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Fecal analysis as an indicator of post-hibernation shifts in the gut microflora of the thirteen-lined ground squirrel (Spermophilus tricecemlineatus).

Abstract. -- Monthly fecal analysis of five specimens of the thirteen-lined ground squirrel (Spermophilus tridecemlineatus) maintained under normal laboratory conditions from November 1997 through 9 April 1998 charted the microfloral responses to emergence of the host animals from semi-torpid conditions approximating hibernation to the active state. Gramnegative taxa included in the study were: Enterobacter hermanii, Klebsiella sp., Proteus mirabilis, Escherichia coli and a miscellaneous assemblage of coliforms. Gram-positive bacteria assessed were: Staphylococcus aureus and Enterococcus sp. Individual ground squirrels maintained unique microflora that varied in diversity throughout the study. General population trends observed over time included a significant February peak of gram-negative taxa and a March-to-April decline of gram-positive cocci. Declines in microbial populations from February numbers coincided with an apparent inability of ground squirrels to digest dietary fat as weight loss occurred through metabolism of body fat.


The vertebrate gut provides a continuum of diverse microhabitats for bacteria that differ in features such as presence or absence of ceca or crypts, pH, osmotic pressure, cellular secretions and varied states of ingested nutrient breakdown (Clark 1977). The extent to which diet influences these internal environmental factors (and thereby the resident microbial flora) has been well documented for laboratory rodents (Smith 1965; Tannock & Savage 1974) and humans (Lee 1966; Franklin & Skoryna 1971; Drasar & Hill 1974; Nord & Kager 1984; Sharma et al. 1995a, 1995b; Sharma & Schumacher 1996). Malnutrition produces even more profound effects on microbial composition than diet alteration in humans (Scrimshaw et al. 1969) and mice (Tannock & Savage 1974). Nevertheless, the intestinal microflora is normally stable, because only extreme changes in diet may significantly influence composition of the gut microflora (Haenel 1961; Drasar & Hill 1974).

Distribution of constituent microbial taxa along the gut are influenced by these internal factors and the peristaltic contractions that force intestinal contents from a mouth-to-anus direction (Donaldson 1968). Peristalsis in mammals is strongest in the jejunum and becomes progressively weaker towards the ileo-caecal valve, where it may be terminated, which may explain the higher concentrations of microorganisms in the terminal ileum and colon (Drasar & Hill 1974). Up to 40% of fecal material in the colon is comprised of bacterial cells representing both indigenous microbes and a mixed sample of taxa carried "downstream" from upper regions of the gut (Savage 1977). Consequently, bacterial composition of defecated fecal matter has been demonstrated to closely mirror the microfloral composition of the cecum in vertebrates (Smith 1965).

Most species of bacteria that have been isolated from the vertebrate intestinal tract are separated into three groups: contaminants from the external environment or other parts of the body (species of Staphylococcus and Bacillus); the indigenous flora which reside in smaller numbers (members of the family Enterobacteriaceae); and the micro-organisms which are present in large numbers (mostly species of Bacteroides) (Drasar & Hill 1974).

The family Enterobacteriaceae includes facultative anaerobic, gramnegative, either motile or non-motile, non-spore forming rods (Kauffman 1969). These microorganisms are constantly present as part of the normal flora (Drasar & Hill 1974). Esherichia coli was considered the predominant organism in the mammalian gut for many years before the discovery of large populations of obligatory anaerobic organisms which proved to be the predominant type of organism (Clarke 1977). Some examples of the family (E. coli, Klebsiella and Proteus) comprise part of a group referred to as coliforms (gram-negative rods that ferment lactose and form gas). These bacteria are facultative anaerobes that do not form spores. Some coliforms, such as E. coli, are known to release colicins into the intestinal lumen, which inhibit the proliferation of other bacteria (Drasar & Hill 1974).

Two families of gram-positive cocci which are facultative anaerobes are common inhabitants of the vertebrate gut (Smith 1965; Drasar & Hill 1974). The Lactobacillaceae includes the genus Streptococcus, which is found in the mouth and intestinal tract, and is one of the dominant groups of facultative anaerobes in the mammalian intestine. The family Micrococcaceae includes the genus Staphylococcus, a typical resident of the mouth and intestinal tract that can also be contracted as an external contaminant from the skin of the animal or the environment.

