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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(3): 797-810, July-September 2021 (Published Aug. 09, 2021)

Floral ontogeny and anatomy of the hemiparasitic shrub

Gaiadendron punctatum and a comparison of floral characters in

the tribes Gaiadendrinae and Nuytsiae (Loranthaceae)

Hernán Darío Suárez

; https://orcid.org/0000-0002-8460-6995

Camila Robayo

; https://orcid.org/0000-0002-1710-9489

Xavier Marquínez

; https://orcid.org/0000-0002-0035-260X

Lauren Raz

; https://orcid.org/0000-0002-3152-4726

1. Departamento de Biología, Facultad de Ciencias, (Sisbio, COL0024669), Universidad Nacional de Colombia-Sede

Bogotá, Colombia, Carrera 30 # 45-03, Bogotá D.C., Colombia; hdsuarezr@unal.edu.co, carobayom@unal.edu.co,

xmarquinezc@unal.edu.co (Correspondence*)

2. Instituto de Ciencias Naturales, Facultad de Ciencias, Universidad Nacional de Colombia-Sede Bogotá, Colombia,

Carrera 30 # 45-03, Bogotá D.C., Colombia; lraz@unal.edu.co

Received 01-III-2021. Corrected 07-V-2021. Accepted 09-VII-2021.

ABSTRACT

Introducction: Gaiadendron punctatum is a hemiparasitic species of Loranthaceae (Tribe Gaiadendreae) that

is widely distributed in mountainous regions of Central and South America. Embryological and phylogenetic

studies in the family indicate a trend towards reduction of the gynoecium and ovules, the morphology of which

supports the current circumscription of Tribe Gaiadendreae (Gaiadendron and Atkinsonia). Molecular phyloge-

netic studies suggest that Nuytsia, Atkinsonia and Gaiadendron diverged successively, forming a grade at the

base of the Loranthaceae, but support values are low.

Objetive: In the present study, the floral anatomy of Gaiadendron punctatum was investigated in order to

provide additional data to permit comparisons among the three basal-most genera in the Loranthaceae and

reevaluate their relationships.

Methods: Flowers of G. punctatum were collected at different developmental stages and serial sections were

prepared and analyzed by light microscopy.

Results: Inflorescence development is acropetal; the flowers are bisexual with an inferior ovary surmounted by

a calyculus, a ring-shaped structure lacking vascular tissue; the ovary is comprised of seven basal locules, each

with an ategmic, tenuinucellate ovule. Above the locules is a mamelon that is fused with the adjacent tissues.

The androecium is comprised of seven epipetalous stamens, the anthers with fibrous endothecium dehiscence

through a single longitudinal slit, releasing tricolpated pollen.

Conclusions: The results of this study show that Gaiadendron and Atkinsonia share versatile, dorsifixed anthers,

while Gaiadendron and Nuytsia share the same mode of anther dehiscence. On the other hand, Gaiadendron

shares with members of Tribe Elytrantheae an amyliferous mamelon and an unvascularized calyculus.

Combined phylogenetic analyses of morphological and molecular data are desirable to determine whether Tribe

Gaiadendreae comprises a clade, a grade or if the two genera are more distantly related.

Key words: Atkinsonia; embryo sacs; floral development; gynoecium; hypostase; mamelon; Nuytsia.

Suárez, H. D., Robayo, C., Marquínez, X., Raz, L. (2021).

Floral ontogeny and anatomy of the shrub Gaiadendron

punctatum and a comparison of floral characters in

the tribes Gaiadendrinae and Nuytsiae (Loranthaceae).

Revista de Biología Tropical, 69(3), 787-810. https://doi.

org/10.15517/rbt.v69i3.46054

https://doi.org/10.15517/rbt.v69i3.46054

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Loranthaceae Juss. is the most diverse fam-

ily of parasitic plants in the tropics, comprising

approximately 73 genera and 960 species (Due-

ñas-Gómez & Franco-Roselli, 2001; Nickrent

et al., 2010; Dueñas-Gómez, 2015). The mono-

phyly of the family is strongly supported by

molecular and morphological evidence, with

the molecular data also providing robust sup-

port for the genus Nuytsia R.Br. as sister to the

rest of the family (Vidal-Russell & Nickrent,

2008a; Nickrent et al., 2010; Su et al., 2015).

In the aforementioned studies, Nuytsia forms

a grade with two other early-diverging genera:

(Nuytsia (Atkinsonia F.Muell. (Gaiadendron

G. Don (rest of the Loranthaceae)))), although

Nickrent et al., (2019) recovered an alternate

topology: (Nuytsia (Gaiadendron (Atkinsonia

(rest of the Loranthaceae)))). Grimsson et al.,

(2017) recovered a completely different topol-

ogy in which Atkinsonia was resolved as sister

to an entirely Australasian clade, but with a

low bootstrap support value. More data are

desirable to resolve the divergence order in this

basal part of the tree.

The current classification of the family

includes five tribes and 11 subtribes (Nickrent

et al., 2010; Kuijt, 2015). This study focuses in

tribe Nuytsieae Tiegh., including only Nuytsia

floribunda (Labill) R.Br., endemic to Western

Australia, and tribe Gaiadendreae Tiegh., com-

prising Atkinsonia ligustrina (Lindl.) F. Muell.,

also from Australia, plus the Neotropical Gaia-

dendron, which includes G. punctatum (Ruiz

& Pav.) G. Don, distributed in high montane

ecosystems of South and Central America,

and G. coronatum Kuijt, narrowly endemic to

high-Andean dwarf forests in Peru (Kuijt &

Graham, 2015). Although Gaiadendreae are not

monophyletic, its two component genera share

the following characters: versatile, dorsifixed

anthers, fruits forming pseudodrupes and seeds

with sulcate endosperm. The inflorescences

of Atkinsonia are composed of monads, while

those of Gaiadendron form triads, but the

floral morphology is similar in both genera

(Nickrent et al., 2010).

