Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 66 2015 SOUTHEASTERN NATURALIST 14(1):66–84 Earthworm Communities in Previously Glaciated and Unglaciated Eastern Deciduous Forests Kristine N. Hopfensperger1,* and Sarah Hamilton2 Abstract - Native earthworms were removed from forested ecosystems during the last glacial advance and have since been replaced with nonnative earthworm species. Nonnative earthworms can cause major changes in microbial and plant communities and nutrient cycling. In this study, we sought to compare the earthworm communities north and south of the last glacial terminus, and to examine correlations between plant communities and soil characteristics. In summer 2011, we measured the earthworm, herbaceous plant, and woody plant communities in 3 forests in southwestern Ohio and 3 forests in northern Kentucky. We also measured soil characteristics including moisture, pH, organic matter, and nitrate and ammonium content. We found no native earthworm species at any of our study sites; however, previously glaciated forests exhibited more diverse earthworm communities and included all ecological groups. Earthworm species richness increased with increased density of invasive woody plant species and decreased with increased soil ammonium. Scientists and managers should continue to survey the earthworm communities in forests to better understand the ranges of nonnative earthworms and the impacts they have on plant communities and nutrient dynamics. Introduction In the US, exotic earthworms are dramatically altering nutrient cycling in forest ecosystems (Bohlen et al. 2004a, c; Fisk et al. 2004; Groffman et al. 2004; Súarez et al. 2004) and have been tied to microbial, plant, and animal community changes in forests (Fisichelli et al. 2013; Frelich et al. 2006; McLean and Parkinson 2000a, b). While studies of nonnative earthworms have become common in the northern hardwood forests of the Northeast and Midwest in the past decades, earthworm surveys and research have been lacking in the Southeastern Plains ecoregion, including areas that span the last glacial terminus, such as southwestern Ohio and northern Kentucky. The most recent glacial advance in North America (the Wisconsin glaciation; 12,000–25,000 y ago) removed native earthworms from areas north of the glacial terminus (Gates 1970, Reynolds et al. 1974). Natural dispersal from unglaciated areas into the northern regions has been slow (Terhivuo and Saura 2006). The introduction of European and Asian earthworm species into these previously glaciated areas started in the 1700s with European settlement (Gates 1966) and continues today (Tiunov et al. 2006). Therefore, there is a mix of native and nonnative earthworm species in areas south of the glacial limits (Reynolds 1970, Reynolds et al. 1974, Stebbings 1962), but northern, previously glaciated regions are dominated by 1Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY 41099. 2Current address - Department of Forestry, University of Kentucky, Lexington, KY 40546. *Corresponding author - hopfenspek1@nku.edu. Manuscript Editor: Lance Williams Southeastern Naturalist 67 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 exotic earthworm species with few isolated observations of native species (Reynolds et al. 2002). An understanding of the dynamics between native and nonnative earthworm species is beginning to take shape (Hendrix et al. 2006, Kalisz and Wood 1995). Although data supporting resistance of native earthworms to invasion by nonnative earthworm species is scarce (Hendrix et al. 2006), studies demonstrating co-occurrence of native and nonnative earthworm species are more common (Abbott 1985, James 1991, Stebbings 1962). However, it has not been determined if co-existence is a persistent or transient state (Hendrix et al. 2006). Exotic earthworms are more prevalent in areas that have been moderately to severely disturbed (Kalisz and Dotson 1989, Kalisz and Wood 1995). Habitat disturbance may increase resource availability, allowing nonnative earthworms to out-compete or co-exist with native species (Fragoso et al. 1999, Winsome et al. 2006). The presence of native and nonnative earthworms can have a dramatic effect on soil-process characteristics, organic matter decomposition, soil structure, and other biota (Bohlen et al. 2004b, Hale et al. 2006, Hendrix et al. 2006); therefore, it is important to characterize the earthworm community to understand forestsoil ecosystem processes. There is no current literature regarding earthworms in southwestern Ohio; however, in 1928, Olson found no exotic Lumbricus terrestris L. (Nightcrawler) or L. rubellus L. (Red Worm) in southwestern Ohio, but did find exotic Allolobophora cholorticus Savigny and Aporrectodea turgida Eisen (Mottled Worm) (Olson 1928). In a more recent study in southeastern