bs_bs_banner

Plant, Cell and Environment (2012) 35, 18981911 doi: 10.1111/j.1365-3040.2012.02524.x

The physiological implications of primary xylem

organization in two ferns pce_2524 1898..1911

CRAIG R. BRODERSEN, LINDSEY C. ROARK & JARMILA PITTERMANN

Department of Ecology & Evolutionary Biology, University of California, 1156 High Street, Santa Cruz, CA 95064, USA

ABSTRACT Ferns have a seemingly primitive vascular system com-

Xylem structure and function are well described in woody
plants, but the implications of xylem organization in less-
derived plants such as ferns are poorly understood. Here,
two ferns with contrasting phenology and xylem organiza-
tion were selected to investigate how xylem dysfunction
affects hydraulic conductivity and stomatal conductance
(gs). The drought-deciduous pioneer species, Pteridium
aquilinum, exhibits fronds composed of 25 to 37 highly
integrated vascular bundles with many connections, high
gs and moderate cavitation resistance (P50 = -2.23 MPa).
By contrast, the evergreen Woodwardia fimbriata exhibits
sectored fronds with 3 to 5 vascular bundles and infrequent
connections, low gs and high resistance to cavitation
(P50 = -5.21 MPa). Xylem-specific conductivity was signifi-
cantly higher in P. aqulinium in part due to its wide, effi-
cient conduits that supply its rapidly transpiring pinnae.
These trade-offs imply that the contrasting xylem organiza-
tion of these ferns mirrors their divergent life history strat-
egies. Greater hydraulic connectivity and gs promote rapid
seasonal growth, but come with the risk of increased vul-
nerability to cavitation in P. aquilinum, while the conserva-
tive xylem organization of W. fimbriata leads to slower
growth but greater drought tolerance and frond longevity.
Key-words: cavitation; hydraulic integration; Pteridium
aquilinum; stomatal conductance; Woodwardia fimbriata.
INTRODUCTION
Xylem organization represents the culmination of over 400
million years of selection for hydraulic systems that balance
transport efficiency with resistance to failure via cavitation
(Tyree & Zimmermann 2002; Sperry 2003; Sperry, Meinzer
& Mcculloh 2008; Pittermann 2010). Coordination between
the supply and demand for water is paramount, as stomatal
conductance (gs) and photosynthesis are directly linked
to hydraulic conductivity (k) (Brodribb & Feild 2000;
Brodribb & Holbrook 2004). Our knowledge of the rela-
tionships between plant hydraulics and photosynthetic per-
formance is based on an accumulation of studies focused on
woody plants, but much less is known about primary xylem
structure and function.
Correspondence: C. Brodersen. E-mail: cbroders@ucsc.edu
1898
posed solely of primary xylem, an assemblage of unicellular
tracheids, and occasionally vessels (Carlquist & Schneider
2001), which act as a markedly different solution to water
transport than the xylem of woody plants, where secondary
growth and multicellular vessels offer both structural
support and wide, efficient conduits. Fern tracheids are
tightly packed, comparatively few in number and have
heavily pitted lateral walls bearing pit membranes that are
often highly porous (White 1963a; Carlquist & Schneider
1997; Pittermann et al. 2011). These combined features
should make ferns extremely vulnerable to cavitation and at
a significant physiological disadvantage because of the inher-
ent risk of embolism spread (Hargrave et al. 1994; Wheeler
et al. 2005; Hacke et al. 2007). Despite these limitations,
ferns have been persistent through their evolutionary
history and represent highly successful forms in both the
past and present (Taylor, Taylor & Krings 2009; Pittermann
2010). Recent research even suggests that many ferns
have surprisingly efficient and cavitation-resistant xylem
(Watkins, Holbrook & Zwieniecki 2010; Pittermann et al.
2011), but to what extent selection has acted to modify the
spatial organization of primary xylem is not well understood.
Recent work in woody plants has shown that xylem
organization and conduit diameter play critical roles in
cavitation resistance (Loepfe et al. 2007; Mencuccini et al.
2010; Savage et al. 2010; Lens et al. 2011), but few studies
have addressed these issues in plants with primary xylem.
Hydraulic integration (i.e. xylem connectivity) in woody
plants is often coupled with small conduit diameter, lower
k and increased vulnerability to the spread of drought-
induced embolism (Zanne et al. 2006; Espino & Schenk
2009). Physical separation of the xylem into discrete
sectors, most often associated with xeric species with wide,
efficient conduits and high k, compartmentalizes water
transport and prevents the systemic spread of embolism
from compromised conduits to functional adjacent ones
(Orians, Babst & Zanne 2005; Salguero-Gmez & Casper
2011). As shown by Schenk et al. (2008), woody species
across aridity gradients display a continuum of xylem sec-
toriality patterns in stem and trunk architecture that help
plants adapt to a wide range of habitats with variable
moisture availability. Similar redundancy and hydraulic
integration in leaves aid in the recovery of gas exchange
and k following xylem damage or drought-induced embo-
lism (Sack et al. 2008; Scoffoni et al. 2011). The question
2012 Blackwell Publishing Ltd

