The Endophytic System of Mediterranean Cytinus (Cytinaceae) Developing On Five Host Cistaceae Species
The Endophytic System of Mediterranean Cytinus (Cytinaceae) Developing On Five Host Cistaceae Species
The Endophytic System of Mediterranean Cytinus (Cytinaceae) Developing On Five Host Cistaceae Species
Received: 28 May 2007 Returned for revision: 10 July 2007 Accepted: 20 July 2007 Published electronically: 5 September 2007
† Background and Aims One of the most extreme manifestations of parasitism is found in the genus Cytinus, a
holoparasite whose vegetative body is reduced to an endophytic system living within its host root. There are two
species of Cytinus in the Mediterranean, C. hypocistis and C. ruber, which parasitize various genera of
Cistaceae, one of the most characteristic families of the Mediterranean scrublands. The aim of this work is to
describe the endophytic systems of C. hypocistis and C. ruber, and their tissue relationships with their host.
† Methods Roots from five different hosts infected with C. hypocistis and C. ruber were harvested, and examined by
anatomical techniques under light microscopy to elucidate the characteristics of the endophytic system of Cytinus,
and to determine if differences in endophytic systems occur between the two species and in response to different
hosts.
† Key Results The endophyte structure is similar in both Cytinus species irrespective of the host species. In the initial
stages of the endophyte, rows of parenchymal cells spread through the host pericyclic derivatives and phloem, and
begin to generate small nodules in the outermost region of the host xylem. Later the nodules anastomose, and bands
of parasitic tissue are formed. The host cambium continues to develop xylem tissue, and consequently the endophyte
becomes enclosed within the xylem. The bands of parasitic tissue fuse to form a continuous sheath. This mature
endophyte has well-developed vascular system with xylem and phloem, and forms sinkers with transfer cells that
grow through the host xylem.
† Conclusions The endophytic system of Cytinus develops in all host root tissues and reaches its most mature stages
in the host xylem. It is more complex than previously reported, showing parenchyma, xylem and phloem tissues.
This is the first report of well-developed phloem in a holoparasitic endophytic species.
Key words: Cistaceae, Cytinaceae, Cytinus hypocistis, Cytinus ruber, endophyte, Mediterranean region, parasitic plant,
sieve elements, sinker, transfer cell.
IN TROD UCT IO N detailed data only for the genus Pilostyles (Dell et al.,
1982; Kuijt et al., 1985). The few existing data indicate
One of the most extreme manifestations of parasitism is
that these endophytes are composed of parenchymal cells,
found in the families of endoparasites Rafflesiaceae,
with xylem elements being observed only occasionally
Mitrastemonaceae, Apodanthaceae and Cytinaceae. These
(Fraysse, 1906; Kuijt, 1969; Dell et al., 1982; Forstmeier
perennial plants, without chlorophyll, are obligate parasites,
et al., 1983; Kuijt et al., 1985). Functional phloem has
and depend on their hosts to obtain water and nutrients
never been observed in the endophytes of these families
(Kuijt, 1969). All show a reduction in their morphological
studied to date, although Kuijt et al. (1985) have described
characters, with scale-like leaves and absence of external
a vestigial phloem tissue in Pilostyles thurberi
roots, and their vegetative body is reduced to a haustorial
(Apodanthaceae). Within the host, the endophytic system
or endophytic system, often compared with that of a
can grow intercellularly between the cambial zone and
fungal plectenchyme. These endophytes live within the
the phloem (Mitrastemonaceae), through the cambial zone
roots or stems of their hosts (Kuijt, 1969; Meijer, 1993),
(Rafflesiaceae, Apodanthaceae), through the phloem
and emerge from the hosts only during the reproductive
(Apodanthaceae), or between the cambial zone and the
period, when the inflorescences arise. Because of this
xylem (Cytinaceae) (Kuijt, 1969; Meijer and Schlauer,
characteristic lifestyle, these endophytic holoparasites
2002).
were long considered to constitute a single family, the
The distribution of the families of the holoparasitic endo-
Rafflesiaceae. However, differences in the morphology of
phytes is mainly tropical or subtropical (Molau, 1995), and
flowers, ovaries and seeds, together with data from recent
in the Mediterranean region there are only two species,
molecular phylogenetic studies, indicate that they are dis-
Cytinus hypocistis and Cytinus ruber (Cytinaceae) both of
tinct families, even belonging to different orders (Bouman
which parasitize exclusively roots of Cistaceae, one of the
and Meijer, 1994; Barkman et al., 2004; Nickrent et al.,
most characteristic families of shrubs in the Mediterranean
2004; Davis et al., 2007).
flora. Cytinus hypocistis has yellow flowers and parasi-
The structure of the haustorial system in these reduced
tizes white or yellow-flowered Cistus, Halimium,
endophytic holoparasites is poorly known, and there are
Helianthemum, while C. ruber has white or
pinkish-white flowers and parasitizes pink-flowered Cistus.
