The objective of the study reported herein was to document the myxomycetes (slime molds) associated with bison dung and cow dung in the temperate prairies of North Dakota. We hypothesized that there is a significant difference between the myxomycetes on bison dung and cow dung. The data were analyzed for any significant statistical differences between the two types of dung, using jmp statistical software. We concluded that there was a significant difference between bison dung and cow dung, with statistical p-value <0.0001*; hence, we rejected the null hypothesis. Reciprocal Simpson’s diversity indices for bison dung and cow dung were 3.01 and 3.03, respectively. The pH recorded for dung samples ranged between 6.48 and 7.41, with the majority of species associated with weak acidic-weak basic conditions.

 Keywords: diversity, coprophilous myxomycetes, bison, cow, watershed, standing rock

The myxomycetes have been mistakenly classified as part of the Kingdom Fungi for a long time, but they are now known to be quite unrelated, since one stage in the myxomycetes life cycle can move, and these organisms lack chitin in their cell walls. The two groups of myxomycetes that have been best described are plasmodial slime molds and cellular slime molds.¹ Slime molds are eukaryotes, and much is not yet known about them, including their association with their various habitats. It is known that the decaying dung of herbivores represents a substrate for myxomycetes.² Though the vast majority of the species associated with dung are presumed to be secondary inhabitants, a few examples appear to be largely or even completely restricted to this substrate.³ There have been relatively few studies of some species, such as coprophilous (sometimes referred to as fimicolous) myxomycetes, so ecological and distributional data for such species are still limited. Myxomycetes diversity varies widely with ecological niches, microhabitats and substrata. Some have narrow ecological niches and are restricted to particular substrata, while others do not. They are important to the environment because they eat decaying vegetation and enhance the nutrient cycle. They also clean the environment by eating bacteria, fungi, and even other slime molds. These characteristics may be beneficial to animals as well as other life forms they associate with. The fact that the decaying dung of herbivores represents a substrate for myxomycetes (plasmodial slime molds or myxogastrids) is well established.² The vast majority of the species associated with dung are presumed to be secondary inhabitants, and a few examples appear to be largely or even completely restricted to this substrate.³

The study was conducted on the Missouri River watershed in the Standing Rock Sioux Reservation of North Dakota. The coordinates of the study site were 45⁰57’35’’ N and 100⁰56’24’’ E, and the site was located within the Sitting Bull College bison pasture and an adjacent cow pasture at Selfridge in the Standing Rock Reservation in Sioux County (Figure 1).

A series of samples of bison dung and cow dung samples were collected from Sitting Bull College bison pasture and an adjacent cow pasture at Selfridge in Standing Rock Reservation, Sioux County. All samples were placed in paper bags, the bags numbered, marked with the type of dung they contained and returned to the laboratory, where they were allowed to air-dry for several days. Later, the entire set of samples was sent to the Eumycetozoan laboratory at the University of Arkansas for processing. At the University of Arkansas, the samples were used to prepare moist chamber cultures. These were prepared in the manner described by Stephenson & Stempen² and consisted of plastic disposable Petri dishes (100mm diameter) lined with filter paper. Two Petri dishes were prepared from each sample giving a total of twenty (20) Petri dishes (10each for bison and cow dung samples) from 5sample plots. A sample plot was represented by grazing fields within the larger pasture. The grazing fields were demarcated by fences. Enough sample material was placed in each dish to cover most of the bottom, and then the material was moistened with distilled water. After a period of approximately 24hours, the pH of each culture was determined with a portable pH meter and then excess water in the Petri dish was poured off. Moist chamber cultures were placed out of direct sunlight and maintained at room temperature. Water was added to these cultures when necessary to maintain moist conditions, and the cultures were checked at least twice each week for evidence (either plasmodia or fruiting bodies) of myxomycetes over a period of three months. Data were collected through observation, and recorded species diversity and abundance on each sample. A hand magnifying lens and microscopes were used for this investigation. Whenever fruiting bodies were observed, they were recorded, removed from the moist chamber culture, air-dried and placed in small pasteboard boxes for long-term storage. A specimen was defined as a record of the occurrence of one or more fruiting bodies of a particular species of myxomycetes in a single culture. Some specimens consisted of only a single fruiting body, whereas others consisted of numerous fruiting bodies in a single culture (Figure 2).

