American Journal of Botany 101(8): 12751285, 2014.

ROOT CONTRACTION IN CYCAS AND ZAMIA (CYCADALES)

DETERMINED BY GELATINOUS FIBERS1

P. BARRY TOMLINSON2,3, TRACY M. MAGELLAN2,4, AND M. PATRICK GRIFFITH2

2Montgomery

Botanical Center 11901 Old Cutler Road, Coral Gables, Florida 33156 USA; and 3The Kampong of the National
Tropical Botanical Garden, 4013 South Douglas Road, Miami, Florida 33133 USA

Premise of the study: Reaction wood (RW) in seed plants can induce late and usually secondary changes in organ orientation.
Conifers produce compression wood (CW), generated by compression tracheids, which generate a push force. Angiosperms
produce tension wood (TW), generated by tension wood fibers (TWF) often described as gelatinous fibers, which exert a pull
force. Usually RW is produced eccentrically, but it can occur concentrically, as in aerial roots of Ficus. However, gymnosperms can produce gelatinous fibers (tension fibers, TF), as in cortical and secondary phloem tissues (Gnetum). TFs are therefore limited neither to wood, xylem, nor angiosperms. Here we demonstrate that TFs in secondary phloem are involved in
contraction of roots of cycads and compare them with TFs of Ficus.
Methods: We sectioned root material of cycads at various stages of seedling development using simple staining and histochemical procedures to follow the course of secondary phloem development. Aerial roots of Ficus were compared with the
cycad root material.
Key results: Tension fibers (gelatinous fibers) occur extensively and continuously in the secondary phloem in roots that undergo contraction. Older tissues, but notably the xylem, become distorted by contraction. TFs in cycads correspond in cell wall
features to TFs that occur in Ficus, but do not occur in secondary xylem. The individual fibers visibly contract.
Conclusions: Tissue contraction in Cycas and Zamia corresponds to that found in angiosperms and Gnetum and further broadens the scope of the activity of tension tissues. This finding possibly indicates that gelatinous fibers originated at a very early
period of seed plant evolution.
Key words: Cycadales; Cycas; gelatinous fibers; reaction wood; root anatomy; root contraction; secondary phloem; Zamia.

Reaction wood has long been examined due to its economic

effects. Reaction tissues in plants produce late changes to either
reorient displaced axes or to maintain them in a fixed position.
The most familiar and widely investigated are those that occur
in secondary xylem (wood) and are produced, but not consistently so, as a presumed gravimorphic response. This response
produces a tissue referred to as reaction wood (RW). Because
RW reduces the commercial value of merchantable tissue, it
has been widely studied by plant anatomists and wood technologists (Hster and Liese, 1966; Wilson and Archer, 1977;
Wardrop, 1964). Reaction wood occurs in two contrasting
forms in seed plants, either as compression wood (CW) in conifers or tension wood (TW) in angiosperms. The former includes
modified tracheids (compression tracheids, CT); the latter includes tension wood fibers (TWF). These cell types are usually
produced eccentrically in relation to a gravimorphic response.
The two cell types are mutually exclusive because conifer wood
does not include fibers. The two cell types also have contrasting
mechanical properties and distributions. CW is produced on the
lower side of leaning axes or branches, and its CTs exert a push
force by cell extension (hence under compression), whereas
1 Manuscript received 11 April 2014; revision accepted 8 July 2014.
The authors thank David Jones (The Kampong), Tiffany Lum (Fairchild
Tropical Botanic Garden), and Chad Husby (Montgomery Botanical
Center) for supplementary plant material. Research was conducted with
support from the Kelly Foundation.
4 Author for correspondence (e-mail: tracym@montgomerybotanical.
org)

doi:10.3732/ajb.1400170

TW is formed on the upper side of leaning axes and branches

with TWFs exerting a pull force by contraction (hence under
tension). In the descriptive literature of wood anatomy, they are
referred to as gelatinous fibers (GFs) from their hydrated and
refractive properties under the light microscope. Our analysis
will show that GFs have a wider distribution than previously
suggested. We demonstrate the presence of GFs in the Cycadales, where they have an important function in root contraction, and we add a third clade to those known to have GFs.
The economic effect of CW in conifers is reflected in a major
review (Timell, 1986) and in its long-term effects on tree form
that were demonstrated in a study lasting over 50 yr (Akachuku,
1993). Tension wood has been studied extensively in relation to
tree architecture (e.g., Fisher and Stevenson, 1981; Fisher,
1985) and experimentally most extensively in seedlings (Wardrop,
1964). Recent research has concentrated on the cellular mechanism that induces cell wall contraction. A number of hypotheses
have been formulated, but the conclusion is that the process of fiber
contraction is still not understood (Du and Yamamoto, 2007;
Schreiber et al., 2010; Mellerowicz and Gorshkova, 2012).
Tension wood may also have important ecological roles. It is
necessary for the erection of viviparous seedlings of the mangrove
Rhizophora and other genera, which are habitually stranded in
a horizontal position (Tomlinson and Cox, 2000; Fisher and
Tomlinson, 2002). It may be an important component of plant
survival in fire-prone habitats because it aids in the burial of
underground parts of seedlings and may occur in stems and
roots (Fisher, 2008). A major recent discovery has been the
demonstration of GFs in the biology of woody lianas through
its formation in tendrils and twining stems (Meloche et al.,
2007; Bowling and Vaughan, 2009; Fisher and Blanco, 2014),

American Journal of Botany 101(8): 12751285, 2014; http://www.amjbot.org/ 2014 Botanical Society of America