The first embryological studies in the

family, including the first interpretation of the

reproductive organs, were published by Treub

(1883). A century later, Bhatnagar and Johri

(1983) studied the floral structure and embryol-

ogy of an expanded sample of 13 genera, cul-

minating in the publication of their series titled

“Morphological and embryological studies in

the family Loranthaceae”. Contemporaneous-

ly, Cocucci (1982) and Venturelli undertook

studies of some Neotropical genera including

Struthanthus Mart. (Venturelli, 1981) and Tri-

podanthus (Eichl.) Tiegh. (Venturelli, 1983).

Based on their work, these authors had differ-

ent interpretations of the process of reduction

of the gynoecium and ovules observed in the

family (Cocucci, 1982; Bhatnagar & Johri,

1983). The “primitive” condition was repre-

sented by Nuytsia (Bhatnagar & Johri, 1983) or

by Tripodanthus and Nuytsia (Cocucci, 1982),

in both cases with ategmic ovules completely

separated from the ovary wall and enclosed

in differentiated locules with the embryo sacs

pointing towards the base of the gynoecium.

These interpretations were based on

detailed studies of embryogenesis in Nuytsia

floribunda (Narayana, 1958a). Detailed embry-

ological studies have also been conducted on

Atkinsonia ligustrina by Prakash (1961), who

suggested that it would be useful to study

the embryology of Gaiadendron in order to

test the relationships of the Gaiadendreae and

Elytrantheae Engl. (considered subtribes at

that time). More recently, Suaza-Gaviria et

al., (2016) studied the floral structure of Gaia-

dendron along with that of other Neotropical

Loranthaceae species, but this work was mostly

focused on inflorescence morphology.

Here we present a detailed study of floral

development in Gaiadendron punctatum in

order to fill in existing gaps in the data and

thereby allow direct comparison of floral mor-

phology and especially embryological char-

acters among the three basal genera (Nuytsia,

Atkinsonia and Gaiadendron) in Loranthaceae,

data that may help shed light on the as-yet

unresolved relationships among these taxa.

MATERIALS AND METHODS

Plant material: Flowers at different pheno-

logical stages (buds, anthetic and post-anthetic

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flowers) were collected in the field and imme-

diately fixed in FAA (formaldehyde: acetic

acid: 70 % ethanol, 10:5:85) from a plant of a

small population of Gaiadendron punctatum

from the following locality: Colombia. Dept.

Cundinamarca, Municipality la Calera, locality

“Los Patios”, North of the tollboth, paramo ca.

3 000 m. (voucher specimen Raz 1742, COL).

The determination was confirmed using keys

and descriptions by Kuijt (1981) and, Dueñas-

Gómez and Franco-Roselli (2001).

In order to facilitate the description of the

floral developmental processes, we established

six stages of flower development taking into

account the phenological phase (bud: 1 to 4;

anthesis: 5; post-anthesis: 6), the total length of

the flower measured in longitudinal section and

the observed phases of microgametogenesis

and megagametogenesis in the androecium and

gynoecium, respectively (Table 1).

The flowers, fixed in FAA for 48 hours,

were subsequently stored in 70 % ethanol,

and treated following the Robles et al., (2016)

protocol. This involved standard methods of

dehydratation, paraffin infiltration, sectioning

with a rotary microtome (820 Spencer, Ameri-

can Optical Company, NY) and attachment

to microscope slides. The slides were stained

with astra-blue and fuchsine and deposited in

the Department of Biology collection at the

Universidad Nacional de Colombia, Bogotá.

The slides were analyzed and photographed

using an Olympus BX-50 microscope with a

Moticam Pro 282B camera. Fixed flowers were

studied with a Leica M205A stereoscope using

multifocus mode and photographed with a

Leica MC 170 HC camera. Digital images were

processed and edited with the software Adobe

Photoshop CC and InDesign.

RESULTS

Flower and inflorescence morphol-

ogy: Gaiadendron punctatum is a profusely

branched shrub with axillary and/or terminal

compound racemose inflorescences, develop-

ing acropetally (Fig. 1A). The partial inflo-

rescences are dichasia composed of a central

flower and two sessile lateral flowers (Fig.

1B). The dichasia are opposite and decussate

along the inflorescence axis, each subtended

by bracts. All the flowers are bisexual and sub-

tended by foliaceous bracts.

The flower of Gaiadendron punctatum is

epigynous, so the ovary is inferior (Fig. 1B,

Fig. 1C) and is surrounded by the hypanthium.

The inferior ovary subtends the other floral

whorls: the calyx (or calyculus), the corolla

and the androecium of epipetalous stamens

(Fig. 1D).

The anatomical description presented here

follows a topological order, from the base to

TABLE 1

Floral developmental stages studied in Gaiadendron punctatum, lengths in mm and characteristics associated

with the androecium and gynoecium

Stage

Length (mm) Characteristics

Inferior

ovary

Calyculus

to apex

Total

Style and

stigma

Androecium Gynoecium

1 1.7 2.5 4.2 1.5

Archesporium, dyads and

tetrads

Ovules

2 2.1 3.4 5.5 2.0 Tetrads Meiosis in the ovule

3 3.1 5.4 8.5 4.2

Tetrads, tetrads with triradiate

pollen

Meiosis in the ovule

4 3.5 6.5 10 4.8

Mature pollen free uninuclear

and binuclear

Lengthening of the embryo sacs

5 3.8 8.1 11.9 6.4

Anthesis flower, anther

dehiscence, mature pollen shed

Lengthening of the embryo sacs

6 4.0 - - 9.5

Post-anthesis flower without

petals and stamens

Mature embryo sacs, Complete

ovular apparatus

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Fig. 1. A. Inflorescence and flower anatomy of Gaiadendron puntantum. A. Inflorescence with flowers developing

acropetally; B. Lateral flowers in side view; C. Detail of flower with inferior ovary; D. Longitudinal section of the flower

(the lines indicate the approximate position of the transverse sections corresponding to Fig. 1E, Fig. 1F and Fig. 1G); E.