remains of whether ferns composed solely of primary
xylem operate under the same set of selective pressures as
woody plants.
Ferns may have overcome the constraints of primary
xylem through the diversification of vascular bundle orga-
nization (i.e. stelar patterns). Stelar patterns within fern
fronds range from the simple, integrated haplostele, to
the more derived and complex eustele, siphonostele or
dictyostele with many discrete vascular bundles, making
them functionally similar to the subdivided stems of xeric
woody plants (Fig. 1; Stein 1993; Schneider et al. 2002;
Zanne et al. 2006; Schenk et al. 2008), thereby mirroring
the sectoriality and integration found in secondary xylem.
Embolism spread within a frond should be determined by
the spatial separation of bundles and the number and fre-
quency of connections within the xylem network. While
xylem organization has been studied in a variety of fern
species (Gibson, Calkin & Nobel 1985), and with specific
focus on hydraulic conductance and photosynthesis in
leaves (e.g. Schulte, Gibson & Nobel 1987; Franks & Far-
quhar 1999; Brodribb et al. 2005), the physiological impli-
cations of xylem organization and connectivity have yet to
be fully explored (Woodhouse & Nobel 1982; Lo Gullo
et al. 2010).
The striking variation in stelar patterns and the ease of
manipulating vascular bundles in ferns (e.g. severing indi-
vidual xylem bundles) present a good opportunity to study
the fundamental relationships between xylem organization
and physiology that would otherwise be difficult or impos-
sible in woody plants. The goal of this study was to deter-
mine what structure-function trade-offs might exist in
ferns as the result of adaptations towards different life
history strategies, where vascular arrangement should
have significant consequences for gas exchange and cavi-
tation resistance. We chose two ferns, Pteridium aquilinum
(L.) Kuhn (Dennstaedtiaceae) and Woodwardia fimbriata
(Sm.) (Blechnaceae), which have differences in phenology,
stelar patterns, conduit morphology and resistance to cavi-
tation, yet frequently occupy similar habitats. P. aquilinum
is a drought deciduous pioneer species known for its
invasiveness (Robinson, Sheffield & Sharpe 2010) and has
moderate resistance to cavitation (Pittermann et al. 2011).
The vascular bundles of P. aquilinum are tightly packed
and appear to fuse readily throughout the frond; an orga-
nization that we hypothesize should favour both efficient
k and the spread of drought-induced embolism (Fig. 1).
In addition, P. aquilinum is one of the few known fern
species to have true vessels in addition to tracheids (Bliss
1939; White 1963b; Carlquist & Schneider 1997, 2007; Pit-
termann et al. 2011). In contrast, W. fimbriata is a slow-
growing evergreen species with fronds that typically last at
least 2 years, is highly resistant to cavitation, and fronds
that have few vascular bundles which appear to be iso-
lated throughout their axial length (Figs 2 and 3).
The focus of this study was to characterize the relation-
ships between xylem organization, cavitation resistance
and gs based on the known differences in anatomy and
phenology.
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Fern xylem and physiology 1899
MATERIALS AND METHODS
Plant material and collection
For this study, we chose two fern species commonly found in
the seasonally moist Sequoia sempervirens understorey. The
deciduous triangular-shaped fronds of P. aquilinum are
3-pinnate pinnatifid and joined together by a long-creeping,
branched rhizome (Fig. 1a) (Calkin, Gibson & Nobel
1985). The evergreen lanceolate fronds of W. fimbriata are
1-pinnate pinnatifid, growing from a short, stout rhizome,
typically around sources of fresh water (Fig. 2a). The orga-
nization of the primary xylem in P. aquilinum was described
by Wardlaw (1950) as a perforated polycyclic solenostele,
with multiple vascular bundles arranged in concentric
circles, while W. fimbriata has a dictyostele consisting of two
large vascular bundles and one to four smaller abaxial
bundles (Beck, Schmid & Rothwell 1982; Schneider et al.
2002) (Figs 1 and 2, Supporting Information Figs S1 and
S2). In both species, each vascular bundle consists of a
central grouping of xylem surrounded by phloem, all of
which are encapsulated by an endodermis. Fully developed
current-year fronds from P. aquilinum and W. fimbriata
were cut from plants growing on the UC Santa Cruz campus
and used for anatomical and hydraulics measurements from
May to June, 2011. Fronds from the same plants were used
for in situ gas exchange measurements in the field during
the same time period.
Anatomical measurements
We characterized xylem anatomy and organization by mea-
suring conduit diameters and the spatial distribution of
bundles from transverse cross sections at three sampling
points on the fronds (Figs 1 and 2, Supporting Information
Figs S1 and S2, Table 1). Transverse sections were made
from three representative fronds from three separate indi-
vidual plants from both species, with each frond subdivided
into three segments from the base of the frond (stipe) at the
soil level, within the rachis above the axis junction (lower
rachis), and from the rachis near the apex (upper rachis,
approximately 90 cm from the soil surface). Freehand sec-
tions were stained with toludine blue, mounted in glycerol
and then photographed at 100 magnification with a digital
camera mounted to a Motic BA400 compound microscope
(Motic Inc., Richmond, British Columbia, Canada). All
measurements were taken from digital images analysed
using ImageJ software (freeware available from the site
http://rsb.info.nih.gov/ij). Conduit diameters, the ratio of
xylem lumen area to cross-sectional area of the stipe or
rachis, were measured. Conduit lumen area was measured
first and then converted to the equivalent circle diameter.
The degree of sectoriality (S) of the xylem bundles was
characterized by calculating the relationship between the
total perimeter of the xylem bundles to the combined cross-
sectional area of the xylem using the following formula
modified from Schenk et al. (2008):
S = p ( A2 ) ,
1900 C. R. Brodersen et al.

(a)

(b)

(c)

(d)

Figure 1. Frond architecture and xylem anatomy of P. aquilinum fronds. The triangular shaped frond of P. aquilinum is 3-pinnate
pinnatifid (a). The number of vascular bundles (b, squares) decreases from the base of the frond (0 cm) to the apex (~120 cm) in three
representative fronds. Representative transverse sections (c) spaced approximately 20 cm apart (see Supporting Information Fig. S1 for
larger images) and the location of connections (d) in three representative fronds show the organization of the vascular bundles. Grey bars
in (a) indicate the three sampling regions of the stipe or rachis used for hydraulics and gas exchange measurements.

2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
 Fern xylem and physiology 1901

(a)

(b)

(c)

(d)

Figure 2. Frond architecture and xylem anatomy in W. fimbriata fronds. The lanceolate frond of W. fimbriata is 1-pinnate pinnatifid (a).
The number of vascular bundles (b, squares) decreases from the base of the frond (0 cm) to the apex (~100 cm) in three representative
fronds. Representative transverse sections (c) spaced approximately 20 cm apart (see Supporting Information Fig. S2 for larger images)
and the location of connections (d) in three representative fronds show the organization of the vascular bundles. Grey bars in (a) indicate
the three sampling regions of the stipe or rachis used in hydraulics and gas exchange measurements.

