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Techniques for Measuring Vessel Lengths and Diameters in Stems of Woody Plants
Author(s): Frank W. Ewers and Jack B. FisherSource: American Journal of Botany , Vol. 76, No. 5 (May, 1989), pp. 645-656
Published by: Botanical Society of America, Inc.
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Amer. J. Bot. 76(5): 645-656. 1989.
TECHNIQUES FOR MEASURING VESSEL LENGTHS AND
DIAMETERS IN STEMS OF WOODY PLANTS
FRANK W. EWERS AND JACK B. FISHER
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824;
and Fairchild Tropical Garden, 11935 Old Cutler Road, Miami, Florida 33156
ABSTRACT
Results were compared between the latex paint and compressed air methods for determining
total vessel lengths, and between the sectioning and maceration methods for determining vessel
diameters. The minimum, mean, median, and maximum vessel diameters were less with the
sectioning method than with the maceration technique. Vessel diameter distributions were
always nonnormal and had roughly similar patterns with the two techniques, but were statistically
different from one another. In all six species where the paint and air methods for determining
vessel length were compared, both methods showed a similar skewed vessel length distribution,
with many short vessels and few long ones. Although there was no consistent pattern to the
difference in results with these two methods, the vessel length frequency distributions were
statistically different from one another. With the paint method, many vessels, especially many
of the narrowest ones, were not paint-filled at the paint infusion port. The air method utilized
the paint method, in part, and, in addition, is based upon the incorrect assumption that all
vessels in the stem are the same diameter. Both techniques tended to exclude vessel lengths of
the narrowest vessels. However, the narrow vessels, although numerous, contributed an insig-
nificant amount to the total theoretical hydraulic conductance in stems.
THERE ARE MANY reports on the diameter and
length of vessel members in plants (e.g., Bailey
and Tupper, 1918; Baas, 1973; van der Graaff
and Baas, 1974; Carlquist, 1975, 1977; van
den Oever, Baas, and Zandee, 1981; Baas and
Carlquist, 1985; Carlquist and Hoekman, 1985;
Rury, 1985), but relatively few reports of total
vessel length. Since a single vessel can consist
of hundreds or thousands of vessel members,
vessel length cannot be easily determined from
conventional microscopic techniques.
In the present report we make comparisons
between the latex paint and compressed air
methods for determining vessel length and be-
tween the sectioning and maceration tech-
niques for measuring vessel diameters. The only
previously published comparison of the paint
and air methods was by Zimmermann and Jeje
(1981), who showed results from two stems of
only one species, Acer saccharum. The sec-
tioning and maceration techniques have not
previously been compared in a manner that
could allow us to determine their appropriate-
ness for xylem structure and function studies.
' Received for publication 29 October 1987; revision
accepted 27 October 1988.
We thank M. Mattmuller, J. S. Sperry, and the late M.
H. Zimmermann for instructions on how to measure vessel
length, S.-T. Chiu and M. Kowalska for technical assis-
tance, and J. S. Sperry, S. Carlquist, P. B. Tomlinson, and
an anonymous reviewer for their many useful comments
on the manuscript. This research was supported by the
National Science Foundation (Grant BSR-8506370).
Our long-term goal is to model water flow in
lianas (woody vines).
Vessel and tracheid diameter are widely rec-
ognized as important for models of xylem
transport (Carlquist, 1975; Zimmermann,
1983; Siau, 1984; Gibson, Calkin, and Nobel,
1985). According to Poiseuille's law for ideal
capillaries, Kh (hydraulic conductance per unit
length in m4 MPa-I sec-') is proportional to
the summation of vessel or tracheid lumen
diameters (d) each raised to the fourth power
(Gibson et al., 1985):
di4
Kh predicted 128= Eq. 1
where: 7 = dynamic viscosity of the fluid (MPa
sec). Due to the fourth power relationship, when
vessel lumens are twice as wide, Kh predicted
is 16 times as great.
Vessel diameters can be measured in sec-
tioned material or from macerations. The sec-
tioning method is more useful for determining
the Kh predicted in a stem since, in transverse
view, the vessel number as well as the diameter
of each vessel lumen can be determined. The
disadvantage in sectioned material is that the
narrowest vessels may be difficult to distin-
guish from tracheids or fibers.