Transverse section (TS) of the floral receptacle; F. TS at the pelvis level; G. TS at the ovary level; H. Detail of the mamelon

in longitudinal section (the lines indicate the approximate position of the transverse sections corresponding to Fig. 1I, Fig.

1J, Fig. 1K, Fig. 2A and Fig. 2B; I. J. and K. TS at different levels of the mamelon immersed in amyliferous tissue. An

= Anther; Cx = Calyculus, Io = Inferior ovary; Ma = Mamelon; Pe = Petal; Pv = Pelvis; Sy = Style; St = Stamen; Ts =

Surrounding tissues; Triangle = Vascular bundle; Triangle with number 1 = External vascular bundle; Triangle with number

2 = Internal vascular bundle; Red asterisks = Locules; D. E. F. G. H. I. J. and K. Astra blue/fuchsine. Scale bars: 2 mm in

A. and B.; 1 mm in C.; 500 µm in D.; 300 µm in E.; 200 µm in F. ; 150 µm in G.; 100 µm in H. I. and J.

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the distal parts of the flower. We begin with

the receptacle, then the inferior ovary and the

hypanthium, following the vascularization as

it continues into the style and stigma and the

perianth: calyculus and corolla, ending with

the epipetalous androecium. The processes of

microsporogenesis and megasporogenesis are

described in association with the description of

the gynoecium and androecium, respectively.

Gynoecium and floral vasculature:

Seven traces enter the sessile flowers via the

receptacle from the inflorescence axis (Fig.

1E). The mature hypanthium has a single

epidermal layer composed of elongate, papil-

lose cells covered by a thick cuticle; after the

epidermis, 6-9 inner layers of tanniniferous

parenchymatic tissue are observed, followed by

10-11 layers of parenchyma towards the central

region; the vascular bundles that irrigate the

petals and stamens are embedded in parenchy-

matic cells (Fig. 1F).

The ovary is situated above the base of

the receptacle, which is in turn composed

of sclerenchymatic tissue (when mature) and

is referred to as the pelvis (Fig. 1F). It is

composed of seven basal locules each with

a single ategmic ovule (Fig. 1G). There is a

non-vascularizated conical structure above the

locules called the mamelon (Fig. 1H) which

is composed of amyliferous parenchyma that

is fused with the surrounding tissues; embryo

sacs will eventually grow through this tissue

(Fig. 1I). The mamelon is separated from the

surrounding tissues just beneath the style (Fig.

1J, Fig. 1K).

At the base of the flower, seven vascu-

lar bundles can be seen forming a ring at the

periphery of the pelvis (Fig. 1E); above this

point, the bundles divide to form two concen-

tric rings (Fig. 1H, Fig. 1I). The interior ring is

made up of seven bundles that correspond to

the seven locules of the gynoecium (Fig. 1H,

Fig. 1J); all or part of these five to seven bun-

dles continue into the style (Fig. 2A, Fig. 2B).

There are seven vascular bundles in the exterior

ring, each one composed of three to five strands

including a large central strand that is external

to the others, plus two to four lateral strands

(Fig. 1I, Fig. 1J, Fig. 1K). The lateral strands

fuse to form a new bundle that enervates the

stamens, while the external strand enervates the

petals. No vascular traces enter the calyculus

(Fig. 1D, Fig. 1E, Fig. 1F, Fig. 1G, Fig. 1H),

in which there is neither evidence of tracheids,

nor any other vascular tissue.

Megasporogenesis: The ovary is com-

posed of seven locules (Fig. 1G), each of

which contains a single ategmic, tenuinucellate

ovule oriented towards the base of the flower

(Fig. 2D). The placentation is axillary. In floral

developmental stages 2 and 3 (Table 1), meio-

sis can be observed, resulting in four cells that

are similar in size and contour (Fig. 2E, Fig.

2F). In each locule, the developing embryo sacs

elongate through the amyliferous tissue of the

mamelon until they reach the base of the style

(Fig. 2C, Fig. 2J; stage 6 in Table 1) without

invading the latter structure.

At the upper end of the mature embryo sac,

a small protuberance is observed. It is called

the caecum, which functions as a haustorium

(Fig. 2G). It is followed by the synergids, the

filiform apparatus, the egg cell and the central

cell (Fig. 2H). The antipodal cells are found at

the base of the embryo sac, near the hypostase

and these tend to degenerate (Fig. 2I).

Style and stigma: The base of the style is

expanded (Fig. 1C, Fig. 1D, Fig. 1H) and seven

projections are observed at the periphery (Fig.

1C); these correspond to the outermost layers

of the tissues that nourish the embryo sacs dur-

ing their development (Fig. 2A). The style is

hollow (Fig. 1D, Fig. 1H) and it is composed

of a single outer epidermal layer interspersed

with stomata and covered by a thick cuticle.

Beneath the epidermis, there are 10-12 layers

of tanniniferous parenchyma and seven collat-

eral vascular bundles are observed towards the

inner part of the tissue, surrounded by smaller

parenchyma cells, which are, in turn, adjacent

to the inner epidermis that surrounds the stylar

canal (Fig. 2B).

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Fig. 2. Floral morphoanatomy of Gaiadendron punctantum, megasporogenesis and perianth. A. Transverse section of the

flower (TSF) at the base of the style; B. TSF at the level of the calyculus; C. Longitudinal section (LS) of the gynoecium,

(the lines indicate the approximate positions of the corresponding transverse sections in A. and B.); D. LS, detail of the

ovary and ategmic ovule; E. LS after first meiotic division, arrows indicate nuclei; F. LS, after second meiotic division,

arrows indicate nuclei; G. and H. LS at the upper level of embryo sac showing the caecum, synergids and egg cell; I. LS at

the lower level of embryo sac showing degenerated antipodal cells; J. Transverse section at the level of the embryo sacs; K.

and L. Detail of the mucilage secreting structures. Ac = Antipodal cells; Ao = Ategmic ovule; At = Amyliferous tissue; Cc

= Caecum; Cx = Calyculus; Ec = Egg cell; Es = Embryo sac; Mc = Mucilage canal; Ms = Mucilage secreting structure; Pe

= Petal; Pv = Pelvis; Sg = Synergids; Sy = Style. Triangle = Vascular bundle; Triangle with number 1 = External vascular

bundle; Triangle with number 2 = Internal vascular bundle; Asterisks = Locules; Red circle = Interdigitated epidermal cells;

2e-3e = Second and third embryo sac divisions; A. B. C. D. E. F. G. H. I. J. K. and L. = Astra blue/fuchsine. Scale bars:

200 µm in A. B. and L.; 500 µm in C; 50 µm in D. G. I. and K.; 30 µm in E. F. and H.; 100 µm in J.