Figure 2 Representative examples of the two types of dung.

A total of 6species were recorded from the thirty dung substrate cultures. Bison dung substrate had a lower mean pH (Figure 3), but higher frequency mean (Figure 4). The species found in bison dung had higher frequency compared to those found in cow dung. Arcyria cinerea, Didymium difforme and Kelleromyxa fimicola do well at a lower pH compared to Perichaena depressa, Perichaena liceoides and Physarum sp. (Figure 5). The term pH refers to the negative logarithm of hydrogen ion concentration (-log [H⁺]). There was significant difference between myxomycetes species found in bison and cow dung substrates with the Wald p-value of less than 0.001 ([Wald p-Value <.0001*] Table 1). The Walden test computes the score test for coefficient of generalized linear model between two means. It is also referred to as chi-square. The analysis of the data revealed that the standard deviation of the sample distribution mean (standard error) remained very low at 0.18, while the percent variance explained by the model value (R-square) was high at 0.86 (86%) (Table 2). This implies that the regression line perfectly fits the real data point. The variation in dependent variable (myxomycetes species) is well explained by independent variable (the subjects). The subjects here refer to bison and cow dung substrates. Root mean square error (RMSE) is a measure of how much error exists between two data sets. It remains low (0.135), indicating a better fit (Table 2). Of the 29 coprophilous myxomycetes recorded, about 55%, 35% and 10% were recorded in the first, second and third months respectively (Table 3). Arcyria cinerea species was the laggard, coming out only in the third month, while Didymium difforme and Perichaena liceoides species were found in each of the three months period. No single species was recorded from cow dung in the third month.

Figure 3 Fit pH by Subject.

Figure 4 Fit Frequency by Subject.

Figure 5 Fit pH by Species.

Species frequency on bison dung were 8, 6, 4 and 1 for Kelleromyxa fimicola, Perichaena liceoides, Didymium difforme, and Arcyria cinerea respectively. On cow dung, the values were 5, 2, 2, and 1 for Perichaena liceoides, Perichaena depressa, Didymium difforme and Physarum species respectively (Table 4 & Figure 4). The result shows that the genus Didymium is the most frequent, while Physarum is the least frequent (Figure 6). Species abundance (the number of individuals per species) was higher on the bison dung, the values being nineteen (19), and only ten (10) on cow dung substrate. Coprophilous myxomycetes biological diversity (biodiversity) refers to the variety of myxomycetes species on the dung substrates. Biodiversity index estimation takes into consideration species richness and species evenness. We defined species evenness as how evenly the myxomycetes species were represented in the dung substrates (Table 4). Both bison dung and cow dung samples had four (4) species each, thus the same species richness (the number of different species present in a substrate) value. Arcyria cinerea, Didymium difforme, Kelleromyxa fimicola, and Perichaena liceoides, were on bison dung substrate while Didymium difforme, Perichaena depressa, Perichaena liceoides, and Physarum species were on cow dung substrate (Table 2 & Table 4).

Figure 6 Fit pH by Species.

The study reveals that there is a significant difference in species composition between bison dung and cow dung substrates. Some myxomycetes species take more than two months to establish on dung substrates. Coprophilous myxomycetes are found in both weak acidic and weak basic substrate conditions. Future study will concentrate on the species’ characterization using molecular tools.^4‒20

The authors wish to acknowledge support provided by the Biological & Environmental Sciences Department of Sitting Bull College, Department of Biotechnology of Shoolini University India, and Eumycetozoan Laboratory, Department of Biological Sciences, University of Arkansas Fayetteville. We thank Thomas DeVille of Sitting Bull College for his participation during field sample collection work. Special appreciation is extended to the US National Science Foundation for supporting this research under EAGER grant #1755745.

Authors declare that there is no conflict of interest.

None.

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