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\begin{document}

\title{Sieve tube structural variation in Austrobaileya scandens and its
significance for lianescence}



\author[1]{Juan Losada}%
\author[2]{Zhe He}%
\author[2]{Noel Holbrook}%
\affil[1]{CSIC}%
\affil[2]{Harvard University}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Lianas combine large leaf areas with slender stems, features that
require an efficient vascular system. The only extant member of the
Austrobaileyaceae is an endemic twining liana of the tropical Australian
forests with well-known xylem hydraulics, but the vascular phloem
continuum aboveground remains understudied. Microscopy analysis across
leaf veins and stems of A. scandens revealed a low foliar xylem to
phloem ratio, with isodiametric vascular elements along the midrib, but
tapered across vein orders. Small sieve plate pore radii increased from
0.08 \selectlanguage{greek}µ\selectlanguage{english}m in minor veins to 0.12 \selectlanguage{greek}µ\selectlanguage{english}m in the petiole, but only to 0.20 \selectlanguage{greek}µ\selectlanguage{english}m at
the stem base, tens of meters away. In searcher branches, phloem
conduits contained a pectin-rich wall and simple plates, whereas in
twinning stems, conduits connected through highly-angled-densely
populated sieve plates. Twisted and elongated stems of A. scandens
display a high hydraulic resistance of phloem conduits, which decreases
from leaves to stems, efficiently delivering photoassimilate from
sources under M\selectlanguage{ngerman}ünch predictions. Sink strength of a continuously growing
canopy might be stronger than in self-supporting understory plants,
favoring resource allocation to aerial organs in angiosperms that
colonized the vertical niche.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Article title:}

Sieve tube structural variation in \emph{Austrobaileya scandens} and its
significance for lianescence

\textbf{Short running::}

Phloem of lianas

Juan M. Losada\textsuperscript{1,2,3*}

Zhe He\textsuperscript{2,3}

N. Michele Holbrook\textsuperscript{2,3}

\textsuperscript{1}Institute for Mediterranean and Subtropical
Horticulture `La Mayora' -- CSIC -- UMA, Avda. Dr. Wienberg s/n, M\selectlanguage{ngerman}álaga,
29750, Spain.

\textsuperscript{2}Department of Organismic and Evolutionary
Biology,~Harvard University, 16 Divinity Avenue, Cambridge, MA, 02138,
USA.

\textsuperscript{3}Arnold Arboretum of Harvard University, 1300 Centre
St., Boston, MA, 02130, USA.

*Author for correspondence:

Phone: + 34 (689) 258686

E-mail:\emph{juan.losada.r@gmail.com}

\textbf{SUMMARY}

\textbf{Lianas combine large leaf areas with slender stems, features
that require an efficient vascular system. The only extant member of the
Austrobaileyaceae is an endemic twining liana of the tropical Australian
forests with well-known xylem hydraulics, but the vascular phloem
continuum aboveground remains understudied.}

\selectlanguage{greek}\textbf{Microscopy analysis across leaf veins and stems of \emph{A.
scandens} revealed a low foliar xylem to phloem ratio, with isodiametric
vascular elements along the midrib, but tapered across vein orders.
Small sieve plate pore radii increased from 0.08 µm in minor veins to
0.12 µm in the petiole, but only to 0.20 µm at the stem base, tens of
meters away. In searcher branches, phloem conduits contained a
pectin-rich wall and simple plates, whereas in twinning stems, conduits
connected through highly-angled-densely populated sieve plates.}\selectlanguage{english}

\selectlanguage{ngerman}\textbf{Twisted and elongated stems of \emph{A. scandens} display a high
hydraulic resistance of phloem conduits, which decreases from leaves to
stems, efficiently delivering photoassimilate from sources under Münch
predictions. Sink strength of a continuously growing canopy might be
stronger than in self-supporting understory plants, favoring resource
allocation to aerial organs in angiosperms that colonized the vertical
niche.}

\textbf{Keywork index:} \emph{Austrobaileya scandens} , lianescence,
pectins, phloem, sieve-tubes, vessels, xylem.

\textbf{1 INTRODUCTION}

Lianas constitute about 40\% of the woody individuals in tropical
forests and make an outsize contribution to forest productivity and
response to disturbance (Gerwing \& Farias, 2000; Schnitzer et al.,
2012) as a result of their ability to elevate a dense leaf canopy tens
of meters aboveground with a minimal investment in self-support (Darwin,
1868; Baillaud, 1962). Anatomical features that allow lianas to meet the
evaporative demands of their leaves despite having slender stems include
large diameter xylem vessels, and thus greater hydraulic efficiency,
compared with co-occurring trees and shrubs (Carlquist, 1991; Ewers et
al., 1991; Isnard et al., 2003; Isnard \& Silk, 2009; Wyka et al., 2013;
Chen et al., 2014; Pace et al., 2015, 2018; Chery et al., 2020). The
allometrical demands of the climbing habit might be expected to extend
to the phloem. Yet, the long-distance effectiveness of carbohydrate
transport remains poorly documented in lianas, due to the scarce
information on the intersection between the vascular structure of the
phloem and lianescence.

Previous anatomical descriptions of the stems of large-bodied vines
belonging to Loganiaceae, Malpighiaceae, Bignoniaceae, Fabaceae or
Sapindaceae emphasize novel topologies with distinctive cambia:
anomalous phloem architectures that evolved with the scandent habit and
represent traits associated with higher flexibility and resilience to
damage (Ewers et al., 1991; Fisher \& Blanco, 2014; Moya et al., 2017;
Pace et al., 2011; 2015; 2018; Chery et al., 2020). Structural variation
of sieve tube elements in relation to axial transport, however, has only
been explored in the long and slender stems of \emph{Ipomoea
nil}(Knoblauch et al., 2016), which showed that while sieve tube
geometry varied little axially, sieve pores connecting conduits
increased in diameter from the top to the base of the stem, in agreement
with the pressure-flow hypothesis developed by Münch in 1930 (Knoblauch
et al., 2016). In other woody life forms such as trees and shrubs, axial
scaling of sieve tubes includes structural variation of phloem conduit
diameter and length, the size of pores, and the number of sieve plates,
all contributing to a more efficient transport at long distances
(Liesche et al., 2017; Savage et al., 2017; Losada \& Holbrook, 2019;
Clerx et al., 2020; Barceló-Anguiano et al., 2021a,b).

