Developing the Tools Necessary to Systematically Quantify the
Vegetative Composition of the Andean Bear Diet
(Tremarctos ornatus)
Tara Ball
Ecology & Conservation Biology
University of Idaho
Supervising Faculty:
Dr. Janet Rachlow (Wildlife Resources) and Dave Tank (Forestry Resources)
University of Idaho
Bruce Davitt (Wildlife Resources)
Washington State University
Rodrigo Cisneros, Nixon Cumbicus, and Jorge Armijos
Universidad Tecnica Particular de Loja
May 2011
Abstract
The páramo ecosystems of Central and South America have recently become an area of
global conservation concern. These ecosystems have become subject to a range of threats
including increased human activity and climate change. The Andean bear (Tremarctos
ornatus) has been identified as a potential flagship species for the overall protection of
these ecosystems, however, more baseline ecological information, such as diet
composition, is needed in order to develop realistic conservation management plans. This
study will build upon existing knowledge of the Andean bear diet in order to develop the
tools necessary to systematically quantify vegetative composition. Specifically, a
reference collection of plants known—or—suspected to be part of the Andean bear will
be produced utilizing microhistological techniques, DNA extraction, and dietary fecal
analysis.
Background and Introduction
Dietary analysis is one of the most important tools managers use today to develop
conservation plans for species of interest. Determining the diet of a species allows
managers to understand the resource and habitat requirements needed for its survival.
Reproductive rates, for example, tend to rely heavily on nutritional status, which in turn,
can be determined by available food resources (Hewitt and Robbins 1996). Conservation
managers thus use food resources as a parameter for determining habitat requirements,
carrying capacity estimates, and future conservation plans (Hewitt and Robbins 1996).
There are several different techniques utilized for dietary analysis, but fecal
analysis tends to be highly favored. Holechek et al., (1982) list multiple advantages of
fecal analysis in a review of Botanical Composition Determination of Range Herbivore
Diets. This review includes three of the most commonly notified advantages of fecal
analysis: the non-interference with study species, the unlimited sampling, and the
minimal requirement of equipment (Holechek et al. 1982).
In this study, we focus on the threatened páramo ecosystems of Central and South
America. A potential flagship species has been identified for the protection of such
ecosystems, however, more baseline ecological information, such as diet composition, is
needed in order to develop realistic management plans for conservation.
The Conservation Issue
The páramo ecosystems of Central and South America are discontinuous
neotropical ecosystems composed of high-altitude, treeless habitat that spans the upper
region of the northern Andes mountains. These ecosystems host a range of unique plant
and wildlife taxa and are home to the Quechua people and other indigenous groups.
Noteworthy for their high levels of biodiversity, uniquely adapted endemic communities,
and key role in the hydrology of the South American continent (Buytaert et al. 2006), the
páramo ecosystems have recently become an area of global conservation concern. The
páramo ecosystems in Ecuador specifically, have become subject to multiple threats
including road development, advancement of agriculture, expanding human population
and climatic change (IUCN 2008, Kattan et al. 2004). Aside from their ecological
importance, these ecosystems also serve as Ecuador’s major public water source for
domestic, agricultural and industrial consumption, and the generation of hydropower
(Buytaert et al 2006, Medina and Vásconez 1999). Researchers propose that protection
of these ecosystems can be established via the conservation of a local inhabitant species,
the Andean bear (Tremarctos ornatus).
The Andean bear is the only bear found in South America, and it is considered an
umbrella species for the páramo ecosystems of the northern Andes (Peyton 1999, Troya
et al. 2004). As an umbrella species, protection of its habitat would indirectly protect
many other species and the overall integrity of these ecosystems (Wilcox 1984). The
broad ecological requirements, seasonal use of different habitats, ecological role, and
profound charisma of the Andean bear make it an appropriate species on which to base
conservation planning to preserve the páramo (Yerena and Torres 1994, Peyton 1999). A
major concern, however, is that the species is currently facing a state of decline.
