in

contrast to Astraea, which has non-vascularised nectaries

(Fig. 5e).

Floral morphology (Figs. 6, 7)

On the sepals of Astraea and Croton, there are both

secretory and non-secretory trichomes (Fig. 6a–d). Non-

secretory trichomes occur over the entire sepal surface

(Fig. 6a, d), whereas secretory trichomes are restricted to

the sepal margins (Fig. 6a–d). Secretory trichomes are

elongated in Astraea (Fig. 6a) and spherical in Croton

(Fig. 6d). They are multicellular, covered by elongated

epidermal cells and contain crystalliferous idioblasts in

Astraea (Fig. 6b, c) and resemble colleters. In C. lundi-

anus, the sepals are covered by an ornamented epidermis

(Fig. 6e).

In staminate flowers of Astraea and Croton, the petals

have only non-secretory trichomes (Fig. 6j, l). In Astraea,

the trichomes are moniliform and occur at the petal base

(Fig. 6j, k); in Croton they are simple and found at both the

base and apex of the petals (Fig. 6l). In C. lundianus the

entire petal surface is papillate (Fig. 6m).

In the pistillate flowers of Astraea, the filamentous

structures are reduced and alternate with the sepals (com-

pare Fig. 6g). The filamentous structures of Croton are

larger than the Astraea trichomes and supplied by a single

vascular bundle (Fig. 6i).

The nectaries, here interpreted as secretory staminodes,

possess a uniseriate epidermis (Fig. 6q–s) with occasional

stomata (Fig. 6n); the epidermis is covered by epicuticular

wax in the shape of bars (Fig. 6o). Internally, Astraea

nectaries consist of thick-walled parenchymatous cells of

dense cytoplasm in pistillate flowers (Fig. 6p) and cells

with amyloplasts in staminate flowers (Fig. 6r). In Croton,

the nectaries of both pistillate (Fig. 6q) and staminate

(Fig. 6s) flowers are formed by three cell types: cells rich

in amyloplasts, cells containing phenolic compounds and

idioblasts containing druses.

Both flower types in Astraea also contain other fila-

mentous structures (Fig. 7a–c), which alternate with the

nectaries in the pistillate flowers (Fig. 7a, b), but lack any

specific pattern of distribution in the staminate flowers

(Fig. 7c).

Homologies of the perianth and floral nectaries in Croton and Astraea 7

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Fig. 5 Vasculature of staminate flowers of Astraea and Croton.

a–f A. praetervisa. a Flower in longitudinal section, indicating

regions where transverse sections were made. b Pedicel with

U-shaped vascular bundle. c Base of receptacle from which the sepal

traces diverge. d Basal region of flower. Divergence of the petal

bundles. e Median region of nectaries and basal region of androphore.

f Median region of flower. g–l Croton piptocalyx. g Flower in

longitudinal section, indicating regions where transverse sections

were made. h Pedicel with eight vascular traces. i Base of receptacle.

j Median region of receptacle. k Detail of base of nectary. l Median

region of nectaries and basal region of androphore. Divergence of the

stamen traces, with the outer whorl opposite the petals. a Stamen, afandrophore, at1 stamen traces of the external whorl, at2 stamen traces

of the second whorl, at3 stamen traces of the internal whorl, n nectary,

nb nectary bundle, p petal, pb petal bundle, pt petal trace, s sepal, sbsepal bundle, st sepal trace. Scale bars 400 lm (g–l), 200 lm (a–f)

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In both Astraea and Croton, the stamen filaments are

curved at early stages (Fig. 7c), becoming erect later

(Fig. 7d), and anthers are tetrasporangiate and introrse

(Fig. 7e). The tricarpellate ovary is glabrous in Astraea

(Fig. 7f) and pilose in Croton (Fig. 7g–i). The carpels are

symmetrical except in C. fuscescens and C. triqueter with

zygomorphic flowers and smaller adaxial carpels (Fig. 7h, i).

The receptive region of the stigma is papillate and

Fig. 6 Detail of mature flowers of Astraea (a–c, f, j–k, p, r) and

Croton (d, e, g–i, l–o, q, s). a Adaxial view of sepal of A. lobata.

b–c A. lobata, detail of trichome indicated in (a) in longitudinal

(b) and transverse section (c) with crystalliferous idioblasts (doublearrowheads). d Adaxial view of sepal of C. lundianus. e Detail of

sepal epidermis of C. lundianus. f Pistillate flower of A. lobata with

one of the filamentous structures that alternate with the sepals (sepals

and nectaries removed). g Filamentous structure in pistillate flower of

C. glandulosus. h–i C. glandulosus, transverse sections in the median

region (h) and basal region (i) of the structure shown in (g).