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There is a trilobate stigma at the end of the

style, with interdigitated epidermal cells at the

edges of the lobes; stigmatic cells are slightly

rounded, whereas the epidermal style cells are

flattened (Fig. 1D).

Perianth - calyculus and corolla: The

calyculus is short and light green in color (Fig.

1C); it forms a ring above the ovary, being

more or less persistent in fruit, with a vari-

able number of irregular teeth at the margin

(Fig. 1C, Fig. 2B, Fig. 2C). The epidermis is

uniseriate abaxially and is made up of radi-

ally elongated cells and structures that secrete

mucilage (Fig. 2B), the latter persisting even in

fixed material (Fig. 1C). The adaxial epidermis

(oriented towards the petals) is composed of

quadrangular cells. The mesophyll is composed

of undifferentiated parenchyma with small

cells and thin walls as well as parenchyma with

larger cells and dark, tanniniferous cytoplasm.

As mentioned above, no vascular traces were

observed (Fig. 2B).

The corolla is composed of seven long,

white petals that measure approximately 3.5

x 1.3 mm, with valvate aestivation (Fig. 1B).

The petals are postgenitally fused in the flower

bud by interdigitation of the external epidermal

cells (Fig. 1D, Fig. 2B, Fig. 3K). The bases of

the petals are narrow and articulated (Fig. 1H).

The corolla lobes are completely free at anthe-

sis, but the basal third of the petals form a tube

that surrounds the style; the petals are reflexed

in the upper two thirds of their length, where

the filaments of the epipetalous stamens are

free from the petals (Fig. 3B). The adaxial epi-

dermis of the petals is papillose, followed by

4-7 layers of tanniniferous parenchyma made

up of very tightly packed cells; then there is a

less compact parenchymatous region toward

the center of the petals in which the vasculature

is embedded, the latter consisting of a central

bundle and one or two lateral bundles. Between

the central and lateral bundles, some packets

of sclerenchymatic cells with not very thick

walls are observed; these are accompanied by

cells that stain densely and form longitudinal

mucilaginous ducts. Exterior to these cells,

another layer of tightly packed parenchymatic

cells is seen, some of which are tanniferous; the

abaxial epidermis at outer edge of the petals is

composed of quadrangular cells and mucilage-

secreting structures (Fig. 2K, Fig. 2L).

Androecium and microsporogenesis:

The androecium is composed of seven stamens

of three different lengths; this variation can be

seen in the mature flower as well as in earlier

developmental stages (Fig. 3A). The filaments

are adnate to the bases of the petals (Fig.

3B) and the anthers are dorsifixed (Fig. 3L,

Fig. 3M), each with two thecae measuring 2-3

mm long (Fig. 3K). Two pollen sacs of similar

size are observed within each theca, each with

a uniseriate exothecium of very thin-walled,

unlignified cells (Fig. 3C). The fibrous endo-

thecium is made up of a single layer of large,

quadrangular cells plus two thinner layers that

do not persist, and a secretory tapetum that per-

sists until maturity. The tapetum is non-inva-

sive (Fig. 3I, Fig. 3K) and is initially composed

of uninucleate cells, becoming binucleate at

the end of their development. No amyloplast

or other type of reserves were observed in the

endothecium, or in any of the other tissues of

the anther during its development.

Microsporogenesis begins with a solid,

diploid archaesporium (Fig. 3C). The uninucle-

ate microspores separate (Fig. 3D) and undergo

two meiotic divisions (Fig. 3E), first forming

dyads (Fig. 3F) and then tetrads (Fig. 3G). The

four nuclei separate simultaneously, creating

primarily tetrahedral tetrads (Fig. 3G), near

which tapetum and orbicules (Ubisch bodies)

can be observed, the latter appearing red in the

figure. In the final stage, four trilobate pollen

grains can be distinguished within the callose

wall of the tetrad (Fig. 3H, Fig. 3I). The free

pollen becomes binucleate just before anthesis

(Fig. 3J).

Anther dehiscence is longitudinal and

occurs via dehydration of the tissues (Fig. 3L,

Fig. 3M). The cells of the exothecium near the

stomium become elongated in the later devel-

opmental stages of the anther to facilitate pol-

len dispersal. Dehiscence occurs without prior

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Fig. 3. Floral morphoanatomy of Gaiadendron puntantum: Androecium and microsporogenesis; A. Anther in longitudinal

section in mature stage; B. Detail of the anther attachment; C. Immature pollen sacs with archaesporium (2n); D. Uninucleate

microspores; E. First meiotic division; F. Dyads; G. Tetrad formation in the pollen sacs; H. Pollen sac with tetrads; I. Detail

of fibrous endothecium, intermediate strata and tapetum; J. Pollen sac with mature pollen and persistent tapetum; K. Mature

anther; L. and M. Dorsifixed anthers showing longitudinal dehiscence. An = Anther; Ar =Archesporium; Ct = Connective

tissue; Da = Dorsifixed anther; En = Endothecium; Ep = Epidermis; Fi = filament of stamen; Is = Intermediate strata; Ld

= Longitudinal dehiscence of the anther; Pe = Petal; g = Pollen grain; Ps = Pollen sac; Se = Septum; t = Stamens; Tp =

Tapetum; Ub = Ubisch bodies (orbicules). C. D. E. F. G. H. I. J. and K. = Astra blue/fuchsine. Scale bars: 2 mm in A.; 500

µm in B., L.; 50 µm in C, J; 20 µm in D. E. G. and I.; 10 µm in F.; 100 µm in H.; 200 µm in K.; 1 mm in M.