Kentucky, the exotic taxa Octolasion tyrtaeum Savigny, L. terrestris, L. rubellus, and L. castaneous Savigny were found on relatively small and scattered disturbed sites (Kalisz and Dotson 1989) in the more mountainous Appalachian Forest ecoregion. We are uncertain if these findings are applicable to the entire region south of the glacial terminus or restricted to the Appalachian Mountains. Characterization of the earthworm community will provide insight into the rates of forest ecosystem processes. For example, the results of many studies in northern hardwood forests indicate that exotic earthworm species significantly alter soil carbon, nitrogen, and phosphorus cycling (Bohlen et al. 2004a; Groffman et al. 2004; Suárez et al. 2004, 2006a). Perhaps the most striking alteration found in northern hardwood forests, is the potential of earthworms to transform these forests from global carbon sinks into carbon sources (Bohlen et al. 2004b, Lubbers et al. 2013). In the short-term, earthworm activity releases nutrients that can increase nutrient-cycling rates (Bohlen et al. 2004b, Groffman et al. 2004) leading to a net increase in carbon dioxide to the atmosphere (Fisk et al. 2004, Li et al. 2003, Lubbers et al. 2013). However, in a meta-analysis, Lubbers et al. (2013) reported that longer-term studies suggested initial carbon dioxide emissions decreased with time leading to stabilization of organic carbon in the soil. Native earthworm species may influence ecosystem processes for more of the year than nonnative species because native earthworms are better adapted to local climatic conditions than nonnative species (Callaham et al. 2001, James 1991). In addition, Lachnicht et al. (2002) found reductions in carbon and nitrogen Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 68 mineralization rates when native and nonnative species were in co-existence compared to when the nonnative species was alone. The nonnative earthworm species colonizing North America have highly invasive characteristics (James and Hendrix 2004) and can cause remarkable changes in soil structure and nutrient cycling depending on the ecological group to which they belong. In general, earthworms prefer moist soil with neutral to basic soil pH (Curry 1998). Earthworms require calcium to supply their calciferous glands (Canti and Pearce 2003), which produce calcium carbonate granules that moderate their blood carbon dioxide levels and can increase soil pH when excreted (Crang et al. 1968). Litter-dwelling epigeic species have minor impacts on soil structure and nutrient concentrations by only mixing the O horizon of the soil (McLean and Parkinson 1997a, b). However, soil-dwelling endogeic species are known to mix surface litter into the upper mineral soil horizons, thereby homogenizing the organic and mineral layers and wholly removing the litter layer (Alban and Berry 1994, Langmaid 1964). When species assemblages include both endogeic and anecic species which burrow up to 2 m in depth, nutrient concentrations change the most, compared to soil without anecic species present (Hale et al. 2005a). Native earthworm assemblages are generally dominated by endogeic species (Fragoso et al. 1999, Kalisz 1993), which may leave the soil surface open to invasion by epigeic exotic species. Earthworm activity can impact forest plant communities in a variety of ways. In field and mesocosm studies of a Pinus contorta Douglas ex Loudon (Lodgepole Pine) forest, earthworms stimulated a shift from a fungal-dominated to bacterialdominated soil (McLean and Parkinson 1998, 2000a, 2000b) causing the loss of important mycorrhizal–plant root relationships (Wardle 2002). However, field studies in a northern hardwood forest found that earthworm-induced elimination of the O soil horizon led to an increase in bacteria over fungi (Dempsey et al. 2011, 2013). Many native understory plants, such as Acer saccharum Marsh. (Sugar Maple), are dependent on mycorrhizal relationships (Brundrett and Kendrick 1988), and decrease in abundance when earthworms are present (Hale et al. 2006, Holdsworth et al. 2007). In fact, Lawrence et al. (2003) found a decrease of mycorrhizal fungi on Sugar Maple roots in earthworm-dominated plots. Earthworms can also alter plant communities by consuming, and thereby reducing, the litter layer (Eisenhauer et al. 2007, Gundale 2002, Hale et al. 2005a), which exposes plants to desiccation and a more mineral-rich soil (Frelich et al. 2006, Heneghan et al. 2007). These changes promoted by earthworms can result in reduced herbaceous-plant cover (Hopfensperger et al. 2011) and a shift toward a more graminoid-dominated plant community (Hale et al. 2006, Holdsworth et al. 2007, Nuzzo et al. 2009). Furthermore, earthworms can directly affect plant communities through seed predation, burial, and inducement or release of seed