\emph{Austrobaileya scandens} is a large-bodied and long-lived liana
native to Queensland (Australia), which reaches more than 15m
aboveground by twining around trees as its only support (Bailey \& Swamy
1949). \emph{A. scandens} exhibits low photosynthetic rates and a slow
stomatal response to drops in environmental humidity (Feild et al.,
2003a,b; Feild \& Arens, 2005, 2007; Feild \& Wilson, 2012; Barral et
al., 2013). Anatomically, the stems of \emph{A. scandens} display wide
xylem vessels (Carlquist, 2001) and a secondary phloem with extremely
angled compound sieve plates (Behnke, 1986). This sieve tube morphology,
which resembles the phloem of gymnosperms, drove early speculations on a
possible transitional tissue toward the `pipe-like´ sieve tubes of most
angiosperms (Bailey \& Swamy, 1949; Srivastava, 1970; Behnke, 1986). An
alternative perspective is that angled plates compensate for small pore
diameters, or that the peculiar morphology of the secondary phloem
of\emph{Austrobaileya} possibly relates to maintaining the functional
demands of the phloem as climbing stems undergo mechanical deformation.
To better understand vascular transport in large-bodied lianas, we
studied the phloem of leaves and stems, so far missing in this species.
The main goal of this study was to understand the structure of the
phloem in relation to 1) axial transport of carbohydrates from the sites
of photosynthesis through the slender stems; 2) mechanical demands of
climbing.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Plant material}

We used the germplasm resources of the Arnold Arboretum of Harvard
University, which includes greenhouse-grown \emph{Austrobaileya
scandens} plants ranging in age from 20 to \textasciitilde{} 25 years
old. The leaf area of seven terminal branches from different plants was
measured by photographing each opposite leaf pair from the branch tip to
the seventh node (total n=84 nodes), above which leaves reached the
maximum size.

\textbf{2.2 Leaf anatomy}

Five mature leaves were used to determine the area occupied by the
vascular elements of the leaf midrib: consecutive hand transverse
sections were cut with a micro scalpel at three different positions in
the leaves: the petiole, the mid part of mid vein, and about 2cm back
from leaf tip. Each section was stained with 0.1\% w/v aniline blue in
PO\textsubscript{4}K\textsubscript{3} (pH 10) (Linskens \& Esser, 1957),
observed with a Zeiss Axiophot epifluorescent microscope using the DAPI
narrow filter band (excitation 365 nm, bandpass 12 nm; dichroic mirror
FT 395 nm; barrier filter LP397 nm), and photographed with an AxioCam
512 Color linked to the AxioVision software (Zeiss, Oberkoche, Germany).
The cross-sectional area of xylem and phloem was measured (totaling 45
measures for each tissue) and, for the 27 cross sections in which all
vascular elements were visible, the number of vascular elements in each
tissue were counted

Five additional leaves were selected for measurements of individual
sieve tubes and xylem vessels. Longitudinal sections of the petiole,
midrib, second, third, and fourth order veins were obtained with a
scalpel, placed in a solution containing acetic acid: hydrogen peroxide
1:1 v/v, left incubating at 60ºC for two days, and then mounted onto
glass slides for microscopy observation and image acquisition
(n\textgreater{}250 total vessel elements measured). Similar areas were
used to obtain fresh longitudinal sections (less than 1mm thick),
mounted onto slides, stained with 0.1\% aniline blue, counterstained
with calcofluor white for cellulose (Hughes \& McCully, 1975),
photographed and measured (at least 35 sieve tubes per vein order,
n=250).

\textbf{2.3 Stem anatomy}

Branches were classified according to both diameter and stiffness (see
Figure 1): \textless{} 2 mm (primary growth, flexible), \textless{} 2 mm
(stiff and twisted around a support), 6-10 mm (green shoots with
secondary growth), 11-15 mm (stems at the base of the vine with highly
twisted-irregular bark). First, we evaluated the general anatomy of the
stems in transverse sections at the top three axial locations (the
widest stems could not be cut transversely to preserve the plants):
flexible searchers (2mm diameter), stiff thin branches (2mm diameter),
and mature stems (6-10mm diameter). Stem material was maintained in 1X
Tris-buffered saline (TBS), sectioned transversally at 50 \selectlanguage{greek}μ\selectlanguage{english}m with a
Reichert-Jung Hn-40 sliding microtome (Austria), mounted onto glass
slides, and stained with aniline blue that binds to callose of the sieve
tubes, or with 0.1\% of acridine orange in TBS buffer, which yields
fluorescence of the lignified tissues (Robertson et al., 1992). To
sample the phloem tissue, the external side of stems containing the bark
were extracted with a sharp knife at the different positions described
above, kept in buffer, sectioned longitudinally with a microscalpel, and
stained with aniline blue. We measured the length and width of at least
30 sieve tubes per sampling location (total n=107), and the number of
sieve areas per compound plate (total n=45).