Since vessels are not ideal capillaries of in-
finite length, the total length of vessels is also
important for models of xylem transport. The
645
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646 AMERCANJOURNAL OF BOTANY [Vo. 76
vessel length represents the maximum distance
that a water molecule can travel without pass-
ing through a pit membrane. A knowledge of
vessel length is important for direct measure-
ments of Kh in isolated stem segments (Zim-
mermann, 1 978; Ewers, 1985; Ewers and Crui-
ziat, in press). Ifthe stem segment used is shorter
than most vessels, some of the resistance to
flow offered by pits would be eliminated. Fur-
thermore, vessel length information is relevant
to studies of xylem dysfunction via emboli-
zation. When water within a vessel is under
sufficient tension, a gas bubble will expand to
the total size of the vessel lumen. Since a gas
bubble cannot easily pass through a wet pit
membrane (Zimmermann, 1983; Newbanks,
Bosch, and Zimmermann, 1983), the longi-
tudinal extent of xylem dysfunction due to an
embolism is equal to the length of the vessel.
At least two major approaches have been
used to quantify vessel length in woody stems.
The first involves infusing the stem with mer-
cury, hot wax, emulsions or colloidal suspen-
sions (e.g., ferric hydroxide, India ink, Magdala
red, lead acetate, latex paint) followed by sec-
tioning to identify filled or marked vessels (Ad-
ler, 1892; Ewart, 1906; Handley, 1936; Skene
and Balodis, 1968; Zimmerman and Jeje, 1981;
Salleo, Lo Gullo, and Siracusano, 1984). The
emulsion and suspension particles are sup-
posed to be of a size range such that they are
small enough to pass through vessel lumens
and perforation plates, but too large to pass
through the pit membranes. Pores in pit mem-
branes of dicotyledons range from about 0.005
to 0.17 Am in diameter depending upon the
species (Siau, 1984).
A second approach involves forcing com-
pressed gas through the stem (Bennett, An-
derssen, and Milad, 1927; Handley, 1936;
Greenidge, 1952; Scholander, 1958; Zimmer-
mann andJeje, 1981; Sperryetal., 1987). This
method depends upon the fact that gas cannot
pass through wet pit membranes and hence,
past vessel ends, except when very high pres-
sures (>2,000 kPa) are used. A vessel that is
cut open at both ends can pass gas even at low
pressures (< 100 kPa).
As these techniques were originally applied,
they could give only the maximum vessel
lengths in a stem. This is because it cannot be
determined whether the infusion surface (xo)
is near the distal, proximal, or median portion
of any particular vessel. Skene and Balodis
(1968) developed a statistical approach to de-
termine vessel length frequency distributions
from raw counts of the number of paint-filled
vessels at regular distances (intervals) from xo
to xn. Their analysis depended upon the as-
sumptions that vessels are randomly distrib-
uted along the stem segment and that individ-
ual vessels do not branch.
Zimmerman and Jeje (1981) modified the
Skene and Balodis (1968) approach to correct
for some of the statistical errors that can arise
from nonrandom distribution of vessel ends.
They experimented with injections of various
substances and found dilute latex paint to be
the most reliable for determining vessel length
distribution. They stressed the importance of
avoiding embolisms prior to perfusing latex
into the xylem.
Zimmermann and Jeje (198 1) also modified
the compressed gas method by repeatedly mea-
suring air conductivity as the stem was trimmed
back at regular intervals. Under these condi-
tions, conductivity is proportional to the num-
ber of open vessels (i.e., vessels continuous
through the remaining segment). This assumes
all vessels have equal lumen diameters.
While using the latex paint method, we found
that a surprising number of the vessels (often
50% or more) were not paint-filled even at the
plane (xo) where the paint was supplied. We
were concerned whether there was a sampling
bias for wide or narrow vessels with this tech-
nique since vessel length distributions were
necessarily determined from paint-filled ves-
sels only. Therefore, in addition to compari-
sons of the paint and air methods, we made
comparisons between the diameter distribu-
tions of paint-filled vessels and of the total
vessel population.
MATERIALS AND METHODS-Plant materi-
al-The tree Bauhinia purpurea L., the shrubs
B. aculeata L. and B. galpinii N.E. Br., and the
lianas B. fassoglensis Kotschy ex Schweinf.,
Hippocratea volubilis L., Passiflora coccinea
Aubl., Pithecoctenium crucigerum (L.) A. Gen-
try [= P. echinatum (Aubl.) Schum.], Saritaea
magnifica Dug., and Stigmaphyllon ellipticum
(HBK) Juss., all growing outdoors at the Fair-
child Tropical Garden in Miami, Florida, were
examined in the summers of 1985 and 1986,
and in the spring of 1988. The stem xylem
diameters, which are shown in the tables and
figure legends, were recorded either at the
transverse plane where the diameter measure-
ments were made, or, in the case of vessel
lengths, at the median portion of the longest
vessels (the plane halfway between xo and xe).
