Techniques for Measuring Vessel Lengths and Diameters in Stems of Woody Plants

Author(s): Frank W. Ewers and Jack B. Fisher

Source: American Journal of Botany, Vol. 76, No. 5 (May, 1989), pp. 645-656
Published by: Botanical Society of America, Inc.
Stable URL: http://www.jstor.org/stable/2444412
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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 insignificant 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 between the sectioning and maceration techniques 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 sectioning and maceration techniques have not
previously been compared in a manner that
could allow us to determine their appropriateness 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 assistance, 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 sectioned material or from macerations. The sectioning 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 distinguish from tracheids or fibers.
Since vessels are not ideal capillaries of infinite length, the total length of vessels is also
important for models of xylem transport. The

645

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646 AMERICAN JOURNAL OF BOTANY [Vol. 76

vessel length represents the maximum distance

that a water molecule can travel without passing through a pit membrane. A knowledge of
vessel length is important for direct measurements of Kh in isolated stem segments (Zimmermann, 1 978; Ewers, 1985; Ewers and Cruiziat, in press). Ifthe stem segment used is shorter
than most vessels, some of the resistance to
flow offered by pits would be eliminated. Furthermore, vessel length information is relevant
to studies of xylem dysfunction via embolization. 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 longitudinal 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 mercury, hot wax, emulsions or colloidal suspensions (e.g., ferric hydroxide, India ink, Magdala
red, lead acetate, latex paint) followed by sectioning to identify filled or marked vessels (Adler, 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 supposed 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 membranes of dicotyledons range from about 0.005

to 0.17 Am in diameter depending upon the

species (Siau, 1984).

A second approach involves forcing compressed gas through the stem (Bennett, Anderssen, and Milad, 1927; Handley, 1936;
Greenidge, 1952; Scholander, 1958; Zimmermann 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 pressures (>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 determine 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 distributed along the stem segment and that individual 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 measuring air conductivity as the stem was trimmed
back at regular intervals. Under these conditions, conductivity is proportional to the number 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 technique since vessel length distributions were
necessarily determined from paint-filled vessels only. Therefore, in addition to comparisons of the paint and air methods, we made
comparisons between the diameter distributions 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. Gentry [= 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 measurements 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 submerged 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 solution 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 paper. 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 solution 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 "paintfilled" 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 pressure applied, as measured with a mercury column. 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 cylinder, 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 successively trimmed off of the experimental stem,
the new end was shaved smooth with a fresh
razor blade, and the flow rates were again measured 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 arbitrarily 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 distributionThe raw vessel count, as determined both from
the air and the paint infusion methods, represents 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 number of increments (steps to zero) gives the number of vessels in that length class. This number
can then be expressed as a percent of the paintfilled 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 AMERICAN JOURNAL OF BOTANY [Vol. 76

TABLE 1. Example of calculation of vessel length distribution. From a Pithecoctinium crucigerum stem with a xylem
diameter of 2.5 mm
Raw vessel First Second Steps No. of Corrected Length class Percent in
Distance in 10-2 m count difference difference to zero vessels vessel no. in 10-2 m length class

160

(x8

Xn)

160-180

140 (x7) 1 1 1 8 8 0.67 140-160 0.7

120(X6)
2
1
0
7
0
0.67
120-140
0.7
100(X5) 2 0 - 1 6 -6 0.67 100-120 0.7
80(X4)
2
0
0
5
0
0
80-100
0
60(X3)
3
1
1
4
4
4
60-80
4.1
40(X2)
8
5
4
3
12
12
40-60
12.4
20

(xl)

42

34

(x0)

97

55

29

21

should equal the raw vessel count at the zero

point.
Negative values in the first difference were
rare, but when they were discovered were attributed to errors in counting (paint-filled vessels 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 vessels in the stem segment. These negative numbers 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 determinations, at xo in stems of Pithecoctenium
crucigerum, Saritaea magnifica, and Hippocratea volubilis, the inner (vessel lumen) di-

58

21

58

21

20-40

0-20

59.8

21.6

ameters of all the paint-filled vessels were measured from drawings of the vessels made with
a camera-lucida device attached to a stereomicroscope. 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 micrometer measurements of all the paint-filled
vessels in a woody stem are almost impossible
without missing some vessels and/or measuring some vessels more than once.