\textbf{2.4 Scanning electron microscopy}

To evaluate the size of pores that make up the sieve plates, samples of
all leaf veins and stem axial areas (see above description) were cut,
frozen in liquid nitrogen, and transferred to super-chilled ethanol,
prior to being sectioned in different angles with respect to the main
axis with the goal of obtaining sections aligned with the highly angled
sieve plates. These sections were incubated within a mixture of 0.1\%
w/v proteinase K dissolved in 50 mM Tris-HCl buffer, 1.5 mM
Ca\textsuperscript{2+} acetate and 8\% Triton X-100, pH 8.0 (Mullendore
et al., 2010), in a water bath at 60\selectlanguage{ngerman}°C for 2 weeks. After rinsing with
ethanol once, and with distilled water thrice, the sections were
incubated with a 1\% w/v aqueous solution of \selectlanguage{greek}α-\selectlanguage{english}amylase for 2 days at
60\selectlanguage{ngerman}°C, then rinsed thrice in water and finally freeze-dried for 24 h with
a Freeze Dyer (Labconco Freeze Dry System). Samples were then mounted on
SEM studs and sputter coated with gold-palladium using a Denton Vacuum
Desk II Sputter Coater for 180 s at 20 V and 6.67 Pa. Samples were
imaged with a JEOL-6010LV scanning electron microscopy (SEM) (JEOL,
Peabody, MA, USA), using high vacuum and an accelerating voltage of
10--15 kV. The size of sieve pores, of sieve areas, and pore density
were measured. Although sieve pore radius was measured for each vein
order, due to the small size of the sieve plates within higher order
veins, the preservation of intact sieve plates was poor. As a result, we
were only able to determine sieve plate size and pore density for the
midrib (n=5). The total number of pores measured was large (n=105 for
petiole; n=262 for primary vein; n= 84 for minor vein orders). In the
thinnest stems, the stiffness of the external fiber layer, along with
the fragility of the sieve tubes, limited preservation --and thus
visualization- of the pores, relative to the base of the stem, where
preservation was much better and resulted in n=600 pores measured.

\textbf{2.5} \textbf{Phloem sap velocity in \emph{Austrobaileya
scandens}}

Phloem transport velocity was measured by tracking the movement of the
fluorescent dye esculin (reviewed by Knoblauch et al., 2015) in the
secondary veins of a five-year-old vine at 11:00 h for several days. A
young plant (five years approximately) with fully formed leaves, was
watered to saturation, and then one of the long branches immobilized on
the microscope stage, with the abaxial surface exposed upward. A 50 \selectlanguage{greek}μ\selectlanguage{english}L
droplet of 5mM aqueous esculin mixture, combined with 0.1\% of the
SilEnergy surfactant (RedRiver Specialties, Shreveport, LA, USA), was
applied with a micropipette to the junction of the second with minor
order veins (adapted from Jensen et al., 2011, Savage et al., 2013). To
allow better permeabilization of the dye, the tip of the pipette was
used to slightly abrade the thick cuticle, and a small window was
opened, and was covered with the liquid during the experiment to prevent
desiccation. To track the movement of the dye, we used a portable Stereo
Microscope Fluorescence Adapter with 510--540 nm excitation wavelength,
and a long pass 405 UV nm filter band (NIGHTSEA, Lexington, MA, USA).
Time-lapse images were obtained every 10 s for 30min with a Zeiss v12
dissecting microscope using the 0.63\selectlanguage{ngerman}× PlanApo objective and an AxioCam
512 Color camera connected to the AxioVision software. Despite multiple
attempts, the movement of the dye could only be observed in two leaves.
In both cases, fluorescence increased gradually downstream the vein a
few minutes after the dye was applied. Velocity was measured by
thresholding the images (Hue 119-188; Saturation: 97-255; Brightness:
0-255nm), cleaning the dark outliers within 2 pixels' radius, and then
calculating the time elapsed by pixel accumulation two centimeters away
from the area where the dye was applied (n=2).

\textbf{2.6 Immunolocalization of a branched pectin epitope}

Immunolocalization of branched 1-4 galactan with the LM26 monoclonal
antibody (PlantProbes, Leeds, UK), was performed in the searcher stems,
but not in twining stems, because they were much stiffer and embedded
poorly or when embedded were difficult to section. Segments of 0.5cm
from flexible branches were fixed with 4\% w/v acrolein (Polysciences,
Warrington, PA, USA) in a modified piperazine-N, N\selectlanguage{english}'-bis
(2-ethanesulfonic acid) (PIPES) buffer adjusted to pH 6.8 (50 mM PIPES
and 1mM MgSO\textsubscript{4} from BDH, London, UK; and 5mM EGTA) for 24
h, then washed and dehydrated through a series of increasing aqueous
acetone concentrations, one hour each: 10\%, 30\%, 50\%, 70\%, 90\%,
100\%. After that, they were incubated in a solution containing the
Technovit 8100 for two weeks, and then hardened in anoxic conditions at
4oC. Longitudinal and transverse 4\selectlanguage{greek}µ\selectlanguage{english}m sections were obtained with a Leica
EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, GE), mounted onto
superfrost slides, and then used for immunolocalization. Pre-incubation
with 5\% bovine albumin serum (BSA), and three washes in 1XPBS, were
followed by incubation with the LM26 monoclonal antibody for 1h. After
washing the primary antibody with PBS, an anti-rat alexa488 secondary
antibody, with a fluorescein isothianate marker-FITC was applied for 1h.
Samples were then washed and observed with a Leica DM2500 microscope
equipped with epifluorescence and a Leica DM600 camera, combining the
405 filter for autofluorescence with the 488 filter for the specific
signal of the FITC.

\textbf{2.7 Image analysis and statistics}

The transverse areas of the leaf vascular tissues were manually
outlined, individual tube number counts and length/width measurements
evaluated on images with the Image J 1.51d software (National Institutes
of Health, Bethesda, MD, USA). Individual images from stem cross
sections were aligned and merged into a composite image with the
Photoshop software (Adobe Systems, Newton, MA, USA).

Averages from geometrical evaluations were compared using a one-way
ANOVA and the post hoc Tukey test at a \emph{p} \textless{}0.05, using
the SPSS software for statistics.

