ORIGINAL PAPER

Ontogenesis of the pseudomonomerous fruits of Acrocomiaaculeata (Arecaceae): a new approach to the developmentof pyrenarium fruits

Hellen Cassia Mazzottini-dos-Santos • Leonardo Monteiro Ribeiro •

Maria Olıvia Mercadante-Simoes • Bruno Francisco Sant’Anna-Santos

Received: 6 January 2014 / Revised: 6 September 2014 / Accepted: 30 October 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract

Key message The formation of an oleaginous palm

fruit was described by anatomical and physiological

evaluations that allowed us to correlate and associate

pericarp and seed ontogenesis and define the develop-

mental phases.

Abstract The pseudomonomerous pyrenarium fruits of

Arecaceae demonstrate complex and slow development

pathways, and accumulate large quantities of energy

reserves, making them of economic interest. Very little is

known about the association between pericarp and seed

development in this family. We characterize here the

ontogenesis of Acrocomia aculeata fruits, a neotropical

oleaginous palm, define their developmental phases,

investigate the relationship between embryogenesis and

fruit development, and describe the formation of struc-

tures related to dormancy and reserve accumulation. The

development of the pistillate flowers and fruit structures

were accompanied over time and evaluated biometrically,

anatomically, and through histochemical tests. Bromato-

logical evaluations were performed on the mesocarp

and seeds during their reserve accumulation phases. A.

aculeata flowers are tricarpellate and syncarpous,

although normally only a single ovule develops; the other

ovules degenerate and become incorporated into the

pyrene. The seed is pachychalazal and embryogenesis is

precocious in relation to fruit development. The exocarp

is the first pericarp structure to attain maturity, while the

pyrene undergoes significant lignification, except for the

region near the abscission zone and acquires a petrous

consistency. The development of the endocarp is restric-

ted to the germination pore plate and seed operculum, and

is associated with dormancy restrictions. The accumula-

tion of lipids in the mesocarp occurs near the time of

abscission. A. aculeata fruits require approximately one

year for full development, which occurs in three phases:

the histo-differentiation of the pericarp; seed maturation;

and mesocarp maturation.

Keywords Fruit ontogenesis � Embryogenesis � Pyrene �Oleaginous plants � Palms

Introduction

Pseudomonomerous fruits, which contain only a single

seed, originated from tricarpellate ovaries, are relatively

common in palms (Mahabale and Biradar 1967; Biradar

and Mahabale 1968; Uhl and Moore 1971; Orozco-Segovia

et al. 2003; Reis et al. 2012). Ontogenetic analyses of the

formation of these characteristic fruits in Arecaceae have

been performed in only a few species of the tribes Boras-

seae (Romanov et al. 2011) and Eugeissoneae(Bobrov et al.

2012). In such cases, the use of term pyrenarium was

proposed for classifying the fruits, when they had a rigid

structure (pyrene) surrounding the seeds and developed

from syncarpous gynoecium. A number of distinct pro-

cesses, such as the complex formation of the pyrene, the

origin of the germination pore, reserve accumulation, and

Communicated by J. Lin.

H. C. Mazzottini-dos-Santos � B. F. Sant’Anna-Santos

Instituto de Ciencias Agrarias, Universidade Federal de Minas

Gerais, Avenida Universitaria, 1000,

CEP: 39.404-006 Montes Claros, Brazil

L. M. Ribeiro (&) � M. O. Mercadante-Simoes

Departamento de Biologia Geral, Universidade Estadual de

Montes Claros, CEP: 39401-089 Montes Claros, Brazil

e-mail: leomrib@hotmail.com

123

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DOI 10.1007/s00468-014-1104-0

seed formation are crucial for the constitution of this type

of fruit but are still poorly understood.

Embryogenesis in Arecaceae is unique and results in the

formation of a microscopic embryonic axis surrounded by

the cotyledonary petiole and a leaf blade with distinct

haustorial functions (Haccius and Philip 1979; DeMason and

Thomson 1981; Genovese-Marcomini et al. 2014). Our

knowledge of these processes is still reasonably scarce,

principally concerning the initial phases of fruit formation

(Mahabale and Biradar 1967; Kulkarni and Mahabale 1974),

and there have been no studies in the family focusing

simultaneously on embryogenesis and fruit development.

Dormancy is commonly observed in palms and has a

strong structural component (Orozco-Segovia et al. 2003;

Neves et al. 2013). Embryo growth and seedling develop-

ment are restricted in many palm species by the tissues that

fill the germination pore in the pyrene (Hussey 1958; Ne-

ves et al. 2013) or by the seed operculum (Myint et al.

2010; Ribeiro et al. 2011, 2013; Oliveira et al. 2013).

Characterizations of the ontogenesis of these structures will

certainly contribute to our knowledge of dormancy in this

family and possibly provide strategies for overcoming it.

Acrocomia aculeata (Arecoideae, Cocoseae), the macaw

palm, is widely distributed in the tropical Americas and well-

known for the high concentrations of high-quality oils stored

in the mesocarp and seeds that can be used to produce bio-

fuels (Hiane et al. 2005; Moura et al. 2010; Manfio et al.