1275

1276

[Vol. 101

AMERICAN JOURNAL OF BOTANY

where GFs can also occur in secondary phloem. Tension wood

also can play an important role in root architecture, especially
in relation to root contraction where it can be developed concentrically. The classic example is found in the pendulous aerial roots of the giant trees of Ficus after they become rooted at
ground level. The induced tension, once the TWFs were identified, was demonstrated by planting descending roots in unanchored plant pots, whereupon they were soon lifted from the
ground by force of contraction (Zimmermann et al., 1968).
These observations have been confirmed most recently by Abasolo et al. (2009). This process is the basis for the so-called
strangling habit of many figs, although its primary function is
to produce straight axes that support an expanding crown.
Although seemingly restricted to the wood of dicotyledons
among angiosperms, gelatinous fibers have been reported in the
leaves of the monocotyledonous Australian grass tree, Xanthorrhoea, where they reposition leaves (Staff, 1974). The GFs in the
leaves of Xanthorrhoea are predominantly located in the abaxial
surface just above the broadened leaf bases. Thus, there is no restriction of gelatinous fibers to wood. We therefore have to redefine
them as tension fibers (TFs). Nor are they restricted to angiosperms. They have been demonstrated in the stems and branches of
the gymnosperm Gnetum (Gnetales), not in the wood, where the
absence of fibers excludes them, but in the primary stem cortex and
secondary phloem. Here TFs are produced eccentrically as a presumed gravimorphic response (Tomlinson, 2001, 2003). The cell
wall is made up of S1 and Sg layers, only the first of which is lignified, the signature characteristic of gelatinous fibers. These fibers
retain the same cell wall features as those of angiosperms, with a
lignified secondary cell wall (S1 layer) and a readily separable cellulosic inner tertiary cell wall layer (Sg layer) (Mellerowicz and
Gorshkova, 2012). They also can occur in the seedling radicle (P.
B. Tomlinson, unpublished data). Consequently, we have evidence
that TFs occur in at least two major clades of seed plants, but not in
conifers, and not necessarily just in wood.
In this article, we now add a third clade in demonstrating the
presence of GFs in the Cycadales, where they have an important function in root contraction. As gymnosperms, the cycads
might be expected to have CW, but Fisher and Marler (2006)
showed that they do not, possibly because their wood is highly
parenchymatous (manoxylic) in contrast to the pycnoxylic condition of conifers. Our results therefore show a new cell type
not previously reported for cycads, in a functional context. We
present our results as a precursor to a more comprehensive survey involving experimental procedures.

are presented in detail for C. taitungensis and Z. vasquezii. To minimize artifacts of fixation and processing that could distort tissue, we cut sections 6090
m thick and examined material in the living condition before using stains and
histochemical reagents. We studied only wet preparations. As a routine, we
used sequential sections along a single organ, although because of variation in
diameter along a single root this sequence could not necessarily be used as a
proxy for developmental changes occurring at one level. A more useful guide
therefore was the changes observed in sections of different diameters. Sections
were cut from unembedded material using a Reichert OME sliding microtome
(Reichert, Vienna, Austria). Initially, sections were examined unstained, i.e., in
as unaltered a state as possible. Subsequently, they could be stained in 0.01%
(w/v) aqueous toluidine blue as a general metachromatic stain, and often after
preliminary bleaching in commercial bleach (5% v/v sodium hypochlorite).
Conventional histochemical tests included those for starch (iodine-potassium
iodide), lignin (phloroglucinol-HCl) and lipids (Sudan IV) (Griffith et al.,
2014). Excessive starch was often removed by concentrated hydrochloric acid.
All photographic documentation was from sections examined as wet, i.e., hydrated specimens to view tissues in as natural a state as possible.
MacerationTo isolate individual cell types by maceration, we cut small slivers, boiled them in 10% w/v KOH for 5 min followed, after washing in tap water,
with 20% w/v chromic acid (aqueous chromium trioxide) for 3060 min or until
material was sufficiently softened for easy cell separation. Material was mounted in
50% glycerin/water (1/1) by volume and teased apart on a slide for viewing.
IllustrationMicrophotographs were taken with a Nikon Coolpix 4500
digital camera with an attached screw-on Leica 10 lens, mounted on either an
Olympus BH2 compound microscope or an Olympus BZH dissecting microscope. Images for polarized light were taken on a Wild M 20 microscope either
black-and-white (crossed polarizer and analyzer) or with an added color filter to
contrast the tissue types in unstained material. Images were minimally transformed in Adobe Illustrator (San Jose, California, USA) for brightness, color
contrast, and cropping.

RESULTS

Material was derived from collections at three institutions: Montgomery Botanical Center and Fairchild Tropical Botanic Garden (Coral Gables) and the
Kampong Garden of the National Tropical Botanical Garden (Miami), South
Florida. It included not only documented collections but also volunteer plants
that reproduced naturally. We studied the descending radicle of seedlings in
their early developmental stages. Some comparison was made to lateral and
stem-borne roots from well-established plants together with the hypocotyl and
stem, but we do not report on these preliminary findings. Whatever their origin,
roots are often irregularly oriented. Root diameter may or may not fluctuate
along the axis. Sequential sections therefore did not necessarily show a consistent increase in diameter. The two genera studied, Cycas and Zamia, show contrasting mechanisms of secondary phloem and hence GF distribution, indicative
of the need for more extended taxonomic consideration.

The seedlingThe morphology of seedlings in Cycas and Zamia contrasts strongly: Cycas has a radicle with uniform taper, is
not fleshy and with a short hypocotyl and an expanded plumular
shoot (Fig. 1A); Zamia has a somewhat fleshy root, variable in
diameter and a swollen hypocotyl (Fig. 1C, D). The later development of branch and stem-borne roots produces a root system
that has been described as fascicular (Bryan, 1936).
Cycad seedlings uniformly exhibit hypogeal germination
with two absorbing cotyledons embedded in the gametophytic
seed storage tissue (often called endosperm). The emergent
seedling includes the eventually long radicle and an erect but
short plumule, and the first (juvenile) leaves have a long petiole
and few (25) terminal leaflets. The hypocotyl, between the
plumule and radicle and completing the seedling axis, is clearly
maintained as a swollen structure. In mature seedlings, the hypocotyl and extended radicle are distinguished, the former with
a smooth, the latter with an increasingly wrinkled surface (cf.
Fig. 1B with D, hyp). Narrow lateral roots (Fig. 1B, lr) arise
progressively as the radicle extends, the upper or earliest often
showing the beginnings of coralloid roots so characteristic of
cycads (Fig. 1A, 1D, cr). In continued diametric expansion of
the radicle through secondary growth from a bifacial cambium
the surface layers are visibly sloughed off as the diameter increases, making the wrinkled surface more obvious (Fig. 1B).
This feature is evidence for organ contraction despite secondary growth. Most evidence for axis contraction comes from
anatomical changes.