\textbf{3 RESULTS}

\textbf{3.1 General anatomy of \emph{Austrobaileya scandens}}

\emph{A. scandens} are evergreen vines with long stems with opposite
leaves and branches (Figure 1a,b; see also Bailey \& Swamy, 1949;
Metcalfe, 1987; Feild et al., 2003b). The self-supported young apical
branches are cylindrical, flexible, and about 2mm diameter (Figure 1c).
These young stems harden around 10cm back from the tip and acquire an
elliptical shape, coincident with their coiling around a solid support
(Figure 1d). Despite the minimal variation of branch diameter, leaves
expand exponentially along the coiled stems, reaching a maximum area at
the seventh and older nodes, thus forming thick leaf canopy (Figure 1e).

\textbf{3.2 Vascular geometry in the leaves of \emph{Austrobaileya
scandens}}

Mature leaves of \emph{A. scandens} (Figure 2a) are coriaceous, with a
short petiole and an entire lamina. The vasculature of the petiole
displayed as adaxial xylem tissue composed mainly of squared tracheids,
and abaxial phloem composed of sieve tubes (identified by the presence
of callose), rays, and parenchymatous tissue (Figure 2b). While fiber
caps were lacking in the petiole, a massive protective perivascular
fiber cap proliferated in the midrib (Figure 2c-e). The cross-sectional
areas of both the xylem and the phloem were greatest in the petiole and
narrowed linearly toward the tip of the major vein {[}xylem:
y=-0.05x+0.20(mm\textsuperscript{2}), phloem:
y=-0.04x+0.15(mm\textsuperscript{2}){]}. Strikingly, conduit diameter of
both phloem and xylem were invariant along the midrib, but the number of
conduits decreased by a factor of 2.6 from the petiole to the midrib
tip.

Xylem and phloem conduits varied in size across vein hierarchies (Figure
3a), gradually decreasing in size from major to minor veins, except in
the petiole, where tracheids were shorter and narrower than those of the
midrib (Figure 3b). Although sieve tube elements were also shorter in
the petiole, they were larger in diameter with respect to midrib (Fig.
3C). Sieve plate pore radii varied from 0.12 \selectlanguage{greek}µ\selectlanguage{english}m (\selectlanguage{ngerman}±0.007 SE) in the
petiole to less than 0.08 \selectlanguage{greek}µ\selectlanguage{english}m (\selectlanguage{ngerman}±0.0006 SE) in the minor veins (Figure
S1). Functionally, foliar phloem architecture correlated with a low bulk
velocity of the mobile dye tracer esculin hydrate \emph{in vivo} (Fig.
4; Video S1), revealing an average rate of 11 \selectlanguage{greek}µ\selectlanguage{english}m s\textsuperscript{-1}in
the second order veins (n=2).

\textbf{3.3 Ontogeny of the phloem in the stems of \emph{Austrobaileya
scandens}}

Cross sections of stems at different axial positions revealed
ontogenetic differences in the eustele, which correlated with stem
mechanical properties. In the flexible searcher stems, a large pith was
surrounded by discontinuous primary xylem traces consisting of only a
few tracheids (Figure 5a), but a more continuous phloem layer (Figure
5b). Twining correlated with a sharp increase in the stiffness due to
lignification of the pith and the development of an extra phloem
pericyclic fiber layer (Figure 5c). In wider stems (the ones that
traverse longest distances), the central pith constituted the largest
fraction of the cross-sectional area, and the axial secondary phloem
displayed a lobed morphology (Figure 5d-e), with sieve tubes closer to
the vascular cambium and separated from the pericyclic fibers by
parenchyma (Figure 5f).

In line with the demands for climbing and the need for flexible searcher
branches, a galacturonan-rich pectic epitope, previously related to the
flexibility of sieve tubes (Torode et al., 2018), localized in the sieve
tube walls of the searcher, flexible stems of \emph{A. scandens} (Figure
6; note that we could not perform immunolocalization in the secondary
phloem due to poor preservation of the twining stems). Different cell
morphologies compose the stems in cross section (Figure 6a), but signal
from this epitope specifically showed the scattered distribution of
sieve tubes (Figure 6b,c). Upon closer inspection, the signal of the
antibody corresponded with the areas of the sieve tubes between the
plasma membrane and the cell walls (Figure 6d-f). In longitudinal
section, the epitope was helpful distinguishing sieve tubes from other
phloem cell types (Figure 6g), which highlighted their longitudinal
silhouette (Figure 6h-i), showing slightly tangential sieve plate
connections (Figure 6j-l).

In the stems supported by twining, and concomitant with leaf expansion,
the morphology of the sieve elements varied, gradually increasing the
number of sieve plate connections at the end of the conduits (Figure
7a,b). In the thickest stems, sieve elements had extremely angled
tangential connections with a great proportion of the tubes covered by
sieve areas (Figure 7b,c). Numerous small pores (0.20\selectlanguage{greek}µ\selectlanguage{english}m on average,
n=600), whose radius was similar to those of the leaf petioles,
populated the sieve areas (Figure 7c,d; Behnke, 1986). In addition to
morphology, sieve conduit dimensions increased from thinner to thicker
stems, but their length was shorter at the base of the vine (Figure 7e).

\textbf{3.4 Phloem hydraulic resistance from leaves to stems
of\emph{Austrobaileya scandens}}