The Andean bear has experienced a major reduction in species number and
geographic range as a result of poaching, agricultural expansion, and increased human
activity (IUCN 2008). The IUCN (International Union for Conservation of Nature) has
placed the Andean bear on its Red List of Threatened Species (a global inventory of the
conservation status of plant and animal species) as a “vulnerable” species, and CITES
(the Convention on International Trade in Endangered Species of Wild Fauna and Flora)
has listed the bear under Appendix I as critically “endangered” (IUCN 2008, CITES
2011). Landscape transformation, fragmentation and the addition of roads have reduced
the bear’s geographic range to 42% of its original area (Kattan et al. 2004). Primary
threats to the future persistence of the Andean bear include habitat reduction and
fragmentation, poaching, and the general lack of knowledge about the distribution and
status of the species (Peyton 1999, Rodriguez et al. 2003). Limited research and lack of
knowledge make it difficult to develop realistic management plans for conservation. In
order to develop a strong foundation for the conservation of the Andean bear, it is critical
to understand and produce baseline ecological information about the species (Troya et al.
2004). The diet of the Andean bear is specifically an area lacking information (Troya et
al. 2004).
Habitat and Dietary Ecology of the Andean Bear
In the quest for food, the Andean bear occupies a large variety of habitats. The
species moves along an altitudinal gradient, following seasonal patterns of food resources
(Cuesta et al. 2003, Ríos-Uzeda et al. 2005). The most common habitats found within the
geographic range of the Andean bear include high elevation elfin forests, upper mountain
humid forests, and humid grasslands (Cuesta et al. 2003, Ríos-Uzeda et al. 2005). Due to
limited research and the tendency for Andean bears to occupy various habitats,
composition of their diet remains unclear.
Current available information of the Andean bear diet suggests that the species is
omnivorous feeding on many kinds of fruits, vegetative material and meat, with the plant
family Bromeliaceae constituting a large percentage (Peyton 1980, Paisley and Garshelis
2006, Mondolfi 1989). Red Tremarctos, the International Network for Andean Bear
Conservation, has produced a document summarizing dietary publications from 1980 to
2001. These studies recorded more than 140 species of plants and 20 species of animals
in the Andean bear diet (Red Tremarctos 1980-2001; see Appendix A). Tools that can
systematically quantify these vegetative components of the Andean bear diet, however,
are lacking.
In collaboration with faculty and researchers from the University of Idaho,
Washington State University, and the Universidad Técnica Particular de Loja (UTPL),
this study will build upon current knowledge of the Andean bear diet to develop tools
necessary to systematically quantify vegetative composition. These tools will then be
organized into a reference bank that will be made available for future researchers
studying animal foraging in the páramo plant communities of southern Ecuador.
Overarching Research Questions
1) What is the relative contribution of different plant species to the diet of the
Andean bear?
2) How does the diet of the Andean bear vary across seasons and its geographic
range?
Project Goal
To establish a reference collection of plants known – or suspected – to be a part of
the Andean bear diet. Collection will consist of multiple parts to establish a robust
resource for future researchers studying animal foraging in páramo plant communities of
southern Ecuador. We are taking the first step in addressing these overarching research
questions by building the foundation information needed for the basis of realistic
conservation management plans for the species and protection of the páramo.
Project Objectives
To develop the following:
1) Voucher collections that will contribute to the ongoing growth and research of
the UTPL herbarium, the national herbarium of Ecuador, and the University of
Idaho Stillinger Herbarium;
2) Silica-gel dried tissue collections to be used for DNA extraction;
3) A microhistological reference collection of plant tissues; and
4) A DNA bank that will be housed at UTPL to be used for future genetic
analyses.
Study Area
Podocarpus National Park, Ecuador
This project will utilize established research sites within and around the
Podocarpus National Park of southern Ecuador. The park straddles the border of the Loja
and Zamora-Chinchipe provinces, encompassing approximately 146,000ha of forested
and páramo communities (Keating 1999). The páramo ecosystems in particular, are
vastly different than others throughout the world. They are unique for their mostlycontinuous expanse, low elevations, high species diversity, high endemism, and extreme
variations in community structure and composition (Keating 1999). The region’s
topography is fairly complex and climate varies with elevation. The upper montane zone,
for example, is noteworthy for its persistent cloud cover (Keating 1999). Along with
diverse flora, the páramo also hosts a range of unique wildlife taxa including the Andean
bear and the mountain tapir (Tapirus pinchaque).