2011); it is considered the second most productive oleagi-

nous plant yet described, and is well-adapted to dry tropical

environments such as the Cerrado (neotropical savanna)

biome, where vast under-used areas of land could be incor-

porated into productive landscapes (Moura et al. 2010;

Ribeiro et al. 2012a; Pires et al. 2013). The endocarp itself

contains high concentrations of lignin, and could be

employed in the production of charcoal for smelting iron and

other metallurgical purposes, and for domestic uses (Pires

et al. 2013). The species is not, however, domesticated, and

only limited information is available concerning its devel-

opmental stages and the characteristics of its fruits and seeds,

including their patterns of maturation and the nature of their

pronounced dormancy, which makes commercial seedling

production quite time-consuming (Clement et al. 2005;

Ribeiro et al. 2012b). Although pericarp development has

been previously investigated in A. aculeata (Reis et al. 2012),

little information is available about the different phases of

fruit development, which requires about one full year from

anthesis to abscission (Scariot and Lleras 1991).

The present work describes the morphoanatomy of A.

aculeata fruits and physiological aspects during develop-

ment, to: (1) define the phases and patterns of fruit and seed

maturation; (2) establish relationships between embryo-

genesis and fruit formation; and (3) characterize the onto-

genesis of the structures related to seed dormancy.

Materials and methods

Botanical material

Three natural populations of A. aculeata (Fig. 1a), located

in the municipalities of Brasılia de Minas (S 16�1205300 W

44�2600800), Mirabela (S 16�1504200 W 44�0901000), and

Montes Claros (S 16�4900900 W 43�5504600), in the northern

region of Minas Gerais State, Brazil, were studied from

November/2011 to November/2012. The native vegetation

of the region is Cerrado (neotropical savanna), although the

collection areas were being used as pasture for cattle, a

typical situation of the occurrence of A. aculeata. Testi-

monial material was deposited in the BHCB Herbarium at

the Federal University of Minas Gerais (register number

149985).

Morphology and physiology

Four palm trees were selected in each population, noting

the dates of opening their bracts and the liberation of the

inflorescences (Fig. 1b). Pistillate flowers were collected

on the same day as the emission of the inflorescences and

five samples of fruits were collected every 10 days for the

first hundred days, and then every 20 days until the initi-

ation of abscission, about 360 days after anthesis (daa). We

measured the lengths and maximum diameters of the fruits

using digital calipers (King Tools, China) as well as their

total fresh masses. Intact and sectioned, by manually

sawing fruits were observed and photographed using a

Motic 7667 L stereomicroscope (Hong Kong) with an

attached Cannon A-650 digital camera (Tokyo, Japan) to

describe and document their external (Fig. 1c) and internal

morphologies (Fig. 1d, e). We evaluated the fresh masses

of the exocarps, mesocarps, pyrenes, and seeds. The dry

masses of those structures were determined after dehy-

dration at 105 �C for 24 h in a forced-air oven, and the

water contents subsequently calculated.

Anatomy and histochemistry

Flowers on the day of anthesis, and fruits at 1, 2, 3, 4, 5, 6,

13, 28, 45, 75, 105, 134, 164, 200, 260, 345 and 360 daa

were collected, cut into pieces approximately 8 mm3, fixed

in FAA50 for 48 h, and stored in 70 % alcohol (Johansen

1940). The material was embedded in glycol-methacrylate

resin (Leica), following Paiva et al. (2011), and cross and

longitudinal sections (5–7 lm thickness) were obtained

using an Atago rotary microtome (Tokio, Japao). The

sections were subsequently stained with toluidine blue, pH

7.4 (O’Brien and McCully 1981, modified), and then

mounted on slides in Itacril acrylic resin (Itaquaquecetuba,

Brazil).

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The following histochemical tests were performed on

fresh and fixed sections: Lugol (Jensen 1962) to identify

starch, and Sudan IV (Pearse 1980) to stain total lipids.

Photographic documentation was obtained using a stereo-

microscope as well as a Lab. AI/Axion Cam ICc 3photo-

microscope (Zeiss, Jena, Germany).

Bromatology

Samples of the mesocarps and seeds, derived from 15 fruits

of A. aculeata from each of the three study areas, were

collected 300 daa, and on the day of abscission, and main-

tained at -20 �C until submitted to bromatological analyses,

following Horwitz (2002). Their protein contents were

determined using the Kjeldahl method, and their lipidic

contents analyzed using the Soxhlet continuous extraction

method with ether as the solvent. The non-nitrogenated

glycidic fraction method was used to determine their car-

bohydrate contents, and a mineral content was obtained after

incineration at 550–570 �C.

Results

Fruit ontogenesis

Complete fruit developments in A. aculeata requires

approximately 1 year, and while each of its structures

Fig. 1 An individual and population of A. aculeata (a). Overlapping

phenophases: persistent bracts, bunches of mature fruits, liberation of

the panicle (b). Fruit with persistent perianth (arrow) (c). Cross

section of the mature fruit with pseudo-pore evident (white arrow)

(d). Region of the germination pore; detail of the pore

plate = endocarp (dashed line) and operculum (red square) (e). Ar

arillode = outer integument, Bt bract, Bu bunches, Ed endosperm, Em

embryo, En endocarp = pore plate, Ex exocarp, Gp germination pore,

In inflorescence, Me mesocarp, Py pyrene, Sc seed coat

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follows a specific developmental pathway (Fig. 1c–e), the

fruits demonstrate three distinct developmental phases

(Fig. 2a–d). During phase I, which covers the period

between anthesis until about 90 daa, the tissues of the

pericarp grow and differentiate, and the water content

remains high.