Crystalliferous idioblasts (double arrowheads). j Petal of A. praeter-visa with hairs along the basal rim. k Detail of (j), showing

moniliform hair. l Petal of C. lundianus with hairs at the base and

apex. m C. lundianus. Detail of papillate petal epidermis. n Nectary

of C. glandulosus with stomata and o epicuticular wax crystals.

p–s Transverse sections of the nectary. p Pistillate flower of A. lobata.Cell with phenolic compounds (arrowhead). q Pistillate flower of C.urucurana. Cells with phenolic compounds (arrowhead), cells rich in

amyloplasts (arrow) and crystalliferous idioblast (double arrowhead).

r Staminate flower of A. praetervisa. Cells with phenolic compounds

(arrowhead) and cells rich in amyloplasts (arrow). s Nectary of the

staminate flower of C. piptocalyx. Cells with phenolic compounds

(arrowhead), cells rich in amyloplasts (arrow) and crystalliferous

idioblast (double arrowhead). cr Crystal, fs filamentous structure,

g gynoecium, n nectary, s sepal, sm stomata, tr trichome. Scale bars500 lm (a, d, l), 250 lm (j), 100 lm (f–g, n), 50 lm (b–c, h–i, q),

25 lm (e, m, p, r, s), 10 lm (k, t), 5 lm (o)

Homologies of the perianth and floral nectaries in Croton and Astraea 9

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Fig. 7 Details of mature flowers of Astraea (a–c, f, j–l) and Croton(d–e, g–i, m–o). A. lobata (a–c, f, j–l), C. glandulosus (d),

C. piptocalyx (e), C. urucurana (g), C. triqueter (h–i), C. lundianus(m–o). a Pistillate flower of A. lobata with two sepals and nectaries

removed. Filamentous structures (arrow) alternating with the nectar-

ies. b Detail of (a) showing filamentous structures. c–d A. lobata,

aspect of young staminate flower (c) and a single mature stamen (d).

Stamen with filamentous structures at the base. e Transverse section

of anther of C. piptocalyx. f–o Pistillate flower. f A. lobata: transverse

section of the ovary. Glabrous surface. g C. urucurana: transverse

section of the ovary. Hirsute surface. h–i C. triqueter: transverse

sections of the ovary in the median region (h) and basal region (i).Arrow pointing to the adaxial smaller carpel. j–l Stigma of A. lobata:

general aspect (j) and its receptive region in frontal view (k) and in

transverse section (l). m–o Stigma of C. lundianus: general aspect

(m) and its receptive region in frontal view with pollen grain and

papillate surface (arrow) (n) and in transverse section (o). g Gynoe-

cium, n nectary, ov ovule, po pollen grain, s sepal. Scale bars 500 lm

(a, c, j), 400 lm (g), 250 lm (d, m), 200 lm (b, e–f, h–i), 100 lm

(k–l), 50 lm (n), 25 lm (o)

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secretory in Croton (Fig. 7m–o), and formed by elongated

non-secretory cells in Astraea (Fig. 7j–l).

Discussion

Homologies of filamentous structures in flowers

of Croton and Astraea (Fig. 8)

Our observations of floral ontogeny and vasculature lead us

to propose new interpretations for the filamentous struc-

tures of the pistillate flowers of Croton and Astraea. Distal

to the sepals and alternating with them, five primordia give

rise to five petals in staminate flowers and to five fila-

mentous structures in pistillate flowers (Fig. 8). In both

Croton and Astraea, these filamentous structures are

unvascularised; traces of associated vascular tissue at their

bases extend into the filamentous structures only in

C. glandulosus. Based on Puri’s (1951) floral vascularisa-

tion model, in which vascular traces that alternate with

sepals are interpreted as petal traces, the filamentous

structures of the pistillate flowers of Croton and Astraea

represent reduced petals. This supports the view of Nair

and Abraham (1962) and Venkata-Rao and Ramalakshmi

(1968), who described the presence of a corolla in pistillate

flowers of Croton. The reduction of the vascular bundles

Fig. 8 Floral diagrams and floral formulae of pistillate and staminate flowers of Croton and Astraea. Floral formulae follow the style proposed

by Prenner et al. (2010)

Homologies of the perianth and floral nectaries in Croton and Astraea 11

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observed here (filamentous structures vascularised in

C. glandulosus but possessing only basal traces in other

species) indicates a reduction of the internal perianth

whorl, which consists of five elements in some Crotonoi-

deae and is entirely absent from others. The inner perianth

whorl is represented by a single petal in C. fuscescens.

