United States

Department of

Agriculture Microhistological

Forest Service

Southern Forest

Experiment Station

New Orleans,

Louisiana

Techniques for Food

Habits Analyses

Research Paper

so-1 99

December, 1983 Mark K. Jo.hnson, Helen Wofford and Henry A. Pearson
CONTENTS

INTRODUCTION .......................................................................................... 1

REFERENCE MATERIALS .......................................................................1

PREPARATION OF DIET SAMPLES ......................................................2

DIAGNOSTIC FEATURES ........................................................................ 2

Micro-anatomy of Monocots .....................................................................2

Coastal Gird& and BuIIiform Cells .......................................................2

Long and.Short CeIIs ............................................................................. 2

Stomata .................................................................................................. 3

Trichomes .............................................................................................. 3

Micro-anatomy of Dicots ........................................................................... 3

Trichomes .............................................................................................. 3

Epidermal Cells ..................................................................................... 4

CrystaIs .................................................................................................. 5

Stomata and Companion Ce& ..............................................................5

IDENTIFICATION ...................................................................................... 6

QUANTIFICATION ..................................................................................... 7

Frequency Sampling.................................................................................. 8

Relative Discernabihty and Digestion ......................................................9

WOOD FIBERS, MUSHROOMS AND MAST ..........................................10

Wood Fibers.. ............................................................................................. 10

Mushrooms ................................................................................................ 10

Mast ........................................................................................................... 12

QUALITY CONTROL .................................................................................. 12

LITERATURE CITED................................................................................. 13

APPENDIX 1-Directions for preparation of mounting media.. ................ 15

APPENDIX II-Drawings of plant fragments under

phase-contrast microscopy.. .............................................17

i

Microhistological Techniques for Food Habits Analyses

Mark K. Johnson, Helen Wofford and Henry A. Pearson

INTRODUCTION

The use of micro-anatomical features for identification of plants in diets of herbivores has received wide

application since the early reports of Baumgartner

and Martin (1939), Norris (1943), and Dusi (1949).

However, detailed descriptions of specific techniques

have not been fully reported. Those attempting to

learn how to quantify diets microhistologically have

great difficulty without help from experienced technicians.

The purpose here is to describe specific techniques

used to prepare and quantify herbivore diet samples

for microhistological analyses. It is beyond our scope

to provide a detailed taxonomy based on micro-anatomy. However, the variety of plants described here

represents probable numbers and anatomical variations that may be normally expected in a diet study.

Additional variations of micro-anatomical features

exist in other plants. The reader should refer to Metcalfe (1960) fqr a complete description of plant microanatomy. Here we describe the types of anatomical

structures useful for identifying plant fragments

regardless of the variation in each.

We thank R. M. Hansen, Colorado State University, L. J. Korschgen, Missouri Department of Conservation, J. L. Holeckek, New Mexico State University, and J. E. Moore, University of Florida, for

reviewing early drafts. This project was supported by

the U.S. Forest Service and Mississippi State University under cooperative agreement No. 19-335.

REFERENCE MATERIALS

The plants described in this report are a select

group of those common on longleaf-slash pine-bluestem range of the lower coastal plain in the southeastern United States. Plant names used in this paper follow those provided in the National List of Scientific

Plant Names (USDA, SCS 1982).

Some researchers prepare referente and study

materials by carefully scraping away tissues to

expose the cutinized epidermis on the opposite side

(Korschgen 1977). This method is preferable to ripping tissues in a blender when intact leaves are

needed for taxonomic description. However, for diet

analysis referente materials must simulate the small

fragments found in samples following mastication

and digestion. Therefore, we prepared referente

plants by mixing about 1 g of dry leaves and 20 ml of

liquid household bleach at high speeds in a blender.

Hertwig’s solution can be used to clear pigments

from plant tissues (Cavender and Hansen 1970, Flinders and Hansen 1972, Todd and Hansen 1973).

However, soaking samples in household bleach

accomplishes the same thing with greater ease (Rey

nolds et al. 1978).

Some researchers stained plant specimens after

decolorizing (Dusi 1949, Voth 1968, Field 1972, Fitzgerald 1976). Although staining aids in distinguishing certain features, some do not stain materials

(Stewart 1967, Cavender and Hansen 1970, Hansen

1971, Westoby et al. 1976, Johnson 1979), and we

believe it unnecessary.

We used Hoyer’s solution for making permanent

slides. However, this solution is hygroscopic so that

in warm, humid climates slides become sticky and

must be stored in low humidity environments for

months before they are dry. An alternative plastic

mounting medium can be made with lactic acid,

phenol crystals, and polyvinyl alcohol. The plastic

medium dries within a few days. (Directions for preparing both Hoyer’s solution and the plastic medium

are in Appendix 1).

When permanent slides are not needed, plant fragments can be mounted in a solution of soapy water or

water mixed with a small amount of gum arabic.

Water infiltrates the tissues quickly and slides can be

examined immediately.

Mark K. Johnson is Associate Professor, School of Forestry and Wildlife Management, Louisiana State University, Baton Rouge, LA 70803.

Helen Wofford is Research Assistant, Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003.

Henry A. Pearson is Chief Range Scientist, Southern Forest Experiment Station, USDA Forest Service, 2500 Shreveport Highway, Pine

ville, LA 71360.

1
PREPARATION OF DIET SAMPLES

Diet samples come from esophageal or rumen fistulated animals, stomachs, rumens, intestines or ceca of

dead animals, or fecal droppings. The condition of

fragments in esophageal samples is similar to reference material making sample preparation relatively

easy. Digested fragments are more difficult to identify since the number of anatomical features per fragment is less usually. Fecal materials exposed to the

environment for long periods may be difficult to identify, but decomposition in arid environments may be

so slow that fossil dung more than 10,000 years old

has been used to obtain a record of an animal’s diet

(Hansen 1978).

Whenever stomach or rumen samples are available, gross examination with dissecting equipment at

low magnifications is recommended before grinding

for micro-analysis. Some foods do not have distinctive micro-anatomical characteristics and can only be

identified before digestion. On the other hand the

presente of some plants can be easily detected by

micro-analysis while they may not be detected by

gross examination (Yarrow 1979).