The pistillate flowers are arranged in triads, generally

flanked by two staminate flowers (Fig. 3a). The pistillate

flowers are tricarpellate and syncarpous (Fig. 3b). Some

pistillate flowers were still receptive on the second day

after inflorescence emission and retained their humid

stigmas and light yellow tones (Fig. 3c). The ovary is

richly vascularized, with three conspicuous ventral bundles

in the middle region (Fig. 3b). In the receptacle, the vas-

cular bundles branch strongly so that it is not possible to

identify the dorsal bundles accurately. The carpel vascular

bundles are collateral (Fig. 3d, e). The protoxylem and

metaxylem have annular and scalariform cell wall thick-

ening, respectively (see Fig. 3e). The phloem present sieve

tube element with transversal to oblique sieve plate and

sieve areas on the wall (Fig. 3e, f). The ovarian wall is

composed of the abaxial epidermis of the carpel as well as

numerous subjacent layers, with bundles of elongated cells;

the mesophyll contains mucilaginous small ducts formed

by the fusion of raphide-containing idioblasts (Fig. 3g).

The anatropous and bitegmic ovules are well-developed in

this phase, each with a conspicuous egg-apparatus. Both

the synergids and the egg cell have a distinctive filiform

apparatus (Fig. 3h, i). The endothelium is composed of

cells with large nuclei and secretory aspect (Fig. 3i).

Fecundation occurs during this period, and can be detected

by observing karyogamy between the sperm and egg cell

nuclei (Fig. 3j). Two days after the emission of the inflo-

rescence, alterations in the colors of the corolla and stigma

could be noted, the former passing from yellow to greenish

and the latter from pinkish to brown (Fig. 3a, k).

After fecundation, a proliferation of idioblasts contain-

ing phenolic compounds could be observed in the meso-

carp, and the mucilaginous ducts increased in volume

(Fig. 3l). The two integuments in the micropylar region

separated, and intense meristematic activity could be

observed in the inner zone of the mesocarp (Fig. 3m). On

the third day, the fruits begin to develop, as seen by the

changing color of the perianth, becoming dark green, while

the persistent trilobate stigma takes on a dark-brown col-

oration (Fig. 3n). The zygote continues between the two

synergids and cells can be observed proliferating in the

seminiferous cavity (Fig. 3o). The outer integument dem-

onstrates meristematic activity in the micropylar region,

with very small cells with large nuclei.

On the fourth day of fruit development, the stigma and

trichomes became visibly dry and dark brown in color

(Fig. 4a). The three ovules have large seminiferous cavities

containing liquid endosperm (Fig. 4b). The synergids in the

micropylar region demonstrate a strongly stained filiform

apparatus, conspicuous nuclei, and granular cytoplasm

(Fig. 4c, d). After 5 days, the stigma has degenerated and

only remnant structures remain (Fig. 4e). The seminiferous

cavities of the three ovules appear well-developed, and

micropilar integument separation proceeds (Fig. 4f, g). The

persistent synergids can be seen flanking the zygote

(Fig. 4f, g), which is now larger and contains large vacu-

oles, with the nucleus in the chalazal position where the

cytoplasm is denser (Fig. 4g). Remnants of the pollen tube

can be seen near one of the synergids. On the sixth day of

development, the ovary has increased in diameter so that

the petals now become positioned near its base; trichomes

continue to surround the fruit (Fig. 4h). The degradation of

any carpel with an unfertilized ovule can be observed

Fig. 2 Fruit developmental phases of A. aculeata (dashed lines):

phase I (histo-differentiation of the pericarp), II (seed maturation),

and III (mesocarp maturation). Length, diameter (a) and fresh mass of

the fruit (b). Dry mass (c) and water contents of the fruit structures (d)

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(Fig. 4i). The seminiferous cavity is now totally filled with

the proliferating cells of the inner integument (Fig. 4j).

All three ovules can be fertilized, and in these cases all

three will develop. However, when one of the ovules

remains unfertilized its degeneration is precocious (Fig. 4i,

j). Normally, after 13 days, one or two of the ovules begin

to degenerate (Fig. 5a) and these seminiferous cavity

become filled. Sometimes remnants of the egg-apparatus

can be seen (Fig. 5b). An arillode develops in the micro-

pylar region, composed of numerous layers of large and

vacuolated cells that were formed by the proliferation and

growth of the outer integument. The outer integument itself

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is composed of a number of layers of small cells with large

nuclei (Fig. 5c, d). One of the synergids, which had been

persistent until this phase, begins to degenerate (Fig. 5d).

After 28 days of development, the exocarp is almost

completely covered by brown trichomes, the persistent

remains of the stigma, and the perianth (Fig. 5e, f). The

pericarp shows three distinct topographic zones. The

inner zone is the most differentiated, being composed of

rounded and vacuolated cells that surround a region

corresponding to the germination pore (Fig. 5g). This

pore is filled with mesocarp tissue, although this tissue is

fibrous and richly vascularized. The endocarp, derived

from the locular epidermis, forms a plate over the pore,

delimiting it internally and separating it from the arillode

(Fig. 5g). The exocarp has fiber bundles that are elon-

gated radially, as well as sclereids (Fig. 5h). The cells of

the external mesocarp are tangentially elongated and

compactly arranged, with few ducts (Fig. 5h). Idioblasts

containing phenolic compounds can be seen in the

median mesocarp, together with large mucilaginous

ducts (Fig. 5i). In this phase, the aborted ovule appears

as only a scar, totally surrounded by cells of the inner

mesocarp or pyrene (Fig. 5j). Phenolic compounds can

be observed in the cells of the inner integument

(Fig. 5k). The endocarp is composed of radially elon-

gated cells that are organized in palisades, with visible

anticlinal divisions that allow their expansion (Fig. 5k).