Filamentous structures similar to those of Astraea and

Croton also occur in the pistillate flowers of Hevea and

Codiaeum (Crotonoideae) and in Micrococca, Acalypha

and Mercurialis (Acalyphoideae). These structures were

interpreted as staminodes in Hevea (Nair and Abraham

1962), reduced petals in Codiaeum (Nair and Abraham

1962; Venkata-Rao and Ramalakshmi 1968) and nectaries

in Micrococca (Venkata-Rao and Ramalakshmi 1968) and

Mercurialis (Baillon 1858).

Sepals and petals are not consistently present in

Euphorbiaceae flowers. In most Crotonoideae there are two

perianth whorls, but staminate flowers of Hevea, Manihot

and Baliaspermum and pistillate flowers of Hevea and

Manihot possess only a calyx whorl (Nair and Abraham

1962; Venkata-Rao and Ramalakshmi 1968), Garcia pos-

sesses a calyx whorl and two-whorled corolla in both

flower types (Baillon 1858), and Croton and Astraea pos-

sess petals transformed into filamentous structures, as

described here. Flowers of subfamilies Cheilosoideae and

(most) Acalyphoideae possess only a calyx (Radcliffe-

Smith 2001). In Euphorbioideae, a perianth is either

entirely absent, or perianth-like structures are present, or

they possess a calyx of 1–8 sepals (in staminate flowers) or

1–6 sepals (in pistillate flowers) (Radcliffe-Smith 2001;

Prenner and Rudall 2007; Prenner et al. 2008). Wurdack

et al. (2005) hypothesised that in Euphorbiaceae the peri-

anth has an important role in floral specialisation. They

highlighted the occurrence of petals in at least one of the

floral types of inaperturate crotonoids as a synapomorphy

for this group. This is corroborated with the evidence

presented here that the filamentous structures of the pis-

tillate flowers of Croton and Astraea represent reduced and

transformed petals.

Homologies of nectaries

Our observations also allow new interpretations for the

nectaries of both pistillate and staminate flowers of Croton

and Astraea. Within the perianth, flowers of both Croton

and Astraea possess structures described either as a ‘‘nec-

tariferous disk’’, based on their morphology (Caruzo and

Cordeiro 2007), or as ‘‘extrastaminal nectaries’’, based on

their position (Webster 1993; Bernardello 2007). Our study

shows that staminate flowers of both Croton and Astraea

possess five free nectaries, which are vascularised by

divergences of the sepal traces in Croton and unvascular-

ised in Astraea. In pistillate flowers of Astraea the nectaries

are free and non-vascularised, but in Croton they are fused

into a nectariferous disk that is vascularised by divergences

of the sepal traces. The nectaries originate from five sep-

arate primordia that become evident only after the other

elements of the flower are formed (Figs. 1g, h, 2d). They

are located opposite the sepals and alternate with the petals

(in staminate flowers) or alternate with the filamentous

structures (in pistillate flowers).

Despite the late initiation of the nectaries, their posi-

tion indicates that they could represent the outer stamen

whorl transformed into secretory staminodes (Fig. 8). Our

observations that in most species the nectaries are vas-

cularised by divergences of the sepal traces and the out-

ermost stamens are located opposite the petals reinforce

this interpretation. Furthermore, occasional pistillate

flowers of C. urucurana develop stamens in positions

opposite the sepals that are normally occupied by nec-

taries (Fig. 1p).

Floral nectaries are relatively common in other Croto-

noideae, occurring in both flower types in Jatropha, Man-

ihot, Baliospermum, Codiaeum and Aleurites (Baillon 1858;

Nair and Abraham 1962; Venkata-Rao and Ramalakshmi

1968) and in the staminate flowers of Ricinocarpus (Baillon

1858). As in Croton and Astraea, the nectaries of the sta-

minate flowers are located opposite the sepals, and the outer

stamens lie in the same sector as the petals. These obser-

vations suggest that in all these genera the nectaries are

homologous with the outer stamen whorl and that secretory

staminodes are a synapomorphy for Crotonoideae.