Anatomical data (average sieve tube radii, and pore radii/length at each
location along the transport pathway in both leaves and stems) was used
to estimate phloem hydraulic resistance, supposing a continuous
pipe-like cylindrical tubing system formed by sieve elements connected
in series, as modelled previously for vines and trees (Knoblauch et al.,
2016; Savage et al., 2017). The Hagen-Poiseuille equation was the
reference to understand the axial resistance of the sieve tube. Due to
the difficulty of measuring the actual length of the twisted stems, we
calculated the total resistance per tube length (Pa s
m\textsuperscript{-4}) at the defined axial positions along the
transport path from leaves to the base of the stem. The poor
preservation the thinner twining stems allowed only a few measurements
of pore size, which revealed an average radius of 0.19\selectlanguage{greek}µ\selectlanguage{english}m, but this was
quite similar to the pore radius at the base of the stem (0.20\selectlanguage{greek}µ\selectlanguage{english}m), where
preservation was much better and resulted in n=600 pores measured. The
following assumptions were made for calculations of the hydraulic
resistance: (1) sieve areas and pore density were equal to the midrib
value for higher vein orders; (2) pore size at the tip of the stems were
similar to those measured at the vine base. The latter assumption is
very conservative, and likely underestimates the resistance of the upper
stem, given that previous reports showed smaller pore sizes at the tips
of vine stems compared with their base (see Knoblauch et al., 2016).
Sieve tube resistance varied about one and a half orders of magnitude
from the branch tips to the base of the stems. Compared with trees of
similar height, the top of the vine displayed similar sieve tube
resistance, but this resistance dropped more moderately than in trees,
resulting in two orders of magnitude higher resistance of \emph{A.
scandens} vines at ground level (Savage et al., 2017; Losada \&
Holbrook, 2019; Clerx et al., 2020). The small pore radii of sieve
plates were a major factor contributing to this high resistance (Figure
8), although compensated by an increasing number of sieve plates from
top to bottom, thus overcoming resistance penalties associated with
length. Monotonic decrease of axial resistance due to axial conduit
structural variation encounters an exception at the base of the vine,
where conduits were shorter.

\textbf{4 DISCUSSION}

\textbf{4.1 Phloem ontogeny and lianescence}

During stem ontogeny, lianas that rely exclusively on twining for
climbing, such as \emph{A. scandens} , transition abruptly from
self-supporting branches, known as searchers, to stiffer ones that grow
in close contact with a solid support. In \emph{A. scandens} , the
photosynthetic, non-lignified searcher branches contain more phloem than
xylem tissue, likely because the immature leaves have little demands of
water, but a significant need of carbohydrates. This limited xylem
implies that pith turgor might be the major contributor to the
mechanical stability of searcher branches, as reported in other twining
vines (Isnard \& Silk, 2009). Morphologically, the tube-like primary
sieve elements that dominated the vasculature of searchers suggested
lack of lateral transport, but directional sap flow toward growing
branch tips. Chemically, sieve tube walls of searcher stems were
composed of a galacturonan-rich pectin, which has been described in a
handful of species such as in leaves of \emph{Beta vulgaris} (Torode et
al., 2018), or in the seasonally renovated sieve tubes of
\emph{Populus}(Ray \& Savage, 2020). These previous works strongly
emphasized that the chemistry of the cell walls connect with mechanics
of the sieve tube wall (Torode et al., 2018). Thus, this wall related
epitope may contribute to the flexibility of searcher branches, and it
may also be present in sieve tubes in the secondary phloem.
Nevertheless, its presence in the primary sieve tubes of \emph{A.
scandens} demonstrates that this distinctive pectin is found in the
sieve tubes of angiosperms with different life histories, growth habits,
and phylogenetic backgrounds.

Despite the conservation of diameter in rigid twining stems, leaf area
increased and reached full expansion. Basipetally, the phloem in the
twining stems acquired a lobed topology, and the sieve tubes an
increasing number of plate connections, features that suggest an
increase in transport efficiency under torsion. In fact, the complex
division patterns (i.e. tangential longitudinal) of the secondary phloem
of \emph{A. scandens} were evaluated in detail about 50 years ago
(Srivastava, 1970), and differed from axial longitudinal divisions
observed in other woody angiosperms, suggesting a correlation with the
helical gyres of the lianoid growth habit. In line with these
observations, we further noticed that, in areas of high rotation at the
base of the stems, the sieve tube elements were shorter (Silk \&
Holbrook, 2005), reinforcing the idea that the vascular cambium activity
is influenced by the mechanics of lianoid stems (Pace et al., 2015,
2018). How this mechanical (un)stability affects function has been
studied for the lianoid xylem, which enhances hydraulic efficiency
through more volumetric vessels despite the strong torsional pressures
such as twisting and bending (Putz \& Holbrook, 1992; Gentry, 1991;
Speck \& Rowe, 1999; Rowe et al., 2006). Our measurements revealed that
sieve tube geometries are more conserved across species, and \emph{A.
scandens} displays radii of sieve elements (7-13 \selectlanguage{greek}µ\selectlanguage{english}m), which fall within
parameters measured in the slender stems of \emph{Ipomoea nil} (10-20
\selectlanguage{greek}µ\selectlanguage{english}m) (Knoblauch et al., 2016), or the trunks of canopy dominant trees
(6-24 \selectlanguage{greek}µ\selectlanguage{english}m) (Liesche et al., 2017; Savage et al., 2017). The radii of the
sieve tube elements increase basipetally at long distances, as observed
in tall trees (Jensen et al., 2012, Savage et al., 2017). Strikingly
though, the average sieve plate pore size of \emph{A. scandens} (0.20\selectlanguage{greek}µ\selectlanguage{english}m
radius) was smaller than in stems of other species, and varied little
axially. Because pore size is the factor that affects phloem hydraulics
the most, \emph{A. scandens} has high phloem resistance in the stem,
compared with other species (Liesche et al., 2017; Savage et al., 2017;
Clerx et al., 2020; Barcel\selectlanguage{ngerman}ó-Anguiano et al., 2021a), but within the
parameters observed in other Austrobaileyales of the understory (Losada
\& Holbrook, 2019). To compensate for the small sieve plate pore size,
the secondary phloem of \emph{A. scandens} has an unusually high number
of sieve plates along the tangential walls of phloem conduits. Compound
sieve plates have been correlated with growth form in height across
woody species that reach the forest crowns (Pace et al., 2015; Knoblauch
et al., 2016; Liesche et al., 2017; Savage et al., 2017; Losada \&
Holbrook, 2019; Clerx et al., 2020; Barceló-Anguiano et al., 2021a,b).
Reasons behind the particular phloem morphology of \emph{A.
scandens}(lobed arrangement in cross sections, tapered tubes with angled
plates and different lengths) may reflect life history traits associated
with the liana growth form, such as the imbalance of carbon allocation
between the leaf canopy and stem tissues. Thus, a reduced number of
below-canopy sink tissues and higher canopy sinks favor a more
functional phloem aboveground, whereas in the understory, the mechanics
of twining constrains the morpho-functional aspects of phloem (for
example, the length of the sieve tubes).