dormancy (Eisenhauer et al. 2009, Hopfensperger et al. 2011, Regnier et al. 2008). As earthworms disturb native plant communities, they may reduce competitive pressure thereby allowing invasive plant species to dominate. Nuzzo et al. (2009) found that nonnative plant cover was positively associated with earthworm biomass; however, there have been very few studies relating earthworms to invasive-plant dynamics. Southeastern Naturalist 69 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 We conducted surveys of the earthworm communities north (i.e., southwestern Ohio) and south (i.e., northern Kentucky) of the last glacial terminus to characterize current earthworm-community composition and to examine correlations between earthworm and plant communities and between earthworm communities and soil characteristics. We expected that: (1) earthworm communities in previously glaciated forests would be dominated by nonnative species, but that native and nonnative species would co-occur in earthworm communities of unglaciated forests; (2) because unglaciated forests have a more diverse earthworm community with increased ecosystem-process rates, these forests would have less soil organic matter and a higher percent cover of invasive plant species; and (3) earthworm density would increase with soil pH and with soil moisture. Methods Site description We sampled in 3 previously glaciated forests in Hamilton and Butler counties, OH (site 1 = Richardson Forest Preserve, site 2 = Winton Woods Park, site 3 = Miami Whitewater Forest) and 3 previously unglaciated forests in Campbell and Kenton counties, KY (site 4 = Hawthorne Crossing Conservation Area, site 5 = AJ Jolly Park, site 6 = Morning View Heritage Land) for a total of 6 sampled forests (Fig. 1 includes geographic coordinates). Both previously glaciated and unglaciated forests lie near the terminus of the last glacial advance (Ray 1974). The glaciated sites are included in the Till Plains section of the Central Lowland Physiographic Province (Fenneman 1916). The previously unglaciated sites are in the Outer Bluegrass Region of the Interior Low Plateau (Brockman 1998). The soils in all study forests are silt loam or silty clay loam and are classified as mesic Typic Hapludalfs (USDA NRCS http://websoilsurvey.nrcs.usda.gov). Climate in the study region is a continental type with cold winters (average January high temperature = -1 °C), hot summers (average July high temperature = 27 °C), and average annual precipitation of ~112 cm (NOAA 2013). Forests of the region have been thoroughly described by Dr. E. Lucy Braun and many others (Braun 1916, 1936, 1950; Bryant 1987, 2004; Kuchler 1964). The area is typical of the mixed mesophytic forest region characterized by dominant species including Fagus grandifolia Ehrh. (American Beech), Fraxinus americana L. (White Ash), Sugar maple, Quercus rubra L. (Red Oak), and Prunus serotina Ehrh. (Black Cherry). None of the forests studied are considered old growth and all of them have had some history of harvesting in the past (K.N. Hopfenperger, pers. observ.). Plant-community sampling We delinated three 400-m2 stands in each of the 6 forest sites and randomly placed one 5-m2 plot within each quadrant of each stand for a total of 12 plots per forest and 72 project plots. We chose stands with tree-canopy species and cover to minimize canopy effect on the earthworm communities and measured soil variables. All stands were dominated by Sugar Maple with a mix of other species, including Fraxinus (ash) and Aesculus (buckeye). We identified all trees within Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 70 each plot and measured their diameter at breast height (1.4 m; DBH) and also identified all saplings, seedlings, and shrubs within each plot. We recorded percent cover (modified methods of Braun-Blanquet 1964) of all herbaceous species within a 0.75-m radius around the center of the plot. We conducted our sampling in July because we thought that would be the time of year when the plant community was at peak biomass. Plant species richness, diversity—using the Shannon diversity index (Shannon and Weaver 1949),—and density of invasive herbaceous plant and woody species were calculated for each plot. Earthworm sampling We sampled earthworm communities in May 2011, when we felt conditions would be optimal for earthworm movement and sampling due to moisture and temperature conditions then. We designated a 30 cm by 30 cm subplot in the center of each plot, and carefully removed litter from the sample area. We slowly poured a solution of 4 L of water mixed with 40 g of ground yellow mustard seed over the plot to stimulate movement of earthworms to the soil surface for collection Figure 1. Map of 6 study sites sampled for earthworm and plant communities and soil characteristics in 2011. Three sites in Ohio