Data Collection Methods
Field Work
Plant collection
To form the voucher collection, we will use standard herbarium protocol (Bridson
1998) to collect plant specimens at the six establish research sites, for each of the species
listed in Appendix A that we can locate and identify. This list was developed by Red
Tremarctos, the International Network for Andean Bear Conservation, summarizing plant
species found in the Andean bear diet from studies conducted in 1980-2001. We will also
be collecting by direct observation. For example, collecting in areas where Andean bear
foraging is evident (i.e., partially chewed vegetation, claw marks on trees, presence of
prints, feces etc.)
Plant specimens will be collected in triplicates in which one will stay at the
UTPL herbarium, one will be sent to Ecuador’s national herbarium, and the other will be
sent to the University of Idaho Stillinger Herbarium. Specimens will be collected,
pressed, dried, mounted, labeled and filed at the UTPL herbarium according to standard
herbarium protocol (Bridson 1998). At the time of collection, specific details for each
specimen will be recorded including the collection number, date, locality, GPS
coordinates, plant associates and specimen details such as flower color, height,
abundance, and other distinguishing characteristics. Additionally photographs will be
taken to assist in identification. For microhistological analysis, it will be most important
to recover multiple plant leaves, buds, stems and flower parts of each different species
(Davitt and Nelson 1980).
Silica-gel dried tissue collection
This collection will occur simultaneously with plant collection. For each of the
species listed in Table 1, several leaves will be removed from a specimen and placed in a
zip-lock bag containing silica gel. Silica gel dries and preserves the field-collected leaves
so that they can later on be used for molecular studies of DNA variation (Chase and Hills
1991). Each bag will be labeled by species name and voucher number (collector’s name
and collection number). The zip-lock bags will then be stored at the UTPL herbarium for
future use of DNA extraction. We chose this preservation method because it is practical,
reliable and fairly inexpensive (Chase and Hills 1991).
Fecal collection
Fecal collection will also occur at the same time of plant collection. For each fecal
sample, a DNA sub-sample will first be collected, approximately 2-3cc in size, and
placed in a 50ml tube with buffer. The remaining sample will be collected in a plastic bag
and used for diet analysis. A preservation substance will not be used therefore samples
will need to be taken to the lab as soon as possible. At the time of collection, the date,
locality, GPS coordinates, and sample number will be recorded. In the lab, each sample
will be measured for wet weight and then dried, at 40ºC for 2-3 days.
Laboratory Work
Preparation of Microhistological Reference Slides
Microhistology is a technique used to identify plant species based on distinctive
cellular characteristics of plant tissues (Holechek et al. 1982). This technique is often
utilized in dietary fecal analysis to determine the plant composition of animal diets
(Holechek et al. 1982).
Using the plant specimens collected in the field, we will build a slide reference
collection by adopting a microhistological technique developed by Bruce Davitt and Jack
R. Nelson of Washington State University. This technique is performed by obtaining a
portion of plant material, either the flowers, stems, leaves, roots, or a combination of all,
and blending them in a household blender to dislodge epidermal fragments. Once
epidermal fragments have been dislodged, they go through a bleaching, staining, and
rinsing process in preparation for mounting, where they are then dried to microscope
slides using a glycerin gel mounting medium (See appendix B for additional details). This
technique is different from other preparation methods of plant reference slides because it
uses a clearing agent, lactophenol, combined with a staining agent, water soluble cotton
blue to stain the plant fragments. This not only increases the quality of plant fragments
that can be identified, but also the clarity.
We will utilize the resulting reference slides most efficiently by making photomicrographs and drawings of each slide. Photo-micrographs are pictures taken through
the microscope. These techniques will not only produce second references, but will
potentially reduce the amount of microscope work, and increase the identification process
during fecal analysis (Dusi 1949).
DNA Bank
Dependent upon the time allotted, the total genomic DNAs will be extracted from
the silica-gel dried plant tissues collected in the field, using the QIAGEN DNeasy Plant
Mini Kit and following the protocol recommended by the manufacturer.