DIAGNOSTIC FEATURES

Leaf fragments in diet samples are identified by

comparison with fragments and drawings made from

referente slides. Drawings can be made by hand or

with the aid of a drawing tube attached to a microscope. This piece of equipment allows non-artists to

accurately outline the micro-anatomy of plant fragments but all structures do not lie on the same plane

so that drawings can not perfectly represent the

appearance of fragments in a microscope (Appendix

II). Descriptions of plant micro-anatomy from Metcalfe (1960) were used to guide our study of those

structures most useful for identification. We generally ignored Metcalfe’s descriptions of relative abundance for certain structures since these characteristics are only apparent on large leaf fragments. Therefore, our descriptions are limited to micro-anatomical

variations of certain structures.

Micro-anatomy of Monocots

The parallel veins of grasses readily separates leaf

tissue into zones, and clearly distinguishes grass leaf

fragments from those of dicots. While identifiable

tissues from sheath, flower, seed, or culm are often

present, leaf blades provide the most useful tissue for

microscopic identification of the grasses (Metcalfe

1960). Minor anatomical differences appear on the

same leaf, among leaves of the same plant and among

2

different plants. However, micro-anatomical characteristics have been used for taxonomic differentiation

of species, genera, and families (Davies 1959, Tateoka

1957, and Metcalfe 1960).

Costal Girder and Bulliform Ce&-The areas

directly over and around the veins are termed costal

zones while areas between the veins are termed intercostal zones (Metcalfe 1960). Schlerenchymous cells

called costal girders surround the veins and are common to grasses from dry habitats (Metcalfe 1960).

Bulliform cells are located intercostally snd have

thinner cell walls than costal girders. They are large,

highly-vaculated, epidermal cells. Their function is

not known but sometimes they are filled with silica

(Esau 1965). Since the arrangement and appearance

of costal girder cells are highly variable, we have not

found costal girder and bulliform cells to be useful

features for identification of grasses.

Long and Short Cells.-There are two other distinct cell types (long and short) in typical epidermis.

Long cells are very narrow and run parallel to the

vascular bundles. They are a major constituent of the

intercostal zone but may also be found over the costal

zone. Cell walls may be sinuous or non-sinuous with

varying degrees of each (fig. 1). Long cells surround

short cells and stomata (Esau 1965). We found that

length and width of long cells provide some clues as

to the identity of a fragment, but the nature of the

cell walls is the primary characteristic we compare

with referente materials when trying to identify leaf

fragments.

Short cells are dispersed among the long cells and

may appear over the costal zones. They are filled with

silica or cork. We have found the shapes and sizes of

silica cells in the costal areas to be particularly diagnostic.

Metcalfe and Chalk (1950) classified about 20 different shapes of silica cells. Three main shapes we

Cell Walls

smooth

wavy

sinuous

Figure l.-Vuriations in cell walls ofgrasses.
have found are dumbbell, non-dumbbell, and dumbbell variations. Non-dumbbell shaped silica cells are

square, cuboid, or irregular cuboid (fig. 2). For dumbbell shaped cells in the costal region, length, width,

and angular characteristics of the middle and ends

aid in identification of species (fig. 3).

For silica cells in the intercostal region, shape and

abundance differ among species. They often occur in

pairs called companions or couples (fig. 4). Comparison of silica cell size is often useful for distinguishing

among species where shapes are similar. However,

when size is used for identification a number of different fragments must be examined to account for variation. Size is most useful for distinguishing between

genera rather than species. For example, the shape of

silica cells found in leaves of Andro@ogon, Schizachyrium and Panicum is bone-like but those in Panicum leaves are generally smaller.

Stomata.-Stomata of grasses occur intercostally.

Openings are surrounded by guard cells that are narrow in the middle and bulbous on the ends. In addition, stomata of grasses are accompanied on their

parallel axes by two subsidiary cells (Metcalfe 1960).

Silicia Cell Variations

square

Q

0

irregular cuboid

000

cuboid

dumbbell

variations of dumbbell shape

Figure Z.-Variations in shapes of silica ce& obserued in grasses.

It is easier to identify grass species whenever both

stomata and silica bodies are present on the same

fragment. The size of stomata is not useful for identification except where they are extremely small or

large.

Z’&homes.-Four types of grass trichomes are

useful for identification. They are macrohairs, microhairs, prickle hairs, and papillae (fig. 5). Macrohairs

are visible with the naked eye or hand lens. Some are

multicellular but the shape of the proximal end is the

most outstanding feature. Of all the hair types,

microhairs are the least diagnostic because in diet

samples the distal cell of bicellular microhairs is often

broken, leaving a blunt end (Metcalfe 1960).

Prickle hairs are very common but there are few

differences in their appearance among species.

Usually located on margins, veins, or between long

cells, they simply aid identification by lending a characteristic appearance to fragments. By contrast, prickle hairs vary in shape, location and distribution on

leaves so that they may be very useful for tsxonomic

characterization of species when complete leaves

rather than small fragments are examined.

Papillae are protrusions of the epidermal surface

which take a variety of shapes. We found that sedges

(Carex spp.) and pineywoods dropseed (Sporobolus

junceus) had numerous papillae present.

Micro-anatomy of Dicots

Z’richomes.-Trichomes provided a primary means

for identifying dicots since they differ more among

species than other epidermal cells, crystals or stoDumbbell Shape Variations

rounded ends notched ends

irregular peanut

cylinder bowtie

Figure 3.-Variations in shapes of dumbbell shaped silica cells

observed in grasses.

3
Cell Pair Variations

silicia cell0companion cell

Figure 4.-Variations in the appearance of silica cell pairs which

occur in grasses.

mata. Metcalfe and Chalk (1950) classified families

into those having glandular or nonglandular trichomes and also according to the numbers of component cells. Trichome anatomy has been used for

generic classification by Rollins (1944), Heintzelman

and Howard (1948), Cowan (1950), and Hummel and

Staesche (1962). While the shape of plant hairs differs

among some taxonomic divisions, there is often considerable uniformity within genera.