Paint infusion method- For each species the
longest unbranched stem segments available
were selected for study. As was determined a
posteriori, these segments were longer than the
longest vessels. Stems were defoliated with
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May 19891 EWERS AND FISHER-MEASURING VESSELS IN WOODY PLANTS 647
shears before they were cut off from the plant
and the cut proximal end immediately recut
under water and the proximal end kept sub-
merged until we began the latex infusion pro-
cess. Within 2 hr the proximal end (xo) was
trimmed with a fresh razor blade and tightly
fitted with clear vinyl tubing to allow for brief
vacuum infiltration with water (5 min at -87
kPa) to remove embolisms that may have been
introduced during handling. A dilute latex so-
lution was then fed into the stem.
The green latex paint initially contained a
wide size range of irregularly shaped pigmented
particles. A 100:1 water: latex paint dilution
was filtered through Whatman no. 1 filter pa-
per. This removed all particles greater than 5
,um in diameter. Filtering with a millipore filter
demonstrated that all pigmented particles were
greater than 0.2 ,m and were thus too large to
pass through pit membranes.
The latex emulsion was gravity fed into the
proximal end of the stem segment from a 2.5
m column. The distal end of the stem segment
was subjected to a -87 kPa vacuum. The so-
lution was allowed to pass through the stem
until flow completely stopped, which took up
to 8 days in some cases.
We cut the stems into n segments of uniform
length x, and stored the segments in a vertical
position with the surface on which vessel counts
would be made facing down on a glass surface.
Within the next 24 hr the stem surfaces (xo to
xJ were shaved smooth with a fresh razor blade,
removing 1 to 2 mm from the surface, and
number of paint-containing vessels counted.
This gave the raw vessel count. Shaving of the
transverse stem surfaces is necessary to remove
surface paint and thus provide a clean and
sharp image. Vessels were counted as paint-
filled even if they were only partially filled
with the latex paint.
Air method-Stems were collected as with
the paint method except that since air was to
be forced through the stems, no special care
was taken to avoid embolisms. Stems were not
cut under water nor were the proximal ends
kept submerged, and vacuum infiltration was
not used. Vinyl tubing was fitted and clamped
to the smoothly shaved basal end (xo) of a
freshly cut stem, and about 60 kPa of air pres-
sure applied, as measured with a mercury col-
umn. The distal end of the stem was dipped
into water and trimmed back until air bubbles
could first be seen to emerge. The distal end
was then shaved smooth with a fresh razor
blade, the air was collected in a graduated cyl-
inder, and the rates of air flow were calculated.
As described by Zimmermann and Jeje (198 1),
in order to calculate the end effect (Pe) and to
calculate flow (F) at a standardized pressure,
P, the air flow rates were measured three times
at each of two different air pressures. Distal
stem segments of length x were then succes-
sively trimmed off of the experimental stem,
the new end was shaved smooth with a fresh
razor blade, and the flow rates were again mea-
sured at two pressures. For each stem length
x., the applied pressure (P) and flow rate (F)
data were fitted with linear regression lines to
obtain the slopes and intercepts. At any arbi-
trarily chosen pressure level, P,
V = Fx,(P - Pe) Eq. 2
where V is a value proportional to the number
of open vessels, F is the calculated flow rate at
P, xn is stem length, and Pe is the y intercept
from the regression equation (i.e., the predicted
flow at P = 0).
After the air flow measurements were made
at the shortest stem length (xl), the remaining
stem segment was vacuum infiltrated with water
for at least 5 min at -87 kPa in order to remove
air emboli. The segment was then perfused
with latex paint at the xo surface until flow
stopped after several days. After shaving the
transverse surfaces, paint-filled vessels were
counted at the distal and proximal surfaces of
the segment to obtain the raw vessel counts at
stem lengths xl and xo, respectively. The raw
vessel count at x1, as determined from paint
infusion, gave the number of open vessels in
the segment. The ratio of this raw vessel count
to the V values, as determined by air flow, gave
the conversion factor. For the remaining stem
lengths (x2 to xn), in which only air flow was
measured, this factor was used to convert V
values into raw vessel counts.
Calculations of vessel length distribution-
The raw vessel count, as determined both from
the air and the paint infusion methods, rep-
resents the number of vessels continuous from
xo. The first difference represents the number
of vessel ends between the distances where the
raw counts were made (Table 1). For vessels
of a particular length class, assuming random
distribution of vessels in the stem, the first
difference will increase linearly towards the zero
point. The second difference represents the rate
of linear increase for vessels of this length class.