where p = xylem perimeter, A = combined cross-sectional
area of the xylem.
Xylem connectivity
To visualize the entire xylem network in the fronds, includ-
ing connections between bundles, we extracted all vascular
bundles in three mature fronds from each species. Fronds
were cut from the field and immediately brought back to
the lab for dissection. Firstly, all of the pinnae were cut from
the fronds so that approximately 1 cm of the midrib
remained attached to the rachis. Next, the stipe and rachis
were gently compressed with a pair of pliers to dislodge the
xylem bundles from the surrounding epidermis and cortex
tissue. The frond was then pinned to a rigid board and the
xylem bundles were carefully extracted with tweezers by
removing all of the tissue not containing xylem such that the
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
xylem connections were preserved and the xylem network
was displayed as a complete unit. The location of every
connection was measured as a distance from the base of the
frond. The total number of xylem bundles was then mea-
sured at 10 cm intervals along the frond. Finally, the xylem
network was photographed with a digital camera and the
images were used to create a xylem network map (Fig. 3).
High-resolution computed tomography (HRCT)
To visualize the three dimensional (3D) organization of
xylem bundles, well-hydrated plants were cut from the UC
Santa Cruz campus and the UC Berkeley Botanical
Garden, placed in plastic bags and transported to the
Lawrence Berkeley National Laboratory Advanced Light
Source, Beamline 8.3.2 microtomography facility. Frond
sections from the lower and upper rachis were cut and
1902 C. R. Brodersen et al.
(a) (b)
Figure 3. Representative xylem network maps for (a)
P. aquilinum and (b) W. fimbriata fronds. Each vertical line
represents a vascular bundle and each white dot represents the
location of a connection. Connections were clustered around the
axis junctions in P. aquilinum while connections were less
frequent in W. fimbriata. The number of bundles decreases
acropetally in both species, with many bundles diverted to the
axis junctions in P. aquilinum, while the pinnae in W. fimbriata
were supplied by short bundles (not shown) bifurcating from the
two large bundles [dark lines in (b)].
allowed to dehydrate under ambient conditions for
approximately 3 h to eliminate the majority of the water in
the xylem but not significantly change the shape of the
samples. Next, stems were wrapped in Parafilm (Peichiney
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Plastic Packaging, Inc., Chicago, IL, USA) to prevent
further dehydration, mounted in the microtomography
instrument and imaged at 15 keV at 0.125 increments over
180 following the methods of Brodersen et al. (2011). The
resulting two-dimensional images were reconstructed into a
3D dataset approximately 1.5 cm in length using Octopus
software (University of Ghent). The datasets were then
visualized with Avizo 6.2 software (Visualization Sciences
Group, Burlington, MA, USA).
Hydraulics measurements
We performed a variety of experiments aimed at studying
cavitation resistance in the xylem network of both species
with and without mechanical damage to the xylem bundles
at different points on the fronds. Fronds were cut from
well-hydrated plants in the field and brought back to the
lab. Sections were recut under water from the stipe, lower
rachis and upper rachis (Figs 1a and 2a). The sections were
then cut to 142 mm in length and the ends were shaved with
a razor blade to expose a smooth transverse surface. If any
axis junctions fell within the excised section, the pinnae
were cut close to the rachis and then sealed with
cyanoacrylic glue (Loctite 409, Henkel Corporation, Rocky
Hill, CT, USA). Sections were then degassed overnight
under vacuum in a 20 mm KCl solution to remove any
native embolism in the xylem.
The k was measured following the methods of Sperry
(1993) and calculated as a function of the flow rate through
the section at a given pressure. Segments (n = 6) were
mounted in a tubing apparatus to direct the flow of a 20 mm
KCl solution through the xylem at a pressure of 68 kPa to
measure maximum k (kmax) after degassing. The flow rate
through the sections without a pressure head was measured
before and after each gravimetric measurement to control
for equilibrium drift in the system. These background mea-
surements were averaged and subtracted from the pressure-
induced flow rates.
The centrifuge method (Pockman, Sperry & OLeary
1995; Alder et al. 1997) was then used to measure the vul-
nerability to cavitation of the xylem at the three sampling
points on the frond in response to a range of xylem pres-
sures. Following the kmax measurement, the sections were
mounted in a custom rotor for a Sorvall RC-5C centrifuge
(Thermo Fisher Scientific, Waltham, MA, USA) and spun
for 3 min at speeds known to induce xylem pressures (Px)
of -0.5, -1.0, -3.0, -5.0, -7.0 and -9.0 MPa. After each
spinning, the k of the sections were measured. The percent
loss of conductivity (PLC) resulting from progressively
negative Px was then calculated as a function of kmax at
Px = 0 MPa:
PLC = 100 (1 k kmax ) ,
A Weibull function was then used to fit the vulnerability
curves from each section to estimate the xylem pressure at
which 50% loss of conductivity (P50) was reached (Neufeld
et al. 1992).
 Fern xylem and physiology 1903

Table 1. Anatomy and sectoriality of P. aquilinum and W. fimbriata fronds at the stipe, lower rachis and upper rachis positions used in
gas exchange and hydraulic conductivity measurements

P. aquilinum W. fimbriata

D NVB S XSA D NVB S XSA

Stipe 9.11 0.81a 33.25 5.56a 12.11 0.47a 0.06 0.01a 10.50 1.64a 5.00 0.00a 9.63 2.86a 0.01 0.01a
Lower rachis 5.52 0.29b 14.66 6.02b 10.62 1.49a 0.09 0.01b 8.25 1.83a 3.50 0.57b 8.84 2.17a 0.02 0.01a
Upper rachis 3.27 0.09c 8.00 2.16b 6.75 1.85b 0.09 0.01b 4.77 1.53b 2.50 0.57c 7.78 3.64a 0.03 0.03a

All values are means SD.

n = 4 for each sampling position. Values with different superscript letters denote statistical significance following anovas with post hoc t-tests
using Bonferroni corrections (P < 0.05).
D, diameter of the stipe or rachis (mm); NVB, number of vascular bundles; S, Sectoriality Index, where increasing S represents increasing
xylem integration; XSA, xylem specific area (xylem lumen area/cross-sectional area of the stem) (%).

A second group of W. fimbriata stipe segments (n = 6)
was then used to estimate the relative hydraulic contribu-
tion of large and small bundles. Segments were degassed as
above and the kmax of each section was measured. Then, k
was measured after individual xylem bundles were sealed
on both ends of the segment with cyanoacrylic glue (Loctite
409). The large number, small size, convoluted shape and
non-symmetric arrangement of P. aquilinum xylem bundles
(Fig. 1) made adequate sealing exceedingly difficult and
prevented these measurements from being made with
consistent treatment between sections. The W. fimbriata
sections, however, had large, discrete bundles that were
easily sealed.
A third group of W. fimbriata stipe segments (n = 12) was
used to test the cavitation resistance of the large and small
bundles. Segments were prepared as above for centrifuga-
tion but half of the segments had large bundles sealed with
glue while the other half had the small bundles sealed. We
then proceeded to measure kmax and k at the same negative
xylem pressures as above.
Another group of stipe segments (n = 6) from both
species was notched at the midpoint of the segments to
sever the xylem bundles and then compared with un-
notched control sections (n = 6) to determine the hydraulic
consequences of air entry. The xylem was notched with a
razor blade perpendicular to the direction of flow through
the segment, and a thin piece of polystyrene plastic was
inserted into the notched area to prevent flow through the
severed xylem. The notched area was then wrapped in Para-
film to prevent the polystyrene from falling out while spin-
ning in the centrifuge. P. aquilinum segments were notched
so approximately 25% of the cross-sectional area was ren-
dered non-conductive, while W. fimbriata segments were
notched so that one large adaxial bundle was rendered
non-conductive.A second group of segments (n = 6) was cut
in the same manner but only to a depth of <1 mm to control
for any wound response effects.
mechanical damage to the xylem network. Thirty minutes
prior to gas exchange measurements, half of the stipes
were notched with a razor blade 10 cm above the soil
surface and perpendicular to transpiration stream. For
P. aquilinum, approximately 25% of the cross-sectional
area of the stipe was notched, while W. fimbriata stipes
were notched such that either one or two large adaxial
vascular bundles were severed. Twelve total P. aquilinum
plants, six control and six notched, and 18 W. fimbriata
plants, six control, six with a single large vascular bundle
notched and six with both large vascular bundles notched,
were used for the gas exchange measurements. Next, a
piece of polystyrene plastic was cut and inserted into the
notched area to block k within the bundle, and then the
notched area was wrapped in Parafilm. Stipes of control
plants were cut such that the epidermis and part of the
cortex were notched to a depth of <1 mm, but without
damaging the vascular bundles to minimize potential
wounding response differences between control and
notched plants. The gs was measured with a Decagon SC-1
Leaf Porometer (Decagon Devices Inc., Pullman, WA,
USA) on consecutive days in June and July 2011 with
cloudless skies. Pinnae from the lower and upper rachis
were measured to determine the effect of vascular bundle
disruption. To capture diurnal changes in gas exchange, gs
was measured on two pinnae, one on each side of the
frond, at both sampling positions at five points during the
day (0900, 1100, 1300, 1500 and 1700 h). Concurrent with
gas exchange measurements, frond water potential was
measured at 0900, 1300 and 1500 h on six plants at the
middle rachis position using a Scholander style pressure
chamber (PMS Instruments, Corvallis, OR, USA). In addi-
tion, water potential measurements were made at the
lower, middle and upper rachis positions at 1300 h on six
fronds from both species to test the water potential gra-
dient within the fronds.