Humans and laboratory mammals afford rather constant optimal conditions for bacterial growth and survival. It is therefore not surprising that some functionally heterothermic mammals, such as ground squirrels (Spermophilus), have attracted the attention of microbiologists. Previous studies (Schmidt 1963, 1967; Barnes & Burton 1970) have dealt wholly or in part with the thirteen-lined ground squirrel (S. tridecemlineatus), because it is an abundant and readily obtainable species throughout the central North American grasslands (Streubel & Fitzgerald 1978), and is easily maintained in captivity.

Hibernation in ground squirrels is preceded by frantic feeding efforts to acquire sufficient fat reserves to last the winter months. Hibernating Spermophilus studied in captivity experienced a metabolic slowdown characterized by loss of appetite, lethargy, minimal respiration and circulation, and a decline in body temperature (Wade 1930; Fisher 1964). Captive ground squirrels are known to become torpid even under normal laboratory conditions in well-lighted rooms with various temperatures and bedding arrangements, and with food and water available. Fisher (1964) found that hibernation was not a continual condition in S. tridecemlineatus, because all animals studied aroused periodically to urinate, eat, drink and defecate on an irregular basis. The intestinal epithelium has also been reported to be active in the arctic ground squirrel (S. parryii) during hibernation (Schmidt 1967).

Previous microbiological investigations of ground squirrels (cecal material removed by dissection from S. tridecemlineatus, Barnes & Burton 1970; fecal analysis of pellets from S. parryii, Schmidt 1963) detailed significant microfloral shifts in animals making the transition from the active state to hibernation that was initiated and maintained by subjecting animals to near-freezing conditions of 3-8[degrees]C. With few exceptions involving generic identifications, bacterial taxa were placed only in general morphological groupings.

It was the purpose of this study to detail the individual and collective microfloral changes in five individuals of S. tridecemlineatus as each reverted from the lethargic state approximating hibernation in captivity to a more active condition. Utilization of a series of recently developed and commercially available media permitted more specific levels of identification of taxa than previous studies, allowing for greater resolution of individual microbial responses associated with the physiological changes that occurred with emergence from hibernation.


Experimental animals. -- Specimens of Spermophilus tridecemlineatus were obtained from natural populations in Wichita Falls, Texas during the month of September 1997 and transported to the Animal Care Facility of Midwestern State University. In an effort to minimize individual variation, only female animals were selected for this study, although Ducluzeau (1984) found that gender was not an important determinant of gut floral diversity in a variety of laboratory animals.

Animals were housed in glass aquaria lined with cedar chips. Floor space dimensions (in cm) ranged from 90 by 43 to 180 by 43. Water and compressed alfalfa pellets were available ad. lib. at all times. Controlled environmental conditions included a constant temperature of 25[degrees]C and 12 h alternating photoperiods.

Collection of fecal specimens. -- Fecal material was collected at approximately one-month intervals, beginning on 10 November 1997 and ending on 9 April 1998. Observations of relative level of activity and alertness of squirrels were noted at the time of fecal specimen collection. Animals were placed in individual holding cages lined with plastic until defecation occurred. Fecal pellet morphology was recorded and pellets were removed as soon as possible, with intervals varying from immediately following defecation to periods of several hours after defecation. Animals were then weighed to the nearest 0.1 g on a triple-beam balance and returned to their cages. Individual samples of two to three pellets (depending on size) were retained from each animal for processing. Fecal samples were weighed with a Denver Instrument precision balance to the nearest 0.001 g.

Processing of fecal specimens. -- The initial series of tests served as a basis for determining standard concentration and serial dilutions for subsequent procedures. The initial fecal sample served as a reference standard for defining subsequent diluent volumes. The initial fecal sample was weighed, added to 2 ml of sterile d[H.sub.2]0, and macerated with a sterile metal spatula until a homogeneous suspension was achieved. Using a sterile 1 ml pipette, 0.25 ml of this undilute suspension was spread onto one of each of three types of selective and differential agar plates. Serial dilutions of 1 X [10.sup.-1] and 1 X [10.sup.-2] were also prepared from subsequent fecal samples. Of the three concentrations, the dilution that provided countable colonies served as a standard. All ensuing fecal samples were then diluted in a volume proportional to the original diluent and fecal sample.