The second difference multiplied by the num-
ber of increments (steps to zero) gives the num-
ber of vessels in that length class. This number
can then be expressed as a percent of the paint-
filled vessels at the zero point. If no mistakes
have been made in calculation, the sum of the
calculated numbers of vessels in each size class
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648 AMERCANJOURNAL OF BOTANY Vo. 76
TABLE 1. Example of calculation of vessel length distribution. From a Pithecoctinium crucigerum stem with a xylem
diameter of 2.5 mm
Rawvessel First Second Steps No of Corrected Lengthclass Percent in
Distance in10-2 mcount difference difference to zero vessels vessel no in10-2 mlengthclass
160 x8 = Xn 0 0 0 0 0 0 160-180 0
140 x7 11188067140-16007
120X 21070067120-14007
100X 20- 16-6067100-12007
0X 20050080-1000
0X 31144460-8041
40X 85 43121240-60124
20 x) 4234292585820-40598
0 x0 9755 21121210-20216
should equal the raw vessel count at the zero
point.
Negative values in the first difference were
rare, but when they were discovered were at-
tributed to errors in counting (paint-filled ves-
sels were then recounted) or to the presence of
branched vessels. As discussed by Zimmer-
mann and Jeje (1981), negative values in the
second difference (which are common) can be
attributed to nonrandom distribution of ves-
sels in the stem segment. These negative num-
bers were almost always confined to the longer
size classes and appear to be an artifact of the
small sample size in the longer classes.
Negative numbers in the No. of vessels
column (Table 1) were removed by grouping
categories to arrive at positive values under
Corrected vessel no. To do this, negative
numbers were averaged with adjacent positive
number(s) in the same column. When a choice
had to be made between averaging with a length
class above or below the length class with the
negative number, the adjacent length class with
the greater positive vessel number was used.
In the example shown in Table 1, -6 was
grouped with 0 and 8 in the No. of vessels
column to obtain an average value of 0.67 for
the Corrected vessel no. in Length classes
100-120, 120-140, and 140-160.
Paint vs. air methods-Matched pairs of
stems from six species of plants were selected
in order to make comparisons between the paint
and air methods. For each species two stems
were selected that were very similar in size,
external morphology, and position on the plant.
The paint method was applied to one stem of
the pair, and the air method to the other.
Vessel diameters: camera lucida -Following
latex paint infusion for vessel length deter-
minations, at xo in stems of Pithecoctenium
crucigerum, Saritaea magnifica, and Hippo-
cratea volubilis, the inner (vessel lumen) di-
ameters of all the paint-filled vessels were mea-
sured from drawings of the vessels made with
a camera-lucida device attached to a stereo-
microscope. The camera-lucida technique was
used in this case, since it was difficult to clearly
photograph all the paint-filled vessels in a
transverse section, and since direct ocular mi-
crometer measurements of all the paint-filled
vessels in a woody stem are almost impossible
without missing some vessels and/or measur-
ing some vessels more than once.
Vessel diameters: sections-To determine the
diameter frequency distribution for all the ves-
sels (paint-filled plus those without paint) in a
transverse view, stems were sectioned with a
sliding microtome at 30 ,um and stained with
safranin and fast green. The sectioning tech-
nique was used since the narrowest vessels (ar-
rows in Fig. 1-3), were difficult to detect with
a stereomicroscope in surface view. A Nikon
photostereomicroscope with transmitted light
capabilities was used to prepare Kodachrome
slides of the stem sections. The slides were
projected onto large sheets of white paper upon
which each vessel was marked off as its lumen
diameter was measured with a ruler. This
method allowed us to quickly measure every
vessel member without counting a member
twice. When a vessel lumen was not circular
in transverse view, the minimum and maxi-
mum diameters were recorded. We measured
the distortion of projected stage micrometer
images throughout the image plane and found
the maximum distortion due to spherical ab-
erration of the projection lens was less than
1 .
In the smaller stems we measured every ves-
sel seen in a transverse section. In stems with
more than a thousand vessels in transverse
view, we measured all the vessels in 4 to 6
evenly-spaced sectors. Each sector had vas-
cular rays for marginal boundaries and the pith
and the vascular cambium as its inner and
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May 1989] EWERS AND FISHER-MEASURING VESSELS IN WOODY PLANTS 649
Fig. 1-3. Transverse sections of stems. 1. Pithecoctenium crucigerum. 2. Saritaea magnifica. 3. Hippocratea volubilis.
Arrows show some of the narrowest vessels. All at same magnification, scale bar = 500 ,um.
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650 AMERICANJOURNAL OF BOTANY[Vol. 76
80 PITHECOCTENIUM
60
H 40
w
0
c c
w
a.
20
0
80 160 240 320 400
40 - SARITAEA
H
w 20
0
c c
w
a.~~~~~~~~~~~~~~~~~~~
20 40 60 80 100120
H20 HIPPOCRATEA
z I
L l
20 l
01
40 80 120 160 200240 280
DIAMETER (pjm)
Fig. 4. Frequency distribution of vessel diameter classes
as determined from the sectioning and maceration tech-
niques. Solid line = sectioning, broken line = maceration.