* For correspondence. E-mail cvega@us.es
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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1210 de Vega et al. — Endophytic System of Mediterranean Cytinus Species
Although Mediterranean Cytinus have been known since specimens of the host plants were deposited in the herbar-
ancient times, and even mentioned by Teofrasto (Villar, ium of the University of Seville.
1997), their life cycle, floral biology, process of infection,
and relationhips with the hosts remain practically
unknown. The anatomy of the endophytic system of R E S U LT S
C. ruber is unknown and that of C. hypocistis poorly docu- Macroscopic morphology of the infection
mented, with almost all the existing studies going back a
greater part of the root, and the vascular elements are het- region of the host xylem, contiguous with the cambial
erogeneous in size. zone (Fig. 2C). Initially, these nodules have only parench-
The development and structure of the endophytic system ymal cells recognizable by their dense cytoplasm, which
of Cytinus are similar in both species studied in their five stains intensely (Fig. 2C). However, they soon thicken,
host species. Thus, the following description is general, and appear as a differentiated central cambial zone sur-
and independent of the host being parasitized. The endo- rounded by parenchyma tissue (Fig. 2D). As the nodules
phytic system of Cytinus grows in all the tissues of the anastomose, bands of parasitic tissue are formed
F I G . 2. Different stages in the development of the endophytic system of Cytinus hypocistis and C. ruber. (A) Cross-section of a root of Cistus salviifolius.
Arrows indicate the parasitic tissue. (B) Magnification of the area marked in red in (A), with the tissue of C. hypocistis coloured pink. Note the parasitic
cells crossing the host pericyclic tissues and phloem. These cells are easily distinguished from those of the host by their great size and large nucleus.
(C) Cross-section of a root of C. salviifolius with nodules of the endophyte in different stages of development. In the nodule on the right there are intensely
stained parenchymal cells (arrowhead) and a cambial zone (arrow). (D) Cross-section of a root of C. salviifolius, showing an endophytic band embedded
in the host xylem, formed by a cambial zone bounded externally and internally by layers of parenchyma (arrows). Arrowheads indicate radial rows of
endophyte cells. (E) Cross-section of a root of C. albidus, showing a mature stage of the endophyte in the host xylem. In the central zone of the endophyte
a cambial zone can be seen, and phloem and xylem vascular tissues. The arrows indicate sinkers. Abbreviations: HPe, host pericyclic tissues; HCa,
Host cambial zone; HP, host phloem; HX, host xylem; PC, parasite cambial zone; PP, parasite phloem; PX, parasite xylem. Scale bars: A ¼ 200 mm;
B–D ¼ 50 mm; E ¼ 100 mm.
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de Vega et al. — Endophytic System of Mediterranean Cytinus Species
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than those of the host, and are more scattered (Fig. 3A, B). In the flowering period of Cytinus, the mature endophyte
As observed under fluorescence, no direct connections were grows towards the outside of the host root, causing a
seen between the phloem of the endophyte and that of the massive distortion in its tissues, to form the inflorescence
host. The sieve elements of the endophyte are long, with (Fig. 4J). From the continuous sheath of the endophyte,
thin walls, and a weakly staining cytoplasm lacking a the bud from which the inflorescence originates begins to
nucleus. The simple sieve plates found in the cross-walls develop, formed by elongated parenchymal cells with
of Cytinus sieve elements are easily observed with fluor- very large nuclei (Fig. 4K, L) and by vascular filaments
F I G . 3. (A–F) Vascular tissues of the endophytic system of Cytinus hypocistis and C. ruber. (A) Longitudinal section of a root of Cistus ladanifer
infected by C. hypocistis, under normal light. (B) The same section, under fluorescence, showing the phloem of C. hypocistis (white arrows), oriented
perpendicular to that of the host (black arrows). (C) Detail of sieve tubes of (B), showing the sieve plates of C. hypocistis. (D) Detail of sieve elements
of C. hypocistis on Halimium halimifolium, showing the sieve plates and lateral sieve areas (arrows). (E) Detail of the xylem vessels of C. ruber on Cistus
albidus, showing the secondary reticulated thickening of the cell wall. (F) Detail of the xylem vessels of C. ruber on Cistus albidus, showing the sec-
ondary helicoidal thickening of the cell wall. (G– I) Mature stages in development of C. hypocistis. (G) Cross-section of a root of Halimium ocymoides,
showing the formation of new endophytic nodules (arrows) and, below, a band of older parasite tissue (arrowhead). (H) Cross-section of a root of
C. salviifolius with various strata of the endophytic system of C. hypocistis alternating with host xylem. The arrows indicate the radial rows of endophyte
cells. (I) Very advanced stage of the infection of C. hypocistis observed in a cross-section of a root of C. salviifolius: the endophyte forms a continuous
sheath embedded in the host xylem. The arrows indicate the radial rows of endophyte cells and the arrowheads the sinkers in the host xylem.