SectioningMaterial of Cycas (C. revoluta, C. taitungensis) and Zamia (Z.

furfuracea, Z. integrifolia [syn. Z. pumila], Z. vasquezii) was examined. Results

Mature plants In well-established seedlings, the hypocotyl

becomes tuberous and together with the stem proper adds to the

MATERIALS AND METHODS

August 2014]

TOMLINSON ET AL.ROOT CONTRACTION IN CYCADS

1277

Fig. 1. Seedling habit. (A. B) Cycas taitungensis. (A) Seedling with uniformly tapered radicle. (B) Wrinkled surface of upper (older) radicle. (C, D)
Zamia integrifolia. (C) Seedling with fleshy irregular radicle. (D) Detail of radicle with wrinkled surface and irregular diameter. Scale bars: A = 5 cm; B =
15 mm; C = 1 cm; D = 5 cm. Abbreviations: cr = coralloid root; hyp = hypocotyl; lr = lateral root.

size of the axis through establishment growth. This addition involves a progressive increase in the primary thickening meristem, which continues for many years in arborescent plants
(Bork, 1990) and establishes the final diameter of tuberous
stems, which often branch dichotomously. The latter condition
typifies Bowenia, Stangeria, and most species of Zamia. Stemborne roots arise at the base of the trunk or tuberous stem and
may repeat the process of secondary enlargement and organ
contraction. In this preliminary report, we restrict our description to the anatomy of the radicle.
(1) CycasWe describe Cycas first because it is the earliestdivergent genus in the whole order (Salas-Leiva et al., 2013;
Griffith et al., 2014) and has been most extensively used as a
type, although this is not our intended approach.
Primary root anatomyThe radicle seen in transverse section (TS) includes a usually diarch stele (Fig. 2A), with two
protoxylem poles (px) and a central group of irregularly distributed metaxylem tracheids (mx). However, triarch and tetrarch
steles are not uncommon, and this condition may change along
a single axis, as reported by Atwood (1936). Secondary growth
appears early in quite narrow roots, resulting in an opposed tissue of secondary xylem (Fig. 2A, xy2) outside of which is
phloem tissue (Fig. 2A, ph)an inner secondary file of radially
arranged cells and an outer layer of irregularly arranged primary phloem cells (Fig. 2I, pf1; pf2). Phloem thus occurs as two
strands parallel to the xylem plate, but essentially alternates
with the protoxylem poles. The cortex (Fig. 1A, co) is thinwalled and without histological differentiation, but separated
from the stele by a thin-walled endodermis (Fig. 2A, end) with
conspicuous Casparian thickenings. The endodermis never becomes thick walled and is early disrupted through secondary

growth. A thin-walled pericycle four to six cells deep separates

the endodermis from the vascular tissues (Fig. 2A, per). A phellogen is developed early in the outer cortex, but periderm does
not accumulate by virtue of the combined effects of secondary
growth and root contraction. Its displaced remains are seen as
strands of collapsed tissue on the root surface. Development of
a vascular cambium between primary xylem and phloem is long
delayed outside the protoxylem so that the elliptical outline of a
diarch root stele persists and is often retained until late into secondary development (Fig. 2B, D).
Secondary root anatomyThe vascular cambium in the
early establishment phase produces xylem of the manoxylic
type, i.e., with narrow radial files of secondary tracheids separated by bands of ray parenchyma. Rays in the xylem are continuous with rays in the secondary phloem (Fig. 2D in Cycas
and Fig. 3D in Zamia). Increase in diameter is accommodated
circumferentially by tangential expansion of outer parenchyma
cells, often accompanied by radial cell division. The cortex initially appears uniform in TS (Fig. 2B, co), but in longitudinal
section (LS), incipient collapse of bands of cortical cells can be
seen, the first indications of root contraction (Fig. 2C, co, arrows). As this process proceeds, the first evidence of stelar disorganization is the axial reorientation of the metaxylem and
early secondary xylem tracheids (Fig. 2D, xy1, xy2, cf. 2G).
Further development produces a marked change in the cortex,
with collapse of bands of cortical cells that are not precisely
horizontal, so that they are seen in both TS and LS (Fig. 2E, 2F,
arrows). That tension is generated from the center is suggested
by the concave shape of individual root sections.
Radial cell numbers in secondary phloem increase faster than
in secondary xylem, so the area ratio of secondary phloem to xylem (measured from the radius) continually increases, (Fig. 2,

1278

AMERICAN JOURNAL OF BOTANY

[Vol. 101

August 2014]