From the stems shown in Fig. 1-3. See summary in Table 2.
outer boundary. Several hundred vessel lumen
diameters were measured in each stem with
this technique.
Vessel diameters: macerations-One worker
measured lumen diameters from projected im-
ages (see above) and another measured lumen
diameters with the maceration technique. To
avoid possible measuring bias, we did not show
the results to one another until all the raw data
were collected. Tissue from a 10 mm length of
iz2O PITHECOCTENIUM
*of
ffi 80 160 240 320 400
SARITAEA
~20L
z
w 0
W I I ,1
20 40 60 80 100 120
40 HIPPOCRATEA
20
z
w
0
w
0
40 80 120 160 200240 280
DIAMETER (pEm)
Fig. 5. Percent total theoretical hydraulic conductance
per unit stem length (Kb predicted) as a function of the
vessel diameter class. From same transverse stem sections
as in Fig. 4.
stem adjacent to the sectioned region was ma-
cerated as follows: all tissues outside the cam-
bium were removed, and the remaining pith,
primary xylem, and secondary xylem were cut
into longitudinal slivers. The material was
treated with Jeifreys's solution (10% chromic
acid + 10% nitric acid) for 2-3 days at room
temperature until it was soft to the touch. The
tissue was washed in water and stained with
aqueous safranin in tubes which were centri-
fuged between solution changes. Cells were sus-
pended in a solution of glycerine jelly, dropped
onto warm slides, and covered with square
cover glasses. Vessel members, as defined by
the possession of at least one perforation plate,
were sampled randomly by including all vessel
members that were visible in the field of view
with a x 10 objective lens. The mechanical slide
stage was moved in a straight line starting from
a random point on the edge of the cover glass.
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My 1989] EWERS AND FISHER-MEASURING VESSELS INWOODYPLANTS 651
TABLE 2. Vessel diameters (,um) in three species of lianas
(woody vines). Two methods were used on a single stem
segment of each species. The diameter distributions
are in Fig. 4
Methods
Vessel diameter Mcera-
Species (xylem diameter) parameter Sectioning tion
Pithcoctenium crucigerum minimum 6 9
6mm x4766
median18 26
maximum 335 360
N762 200
Saritaea magnifica (7 mm) minimum 8 9
x3724
mdian 24 30
maximum 126 128
N1,206 200
Hippocratea volubilis minimum 12 16
7mm x90 127
median94 130
maximum 196 286
N506 200
Horizontal and vertical edges were used on
alternate slides. Vessel member length and lu-
men diameter were measured directly with an
ocular micrometer with a 20 and x 40 objec-
tive, respectively. Lumen diameter was mea-
sured at the median portion of each vessel
member. A total of 200 vessel members was
sampled for each stem.
Kh predicted-This was determined in sec-
tioned material from Equation 1 with the fol-
lowing modifications for vessel lumens that
were elliptic rather than circular in transverse
outline. First, d was calculated as the diameter
that a circle of equal transverse area would
have, d = ab, where a and b are the diameters
of the major and minor axes. For each vessel,
the Kh predicted was then multiplied by the
following factor (Calkin, Gibson, and Nobel,
1986) to correct for the effect of noncircularity
on water flow:
2ab
Eq. 3
However, for graphic representation (e.g.,
Fig. 4, 5) vessel diameter refers simply to the
average diameter (0.5[a + b]) of each elliptic
vessel.
Statistical tests -These were carried out us-
ing the computer program package BIOSTAT
I (Pimentel and Smith, 1986). Significant x2
values were taken from Steel and Torrie (1 9 80).
RESULTS-The pattern of vessel diameter
frequency distributions appeared to be similar
for the maceration and sectioning techniques
(Fig. 4), but the distributions were statistically
different from one another based upon the x2,
D, and G tests of goodness-of-fit at the 0.95
level. In all three species the minimum, mean,
median, and maximum vessel diameters were
smaller with the sectioning technique than with
the maceration method (Table 2).
The narrower vessels in stems, although quite
numerous (Fig. 4), contributed an insignificant
amount to the Kh predicted for each stem (Fig.
5). For instance, in Pithecoctenium crucigerum,
68.5% of the vessels were less than 35 gm in
diameter (Fig. 4), but these contributed only
0.07% of the total Kh predicted (Fig. 5).