We found shape and number of cells making up a

trichome to be the best starting point for identifying

dicot species. We observed 4 basic kinds of trichomes:

ligulate, branched, compound and papillate (fig. 6).

Characteristics such as size of the base, shape of the

apex, texture of the surface and degree of tapering are

useful for identification (fig. 7).

Epidermal Ceh-Unless otherwise indicated, epidermal ce11 as used here refers only to epidermis of

leaves including cells of the upper leaf surface, lower

leaf surface, trichome attachment cells, and specialized cells such as glands. Shapes of epidermal cells

vary among families, genera, species, and parts of the

same plant. Their form is affected by factors such as

light intensity and atmospheric humidity. Thus, the

size, shape, and walls of epidermal cells may vary

4

Trichome Types-Grass

macrohair

prickie hair papillae

-L

Figure 5.-Four basic shapes of trichomes commonly found on

gmss leaves.

from year to year and place to place (Metcalfe and

Chalk 1950). In addition, some plants have multiple

epidermal layers (Esau 1965).

Despite variation, characteristics of epidermal

cells are highly useful for plant fragment identification. Notable features include wall structure, relative

size, and relative thickness. Two distinct shapes are

angular and contour. Angular cells have pointed or

rounded corners, and five, six, or eight straight sides

which give the tissue a honeycomb appearance. By

comparison, the walls of contour cells fit together like

those of a jigsaw puzzle. We found the degree of contour in the walls to be characteristic (fig. 8).

Trichome attachments have different shapes than

other epidermal cells, and there is usually a constar&
number of cells surrounding each attachment (fig. 9).

Glands occur on epidermal tissues of many plants

and are usually smaller than other trichomes (fig. 10).

CrystuZs.-Crystals are formed from secretions of

calcium carbonate, calcium oxalate, starch, or silica.

While leaves of Rubus contain large crystals called

druses, those produced by Loniceru are usually larger

and fewer. Crystals are located in epidermis, vascular

tissue, and stemlike tissues around trichome attachment cells. Types of crystals are druses (starshaped),

raphides (needlelike), various square or rectangular

crystals, and intermediates of druses and squares

(fig. ll).

Stomata and Companion Cells.-Stomata are

usually most abundant on the lower surface of leaves.

However, they may also be located on upper surfaces

or on the epidermis of petioles, stems and flowers.

The companion cells that surround the guard cells are

not generally useful for identification. Exceptions are

those that appear to have comer cells at the point

where two guard cells meet. These tiny triangular

cells are common to Quercus, but they are also found

in most conifers and fems.

Frequency and size of stomata are highly variable

characteristics that depend on environment, geography, and location on the plant. These characteristics

are not useful for identifying small fragments. More

useful for identification is the number of epidermal

cells that surround stomata. Among the plants in our

collections are the pattems outlined by Metcalfe and

Chalk (1950): irregular-celled (four or more cells surround the stomata), three unequal-celled (three cells

surround stomata, one may be distinctly smaller than

the other two), parallel-celled (two cells surround

Trichome Types-Dicot

ligulate one-celled base textured, one-celled base

branched

Figure ô.-Basic kinds of trichomes commonly found on leaves of dicots.
stomata and their common side is parallel to the long

axis of the pore), and cross-celled (two cells surround

the stomata and their common wall is at right angles

to the long axis of the pore)(fig. 12).

IDENTIFICATION

Whether plant fragments are from referente material or diet samples, they are not uniform in size,

shape, or thickness. We mount samples on glass

slides (3” x 1”; cover slips, 20 x 40 mm) in a layer of

medium about 1.0 mm thick. All of the fragments do

not lie in the same plane, and some individual fragments curl so different anatomical structures do not

lie on the same plane of focus. In addition, tissue

thickness causes anatomical structures to be on different planes of focus. Complete view of 8 fragment

requires focusing through tissues until all features

are examined. Composite drawings such as those published here do not represent the true appearance of

fragments under a microscope, and the necessity of

focusing through tissues limits the use of photomicrographs for illustration or as referente material. However, after study, drawings provide excellent reference with reduced effort compared to constant examiG

Trichome Variations

wavy textured

spined

B

/

/5/

-th pitted ridged

bases and tips

blunt point.ed mucronate

Figure 7.-Ba& characteristics of tips and bases of trichomes usually found on leaves of dicots.

6
nation of referente slides which are only needed for

confirming uncertain identifications.

Distinguishing monocots from dicots is simple

because of the pronounced difference in venation.

However, distinguishing among different dicots

requires recognition of cell patterns and anatomical

structures. Often the presente of a distinctive trichome is sufficient evidente for identifying a fragment. However, some trichomes separate from fragments and in these cases accurate quantification

requires association of trichomes with epidermal fragments. For species or parts of leaves without distinctive trichomes, identification depends upon study of

ce11 shape, characteristics of ce11 walls, the form of

stomates, and the presente of crystals. Usually, only

one or two micro-anatomical features are needed for

identification of dicots.

Identification of grasses, requires study of the cell

walls, stomates, and silica cells. In general, fragments

of different grasses appear more similar to each other

than fragments of dicots. When a sample contains

two or more grasses, fragment identification usualIy

requires the presente of at least two distinctive features.

For a large collection of species, it would be difficult to identify plants without large pieces of leaves

so that a complete pattern of structures could be

examined compared to what can be seen on small

fragments. However, practical food habits studies

deal with a limited list of plants from which herbivores usually select a few staples during any season.

By the process of elimination and intense study of

referente plants, small fragments of leaves can be

used to identify the foods selected by a herbivore.

