Investigating the Effects of Agricultural Nutrient Pollution on a Semi-aquatic. Bryophyte. Morgan B. Elfelt 1. (Morgan Elfelt:

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1 Investigating the Effects of Agricultural Nutrient Pollution on a Semi-aquatic Bryophyte Morgan B. Elfelt 1 1 Minnesota State University Moorhead, th Ave. S. Moorhead, MN USA. (Morgan Elfelt: elfeltmo@mnstate.edu) Abstract Bryophytes, mosses, liverworts and hornworts, are a group of non-lignified plants that have been found in many habitat types on all seven continents. Here in centralwestern Minnesota mosses are common in wetlands that are surrounded by agricultural fields where soils are typically high in nitrogen. The effect of this nitrogen pollution on growth morphology of a moss was investigated to determine whether plants in these areas are adapted to high levels of nutrients. Multiple individuals of the moss Brachythecium acuminatum were collected from five wetlands in agricultural and prairie areas with high and low concentrations of soil nitrogen, respectively. Plants were cultured in three levels of nitrogen to determine the degree to which differences in growth rates between the two population types is due to genes or phenotypic plasticity. The treatments were shown to have little effect on growth, but the plants collected from high nutrient levels outgrew those plants collected from lower nutrient levels in a common garden. This along with calculations of heritability of branch length suggests that these plants are locally adapted to their higher nitrogen conditions in wetlands impacted by agriculture. Keywords: Brachythecium, bryophyte adaptation, morphology, heritability

2 Introduction Bryophytes are a group of non-vascular plants that have been extensively studied for years by botanists and other scientists around the world. This ability to survive and reproduce in such a wide variety of habitats may be related to high phenotypic plasticity (Bates 1998). Some of the many environments they inhabit are in areas with large amounts of land area under agricultural cultivation. Fertilizers commonly used in agriculture contain high levels of nitrogen, phosphorus, and potassium to promote higher yield of harvested plants. However, the nutrients that are not absorbed by plants run off into surrounding areas, causing elevated levels of these nutrients (Carpenter et al. 1998). Especially in wetland areas, this nutrient pollution greatly alters the species distribution and abundance of plants. Previous studies have found that the effects of different nutrient levels vary considerably across bryophyte species. For example, Bergamini and Peintinger (2002) found that increased nitrogen levels did not have a significant effect on the growth of the moss, Calliergonella cuspidata. Other studies found that increased nutrient levels led to decreased bryophyte biomass as well as decreased species diversity in natural fens in Switzerland over time (Bergamini and Pauli 2001) and decreased bryophyte richness in a dry grassland (Aude and Ejrnaes 2005). Bates (1994) found that growth of the moss, Pseudoscleropodium purum, was stimulated by the addition of nutrients, whereas the growth of the moss, Brachythecium rutabulum, was not stimulated. None of these papers have investigated whether these bryophyte populations were locally adapted to nutrient levels. Another aspect of bryophyte growth that could be impacted by increased nitrogen levels is the growth strategy or life-form of the bryophytes. In a mini-review of

3 the literature on this topic, Bates noted the correlation between life-form and water and light levels in natural habitats (1998). However, few studies have been conducted to determine the impact of environmental nutrient levels on growth strategies. A similar review done by During discusses the competitive advantages of moss adopting a spreading growth form (1979), which would suggest that plants in an area with limited nutrients would benefit by employing an extensively branched growth form. This experiment was designed to test whether plants collected from locations with high and low levels of nitrogen will grow equally well when cultured in vitro in varying nitrogen levels. I tested the hypothesis that plants collected from areas with low nitrogen content would grow better in vitro in environments with low nitrogen and plants collected from areas with high nitrogen content would grow better in vitro in environments with high nitrogen content, indicating local adaptation Methods Study Organism. Brachythecium acuminatum (Hedw.) Aust. is a pleurocarpous moss native to North America and characterized by a short, thick capsule. This moss is abundant in the wetlands of northwestern Minnesota, and ranges from Saskatchewan to Texas and east to New Brunswick and Florida, often found at the base of trees and on rocks and soil (Crum 1983). In northwestern Minnesota, B. acuminatum grows abundantly on the moist soil at the base of cattails and sedges in the wetlands found across the prairie pothole region. Collection and maintenance. Specimens of B. acuminatum were collected from five state owned wildlife management and waterfowl production areas in Clay County,