Bacterial growth and identification media. -- Identification of bacterial taxa was accomplished with two proprietary chromogenic agars obtained from CHROMagar (198 North Queens Ave., North Massapequa, New York 11758), which were prepared according to the manufacturer's instructions 24 h before use. Selection of the two agar types used in this study was based on taxonomic breadth afforded by each. CHROMagar media has been shown to be effective in identification and discrimination of various populations in microbial cultures (Odds & Bernaerts 1994; Beighton et al. 1995; Merlino et al. 1996; Pfaller et al. 1996). CHROMagar Orientation media permitted identification by the selective growth of color-coded colonies of Escherichia coli (pinkish-red colonies), Proteus mirabilis (brown colonies), Staphylococcus aureus (opaque-cream colonies), S. saprophyticus (pink colonies), the genera Klebsiella (metallic blue colonies) and Streptococcus (turquoise colonies). The 0157 media identified Enterobacter hermanii (white colonies), Proteus mirabilis (brown colonies) and general coliforms (blue colonies). A third medium which selected for Salmonella was discontinued after two months because of negative findings in each of the squirrels. Plates were incubated at a temperature of 37[degrees]C for 24 h. Upon removal from incubation, plates were scored with the aid of a Quebec darkfield colony counter and the colors of the colonies were noted.

Statistical analyses. -- Colony count data were log-transformed prior to statistical analyses. Analyses-of-variance (ANOVAs) and Duncan's multiple means tests were run to determine the presence of any statistically significant variation (at the P [less than or equal to] 0.05 level) between samples in this study. All statistical operations were performed using the NCSS 97 statistical package (Hintze 1997).


Ground squirrels had already acquired a noticeable accumulation of body fat by the onset of the study, and the mean body mass remained fairly constant through the first three sampling periods of November, December and February (174.6 g, 182.2 g and 184.1 g, respectively). Significant weight fluctuations (at P [less than or equal to] 0.05 level, Duncan's test) were experienced during the last two sampling periods.

March weighings indicated a significant decline in mean body mass to 147.9 g (r = -0.51; ANOVA, P < 0.0001) and subsequent April increase to 164.4 g. Fecal pellet morphology collected of each animal during early March differed strikingly from the dark and tightly compacted pellets taken both before and after this sampling period. Consistency was soft and pasty, and coloration was of a distinct yellowish color, although presence of roughage indicated that animals were still feeding on the alfalfa pellets.

General microbial population trends. -- Five categories of gram-negative bacteria (Enterobacter hermanii, Klebsiella sp., Proteus mirabilis, Escherichia coli and the assemblage of general coliforms) and two gram-positive taxa (Staphylococcus aureus and Enterococcus sp.) were detected with the CHROMagar media (Table 1).

The entire bacterial population in each squirrel experienced significant temporal fluctuations (ANOVA, P < 0.001) with an overall decline (r = - 0.43) during the course of the study. Colony numbers from the first two sampling periods did not differ significantly (at P [less than or equal to] 0.05 level, Duncan's test), but the dramatic February increase and successive sharp declines in March and April were each significant.

Pooled samples of gram-negative and gram-positive taxa experienced gradual overall declines during the study period (Fig. 1), although patterns of decline differed. Gram-negative bacteria experienced significant population growth during the February sampling period (Fig. 1a), but colony counts did not otherwise vary significantly (at P [less than or equal to] 0.05 level, Duncan's test). Numbers of gram-positive taxa remained steady through the February sampling period (Fig. 1b), before experiencing two successive declines (at P [less than or equal to] 0.05 level, Duncan's test).

Individual microbial trends. -- Individual categories of both gram-negative and gram-positive bacteria responded similarly to pooled samples (Fig. 2); gram-negative taxa demonstrated prominent February peaks and gram-positive taxa experienced a steady and pronounced decline from stable levels of November-February. However, individual gram-negative taxa were more erratic in distribution and less stable in numbers (Fig. 2a-d) than gram-positive species (Fig. 2e-f), and appearances and disappearances of detectable colonies of gram-negative bacteria from the microflora of individual ground squirrels were not uncommon (Table 1).



Gram-negative taxa. -- Each gram-negative taxon experienced an overall decline in numbers during the course of the study, but two taxa exhibited temporal patterns worthy of note. Enterobacter hermanii (Fig. 2a) was the prevalent gram-negative taxon, having been documented in four of five squirrels during four or more sampling periods. Klebsiella (not figured) was the rarest of surveyed bacteria in this study. It was detected only during the first and last sampling periods, was never documented in two ground squirrels, and was identified during only a single sampling period in two others.