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coalescence of the pollen sacs in each theca, via

rupturing of the central septum and the walls

of the two pollen sacs along their entire length

(Fig. 3J).

DISCUSSION

Given the lack of robust support for a

specific branching order among the three basal

genera in the Loranthaceae (Gaiadendron,

Nuytsia and Atkinsonia) according to the phy-

logenetic proposals available for the family,

we consider the utility of comparative floral

morphoanatomy to help resolve these relation-

ships. With the data reported here for Gaiaden-

dron, such a comparison is now possible and

is presented below. Table 2 summarizes some

of the characters and their states in the basal

genera of Loranthaceae: Nuytsia, Atkinsonia

and Gaiadendron. Comparisons with some

members of the nearby tribe Elytrantheae are

also considered here.

The inflorescences of the three basal genera

in the Loranthaceae are indeterminate racemes;

the parcial inflorescences are dichasia in Gaia-

dendron, but these are reduced to monads in

Atkinsonia. The flowers are generally bisexual,

TABLE 2

Comparison of characters among the three basal genera of Loranthaceae

Character

Nuytsia Atkinsonia Gaiadendron

Inflorescence Racemose Racemose Racemose

Parcial inflorescence Dichasium with central

flower bisexual, laterals

pistillate

Monad subtended by three

bracts, bisexual flowers

Dichasium of

bisexual flowers

Central mamelon Present Present (¿?) Present

Number of locules 3 4 7

Fusion of the mamelon and

adjacent tissues

No ¿? Yes, in the middle

Tracheids in the mamelon Yes ¿? No

Number of vascular bundles

at the base

7 7 7

Number of bundles in the

internal ring of the ovary and

(style)

6 (6) 4 (4) 7 (5-7)

Number of bundles in the

internal ring of the petals and

(stamens)

7 bundles each with:

1 external

(2 laterals that fuse)

7 bundles each with:

1 external

(2 laterals that fuse)

7 bundles each with:

1 external

(2 to 4 laterals that fuse)

Vascularization of the

calyculus

Yes, 5 initial traces that

anastomose

Yes; no traces enter from

the main stele, but tracheids

differentiate in each lobe

Ovules Ategmic, oriented towards the

base, tenuinucellar

¿? Ategmic, oriented towards

the base, tenuinucellar

Style With amyliferous tissue ¿? Without amyliferous tissue

Intrusion of the embryo sacs Ovary and style Ovary Ovary

Hipostase composition Collenchyma Collenchyma Sclerenchyma

Base of the style With angular thickenings With slightly rounded

thickenings

With angular thickenings

Anthers Epipetalous, Basifixed Epipetalous, dorsifixed,

Versatile

Epipetalous, dorsifixed,

Versatile

Sources Narayana (1958a)

Narayana (1958b)

Prakash (1961)

Garg (1958)

Cocucci (1982)

This study

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except in Nuytsia, where the central flower is

bisexual and the lateral flowers are pistillate or

staminate (Narayana, 1958a); lateral flowers

abort in some cases in this genus.

Gaiadendron punctatum has a septilocular

ovary, which is the greatest number of loc-

ules in the Loranthaceae. Nuytsia (Narayana,

1958a) and Lepeostegeres (Dixit, 1958) each

have three locules, while Atkinsonia, Amylothe-

ca (Raj, 1970), Lysiana (Narayana, 1958b) and

Peraxilla (Prakash, 1960) each have four. The

ovary is unilocular in the remaining genera of

the family (Kuijt, 2015).Nuytsia, Gaiadendron

and the genera of the tribe Elytrantheae all have

a mamelon, being amyliferous in G. punctatum

and in the tribe Elytrantheae (Johri et al., 1992)

and not being amyliferous in Nuytsia (Nara-

yana, 1958a). The mamelon in G. puntatum

is retained within the ovary without entering

the style, a condition also reported in Perax-

illa Tiegh. and Amylotheca Tiegh. (Prakash,

1960; Raj, 1970), while in Nuytsia, Lysiana

Tiegh. and Lepeostegeres Blume (Dixit, 1958;

Narayana, 1958a; Narayana, 1958b), the mam-

elon invades the hollow style. In Nuytsia the

mamelon remains separate from the adjacent

tissue, but in Lysiana and Lepeostegeres this

structure is fused with the ovary wall and the

tissues of the stigma.

The mamelon of Nuytsia does not have

amyliferous tissue, but the style does have

amyliferous parenchyma. Thus, the embryo

sacs can invade the style for quite a good

part of its length. The embryo sacs of Lysiana

and Lepeostegeres also enter the style due to

the invasion of the amyliferous tissue of the

mamelon into the lumen of the stylar canal

(Bhatnagar & Johri, 1983). The embryo sacs

of Gaiadendron, just like those of Peraxilla y

Macrosolen (Blume) Rchb., develop only up

to the base of the style because the mamelon

is short and there is no amyliferous tissue in

the style. The mamelon is fused with the ovary

wall in Gaiadendron, a condition also seen in

Lysiana and Lepeostegeres (Dixit, 1958; Nara-

yana, 1958b) but not in Nuytsia. The aforemen-

tioned characters suggest that Gaiadendron has

a greater affinity with the tribe Elytrantheae

than with Nuytsia.

Although Cocucci (1982) indicated that

Atkinsonia ligustrina has an amyliferous ovary

like that found in genera of the tribe Elytranthe-

ae, there is no direct evidence of this condition

in Atkinsonia according to the studies cited by

Cocucci (Garg, 1958; Prakash, 1961).