Vessel diameters: sections-To determine the

diameter frequency distribution for all the vessels (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 technique was used since the narrowest vessels (arrows 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 maximum diameters were recorded. We measured
the distortion of projected stage micrometer
images throughout the image plane and found
the maximum distortion due to spherical aberration of the projection lens was less than
1%.
In the smaller stems we measured every vessel 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 vascular 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 AMERICAN JOURNAL OF BOTANY [Vol. 76

80 PITHECOCTENIUM

iz2O PITHECOCTENIUM

60

*of
ffi
~20L

H40

80 160 240 320 400

SARITAEA

w 0

cc
w
a.

20

W0

..,1

20 40 60 80 100 120

80 160 240 320 400

40 - SARITAEA

40 HIPPOCRATEA
0

w 20

20

cc
w
a.~~~~~~~~~~~~~~~~~~~

20 40 60 80 100120

40 80 120 160 200240 280

DIAMETER (pEm)

zI

H20 HIPPOCRATEA

20Ll l

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.

01

40 80 120 160 200240 280

DIAMETER (pjm)
Fig. 4. Frequency distribution of vessel diameter classes
as determined from the sectioning and maceration techniques. 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 images (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

stem adjacent to the sectioned region was macerated as follows: all tissues outside the cambium 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 centrifuged between solution changes. Cells were suspended 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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May 1989] EWERS AND FISHER-MEASURING VESSELS IN WOODY PLANTS 651

TABLE 2. Vessel diameters (,um) in three species of lianas

(woody vines). Two methods were used on a single stem

2ab

Eq. 3

segment of each species. The diameter distributions

are in Fig. 4
Methods

Vessel diameter Macera-

Species (xylem diameter) parameter Sectioning tion

Pithcoctenium crucigerum minimum 6 9

(6 mm) x 47 66
median 18 26
maximum 335 360
N

762

200

Saritaea magnifica (7 mm) minimum 8 9

x

37

24

median 24 30
maximum 126 128
N 1,206 200

Hippocratea volubilis minimum 12 16

(7 mm) x 90 127
median 94 130
maximum 196 286
N 506 200

Horizontal and vertical edges were used on

alternate slides. Vessel member length and lumen diameter were measured directly with an
ocular micrometer with a 20 and x 40 objective, respectively. Lumen diameter was measured 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 sectioned material from Equation 1 with the following 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:

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 using 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 measurements between the two methods in the six
species where this was examined (Fig. 6, Table
3). The paint method showed a higher frequency 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

Xylem

diameter

Species

(mm)

Median

Air

Max

Paint

Median

Max

Bauhinia aculeata 6 3 2.5 34 642 5 2.5 47 780

Bauhinia fassoglensis 3 27 10 65 100 11 5 65 98
Bauhinia galpinii 4 7 5 44 297 9 5 55 414
Bauhinia purpurea 7 17 10 48 425 8 5 65 330
Passiflora coccinia 1 16 5 52 641 16 5 52 168
Stigmaphyllon elipticum 4 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 AMERICAN JOURNAL OF BOTANY [Vol. 76

PASSIFLORA B. ACULEATA

80 4

80

z 60
w

w Z~~~~~~~~~~~~~~ 60

cr40
40
a.
150
w
40
20

0~~~~~~~~2

STIGTH(102m)PYLONGH L H20 30 102 20 0 04050

80 SGA Y O80 B. GALPINII

60

z
w

040
w

~20

60z
w

040
w

4,-,

0
10'0
150
050
20
4060

20:4

50
150o
20 00
41020
6'0 40642'008
20 40 60
80100
2040
60

B. FASSOGLENSIS B. PURPUREA

80

wz

60-

a.

80

60-

w
0.