TOMLINSON ET AL.ROOT CONTRACTION IN CYCADS

cf. B with D) even though the older, originally primary, phloem

becomes progressively disorganized (Fig. 2I, pf1). Parallel to
the rays in the phloem are radiating bands of alternate conducting and mechanical tissues (Fig. 2I, pf2). The former are very
uniform and include parenchyma and presumably sieve cells,
although the latter could not be diagnosed with our methods.
The mechanical tissue consists of irregular radial files of gelatinous fibers (GFs) several cells wide. The gelatinous fibers are
recognized by their lignified secondary walls (S1) and unlignified and refractive tertiary walls (Sg), the latter identical with
the Sg layer of known tension fibers (Fig. 2J). We feel justified
therefore in referring to them as tension fibers (TFs) even
though a confirmatory character of nearly axial microfibril angle could not be verified by our exploratory methods. However,
later evidence indicates how they contract. An added feature
frequent in later generated secondary phloem is the presence of
radial files of parenchyma with included druse-like crystals.
Mechanism of root contractionIn transverse view as the
root diameter increases with addition of secondary tissues,
which is largely secondary phloem, there is conspicuous disruption of older vascular tissues (Fig. 2D). In the outer secondary phloem, the gelatinous fibers lose their regular radial
seriation and are seen in various oblique or even horizontal positions. The surrounding parenchyma and conducting cells are
similarly disorganized or collapsed. The original central primary xylem (xy1) and progressively the first formed (inner) secondary xylem (xy2) also become disorganized, with the loss of
radial seriation (Fig. 2G). This disruption is most obvious in
longitudinal section, the tracheary elements in particular appearing sinuous. Here the contrast between axially oriented
outer (newly formed) tracheids just inside the cambium (Fig.
2G, xy2) and the inner older xylem region (xy1) is very clear.
Similarly, the outer (older) phloem fibers (pf1) become shortened, twisted, and variously oriented, in contrast to the new axially oriented and noncontracted inner fibers (Fig. 2G, pf2).
Macerated material most clearly illustrates this change, especially where straight fibers and contorted fibers appear in the
same field of view (Fig. 4D). We consider this late development
of mature fibers, combined with their cell wall characteristics
and distinctive shortening justification for referring to them as
tension fibers (TFs). A feature of the more parenchymatous cortical tissues in initial stages of this process is their frequent collapse to form flattened plates of cells visible in both longitudinal
and transverse view (Fig. 2E, 2F, arrows). We interpreted this
feature as a result of the action of TFs, i.e., a consequence, not
a cause, of tissue and organ contraction. Direct evidence is seen
in the close-up detail of the outer stele in LS in Fig. 2H. Here
the contraction of the outer phloem fibers (pf1) has induced a

1279

corresponding shortening of pericycle and endodermal (end)

cells transmitted to the cortex and accommodated by the formation of regular plates of collapsed cells (Fig. 2H, arrows).
(2) ZamiaPrimary structureThe primary construction in
Zamia integrifolia is represented by a triarch root (Fig. 3A),
although this is not necessarily continued into later development. Three protoxylem poles (px) alternate with bands of primary xylem and most obviously primary phloem (pf1), which
includes numerous GFs.
Secondary developmentAn older root of Z. integrifolia
(Fig. 3B) shows progressive development of secondary tissues,
producing much more extensive phloem (ph) than xylem (xy),
a feature that becomes distinctive for Zamia. In a comparable
root of Zamia vasquezii (Fig. 3C), the diarch condition is evident and the secondary tissues still compact; the polarized part
of the image shows this most clearly. In particular, the cortex
(co) is still an evident component of root anatomy, a situation
that changes with root contraction. At an early stage (Fig. 3D),
the central tissues remain undisrupted, with protoxylem (px)
still distinct from secondary xylem (xy2) and radial continuity
from xylem to phloem still precise.
With increasing root diameter, occasioned largely by increase in the amount of secondary phloem, root contraction disrupts the regularity of early-formed tissues (Fig. 3F), and the
area ratio of phloem (ph) to xylem (xy) increases to values
much greater than 1 : 1. The boundary between stele and cortex
(co), originally represented by the endodermis, becomes obscure, and the cortex narrows as a periderm encroaches upon it.
At a later stage (Fig. 3F), the cortex disappears, and the outermost part of the stele is represented by disrupted phloem cells.
The central part of the xylem is then represented by a mass of
disrupted older xylem elements (Fig. 3E, xy). All these changes
are best seen in LS as in Fig. 3GL.
The root in a noncontracted state (Fig. 3G) shows the axial
orientation of the vascular tissues, both the central xylem (xy)
and phloem (ph), included GFs clearly demarcated in polarized
light. The cortex (co) is pronounced. Further development
shows the beginnings of fiber contraction, as seen in tangential
LS (Fig. 3H, ph) and the beginnings of cortical disorganization
as parenchyma cells (par) become realigned. Older tissue of the
primary and secondary phloem when macerated and cells separated shows the crumpled appearance of GFs (Fig. 3I), made
apparent in polarized light. An overall radial view (Fig. 3J)
shows the central tracheal elements of the xylem (xy) in a crumpled condition, which must be a passive response to contraction. This state is seen most clearly in a nonpolarized LS
(Fig. 3K, xy) stained with phloroglucinol-HCl. The more precise

Fig. 2. Cycas taitungensis. Noncontracted and contracted roots in section. (A) Tangential section (TS) diarch stele of noncontracted root (unstained).
(B) TS of older root with more extensive secondary xylem and phloem. (C) Radial longitudinal section (LS) of same root as (B); arrows indicate incipient
collapse of bands of cortical cells. (D) TS of stele of older root as in (B), with secondary xylem and phloem still in approximately equal proportions. Primary xylem (xy1) = metaxylem (mx) and protoxylem poles (px). (E) TS of older, wider root as in (D) with collapsed bands of cortical cells (arrows). (F)
Corresponding LS of similar root with collapsed bands of cortical cells (arrows). (G) LS of stele of contracted root; older (inner) primary (xy1) and secondary (xy2) xylem tracheids crumpled passively by tissue contraction, younger tracheids of xy2 uncrumpled; inner (younger) fibers of primary phloem (pf1)
contracted; arrows indicate collapsed bands of cortical cells. (H) Radial LS of detail of outer stele; contraction of primary phloem fibers (pf1) induces
shortening of endodermal cells (end) and collapse of bands of cortical cells (arrows). (I) TS of phloem tissue; GFs of primary phloem (pf1) irregularly
oriented; GFs of secondary phloem (pf2) in radial files. (J) Gelatinous fibers of primary phloem (pf1) with lignified S1 layer and unlignified Sg layer. Scale
bars: A = 1 mm; B = 1 mm; C = 100 m; D = 1 mm; E = 2 mm; F = 3 mm; G = 400 m; H = 100 m; I = 100 m; J = 50 m. Abbreviations: c = cambium;
co = cortex; end = endodermis; mx = metaxylem; per = pericycle; pf1 = primary phloem fibers; pf2 = secondary phloem fibers; ph = phloem; px = protoxylem pole; S1 = outer (lignified) layer of secondary wall; Sg = inner (unlignified) gelatinous layer of tertiary wall; xy1 = primary xylem, which is made up
of protoxylem poles (px) and metaxylem (mx); xy2 =secondary xylem; arrows in C, EH = collapsed plates of cortical parenchyma.

