Southeastern Naturalist 681 A.W. Rollins and S.L. Stephenson 22001166 SOUTHEASTERN NATURALIST 1V5o(4l.) :1658,1 N–6o8. 84 Myxomycete Assemblages Recovered from Experimental Grass and Forb Microhabitats Placed Out and Then Recollected in the Tallgrass Prairie Preserve, OK Adam W. Rollins1,* and Steven L. Stephenson2 Abstract - Results obtained in studies of grassland myxomycetes (plasmodial slime molds or myxogastrids), based on the species appearing in moist-chamber cultures, have indicated that forb microhabitats yield considerably more collections than grass microhabitats. We experimentally evaluated this pattern at the Tall Grass Prairie Preserve in Oklahoma by using litterbags prepared with autoclaved samples of grasses and forbs. We obtained a total of 162 collections representing 20 species; Perichaena pedata and Diderma effusum were the dominant species present. Total number of collections, species richness, and species diversity were significantly higher for forb microhabitats when compared to grass microhabitats. These results corroborate previous reports and demonstrate the utility of using litterbags as an experimental approach to assess myxomycete diversity and to confirm or refute observations from previous ecological studies. Introduction Ecological studies of myxomycetes (also known as plasmodial slime molds or myxogastrids) have been biased toward forest ecosystems in the northern hemisphere, with particular emphasis placed on temperate deciduous forests (Rollins and Stephenson 2011). In an effort to develop a more complete understanding of the distribution and ecology of these organisms, a number of recent investigations have been directed towards less-studied ecosystems such as Neotropical forests (Rojas and Stephenson 2007, Schnittler and Stephenson 2000), Old World tropical forests (Tran et al. 2006, Wrigley de Basanta et al. 2013), boreal forests (Schnittler and Novozhilov 1996), tundra (Stephenson et al. 2000), deserts (Lado et al. 2007), and grasslands (Rollins and Stephenson 2013). Remarkably, studies of desert and grassland ecosystems have revealed that myxomycetes can be quite abundant and diverse even under rather xeric conditions. Decomposing forbs (broadleaf plants) and grasses represent microhabitats from which myxomycetes can be recovered using the moist-chamber culture technique (Fischer and Stephenson 2014, Gabel et al. 2010, Kilgore et al. 2009, Rollins 2007, Saunders and Saunders 1900). Interestingly, cultures prepared with forbs have been reported to be considerably more productive than those prepared with grasses. For example, this pattern was consistent across 9 grassland study areas in the midwestern US, where 65% of the collections from moist chambers (prepared with forbs and grasses) were obtained from forbs (Rollins and Stephenson 2013). In another study, 1Department of Biology’s Cumberland Mountain Research Center, Lincoln Memorial University, Harrogate, TN 37752. 2Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72710. *Corresponding author - Adam.Rollins@LMUnet.edu. Manuscript Editor: Richard Baird Southeastern Naturalist A.W. Rollins and S.L. Stephenson 2016 Vol. 15, No. 4 682 which used a combination of field surveys and moist-chamber cultures, Gabel et al. (2010) recovered only 9% of their collections from grass litter, further suggesting that grass microhabitats represent a poor substrate for many myxomycetes. It seems that fundamental ecological differences exist between decomposing grasses and forbs that affect their suitability as substrates for myxomycetes. These differences may be related to a wide range of variables such as the respective rates of decomposition for forbs and grasses, quantitative differences in the nutrient pools associated with each microhabitat, or differences in the types and/or abundances of the microbes myxomycetes utilize as a food resource. Moore and Spiegel (1995, 2000) examined the distribution patterns of protosteloid amoebae (often referred to as protostelids) by placing autoclaved sections of wheat straw into a series of study areas. After a period of time, the straws were collected, placed in laboratory culture, and monitored for the occurrence of protosteloid amoebae. The results obtained through the use of this technique were comparable to results obtained by culturing naturally occurring substrates. The objective of the present study was to assess the differences in the assemblages of myxomycetes associated with the 2 microhabitats represented by the sterilized litter of forbs