QUANTIFICATION

Anthony and Smith (1974) visually estimated the

contribution of each species to the numbers of fragments observed on microscope slides. Although this

method seems efficient and practica& there is no control over observer bias. Most quantification of plant

fragments identified on a microscope slide has been

performed by counting fragments or by using some

variation of simple counting. Voth (1968) measured

the dimensions of each fragment to calculate an area

Variations in Cell Wall Contour

Q 0 OH

angular

.o

Figure 8.- Variations in the contour of cell walls in leaves of dicots.
Trichome Attachments

8-celled 1 0-celled

Figure 9.-Examples of trichome attachments observed on leaves

of dicots.

for each one. Other researchers have counted the

numbers of fragments and calculated the relative

number for each species (Storr 1961, Ward and Keith

1962, Myers and Vaughan 1964, Bear and Hansen

1966, Sparks 1967, Stewart 1967).

glandular cells in epidermal tissue (surface view)

erect glandular trichome (side view)

Frequency Sampling

Frequency sampling is nothing more than recording the presente or absence of an item in a sampling

unit. The method has been employed for study of wild

plant distributions and abundance (Fracker and

Brischle 1944), and relative abundance of wild animals (Caughley 1977). Sparks and Malechek (1968)

demonstrated that frequency sampling was an accurate alternative to counting each plant fragment when

quantifying botanical compositions on microscope

slides. As a result the procedure has been used for

estimating herbivore diets by a variety of researchers

(Flinders and Hansen 1972, Todd and Hansen 1973,

Hansen et al. 1973, Dearden et al. 1975, Johnson

1979).

The technique is relatively simple. Microscope

slides are made with mixtures of plant fragments

ground in a Wiley Mill over a 1.0 mm mesh sieve so

that all particles average about the same size. A predetermined number of fields are systematically examined at the same magnification, and the presente of

each species is recorded.

Frequency of occurrence is calculated for each species. As long as the amount of ground plant material

on each slide is relatively small (1 - 3 fragments per

field), average relative frequency of occurrence represents average relative abundance of the different species in the mixture. Since bulk densities of leaves

from different plants are about equal, relative abundance provides an estimate of relative dry weight for

Figure lO.-Examples ofglands observed on leaves of dicots.

each food in a herbivore’s diet (Johnson 1982). A

number of published reports demonstrate that, in

general, this technique provides accurate estimates of

herbivore diets whether study ‘materials are stomach

contents or fecal pellets (Hansen et al. 1973, Dearden

et al. 1975).

Under the following assumptions each microscope

field is treated as a sampling unit: (1) microfragments

of plants are randomly distributed on microscope

slides, (2) microfragments from different plant taxa

are the same average size, (3) dry weight bulk densities of different plant taxa are the same. These

assumptions are valid since the distribution, size and

average number of fragments per microscope field is

controlled in slide making and since there are no significant differences in dry weight bulk densities

among the leaf tissues from different plants.

The relationship between particle density and fre

quency of occurrence is based on finite numbers of

plant fragments distributed at random over a microscope slide. A detailed description of the mathematical theory is available in Johnson (1982).

The standard form of the realtionship between frequency and density is

F=l-e -d, (1)

where F is relative frequency, e is the natural logarithm and d is the mean particle density determined

8
by the number of fragments (n) and number of microscope fields examined (k) so that

d=“lk (2)

If fragments from m different plant species are

randomly distributed in the microscope fields, the

particle density (average number of fragments per

field) of each is independent from the others. Relative

particle densities (ri), which are estimates for the relative dry weights of each plant in the diet sample, can

be calculated:

r.=-’ di

1 m

C di

i=l (3)

where i=l, . . . , m and di’s are the particle densities

for each species. For example, we may simultaneously count the presente or absence of several different taxa in a microscope analysis. Since proper mixing and spreading of microfragments assures randomness and independence of particle distributions,

separate estimates for each taxon’s particle density

may be obtained and relative particle densities calculated.

In practical applications one seeks to estimate the

average particle density, which is unknown and not

Crystal Shapes

druse square

rectangular raphide

Figure ll.-Basic examples of crystals observed in leaves of dicots.

easily estimated. This is done by estimating frequency of occurrence, which is also unknown but easily estimated by recording particle presente or

absence, and then estimating density from frequency

using equation (1). Estimates for frequency of occurrence are subject to sampling variability.

Relative precision of the estimate for density is

also dependent on average frequency (Curtis and

McIntosh 1950). As average frequency increases,

relative precision increases up to an optimum. Precision decreases for larger particle densities because

most or all of the fields contain at least one identifiable plant fragment. This result imposes constraints

on the maximum particle density which is allowable

on microscope slides. Conversley, low particle densities of rare taxa, which result when microscope,slides

are made to avoid high particle densities for abundant taxa, require intensive sampling to yield precise

estimates of these particle densities. We obtain accurate results in quantifying mixtures when slides are

made so that microscope fields contain from 3-5 identifiable fragments.

Relative Discernability and Digestion

Since all fragments can not be identified, there

have been attempts to account for differences among

species as to proportions of fragments that can be

Stomatal Arrangement in Leaf Epidermis

/

stomatal pore

irregular-celled cross-celled

parallel-celled three unequal-celled

Figure 12.-Basic arrangements of stomata and surrounding qpidermal cells observed in leaves of dicots.
identified, and to account for the affects of digestion

on the discernability of fragments. In addition, for

the same weight of leaf material some plants fragment during mastication or sample preparation into

more than twice the number of pieces as other plants

(Johnson et al. 1983).

Differential digestibility has been widely discussed

as introducing bias into diet estimates when fecal

samples are used. Some researchers have gone to

great lengths to adjust data to account for effects of.

digestion (Voth and Black 1973) even though reports

were not available to suggest that digestion significantly biased analyses. There are no known microorganisms that possess cutin degrading enzymes

(Frey-Wyssling and Muhlentahler 1959). The equivalence factors reported by Voth and Black (1973) were

related to digestion of tissues underlying the cuticle

rather than reduction of the cuticle. Plant fragment

identification is based on micro-anatomical characteristics of an indigestible cuticle and cells underlying

the cuticle that escape digestiofl. Leaves are made of

epidermis, mesophyll, and vascular bundles. Epidermis is covered by indigestible cuticle which inhibits

digestion of mesophyll by blocking passage of

microbes into the leaves (Harbers et al. 1981). However, once passage is gained by maceration, mesophyll is readily degraded while epidermis and cells

surrounding vascular bundles are degraded next

(Aiken 1979). Generally, lignified vascular tissues

resist digestion. Theoretically, identification is made

only from cutin which retains the impression of epidermal tissues. But undigested groups of cells

enhance the visibility of cutinized fragments because

the increased thickness causes greater refraction of

light. Microhistological identification is easier when

both the epidermis and underlying tissues are present.