\textbf{4.2 Vascular scaling in leaves of Austrobaileyales and other
angiosperms}

Within leaves of \emph{A. scandens} , the geometry of vascular elements
(tracheids and sieve tube elements) vary across vein orders.
Hierarchical scaling of the xylem has been documented in the reticulate
leaves of angiosperms, suggesting that the efficiency of water
distribution across the continuous xylem conduits follows laws of energy
conservation, such as Murray´s law (Murray, 1926; McCulloh et al., 2003;
Carvalho et al., 2017a, Scoffoni et al., 2017). In contrast, the scaling
of sieve tube elements in leaf veins of woody plants remains poorly
characterized. Recent work in leaves with different branching patterns,
such as the dichotomously branched veins of \emph{Ginkgo} (Carvalho et
al., 2017b), or reticulate-veined leaves of \emph{Populus}
and\emph{Illicium} (Carvalho et al., 2017a; Losada \& Holbrook, 2019),
strongly suggest universal variation of the geometry of phloem conduits
across leaf vein orders. Yet, an unexplored feature in the leaves of
angiosperms is the size of pores connecting sieve tubes in the leaves.
Our work provides evidence that pore sizes vary in accordance with sieve
tube dimensions, from 0.08\selectlanguage{greek}µ\selectlanguage{english}m in minor veins to 0.12\selectlanguage{greek}µ\selectlanguage{english}m in the petiole. As
a result, variation of both the geometry of tubes and size of pores
enhance bulk export of photoassimilates toward the petiole under the
pressure-flow predictions (M\selectlanguage{ngerman}ünch, 1930).

We further report a 1:1.25 xylem to phloem ratio of areas of the major
vein in \emph{Austrobaileya} leaves, balanced in favor of the phloem
compared to the general range of 1:4 to 1:10 reported in leaves of
deciduous trees (Artschwager, 1926; Waisel et al., 1966). Additionally,
the isodiametric sieve tube elements in the midrib linearly increased in
number toward the petiole. The dimensions of sieve elements in the major
leaf veins remains poorly explored in angiosperms, but they are well
documented in the single veined needles of conifers (Ronellenfitsch et
al., 2015), in which photo assimilate export is favored by the
increasing number of isodiametric phloem conduits from the tip to the
base of the needles. This suggests a convergent strategy between conifer
needles and \emph{A. scandens} midrib, but whether this is
well-maintained in other angiosperm leaves, needs further testing.
Furthermore, the isodiametric tracheids of the midrib linearly increased
in number toward the petiole, supporting predictions on the uniformity
of xylem conduit diameter within the same vein order (McCulloh et al.,
2003, 2009; Gleason et al., 2018). Among the scarce studies documenting
xylem allometry in single veins, variation in conduit diameter along the
midrib has been studied in \emph{Fraxinus} (Petit et al., 2016)
and\emph{Acer} (Lechthaler et al., 2019). The shortening of tracheids
toward the petiole of \emph{A. scandens} suggests that conductivity of
water uptake by leaves is lower, in sharp contrast with the highly
conductive stems, pointing to hydraulic segmentation of the xylem
between the stems and the leaves. In contrast, the sieve tube elements
of the petiole reduced their length, but not their diameter, thus
maintaining a stable transport capacity. We previously showed a similar
trait in the leaves of \emph{Illicium parviflorum} (Losada \& Holbrook,
2019; Carvalho et al., 2018), a pattern confirmed in leaves of five tree
species: \emph{Acer saccharum} , \emph{Liriodendron tulipifera}
,\emph{Catalpa speciosa} , \emph{Liquidambar styraciflua} ,
and\emph{Quercus rubra} (unpublished).

The special characteristics of the petioles have been previously put
forward in a number of species such as in the genera \emph{Beta} (Geiger
et al, 1969), \emph{Cyclamen} (Grimm et al., 1997), or\emph{Pelargonium}
(Ray \& Jones, 2018), suggesting architectural plasticity that is
uncoupled from the leaf lamina. Strikingly, we found that the petiole of
\emph{A. scandens} is fiberless, in sharp contrast with the massive
presence of perivascular fibers in the leaf lamina. Fiberless petioles
were further observed in \emph{Illicium parviflorum}(Losada \& Holbrook,
2019), and suggest that these short petioles require flexibility,
especially in understory plants such as the majority of members of the
\emph{Austrobaileyales,} which need to orientate their leaves toward sun
flecks. In addition, the petioles of\emph{A. scandens} may aid with
twining, as previously suggested (Feild et al., 2003b), in agreement
with previous evidence of a pivotal role of petiole reorientation in
generating the squeezing force that stabilize twining stems (Isnard et
al., 2009).

\textbf{4.3 Contrasting vascular strategies between leaves and stems of
lianas in the understory}

Our measurements, although limited in number, suggest that the velocity
of the phloem in \emph{A. scandens} is one order of magnitude slower
than \emph{Ipomoea} and in trees (Windt et al., 2006; Babst et al.,
2013; Knoblauch et al., 2016), but similar to the rates observed
in\emph{Illicium parviflorum} (Losada \& Holbrook, 2019). While the
radii of the phloem conduits were similar in petioles and searcher
branches, the hydraulic resistance from leaves to stems followed a
continuum, differing by three orders of magnitude from the minor veins
to the base of the plants. Yet, this is a smaller difference than that
reported between the top and the bottom of tree stems of comparable
heights (Savage et al., 2017). Our calculations, which assume a constant
viscosity of 1.7 mPa s\textsuperscript{-1}, would not allow transport at
long distances with faster velocities. For example, the pressure
required to transport the sap 3 m from the leaves would be 1.4 MPa,
suggesting that longer distances could impair transport. In conclusion,
with constant viscosity and velocity, the export of carbohydrates from
the leaves is facilitated by the geometry of the phloem, but encounter
architectural limitations in the stems (i.e. tiny pores). What this may
imply is that the export of photoassimilates may easily be redirected
toward the continuously growing canopy. This is possible because the
stem girth is maintained constant for long distances, but enlarges
typically at the base of the vine. Additionally, the stems of \emph{A.
scandens} are photosynthetic along their length, pointing to the
possibility of self-sustaining, at least partially, the limited
secondary growth, and root elongation.