and grasses placed in nylon-mesh bags and subjected to field conditions in the Tallgrass Prairie Preserve in Oklahoma. Study Site The study was carried out in the 15,432-ha Tallgrass Prairie Preserve (36°51'40.17"N, 96°25'15.93"W), located in the Osage Plains section of the Central Lowland physiographic province of Oklahoma. The underlying geology is characterized by chert-bearing limestone and shale, with rocky soils that have prevented extensive farming, making this area the largest remaining tract of undisturbed tall grassland in North America. The study site has an elevation of 322 m and receives an average of 1118 mm annual precipitation. The dominant vegetation consists of Andropogon gerardi Vitman (Big Bluestem), Sorghastrum nutans (L.) Nash (Indian Grass), Panicum virgatum L. (Switchgrass), and Schizachyrium scoparium (Michx.) Nash var. scoparium (Little Bluestem) (Coppedge and Shaw 1998). The preserve is grazed by Bos taurus L. (Cattle) and Bison bison Hamilton Smith (American Bison), and prescribed burning is utilized at various scales and frequencies. Methods In May 2007, we collected samples of the dead and decomposing litter of forbs and grasses from the Tallgrass Prairie Preserve study site. We placed the samples in brown paper bags and transported them to the laboratory at the University of Arkansas, where we autoclaved all samples and stored them in plastic freezer-bags. We constructed from a nylon-mesh hardware cloth (1-mm2 openings) fifty-four 12 cm x 15 cm litterbags, similar in design to those used in litter-decomposition studies (e.g., Bradford et al. 2002). We filled 27 bags each with grass litter or forb litter. We closed the bags with a glue sealant and numbered each with fabric paint and a Southeastern Naturalist 683 A.W. Rollins and S.L. Stephenson 2016 Vol. 15, No. 4 permanent black marker, autoclave-sterilized, and stored them in plastic freezerbags for transport to the field-study site. In June 2007, we placed the bags in a grid pattern with alternating rows of grass litter and forb litter at the Tallgrass Prairie Preserve study site. Each row contained 9 bags, with each bag separated, in all directions by 1 m. We secured every bag to the ground with a metal landscaping staple (Fig. 1). The litterbags represented experimental implants of the grass-ground and forb-ground microhabitats. At 3, 6, and 9 months, we relocated and collected a randomly predetermined subset consisting of 12 litterbags (6 forb and 6 grass). We took the bags to the laboratory at the University of Arkansas and allowed them to air dry for ~1 week. We prepared 3 moist-chamber cultures from each litterbag as described by Stephenson and Stempen (1994); thus, 18 forb-litter and 18-grass litter cultures were prepared during each collection period, resulting in an overall total of 108 cultures prepared from the implanted litterbags. As a control, we prepared an additional 20 cultures (10 forb and 10 grass) with sterilized substrate material that had never been placed out in the field. We ranked species of myxomycetes detected in the moist-chamber cultures as A = abundant (>3% of total collections), C = common (>1.5%–3%), O = occasional (>0.5%–1.5%), and R = rare (≤0.5%) as described by Stephenson et al. (1993). We pooled values obtained from each triplicate set for subsequent analysis. We calculated relative abundance, diversity indices, and percent similarity as described in Rollins et al. (2010). We employed the nonparametric Mann-Whitney U test and the parametric unpaired 2-sample t-test to evaluate the statistical significance between Figure 1. (A) The general aspect of the Tallgrass Prairie Preserve study site, and (B) a litterbag secured to the ground by a metal landscaping staple. Southeastern Naturalist A.W. Rollins and S.L. Stephenson 2016 Vol. 15, No. 4 684 treatments at each time interval (i.e., 3, 6, 9 months). The data often failed to approximate a normal distribution and the sample size for each pooled treatment was small (n = 6) (i.e., 3, 6, 9 months); thus, statistical significance was recognized only when both tests indicated this outcome. We applied diversity t-tests according to Poole (1974) and conducted all data analyses using the Past 3.06 statistical software package (Hammer et al. 2001). Myxomycete nomenclature follows the morphospecies concepts of Lado (2016). Results The 20 control cultures did not produce a single plasmodium or fruiting body. We obtained 162 collections representing 20 species from the moist-chamber cultures prepared with material