1280

AMERICAN JOURNAL OF BOTANY

[Vol. 101

August 2014]

TOMLINSON ET AL.ROOT CONTRACTION IN CYCADS

location of GFs in Fig. 3J and L contrasts inner noncontracted

fibers of the younger phloem (ipf) with the older, outer contracted fibers (opf) of the outer phloem. Because of their contorted, corkscrew appearance, as seen in Fig. 3I, only short
segments of individual fibers appear in section. Further evidence for the modification resulting from overall contraction is
seen in Fig. 3K and 3L.
Gelatinous fibersThe development of GFs shows a close
association with that of tracheids. For each narrow file of tracheids on the xylem side of the cambium, there is a corresponding series of cells on the phloem side (Fig. 4A, B). This series
is maintained over an extended period of cambial activity and
indicates that a series of initials within the dividing cambium is
producing corresponding specialized cells in its inner and outer
derivatives, those of the xylem becoming tracheids and those in
the phloem becoming in part fibers. This specialization may
proceed without initiation of a new series of such cells so that
the ever-widening circumference of the cambium must be accommodated by radial divisions in derivative ray cells, accompanied by tangential cell expansion (Fig. 4B). The process is
particularly striking in Zamia species where phloem tissues
come to dominate the whole of the root (Fig. 3E). Accompanying this expansion is the contraction of the older largely primary GFs at the periphery of the phloem, seen in tangential
section (TS) as disoriented cells (Fig. 4C). Progressively, the
whole of the outer phloem tissue eventually consists of contracted and wrinkled fibers (Fig. 3F) set among a ground tissue
of irregular parenchyma cells. The GFs of the original primary
phloem are not identifiable, and the surrounding endodermis is
totally disrupted and no longer recognizable. Ultimately, outer
tissues of the stele become incorporated in a periderm and the
cortex no longer exists. This massive accumulation of parenchyma accounts for the fleshy nature of older roots in Zamia
and its variable expression in an axial direction to produce the
irregular diameter of old roots (Fig. 1D). Cells may accumulate
considerable amounts of stored starch, but the tissues have a
high water content.
Fiber wallsIt is clear from these topographic considerations that the axial shrinkage of roots can only be brought
about by the collective tension induced by the older GFs. This
contraction is shown in situ in polarized light, where inner (ipf)
and outer (opf) fibers can be contrasted (Fig. 4D). This observation raises questions of the association between individual GFs
within a file of phloem cells. Adjacent fibers are not necessarily
sister cells, as might be expected if they arose from the same
cambial initial. Paired GFs overlap without common end walls
(Fig. 4G), but they remain adherent and show the same pattern
of contraction, meaning that the tension generated can be transmitted along a linear series of individual GFs. The actual wall
properties of the GFs are distinctive. Under polarized light,

1281

their crystalline nature is indicated by a kaleidoscopic effect

(Fig. 4E), particularly striking at the highest magnifications
(Fig. 4F). We draw attention to this phenomenon although it is
beyond the scope of this paper to investigate further.
(3) FicusWe compare cycad roots with the aerial roots of
Ficus altissima to provide a more convincing analogy for the
function of GFs in roots of gymnosperms as well as angiosperms. This genus was the source of the classical study by
Zimmermann et al. (1968) that established the method of contraction of aerial roots in the genus, an important component of
fig trees ecological success. Zimmermann et al. (1968) thus
established that GFs could occur in roots, concentrically and in
the absence of a gravimorphic response. Tension in such roots
can be demonstrated by severing an attached root, whereupon
the cut ends separate by a considerable distance as the original
tension is released (Fig. 5A before the cut; Fig. 5B after the
cut). Sections of the cut root show the central primary root construction of the original unattached aerial root and the extensive
development of secondary xylem formed after the root reaches
the ground (Fig. 5C). The ground tissue is composed of alternating tangential bands of fibers and lignified parenchyma (Fig.
5D, triangles vs. asterisks). The fibers, almost without exception, have the characteristics of tension fibers, i.e., a lignified S1
layer and the cellulosic Sg layer that tends to separate from the
primary wall (Fig. 5E). Such fibers are identical with those seen
in cycad roots (Fig. 5F cf. Fig. 2J). As with cycad roots, secondary growth in diameter together with root contraction causes the
surface periderm to be shed continuously (cf. Fig. 2A with Fig.
5G). A curious feature of these roots is the development of cellulosic unlignified fibers in the secondary phloem, although
these lack the lignified S1 layer (Fig. 5H).
DISCUSSION
The early literature on cycad roots has no mention of gelatinous fibers in cycad phloem. They can be seen even in the primary phloem of roots prior to the onset of secondary growth
(Fig. 3A). Dorety (1909) did not mention them, largely because
her section said to be of the root was from the hypocotyl. Cellulosic fibers have been reported in the secondary phloem of
cycad roots and stems. Atwood (1936) referred to them as bast
fibers, as did Terrazas (1991) in stems, whereas Chamberlain
(1934), for some reason, described them as tracheids. Fisher
and Vovides (2004) briefly mentioned gelatinous fibers in their
study of mycorrhizae in cycad roots, but the roots studied were
necessarily ultimate roots. Fisher (2008) referred to them as
gelatinous fibers in seedling roots of Zamia integrifolia (= Z.
pumila) and by implication associated them with root contraction along with the angiosperms he described, but made no further