4 MN, USA between 4 September and 24 October 2009 (Table 1). Each location was classified as either agricultural (A) or prairie (P) based on the distance to the closest agricultural field that could act as a source of nutrient runoff. Locations greater than 40 meters from an agricultural field were considered prairie, and locations less than 40 meters away were considered agricultural. At each location, B. acuminatum samples of approximately 50 cm 2 were collected at least 5 meters apart to ensure genetic individuality. The number of samples taken at each site ranged from 8 to 17 based on abundance. The specimens were maintained in a growth chamber under 12:12 photoperiod of Minnesota State University Moorhead (MSUM) in Moorhead, MN for 5 months in 50-pot greenhouse trays, with pots approximately 5 cm in diameter and 7 cm deep. Pots were filled half way with clay cat-litter, and plugs of moss were placed on top of the clay. The pots were set in trays filled approximately cm deep with distilled water to provide moisture to the plants. Each individual was divided and grown in two adjacent pots. Water levels were checked twice weekly and supplemented as needed. Two soil samples were also collected at each site and analyzed for nitrogen and phosphorus content at the soil testing lab of North Dakota State University in Fargo, ND, USA. After approximately 4 months of growth to minimize parental environmental effects, replicate individual mosses from prairie and agriculture sites were planted on agar media containing different levels of nitrogen. The agar solution was prepared using 1% Bactoagar with distilled water, and Hoagland s Basal salt mixture without Nitrogen (Caisson Labs catalog number HOP03-1LT). The nitrogen levels in the media were

5 varied at 0, 3 ppm, and 7.5 ppm using solid ammonium nitrate (NH 4 NO 3 ), and ph was equilibrated to the control level (8.0) with NaOH. Petri plates 2 cm deep with a 5 cm radius were filled approximately 0.5 cm deep with media. At the time of experiment start on 15 Feb 2010, some moss had died and enough were living to use 6 individuals from 4 locations (C, U, H, and W) for a total of 24 individuals. Based on collection methods, each sample could be considered to be a genetic individual made up of a branching clone with all branches having identical genetic material. Forty-five 0.8 cm long apices were cut from each individual specimen and kept in a damp cloth until all specimens could be planted. For each individual, fifteen stems were planted in each of the three treatments. Because of the genetic identicalness of the stems, the observed differences in growth should result from environmental factors. Each treatment plate was split into 3 sections, and 5 stems were planted in each section by poking a small 2 mm section of the stem into the agar so the stems stood upright. All plates were covered, sealed with parafilm, and placed under artificial lights with a 12:12 photoperiod. Plate locations were shuffled every week to insure identical growth conditions. At the end of four and eight weeks, data were collected to quantify growth. At four weeks, five plant stems were randomly selected, and the number of growing branches was recorded as an indicator of growth. Branching data were analyzed using a nested analysis of variance (ANOVA) with population and treatment nested within habitat. Five plant stems were also removed (from one section of the plate). These stems were dried and weighed to calculate the growth of the plants in grams of dry mass gained. A nested ANOVA was again used with habitat and treatment as main effects to determine whether collection location (agriculture or prairie) had an effect on

6 the growth of the moss across treatments. At week 8, dry mass was again measured using a randomly harvested section of each plate. The longest branch was also measured on each of the harvested stems and branch length was analyzed using the nested ANOVA design. Broad-sense heritability (H 2 ) was calculated using variation among populations variation among and within populations using results of an ANOVA with branch length as the independent variable and population as a fixed factor. This was calculated from the mean square of populations (MS P ) and the mean square 116 of error (MS e ) using the formula H 2 MSP = MS + MS P e as described by Lynch and 117 Walsh (1998). 118 Results Soil from agriculture sites had significantly higher nitrogen levels than that from prairie sites (two-sample t 2 = , p = 0.04; Figure 1). At week four, dry mass did not differ significantly between populations or treatments (F 1,10 = 0.968, P = 0.480), but dry mass was significantly higher in plants from agricultural sites compared to prairie sites (F 1,1 = 6.016, P = 0.017; Figure 2). Branching did not differ significantly between populations or treatments (F 1,10 = 1.364, P = 0.219) or between habitat types (F 1,1 = 0.162, P = 0.689; Figure 3). However, the dry mass data has high standard error as many of the samples did not have enough mass to register on the most precise balance available. Dry mass data collected at week 8 similarly showed significantly higher values in agricultural plants compared to prairie plants (F 1,1 = , p < 0.001) but not between populations or treatments (F 1,10 = 0.831, p = 0.601; Figure 4). Branch length was significantly higher in plants from agricultural areas (F 1,1 = , p < 0.001) as