from the recovered litterbags (Table 1). The forb microhabitat was the most productive—116 collections assigned to 18 species representing 72% of the total number of collections. As assessed by percent similarity, the myxomycete assemblages recorded from the 2 microhabitats were rather dissimilar (Table 1). Perichaena pedata and Diderma effusum were the most frequently recovered species, accounting for 54% of the total collections obtained in the study (Table 2). These 2 species were common to both microhabitats, but represented 84% of the collections obtained from grasses, where no other species had a relative abundance of ≥5% . Forb microhabitats were characterized by a significantly greater (a) number of collections, (b) species richness, and (c) species diversity when compared to grass microhabitats (Fig. 2). Species richness and diversity did not vary appreciably between the 3- and 6-month samples, but there was a noticeable decrease for the 9-month samples. The recovery of Arcyria afroalpina and an unidentified Lepidoderma (both from forbs) was particularly noteworthy. Discussion The results of the present study support previous reports that forb microhabitats are considerably more productive for myxomycetes than are grass microhabitats (e.g., Rollins and Stephenson 2013). The mechanism(s) resulting in this consistent and recurring pattern are currently unknown. However, the impacts of differential decomposition rates, nutrient quality and availability, and the types and/or abundances of food organisms available represent areas that warrant further study. Table 1. Summary data for each microhabitat sampled in each collection interval and pooled data for the entire study. 3 Months 6 Months 9 Months Forbs Grasses All Forb Grass Forb Grass Forb Grass pooled pooled pooled Number of collections 42 24 50 19 24 3 116 46 162 % of collections 63.6 36.4 72.5 27.5 88.9 11.1 71.6 28.4 100 Species richness 11 5 13 5 8 2 18 8 20 Percent similarity 57.1 38.5 37.5 52.2 - Southeastern Naturalist 685 A.W. Rollins and S.L. Stephenson 2016 Vol. 15, No. 4 Prior to carrying out the study, we hypothesized that species diversity and richness would increase with time for each of the 2 types of microhabitats. The forb-litter samples displayed a slight increase in species richness and diversity between the 3- and 6-month time intervals, whereas the values recorded for the grass-litter samples remained relatively unchanged. However, both species richness and diversity decreased for both sets of samples at the 9-month interval. Although this result was unexpected, it is noteworthy that during the week prior to the 9-month collection interval, the study area experienced torrential downpours and flooding. It seems possible that these conditions could have washed away or otherwise depressed the numbers of propagules (i.e., myxamoebae and microcysts) present at the time we collected the samples. These data might provide some insight into the short-term effects of extreme rain events on myxomycete dynamics at a given locality. This possibility is particularly interesting because it was proposed (Alexopoulos 1970) that the tropics might be characterized by a lower richness and diversity of myxomycetes due to the impact of rainfall as a disturbance factor. The assemblages at both substrates were most similar at the 3-month sampling period (PS = 57.1). We obtained this result because the 2 most-common species, Table 2. The overall relative abundance (RA) and ranking of the species of myxomycetes obtained from the experimentally implanted forb and grass microhabitats at the Tallgrass Prairie Preserve. Perichaena pedata and Diderma effusum were the dominant species, accounting for 54% of all collections obtained in the entire study. Ranks: A = abundant (>3% of total collections), C = common (>1.5%-3%), O = occasional (>0.5%-1.5%), and R = rare (≤0.5%) as described by Stephenson et al. 1993. A dash indicates that the species was not present. Forb Grass Substrates Rank Species Forb RA Rank Grass RA Rank pooled pooled Perichaena pedata (Lister & G. Lister) G. 25.9 A 54.3 A 34.0 A Lister Diderma effusum (Schwein.) Morgan 16.4 A 30.4 A 20.4 A Arcyria cinerea (Bull.) Pers. 16.4 A 0.0 - 11.7 A Lamproderma scintillans (Berk. & Broome) 14.7 A 4.3 A 11.7 A Morgan Didymium difforme (Pers.) S.F. Gray 4.3 A 2.2 C 3.7 A Perichaena depressa Libert 5.2 A 0.0 - 3.7 A Didymium ochroideum G. Lister 1.7 C 2.2 C 1.9 C Perichaena chrysosperma (Currey) Lister 1.7 C 2.2 C 1.9 C Physarum cinereum (Batsch) Pers. 2.6 C 0.0 - 1.9 C Arcyria afroalpina Rammeloo 1.7 C 0.0 - 1.2 O Hemitrichia pardina (Minakata) B. Ing 1.7 C 0.0 - 1.2 O Lepidoderma sp. A 1.7 C 0.0 - 1.2 O Perichaena corticalis (Batsch) Rostaf. 