We recorded numbers of fragments and proportions identifiable for a variety of undigested and digested plants (Johnson et al. 1983). For 47 species

tested, digestion increased discernability for 3 and

decreased discernability for 9 by more than 10 percent while the other 35 plants were little affected.

Digestion improved discernability for plants having highly suberized, pigmented cell walls, and few

trichomes by clearing pigments from tissues making

cells easier to see. Digestion decreased discernability

for plants having thin cell walls and few stomates or

trichomes. Easily identified plant fragments generally had an abundance of features that were easily

recognized. Regardless of these affects, we found that

they were not dramatic enough to have very much

influente on estimates of botanical composition

(Johnson et al. 1983). This work helped to explain

why estimates of botanical compositions for feeds

and feces have usually been significantly similar

(Todd and Hansen 1973, Hansen et al. 1973, Johnson

and Pearson 1981.)

10

WOOD FIBERS, MUSHROOMS AND MAST

Wood Fibers

Digesta from animals that browse or gnaw wood

may contain fragments that cannot be identified by

gross. analysis. However, wood fibers can be identified microscopically and often occur in slides made

from fecal materials. Identification is performed by

comparison with referente materials in the same

manner as leaf fragments are identified.

Preparation of referente fibers or wood fragments

taken from food habits samples is performed by

maceration in a 50:50 mixture of glacial acetic and

30% hydrogen peroxide. Wood fragments should be

refluxed in the solution for 1 to 2 hours. The specific

time needed should be established through practice;

some woodwill take longer than others. Variations on

this procedure are also available (Panshin and

deZeeuw 1980). Materials should be rinsed thoroughly after maceration. Microscope slides can be

made and examinations performed as they are for

other plant fragments.

Diagnostic features are size, shane, and pittinn of

fibers.l”racheids are the only cells useful foi ideniification of softwoods. Various features of vessel elements and tracheids as well as the presente or

absence of tracheids are diagnostic for hardwoods

(Carpenter and Leney 1952, Panshin and deZeeuw

1980).

We know of no authors employing these procedures in food habits studies. We have observed the

fibers in fecal pellets of a variety of herbivores but

have never attempted quantifying them.

Mushrooms

Herbivores, as well as other animals, eat mushrooms, and proportions in the diet may be significsnt.

Many animals consume mushrooms that are poisonous to humans. Mushrooms tend to’concentrate phosphorous and are rich in protein (Miller and Halls

1969, Fogel and Trappe 1978).Both of these nutrients

may be lacking in the vegetation of some regions.

The presente of mushrooms in food habits samples

complicates quantification. The point analysis

method useful for quantifying relative dry weights of

macroscopic fragments in stomachs has two requirements; bulk densities of materials should not differ

greatly and the form of different items must be similar (Chamrad and Box 1964). For example, the dimensionless quality of point methods is destroyed if large

flat pieces of leaves are analyzed in mixtures with

small seeds which have a much higher bulk density

but a lower probability of occurrence at a point.

These problems can be overcome by hand separation of mushrooms for separate weighing or volume-
tric study, or by microscopic analyses of ground

samples. Regression equations can be developed to

relate occurrences of mycelia in stomachs to occurrences of plant fragments.

Digestibility of fungal mycelia is nearly complete

except where large amounts are ingested ín a single

meal. We have observed intact mycelia in feces in

these cases. However, when mushrooms are eaten in

smaller amounts, only spores are present in fecal

material. Although indigestible (Fogel and Trappe

1978), spores may not be detected in fecal samples

during routine analysis because they are generally

tiny. Some can be detected at 100 magnifications but

others require 500 to 1000 magnifications before they

can be seen.

Proportions of fecal samp,les made up by spores

might be large for small, mycophagous mammals

(Tevis 1952), but insignificant for other wildlife; while

the proportion of the diet they represent can be more

than 30% (unpublished data). Therefore, accurate

diet quantification requires developing correlations

between amounts eaten and number of spores.

To demonstrate this procedure we separately

ground dry mushrooms (Amantiu sp.) and plairt

leaves in a Wiley mill through a 20 mesh sieve. Microscope slides were prepared with 14 different amounts

of pure plant fragments ranging from 6 - 60 mg. Ten

slides were made for each different weight so that 140

slides were used. Slides were also made with 8 different amounts of pure ground mushrooms ranging from

1 - 40 mg and 10 replications were made so that 80

slides were used. We then counted the number of

plant fragments (125 x ) (regardless of whether they

were discernable fragments) (fig. 13) and the number

v 6 7 S 9 10 20 3 0 4 0 50 60

DRY WEIGHT OF LEAF FRAGMENTS

of spores (500 x ) in each microscope field (fig. 14).

Twenty fields were examined for each slide, averages

were calculated and grand averages were calculated

among the slides for each different weight of material.

Five different test mixtures were made by mixing

different proportions of ground mushroom (5 - 30%)

with different proportions of leaves from Schizachyr

ium tenerum, Eragrostis spectabilis, Desmodium ciliare and Lonicera japonica. Five microscope slides

were made for each test mixture. Quantification was

performed in two steps. First, the slides were examined separately at 125 x for plant leaf fragments and

at 500 x for spores; 20 fields were examined in each

case. Al1 plant fragments or spores occurring in each

field were counted and an average particle density

Y

0 I 2 3 4 1 0 2 0 30 4 0

DRY WEIGHT OF MUSHROOM

Figure 13.-Relationship between number of leaf fragments per

microscope field (125 X) to dry weight (g x 10P3) of leaf

fragments used to prepare slides having 22x40 mm

cover slips.