were previously glaciated forests, and 3 sites in Kentucky were unglaciated forests. Southeastern Naturalist 71 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 (Lawrence and Bowers 2002); earthworms were collected from each plot for 15 minutes. Upon collection, we rinsed each earthworm with water and placed it in 70% isopropyl alcohol for transportation back to the lab. Within 24 hours of being extracted from the field, the earthworms were placed in formalin to fix their tissues. After fixation in formalin, we placed earthworms back into 70% isopropyl alcohol for long-term storage. We identified adult earthworms to species, categorized immature earthworms as either “Lumbricus immature” or “other immature”, and calculated percent of immature earthworms for each plot. Earthworms were separated by species per plot and dried at 60 °C for 48 h. We determined ash-free dry biomass (AFDM) by ashing the worms in a muffle furnace at 500 °C for 4 h. For each plot, we calculated earthworm species richness, diversity, and density (including numbers of immature earthworms), as well as the percent immature. Soil sampling We collected 3 replicate soil cores (2.54 cm diameter x 10 cm depth) adjacent to each subplot on the same day as we extracted worms. Soil temperature was recorded in the field for each plot during worm and soil-core extraction. Samples were stored and transported on ice in the field and then stored in a cooler at 4 °C until processing. We homogenized soil cores from each plot and passed the samples through a 2-mm-mesh sieve to remove large roots and rocks. We measured the pH of 2 subsamples from each plot and averaged them following the protocol of Robertson et al. (1999). Soil samples were then dried at 70 °C to a constant mass to obtain gravimetric water content (i.e., soil moisture; Jarrell et al. 1999). Soil organic matter content was obtained using the loss-on-ignition technique (Nelson and Sommers 1996). Soil nitrate-N (NO3 --N) and ammonium-N (NH4 +-N) were extracted from each sample with 2M KCl. We measured NH4 +-N colorimetrically using a microplate reader following a method that replaces phenol with the sodium salt of 2-phenylphenol (PPS) as the substrate for the Berthelot reaction (Rhine et al. 1998, Sims 2006, Sims et al. 1995). We also measured NO3 --N colorimetrically on a microplate reader using an enzyme method to convert NO3 - to NO2 - (Campbell et al. 2006, Ringuet et al. 2011). In this process, AtNaR2 (acquired from NECi, Lake Linden, MI) quantitatively reduces nitrate to nitrite in a phosphate buffer. All nitrite then diazotizes with sulfanilamide and then reacts with N-(1-Napthyl)ethylenediamine to form a pink color absorption that is read at 540 nm (Patton and Kryskalla 2011). Statistical analyses To test for differences between previously glaciated and unglaciated forests, we used nested mixed-model analysis of variance. Results from the 3 stands (12 plots total) within each of the 6 forest sites were averaged and nested within the 2 treatments (glaciated vs. unglaciated forests). In the mixed models, random effects were all the possible forests within the treatments, and the main effect (independent variable) was treatment. We ran nested mixed-models for all measured (dependent) variables (i.e., plant-community metrics, earthworm-community metrics, and Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 72 soil characteristics). Using Pearson correlation analysis, we tested relationships between earthworm metrics and the plant community and soil variables. We chose correlation analysis over regression because the study was not designed to test cause and effect and there are plausible reasons for both earthworms affecting soil and plant variables and vice versa. Correlation analyses were performed on the average of all replicate plots within each forest for each measured variable (n = 6). We checked all measured variables to verify that they met the assumption of normality. Statistical analyses were conducted in SAS system for Windows (SAS Institute v8). Results Differences between glaciated and unglaciated forests We did not find distinct differences between the earthworm communities of previously glaciated and unglaciated forests. Earthworm communities in previously glaciated forests had slightly greater earthworm biomass than sampled plots from unglaciated forests (F = 5.81, P = 0.07); however, earthworm density, species richness, and diversity did not differ between the treatments (Table 1). We found 11 different earthworm species from all sampled locations; none were native (Table 2). Four earthworm species were of the epigeic ecological group, 6 species were endogeic, and 1 species sampled, L. terrestris, was anecic. Earthworm species with the greatest densities found during the study included L. rubellus, L. terrestris, and