7 well as treatment/population (3.589, p = 0.001; Figure 5). Tukey homogenous subsets were also determined for branch length data by population (Figure 6). These subsets indicate similarities in branch length between the populations across all of the treatments. These results prompted further investigation into the heritability of these traits to determine if the observed effects were due to high plasticity or genetic factors. The broad-sense heritability of branch length was calculated using mean-square values from the ANOVA with branch length as the independent variable and population as a fixed factor. The resulting heritability was Discussion Growth patterns of B. acuminatum gathered from environments with differing proved to be significantly different. Plants from areas with elevated nitrogen levels from agricultural run-off had higher dry mass and longer branches on average than those plants collected from areas without runoff. These results combined with calculated heritability of branch length suggest local adaptation is occurring. These adaptations may be in response to differences in light availability between habitats with high and low nitrogen content. According to these results, the null hypothesis would be true, suggesting that B. acuminatum did not have high plasticity in differing growth conditions. However, the data also seem to indicate a difference in growth between the agricultural and prairie plants that was not dependent on the treatment. The calculated broad-sense heritability for branch length was shown to be very high, implying genetic differences between

8 populations in these two habitat types. This genetic difference could be the result of prolonged exposure to high levels of nutrients in agricultural fertilizers, influencing growth strategies. Experimental results for branch length are contrary to the nutrient competition based hypothesis (During 1979), however another possible explanation for this behavior is one proposed by Rincon and Grime where in areas with high nutrient levels, the vascular plants in the community are able to grow more productively. This growth blocks more sunlight than in areas where vascular plants are not as substantial and bryophytes are better able to use the scarce amount of sunlight reaching the ground by adopting a spreading growth strategy (1989). Further experiments incorporating light limitations would need to be carried out to determine if this effect could be occurring. Incorporating other pleurocarpous mosses into similar studies could determine if this adaptation is an isolated event or is occurring across many species in the prairie pothole region.

9 Literature Cited Aude, E. & R. Ejrnaes Bryophyte colonisation in experimental microcosms: the role of nutrients, defoliation and vascular vegetation. Oikos 109: Bates, J. W Responses of the mosses Brachythecium rutabulum and Psuedoscleropodium purum to a mineral nutrient pulse. Functional Ecology 8: , Is life-form a useful concept in bryophyte ecology? Oikos 82: Bergamini, A. & D. Pauli Effects of increased nutrient supply bryophytes in montane calcareous fens. Journal of Bryology 23: , & M. Peintinger Effects of light and nitrogen on morphological plasticity of the moss Calliergonella cuspidata. Oikos 96: Carpenter, S. R., N. F. Caraco, D. L. Correll, R. W. Howarth, A. N. Sharpley & V. H. Smith Nonpoint pollution of surface waters with phosphorus and nitrogen. Ecological Applications 8(3): Crum, H Mosses of the Great Lakes forest. 3 rd ed. Ann Arbor, MI: University of Michigan Herbarium. 184 During, H. J Life strategies of bryophytes: a preliminary review. Lindbergia 5: Lynch, M. & Walsh, B Genetics and analysis of quantitative traits. Sunderland, MA: Sinauer Assocaiates.

10 Rincon, E. & J. P. Grime Plasticity and light interception by six bryophytes of contrasted ecology. Journal of Ecology 77: Figure Legends Figure 1. Average nitrogen levels in soil at agricultural (A) and prairie (P) sites where moss were collected, showing a significant difference in mean. Figure 2. Average Dry Mass +/- SE for five stems of B. acuminatum harvested after 4 weeks of growth. Figure 3. Average Number of Branches +/- SE for five stems per plate of B. acuminatum counted after 4 weeks of growth. Figure 4. Average Dry Mass +/- SE for five stems of B. acuminatum harvested after 8 weeks of growth. Figure 5. Length of longest branch +/- SE for five stems of B. acuminatum harvested after 8 weeks of growth. Figure 6. Letters above bars indicate Tukey homogenous subsets of branch length data collected after 8 weeks of growth. Capital letters (C, U, W, and H) are used for labelling of four populations. 203

11 Table 1. GPS coordinates of collection locations of B. acuminatum and observational nutrient level classification based on distance to agricultural fields. Each location was labeled with a letter for easier identification. Location Latitude and Longitude Coordinates Classification Gruhl Wildlife N W Agricultural Management Area (G) Hoykens Waterfowl N W Agricultural Production Area (C) Highland Grove Wildlife N W Prairie Management Area (H) Ashmore Waterfowl N W Agricultural Production Area (U) Lofgren Waterfowl N W Prairie Production Area (W)

12 Figure

13 213 Figure Figure 3.

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15 220 Figure Figure 6.