Gas exchange measurements
To evaluate the role of redundancy in xylem organiza-
tion in situ we measured gs in plants with and without
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Statistical analysis
The P50, gs, anatomical measurements and water potential
values were compared using analyses of variance (anovas)
and post hoc t-tests with Bonferroni corrections to test
1904 C. R. Brodersen et al.

for statistical significance between multiple treatments

(P < 0.05) using SPSS software (v. 20.0.0; SPSS Inc., (a) (e)
Chicago, IL, USA).

RESULTS
Frond anatomy and architecture
We observed two extremes of xylem organization after dis-
secting the fronds of P. aquilinum (complex and convo-
luted) and W. fimbriata (simple and discrete) (Figs 13,
Table 1). The basal end of P. aquilinum stipes had an
average of 34 discrete vascular bundles that anastomose
along the frond to approximately eight bundles at the apex (b) (f)
(Figs 1b,c, Supporting Information Fig. S1; Table 1). Con-
nections in P. aquilinum were concentrated at the axis junc-
tions where pinnae emerge from the rachis, and decreased
in frequency towards the apex (Figs 1d & 3a). Such connec-
tions typically consisted of the fusion of two large bundles
or the bifurcation of an axial bundle, with the secondary
bundle leading to pinnae (Fig. 3a). The basal end of W. fim-
briata stipes had an average of five vascular bundles con-
sisting of two large adaxial bundles and one to four smaller (c) (g)
abaxial bundles (Fig. 2b,c). Large bundles spanned the
entire frond, eventually merging in the apex, and solely
supplied the pinnae through bifurcations from the main
axial bundles (Figs 2b,c & Supporting Information Fig. S2).
The small bundles made infrequent connections to other
small bundles, and occasionally to the large bundles. We
observed increasing vascular integration from the base to
the apex in P. aquilinum fronds, while W. fimbriata showed
nominal changes in integration (Table 1).The ratio of xylem
lumen area to cross-sectional area increased along the
frond in both species, with the greatest axial increase in
W. fimbriata (Table 1). (d) (h)

HRCT imaging
The 3D HRCT images confirmed the results from the
manual dissection of vascular bundles, with the benefit of
preserving the spatial orientation of the bundles in situ
(Fig. 4 & Supporting Information Fig. S3, Supporting
Information Movies S1 & S2). In the upper rachis of the P.
aquilinum segment (Fig. 4a,b), no axial bundles merged;
however, several secondary bundles merged after bifurcat-
ing into axis junctions leading to the pinnae. In the upper
rachis of W. fimbriata, there were no connections between
axial bundles, yet both bundles did bifurcate with the sec- Figure 4. High-resolution computed tomography volume
ondary bundles leading to the pinnae (Fig. 4e,f). In the lower renderings of P. aquilinum (ad) and W. fimbriata (eh). Frond
segments 1.5 cm in length from the upper (a, b, e, f) and middle
rachis segment of P. aquilinum, there were four connections
rachis (d, c, h, g) are viewed in longitudinal plane and then
between the axial bundles (Fig. 4c,d), while there was only rotated in 45 towards the reader, revealing primary xylem
one connection in W. fimbriata between two small bundles bundles (blue) and the surrounding tissue (green). The upper and
(Fig. 4g,h). The large axial bundles of W. fimbriata remained middle rachis of P. aquilinum consists of many xylem bundles
isolated throughout the segment. with multiple connections located in the axis junctions. The upper
and middle rachis of W. fimbriata consists of two and five xylem
bundles, respectively, with few connections. Scale varies with
Hydraulic measurements perspective. Bar = 5 mm.
Xylem integration varies along the frond; so to characterize
the susceptibility of different frond segments to cavitation,
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911

(a)
(b)
Figure 5. Vulnerability to cavitation at three sampling points
on fronds of (a) P. aquilinum and (b) W. fimbriata, measured at
the base of the stipe (black symbols), the lower rachis (light grey
symbols) and the upper rachis (white symbols). Bars SE,
n = 6. Mean P50 values are reported in Table 2.
we generated vulnerability curves from the stipe, lower
rachis and upper rachis positions from both species. Vulner-
ability curves for P. aquilinum were limited to centrifuga-
tion speeds that generated up to -7.0 MPa, as segments
spun at greater speeds had a tendency to disintegrate in the
centrifuge rotor. By -7.0 MPa, most P. aquilinum segments
had reached at least 90% loss of conductivity. Vulnerability
curves from P. aquilinum fronds revealed that segments
from all three frond positions were equally resistant to cavi-
tation (no significant difference between mean P50 values,
P > 0.05) (Fig. 5a, Table 2). Vulnerability curves in W. fim-
briata revealed that this species was much more resistant
to cavitation, yet became significantly more vulnerable
towards the apex (Fig. 5b, Table 2). We then compared the
vulnerability curves of large and small vascular bundles in
W. fimbriata stipes and found no statistically significant dif-
ferences in P50 (-5.94 MPa 3.52 SD and -6.19 MPa
2.32 SD for small and large bundles, respectively; Fig. 5a).
We found a direct trade-off between safe and efficient water
transport, with xylem-specific conductivity of P. aquilinum
stipes being significantly higher than W. fimbriata (67.1
13.2 SD and 41.6 6.0 SD mg mm-1 kPa-1 s-1, respectively,
P < 0.05).
To assess how quickly air can spread through an inte-
grated versus sectored xylem network, we severed either
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Fern xylem and physiology 1905
one quarter of the cross-sectional area of P. aquilinum seg-
ments and one large xylem bundle in W. fimbriata segments,
and then generated vulnerability curves. When the xylem
was cut in the integrated P. aquilinum fronds, vulnerability
curves became steeper; however, the P50 values were not
significantly different from uncut segments (Fig. 6b,
Table 2). In W. fimbriata, however, segments with one large
bundle cut became significantly more resistant to cavitation
compared with uncut segments (P50 = -5.21 MPa 2.11 SD
and -2.00 1.21 MPa SD for cut and uncut stipes, respec-
tively; Fig. 6c, Table 2). When analysing the relative contri-
butions of large and small vascular bundles in W. fimbriata,
we found that large bundles were responsible for the bulk
of water transport (45.0% 0.25 SD of kmax per large
bundle), with minimal contribution from the small bundles
(4.98% 0.02 SD of kmax per small bundle).
Tracheid diameter distribution
We then measured conduit lumen diameters in the stipe,
lower rachis and upper rachis to test whether the differ-
ences we observed in higher resistance to cavitation along
the frond could be explained by variations in conduit diam-
eter distributions. P. aquilinum xylem had a broader range
in conduit diameters than W. fimbriata, and also showed a
shift towards smaller mean tracheid diameter towards the
apex while W. fimbriata tracheids maintained a constant
mean tracheid diameter at all sampling positions (Fig. 7 &
Supporting Information Fig. S4). An analysis of tracheid
diameters in large and small xylem bundles of W. fimbriata
showed a slight shift towards larger diameter tracheids in
the large bundles (Fig. 7c).
Gas exchange measurements
To test the functional relationship between xylem integra-
tion and gas exchange, we measured diurnal changes in gs in
plants with and without mechanical damage to the xylem
network. By severing the xylem bundles, our goal was to
Table 2. Vulnerability to cavitation as measured by the percent
loss of conductivity in fronds of P. aquilinum and W. fimbriata
P. aquilinum W. fimbriata
P50 P50
Stipe -2.23 1.64a -5.21 2.11a
Middle rachis -1.47 0.64a -3.71 3.09a
Upper rachis -1.23 0.59a -1.11 0.72b
Stipe 1/4 cut -2.11 0.62a NA
Stipe 1 LB cut NA -2.00 1.21b
All values are means SD.
n = 6 for all treatments. Values are means SD. Values with differ-
ent superscript letters denote statistical significance following
anovas with post hoc t-tests using Bonferroni corrections
(P < 0.05).
P50, 50% loss of conductivity; 1/4 cut, one quarter of the xylem
bundles severed; 1 LB cut, one large bundle cut.
1906 C. R. Brodersen et al.

