Ewers Fisher 1989

8/16/2019 Ewers Fisher 1989

1/13

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.

Stable URL: http://www.jstor.org/stable/2444412

Accessed: 08-04-2016 00:13 UTC

R F R N S

Linked references are available on JSTOR for this article:http://www.jstor.org/stable/2444412?seq=1&cid=pdf-reference#references_tab_contents

You may need to log in to JSTOR to access the linked references.

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at

http://about.jstor.org/terms

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted

digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about

JSTOR, please contact [email protected].

Botanical Society of America, Inc. is collaborating with JSTOR to digitize, preserve and extend access toAmerican Journal of Botany

This content downloaded from 132.248.193.60 on Fri, 08 Apr 2016 00:13:47 UTCAll use subject to http://about.jstor.org/terms


2/13

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



3/13

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



4/13

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



5/13

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



6/13

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.



7/13

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.



8/13

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



9/13


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-



10/13


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



11/13


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



12/13


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.

LITERATURE CITED

ADLER, A. 1892. Untersuchungen uber die Langenaus-

dehnung der Gefassraume. Inaug. Diss., Jena.

BAAS, P. 1973. The wood anatomical range in Ilex (Aq-

uifoliaceae) and its ecological and phylogenetic sig-

nificance. Blumea 21: 193-258.

, AND S. CARLQUIST. 1985. A comparison of the

ecological wood anatomy of the floras of southern

California and Israel. Int. Assoc. Wood Anat. Bull.

n.s. 6: 349-353.

BAILEY, I. W., AND W. W. TUPPER. 1918. Size variation

in tracheary cells: I. A comparison between the sec-

ondary xylems of vascular cryptogams, gymnosperms,

and angiosperms. Amer. Acad. Arts Sci. Proc. 54:

149-204.

BENNETT, J. P., F. G. ANDERSSEN, AND Y. MILAD. 1927.

Methods of obtaining tracheal sap from woody plants.

New Phytol. 26: 316-323.

CALKIN, H. W., A. C. GIBSON, AND P. S. NOBEL. 1986.

Biophysical model of xylem conductance in tracheids

of the fern Pteris vittata. J. Exp. Bot. 37: 1054-1064.

CARLQUIST, S. 1975. Ecological stategies of xylem evo-

lution. University of California Press, Berkeley.

1977. Ecological factors in wood evolution: a

floristic approach. Amer. J. Bot. 64: 887-896.

, AND D. A. HOEKMAN. 1985. Ecological wood

anatomy of the woody southern California flora. Int.

Assoc. Wood Anat. Bull. n.s. 6: 319-347.

EWART, A. J. 1906. The ascent of water in trees. I. Philos.

Trans. B 198: 41-85.

EwERs, F. W. 1985. Xylem structure and water conduc-

tion in conifer trees, dicot trees, and lianas. Int. Assoc.

Wood Anat. Bull. n.s. 6: 309-317.

, AND P. CRUIZIAT. In press. Water transport and

storage in trees. In T. M. Hinckley and J. P. Lassoie

[eds.], Techniques and approaches in forest tree eco-

physiology. CRC Press, Boca Raton, FL.

FISHER, J. B. 1970. Xylem derived from the intercalary

meristem of Cyperus alternifolius. Bull. Torrey Bot.

Club 97: 58-66.

GASSON, P. 1987. Interpretation and choice of vessel

characters in the IAWA standard list. Int. Assoc. Wood

Anat. Bull. n.s. 8: 233-235.

GIBSON, A. C., H. W. CALKIN, AND P. S. NOBEL. 1985.

Hydraulic conductance and xylem structure in tra-

cheid-bearing plants. Int. Assoc. Wood Anat. Bull.

n.s. 6: 293-302.

GRAAFF, N. A. VAN DER, AND P. BAAs. 1974. Wood

anatomical variation in relation to latitude and alti-

tude. Blumea 22: 101-121.

GREENIDGE, K. N. H. 1952. An approach to the study

of vessel length in hardwood species. Amer. J. Bot.

39: 570-574.

HANDLEY, W. R. C. 1936. Some observations on the

problem of vessel length determination in woody di-

cotyledons. New Phytol. 35: 456-471.



13/13

656 AMERCANJOURNAL OF BOTANY Vo. 76

NEWBANKS, D., A. BOSCH, AND M. H. ZIMMERMANN. 1983.

Evidence for xylem dysfunction by embolization in

Dutch elm disease. Phytopathology 73: 1060-1063.

OEVER, L. VAN DEN, P. BAAS, AND M. ZANDEE. 1981.

Comparative wood anatomy of Symplocos and lati-

tude and altitude of the provenance. Int. Assoc. Wood

Anat. Bull. n.s. 2: 3-24.

PIMENTEL, R. A., AND J. D. SMITH. 1986. Biostat I: a

univariat statistical toolbox. Sigma Soft, Placentia,

CA.

RURY, P. M. 1985. Systematic and ecological wood anat-

omy of the Erythroxylaceae. Int. Assoc. Wood Anat.

Bull. n.s. 6: 365-397.

SALLEO, S., M. A. Lo GULLO, AND L. SIRACUSANO. 1984.

Distribution of vessel ends in stems of some diffuse-

and ring-porous trees: the nodal regions as 'safety zones'

of the water conducting system. Ann. Bot. 54: 543-

552.

SCHOLANDER, P. F. 1958. The rise of sap in lianas. In K.

V. Thimann [ed.], The physiology of forest trees, 3-

17. Ronald Press, New York.

SIAU, J. F. 1984. Transport processes in wood. Springer-

Verlag, Berlin.

SKENE, D. S., AND V. BALODIS. 1968. A study of vessel

length in Eucalyptus obliqua L Herit. J. Exp. Bot. 19:

825-830.

SPERRY, J. S., N. M. HOLBROOK, M. H. ZIMMERMANN, AND

M. T. TYREE. 1987. Spring filling of xylem vessels

in wild grapevine. P1. Physiol. 83: 414-417.

STEEL, R. G. D., AND J. H. TORRIE. 1980. Principles and

procedures of statistics. 2d ed. McGraw-Hill, New

York.

ZIMMERMANN, M. H. 1971. Dicotyledonous wood struc-

ture made apparent by sequential sections. Film E

1735 (Film data and summary available as a reprint).

Inst. wiss. Film, Nonnenstieg 72, 34 Gottingen, West

Germany.

1978. Hydraulic architecture of some diffuse-

porous trees. Canad. J. Bot. 56: 2286-2295.

1983. Xylem structure and the ascent of sap.

Springer-Verlag, Berlin.

, AND A. A. JEJE. 198 1. Vessel-length distribution

in stems of some American woody plants. Canad. J.

Bot. 59: 1882-1892.

,AND P. B. TOMLINSON. 1966. Analysis of complex

vascular systems in plants: optical shuttle method.

Science 152: 72-73.

Date post:	05-Jul-2018
Category:	Documents
Upload:	gilberto-aleman-sancheschulz
View:	216 times
Download:	0 times

Ewers Fisher 1989

Documents