Aporrectodea rosea Savigny; however, L. terrestris was more commonly found in the previously glaciated forests, while L. rubellus was more commonly found in the unglaciated forests (Fig. 2). Species richness and diversity of the herbaceous and woody plant communities of glaciated and unglaciated forests did not differ from each other (Table 1). Overall, we found a total of 58 plant species in the herbaceous plant-cover survey of all sites. The 2 dominant herbaceous species were Allaria petiolata (M. Bieb.) (Garlic Table 1. Nested mixed-model results given as mean ± standard error for measured variables in previously glaciated and unglaciated forest plots. df1, df2 = 1, 4. Dependent variable Glaciated Unglaciated F-statistic P-value Earthworm biomass (AFDM m-2) 11.90 ± 2.28 5.17 ± 1.21 5.81 0.07 Earthworm density (worms m-2) 184.00 ± 33.20 140.00 ± 36.70 0.26 0.64 Earthworm species richness 23.90 ± 0.34 1.64 ± 0.24 1.63 0.27 Earthworm diversity 0.73 ± 0.13 0.38 ± 0.09 3.49 0.14 Percent immature earthworms 63.40 ± 8.61 75.40 ± 6.35 0.47 0.53 Herbaceous species richness 5.56 ± 0.49 6.00 ± 0.50 0.17 0.70 Woody species richness 5.42 ± 0.57 3.56 ± 0.41 2.87 0.17 Total vegetative cover 17.90 ± 1.68 19.90 ± 3.14 0.10 0.77 # herbaceous invasive species 1.33 ± 0.24 1.08 ± 0.18 0.21 0.67 # woody invasive species 0.78 ± 0.18 0.61 ± 0.16 0.25 0.64 Soil pH 6.02 ± 0.07 6.33 ± 0.12 1.57 0.28 Soil temperature (°C) 19.50 ± 0.58 18.80 ± 0.57 0.97 0.38 Soil moisture (in situ, %) 57.00 ± 4.92 53.20 ± 4.35 0.12 0.75 Soil nitrate (mg N kg-1) 2.16 ± 1.05 6.05 ± 1.38 2.30 0.20 Soil ammonium (mg N kg-1) 9.01 ± 0.94 11.80 ± 0.92 2.43 0.19 Southeastern Naturalist 73 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 Mustard) and Potentilla simplex Michx. (Common Cinquefoil). Garlic mustard was the only herbaceous species found at every study site. We recorded a total of 34 woody species among all surveyed forests; Sugar Maple was dominant by threefold, followed by Fraxinus pennsylvanica Marshall (Green Ash) and Acer negundo L. (Boxelder). Less-common woody species included White Ash, Lonicera maackii (Rupr.) Herder (Amur Honeysuckle), and Black Cherry. Sugar Maple and Amur Honeysuckle were the only woody species found at every study site. We recorded in the plant surveys 5 species commonly thought of as invasive including Garlic Mustard, Euonymus fortunei (Turcz.) Hand.-Maz. (Wintercreeper), Lysimachia nummularia L. (Moneywort), Rosa multiflora Thunb. (Multiflora Rose), and Amur Honeysuckle (Table 3). We recorded 5 invasive plant species in 2 of the previously glaciated forests and 3–4 in the unglaciated forests (Fig. 3). Soil characteristics Table 2. Ecological group and average density (worms m-2) of all earthworm species collected during sampling at each forest site (n = 12). Glaciated Unglaciated Ecological Earthworm species group Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Aporrectodea rosea Epigeic 2.7 17.7 0.0 0.0 1.4 13.6 Dendrobaena octaedra Epigeic 2.0 0.7 0.0 0.0 0.0 0.0 Dendrodrilus rubidus Epigeic 0.0 0.0 0.7 1.4 0.0 0.0 Lumbricus castaneus Epigeic 3.4 0.7 0.0 0.0 0.0 0.7 Allobophora chlorotica Endogeic 0.0 12.9 7.5 3.4 4.1 0.0 Aporrectodea caliginosa Endogeic 0.0 1.4 0.0 0.0 0.7 0.0 Aporrectodea trapezoides Endogeic 0.0 5.4 2.7 0.0 2.0 1.4 Aporrectodea turgida Endogeic 17.0 2.7 2.0 0.7 0.0 0.0 Lumbricus rubellus Endogeic 0.7 6.8 8.2 0.0 10.2 27.9 Octolasion tyrtaeum Endogeic 0.0 2.0 4.8 0.0 2.0 6.8 Lumbricus terrestris Anecic 21.8 10.2 0.7 1.4 1.4 0.0 Lumbricus immature 46.9 71.4 207.5 0.0 101.4 209.5 Other immature 12.2 34.0 47.6 10.2 12.2 12.9 Figure 2. Average density of worms m-2 at each sampled forest site for the 3 most-dominant earthworm species collected during the study. Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 74 including soil temperature, moisture, pH, and inorganic nitrogen did not vary between previously glaciated and unglaciated forests (Table 1). Interactions between earthworms and their environment We found many significant correlations between earthworms and their physical, chemical, and biological environment (Table 4). Earthworm species richness and diversity were positively correlated with abundance of invasive woody species (Amur Honeysuckle and Multiflora Rose) (P < 0.05; Table 4). In addition, the percent of immature earthworms was negatively correlated with the abundance of herbaceous invasive species (P < 0.05; Table 4). Earthworm density and percent of immature earthworms were negatively correlated with soil moisture (P < 0.03; Fig. 4). Percent of immature earthworms was also negatively correlated with soil temperature (P < 0.01; Table 4). Earthworm diversity was negatively correlated with soil ammonium (P < 0.05; Fig. 4). We found no significant correlations between any earthworm metrics and soil pH or soil nitrate level. Table 3. Average