disrupt the transpiration stream and introduce air into the

(a) xylem network. We observed gs rates up to threefold higher
in P. aquilinum compared with W. fimbriata, but P. aquili-
num suffered greater decreases in gs following xylem
damage (Fig. 8).
In W. fimbriata the effects of severing a single large xylem
bundle, representing approximately 45% of the total k,
were not significant at any point during the day in either the
lower or upper rachis (Fig. 8c,d). Severing both large xylem
bundles in W. fimbriata, which represented approximately
90% of the total k, resulted in significant declines in gs
during the 0900 and 1100 h sampling points, yet the
maximum loss in gs was only 49%. In P. aquilinum, where
approximately 25% of the k was severed, losses in gs were
(b) more substantial, up to 56% in the lower rachis during the

(a)

(c)

(b)

(c)

Figure 6. Vulnerability to cavitation of xylem in stipes of

P. aquilinum and W. fimbriata. In (a), W. fimbriata xylem in large
bundles (black circles) was more vulnerable to cavitation than
xylem in small bundles (white circles). Vulnerability to cavitation
increased in (b) P. aquilinum stipes with one quarter of the xylem
bundles severed (white circles) compared with intact stipes
(black circles). Vulnerability to cavitation decreased in
(c) W. fimbriata, with one large xylem bundle severed (black
circles) compared with intact stipes (white circles). Vulnerability
curves were normalized to data after the first centrifugation at
-0.5 MPa. Bars SE, n = 6 in all figures. Mean P50 values are
reported in Table 2.
Figure 7. Tracheid diameter distributions in (a) P. aquilinum
stipes, (b) W. fimbriata stipes, and (c) large (grey bars) and small
(white bars) xylem bundles in W. fimbriata stipes. Bars SE,
n = 4. Arrows show bins with low frequency for large and small
bundles.
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
 Fern xylem and physiology 1907

(a) (c)

(b) (d)

Figure 8. Diurnal trends in mean stomatal conductance and mean frond water potential in P. aquilinum (a,b) and W. fimbriata (c,d)
fronds measured at upper (a,c) and lower (b,d) rachis positions. Stomatal conductance of control plants (black circles) was typically
higher than plants with one quarter of the stipe severed (a,b; grey circles), one large vascular bundle severed (c,d; grey circles) or fronds
with two large vascular bundles severed (c,d; white circles). Frond water potential measured at the lower rachis (b,d; grey triangles)
declined at midday and recovered by 1700 h in P. aquilinum (b), while W. fimbriata values declined throughout the day. Bars SE, n = 9.
Superscript letters denote statistical significance between control and experimental pinnae within a sampling period following anovas with
post hoc t-tests using Bonferroni corrections (P < 0.05).