Abbreviations: HPe, host pericyclic tissues; HP, host phloem; HX, host xylem; PPa, parasite parenchyma. Scale bars: A, B, G ¼ 100 mm; C, E, F ¼
10 mm; D ¼ 5 mm; H, I ¼ 200 mm.
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only a cortical outer layer, a cambial zone, and a medullary mature endophyte bands of Cytinus and the host xylem.
inner layer with xylem conductive elements (Solms- This seems to be a common circumstance, because although
Laubach, 1867; Fraysse, 1906; Forstmeier et al., 1983). xylem – xylem contacts have been reported in other parasitic
This was probably because fluorescence was not used in angiosperms (e.g. Calvin, 1967; Kuijt, 1977; Sallé, 1979;
those studies and, despite the size of the phloem elements, Heide-Jørgensen and Kuijt, 1995; Pate, 1995; Calladine
they are barely perceptible without this technique (see and Pate, 2000), a luminal continuity was actually seen in
Fig. 3A and B). Likewise, phloem has not been reported very few cases (e.g. Pate et al., 1990; Dörr, 1997).
F I G . 4. (A–H) Structure and development of the sinkers of Cytinus hypocistis and C. ruber. (A) Detail of C. ruber on Cistus albidus in a cross-section,
showing sinkers (arrows) being formed from the cambial zone of endophyte bands growing among the host xylem. (B) Sinkers of C. hypocistis invading
the xylem of Halimium halimifolium, in a cross-section. (C) Detail of sinkers of C. hypocistis, with thick cell walls and pointed apical cells (arrows), in the
xylem of Halimium ocymoides. (D, E) Sinkers of C. hypocistis on H. halimifolium. Note the presence of thick accumulations of wall material (D) and
differently thickened walls (E) in sinkers cells abutting host vessel elements (arrows). (F) Detail of a uniseriate sinker of C. hypocistis in the xylem of
H. ocymoides, showing a vascular element (arrow) between two parenchymal cells (arrowheads). (G) Longitudinal section of the xylem of H. halimifolium
thoroughly permeated by sinkers of C. hypocistis with cells having a large nucleus. (H) Longitudinal section of the xylem of H. halimifolium, showing a
sinker of C. hypocistis (arrow) that seems to invade two vascular elements of the host. (I) Longitudinal section of the xylem of H. halimifolium with
sinkers of C. hypocistis (arrows) that seems to penetrate the host vessels. (J–N) Details of the endophyte of Cytinus hypocistis and C. ruber forming
flower buds. (J) Cross-section of a root of Cistus albidus, showing the growth of the endophyte to form the inflorescence. (K) Detail of the large
nuclei of the endophyte cells from which the floral buds of C. hypocistis originate, in a longitudinal section of a root of C. salviifolius. (L)
Magnification of the marked area in (J) showing a parenchyma composed of long cells and vascular tissues (arrow). (M) Magnification of the marked
area in (L) showing xylem and phloem. In the xylem, the helicoidal thickenings of the walls can be observed, and in the phloem, the sieve elements
(arrows) and their associated parenchyma are evident. (N) Detail of the phloem, under fluorescence, of a floral bud of C. hypocistis on Cistus ladanifer,
showing sieve cells with a large amount of callose on their sieve plates. Abbreviations: HPe, host pericyclic tissues; HP, host phloem; HX, host xylem; PE,
endophytic system of Cytinus; PI, parasite inflorescence; PP, parasite phloem; PPa, parasite parenchyma; PX, parasite xylem. Scale bars: A, B, G, H, I, L,
N ¼ 50 mm; C, E, F, K, M ¼ 20 mm; D ¼ 10 mm; J ¼ 500 mm.
1216 de Vega et al. — Endophytic System of Mediterranean Cytinus Species
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