The frequency distribution of vessel lengths
using both the air and paint methods produced
similar, highly skewed, nonnormal distribu-
tions with a high frequency of short vessels
(Fig. 6, 7). The air and paint methods produced
statistically significant different distributions
as determined by x2, D, and G statistics (at the
0.95 level). However, there was no consistent
pattern of difference in vessel length measure-
ments between the two methods in the six
species where this was examined (Fig. 6, Table
3). The paint method showed a higher fre-
quency of short vessel classes than did the air
method in Passiflora coccinea, Bauhinia fas-
TABLE 3. Summary of vessel lengths (10-2 m) for paired stems of each species by the paint and air methods. Frequency
distributions shown in Fig. 6
XymArPn
diameter
Speces (mm xMdian MxNxMdian MxN
Bauhnia aculeata 6 3 2.5 34 642 5 2.5 47 780
Bauhinia fassoglensis 3 27 10 65 100 11 5 65 98
Bauhnia galpnii 4 7 5 44 297 9 5 55 414
Bauhnia purpurea 7 17 10 48 425 8 5 65 330
Passiflora coccnia 1 16 5 52 641 16 5 52 168
Stigmaphyllonelipticum4 27 12 87 89 43 37 162 50
Means ? SE 14 ? 6 10 ? 5 73 ? 18 16 ? 4 7 ? 2 55 ? 8
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652 AMERCANJOURNAL OF BOTANY [Vo. 76
PASSIFLORA B. ACULEATA
80 4
80
z 60
w Z~~~~~~~~~~~~~~ 60
cr4 4
w
150w40
20
0~~~~~~~~2
0 20 4060 80 2040 60 00 20 40642008
STIGTH(102m)PYLONGH L H20 30 102 20 0 04050
80 SGAY O80 B GALPINI
60 60
zz
ww
ww
~204- 204
50 100 150 50 100 150o 20 410 60 20 40 60
B. FASSOGLENSIS B. PURPUREA
88
w 60- 60-
0.
2 0 2, 4
0 1 1 t I ~ ~~~~04 ,
20406080 20 40608 2000 2040608
LEGH(10j m) LENGTH (102 m) LENGTH (102 m) LENGTH (10-2m
Fig. 6. Frequency distributions of vessel length for paired stems of 6 species by air method (light bars) and paint
method (dark bars). Arrow = longest vessel. See summary in Table 3.
soglensis, and B. purpurea. However, this sit-
uation was reversed in Stigmaphyllon ellipti-
cum, B. aculeata, and B. galpinii. Maximum
vessel lengths were the same for both methods
in B. fassoglensis, slightly longer with air in
Passiflora, and longer with paint in the re-
maining species (Table 3).
In the latex paint infusion technique the
heartwood vessels of large stems were not paint-
filled even at xo. In addition, often more than
50% ofthe sapwood vessels were without paint.
Some of these had gums, tyloses, or other ob-
vious obstructions, but most did not. Figure 8
shows that the diameter frequency distribu-
tions for paint-filled vessels were much closer
to a normal distribution than were the total
vessel distributions (paint-filled plus those
without paint). Total vessel distributions tend-
ed to be highly skewed with many more narrow
than wide vessels (Fig. 4, 8).
The maceration technique revealed that 9 of
the 200 sampled vessel members of both Sar-
itaea magnifica and Pithecoctenium cruciger-
um were 4vessel ends as indicated by the pos-
session of only one perforation plate. These
vessel ends were much more common for nar-
row elements than wide elements. For S. mag-
nifica, while 15% of the vessel members were
< 18 ,tm in diameter, this diameter class con-
tained 55% of the vessel ends. Similarly, this
narrowest diameter class in P. crucigerum con-
tained 19% of the total vessel members but
78% of the vessel ends. Based upon the fre-
quency of vessel ends, Fisher (1970) used the
following equation to calculate mean vessel
length: 2 + [No. vessel members with 2 per-
forations/0.5(No. vessel ends)]. Using this
equation and assuming that vessels do not vary
in diameter class along their length, for S. mag-
nifica and P. crucigerum, mean vessel lengths
for the narrowest diameter class would be 14
vessel members and 13 vessel members, re-
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8/16/2019 Ewers Fisher 1989
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PITHECOCTENIUM
H 40
z
0 20 \
40 80 120 160200
SARITAEA
40 -
z
20
w
00
5 10 15 20 25
HIPPOCRATEA
80
660
z
40
20
80 160 240 320
2
LENGTH (10 m)
Fig. 7. Vessel length frequency distributions based upon
the paint method in a stem of Pithecoctenium crucigerum,
Saritaea magnifica, and Hippocratea volubilis. Vessel di-
ameters for these same stems shown in Fig. 8. In both Fig.