Preliminary Diet Analyses
Dependent upon time and completion of the listed objectives, we will additionally
process and analyze 5 fecal samples, utilizing the developed reference bank. Fecal
samples that were collected in the field and then preserved will be broken down and
prepared on microscope slides for diet analyses. Preparation will occur in the exact same
manner as plant voucher slides (See appendix B for additional details).
The vegetative composition of each sample will be quantified using a grid
technique adopted by Bruce Davitt. A 10 X 10 square grid will be placed in the eyepiece
of the microscope. For each reference slide, the relative plant cover will be quantified for
25 randomly located microscope views. For each plant species present in a view, area of
coverage will be recorded. The percent diet composition of a species can then be
calculated by dividing that species cover by total cover of all species, and then
multiplying by 100 (See appendix C for additional details).
Relevance
This project aims to develop the baseline tools necessary for future diet analyses
of the Andean bear. It is the first step required in addressing overarching dietary research
questions such as the relative contributions of different plant species and the variation in
diet across different seasons and the species geographic range. This information is greatly
needed in order to develop realistic management plans for the conservation of the species,
and in turn, protect the threatened páramo ecosystems of Central and South America.
Synergistic Ties
Working among a small, interdisciplinary team, this project will be one of four,
addressing individual research questions that tie into an overarching theme of
biodiversity conservation and the importance of páramo ecosystems in southern Ecuador.
Specific projects include assessing plant species diversity and composition across a
páramo gradient, developing a plant reference collection for the diet of the Andean bear,
evaluating Andean bear foraging preferences on bromeliad species, and evaluating the
social and cultural importance of the páramo. These projects will contribute to future
conservation planning in the United States and other countries, and will also allow
participants to gain experience with the scientific method, different cultural perspectives
and the collaborative process.
Time table
February 27th 2011—May 6th 2011
April 15th 2011
April 21st 2011
April 28th 2011
May 11th 2011
May 17th 2011
May 19th 2011
May 19th—May 30th 2011
May 30th—July 23rd 2011
July 23rd 2011
September 2011—December 2011
December 2011
Complete written proposal
Complete “Responsible and Ethical
Conduct of Research” Training
Visit Bruce Davitt’s Nutritional Lab
(WSU) to develop microhistological
techniques
Present proposal to faculty panel and ECB
mentor
Present proposal to UTPL participants
Depart for Quito, Ecuador
Depart for Loja, Ecuador
Project Training
Project field and laboratory work
Depart for USA
Complete final report and project poster
Present project
Support and Feasibility
This project is a collaborative effort among Dr. Janet Rachlow (Wildlife
Resources University of Idaho), Dave Tank (Forest Resources University of Idaho),
Bruce Davitt (Wildlife Nutritional Lab Washington State University), Doctoral Candidate
Rodrigo Cisneros (UTPL), Field Botanist Nixon Cumbicus (UTPL), Jorge Armijos
(UTPL) and undergraduate Tara Ball (Conservation Biology University of Idaho).
The National Science Foundation has awarded this project, along with seven
others, $144,883 to work among interdisciplinary teams with common overarching
research themes. This grant will cover travel and board, a stipend, and any extra funds
that arise for research support.
This project will be beneficial for future research and growth of the UTPL and
University of Idaho along with future conservation planning of the United States and
other countries. It is important to acknowledge, however, that completion of all
objectives will be time-dependent. Building a microhistological plant reference collection
may potentially consume more time then allotted, therefore, the status of the DNA bank
and preliminary diet analyses will depend on completion of the collection.
Budget
Airfare
Subsistence (Housing/Food)
Research Support
(Reagents/Supplies/Travel to Sites)
Total
Cost
$1461
$1000
$1000
$3461
Literature References
Bridson, D. 1998. The Herbarium Handbook 3rd edition. L. Forman editor. Royal
Botanical Gardens, Kew.
Buytaert, W., R. Célleri, B.D. Bièvre, F. Cisneros, G. Wyseure, J. Deckers, and
R. Hofstede. 2006. Human impact on hydrology of the Andean páramos. Earth
Science Reviews 79:53-72.