Figure 14.-Relationship. between number of Amanita sp. spores

per microscope field (500x) to dry weight (g ~10~~) of

dry mushroom used to prepare slides having 22x40

mm cover slips.

l l
Table l.-Comparison of known proportions (%) ofplant leaves and mushroom in five test mixtures with estimates obtained from particle

densities of leaf fragments and spores

Species

in test

mixture Test Estimate Test Estimate Test Estimate Test Estimate Test Estimate

Schizachyrium tenerum 50 35 40 35 30 18 40 . 25 20 10

Eragrostis spectabilis 10 10 20 l l 15 5 10 6 10 5

Desmodium ciliare 25 34 20 37 15 14 10 24 10 3

Lonicera japonica 10 10 10 4 20 49 10 17 10 4

Amanita sp. 5 l l 10 13 20 14 30 28 60 78

was calculated by dividing the total number of plant

leaf fragments or spores by the number of fields

examined. The dry weight represented by each particle density was estimated by using figure 13 for plant

leaves and figure 14 for mushrooms. Relative dry

weights of mushroom and plant leaves in the mixtures were estimated by calculating the relative contribution of each from estimates of their dry weights

on slides.

The second step in quantification was performed

by calculating relative particle densities of leaves

according to the procedures described earlier (Sparks

and Malechek 1968), and multiplying by the proportion of leaf fragments in each mixture.

Using this procedure known and estimated proportions for mushrooms in the simulated diet were similar except when the proportions of mushrooms were

50% (table 1). At this leve1 the number of spores per

field became too numerous for accurate estimates.

Based on earlier analyses the number of spores per

field from diet sample slides should remain below 20.

The accuracy of estimating plant leaf compositions

using the same slides is not affected.

Data for leaf particle densities should apply to

other situations while data for mushrooms applies

only to the species we used. This procedure should be

repeated for each mushroom species and each phenological stage.

Mast

Most wildlife husk nuts before eating them, and

even though deer often swallow acorns whole, the

shells may be regurgitated and spit out. In fragmented form, acorn hulls are difficult to identify and

mast (endosperm) is amorphous. Since mast is highly

digestible, it is rarely found in fecal material. We have

observed mast in fecal pellets of deer and goats that

feasted on abundant supplies. In these cases the bulk

of material ingested was probably too much for complete digestion.

Regardless of these problems, there are ways to

identify the tree that produced the mast found in

stomach, rumen or fecal samples. First, all acorns

have some trichomes on the hulls. These are brittle,

become detached and remain in the digestive tract

12

passing through to the feces. Microhistological examination and comparison with referente material

allows identification of species when trichomes are

not similar. Quantification can be performed by learning to estimate percentages of mast in mixtures from

frequency counts of trichomes on slides (table 2).

Differences in shapes of starch cells in the mast

from different acorns have been detected (Korschgen

1981). Referente slides can be made from scrapings of

the endosperm and mast in stomachs can be identified from water mounts of the semi-digested starch.

This method has not been widely investigated but

may be useful for food habits work with a variety of

granivores.

QUALITY CONTROL

A technician’s behavior and performance can

change from day to day because identification is basically a value judgment. This human source of error

cannot be overcome through the employ of correction

equations or equivalence factors regardless of how

they are calculated.

Most plants can be identified through some of the

fragrnents. However, we found it necessary to ignore

the presente of any trichomes that were not attached

to fragments to avoid over estimation of such species’

contribution to diet samples.

Different strategies may be needed for each situation. For example, every fragment of a grass could be

identified if it was mixed with dicots. Test mixtures

of 2 to 6 different plants are adequate to periodically

check a technicians accuracy. Test slides are also valuable for building technician confidente during training periods. There are so many ways to combine

plants in mixtures that it is impractical to produce

enough tests to simulate every possible combination

of foods in actual diet samples. Tests sometimes have

to be made after the plants in a mixture are identified,

but before they are quantified. Sources of error and

uniqueness of mixtures make microhistological techniques both an art and a science (Johnson and Pearson 1981), but a regimented sampling procedure is

necessary to provide consistent results.
LITERATURE CITED

Akin, D. E. 1979. Microscopic evahration of forage

digestion by rumen microorganisms-a review.

J. Anim. Sci. 48:701- 710.

Anthony, R. B., and N: S. Smith. 1974. Comparison

of rumen and fecal analysis to describe deer diets.

J. Wildl. Manage. 38(3):535 - 540.

Baumgartner, L. L., and A. C. Martin. 1939. Plant

histology as an aid in squirrel food habits studies.

J. Wildl. Manage. 3:266 - 268.

Bear, G. D., and R. M. Hansen. 1966. Food habits,

growth and reproduction of white tailed jackrabbits in Colorado. Colorado State Univ. Agri. Exp.

Sta. Tech. Bull. No. 90.59 p.

Carpenter, C. H., and L. Leney. 1952. 382 photomicrographs of 91 paper making fibers. Dept. Wood

Tech. State Univ. College For. Syracuse, New

York. 152 p.

Caughley, G. 1977. Analysis of vertebrate populations. John Wiley and Sons, New York. 234 p.

Cavender, B. R., and R. M. Hansen. 1970. The microscopie method used for herbivore diet estimates and

botanical analysis of litter and mulch at the Pawnee Site. IBP Tech. Rep. No. 18. Nat. Res. Ecol.

Lab., Colorado State University. Fort Cohins, CO.

6 P.

Chamrad, A. D., and T. W. Box. 1964. Food habits of

white-tailed deer in South Texas. J. Range Mange.

21:158 - 164.

Cowan, J. M. 1950. The Rhododendron leaf: a study

of the epidermal appendages. Edinburgh, Oliver

and Boyd. London. 115 p.

Curtis, J. T., and R. P. McIntosh. 1950. The interrelations of certain analytic and synathetic phytosociological characteristics. Ecol. 31:434 - 455.

Davies, 1. 1959. The use of epidermai characteristics

for the identification of grasses in the leafy stage.