2,

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 situation was reversed in Stigmaphyllon ellipticum, 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 remaining 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 obvious obstructions, but most did not. Figure 8
shows that the diameter frequency distributions 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 tended 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 crucigerum were "4vessel ends" as indicated by the possession of only one perforation plate. These
vessel ends were much more common for narrow elements than wide elements. For S. magnifica, while 15% of the vessel members were
< 18 ,tm in diameter, this diameter class contained 55% of the vessel ends. Similarly, this
narrowest diameter class in P. crucigerum contained 19% of the total vessel members but
78% of the vessel ends. Based upon the frequency of vessel ends, Fisher (1970) used the
following equation to calculate mean vessel

length: 2 + [No. vessel members with 2 perforations/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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May 1989] EWERS AND FISHER-MEASURING VESSELS IN WOODY PLANTS 653

PITHECOCTENIUM

40

H 40

PITHECOCTENIUM

0 20 \

20
ix 20 r-40 80 120 160200

~~~~~~~r--I

SARITAEA

20 40 60 80 100120

40 z

20

.I'

40 SARITAEA

00
5 10 15 20 25

20
80

HIPPOCRATEA

a.

o
0

20 40 60 80 100120140

660
z

HIPPOCRATEA

40

20

,, 15
0.~~~~~~~~~~~~.

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 diameters 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 sampling too limited to allow for meaningful calculations 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

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 diameters. There appears to be a shift in the

distribution pattern to wider vessel measurements with the maceration method (Fig. 4;
Table 2).
We expect that the maceration technique is
subject to bias towards larger diameter measurements for three or more reasons: 1) Crushing of large cells by the cover slip would lead
to greater diameter measurements, especially
maximum diameters, in the maceration but

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654 AMERICAN JOURNAL OF BOTANY [Vol. 76

not in the transverse sectioning method. Of

vessel members wider than 100 ,um, about 12%

be taken not to overlook the narrowest vessels

(Fig. 1-3) which could be confused with tra-

in Pithecoctenium crucigerum and 4% in Saritaea magnijica were torn or obviously damaged during processing and could not be measured. 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

cheids.

be included in calculating V, since, in his opinion, Pe is not a true end effect but instead represents the pressure required to prevent meniscus formation in the air-conducting vessels.
However, exclusion of Pe makes little differ-

be measured.

ence in the final results.

The sectioning technique is clearly superior

to the maceration method for calculations of
Kh predicted. Aside from the above considerations, 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 conductive pathway. Maceration washes out these
dyes. Lastly, the biggest potential problem with
the sectioning technique, that some of the narrow vessels may be excluded from consideration (which does not seem significant for the
three species we examined-Fig. 4), has virtually 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 anatomists is that when diameter distributions are
not normally distributed, as is often the case
(Fig. 4, 8), mean values are misleading. In addition, 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 approach for both comparative and physiological
wood anatomical studies would be to incorporate entire vessel diameter distributions into
the analyses. In comparative studies, care must

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 intensive by both methods. Neither method can be
used to determine the absolute minimum vessel 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 narrower vessels. The air method makes the obviously incorrect assumption that vessels are
all the same diameter. One might expect the
air method to reflect results mostly for the widest vessels, since these have the greatest air
conductivity. However, this bias is tempered
somewhat by the fact that the air method depends upon latex paint infusion for raw vessel

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

counts at x0 and xl. The counts at x0 and xl

are particularly critical since they greatly influence 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 experimentally induced embolism. Some of the vessels without paint had obvious tyloses and/or
gums. However, this would not normally explain why the narrow vessels in particular tended 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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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 surface 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 trimming 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 approximately 2.4 and 3.1 mm long based upon
our data from macerated tissue. Assuming random vessel distribution within the stem, trimming away 1.5 mm of tissue would exclude
50% or more of the narrowest paint-filled vessels. Unfortunately, this trimming and resulting 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 narrowest vessels appear to have been excluded
by the paint and air methods, the actual vessel
length distribution patterns, which would include 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 labrusca and Tetracera based upon measurements of the water volume released by vertically 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 capillarity, 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 vessels (about 12 and 1.7 mm, respectively). Vessels 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 (personal communication) are presently attempting to use cinematographic analysis to measure

vessel lengths in Vitis labrusca. This method,

which requires using a movie camera to photograph serial microscopic sections (Zimmer-

mann and Tomlinson, 1966; Zimmermann,

1971), may be quite accurate but is too laborious to be practical in studies of many stems
with long vessels.
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