Calyculus, base of style and nectary: The

vascularization of the calyculus is different in

each of the three basal genera of Loranthaceae

(Table 2). The absence of bundles or tracheids

in the calyculus of Gaiadendron punctatum is

the state found in the rest of the Loranthaceae,

while only Nuytsia and Atkinsonia present

alternative states. This result supports the Su

et al., (2015) topology of (Nuytsia (Atkinsonia

(Gaiadendron (rest of the Loranthaceae)))).

It should be noted that the inconspicuous

calyculus of G. punctatum contrasts with that

of G. coronatum, in which the lobes are den-

tate, well differentiated, and persistent in fruit

(Kuijt, 2015).

Diverse types of nectaries have been

reported in Loranthaceae. In the Neotropical

clade Psittacanthinae, (sensu Nickrent et al.,

2010), the nectaries often form a disk or ring

between the base of the style and the base of

the petals. This ring usually has stomata and

subepidermal parenchyma with dark-staining

cellular contents, as can be seen in Peri-

stethium leptostachyum Kunth (Tiegh.) (Robles

et al., 2016), Psitacanthus Mart. (Robayo et

al., 2020), Passovia Karst., Aetanthus (Eichl.)

Engl. and Oryctanthus Eichl. (Suaza-Gaviria

et al., 2016). A different type of nectary found

at the base of the petals has been described in

Cladocolea Tiegh. (Cid-Villamil, 2006).

Nevertheless, it has been reported that

the basal genera Nuytsia (Narayana, 1958a),

Atkinsonia (Prakash, 1961) and few species of

the Tribe Elytrantheae have thickenings at the

base of the style (Amylotheca, Lepeostegeres:

respectively, Dixit, 1958; Raj, 1970). In some

of these cases, they have been interpreted

as possible nectaries (Lepeostegeres, Nuyt-

sia: respectively Narayana, 1958a; Raj, 1970)

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and only in Lepeostegeres, the presence of a

dark-staining subepidermal tissue has been

confirmed (Schaeppi & Steindl, 1942). Also,

Lamilla et al., (2020) recently found that the

base of the style conforms a nectary in Tristerix

longebracteatus (Desr.) Barlow & Wiens, a

basal member of the tribe Psittacantheae Horan

Suaza-Gaviria et al., (2016) suggested that

G. punctatum presents a nectary disk. How-

ever, no disk was observed in the present study

(Fig. 1D, Fig. 1E, Fig. 1F, Fig. 1G, Fig. 1H;

Fig. 2B, Fig. 2C). Our longitudinal sections

of G. punctatum found a widened base of the

style with some stomata in the epidermis and

typical parenchyma beneath the epidermis (Fig.

1D, Fig. 2B); nor did we find sunken stomata

at the base of style, and therefore the evidence

is insufficient to confirm or reject that this

structure is a nectary. In contrast, our research

team found a widened style in Tristerix formed

by an epidermis with abundant sunken stomata,

underlain by parenchymatic tissue with dense

cytoplasm and abundant intercellular spaces

surrounding the vascular bundles (Lamilla et

al., 2020). Nevertheless, the dark-staining sub-

epidermal parenchyma at the base of the petals

and stamens suggests the possibility of a differ-

ent type of nectary (Fig. 2B).

Anther dehiscence: The endothecium in

Loranthaceae can be fibrous or non-fibrous.

When is it fibrous, it is composed of thickened

cells that surround the pollen sacs, interrupted

by cells that lack thickenings. The position of

these weaker cells determines the type of dehis-

cence (Table 3).

In type A dehiscence, present in Atkinso-

nia ligustrina (Prakash, 1961), the two pol-

len sacs of each theca undergo coalescence

due to the rupturing of the septum between

them, followed by a single longitudinal rupture

of the stomium.

In type B dehiscence, present in Nuyt-

sia (Narayana, 1958a) and Gaiadendron (this

study), the septum that divides the pollen sacs

within each theca remains intact initially, and

dehiscence occurs via simultaneous rupture

of the septum and the outer wall of the theca

(stomium), at the precise point where they

connect, thereby creating a single longitudinal

opening (Table 3).

TABLE 3

Anther dehiscence in Nuytsia, Atkinsonia, Gaiadendron and some genera of the tribe Elytrantheae

Genus Endothecium Dehiscence type Scheme of dehiscence Source

Atkinsonia

Fibrous A Prakash, 1961

Nuytsia

Fibrous B Narayana, 1958a

Gaiadendron

This study

Lysiana

Fibrous C Narayana, 1958b

Peraxilla

Prakash, 1960

Lepeostegeres

Non-Fibrous D Dixit, 1958

Amylotheca

Raj, 1970

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In the genera Lysiana and Peraxilla of

the tribe Elytrantheae, the interruption of the

fibrous endothecium occurs in the middle of

each pollen sac (Table 3) such that each theca

opens via two longitudinal slits (type C dehis-

cence). On the other hand, in Amylotheca and

Lepeostegeres, also of the tribe Elytrantheae,

the endothecium has no thickened zones and

therefore dehiscence may occur at any point

(type D dehiscence).

According to Fig. 7F of Suaza-Gaviria et

al., (2016), anther dehiscence in Gaiadendron

punctatum occurs via two longitudinal slits

in each theca, one for each pollen sac, corre-

sponding to our type C. The results of the pres-

ent study do not corroborate type C dehiscence

in this taxon. The septum that separates the

two pollen sacs within each theca is thickened

and the weakest zone (Fig. 3K) is localized to

the point where it fuses with the outer wall of

the theca (the stomium, Table 3). Dehiscence

occurs via a single longitudinal slit between the

two pollen sacs (Fig. 3M). Therefore, Gaiaden-

dron punctatum has type B anther dehiscence,

just like Nuytsia floribunda.

Atkinsonia and Gaiadendron: clade

or grade: In traditional classifications of

Loranthaceae as well as in recent molecular

systematic studies, Nuytsia, Atkinsonia and

Gaiadendron have been considered basal gen-

era. The character traditionally used to support

this position is root parasitism through second-

ary haustoria, whereas in the rest of the family,

it is the primary haustorium that parasitizes

the stems of the host plants (Vidal-Russell &

Nickrent, 2008b). It should be noted, however,

that Gaiadendron punctatum can occasionally

parasitize epiphytes (Kuijt, 2015).