Floral nectaries of unknown nature occur in other

Euphorbiaceae, such as Acalyphoideae (Clutia, Micrococca,

Chrozophora, Argythamnia and Ditaxis) and some

Cheilosoideae (Wurdack et al. 2005). Within Euphorbioi-

deae, the nectaries related to reproduction (nuptial nec-

taries) are apparently not of floral origin, but are strictly

extrafloral because they are associated with the cyathium,

which is normally interpreted as an inflorescence (Prenner

and Rudall 2007). Staminodial nectaries are also common

in other families of the order Malphigiales, such as Bon-

netiaceae (Dickinson and Weitzman 1998), Caryocaraceae

(Prance and Freitas da Silva 1973; Dickinson 1990),

Clusiaceae (Robson 1961; Ronse Decraene and Smets

1991), Chrysobalanaceae (Matthews and Endress 2008),

Linaceae (Brown 1938; Cronquist 1981), Passifloraceae

(Cronquist 1981; Bernhard 1999) and Violaceae (Smets

1986; Vogel 1998; Freitas and Sazima 2003). However,

staminodial nectaries are absent from Rafflesiaceae and

Peraceae, the putative sister families of Euphorbiaceae

(Davis et al. 2007; Wurdack and Davis 2009).

In addition to staminodial nectaries, nectariferous

structures occur in pistillate flowers of most Croton spe-

cies, resulting in flowers with two secretory whorls, as

Freitas et al. (2001) reported for Croton sarcopetalus. In

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these cases, the outer secretory whorl is filamentous and

formed by transformed petals, and the inner one is necta-

riferous and formed by modified staminodes. C. triqueter

possesses a single filamentous structure between the two

adaxial sepals, but this structure is lacking in C. fuscescens,

in which a continuous horseshoe-shaped nectary is formed

internally and in an abaxial position. In these cases, the

early developing nectary lobes alternate with the sepal

traces (Fig. 4k) so the nectaries are probably petal-derived

structures, though this interpretation requires further

investigation.

The function of the floral nectaries in Croton is not yet

fully understood. In C. suberosus, a supposed anemophi-

lous species (Domınguez and Bullock 1989), the nectaries

do not appear to be essential for pollination. However, in

the entomophilous C. sarcopetalus, both the outer floral

nectaries (here interpreted as petals) and the inner nectaries

(here interpreted as staminodes) secrete fructose and glu-

cose at anthesis. Furthermore, the external nectaries remain

secretory in the fruit when they secrete glucose only

(Freitas et al. 2001).

Segregation of Astraea from Croton is supported

by floral structure

Although Astraea and Croton are sister genera and could

be placed together without loss of monophyly, our results

support the recent segregation of Astraea species from the

larger genus Croton because there are considerable mor-

phological and anatomical differences between the two

genera. These differences include the presence of colleters

in Astraea (absent in Croton), moniliform trichomes on

petals in Astraea (simple in Croton) and non-vascularised

nectaries (vascularised in Croton). Additionally, the fila-

mentous structures of Astraea, which we interpret as

petals, are highly reduced and non-secretory, in contrast

to Croton with well-developed, secretory filamentous

structures.

In Astraea, besides the filamentous petals, other fila-

mentous structures are initiated after the staminodial nec-

taries (Fig. 1n, o) and alternate with them (Fig. 7a–c).

These filamentous structures could represent an additional

whorl of staminodes, but the absence of vasculature does

not fully support this hypothesis. Endress (2008) noted that

organs that have decreased in size during evolution and lost

their original function frequently become labile in number.

This could be the case in some instances in the current

study (e.g. in Astraea lobata), where the number of fila-

mentous structures does not correspond with the original

petal number.

Acknowledgments The authors thank J. Marzinek, C.J. Campos,

J. Lombardi and L.C. Bernacci for their help with field work. O.C.

De-Paula acknowledges financial support (2006/61641-0) from the

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)

and the Kew Latin American Research Fellowship scheme. We thank

Peter Endress and an anonymous reviewer for their critical and

helpful reviews.

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