herbaceous percent cover and total number of woody stems for all invasive plant species found during peak growth 2011. Twelve 5-m2 plots were sampled per site. Average % cover per plot given for herbaceous species, and total # of stems given for woody species. Glaciated Unglaciated Species Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Invasive herbaceous species Allaria petiolata 4.54 1.22 0.75 6.0 0.08 3.08 Euonymus fortunei 0.04 1.04 0.00 0.00 0.00 0.00 Lonicera maackii 4.08 0.46 0.00 0.00 0.17 0.00 Rosa multiflora 0.04 0.00 0.00 0.04 0.00 0.00 Lysimachia nummularia 0.00 0.00 0.00 28.4 0.00 0.04 Invasive woody species Lonicera maackii 55 50 11 0 23 22 Rosa multiflora 0 10 1 0 0 16 Figure 3. Invasive plant species richness for each sampled forest site. A total of 7 invasive herbaceous and woody species were found throughout the project sites. Southeastern Naturalist 75 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 Discussion Earthworm density and richness did not differ between previously glaciated and unglaciated forests, and most interestingly, we found only nonnative earthworm species at all sampled sites. Earthworm communities at our sites were more similar to those found in the northern hardwood, glaciated forests of Minnesota (Hale et al. 2005b, Reynolds et al. 2002) and New York (Stoscheck et al. 2012, Suarez et al. 2006b), where no native species were found, than to those found in the southeastern US (Hendrix et al. 1992, Kalisz and Dotson 1989). Native earthworm species including Sparganophilus eiseni Smith and Diplocardia singularis Ude were once found in the region (Olson 1928); therefore, our current data suggests that native species have been displaced by exotics like Lumbricus rubellus. Others suggest that Table 4. Significant correlations between measured earthworm variables with plant community variables and soil characteristics. Correlated metric Earthworm metric r value P-value Earthworm correlations with plant variables # invasive herbaceous species Percent immature -0.83 0.04 # invasive woody species Earthworm species richness 0.87 0.02 # invasive woody species Earthworm diversity 0.82 0.04 Earthworm correlations with soil variables Soil ammonium Earthworm diversity -0.83 0.04 Soil moisture Earthworm density -0.85 0.03 Soil moisture Percent immature -0.93 0.01 Soil temperature Percent immature -0.96 less than 0.01 Figure 4. Earthworm density and percent of immature earthworms (measured per m2 and averaged per site) decreased with soil moisture, percent of immature earthworms decreased with soil temperature, and earthworm diversity (measured using the Shannon index) decreased with soil ammonium among the study sites. Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 76 the same phenomenon of exotic earthworm species replacing natives has occurred in the southeastern US (Kalisz and Dotson 1989, Reynolds 1972). Site variability may have masked differences in earthworm communities that might have been apparent if the soils and vegetation at our sites had been more similar. For example, we found differences in dominant earthworm species, dominant invasive plant species, and soil parameters among forest sites within the previously glaciated and unglaciated treatments. Although earthworm communities found in both the previously glaciated and unglaciated forests contained all three ecological earthworm groups (i.e., epigeic, endogeic, and anecic), the communities in previously glaciated sites had higher densities of anecic earthworms, and the unglaciated forests had higher densities of endogeic earthworms (Fig. 2). High densities of endogeic earthworms are characteristic of soils dominated by native earthworms (Fragoso et al. 1999, Kalisz 1993). We predicted, but did not find to be true, that forests on unglaciated sites would have a more diverse earthworm community with a mixture of native and nonnative species occupying more niche spaces than in the forests at previously glaciated sites. Earthworm communities that occupy multiple niches have a greater impact on forests by changing soil structure and altering soil nutrients with the relocation of organic matter in multiple soil layers (Bohlen et al. 2004c, Frelich et al. 2006, Hale et al. 2005a, Hopfensperger et al. 2011). When a multi-species earthworm community removes soil organic matter from the surface, the loss of available nutrients is magnified compared to single-habit earthworm invasions (Hale et al. 2005a, Sackett et al. 2012, Suarez et al. 2004). In fact, we found that forests in both previously glaciated and unglaciated areas with all ecological earthworm groups represented had lower soil-N concentrations (Fig. 4C). In addition to impacting soil dynamics, earthworm communities containing all ecological groups have also been found to have a greater effect on plant communities than