The grouping of the genera Atkinsonia and

Gaiadendron in the tribe Gaiadendreae was

based on the following characters: vascular

bundles in the calyculus, drupaceus fruit, fur-

rowed endosperm and dorsifixed and versatile

anthers (Nickrent et al., 2010). Regarding

the first character, the vascularization of the

calyculus occurs in three different states in

each of these three basal genera (Table 2)

with Gaiadendron punctatum lacking vascular

traces or tracheids, a state shared with the rest

of the Loranthaceae. It would be interesting to

study this character in Gaiadendrum corona-

tum, the calyculus of which is much larger and

persists in the fruit (Kuijt, 2015). Regarding the

drupaceous fruit, Atkinsonia and Gaiadendron

have pseudodrupes in which the embyro is sur-

rounded by a hard endocarp. This condition is

also found in fruits of the tribe Elytrantheae,

including the genera Amylotheca (Raj, 1970),

Lepeostegeres (Dixit, 1958), Lysiana (Naraya-

na, 1958b) and Peraxilla (Prakash, 1960). The

fruit of Nuytsia is completely distinct, being a

winged nut (Kuijt, 2015). Discarding the first

two characters, only furrowed endosperm and

dorsifixed versatile anthers remain as possible

synapomorphies of tribe Gaiadendreae as it is

currently circumscribed.

In most molecular phylogenetic studies

published to date (Nickrent et al., 2010; Su et

al., 2015; Nickrent et al., 2019), Gaiadendron

and Atkinsonia form a grade rather than a

clade, although Grimsson et al., (2017) recov-

ered Atkinsonia as sister to a clade comprising

mostly genera of the tribe Elytrantheae. As

can be seen, the morphological evidence is

not unequivocal either, with some characters

supporting Gaiadendron as sister to Atkinsonia

(dorsifixed versatile anthers) and/or to Nuytsia

(type of anther dehiscence), while the combina-

tion of characters 1. non-vascularized calyculus

and 2. amyliferous mamelon would appear

to support a sister relationship of Gaiaden-

dron to the tribe Elytrantheae or to rest of the

Loranthaceae. In order to definitively resolve

the matter, it will be necessary to collect the

last bits of missing data for Atkinsonia ligus-

trina (Table 2) and ideally, perform a combined

analysis of molecular and morphological data

to determine if the two genera of Gaiadendreae

really do form a clade, a grade or are more

distantly related.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we

809

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followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We thank the Fundación para la Promoción

de la Investigación y la Tecnología “Banco de

la República” for the partial financing of Proj-

ect 3758 (Agreement 201618 with the U.N.C.)

and the Universidad Nacional de Colombia

for matching funds. The authors thank Diego

Andrés Benítez for helping to process the

plant material.

RESUMEN

Ontogenia floral y anatomía del arbusto

hemiparasítico Gaiadendron punctatum y comparación

de caracteres florales en las tribus Gaiadendrinae

y Nuytsiae (Loranthaceae)

Introducción: Gaiadendron punctatum es una especie

hemiparásita perteneciente a uno de los tres géneros basa-

les de la familia Loranthaceae, siendo los otros dos Nuytsia

y Atkinsonia. El género está conformado por dos especies

distribuidas en regiones montañosas de Sudamérica y

Centroamérica. Tanto los estudios embriológicos, como

los filogenéticos, indican una tendencia hacia la reducción

del gineceo y de los óvulos en la familia, cuya morfolo-

gía respalda la circunscripción de la tribu Gaiadendreae

(Gaiadendron y Atkinsonia). Estudios filogenéticos mole-

culares sugieren que Nuytsia, Atkinsonia y Gaiadendron

divergieron sucesivamente, formando un grado en la base

de la familia Loranthaceae, pero los valores en los que se

sustenta son bajos.

Objetivo: En el presente trabajo se describe la anatomía

floral de la especie Gaiadendron punctatum con el objetivo

de complementar la información embriológica, de manera

que se pueda comparar directamente la morfología floral y

los caracteres embriológicos entre los tres géneros basales

de la familia Loranthaceae y reevaluar sus relaciones.

Métodos: Las flores de G. punctatum fueron recolectadas

en diferentes estados de desarrollo, se realizaron cortes

histológicos seriados, se tiñeron con azul de astra y fucsina,

y se analizaron mediante microscopía óptica.

Resultados: Las inflorescencias mostraron un desarrollo

acrópeto, las flores bisexuales presentaron ovario ínfe-

ro con presencia de una estructura en forma de anillo,

carente de tejidos vasculares llamada calículo; el ovario

se compone por siete lóculos basales, cada uno con un

óvulo atégmico tenuinucelar. Por encima de los óvulos, el

mamelón se fusiona con los tejidos adyacentes. El andro-

ceo está conformado por siete estambres epipétalos, las

anteras presentan un endotecio fibroso y granos de polen

tricolpados. La dehiscencia de las anteras es mediante una

sola apertura longitudinal.

Conclusiones: Los resultados del presente trabajo demues-

tran que Gaiadendron y Atkinsonia comparten anteras

dorsifijas y versátiles, mientras Gaiadendron y Nuytsia

comparten el tipo de dehiscencia anteral y por otro lado

Gaiadendron comparte los caracteres de mamelón amilí-

fero y calículo no vascularizado con la tribu Elytrantheae.

La clasificación del género Gaiadendron con respecto a los

dos géneros basales de la familia debe ser objeto de inves-

tigación (análisis filogenético combinado) que permita

dirimir si la tribu Gaiadendrae es un clado, un grado o dos

géneros más lejanamente emparentados.

Palabras clave: Atkinsonia; sacos embrionarios; desarrollo

floral; gineceo; hipostasa; mamelón; Nuytsia.