1100 h sampling point. We then observed diminishing
effects of xylem damage as the day progressed beyond
1300 h. The greatest decreases in gs for both species
occurred in pinnae from the lower rachis, while the highest
gs rates were observed in the upper rachis (Fig. 8). Lower
rachis W. fimbriata pinnae showed little diurnal variation in
gs with two large bundles severed (Fig. 8d).
Frond water potential
The water status of P. aquilinum fronds measured on
pinnae at the middle rachis decreased from 0900 to 1300 h,
followed by a recovery by 1700 h (Fig. 8b). Frond water
status declined modestly throughout the day in W. fim-
briata, with no recovery at the 1700 h sampling point. The
lowest water potential measurement at 1300 h, -1.4 MPa,
fell very close to the P50 value for P. aquilinum. Midday
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
(1300 ) water potentials in P. aquilinum were significantly
lower in the middle and upper rachis compared with the
lower rachis (-1.4 0.21a, -2.05 0.19b, -2.07 0.21b MPa
for the lower, middle and upper rachis, respectively,
P < 0.05). The midday frond water potential was signifi-
cantly lower at the upper rachis compared with the lower
and middle rachis in W. fimbriata (-0.5 0.14a, -0.67
0.31a, -0.75 0.12b MPa for the lower, middle and upper
rachis, respectively, P < 0.05).
DISCUSSION
This study illustrates how the balance between safe and
efficient water transport directly influences gas exchange in
two ferns with markedly different life history strategies.
P. aquilinum has adopted a phenology optimized for rapid
early season growth prior to the onset of late season
1908 C. R. Brodersen et al.
drought. To support high rates of gs, P. aquilinum has wide,
efficient conduits within a highly integrated xylem network.
While the xylem of P. aquilinum may be more efficient in
delivering water to the transpiring pinnae, the interconnec-
tions within the xylem ultimately make it more vulnerable
to embolism spread. The opposite and more conservative
strategy was seen in W. fimbriata, which favours perennial
growth, lower gs, drought-resistant xylem and a habitat pref-
erence for consistently moist areas. This strategy allows
W. fimbriata to tolerate the seasonal fluctuations in tem-
perature and water availability across its broad habitat
range, including freezing temperatures.
While P. aquilinum may appear to be highly sectored in
transverse cross sections with many discrete vascular
bundles (Fig. 1 & Supporting Information Fig. S1), the con-
nections between bundles increase the total integration of
the frond (Figs 3 & 4, Table 1). The degree of integration is
not immediately apparent from transverse sections (Fig. 1
& Supporting Information Fig. S1), but when viewing the
xylem network as a whole or in 3D (Figs 3 & 4, Supporting
Information Fig. S3, Supporting Information Movies S1 &
S2), the connections between xylem bundles are revealed,
and highlight the benefits of both of the techniques used to
study xylem organization. While the physical dimensions of
the HRCT instrument and our limited data processing
capabilities prevented the 3D analysis of longer segments,
HRCT analysis is clearly superior for visualizing xylem net-
works in 3D and preserving the spatial orientation of the
xylem in situ (Fig. 4, Supporting Information Fig. S3, Sup-
porting Information Movies S1 & S2). However, manual
dissection of the xylem bundles was easy to perform, did not
require any specialized equipment and yielded network
connectivity data on a much larger scale (whole fronds
versus short segments) (Fig. 3). While manual dissection of
the xylem network was only performed on the two species
described in this study, we have subsequently found that the
technique works well for most ferns and is a good alterna-
tive to HRCT imaging.
In P. aquilinum fronds, comparable vulnerability curves
and P50 values when coupled with negligible differences in
conduit diameters between frond positions all strongly
suggest that vascular integration influenced embolism
spread in this species (Fig. 5a). We suspect that increasing
xylem integration along W. fimbriata fronds contributed to
decreases in cavitation resistance, specifically the fusion of
the large bundles near the apex and the loss of the more
resistant small bundles (Figs 2c, 5b, 6a, Supporting Informa-
tion Fig. S2, Tables 1 & 2). Fern tracheids have at least one
side of their lateral walls completely pitted (Bierhorst 1960;
White 1963b; Evert 2006), and there is a higher likelihood
of inter-tracheid contact in large bundles than in small
bundles. Given the propensity in this species for greater
integration and higher vulnerability to cavitation in more
distal parts of the frond, it may be that the proportionality
between pit area and tracheid volume also increases. Con-
sequently, the higher probability of the presence of a weak,
rare pit may contribute to cavitation resistance in some
ferns (Christman, Sperry & Adler 2009; Christman, Sperry
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
& Smith 2012) though membrane thickness and porosity
(Lens et al. 2011) may also play an important role.
Gas exchange in P. aquilinum was more vulnerable to
mechanical damage than in W. fimbriata with its sectored
xylem network. The gs measurements in situ were consis-
tent with frond hydraulics, with both experiments showing
a higher propensity for embolism spread in the integrated
xylem of P. aquilinum. However, xylem connectivity may
provide some benefit when sections of the network
become dysfunctional. Xylem bundles within P. aquilinum
frequently merge in the upper rachis (Fig. 3) and those
connections appear to facilitate hydraulic redistribution
where water is rerouted around the severed bundles
through alternate pathways, similar to what has been
reported in the leaves of woody plants (Sack et al. 2008;
Scoffoni et al. 2011). Support for this hypothesis can be
seen in the declines in gs between the upper and lower
rachis following xylem damage (Fig. 8a,b). The greatest
differences in gs between cut and uncut fronds were at
1100 h in both the upper and lower rachis, but the pinnae
closest to the cut (lower rachis) showed the greatest
decline. As shown in other ferns (e.g. Calkin et al. 1985),
we observed increasing xylem tension towards the frond
apex, a driving force that would facilitate the distal move-
ment of water around dysfunctional bundles through func-
tional secondary pathways.
Because the two large W. fimbriata bundles each contrib-
ute approximately 45% of the k, we expected that severing
one large bundle would lead to a comparable decrease in gs.
Instead, severing a single bundle resulted in no significant
decline in gs, and only when two large bundles were severed
(estimated 90% loss in conductivity) was there a significant
decrease in gs, causing gs to fall by up to 49% compared with
control plants (Fig. 8c,d). Hydraulic redistribution is likely
more difficult in W. fimbriata due to the limited number of
connections compared with P. aquilinium. For redistribu-
tion to work in W. fimbriata, water would have to travel the
entire length of the frond in the functional bundle to the
union of the two large bundles near the apex and then flow
basipetally (against the water potential gradient) to the
transpiring pinnae through the severed bundle, a highly
unlikely scenario. Alternatively, water could flow through
the few connections between small bundles, but their
infrequency would limit the effectiveness of this pathway
(Fig. 3). Instead, there may be internal capacitance pro-
vided by water stored in the pinnae, cortex or rhizome that
compensates for short-term xylem dysfunction (Meinzer
et al. 2009), a hypothesis consistent with the isohydric
pattern in diurnal frond water potential (Fig. 8d). Having all
of the pinnae directly connected to the two large vascular
bundles appears to be a risky strategy, but the narrow,
cavitation-resistant tracheids and internal capacitance
appear to buffer the fronds from hydraulic dysfunction.
In comparison with xylem integration trends observed in
woody plants (e.g. Zanne et al. 2006; Schenk et al. 2008),
these two ferns present an interesting contrast. Following
the woody plant model, for these mesic fern species one
would expect integrated xylem with low conductivity, small