The proembryo can be observed dividing, while only one

of the synergids is persistent, and in an advanced stage

of degeneration (Fig. 5k, l).

After 45 days of development, the structures composing

the pericarp can be detected with the naked eye, although

the seed is still largely undeveloped and has a nuclear

endosperm (Fig. 6a). In the exocarp, agglomerations of

sclereids can be seen between the fiber bundles (Fig. 6b).

The pyrene, derived from the inner mesocarp, is not yet

lignified and its cells are oriented in many different planes

(Fig. 6a, c). The arillode has expanded due to the elonga-

tion of vacuolated parenchymatous cells with pectic con-

tents and to the formation of intercellular spaces (Fig. 6d).

The embryo is globular in this phase, with a multicellular

suspensor (Fig. 6d, e), and the cellularization of the

endosperm has begun in the micropylar region (Fig. 6d).

Occasionally, more than one seed develops (Fig. 7a).

After 75 days of development, the fruit exocarp is thick

and green. The mesocarp has a whitish color and the pyr-

ene is becoming sclerified (Fig. 7a). The mesocarp is

composed of parenchymatous cells, idioblasts containing

phenolic compounds, fiber bundles, and large ducts, the

latter being formed by the degradation of their transversal

walls (Fig. 7b). The walls of the cells composing the pyr-

ene have become thickened and lignified and oriented in

many planes (Fig. 7c). Adjacent to the pyrene are some

vascularized pachychalaza layers, especially near the aril-

lode (Fig. 7c). The vascular bundles are well-differenti-

ated. The seed coat is well-developed and has two distinct

regions: an outer composed of dead cells and containing

phenolic compounds and an inner composed of living cells

corresponding to the endothelium (Fig. 7d, e). There was a

proliferation of the cells in the micropylar region. A cel-

lular endosperm, whose occurrence is restricted to the

micropylar region, surrounds the embryo (Fig. 7d). The

endocarp cells developed thickened and lignified walls,

establishing a band of resistance in the limits of the ger-

mination pore (Fig. 7c, d). In cross section, the embryo

shows two cotyledon lobes, as in the cordiform phase of

eudicotyledons, and periclinal and anticlinal divisions are

proceeding in the protoderm (Fig. 7d, e). The precocious

establishment of haustorial activity in the embryo is indi-

cated by the presence of collapsed endospermic cells sur-

rounding the cotyledonary lobe (Fig. 7e). Approximately,

90 days after anthesis, which corresponds to the end of

phase I (Fig. 2a), the dimensions of a mature fruit has been

fully attained.

During phase II, water is substituted by structural sub-

stances and reserves in all of the fruit structures (Fig. 2c-d).

At 105 days, the embryo demonstrates quite pronounced

growth (Fig. 8a). The cellular endosperm surrounding the

embryo is being actively consumed during embryo expan-

sion and it does not yet fill the seminiferous cavity. The

embryonic axis has differentiated, with the formation of an

evident radicule, a plumule showing leaf primordia, and

procambial strands are being emitted from the cotyledonary

b Fig. 3 Flower and fruit development in A. aculeata. Cross sections

(b, d, g, i, j, l, m, o). Longitudinal sections (e, f, h). Triad (a).

Tricarpellary and syncarpic ovary; ventral vascular bundles (arrow-

heads) (b). Receptive flower 2 daa (c). Collateral vascular bundle (d).

Vascular bundle (e, f). Protoxylem and metaxylem with annular and

scalariform cell wall thickening, respectively (e). Phloem with

transversal to oblique sieve plates (f). Mesocarp with mucilaginous

ducts (asterisk) (g). Scattered vascular bundles (arrowheads) in

anatropous ovules with evident synergids (white arrow) (h). Ovule

with evident egg cell (i). Micropylar region showing zygote (black

arrow) (j). Fruit on second day of development (k-m), demonstrating

increases in the numbers of idioblasts containing phenolic compounds

and the volumes of the mucilaginous ducts (asterisk) in the mesocarp

(l). Dislocation of the integuments in the micropylar region (red

square); the arrowheads indicate vascular bundle (m). Fruit after

3 days of development (n). Zygote flanked by two persistent

synergids (white arrows); micropylar region showing meristematic

activity, with cells with large nuclei and dense cytoplasm (o). Ec egg

cell, Et endothelium, Ex Exocarp, Ii inner integument = seed coat, Ip

idioblasts containing phenolic compounds, Le locular epider-

mis = endocarp = pore plate, Mx metaxylem, Mz meristematic zone,

Oi outer integument = arillode, Ol ovule, Ov ovary, Pf pistillate

flower, Ph phloem, Pt petal, Px protoxylem, Sa sieve area, Sf

staminate flower, Sl style, Sp sieve plate, St stigma, Tr trichomes, Vb

vascular bundle, Xy xylem, Zy zygote

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node towards the distal region (Fig. 8a, b). Collapsed cells

have accumulated in the micropylar endosperm around the

cotyledonar petiole, forming a zone of weakness and

delimiting the operculum region (Fig. 8a), by which the

suspensor remains affixed to the embryo (Fig. 8c).