Gram-positive taxa. -- Two cocci were the most consistently documented bacterial taxa in this study, and each displayed similar population growth patterns (Fig. 2 a-b). Enterococcus (= Streptococcus) was the only truly ubiquitous component of the squirrel microflora assessed in this study, being documented in each ground squirrel during each sampling period (Table 1). Staphylococcus aureus was nearly so, found missing only once in each of the five sampling periods of three ground squirrels.

Individual variation of squirrel microflora. -- All ground squirrels experienced some decline in collective colony counts over the study period, but the decrease was significant only for squirrels #1 (r = -0.50, P < 0.05), #3 (r = - 0.53, P < 0.01) and #5 (r = - 0.65, P < 0.001). In each of these instances, the animals supported high populations during the February sampling period, followed by significant declines until termination of the study (at P [less than or equal to] 0.05 level, Duncan's test).

Each of the five ground squirrels supported a unique microflora that commonly varied between sampling periods (Table 1). Given the ubiquitous nature of the gram-positive cocci, it is the varying presence or absence of gram-negative bacteria that accounted for overall microfloral differentiation among ground squirrels and between sampling periods.

Frequency of positive scorings for each of the seven bacterial entities from the five sampling periods was used as a simple microfloral diversity index. High overall indices were sustained by three squirrels throughout the study (#1=0.71; #3=0.68; #5=0.71), compared to low indices of the remaining two animals (#2=0.46; #4=0.43). Only during the April sampling period were all surveyed bacterial taxa present in a single animal (squirrel #3), in contrast to the instance where only the ubiquitous Enterococcus was detected in squirrel #4.


Schmidt's (1963) analysis of fecal pellets from 15 Arctic ground squirrels (Spermophilus parryii) documented a 1000 fold increase in psychrophilic bacteria (microbes exhibiting an optimum temperature of 15[degrees]C and an inability to grow above 20[degrees]C) and a 1000 fold reduction in coliform bacteria, while numbers of enterococci remained rather constant. Barnes & Burton (1970) performed a comparable study on the thirteen-lined ground squirrel (S. tridecemlineatus) by dissection of fecal material from the cecum of eight animals from both active and torpid states. Among their findings were that most of the decline in microbial numbers and taxa occurred within the first six days of hibernation, anaerobic gram-positive rods and coccobacilli either became rare or disappeared from the ceca of hibernating animals, and populations of presumptive Proteus and Enterococcus (documented in two-thirds of the animals) remained constant.

Each of the above studies of ground squirrels (Spermophilus) entering hibernation have demonstrated profound shifts in microbial population numbers as the animals completed the transition from active to hibernating states at near-freezing temperatures of 3-8[degrees]C. Only the enterococci populations remained stable. Among factors implicated in microbial declines cited by Schmidt (1963) and Barnes & Burton (1970) are: (1) lowered temperatures which provide less-than-optimum growing conditions for many microbial taxa; (2) loss or decrease of necessary nutrient requirements (e.g. typical microbial fare of sloughed epithelial cells and glycoproteins from the intestinal mucus, Lee 1966); and (3) perhaps also a shift in the competitive balance with psychrophilic bacteria for those diminished resources.

Emergence from hibernation presents the opportunity for restoration of the pre-hibernation microflora through return of optimal conditions and reintroduction of bacterial taxa by ingestion where local extinction may have occurred. The importance of temperature as a preeminent factor influencing microbial fluctuations in this study was largely negated by maintenance of the gut environment within the comparatively narrow temperature ranges of 25-38[degrees]C, as defined by ambient room temperatures and the normal body temperature of an active ground squirrel.

The sharp decline of early March samples of both gram-positive and gram-negative microbial populations from February peaks (Fig. 1) coincided with dramatic weight loss in ground squirrels. Accelerated metabolism of body fat accounting for loss of body mass was evident in the lean physiques of increasingly active ground squirrels. The atypically pasty and yellow fecal pellets produced during the March sampling period is attributed to the presence of dietary fat passing undigested through the gut as animals metabolized body fat. Of particular interest is the continued depression of gram-positive microbe numbers noted at the conclusion of the study in April, while populations of gram-negative taxa stabilized following the March decline.