sites with less diverse earthworm communities (Hale et al. 2006, Hopfensperger et al. 2011). However, Hale et al. (2006) found that the presence of a specific species, Lumbricus rubellus, was most closely tied to sites where the plant community was dominated by invasive and nonnative plant species. We found higher densities of L. rubellus in 2 of the 3 unglaciated forest plots; however, these plots did not have greater invasive plant density. When we combined data from previously glaciated and unglaciated forests, we found that earthworm species richness and diversity increased with number of invasive woody plant species. Though we did not find our prediction of a co-occurrence of native and nonnative species resulting in greater earthworm diversity in unglaciated forests to be true, we predicted and found evidence of an increase of invasive plants with increased earthworm diversity; however, we obtained this result only for our previously glaciated sites. Perhaps as others have suggested, increased disturbance allowed for greater diversity of nonnative earthworms and higher densities of invasive plants at these sites (Kalisz and Dotson 1989, Kalisz and Wood 1995). Nuzzo et al. (2009) found that earthworm biomass increased with greater nonnative plant cover, which they attributed to earthworms altering Southeastern Naturalist 77 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 soil nutrients and/or disrupting plant mycorrhizae, creating an ideal environment for nonnative plants to dominate. Another way earthworms may facilitate invasion of nonnative plants is through direct removal of the O horizon and soil organic matter, leading to a decline in native herbaceous cover (Alban and Berry 1994; Bohlen et al. 2004a, c; Hale et al. 2004, 2005b), which could open-up the forest floor to colonization by new and/or invasive species. Indeed, we observed that previously glaciated sites with high earthworm diversity also had high densities of invasive Garlic Mustard, Amur Honeysuckle, and Multiflora Rose, which exploit disturbed areas (Doll 2006, Luken 1988, Szafoni 1991). In addition, these latter 2 are woody invasive species that bear fruit eaten and readily dispersed by wildlife and that commonly become established in forests with sparse ground cover and a thin O horizon (Baskin and Baskin 1998, Grime 1979). In contrast to the positive correlation between woody invasive species and earthworm diversity and richness, we found that the percent of immature earthworms in the earthworm community was negatively correlated with invasive herbaceous plant cover. The dominant invasive herbaceous species at our sites was Garlic Mustard, which is known to exhibit allelopathic properties (Prati and Bossdorf 2004). Plants that contain herbivore-repellant secondary compounds may also be avoided by earthworms. For example, Hale et al. (2006) found that earthworms avoided consuming toxic compounds produced by Arisaema triphyllum (L.) Schott (Jack-in-the-Pulpit) and Allium tricoccum Aiton (Wild Leek). Currently, there is no published data linking allelopathy from Garlic Mustard to earthworms; however, Garlic Mustard is known to contain compounds that inhibit feeding by butterfly larvae (Haribal et al. 2001). Perhaps immature earthworms at our study sites avoid occupying areas that have a high percent cover of Garlic Mustard due to the same compounds that inhibit butterfly larvae. Indeed, we found the lowest earthworm density at site 4 (Fig. 2), which had the greatest percent cover of Garlic Mustard (Table 3). In addition, the dominant species found at site 4 was L. terrestris, an anecic species whose deep burrows may help them to avoid the allelopathic effects of Garlic Mustard. Another native but invasive species known to increase with earthworm biomass, Carex pensylvanica Lam. (Pennsylvania Sedge), is known to invade forest floors (Aikens et al. 2007, Hopfensperger et al. 2011). Pennsylvania Sedge responds to the presence of endogeic earthworm species such as L. rubellus by creating new root systems via basal meristems and spreading vegetatively through the developing A horizon (Hale et al. 2006). Pennsylvania Sedge was a dominant understory species at sites 3 and 5, which were also dominated by endogeic earthworm species (Fig. 2). We found aspects of earthworm communities from both previously glaciated and unglaciated forests correlated with soil moisture, temperature, and soil ammonium at our study sites. We observed high earthworm diversity in areas with low soil ammonium (Fig. 4C), which may indicate that diverse earthworm communities occupying multiple soil niches may increase soil-N cycling. Results of published studies concerning earthworms and soil N dynamics are mixed. For example, accelerated N-process rates in the presence of nonnative earthworms may both increase and decrease