Convention on International Trade in Endangered Species of Wild Fauna and Flora
(CITES). 2011. United Nations Environmental Program (UNEP). Available at
http://cites.org/
Chase, M. W., and H. H. Hills. 1991. An Ideal Material for Field Preservation of Leaf
Samples for DNA Studies. Taxon 40:215-220. International Association for Plant
Taxonomy (IAPT).
Cuesta, F., M.F. Peralvo, and F.T. van Manen 2003. Andean bear habitat use in the
Oyacachi River Basin, Ecuador. Ursus 14:198-209.
Davitt, B. B., and J. R. Nelson. 1980. A Method of Preparing Plant Epidermal Tissue for
Use in Fecal Analysis. Washington State University CIRCULAR 0628.
Dusi, J. L. 1949. Methods for the Determination of Food Habits by Plant
Microtechniques and Histology and Their Application to Cottontail Rabbit Food
Habits. The Journal of Wildlife Management 13:295-298.
Holechek, J. L., M. Vavra, and R. D. Pieper. 1982. Botanical Composition Determination
of Range and Herbivore Diets: A Review. Journal of Range Management 35:309315.
Hewitt, D. G., and C. T. Robbins. 1996. Estimating Grizzly Bear Food Habits from Fecal
Analysis. Wildlife Society Bulletin 24:547-550.
IUCN Red List of Threatened Species (IUCN). 2008. Version 2010.4. Tremarctos
ornatus. Available at http://www.iucnredlist.org/apps/redlist/details/22066/0
Kattan, G., O. L. Hernández, I. Goldstein, V. Rojas, O. Murillo, C. Gomez, H. Restrepo,
and F. Cuesta. 2004. Range fragmentation in the spectacled bear Tremarctos
ornatus in the northern Andes. Oryx 38:155-163.
Keating, P. L. 1999. Changes in Paramo Vegetation Along and Elevation Gradient in
Southern Ecuador. Journal of the Torrey Botanical Society 126:159-175.
Medina, G., and P. M. Vasconez. 1999. Proyecto paramo. Available at
http://www.puce.edu.ec/zoologia/vertebrados/personal/sburneo/cursos/EcologiaII/
11%20Paramo.pdf
Mondolfi, E. 1989. Notes on the distribution, habitat, food habits, status and conservation
of the spectacled bear (Tremarctos ornatus) in Venezuela. Mammalia. 53:525544.
Paisley, S., and D. L. Garshelis. 2006. Activity patterns and time budgets of Andean
bears (Tremarctos ornatus) in the Apolobamba Range of Bolivia. Journal of
Zoology 268:25 – 34.
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Cambridge, UK.
Ríos-Uzeda, B., H. Gómez, and R. Wallace. 2005. Habitat preferences of the Andean
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EcoCiencia, Wildlife Conservation Society, and Red Tremarctos.
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Appendix A
List of Plant Species for Collection
Appendix B
Microhistological Protocol for Plant and Fecal Reference Slides
From: Davitt, B. B., and J. R. Nelson. 1980. A Method of Preparing Plant Epidermal Tissue for
Use in Fecal Analysis. Washington State University CIRCULAR 0628.
Glycerin gel mounting medium
Prepare glycerin gel, a widely-used mycological mounting medium, by slowly dissolving
21 grams of powdered gelatin in 125 ml of water at about 60oC. Put a small beaker
containing the water and gelatin inside a larger beaker that is partly filled with water.
Cover small beaker with a watch glass to reduce evaporation during the heating period.
Place this apparatus on a hot plate and heat to 60oC for 2 to 3 hours. When the gelatin
has dissolved, add 3 grams of phenol and 125 ml of glycerol (glycerin) to the solution
and stir slowly. Filter the hot mixture through four layers of cheesecloth and pour into
empty petri dishes. Then refrigerate the gel to solidify the solution. Once prepared, the
gel can be reliquified and reset repeatedly by changing the temperature above or below
40oC. The gel can be stored for several months.