After 134 days of development, the mesocarp has a pale

yellow color and the pyrene is black, with a petrous

constitution (Fig. 8d, e). The seed is seen to be well-

developed, with a cordiform shape and a cavity in the

central region. The germination pore is well-defined and

filled with mesocarp tissue (Fig. 8d). The ovules which

have not formed seeds can be identified as clear regions;

they are surrounded by a pyrene. They do develop a seed

coat, arillode, endocarp, and a pseudo-pore that does not

Fig. 4 Initial phases of fruit development in A. aculeata. Cross

sections (b–d, i–j). Longitudinal sections (f–g). Fruit after 4 days of

development (a–d). Ovary with three well-developed gametophytes

and three ventral bundles (arrowheads) (b). Persistent synergids, with

evident nuclei and filiform apparatus (c, d). Fruit after 5 days of

development (e–g). Dislocation of the integuments in the micropylar

region (red square), where the zygote and synergids (white arrow) are

attached; note the vascularization of the pachychalaza (white

arrowheads) (f). Zygote, persistent synergids, and synergid at the

site of the pollen tube discharge (white arrow) (g). Fruit after 6 days

of development (h–j). Two well-developed gametophytes and one

degrading gametophyte (black arrow) (i). Aborted gametophyte,

filling the seminiferous cavity (black arrow) (j). Ar arillode, En

endocarp = pore plate, Et endothelium, Ex exocarp, Fa filiform

apparatus, Gp germination pore, Me mesocarp, Mz meristematic zone,

Nu nuclei, Pc pachychalaza, Pt petal, Py pyrene, Sc seed coat, Sl

style, St stigma, Sy synergid, Tr trichomes, Zy zygote

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enter into contact with the seed endosperm (Fig. 8e). The

embryo is positioned in a cavity formed by the degradation

of the adjacent endosperm, with its distal portion

corresponding to the cotyledonary leaf blade and proximal

portion corresponding to the cotyledonary petiole, sur-

rounding the microscopic embryonic axis (Fig. 8f). The

Fig. 5 Development of the A. aculeata fruit. Longitudinal section

(a). Cross sections (b–d, g–l). Fruit after 13 days of development (a–

d). The pseudomonomerous condition is attained; note degeneration

of one of the gametophytes (black arrow) and vascularization of the

pachychalaza (white arrowheads) (a). Fertilized gametophyte degen-

erating with remnants of egg-apparatus (red circle) (b). Initial phase

of seed development (c). Persistent synergid and another in initial

phase of degeneration (arrow) (d). Fruit after 28 days of development

(e–l). Fruit with persistent stigma and style (e). Fruit covered by

trichomes, with persistent perianth (f). Pericarp with three distinct

zones: exocarp, external mesocarp, and inner mesocarp; differentia-

tion of the inner mesocarp into the pyrene, the germination pore, the

endocarp in the pore plate, the outer integument in the arillode, the

integuments in the seed coat, and the locular epidermis in the

endocarp = pore plate; note vascularized pachychalaza (white arrow-

heads) (g). Exocarp (h). Outer mesocarp with mucilaginous ducts

(asterisks) (i). Aborted ovule; note the regions of the gametophyte

(arrow) and pachychalaza (arrowheads) (j). Detail of the region near

the germination pore (k). Proembryo (red rectangle in k) and

persistent synergid (l). Ar arillode = outer integument, En endo-

carp = pore plate, Ex exocarp, Fb fiber bundle, Gp germination pore,

Me mesocarp, Mz meristematic zone, Pc pachychalaza, Pe proem-

bryo, Py pyrene, Sc seed coat, Sd sclereids, Sm seminiferous cavity,

Sy synergid, Vb vascular bundle

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hypocotyl–radicule axis has an oblique orientation in

relation to the cotyledon axis. Two leaf primordia and a

promeristem can be seen on the plumule (Fig. 8g). The

haustorium has invaginations and numerous procambial

strands (Fig. 8h).

After 164 days of development, the embryo fills the

cavity formed by the degradation of endosperm cells, and

haustorium growth is noted, enlarging the invaginations in

the protoderm (Fig. 8i). The embryo is composed of vac-

uolated cells, with the initiation of reserve material allo-

cation. A meristematic zone is formed adjacent to the

radicule and the cotyledonal groove is now well-developed

adjacent to the plumule (Fig. 8j). The protoderm is com-

posed of cubical cells with conspicuous nuclei. The

endosperm cells adjacent to the haustorium continue to be

degraded during embryo development (Fig. 8k).