The vesselless leaves of \emph{A. scandens} were previously suggested as
having a simpler anatomy than other angiosperms, such as the absence of
palisade parenchyma, low stomatal density with slow responses to VPD
changes (Feild et al., 2003), which correlated with a physiology
associated to the understory environment (Brodribb \& Feild, 2010).
Similarly, woody members of the ancestral ANA grade, such
as\emph{Amborella trichopoda} , is vessel free and lacks reaction wood.
(Feild et al., 2005; 2012). Altogether, these results that include a
strong hydraulic segmentation of the xylem, and a high resistance of
phloem to sap transport, correlate with the slow growth rate of this
species in the understory conditions (Feild et al., 2012).

Exploring the phloem of extant members of the ancestral angiosperm grade
ANA (Amborellales, Nymphaeales, Austrobaileyales) is particularly
relevant, given that fossils, when available, rarely preserve this
tissue. Thus, living members allow for the inference of the varied
hydraulic solutions evolved -and perhaps maintained- by angiosperms
during their initial radiation. \emph{A. scandens} , the only extant
member of the Austrobaileyaceae family, itself one of the three lineages
composing the ANA grade, a sister grade to all flowering plants (Mathews
\& Donoghue, 1999; Parkinson et al., 1999; Qiu et al., 2000; Soltis et
al., 1999, 2018), has been historically used to speculate on the
primitive growth habits of the first angiosperms. In particular, one of
the critical questions is when and how flowering plants reached the
canopy (Judd et al., 2018). Our work provides the first empirical
interpretation of long distance transport in woody lianas with simple
body plans, and the constraints associated with their life histories.
Woody lianas, semi climbers and shrubs (rarely trees) dominate the
extant forms of the earliest angiosperm lineages Amborellales and
Austrobaileyales. Woodiness, considered the symplesiomorphic condition
of angiosperms as a whole, was likely required to colonize the vertical
niche during the Cretaceous, previously dominated through millions of
years by gymnosperms. The question remains as to whether the great
anatomical diversity of climbers, typically considered as highly
derived, could have been the ancestral condition of angiosperms.

\textbf{Acknowledgements:}

We are grateful to William E. (Ned) Friedman for kindly sharing plant
material and lab facilities and to the staff of the Arnold Arboretum of
Harvard University for continuous support. This work was funded by the
National Science Foundation IOS 1456845 research grant to N.M.H. Z.H.
was awarded with a research internship program from the DaRin Butz
Foundation at the Arnold Arboretum of Harvard University. JML is a
ComFuturo researcher at the IHSM La Mayora, funded by FGCSIC, a
RTI2018-102222-A-I00 grant from the Spanish Ministry of Science and
Universities, and a LINKB20067 from CSIC.

\textbf{Conflict of interest}

The authors declare there is no conflict of interest.

\textbf{Data availability}

Data are available upon request to the corresponding author.

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\textbf{FIGURES AND LEGENDS}

\textbf{FIGURE 1. (A)} Adult vines of \emph{Austrobaileya scandens} in
the greenhouse. \textbf{(B} ) \emph{A. scandens} vines at the canopy
layer in the tropical rainforest of Queensland (Australia). (\textbf{C}
) Apical part of a `searcher' branch in the greenhouse. (\textbf{D} )
Branches twisted around a stable support without increasing their
diameter. (\textbf{E} ) The average leaf area at each particular node
increased by five fold up to the fully expanded leaves of the seventh
node (\emph{p} \textless{}0.05).