diameter tracheids and low resistance to cavitation. Xylem
within the individual bundles of both species was highly
integrated (Supporting Information Figs S1 & S2), but in
ferns integration must also be assessed at the scale of the
frond. W. fimbriata utilized the beneficial safety features of
both integrated and sectored xylem. Within the bundles
conduits are small and redundant, while the bundles were
spatially discrete to limit embolism spread. As a result, gs
was low, but the xylem network was significantly more
resistant to cavitation and less susceptible to mechanical
damage. In contrast, P. aquilinum relied on a highly inte-
grated xylem network within and between bundles; with
wide, efficient conduits to maintain high gs but at the cost of
low cavitation resistance. Indeed, there was a clear trade-off
between safety and efficiency, as xylem-specific conductivity
was significantly higher in P. aqulinum, which also suffered
from lower cavitation resistance, similar to trade-offs
present in woody plants (Wheeler et al. 2005; Loepfe et al.
2007). To overcome the effects of drought, W. fimbriata
relies on safe xylem networks and hydraulic capacitance of
the surrounding tissue rather than hydraulic redistribution
and a phenology targeted at early season growth prior to
the seasonal drought common in this Mediterranean
climate as seen in P. aquilinum.
Recent research into the evolution of stomatal control
(Brodribb & McAdam 2011) raises a number of questions
related to fern physiology. Evidence for stomatal control in
ferns is mixed, with reports of active stomatal control in
P. aquilinum to avoid excessive water loss in open high-light
areas, but not in ferns in occupying moist, shaded areas
(Nobel, Calkin & Gibson 1984). Conversely, others report
minimal stomatal response to changes in vapour pressure
deficit in ferns (including P. aquilinum) (Roberts,Wallace &
Pitman 1984; Franks & Farquhar 1999). The most recent
research suggests a passive, hydraulic response in ferns,
likely the result of the lack of an ABA signalling mechanism
in fern stomata common among angiosperms (Brodribb &
McAdam 2011). Our gas exchange experiments showed
dramatic decreases in gs in response to mechanical damage
to the xylem in the drought-deciduous P. aquilinum fronds,
and suggest a passive, hydraulic response in the control of
stomata closure that is consistent with the findings of
Brodribb & McAdam (2011). An ABA-regulated stomatal
response may not be as critical for ferns because many are
understorey species that prefer moist habitats.
This study illustrates the functional consequences of
xylem organization on cavitation resistance and gas
exchange and highlights two extremes in phenology in two
coexisting ferns. Our results raise a number of questions to
be addressed in future research. Because pit membranes
play such a critical role in cavitation resistance, research is
needed to determine what pit membrane characteristics are
important to cavitation resistance in ferns (Wheeler et al.
2005; Choat & Pittermann 2009).The weak or highly porous
pit membranes reported for a number of ferns (Carlquist &
Schneider 2007) suggest that xylem organization may be
even more important for fern cavitation resistance com-
pared with angiosperms, particularly in light of the higher
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Fern xylem and physiology 1909
safety margins and conservative drought-resistance strate-
gies employed by ferns when faced with desiccation (Bro-
dribb & Holbrook 2004).
ACKNOWLEDGMENTS
The authors would like to thank the NSF (IOS-1027410 to
J.P.) for supporting this research. We would also like to
thank E. Limm, C. Rico and S. Bastami for their assistance
in the collection of hydraulics and anatomical pilot data.
Holly Forbes at the UC Berkeley Botanical Garden kindly
provided access to plant material for 3D imaging. We also
thank Alastair MacDowell and Dula Parkinson of the
Lawrence Berkeley National Laboratory Advanced Light
Source microtomography beamline 8.3.2 for their assis-
tance in 3D imaging. The Advanced Light Source is sup-
ported by the Director, Office of Science, Office of Basic
Energy Sciences, of the US Department of Energy under
contract no. DE-AC02-05CH11231.
REFERENCES
Alder N., Pockman W., Sperry J. & Nuismer S. (1997) Use of cen-
trifugal force in the study of xylem cavitation. Journal of Experi-
mental Botany 48, 665674.
Beck C.B., Schmid R. & Rothwell G.W. (1982) Stelar morphology
and the primary vascular system of seed plants. Botanical Review
48, 691815.
Bierhorst D.W. (1960) Observations on tracheary elements. Phyto-
morphology 10, 249305.
Bliss M.C. (1939) The tracheal elements in the ferns. American
Journal of Botany 26, 620624.
Brodersen C., Lee E., Mcelrone A., Shackel K., Choat B., Phillips
R. & Matthews M. (2011) Analyzing 3D xylem networks in Vitis
vinifera using high resolution computed tomography (HRCT).
New Phytologist 191, 11681179.
Brodribb T. & Feild T. (2000) Stem hydraulic supply is linked to
leaf photosynthetic capacity: evidence from New Caledonian
and Tasmanian rainforests. Plant, Cell & Environment 23, 1381
1388.
Brodribb T.J. & Holbrook N.M. (2004) Stomatal protection against
hydraulic failure: a comparison of coexisting ferns and
angiosperms. New Phytologist 162, 663670.
Brodribb T.J. & McAdam S.A.M. (2011) Passive origins of stomatal
control in vascular plants. Science 331, 582585.
Brodribb T.J., Holbrook N.M., Zwieniecki M.A. & Palma B. (2005)
Leaf hydraulic capacity in ferns, conifers and angiosperms:
impacts on photosynthetic maxima. New Phytologist 165, 839
846.
Calkin H.W., Gibson A.C. & Nobel P.S. (1985) Xylem water poten-
tials and hydraulic conductances in eight species of ferns. Cana-
dian Journal of Botany 63, 632637.
Carlquist S. & Schneider E.L. (1997) SEM studies on vessels in
ferns. 2. Pteridium. American Journal of Botany 84, 581.
Carlquist S. & Schneider E.L. (2001) Vessels in ferns: structural,
ecological, and evolutionary significance. American Journal of
Botany 88, 113.
Carlquist S. & Schneider E.L. (2007) Tracheary elements in ferns:
new techniques, observations, and concepts. American Fern
Journal 97, 199211.
Choat B. & Pittermann J. (2009) New insights into bordered pit
structure and cavitation resistance in angiosperms and conifers.
New Phytologist 182, 557560.
1910 C. R. Brodersen et al.
Christman M.A., Sperry J.S. & Adler F.R. (2009) Testing the rare
pit hypothesis for xylem cavitation resistance in three species of
Acer. New Phytologist 182, 664674.
Christman M.A., Sperry J.S. & Smith D.D. (2012) Rare pits, large
vessels, and extreme vulnerability to cavitation in a ring-porous
tree species. New Phytologist 193, 713720.
Espino S. & Schenk H.J. (2009) Hydraulically integrated or
modular? Comparing whole-plant-level hydraulic systems
between two desert shrub species with different growth forms.
New Phytologist 183, 142152.
Evert R.F. (2006) Esaus Plant Anatomy: Meristems, Cells and
Tissues of the Plant Body: Their Structure, Function and Devel-
opment. John Wiley & Sons, Hoboken, NJ, USA.
Franks P. & Farquhar G. (1999) A relationship between humidity
response, growth form and photosynthetic operating point in C3
plants. Plant, Cell & Environment 22, 13371349.
Gibson A.C., Calkin H.W. & Nobel P.S. (1985) Hydraulic
conductance and xylem structure in tracheid-bearing plants.
International Association of Wood Anatomists Bulletin 6, 293
302.
Hacke U., Sperry J., Feild T., Sano Y., Sikkema E. & Pittermann J.
(2007) Water transport in vesselless angiosperms: conducting
efficiency and cavitation safety. International Journal of Plant
Sciences 168, 11131126.
Hargrave K., Kolb K., Ewers F. & Davis S. (1994) Conduit diameter
and drought induced embolism in Salvia mellifera Greene
(Labiatae). New Phytologist 126, 695705.
Lens F., Sperry J.S., Christman M.A., Choat B., Rabaey D. & Jansen
S. (2011) Testing hypotheses that link wood anatomy to cavita-
tion resistance and hydraulic conductivity in the genus Acer.
New Phytologist 190, 709723.
Lo Gullo M.A., Raimondo F., Crisafulli A., Salleo S. & Nardini A.
(2010) Leaf hydraulic architecture and water relations of three
ferns from contrasting light habitats. Functional Plant Biology
37, 566574.
Loepfe L., Martinez-Vilalta J., Piol J. & Mencuccini M. (2007) The
relevance of xylem network structure for plant hydraulic effi-
ciency and safety. Journal of Theoretical Biology 247, 788803.
Meinzer F.C., Johnson D.M., Lachenbruch B., Mcculloh K.A. &
Woodruff D.R. (2009) Xylem hydraulic safety margins in woody
plants: coordination of stomatal control of xylem tension with
hydraulic capacitance. Functional Ecology 23, 922930.
Mencuccini M., Martnez-Vilalta J., Pinol J., Loepfe L., Burnat M.,
Alvarez X., Camacho J. & Gil D. (2010) A quantitative and
statistically robust method for the determination of xylem
conduit spatial distribution. American Journal of Botany 97,
12471259.
Neufeld H.S., Grantz D.A., Meinzer F.C., Goldstein G., Crisosto
G.M. & Crisosto C. (1992) Genotypic variability in vulnerability
of leaf xylem to cavitation in water-stressed and well-irrigated
sugarcane. Plant Physiology 100, 10201028.
Nobel P.S., Calkin H.W. & Gibson A.C. (1984) Influences of PAR,
temperature and water vapor concentration on gas exchange by
ferns. Physiologia Plantarum 62, 527534.
Orians C.M., Babst B. & Zanne A.E. (2005) Vascular constraints of
long distance transport in dicots. In Vascular Transport in Plants
(eds N.M. Holbrook & M.A. Zwieniecki), pp. 355374. Elsevier
Inc., Burlington, MA, USA.
Pittermann J. (2010) The evolution of water transport in plants: an
integrated approach. Geobiology 8, 112139.
Pittermann J., Limm E., Rico C. & Christman M.A. (2011)
Structure-function constraints of tracheid based xylem: a com-
parison of conifers and ferns. New Phytologist 192, 449461.
Pockman W.T., Sperry J.S. & OLeary J.W. (1995) Sustained and
significant negative water pressure in xylem. Nature 378, 715
716.
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Roberts J., Wallace J.S. & Pitman R.M. (1984) Factors affecting
stomatal conductance of bracken below a forest canopy. Journal
of Applied Ecology 21, 643655.
Robinson R.C., Sheffield E. & Sharpe J.M. (2010) Problem ferns:
their impact and management. In Fern Ecology (eds K. Mehl-
treter, L.R. Walker & J.M. Sharpe), pp. 255310. Cambridge
University Press, Cambridge, UK.
Sack L., Dietrich E., Streeter C., Sanchez-Gomez D. & Holbrook N.
(2008) Leaf palmate venation and vascular redundancy confer
tolerance of hydraulic disruption. Proceedings of the National
Academy of Sciences of the United States of America 105, 1567
1572.
Salguero-Gmez R. & Casper B. (2011) A hydraulic explanation
for size-specific plant shrinkage: developmental hydraulic secto-
riality. New Phytologist 189, 229240.
Savage V., Bentley L., Enquist B., Sperry J., Smith D., Reich P. &
Von Allmen E. (2010) Hydraulic trade-offs and space filling
enable better predictions of vascular structure and function in
plants. Proceedings of the National Academy of Sciences of the
United States of America 107, 2272222727.
Schenk H.J., Espino S., Goedhart C.M., Nordenstahl M., Cabrera
H.I.M. & Jones C.S. (2008) Hydraulic integration and shrub
growth form linked across continental aridity gradients. Proceed-
ings of the National Academy of Sciences of the United States of
America 105, 1124811253.
Schneider H., Pryer K.M., Cranfill R., Smith A.R. & Wolf P.G.
(2002) Evolution of vascular plant body plans: a phylogenetic
perspective. Developmental Genetics and Plant Evolution 65,
330364.
Schulte P.J., Gibson A.C. & Nobel P.S. (1987) Xylem anatomy and
hydraulic conductance of Psilotum nudum. American Journal of
Botany 74, 14381445.
Scoffoni C., Rawls M., Mckown A., Cochard H. & Sack L. (2011)
Decline of leaf hydraulic conductance with dehydration: rela-
tionship to leaf size and venation architecture. Plant Physiology
156, 832843.
Sperry J.S. (1993) Winter embolism and spring recovery in Betula
cordifolia, Fagus grandifolia, Abies balsamifera, and Picea
rubens. In Water Transport in Plants under Climatic Stress, pp.
8698. Cambridge University Press, Cambridge, UK.
Sperry J.S. (2003) Evolution of water transport and xylem struc-
ture. International Journal of Plant Sciences 164, S115S127.
Sperry J.S., Meinzer F.C. & Mcculloh K.A. (2008) Safety and effi-
ciency conflicts in hydraulic architecture: scaling from tissues to
trees. Plant, Cell & Environment 31, 632645.
Stein W. (1993) Modeling the evolution of stelar architecture in
vascular plants. International Journal of Plant Sciences 154, 229
263.
Taylor T.N., Taylor E.L. & Krings M. (2009) Paleobotany: The
Biology and Evolution of Fossil Plants. Academic Press, Burling-
ton, MA, USA.
Tyree M.T. & Zimmermann M.H. (2002) Xylem Structure and the
Ascent of Sap. Springer Verlag, New York, NY, USA.
Wardlaw C.W. (1950) The comparative investigation of apices of
vascular plants by experimental methods. Philosophical Trans-
actions of the Royal Society of London. Series B, Biological
Sciences 234, 583602.
Watkins J.E., Holbrook N.M. & Zwieniecki M.A. (2010) Hydraulic
properties of fern sporophytes: consequences for ecological and
evolutionary diversification. American Journal of Botany 97,
20072019.
Wheeler J.K., Sperry J.S., Hacke U.G. & Hoang N. (2005)
Inter vessel pitting and cavitation in woody Rosaceae and
other vesselled plants: a basis for a safety versus efficiency
trade off in xylem transport. Plant, Cell & Environment 28, 800
812.