It does not seem likely that the coincidence of microbial population declines, squirrel weight losses and apparent inhibition of intestinal fat metabolism can be assigned to chance alone. Rather, it appears that certain physiological factors adversely influence populations of those bacterial taxa included in this study, as squirrels make the transition from lethargy to the active state. Avenues for future study include specific determination of factors responsible for microbial declines in presence of body fat metabolism, and the influence of those factors on the differential responses of gram-positive and gram-negative bacteria.
Table 1. Monthly microbial colony counts of fecal samples expressed in
number of colonies per gram from five specimens of Spermophilus
tridecemlineatus, as determined by cultures on CHROMagar media.

Bacteria Dates of Collection (1997-98)
 10 November 1 January

Squirrel #1
 Enterobacter hermanii 2.98 X [10.sup.2] 1.00 X [10.sup.2]
 Klebsiella spp. -- --
 Proteus mirabilis 5.41 X [10.sup.2] 2.11 X [10.sup.3]
 Escherichia coli 1.08 X [10.sup.2] 2.17 X [10.sup.2]
 General coliforms 8.10 X [10.sup.1] 1.60 X [10.sup.1]
 Staphylococcus aureus 8.10 X [10.sup.1] 3.52 X [10.sup.2]
 Enterococcus sp. 7.85 X [10.sup.2] 3.68 X [10.sup.3]
Squirrel #2
 Enterobacter hermanii 1.10 X [10.sup.1] 2.70 X [10.sup.1]
 Klebsiella sp. -- --
 Proteus mirabilis 2.70 X [10.sup.1] --
 Escherichia coli 1.73 X [10.sup.5] --
 General coliforms -- --
 Staphylococcus aureus -- 1.63 X [10.sup.2]
 Enterococcus sp. 8.02 X [10.sup.3] 6.50 X [10.sup.2]
Squirrel #3
 Enterobacter hermanii 5.40 X [10.sup.1] --
 Klebsiella sp. 4.33 X [10.sup.4] --
 Proteus mirabilis -- --
 Escherichia coli 1.08 X [10.sup.2] --
 General coliforms 1.57 X [10.sup.2] --
 Staphylococcus aureus 2.71 X [10.sup.2] 1.33 X [10.sup.3]
 Enterococcus sp. 1.89 X [10.sup.2] 1.35 X [10.sup.3]
Squirrel #4
 Enterobacter hermanii -- --
 Klebsiella sp. 1.63 X [10.sup.2] --
 Proteus mirabilis -- 8.10 X [10.sup.1]
 Escherichia coli 5.40 X [10.sup.1] --
 General coliforms -- --
 Staphylococcus aureus 8.20 X [10.sup.1] 2.44 X [10.sup.2]
 Enterococcus sp. 6.00 X [10.sup.1] 1.71 X [10.sup.3]
Squirrel #5
 Enterobacter hermanii 5.20 X [10.sup.1] --
 Klebsiella sp. 1.76 X [10.sup.3] --
 Proteus mirabilis -- --
 Escherichia coli 8.10 X [10.sup.1] --
 General coliforms 2.98 X [10.sup.2] --
 Staphylococcus aureus 1.36 X [10.sup.2] 2.70 X [10.sup.1]
 Enterococcus sp. 1.76 X [10.sup.3] 5.13 X [10.sup.2]