REFERENCES

Bhatnagar, S., & Johri, B. M. (1983). Embryology of

Loranthaceae. In M. Calder & B. Peter (Eds.), The

biology of mistletoes (pp. 129–143). University of

Melbourne, Australia.

Cid-Villamil, R. M. (2006). Biología del desarrollo de Cla-

docolea loniceroides (Van Tieghem) Kuijt (Lorantha-

ceae). Tesis de Doctorado, Universidad Nacional

Autónoma de México, México.

Cocucci, A. E. (1982). El ovulo y el gineceo en Lorantha-

ceae. Boletín de la Sociedad Argentina de Botánica,

21, 131–141.

Dixit, S. (1958). Morphological and Embryological Studies

in the Family Loranthaceae IV. Amyema Van Tiegh.

Phytomorphology, 8, 346–364.

Dueñas-Gómez, H. C. (2015). Loranthaceae. En R. Bernal,

S. Gradstein, & M. Celis (Eds.), Catálogo de plantas

y líquenes de Colombia (pp. 1487–1493). Editorial

Universidad Nacional de Colombia.

Dueñas-Gómez, H. C., & Franco-Roselli, P. (2001). Sinop-

sis de las Loranthaceae de Colombia. Caldasia, 23,

81–99.

Garg, S. (1958). Embryology of Atkinsonia ligustrina (A.

Cunn. ex F. Muell.) F. Muell. Nature, 182(4649),

1615–1616.

Grimsson, F., Grimm, G. W., & Zetter, R. (2017). Evolu-

tion of pollen morphology in Loranthaceae. Grana,

57, 16–116.

810

Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(3): 797-810, July-September 2021 (Published Aug. 09, 2021)

Johri, B. M., Ambegaokar, K. B., & Srivastava, P. S.

(1992). Comparative embryology of angiosperms.

Springer-Verlag.

Kuijt, J. (1981). A rejoinder on Oryctina (Loranthaceae).

Plant Systematic and Evolution, 137, 215–219.

Kuijt, J. (2015). Santalales. In J. Kuijt & B. Hansen (Eds.),

The families and genera of vascular plants, Flowe-

ring plants: Eudicots; Santalales, Balanophorales

(pp. 1–189, Vol. 12). Springer.

Kuijt, J., & Graham, J. C. (2015). Two New Species of

Loranthaceae from Central Peru. Novon A Journal for

Botanical Nomenclature, 24, 173–178.

Lamilla, L. A., Robayo, C. A., Castaño, F., Marquínez,

X., & Raz, L. (2020). Floral anatomy of Tristerix

longebracteatus (Loranthaceae). Revista de Biología

Tropical, 68, 87–98.

Narayana, R. (1958a). Morphological and Embryological

Studies in the Family Loranthaceae - III Nuytsia flo-

ribunda (Labill) R.Br. Phytomorphology, 8, 306–323.

Narayana, R. (1958b). Morphological and embryological

studies in the family Loranthaceae - Lysiana exocarpi

(Behr) Van Tieghem. Phytomorphology, 15, 146–168.

Nickrent, D. L., Malécot, V., Vidal-Russell, R., & Der, J.P.

(2010). A revised classification of santalales. Taxon,

59, 538–558.

Nickrent, D. L., Anderson, F., & Kuijt, J. (2019). Inflo-

rescence evolution in Santalales: Integrating mor-

phological characters and molecular phylogenetics.

American Journal of Botany, 106(3), 402–414.

Prakash, S. (1960). Morphological and embryological

studies in the family Loranthaceae - VI. Peraxilla

tetrapetala (Linn. F.) van Tiegh. Phytomorphology,

10, 224–234.

Prakash, S. (1961). Morphological and Embryological

Studies in the Family Loranthaceae - VII Atkinsonia

Ligustrina (Cunningh) FV Muell. Phytomorphology,

11, 325–335.

Raj, B. (1970). Morphological and Embryological Studies

in the Family Loranthaceae-XIII. Amylotheca dic-

tyophleba Van Tiegh. Öesterreichische Botanische

Zeitschrift, 118, 417–430.

Robayo, C. A., Marquínez, X., Raz, L., & Nickrent, D.

L. (2020). Floral anatomy of the plant Psittacanthus

schiedeanus (Loranthaceae). Revista de Biología

Tropical, 68, 1–11.

Robles, A., Raz, L., & Marquínez, X. (2016). Anatomía

floral de Peristethium leptostachyum (Loranthaceae).

Revista de Biología Tropical, 64, 357–368.

Schaeppi, H. & Steindl, F. (1942). Blütenmorphologische

und embryologische Untersuchungen an Loranthoi-

deen. Vierteljahrsschr Naturf Ges Zürich, 87,

301–337.

Su, H. J., Hu, J. M., Anderson, F. E., Der, J. P., & Nickrent,

D. L. (2015). Phylogenetic relationships of Santalales

with insights into the origins of holoparasitic Balano-

phoraceae. Taxon, 64, 491–506.

Suaza-Gaviria, V., Pabón, N., & González, F. (2016). Deve-

lopment and morphology of flowers in Loranthaceae.

International Journal of Plant Science, 177, 559–578.

Treub, M. (1883). Observations sur les Loranthacees. IV.

Loranthus pentandrus L. Annales du Jardin Boanique

de Buitenzorg, 3, 184–189.

Venturelli, M. (1981). Embriologia de Struthanthus vul-

garis (Loranthaceae-Loranthoideae). Kurtziana, 14,

73–100.

Venturelli, M. (1983). Estudos embriológicos em Lorantha-

ceae: Gênero Tripodanthus. Kurtziana, 16, 71–90.

Vidal-Russell, R., & Nickrent, D. L. (2008a). Evolutio-

nary relationships in the showy mistletoe family

(Loranthaceae). American Journal of Botany, 95,

1015–1029.

Vidal-Russell, R., & Nickrent, D. L. (2008b). The first

mistletoes: Origins of aerial parasitism in Santalales.

Molecular Phylogenetic and Evolution, 47, 523–537.