After 260 days of development, the exocarp is lignified

and is composed of an ovarian epidermis and various

subjacent layers composed of fiber bundles and sclereids

(Fig. 9a). Groups of parenchymatous cells undergoing

lignification surround the ducts located in the outer meso-

carp. In spite of the fact that the embryo appears well-

developed, it can be noted that reserve accumulation is

incomplete, with vacuolated cells still visible near the

plumule (Fig. 9b). The operculum is now well-defined, and

composed of opercular seed coat tissue and micropylar

endosperm. Adjacent to the operculum is the arillode,

composed of parenchymatous cells (Fig. 9b). The

Fig. 6 Fruit of A. aculeata after

45 days of development. Cross

sections. Region of the

germination pore (a). Fiber

bundles and sclereids in the

exocarp (b). Differentiation of

the pyrene, with cells oriented

in many different planes that

contribute to its structural

rigidity (c). Endocarp delimiting

the arillode and the germination

pore; embryo evident (red

square) and the initiation of

cellularization of the endosperm

(white arrow) (d). Globular

embryo, with multicellular

suspensor (e). Ar

arillode = outer integument,

Em embryo, En

endocarp = pore plate, Et

endothelium, Ex exocarp, Fb

fiber bundle, Gp germination

pore, Ip phenolic idioblast, Me

mesocarp, Mz meristematic

zone, Py pyrene = inner

mesocarp, Sc seed coat, Sd

sclereids, Sm seminiferous

cavity, Sp suspensor

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physiological maturity of the seed, from which there is no

increase in dry mass, is attained approximately 300 days

after anthesis and defines the end of phase II (Fig. 2c).

In phase III, the mesocarp matures, associated with the

continuous deposition of metabolic reserves, a process that

continues until abscission (Fig. 2c-d). Near 345 daa, the

mesocarp cells do not show high levels of reserve com-

pounds, although starch grains can be observed (Fig. 9c).

The embryo, on the other hand, demonstrates cells with

ample reserve accumulations (Fig. 9d). Exocarp maturation

is more precocious than that of the other structures, while

pyrene maturation is attained only just a few days before

abscission (Fig. 2c-d).

The mature fruits of A. aculeata are globular, with

persistent remnants of the perianth and stigma (Fig. 1c).

The exocarp is green-colored with brown splotches, and is

quite thick (Fig. 1c, d). The mesocarp is yellow/orange and

surrounded by a thick, petrous, and black pyrene. The

endocarp forms a plate over the germination pore, delim-

iting the mesocarp and arillode (Fig. 1e). The pachychalaza

is observed to be collapsed around the seed, and its vas-

cular bundles can be seen trapped internally to the pyrene

when the seed is removed. The seed is rigid and white, and

its endosperm surrounds the linear embryo, which is

positioned next to the germination pore (Fig. 1d, e).

Histochemistry and bromatology

Reserve compounds are not stored in mesocarp cells in the

initial phases of fruit development but, near 200 days of

development, starch grains begin to be deposited there

(Fig. 10a), accompanied by increases in the dry mass of the

fruit and reductions in its water content (Fig. 2c-d). Lipid

accumulation also initiates in this same phase.

Fig. 7 A. aculeata fruits after 75 days of development. Cross

sections. Fruit with two seeds being formed (a). Mesocarp with large

ducts (asterisk) and fiber bundles (b). Endocarp = pore plate and

pyrene = inner mesocarp in the process of lignification; vascularized

pachychalaza surrounding the seed (c). Cellularization and multipli-

cation in the endosperm; globular embryo (d, e). Embryo with

dividing cells (white arrows) and conspicuous haustorial function

(black arrow); note internal seed coat region corresponding to the

endothelium (e). Ar arillode, Ed endosperm, Em embryo, En

endocarp = pore plate, Et endothelium, Ex exocarp, Fb fiber bundle,

Gp germination pore, Me mesocarp, Pc pachychalaza, Py pyrene, Sc

seed coat, Sp suspensor, Vb vascular bundle

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Until approximately 260 days of development, lipid

deposition is reduced in both inner and outer mesocarp

cells (Fig. 10b), but by 342 days, the entire mesocarp

showed a significant increase in lipid storage (Figs. 2c, d,

10c), although the greatest accumulations are only

observed after fruit abscission, approximately 360 daa

(Fig. 10d).

The bromatalogical analyses indicated that lipids are the

principal substances stored by the fruits (Fig. 11). Moder-

ate quantities of carbohydrates and proteins, in the case of

the seeds, also contribute to fruit composition, while its

mineral content is quite low. The deposition of lipids and

carbohydrates is responsible for the significant increase in

the dry mass of the mesocarp in the final phase of fruit

Fig. 8 Fruit of A. aculeata after

105 (a–c), 134 (d–h), and 164

(i–k) days of development.

Cross sections. Micropylar

region indicating the zone of

weakness (black arrows) (a).

Plumule (b). Differentiated

radicule and persistent

suspensor (c). Developed seed

and embryo (d); aborted seed

(red circle) (e). Embryo in the

endosperm cavity (f).Embryonic axis with

differentiated plumule and

radicule (g). Endosperm cavity

(Ca); conspicuous invaginations

in the protoderm (h). Immature

embryo (i). Cotyledonary

groove (black arrow), note

meristematic zone (j).Haustorium and collapsed

endosperm (star) (k). Cn

cotyledonary node, Cp

cotyledonary petiole, Ed

endosperm, Gp germination

pore, Ha haustorium, Hr

hypocotyl–radicle axis, Li

ligule, Mi micropylar

endosperm, Mz meristematic

zone, Op operculum, Pd

protoderm, Pm promeristem, Pr

procambium, Rd radicle, s1 and

s2 leaf primordia

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development, while no significant changes occur in the

seed (Figs. 2c, 11).