Fig. 3. (A, B) Zamia integrifolia and (CL) Z. vasquezii. Tangential section (TS) in (AF); longitudinal section (LS) in (GL); (F, J, L) polarized light.
(A) Primary triarch root, toluidine blue. (B) Older but diarch version of (A) but with limited amount of phloem fibers. (C) Split image of older root, upper
unstained, lower in polarized light. (D) Center of older root, xylem still organized. (E) Same root to show increased secondary phloem relative to xylem. (F)
Older and slightly eccentric root, xylem disorganized (cf. Fig. 3K), the outer tissue completely secondary phloem as far as the periderm. (G) Young root
without evidence of contraction. (H) Tangential LS to show contracted GFs of one phloem band (ph). (I) Macerated GFs to show individual fiber contraction.
(J) Median radial TS with contracted xylem (xy), inner phloem fibers (ipf) uncontracted, outer GFs (opf) contracted. (K) Xylem tissue (xy) stained in
phloroglucinol-HCl with extreme passive contraction. (L) Radial LS of secondary phloem with inner GFs (ipf) uncontracted, outer GFs (opf) contracted, but
without collapse of adjacent parenchyma. Scale bars: A = 200 m; B = 1 mm; C = 1 mm; D = 500 m; E = 1 mm; F = 2 mm; G = 500 m; H = 500 m; I =
250 m; J = 1 mm; K = 250 m; L = 500 m. Abbreviations: c = cambium; co = cortex; ipf = inner phloem fibers; mx = metaxylem; opf = outer phloem fibers;
par = parenchyma; pf1 = primary phloem fibers; ph = phloem; ph2 = secondary phloem; px = protoxylem pole; xy = xylem; and xy2 = secondary xylem.

1282

AMERICAN JOURNAL OF BOTANY

[Vol. 101

Fig. 4. Fiber details in Zamia. (AC) Zamia integrifolia; (DG) Z. furfuracea. (A) Tangential section (TS) root to show concordance of tracheid and
GF files across the cambium. (B) Detail, section in phloroglucinol-HCl) with tangential expansion of ray parenchyma cells. (C) TS of early formation of
secondary phloem (toluidine blue), older (outer) GFs of primary phloem (pf1) being distorted, and young fibers of secondary phloem (pf2) not disoriented.
(D) Longitudinal section (polarized light) inner phloem fibers (ipf) and xylem tracheids (xy) noncontracted, older phloem fibers (opf) contracted and with
disorganized wall construction. (E) Contracted GFs with cell wall irregularity seen as a kaleidoscopic effect in polarized light. (F) Close up of a contracted
GF as mentioned in (E). (G) Macerated fibers from region of contraction. Single wrinkled fiber (left). GFs juxtaposed with corresponding outline postcontraction (center). Overlap of fiber end (right); GFs are not sister cells in a tangential direction. Scale bars: A = 1 mm; B = 200 m; C = 100 m; D = 200 m;
E = 200 m; F = 100 m; G = 200 m (left), 100 m (center), 50 m (right). Abbreviations: c = cambium; co = cortex; ipf = inner phloem fibers; mx =
metaxylem; opf = outer phloem fibers; par = parenchyma; pf1 = primary phloem fibers; pf2 = secondary phloem fibers; ph = phloem; px = protoxylem pole;
xy = xylem; and xy2 = secondary xylem.

detailed investigation. Pesacreta and Purpurea (2014) described

basic differences between angiosperms and gymnosperms in
terms of protophloem development and included cycad examples in Dioon and Zamia. The stages they described are much
earlier than we have studied, and they did not show the later
stages that would include fiber development.
Our results, which are clearly preliminary, establish that cycad roots include GFs, and we interpret them as being responsible for root contraction, based on the structural similarity of
GFs in Cycas and Zamia with those found in angiosperms, as
seen in Ficus. Furthermore, we describe contrasting tissue configurations of the secondary phloem between Cycas and Zamia,
although the underlying function in contraction is the same. In
particular in both genera, in both in situ and macerated preparations, the contractile nature of fibers, either individually or in
concert and resulting in contorted end products is abundantly
clear (Figs. 24).

Rimbach (1929) provided an overview of root contraction in

seed plants, presenting information on 450 species in 315 genera
within 82 families, i.e., 15 monocotyledonous and 66 dicotyledonous angiosperms, but only two gymnosperm species of the
cycads (Cycas rumphii and Zamia muricata). His results were
presented as percentage decrease in root length. Seedling, adventitious, and lateral roots were included. Values for the cycads were 60% for Cycas and 50% for Zamia, among the
highest values reported overall. Rimbach made no suggestions as to the mechanism causing the contraction. Stevenson
(1975, 2013) described the mechanism of root contraction in
Botrychium as lysigenous cavities forming and then collapsing.
Stevenson (1980, 2013) also described root contraction in terms
of the processes reported in angiosperms such as Oxalis (cf. Ptz,
2002), where root contraction is a result of the collapse of horizontal rows of parenchyma cells in the cortex and pith. For cycads, his evidence for contraction was the development of a

August 2014]

TOMLINSON ET AL.ROOT CONTRACTION IN CYCADS

1283

Fig. 5. Ficus altissima. Anatomy of contractile roots. (A, B) Complete and severed grounded roots. (A) Complete root. (B) Severed root with tension released, cut surfaces separated a distance of 3 cm (double-headed white arrow), arrows indicate identifying marks. (CH) Tangential section (TS) of severed
root; (CF) stained in phloroglucinol-HCl. (C) TS of root center, primary root (p.r.) marks the portion of the root developed prior to secondary growth and attachment, while the aerial roots are unattached. (D) Secondary, banded xylem, triangles indicate GFs, asterisks indicate parenchyma, also in (E). (E) Band of
gelatinous fibers (GFs) with bands of parenchyma cells. (F) Details of GFs. (G) Similar fibers stained in toluidine blue. (H) Detail of outer root layers in polarized light; secondary phloem also with cellulosic fibers (but not GFs). Scale bars: A = 1 cm; B = 1 cm, C = 1 mm; D = 200 m; E = 100 m; F = 50 m; G = 1
mm; H = 500 m. Abbreviations: p.r. = primary root; xy2 = secondary xylem; * = parenchyma;  = gelatinous fibers; S1 = outer (lignified) layer of secondary
wall; Sg = inner (unlignified) gelatinous layer of tertiary wall; A = mature secondary xylem; B = maturing secondary xylem; and C = secondary phloem.