7 and 8, N = 76 (P. crucigerum), 212 (S. magnifica), and
279 (H. volubilis).
spectively. With mean vessel member lengths
(perforation to perforation) of 221 (SE = 13)
and 187 (SE = 14) ,m, respectively, the mean
total vessel lengths would be 3.1 and 2.4 mm,
respectively, for the narrowest diameter class
of these species. For the wider diameter classes
vessel ends were too infrequent and our sam-
pling too limited to allow for meaningful cal-
culations of vessel lengths by this method. For
Hippocratea volubilis, there were no vessel ends
among the 200 vessel members sampled.
DISCUSSION-There are limitations to the
data derived from the maceration as well as
PITHECOCTENIUM
40
z
w
20
ix 20 r--
I ~~~~~~~r--I
0
20 40 60 80 100120
40 SARITAEA
H
20
o
a.
0
20 40 60 80 100120140
HIPPOCRATEA
,, 15
0.~~~~~~~~~~~~.
60 120 180 240
DIAMETER (jim)
Fig. 8. Diameter frequency distributions for paint-filled
vessels (broken line) and the total vessel population (solid
line). Total vessel population diameters were determined
by the sectioning technique. Results were from a different
set of stems than in Fig. 1-4. Stem xylem diameters and
N (for total vessels): Pithecoctenium crucigerum 2.5 mm
(420), Saritaea magnifica 6 mm (815), and Hippocratea
volubilis 6 mm (278). For N of paint-filled vessels see Fig. 7.
sectioning methods of determining vessel di-
ameters. There appears to be a shift in the
distribution pattern to wider vessel measure-
ments with the maceration method (Fig. 4;
Table 2).
We expect that the maceration technique is
subject to bias towards larger diameter mea-
surements for three or more reasons: 1) Crush-
ing of large cells by the cover slip would lead
to greater diameter measurements, especially
maximum diameters, in the maceration but
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8/16/2019 Ewers Fisher 1989
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654 AMERCANJOURNAL OF BOTANY [Vo. 76
not in the transverse sectioning method. Of
vessel members wider than 100 ,um, about 12%
in Pithecoctenium crucigerum and 4% in Sar-
itaea magnijica were torn or obviously dam-
aged during processing and could not be mea-
sured. Fewer narrow vessel members showed
any damage. Many of the measurements may
have been on vessels that were partially crushed,
but lacking in obvious rips or distortions. 2)
The maceration technique does not involve
representative sampling of vessel diameter
along the length of a vessel. Instead, the vessel
member is measured only at the midpoint of
each vessel member. In contrast, the sectioning
technique serves to randomly sample along the
length of vessel members and thus includes
tapered ends, which would account for smaller
minimum diameters. 3) In the maceration
technique only one diameter can be measured
in each cell, since the macerated cells are always
oriented with their longitudinal axis more or
less parallel to the plane of the slide. In section
the minimum and maximum diameters of cells
that are non-circular in transverse outline can
be measured.
The sectioning technique is clearly superior
to the maceration method for calculations of
Kh predicted. Aside from the above consider-
ations, the maceration technique, by itself, gives
no idea of the absolute number of vessels of
each diameter that would occur in transverse
view. In addition, corrections for vessels that
are noncircular in transverse outline can only
be made from sections. Another advantage of
the sectioning technique is that it can be used
in conjunction with dyes that marked the con-
ductive pathway. Maceration washes out these
dyes. Lastly, the biggest potential problem with
the sectioning technique, that some of the nar-
row vessels may be excluded from consider-
ation (which does not seem significant for the
three species we examined-Fig. 4), has vir-
tually no effect on the Kh predicted of a stem.
Due to the fourth power relationship to vessel
lumen diameter (Eq. 1), the narrowest vessels,
although often numerous (Fig. 4), contribute
very little to the total Kh predicted (Fig. 5).
A problem for comparative wood anato-
mists is that when diameter distributions are
not normally distributed, as is often the case
(Fig. 4, 8), mean values are misleading. In ad-
dition, as mentioned recently by Gasson (1987),
the common practice in comparative wood
studies of giving mean vessel diameters of the
larger vessels lacks objectivity. The ideal ap-
proach for both comparative and physiological
wood anatomical studies would be to incor-
porate entire vessel diameter distributions into
the analyses. In comparative studies, care must
be taken not to overlook the narrowest vessels
(Fig. 1-3) which could be confused with tra-
cheids.
A justification for both the paint infusion
and air methods of determining vessel lengths
is that they lead to roughly similar results, with
many short vessels and few long ones (Fig. 6;
Zimmermann and Jeje, 1981). Although vessel
length distributions in the matched pairs of
stems were significantly different from one
another, there was no consistent direction to
the differences (Table 3). Given that there is
much variation in vessel length within these
species (Ewers and Fisher, unpublished), the
differences in results shown in Fig. 6 may reflect
actual differences in vessel length between stems
rather than differences due to the techniques.