\textbf{FIGURE 2.} Vascular anatomy of the continuum petiole-mid vein
of\emph{Austrobaileya scandens} leaves\textbf{.} (\textbf{A} ) Mature
leaf of \emph{A. scandens} . (\textbf{B} ) Vascular tissues of the fiber
less petiole. \textbf{(C} ) Vasculature in the mid part of the major
vein showing a massive layer of pericyclic fibers surrounding the xylem
and the phloem. (\textbf{D} ) Vasculature at the tip of the mid vein.
(\textbf{E} ) Cross sectional areas of the vascular tissues at the three
positions in the midvein: while the cross-sectional areas of xylem (red
squares) and phloem (yellow circles) decreased linearly toward the tip
(\emph{p} \textless{}0.05), the fibers (grey ribbons) occupied a wide
cross-sectional area in the leaf lamina, but not in the
petiole.\textbf{B-D} . Cross sections stained with aniline blue to
detect callose of the sieve tube elements. Scale bars: \textbf{A} =
1cm;\textbf{B} -\textbf{D} = 200 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 3.} Geometrical scaling of the vascular elements in the
leaves of \emph{Austrobaileya scandens} . (\textbf{A} ) General view of
the leaf veins showing the sampling areas (pink hexagons) and closeup of
the minor veins. (\textbf{B-C} ) Length (squares) and radius (circles)
of the individual vessel elements (red), and sieve tube elements
(yellow) across vein orders; error bars represent standard error at
a\emph{p} \textless{}0.05. Inset in panel (\textbf{A)} shows a
fluorescence image section of the leaf stained with the Feulgen
reactive, cleared, and cuticle dissected. Fou, fourth order veins; pet,
petiole; pri, primary vein; sec, secondary vein; ter, tertiary vein.
Scale bar = 500\selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 4.} Velocity of the phloem sap in \emph{Austrobaileya
scandens} leaves. Time-lapse images of a secondary vein showing a 2 min
advancement of esculin hydrate dye from 0min (\textbf{A} ) to 6min
(\textbf{D} ). Insets show the thresholding applied to the sequential
images for the calculation of velocity, with a gradual accumulation of
the dye within the veins. Scale bars = 1000\selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{FIGURE 5.} Anatomy of the \emph{Austrobaileya scandens} stems.
(\textbf{A} ) Cross section of a 2mm diameter `searcher' branch tip with
primary growth, with an extensive central pith. (\textbf{B} ) Same stem
showing callose in the continuous phloem ring (fluorescent green).
(\textbf{C} ) Cross section of a stiff 2mm diameter branch showing a
dramatic increase in lignification of the pith, the xylem, and a
pericyclic fiber cap (fluorescent green). (\textbf{D} ) The phloem forms
a thin layer between the xylem and the fiber cap. (\textbf{E} ) Wider
stems (8mm diameter) with a high degree of lignification in all central
tissues, wide vessels of the xylem, and enlarged areas between the xylem
and the fiber cap. (\textbf{F} ) The fascicular phloem tissue was
separated by axial multilayered phloem parenchyma and the active tubes
were in the vicinity of the vascular cambium. \textbf{A,C,E:} cross
sections of stems stained with acridine orange, which displays
fluorescence of the lignified tissues in fluorescent green and living
tissues in reddish color; \textbf{B,D,F:} cross sections of the stems
stained with aniline blue for callose in the sieve tube elements of the
phloem. F, fibers; p, pith; phl, phloem; xyl, xylem. Scale
bars:\textbf{A-F} = 1mm; insets = 100\selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 6.} Immunolocalization of LM26 epitope in the sieve tube
elements of searcher branches of \emph{Austrobaileya scandens} .
(\textbf{A} ) Transverse section showing multicellular arrangement in
the different tissue layers, including three isolated tracheids that
compose the primary xylem (red arrowheads). (\textbf{B} ) Same section
displaying the sieve tube elements of the primary phloem forming a
continuous ring (yellow arrows). (\textbf{C} ) Merged images.
(\textbf{D} ) Close up of the phloem in cross section. (\textbf{E} ) The
profile of the sieve tubes in cross section after immunolocalization
(yellow arrows). (\textbf{F} ) Merged images. (\textbf{G} ) Longitudinal
section of the branch displaying he external part in the top and the
internal tissues at the bottom. (\textbf{H} ) Longitudinal profile of
the sieve tube element wall after immunolocalization, yellow arrowheads
define the connection between tubes. (\textbf{I)} Merged images.
(\textbf{J)} Detail of the sieve tube connections\textbf{. (K)} Sieve
plate in the primary sieve tubes are slightly tangential (yellow
arrowhead). \textbf{(L)} Merged images. 4\selectlanguage{greek}µ\selectlanguage{english}m thick transverse
(\textbf{A-F} ) or longitudinal (\textbf{G-L} ) sections of the flexible
branches displaying autofluorescence with the 405nm filter
(\textbf{A,D,G,J} ), immunolocalized with the LM26 monoclonal antibody
(\textbf{B,E,H,K} ), and merged images (\textbf{C,F,I,L} ). Ct, cortical
tissue; phl, phloem; sp, sieve plate; vc, vascular cambium; xyl, xylem.
Scale bars:\textbf{A-C} = 50 \selectlanguage{greek}µ\selectlanguage{english}m; \textbf{G-I} = 25 \selectlanguage{greek}µ\selectlanguage{english}m; \textbf{D-F} ,
\textbf{J-L} = 10 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 7} . Morphology of the sieve tube elements in the stems
of\emph{Austrobaileya scandens} . \textbf{(A} ) Longitudinal view of a
sieve tube element from a flexible branch, 2mm diameter, showing either
simple or compound sieve plate connections (arrows). (\textbf{B} ) Sieve
tube element of an 8mm diameter stem showing numerous sieve plate
connections between tubes (arrows). (\textbf{C} ) Longitudinal view of a
sieve tube element with scanning electron microscopy displaying numerous
sieve plates and sieve areas all along the tube wall (arrows).
(\textbf{D} ) Close ups of three sieve plates (analogous to the white
dotted square in figure C), with details of pores. (\textbf{E} ) Sieve
tube element length (squares), and width (circles) across axial stems of
different diameter ranges; bars represent the standard error at a
\emph{p} \textless{}0.05.\textbf{A, B} , longitudinal hand sections of
the stems stained with aniline blue to detect callose (bright
fluorescence); \textbf{C,D} , scanning electron microscopy images
showing the sieve plates and the sieve pores in detail. Scale bars:
\textbf{A-C} = 20um; \textbf{D} = 2\selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 8.} Sieve tube hydraulic resistance across the aerial
organs of \emph{Austrobaileya scandens} . The sieve tube resistance
drops from 1.5 to 2 orders of magnitude from the minor leaf veins
(squares) to the base of the stems (circles), even though the distance
between them could be up to 20m. Numbers indicate the relative position
along the transport pathway, noted in the figures, but they do not
represent real distances.

\textbf{SUPPORTING INFORMATION}

\textbf{Figure S1.} Pore radii in the leaves of \emph{Austrobaileya
scandens} . X axis displays the different vein orders: pet, petiole;
pri, primary vein; sec, secondary vein; ter, terciary vein; fou, fourth
vein order; fiv, fifth vein order. Bars represent standard error at a
p\textless{}0.05.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1-Vine-and-leaf-areaB/Figure-1-Vine-and-leaf-areaB}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-2-Leaf-midrib2/Figure-2-Leaf-midrib2}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-3-STE-leaves/Figure-3-STE-leaves}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-4-Leaf-VELOCITY-C/Figure-4-Leaf-VELOCITY-C}

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\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-5-ImmunoC/Figure-5-ImmunoC}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-6-Stems-CrossSect1/Figure-6-Stems-CrossSect1}

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\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-7-STE-stemB/Figure-7-STE-stemB}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-8-Resistance/Figure-8-Resistance}

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