1284

[Vol. 101

AMERICAN JOURNAL OF BOTANY

sinuous course of vascular tissues as seen in longitudinal section,

the wrinkled surface of roots, and the apparent shortening of leaf
scars on the stem surface, thus adding stems to the concept of
axis shortening. In following earlier reports, especially of monocotyledons, he invoked the collapse of ground tissue cells to form
flattened plates of cells. Stevenson (1980) did not describe fibers
in the phloem. Watanabe (1925) made a similar description of
cell collapse accounting for root contraction in Cycas, but again
without describing the existence of phloem fibers.
We present an alternative scenario in which the gelatinous
fibers are the source of root contraction, collapse of ground tissue cells is a consequence and not a cause of root contraction,
and there is no contraction of stems other than that might be
induced by the downward pull of contracting roots. Our suggestions are supported by the major difference between Cycas
(with collapsed cortical tissues) and Zamia (without collapsed
cortical tissues). Both have contractile GFs, which can readily
explain collapsed ground cell tissues, while it is difficult to understand how collapse of parenchyma cells alone can generate
the force necessary to shorten roots. Emphasis needs to be
placed on the common occurrence of GFs in both genera. The
existence of cells with the same structural features as tension
fibers in angiosperms, here largely confined to roots and produced in large numbers, provide a logical alternative to prior
interpretations (Watanabe, 1925; Stevenson, 1980). The contraction of these fibers is unequivocal in Ficus roots, where the
forces generated by tension fibers can be enormous (Fig. 5).
Here the position where an attached root is severed is indicated
before and after a cut is made (Fig. 5A, B) and shows a separation of 3 cm when the tension is released. This force is such that
it is then impossible by hand to restore the cut ends to their
original position and so recreate the tension. In this root, the fibers are all TWFs, forming plates of tissue alternating with tangential bands of xylem parenchyma and delimited by rays (Fig.
5). The microscopic details can be compared to similar fibers in
cycads, but with a different arrangement. It is therefore logical
to apply the same function to TFs, despite the wide phylogenetic separation.
Seed plant evolution The consequences of our claimed
discovery are of some significance in interpreting the evolutionary diversification of seed plants. If tension fibers are homologous cells in seed plants we now know that they occur in
three major clades, including not only angiosperms (Magnoliophytes), but Gnetophytes and Cycadophytes. The former have
pycnoxylic secondary xylem, the latter are manoxylic. More
significantly, Cycadales is the closest extant lineage to the
deeper Pteridospermatophyta (e.g., Lyginopteris, Callistophyton; Doyle, 2006) so this extension for the distribution of gelatinous fibers could place the origin of this type of cell at a much
earlier date of seed plant diversification than is represented by
Gnetum and angiosperms. Conventional wisdom might not accept a close association between angiosperms and cycads, except for the statement in Brenner et al. (2007, p. 183) that a
surprising large number of cycad contigs had homology to
genes in angiosperms with roles in development and signaling
pathways. However, one might temper that statement with the
observation that molecular sequence homology does not necessarily translate into morphogenetic or organographic homology (Niklas, 2014, p. 21).
The fossil record should be the ultimate arbiter in a search for
gelatinous fibers in early lignophytes, but permineralized secondary phloem tissues are rare. Fossil phloem was reviewed by

Decombeix and Meyer-Berthaud (2013), in which they show in

Callixylon (a progymnosperm) unusual secondary phloem cells
with thick walls are interpreted as fibers, but in tangential, not
radial bands. Nevertheless, the preservation of cellulosic, unlignified gelatinous fibers in the fossil record seems remote. The
added problem is, of course, the unlikely ability to recognize
the characteristic microfibrillar organization of the cell wall (cf.
Lautner et al., 2012).
ConclusionOur observations show that GFs occur in the
roots of cycads. It remains curious that such a distinctive feature
of cycad anatomy should have remained undiscovered until the
present, but we now have a signpost leading to future discoveries
about this ancient and enigmatic group. The availability of large
living collections and more advanced techniques cannot fail to
produce a better understanding of cycad structural biology.
LITERATURE CITED
ABASOLO, W. P., M. YOSIDA, H. YAMAMOTO, AND T. OKUYAMA. 2009. Stress
generation in aerial roots of Ficus elastic (Moraceae). International
Association of Wood Anatomists Journal 30: 216224.
AKACHUKU, A. E. 1993. Recovery and morphology of Pinus resinosa
Ait. trees 50 years after they were displaced by a hurricane. Forest
Ecology and Management 56: 113129.
ATWOOD, S. 1936. The anomalous root structure of Cycas revoluta.
American Journal of Botany 23: 336340.
BORK, J. 1990. Developmental cycles in shoot growth of male Cycas circinalis. American Journal of Botany 77: 981985.
BOWLING, A. J., AND K. J. VAUGHAN. 2009. Gelatinous fibers are widespread in coiling tendrils and twining vines. American Journal of
Botany 96: 719727.
BRENNER, E. D., D. W. STEVENSON, M. S. KATARI, R. W. MCCOMBIE, S. A.
RUDD, S. J. RUNKO, AND R. A. MANTIENSSEN. 2007. Genomic studies
in Cycas rumphii Miquel. Memoirs of the New York Botanical Garden
97: 182193.
BRYAN, G. S. 1936. Fascicled roots of Cycas revoluta. American Journal
of Botany 23: 334335.
CHAMBERLAIN, C. J. 1934. GymnospermsStructure and evolution.
Chicago University Press, Chicago, Illinois, USA. [Reprint 1957,
Johnson Reprint Corp., New York, New York, USA.]
DECOMBEIX, A.-L., AND B. MEYER-BERTHAUD. 2013. A Callixylon
(Archaeopteridales, Progymnospermopsida) trunk with preserved
secondary phloem from the Late Devonian of Morocco. American
Journal of Botany 100: 22192230.
DORETY, H. A. 1909. Vascular anatomy of the seedling of Microcycas
calocoma. Botanical Gazette 47: 139147.
DOYLE, J. A. 2006. Seed ferns and the origin of angiosperms. Journal of
the Torrey Botanical Society 133: 169209.
DU, S., AND F. YAMAMOTO. 2007. An overview of the biology of reaction wood formation. Journal of Integrative Plant Biology 49:
131143.
FISHER, J. B. 1985. Induction of reaction wood in Terminalia (Combretaceae):
Roles of gravity and stress. Annals of Botany 55: 237248.
FISHER, J. B. 2008. Anatomy of axis contraction in seedlings from fireprone habitats. American Journal of Botany 95: 13371348.
FISHER, J. B., AND M. A. BLANCO. 2014. Gelatinous fibers and variant secondary growth related to stem undulation and contraction in a monkey
ladder vine, Bauhinia glabra (Fabaceae). American Journal of Botany
101: 608616.
FISHER, J. B., AND T. E. MARLER. 2006. Eccentric growth, but no compression wood in a horizontal stem of Cycas micronesica. International
Association of Wood Anatomists Journal 27: 377382.
FISHER, J. B., AND J. W. STEVENSON. 1981. Occurrence of reaction wood
in branches of dicotyledons and its role in tree architecture. Botanical
Gazette 142: 8295.