J. British Grassland Society. 14:7 - 16.

Dearden, B. L., R. E. Pegau, and R. M. Hansen. 1975.

Precision of microhistological estimates of rumiTable 2.-Compatison of known and estimated proportions (%) of mast in fue mixtures with plant leaf fragments. Mast was quantified by

counting acorn trichomes

Species

in test

mixture Test

Quercus rubra (acoms) 5 0

Quercus rubm (leaves) . . . .

Rubus sp. . . . .

Lonicera japonica . . . .

Gelsemium sempervirens . . . .

Smilax mtundifolia . . . .

Toxicodendron radicans 50

Estimate Test

54 25

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . 25

4 6 50

Estimate

19

. . . .

. . . .

. . . .

. . . .

34

47

Test

25

25

25

. . . .

2 5

. . . .

. . . .

Estimate

21

22

29

. . . .

2 6

. . . .

. . . .

Test

40

. . . .

. . . .

25

15

20

. . . .

Estimate

40

. . . .

. . . .

27

16

17

. . . .

Test Estimate

10 20

. . . . . . . .

40 39

10 17

4 0 2 4

. . . . . . . .

. . . . . . . .

nant food habits. J. Wildl. Manage. 39:402 - 407.

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and Sons, New York. 761 p.

Field, R. 1972. The food habits of wild ungulates in

Uganda by analyses of stomach contents. E. Afr.

Wildl. J. lo:17 - 42.

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Wellington, New Zealand, in relation to food plant

availability. N. Z. J. Zool. 3:339 - 419.

Flinders, J. T., and R. M. Hansen. 1972. Diets and

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Univ., Fort Collins, CO. 29 p.

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Hansen, R. M. 1971. Drawings of tissues of plants

found in herbivore diets and in litter of grasslands.

U. S. IBP Grasslands Biome Tech. Rep. No. 70.

Natural Resources Ecology Colorado State University Lab., Fort Collins, Col. 69 p.

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13
The comparative morphology of the Icacinaceae.

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14
15
Directions for Preparing Hoyer’s Soluti’on

Chloral hydrate crystals 200 g

Glycerine 2oml

Gum arabic (photopurified) 3og

Tap water 5oml

Combine chloral hydrate and glycerine. Add gum arabic and water. Place

container in a hot water bath and stir until ingredients are combined.

Directions for Preparing Plastic Mounting Medium

Phenol crystals 400 g

Lactic acid / 440 n-ll

Polyvinyl alcohol beads 150g

Distilled water 1120 ml

Add phenol crystals to lactic acid and stir until dissolved. Work should be

performed in a ventilated hood. Mix polyvinyl alcohol beads with water in a

hot water bath. Combine the two solutions and stir while keeping the mixture

warm in a hot water bath.
1 7
Grasses and Grasslikes

b

,

Purple lovegrass (Eragros tis spectabilis) (a) and tridens (Ttidens

spp.) (b)-Stomata are small

and have rounded domes. Fragments may have prickles but

are without other types of trichomes.

Bearded skeletongrass (Gymnopogon ambiguus) (a) and switchgrass (Panicum virgatum) (b)-

Silica bodies are dumbbellshaped but compared to

bearded skeletongrass, switchgrass stomata are larger and

adjoining ce11 walls are tightly

sinuous or scalloped.

Sedges (Carex spp.)-Shiny, pappillae are common. Cells are

square or rectangular with

wavy walls.

Paspalums (Paspalum spp.)-Silica bodies resemble butterflies.

Some have cuboid companion

cells.
Cutover muhly

expansa)-Stomata are small

with peaked domes. Fragments

may have many short trichomes. Costal girders are

square.

Green silkyscale (Anthaenantia

villosa)-Stomates are mostly

rounded but some are slightly

peaked. Most silica bodies look

like peanuts.

Panic grasses (Punicum spp.)-

Relatively long bone-shaped silica bodies resemble 8’s.
Threeawns (Aristida spp.)-Sihca Pineywoods dropseed (Sporobolus

bodies are dumbbell-shaped junceus)-Papillate protrusions

with rounded ends. Stomata of epidermal cells are similar to

have peaked domes and look those of sedges. Vein silica bodnearly triangular. Cell walls in ies and companion ce& form

the intercostal area are deeply couples. Stomates are small

sinuous. and round.

Q

b

Common carpetgrass (Axonopus

affinis) (a) and yellow indiangrass (b) (Sorghastrum nutans)

-Dumbbell-shaped silica bodies are deeply notched. Carpetgrass stomates are triangular

while those of indiangrass are

oval-shaped.

20
Bluestems (Andropogon spp.,

Schizachytium spp.)-Stomata

are triangular. Silica bodies are

dumbbell-shaped, with notched

ends.

Spike uniola (Chasmanthium

laxum)-Bulliform cells may

contain crystals. Silica bodies

are short, small dumbbells that

may look like bow ties.

,

21 ’
Legumes

Partridgepea fuscicuhtu)

-Unicellular str,ap-shaped trichomes are internally segmented. Square crystals are

most abundant over veins.

22,
Pigeonwings (Clitoria mariana)-

Fragments are without trichomes and numerous square

crystals cover the veins.

i

Littleleaf tickclover Desmodium

ciliare)-Three types of trichomes can be observe&, one

has a mucronate tip, two have

hooked tips. All trichome bases

are separate, single cells.

Square crystals cover the veins. 2 3
Virginia tephrósia (Tephrosia virginiana)-Trichomes are stiff

and tapered with mucronate

tips, and have a 1-celled, round

base. Square and rectangukr

crystals are abundant.

24

Lespedezas (Lespedeza spp.)-Trichomes are flexible with

mucronate tips. Surfaces of trichomes are often ciliate in L.

virginica; sometimes ciliate in

L. hirta.
Downy milkpea (Galuc tia volubilis)-Trichomes are stiff with

a mucronate tip and 1-celled

base. Intercostal cells are puzzle-like. Square crystals are

mostly over the veins.

Pencilflower (Stylosanthes bifloru)

-Trichomes appear wilted and

have a 1-celled base. Square

crystals are very abundant.