1.7 C 0.0 - 1.2 O Didymium anellus Morgan 0.0 - 2.2 C 0.6 O Didymium sp. A 0.0 - 2.2 C 0.6 O Licea operculata (Wingate) G.W. Martin 0.9 O 0.0 - 0.6 O Perichaena liceoides Rostaf. 0.9 O 0.0 - 0.6 O Perichaena vermicularis (Schwein.) Rostaf. 0.9 O 0.0 - 0.6 O Physarum notabile T. Macbr. 0.9 O 0.0 - 0.6 O Trichia contorta (Ditmar) Rostaf. 0.9 O 0.0 - 0.6 O Total 100.0 100.0 100.0 Southeastern Naturalist A.W. Rollins and S.L. Stephenson 2016 Vol. 15, No. 4 686 Diderma effusum and Perichaena pedata, colonized both substrates at a relatively equal frequency. However, after 6 months, the frequency of recovery of these 2 species decreased substantially for grasses, while they remained consistent on forbs, and forbs also continued to gain myxomycete species. It seems that the mostcommon species in the area quickly colonized the sampled substrates, but only persisted on the most suitable substrate represented by forbs. Throughout the course of the study, the assemblages of species associated with forb litter remained fairly similar, while the assemblages associated with grass litter were rather dissimilar. These results suggest that the assemblages of myxomycetes associated with forbs may be more stable, whereas those associated with grasses may be more transient. The possible stability associated with a particular microhabitat warrants further investigation. As mentioned earlier, this situation may be related to the fundamental biotic and abiotic differences that exist between the 2 microhabitats. We obtained what appears to represent an undescribed species of Lepidoderma from the forb samples in the current study as well as from the samples processed during a previous study carried out in the Sheyenne National Grasslands (Rollins Figure 2. Comparisons of (A) mean number of collections per sample, (B) mean species richness per sample, (C) Shannon diversity index, and (D) Simpson’s index of diversity for forb microhabitats (shaded bars) and grass microhabitats (open bars) at each collection interval and all intervals pooled. Forb microhabitats had significantly greater values for all comparisons with the exception of the 9-month Simpson index of diversity (D). Error bars represent standard error of the mean (A and B) and standard deviation (C and D). We set an α value of 0.05 for all tests. * indicates a significant difference between microhabitats. Southeastern Naturalist 687 A.W. Rollins and S.L. Stephenson 2016 Vol. 15, No. 4 and Stephenson 2013). These collections require further study in order to confirm their taxonomic status. It is also noteworthy that the 2 collections of Arcyria afroalpina represent a rare species typically recorded only from tropical regions (Lado and Wrigley de Basanta 2008, Rojas et al. 2010). Numerous descriptive studies have focused on myxomycete occurrence patterns across various biomes and microhabitats. However, very little work has been done that utilizes experimental manipulation to evaluate fundamental ecological hypotheses. Myxomycetes are abundant in virtually every terrestrial microhabitat and produce macroscopic fruiting bodies that are easily collected and cultured; thus, they represent excellent model organisms to study microbial ecology. Although myxomycete studies have been biased toward forested ecosystems, grassland ecosystems represent a simpler system in which to utilize myxomycetes to address broad ecological questions. Grasslands hold great potential to study the role of microorganisms in ecosystem function, biogeochemical processes, and functional traits. The results presented herein are drawn from 54 independent samples placed across a single location covering an area less than 54 m2 and provide a limited ability to make large-scale inferences across the grassland biome. The ultimate significance of our study is that it demonstrated the utility of using litterbags as an experimental approach for assessing myxomycete diversity. This technique has great promise as a tool to support or refute observations made as part of previous ecological studies. Because myxomycetes are relatively easy to isolate from samples of dead plant material under laboratory conditions, a similar approach could be employed to investigate a number of other aspects of the distribution and ecology of this group of organisms. 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