Bacteria Dates of Collection (1997-98)
 4 February 5 March

Squirrel #1
 Enterobacter hermanii 8.90 X [10.sup.5] 5.70 X [10.sup.3]
 Klebsiella spp. -- --
 Proteus mirabilis 2.00 X [10.sup.5] --
 Escherichia coli 1.62 X [10.sup.4] --
 General coliforms 4.05 X [10.sup.4] 1.35 X [10.sup.4]
 Staphylococcus aureus 4.60 X [10.sup.4] --
 Enterococcus sp. 1.30 X [10.sup.5] 1.06 X [10.sup.4]
Squirrel #2
 Enterobacter hermanii 2.30 X [10.sup.6] 2.70 X [10.sup.4]
 Klebsiella sp. -- --
 Proteus mirabilis -- --
 Escherichia coli -- --
 General coliforms -- --
 Staphylococcus aureus 2.00 X [10.sup.6] 2.70 X [10.sup.6]
 Enterococcus sp. 4.32 X [10.sup.5] 3.82 X [10.sup.3]
Squirrel #3
 Enterobacter hermanii 5.18 X [10.sup.4] 2.09 X [10.sup.4]
 Klebsiella sp. -- --
 Proteus mirabilis 1.04 X [10.sup.5] --
 Escherichia coli 2.72 X [10.sup.4] --
 General coliforms -- 1.80 X [10.sup.3]
 Staphylococcus aureus 5.50 X [10.sup.3] 2.69 X [10.sup.3]
 Enterococcus sp. 2.15 X [10.sup.5] 2.18 X [10.sup.3]
Squirrel #4
 Enterobacter hermanii 9.70 X [10.sup.4] --
 Klebsiella sp. -- --
 Proteus mirabilis 5.65 X [10.sup.4] --
 Escherichia coli -- --
 General coliforms 5.40 X [10.sup.3] --
 Staphylococcus aureus 2.15 X [10.sup.4] 2.29 X [10.sup.3]
 Enterococcus sp. 2.04 X [10.sup.5] 3.60 X [10.sup.4]
Squirrel #5
 Enterobacter hermanii 1.06 X [10.sup.5] 7.10 X [10.sup.3]
 Klebsiella sp. -- --
 Proteus mirabilis 5.44 X [10.sup.4] 3.60 X [10.sup.3]
 Escherichia coli 2.34 X [10.sup.5] 1.89 X [10.sup.3]
 General coliforms 3.65 X [10.sup.4] 2.68 X [10.sup.2]
 Staphylococcus aureus 2.45 X [10.sup.4] 3.29 X [10.sup.3]
 Enterococcus sp. 3.27 X [10.sup.4] 6.10 X [10.sup.3]

Bacteria Dates of Collection (1997-98)
 9 April

Squirrel #1
 Enterobacter hermanii 7.40 X [10.sup.2]
 Klebsiella spp. --
 Proteus mirabilis 1.30 X [10.sup.3]
 Escherichia coli --
 General coliforms --
 Staphylococcus aureus 1.25 X [10.sup.2]
 Enterococcus sp. 4.20 X [10.sup.3]
Squirrel #2
 Enterobacter hermanii 9.80 X [10.sup.3]
 Klebsiella sp. --
 Proteus mirabilis --
 Escherichia coli --
 General coliforms --
 Staphylococcus aureus 7.13 X [10.sup.3]
 Enterococcus sp. 2.10 X [10.sup.3]
Squirrel #3
 Enterobacter hermanii 2.10 X [10.sup.3]
 Klebsiella sp. 4.12 X [10.sup.3]
 Proteus mirabilis 1.70 X [10.sup.3]
 Escherichia coli 4.30 X [10.sup.3]
 General coliforms 1.20 X [10.sup.3]
 Staphylococcus aureus 8.81 X [10.sup.2]
 Enterococcus sp. 1.52 X [10.sup.3]
Squirrel #4
 Enterobacter hermanii --
 Klebsiella sp. --
 Proteus mirabilis --
 Escherichia coli --
 General coliforms --
 Staphylococcus aureus --
 Enterococcus sp. 1.62 X [10.sup.3]
Squirrel #5
 Enterobacter hermanii 1.12 X [10.sup.3]
 Klebsiella sp. --
 Proteus mirabilis --
 Escherichia coli 1.22 X [10.sup.3]
 General coliforms 6.81 X [10.sup.1]
 Staphylococcus aureus 3.72 X [10.sup.1]
 Enterococcus sp. 2.47 X [10.sup.1]


An earlier version of this manuscript by the first author fulfilled part of the requirements for the Master of Science degree in biology from Midwestern State University. We thank graduate committee members Marla Stevens and Bill Cook for their expertise offered during the course of the investigation, and for their constructive comments on earlier drafts of the manuscript. Clyde Jones and Jim Goetze critically reviewed a later draft of the work.


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Bonnie L. Blossman-Myer and Frederick B. Stangl, Jr.

Department of Biology, Midwestern State University

Wichita Falls, Texas 76308.

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Author:Blossman-Myer, Bonnie L.; Stangl, Frederick B., Jr.
Publication:The Texas Journal of Science
Geographic Code:1USA
Date:May 1, 1999
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