Discussion

Acrocomia aculeata fruits require about one complete year

for full development, and this period can be divided into

three phases: histo-differentiation of the pericarp

(1–90 daa), seed maturation (90–300 daa), and mesocarp

maturation (300–360 daa). The exocarp attains physiolog-

ical maturity rapidly, while lignification of the pyrene

becomes well-established only at 105 daa, although this

process is continuous until just a short time before final

fruit development, corroborating the hypothesis that the

sclerification of the pyrene defines the final size of the palm

fruit (Murray 1973; Reis et al. 2012). The precocious dif-

ferentiation of the pericarp in A. aculeata possibly has an

important role in protecting the fruit, considering the high

rate of predation by insects during early stages of devel-

opment (Ramos et al. 2001; Pereira et al. 2014). The same

pattern of differentiation was observed in pericarp of other

palm species belonging to tribes Borasseae and Eugeisso-

neae (Romanov et al. 2011; Bobrov et al. 2012).

Fig. 9 A. aculeata fruits after 260 (a, b) and 345 (c, d) days of

development. Cross sections. Mature exocarp and mesocarp showing

ducts (asterisk) surrounded by cells with thick, lignified walls (white

arrow) (a). Opercular region: pore plate, arillode, seed coat, and

micropylar endosperm. Embryo with vacuolated cells and differen-

tiated plumule (b). Mesocarp with starch accumulations (black

arrows) (c). Embryo with maximum accumulation of reserves (d).

Ar arillode, Ed endosperm, Em embryo, En endocarp, Ex exocarp, Fb

fiber bundle, Me mesocarp, Mi micropylar, endosperm, Op opercu-

lum, Pp pore plate = endocarp, Pr procambium, Sc seed coat, Sd

sclereids, s1 and s2, leaf primordia

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The establishment of the pseudomonomerous condition

in A. aculeata occurs approximately 13 daa, followed by

the degeneration of the other ovules, even though they may

have been fertilized. This same characteristic was also

observed in species of Eugeissona (Bobrov et al. 2012) and

may be related to the programmed abortion of certain

ovules, as occurs in many plant species (Lersten 2004). The

carpels that do not develop will be incorporated into the

sclerenchyma tissue of the pyrene and contribute to fruit

formation. The aborted seeds become incorporated into the

pyrene. This same process has been described only for

Phoenix sylvestris and Arecastrum romanzoffianum among

the palms (Mahabale and Biradar 1967; Murray 1973).

Romanov et al. (2011) proposed the use of the term

pseudomonomerous pyrenarium for the classification of

this kind of fruit and di- or tri-pyrenarium when two or

three seed develop, respectively.

The differentiation of the mesocarp into distinct topo-

graphic zones has been described for other species of the

Arecaceae family (Romanov et al. 2011; Bobrov et al.

2012). In A. aculeata, the median region of the mesocarp is

composed of a tissue type that appears to be spongy due to

the presence of large numbers of mucilaginous ducts,

which probably have important role in water storage (Reis

et al. 2012). The pyrene, which is derived from various

layers of parenchymatous cells from the inner mesocarp,

can be classified as complex endocarps of mixed origin

(Murray 1973) or as endocarps sensu lato (Roth 1977; Reis

et al. 2012). However, as we are dealing here with a

structure that originated from the mesocarp region and

composes the pericarp, the recommended classification is

that of a pyrene (Romanov et al. 2011). The germination

pore plate (Neves et al. 2013), on the other hand, formed by

the sclerification of the locular epidermis, and therefore

formed independent of the pyrene, constitutes the endocarp

sensu stricto (Roth 1977). The great accumulations of oils

and carbohydrates in the mesocarp occur only after the

completion of embryogenesis and considerable sclerifica-

tion of the pyrene. This strategy avoids potential losses of

reserves if normal and viable seed development does not

occur.

The seeds of A. aculeata are pachychalazal, feature

reported for palms only in Syagrus inajai (Genovese-

Marcomini et al. 2013). Pachychalazal seeds form by the

growth of the chalaza, which comes to constitute a large

part of the seminal covering (Werker 1997) and is associ-

ated with seeds with bitegumented ovules, nuclear endo-

sperm, and tropical habitats (Corner 1976; Von Teichman

and Van Wyk 1991). The spreading of the two integuments

in the micropylar region, seen in the very initial phases of

fruit development in A. aculeata, indicates the formation of

the arillode from the outer integument. The occurrence of

an arillode in the ovule, as well as variations in the shapes

Fig. 10 Histochemical tests of the mesocarp of A. aculeata. Fruit

after 200 days of development showing significant accumulations of

starch (black staining with Lugol) and inconspicuous lipidic reserves

(red staining with Sudan red) (a). Progressive increases in

accumulated lipidic reserves (b–d). Fruit after 260 days of develop-

ment (red staining with Sudan red) (b). Fruit after 342 days of

development (c). Mature fruit after abscission (red staining with

Sudan red) (d). Lp lipids, Sr starch

Fig. 11 Lipid, carbohydrate, protein, and mineral contents of the

mesocarps and seeds of A. aculeata 300 days after anthesis, and at the

moment of abscission

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of the integuments and their fusion, could be seen, but

these features are not yet well-known in all palm genera

(Moore and Uhl 1982). There have been reports of arils in

Livistona chinensis (Kulkarni and Mahabale 1974) and

Butia capitata seeds (Oliveira et al. 2013), although with-

out any ontogenetic observations; these seminal out

growths may be related to protecting the embryo against

impacts or desiccation, as their cells are quite large and

store large quantities of water (Perez 2004). The endothe-

lium observed in A. aculeata is formed by the inner epi-

dermis of the seed coat and may play role nutrition or

physical barrier (Werker 1997). Seed coat composed of

distinct regions is reported for Arecaceae only in Mauritia

flexuosa (Silva et al. 2014) and future studies may check if

the pattern observed in the present work has widespread

occurrence in the family.