inorganic N concentrations (Bohlen et al. 2004c). Sackett et al. (2012) Southeastern Naturalist K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 78 found that soil NH4 +-N increased with soil pH and offset the amount of N lost from increased decomposition and organic matter removal by earthworms. We predicted an increase in soil pH with earthworm density; however, we did not find any correlations between earthworm community metrics and soil pH. The absence of a clear relationship between earthworms and soil pH in our study is noteworthy because many others have found that earthworm density increases with soil pH (Burtelow et al. 1998, Fisichelli et al. 2013, Hopfensperger et al. 2011); acidic soil inhibits many earthworm species because of their need for calcium (Canti and Pearce 2003, Curry 2004). Perhaps soil pH was not variable enough among our sites to determine a trend in the data (Table 1). Lastly, at our study sites, earthworm density was highest at moderate levels of soil moisture (between 25% and 60%), but decreased at higher moisture levels, which is counter to what we predicted. We recorded no earthworms when soil moisture was below 25% (Fig. 4). This observation suggests that there is a threshold beyond which soil can become too moist for earthworms, resulting in less than optimal conditions with lower earthworm densities. Previously glaciated forests with earthworm communities containing all 3 ecological groups had lower soil pH and lower N concentrations than the unglaciated forests that lacked anecic earthworms. When anecic earthworms transport fresh organic litter from the forest floor to the A horizon, they may have a great impact on soil-nutrient dynamics (Sackett et al. 2012; Suarez et al. 2006a, b). We conducted our soil pH and nutrient-content measurements on samples taken in the top 20 cm of the soil; anecic earthworms relocate fresh litter to well below that soil depth. The removal of the fresh litter from the surface relocates the ecosystem’s nutrient source to a depth below which our soil samples were collected, which may explain the lower inorganic N concentrations recorded in the surface soils (Bohlen et al. 2004a, Sackett et al. 2012). N loss may also occur by leaching from the soil surface down the flowpaths of anecic earthworm burrows (Suarez et al. 20 04, Subler et al. 1997). In addition, the movement of organic matter away from the surface by anecic earthworm species results in much greater mixing of the soil compared to soil found in areas without anecic earthworms (Bohlen et al. 2004a, Suarez et al. 2004). Greater soil mixing in the presence of anecic species may prevent the maintenance of a higher soil pH than would occur in earthworm communities without anecic earthworms (Burtelow et al. 1998, Hopfensperger et al. 2011). Perhaps the prevalence of anecic earthworms at our previously glaciated study sites was due to past land use. Non-native earthworm establishment is related to degree of disturbance and human activity; future studies could investigate and quantify the difference in the degree of disturbance between the previously glaciated and unglaciated sites. The 3 previously glaciated forests we studied are in protected forest preserves, 1 unglaciated forest is in a protected forest preserve and the other 2 unglaciated sites are recently protected forests that were once farmsteads. Summary We studied earthworm communities in forests directly north and south of the last glacial terminus in a region of the US that had not been surveyed for earthworms in almost 100 years. We did not observe native earthworm species during our study, Southeastern Naturalist 79 K.N. Hopfensperger and S. Hamilton 2015 Vol. 14, No. 1 and earthworm density and species richness did not differ between previously glaciated and unglaciated forests, perhaps due to the variability among the study sites within the treatments. However, previously glaciated forests contained earthworm communities with species occupying more niche spaces than we observed in unglaciated forests. Specifically, the high density of anecic earthworms we recorded in previously glaciated forests may be an artifact of site disturbance and human activity. The presence of anecic earthworms in the community was associated with and may have generated lower soil pH and lower soil N concentrations. It is possible that the effect of anecic earthworms on the soil created conditions conducive for the establishment of invasive plant species. Due to the major impacts nonnative earthworms can have on the plant communities and nutrient dynamics of our forest ecosystems, scientists and managers should continue to survey the earthworm and plant communities throughout the US to better understand the movement of native and exotic earthworm species. 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