Butterfly pea (Centrosema uirginiunum)-This plant resembles

Desmodium and Guluctiu. Two

types of trichomes are present;

one is large with mucronate

tips, the other is smaller with

hooked tips. Square and rectangular crystals are present. 2 5
Composites

Hymenopapus (Hymenopappus

artemisiifolius)-Flattened,

ribbon-like trichomes look like

spaghetti. No crystals appear

in tissue. Cells walls are very

thin so that tissues are difficult

to see.

Goldenrod (S&dago rugosa)-

Ligulate, segmented trichomes

have thick walls. Flared bases

are usually broken off. Intercostal cells are angular.

26

_ ..__ .__. --_.----_l_^^__lll____--l--l__----.-..
Thickleaf coreopsis (Coreopsis

lanceolata)-Segmented trichomes are ligulate. Small

druses have slightly lobed cells.

Grassleaf goldaster (Heterotheca

graminifolia)-Segmented trichomes are ligulate. Very small

druses occur intercostally with

slightly lobed cells.

2 7
Eupatoriums (Eupatorium spp.)-

Ligulate, segmented trichomes

and silica flowers are present.

Intercostal cells are angular

with wavy walls.

28

Hender rosinweed (Silphium graci¿e)-Trichomes and tissues

area similar to those of swamp

sunflower.
Swampsunflower (Helianthus

angustifoZius)-Large, segmented trichomes have thick

bumpy surfaces. Distinct

attachment cells in tissue are

made up of large thin-w&d

cells and druses are present.

Blackeyed susan (Rudbeckia hirta)

-Large, ligulate, segmented

trichomes are present.
Plantainleaf coneflower (Rudbeckia grandiflora)-Medium,

ligulate, segmented trichomes

are present. Small raphides are

very abundant.

Daisy fleabane (Erigeron strigosus)-Segmented trichomes

appear jointed. Leaf cells are

usually puzzle-like while stem

cells are rectangular and parallel.

30
Savoryleaf aster (Aster linariifolius)-Short, fat segmented trichornes, and some nipple-like

pappillae are present. Intercostal cella are angular and occur

in layers so that cell walls overlap each other.

Elephantsfoot (Elephantopus

tomentosus)-Long, tapered,

thin trichomes have 2-celled

bases. Puzzle-like intercostal

cells contain small druses.

3 1
Miscellaneous Forbs

Flowering spurge (Euphoria corollata)-There are two types of

trichomes; one is branched and

similar to dogwood trichomes.

Trichome attachments are surrounded by mky cells.

32

Croton (Croton spp.)-Compound,

stellate trichomes are often

attached by a common stalk.

Angular cells have very thin

walls so that tissues are hard to

see.
Poor-joe (Diodia teres)-Few trichomes occur on leaf margins.

Each intercostal cell contains a

nipple-like papilla. Raphides

are present.

Bracken fern (Pteridium aquilinum)-This distinctive tissue

is made of deeply lobed cells.

Stomata corners have triangular cells.

33
Browse

Southern waxmyrtle (Myrica ceriferal-Trichomes are not abundant and usually occur only on

leaf margins. Druses are common.

Flowering dogwood (Cornus flor

ida)-Trichomes branch to fox-m

a ‘t’ and attachment ce& are

mostly 8- sided. Druses are

common.
Poison ivy (Toxicodendron radicans)-Ligulate and glandular

trichome bases are usually 6-

sided. Druses occur over veins

and square crystals occur

between the veins.

Carolina jessamine (Gelsemium

sempervirens)-There are few

trichomes so that tissues are

essentially glabrous. Crystals

are small and shaped like

prisms.

Rattanvine (Berchemia scandens)

-There are no trichomes.

Druses occur intercostally and

square crystals are found over

the veins. Woody tissue has

small round cells and contains

both types of crystals.

3 5
Sumac (Rhus copallina)-Ligulate,

internally segmented trichomes

have swollen bases that are

usually 6-sided. Cells fit

together to resemble alligator

skin.

Japanese honeysuckle (Loniceru

juponica)-Ligulate, hollow trichomes have 6-sided attachment cells. Large crystals are

not abundant.

36
Oaks (Quercus spp.)-All snecies

have compouñd stellate - hairs

that vary in size. Small druses

are usually abundant. Some

species have a few square crystals.

Ameritan beautyberry (Callicarpa

americana)-Some trichomes

are branched or compound but

most are stellate.

3 7
Wild grape Wild grape leaves (Vitis spp.)-

Trichomes are ribbon-like.

Square crystals and druses are

usually along the veins, and

large raphides are present intercostally.

Dewberries (Rubus spp.)-

Medium to small druses occur

in tissues with angular cells.

Trichomes are ligulate.

Pine (Pinus spp.)-Large stoma

are deployed in rows with paralle1 intercostal cells. There are

triangular cells in stoma corners.

38
Yaupon (Ilex vomitoria)-Short,

ligulate trichomes are present

on leaf margins, but not abundant. Medium and small druses

are present.

Greenbriars (Smilax spp.)-S.

glauca has small pappilae while

S. pumila has long, ligulate trichomes with Z-celled bases.

Raphides are present in both

species.

39
Blueberries (Vuccinium spp.)-

Fragments are without trichomes. Druses are present

intercostally with square crystsls over the veins.

4 0
Johnson, Mark K.; Wofford, Helen; Pearson, Henry A. Microhistological techniques for food habits analyses. Res. Pap.

SO-199. New Orleans, LA: U.S. Department of Agriculture,

Forest Service, Southern Forest Experiment Station; 1983.

40 p.

Techniques used to prepare and quantify herbivore diet

samples for microhistological analyses are described. Plant

fragments are illustrated for more than 50 selected plants

common on longleaf-slash pine-bluestem range in the southeastern United States.

Keywords: Herbivore diets, plant fragments, micro-anatomy, longleaf-slash pine-bluestem range.

* U.S. GOVERNMENT PRINTING OFFICE: 1984-769-089:8