Embryogenesis in A. aculeta is precocious and dem-

onstrates a number of peculiarities in comparison with

the patterns most commonly seen. Both synergids remain

intact until the 13th daa, to the contrary of what is

commonly seen in gametophytes after fertilization (Ler-

sten 2004), and one of them persists until 28 daa, as was

also seen in Cocos nucifera (Haccius and Philip 1979)

and in S. inajai (Genovese-Marcomini et al. 2014). The

zygote remains undivided for a relatively long period of

time (until 28 daa) as compared to most species (Lersten

2004). After the globular phase, the embryo takes on a

cordiform shape, typical of Eudicotyledon embryos, as

has been observed in other palms (Mahabale and Biradar

1967; Biradar 1967; Biradar and Mahabale 1968; Kulk-

arni and Mahabale 1974; Genovese-Marcomini et al.

2014); this condition is associated with the formation of

the ligule that surrounds the apical meristem, placing the

embryonic axis internal to the cotyledonary petiole

(Haccius and Philip 1979). The suspensor is multicellular

and persistent, as seen in C. nucifera (Haccius and Philip

1979), and contributes to anchoring the mature embryo

to the operculum, as observed in B. capitata (Oliveira

et al. 2013). Beginning at the globular phase of the

embryo the protoderm demonstrates a notable haustorial

function associated with the degradation and consump-

tion of the adjacent cellularized endosperm, the principal

source of nutrition for the developing embryo. This

function has only been reported in palm seedlings when

the cotyledon leaf blade develops into the haustorium, an

organ specialized to digest and absorb endosperm

reserves during post-germinative development (DeMason

and Thomson 1981; Sekhar and DeMason 1988; Panza

et al. 2004; Iossi et al. 2006; Moura et al. 2010; Ribeiro

et al. 2012b). The precocious maturity of the embryo of

A. aculeata observed in the present work corroborates

evaluations of Pritchardia remota, whose embryos

demonstrated net accumulations of dry mass before

either the fruits or the seeds (Perez et al. 2012), and

studies of the in vitro development of seedlings from

cultivated embryos excised from immature B. capitata

(Neves et al. 2010) and A. aculeata fruits (Silva et al.

2013).

The structures related to the pronounced dormancy of A.

aculeata diaspores have diverse origins and also function

to protect the embryo. Dormancy in palm seeds has a

strong structural component that is usually associated with

the tissues adjacent to the embryo (Oliveira et al. 2013;

Neves et al. 2013). The germination pore plate, repre-

senting the sclerified endocarp, has an important role in A.

aculeata in protecting the embryo and mechanically

restricting seedling protrusion (Ribeiro et al. 2011). The

operculum, composed of the opercular seed coat and the

micropylar endosperm, not only mechanically restricts the

embryo but quite likely also slows gas exchange, thus

limiting its germinative capacity (Ribeiro et al. 2013). A

zone of weakness at the micropylar endosperm composed

of thin-walled cells is present in the initial phases of seed

development and has been associated with facilitating the

displacement of the operculum during germination (Gong

et al. 2005), thus playing a relevant role in overcoming

dormancy (Neves et al. 2013; Oliveira et al. 2013).

The results of the present work have allowed us to:

establish phases describing the development of A. aculeata

fruits; to determine that the pyrene, the rigid structure that

envelops the seed, originates from the inner mesocarp; to

characterize the ontogenesis of the endocarp and opercu-

lum as being related to dormancy and embryo protection;

to determine that embryonic development is precocious

and associated with the consumption of the cellularized

endosperm; and to determine that the principal phase of

lipidic allocation in the mesocarp occurs very near the time

of abscission.

Author contribution statement Hellen Cassia Mazzottini-dos-

Santos: Collection and processing of plant material. Preparation and

analysis of histological slides. Development of anatomical plates.

Preparation of the initial text. Leonardo Monteiro Ribeiro: Proposi-

tion of the work. Physiological analysis and interpretation of bio-

metric data. Contribution to the anatomical assessment. Drafting of

the final text. Maria Olıvia Mercadante-Simoes: Histochemical and

bromatological analyzes. Photographic records and contribution to the

writing of the final text. Bruno Francisco Sant’Anna-Santos: Ana-

tomical evaluation and drafting of the final text.

Acknowledgments The authors would like to thank Petrobras for

the grant conceded to the first author and financially supporting this

research project, as well as Fundacao de Amparo a Pesquisa do Es-

tado de Minas Gerais (FAPEMIG) for the BIPDT grants awarded to

L.M. Ribeiro and M.O. Mercadante-Simoes.

Conflict of interest The authors declare that they have no conflict

of interest.

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123

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