Dr. John Sperry (personal communication)
has argued that in Equation 1, Pe should not
be included in calculating V, since, in his opin-
ion, Pe is not a true end effect but instead rep-
resents the pressure required to prevent me-
niscus formation in the air-conducting vessels.
However, exclusion of Pe makes little differ-
ence in the final results.
Although the air method is much faster than
the paint method for determining maximum
vessel lengths, determination of vessel length
frequency distribution is similarly labor inten-
sive by both methods. Neither method can be
used to determine the absolute minimum ves-
sel length, which may be equal to the length
of two vessel members.
Both the paint and air methods appear to be
biased towards excluding lengths of the nar-
rower vessels. The air method makes the ob-
viously incorrect assumption that vessels are
all the same diameter. One might expect the
air method to reflect results mostly for the wid-
est vessels, since these have the greatest air
conductivity. However, this bias is tempered
somewhat by the fact that the air method de-
pends upon latex paint infusion for raw vessel
counts at x0 and xl. The counts at x0 and xl
are particularly critical since they greatly in-
fluence the shape of the entire vessel length
distribution.
With the paint method the nonfilling of many
vessels at the infusion port (x0) may have been
due to either naturally occurring or to exper-
imentally induced embolism. Some of the ves-
sels without paint had obvious tyloses and/or
gums. However, this would not normally ex-
plain why the narrow vessels in particular tend-
ed to lack paint at x0 (Fig. 8).
There are at least three possible reasons for
the observed scarcity of narrow paint-filled
vessels: 1) In the case of Hippocratea volubilis,
the narrower vessels are most abundant in the
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8/16/2019 Ewers Fisher 1989
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May 1989] EWERS AND FISHER-MEASURING VESSELS IN WOODY PLANTS 655
inner xylem (Fig. 3), which is the first xylem
to become heartwood. 2) Wound response of
living xylem parenchyma cells at the cut sur-
face may cause a coagulation of latex particles
and clog the narrowest vessels. 3) In the case
of Pithecoctenium crucigerum and Saritaea
magnifica, many of the narrow vessels may
have been paint-filled, but the routine trim-
ming of 1 to 2 mm of tissue from the xylem
surface at xo for surface observation of the ves-
sels may exclude them from consideration. The
narrowest vessels in these species were ap-
proximately 2.4 and 3.1 mm long based upon
our data from macerated tissue. Assuming ran-
dom vessel distribution within the stem, trim-
ming away 1.5 mm of tissue would exclude
50% or more of the narrowest paint-filled ves-
sels. Unfortunately, this trimming and result-
ing artifact cannot be avoided.
The vessel length frequency distributions
(Fig. 7) were more skewed than the diameter
distributions of the same paint-filled vessels
(Fig. 8). Since many of the shortest and nar-
rowest vessels appear to have been excluded
by the paint and air methods, the actual vessel
length distribution patterns, which would in-
clude vessels of all diameters, may be even
more skewed than indicated in Fig. 6, 7.
Scholander (1958) calculated mean vessel
lengths in the lianas (woody vines) Vitis la-
brusca and Tetracera based upon measure-
ments of the water volume released by verti-
cally held fresh stem segments which were
trimmed back at measured intervals. This
technique gives no indication of maximum and
minimum vessel length and is probably even
more biased against incorporating information
on the narrow vessels than are the paint and
air methods. The wider vessels obviously would
contain much greater volumes of water to be
released upon cutting than would the narrow
ones. The narrow vessels also tend to hold on
to their diminutive water volume due to cap-
illarity, which is probably why this technique
does not work at all for most species.
Fisher (1970) used the maceration technique
to estimate mean vessel length in the monocot
Cyperus alternifolius. This technique is most
appropriate for plants, such as Cyperus, with
readily distinguishable vessel types (early and
late metaxylem) and with extremely short ves-
sels (about 12 and 1.7 mm, respectively). Ves-
sels greater than 1 m long, such as occurred in
some of the stems we examined (Fig. 6, 7),
could have more than 1,000 vessel members
per vessel, meaning that many thousands of
macerated vessel members would have to be
sampled to accurately determine mean vessel
length.
Drs. P. B. Tomlinson and A. M. Lewis (per-
sonal communication) are presently attempt-
ing to use cinematographic analysis to measure
vessel lengths in Vitis labrusca. This method,
which requires using a movie camera to pho-
tograph serial microscopic sections (Zimmer-
mann and Tomlinson, 1966; Zimmermann,
1971), may be quite accurate but is too labo-
rious to be practical in studies of many stems
with long vessels.
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