White R.A. (1963a) Tracheary elements of the ferns. II. Morphol-
ogy of tracheary elements; conclusions. American Journal of
Botany 50, 514522.
White R.A. (1963b) Tracheary elements of the ferns. I. Factors
which influence tracheid length; correlation of length with
evolutionary divergence. American Journal of Botany 50, 447
455.
Woodhouse R.M. & Nobel P.S. (1982) Stipe anatomy, water
potentials, and xylem conductances in seven species of ferns
(Filicopsida). American Journal of Botany 69, 135140.
Zanne A., Sweeney K., Sharma M. & Orians C. (2006) Patterns
and consequences of differential vascular sectoriality in 18
temperate tree and shrub species. Functional Ecology 20, 200
206.
Received 22 October 2011; received in revised form 3 April 2012;
accepted for publication 16 April 2012
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Representative freehand transverse sections
through a P. aquilinum frond at 20 cm intervals.
Bar = 1 mm.
Figure S2. Representative freehand transverse sections
through a W. fimbriata frond at 20 cm intervals. Bar = 1 mm.
Figure S3. High-resolution computed tomography images
of P. aquilinum (a,c) and W. fimbriata. (b,d). Each column
shows the 1.5 cm long frond segments rotated in 45
2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 18981911
Fern xylem and physiology 1911
increments over 180, revealing primary xylem bundles
(blue) and the surrounding tissue (green). The upper and
middle rachis of P. aquilinum (a and c, respectively) consist
of many more xylem bundles with multiple connections,
typically located in the axis junctions. The upper and middle
rachis of W. fimbriata (b and d, respectively) consist of two
and five xylem bundles, respectively, with few connections.
Figure S4. Conduit diameter distributions in P. aquilinum
(ac) and W. fimbriata (df) at upper rachis (a,d), the
middle rachis (b,e) and the lower rachis (c,f). Bars SE,
n = 4. Arrows show bins with low frequency.
Movie S1. Three-dimensional HRCT volume renderings
of the xylem network of 1.5 cm of lower rachis tissue in
P. aquilinum and W. fimbriata fronds. This movie shows the
interconnected P. aquilinum xylem network and the sec-
tored W. fimbriata xylem network with a single connection
between two small bundles.
Movie S2. Three-dimensional HRCT volume renderings of
the xylem network of 1.5 cm of upper rachis tissue in P. aq-
uilinum and W. fimbriata fronds.This movie shows the inter-
connected P. aquilinum xylem network and the sectored
W. fimbriata xylem network. In W. fimbriata, the bifurca-
tions of the two large bundles that supply the pinnae are
visible.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing mate-
rial) should be directed to the corresponding author for the
article.