August 2014]

TOMLINSON ET AL.ROOT CONTRACTION IN CYCADS

FISHER, J. B., AND P. B. TOMLINSON. 2002. Tension wood fibers are related
to gravitropic movement of red mangrove (Rhizophora mangle) seedlings. Journal of Plant Research 115: 3945.
FISHER, J. B., AND A. P. VOVIDES. 2004. Mycorrhizae are present in Cycad
roots. Botanical Review 70: 1623.
GRIFFITH, M. P., T. M. MAGELLAN, AND P. B. TOMLINSON. 2014. Variation
in leaflet structure in Cycas (Cycadales: Cycadaceae): Does anatomy
follow phylogeny and geography? International Journal of Plant
Sciences 175: 241255.
HSTER, H. R., AND W. LIESE. 1966. ber das Vorkommen von
Reaktionsgewebe in Wurzeln und sten der Dikotyledonen. Holzforschung 20: 8090.
LAUTNER, S., C. ZOLLFRANK, AND J. FROMM. 2012. Microfibril angle distribution of poplar tension wood. International Association of Wood
Anatomists Journal 33: 431439.
MELLEROWICZ, E. J., AND T. A. GORSHKOVA. 2012. Tensional stress generation in gelatinous fibers: A review and possible mechanism based on
cell-wall structure and composition. Journal of Experimental Botany
63: 551565.
MELOCHE, C. G., J. P. KNOX, AND K. C. VAUGHAN. 2007. A cortical band of gelatinous fibers causes the coiling of redvine tendrils: A model based upon
cytochemical and immunocytochemical studies. Planta 225: 485498.
NIKLAS, K. J. 2014. The evolutionary-developmental origins of multicellularity. American Journal of Botany 101: 625.
PESACRETA, T. C., AND M. A. PURPUREA. 2014. Light microscopy survey
of extant gymnosperm root protophloem and comparison with basal
angiosperms. Botany 92: 388401.
PTZ, N. 2002. Contractile roots. In Y. Waisel, A. Eshel, and U. Kaflafi
[eds.], Plant roots: The hidden half, 3rd ed., 975987. Marcel Decker,
New York, New York, USA.
RIMBACH, A. 1929. Die Verbreitung der Wurzelverkurzung im Pflanzenreich. Berichte der deutschen Botanischen Gesellschaft 47:
2231.
SALAS-LEIVA, D. E., A. W. MEEROW, M. CALONJE, M. P. GRIFFITH, J.
FRANCISCO-ORTEGA, K. NAKAMURA, D. W. STEVENSON, ET AL. 2013.
Phylogeny of the cycads based on multiple single-copy nuclear genes:
Congruence of concatenated parsimony, likelihood and species tree
inference methods. Annals of Botany 112: 12631278.

1285

SCHREIBER, N., N. GIERLINGER, N. PTZ, P. FRATZL, C. NEINHAUS, AND I.

BURGERT. 2010. G-fibres in storage roots of Trifolium pratense
(Fabaceae): Tensile stress generators for contraction. Plant Journal
61: 854861.
STAFF, I. A. 1974. The occurrence of reaction fibers in Xanthorrhoea australis L. Protoplasma 61: 854861.
STEVENSON, D. W. 1975. Taxonomic and morphological observations on Botrychium multifidium (Ophioglossaeae). Madroo 23:
198204.
STEVENSON, D. W. 1980. More observations on root and stem contraction in cycads (Cycadales) with special reference to Zamia pumila L.
Botanical Journal of the Linnean Society 81: 275281.
STEVENSON, D. W. 2013. Gymnosperms. In B. A. Ambrose and M.
Purugganan [eds.], Annual Plant Reviews vol. 45, The evolution of
plant form, 141162. Blackwell, Ames, Iowa, USA.
TERRAZAS, T. 1991. Origin and activity of successive cambia in Cycas
(Cycadales). American Journal of Botany 78: 13351344.
TIMELL, T. E. 1986. Compression wood in gymnosperms, vols. 13.
Springer Verlag, New York, New York, USA.
TOMLINSON, P. B. 2001. Reaction tissues in Gnetum gnemon (Gnetales):
A preliminary report. International Association of Wood Anatomists
Journal 22: 401413.
TOMLINSON, P. B. 2003. Development of gelatinous (reaction) fibers in stems
of Gnetum gnemon (Gnetales). American Journal of Botany 90: 965972.
TOMLINSON, P. B., AND P. A. COX. 2000. Systematic and functional anatomy of seedlings in mangrove Rhizophoraceae: Vivipary explained.
Botanical Journal of the Linnean Society 134: 215231.
WARDROP, A. B. 1964. Reaction anatomy of arborescent angiosperms.
In M. H. Zimmermann [ed.], The formation of wood in forest trees,
405456. Academic Press, New York, New York, USA.
WATANABE, K. 1925. ber die Kontraction und daraus versuchte
Anomalie in der Wurzul von Cycas revoluta. Japanese Journal of
Botany 2: 293297.
WILSON, B. F., AND R. R. ARCHER. 1977. Reaction wood: Induction and
mechanical action. Annual Review of Plant Physiology 28: 2343.
ZIMMERMANN, M. H., A. B. WARDROP, AND P. B. TOMLINSON. 1968. Tension
wood in aerial roots of Ficus benjamina L. Wood Science and
Technology 2: 95104.