Lactophenol blue clearing and staining agent
A clearing agent, lactophenol, is combined with a staining agent, water soluble cotton
blue, to prepare plant and fecal microscope slides. Prepare lactophenol with 20 ml liquid
phenol, 40 ml lactic acid, 20 ml water and 40 ml glycerol. Add 100-ml aliquot of
lactophenol to 20 ml glacial acetic acid and 2 ml cotton blue dye (1% solution). This
clearing-staining agent improves microscopic resolution of epidermal fragments. Plant or
fecal specimens can be left in the lactophenol blue solution for 7 to 10 days at room
temperature or for 2 to 4 days while being heated at 60oC. Pressed or dried plant
specimens can be restored by immersion in lactophenol blue for several days because the
glycerin in the solution will soften them.
Plant voucher material
Put plant leaf, bud, stem and flower parts in 125 ml, widemouth glass or plastic bottles
filled with 95% ethyl alcohol, for approximately 1 week, to extract plant pigments. Then
beach plant parts 5 to 20 minutes with 5% sodium hypochlorite solution and thoroughly
wash with water through a 75-micrometer mesh screen (U.S.A. standard testing sieve no.
200 Tyler equivalent 200 mesh). Following this, return the specimens to a bottle filled
with enough lactophenol blue to cover the plant material and store for 7 to 10 days at
room temperature.
After bleaching and staining, wash the plant parts with water, transfer to a household
blender with about 200 mls water, and agitate for 3 to 5 minutes to dislodge epidermal
fragments. Then pour water and fragments into a large watch glass. Recover epidermal
fragments with a paintbrush or forceps and place on a microscope slide with a few drops
of water. Coarse or woody plant stems can also be scraped with a razor blade and the
epidermal fragments mounted on a microscope slide as described above. Dry slides on a
hot plate and melt glycerin gel over the plant fragments. To make the mounts permanent,
seal the edge of the cover slip with nail polish or other sealing compounds. Cool the
slides at room temperature, label and store in microscope slide boxes.
Fecal material
Break down fecal pellets by several minutes of agitation at low speed in a household
blender. You can take a subsample if large amounts of fecal material are available. To
do so, withdraw about a 40-gram sample (100 ml) of the fecal material while it is
agitating in the blender. Then wash the fecal material with water in a 75-micrometer
mesh screen to remove foreign materials, and store in a 125-ml bottle with 95% ethyl
alcohol.
After 1 week in alcohol, prepare the fecal material in the same manner as described for
plant voucher material. Bleach the fecal sample for 5 to 20 minutes with a 5% sodium
hypochlorite solution and wash thoroughly with water in a 75-micrometer mesh screen.
Then return specimen to a bottle filled with enough lactophenol blue to cover the sample
and store for 7 to 10 days. When the staining period is completed, wash the fecal
material with water and transfer to a watch glass where epidermal fragments are then
suspended in water. Use a forceps or paintbrush to recover the fragments and put them
on a microscope slide with a few drops of water. Dry slides on a hot plate and melt
glycerin gel over the fragments. Put a cover slip on the slide and seal as described
before. Cool and label the slides and store them in microscope boxes.
Appendix C
Protocol for Determining Botanical Composition of Diet
From: Bruce Davitt Wildlife Habitat/Nutrition Lab, Dept. Natural Resources Sciences,
Washington State Univ., Pullman WA 99164-6410 (509)-335-2318
Botanical composition was determined microhistologically by modifying existing
frequency - density conversion sampling procedures of Sparks and Malechek (1968), and
Flinders and Hansen (1972), Holechek and Vavra (1981) and Holechek and Gross (1982).
Relative cover (Korfhage 1974, Davitt 1979) of plant cuticle and epidermal fragments are
quantified for 25 randomly located microscope views on each of eight slides (total 200
views), (Note: 4 slides for 100 views,etc.) but various numbers of slides and views may
be applied. A 10 square x 10 square grid mounted in the eyepiece of the microscope is
used to measure area covered by each positively identified fragment observed at 100x
magnification. Larger magnification (200x to 450x) are used to aid in identification of
discernable fragments (Holechek and Valdez 1985). Measurements of area covered are
recorded by plant species, genus, or forage class category as desired. Percent diet
composition is calculated by dividing cover of each plant by total cover observed for all
species, then multiplying by 100.