2009 SOUTHEASTERN NATURALIST 8(2):325–334 Soil Moisture and Temperature: Tolerances and Optima for a Non-native Earthworm Species, Amynthas agrestis (Oligochaeta: Opisthopora: Megascolecidae) D. Russell Richardson1, Bruce A. Snyder1, and Paul F. Hendrix1,2,* Abstract - Field observations indicate an invasion by the non-native, Asian earthworm species Amynthas agrestis (Goto and Hatai 1899) in the Great Smoky Mountains National Park (GSMNP). The aim of this study was to determine if A. agrestis was capable of surviving in the ridge-top soil along an invasion front in a mesic–xeric habitat gradient in GSMNP. Additionally, this study sought to determine optimum and tolerance conditions for A. agrestis within a range of soil moistures and temperatures. Investigating soil temperature and moisture as parameters of earthworm survivability will allow for more predictive power when investigating the GSNMP invasion. Although A. agrestis invasions are widespread in eastern North America, few studies have addressed factors that may infl uence their distribution and their potential impacts on ecosystem processes. Using incubators and PVC tube microcosms, it was determined that A. agrestis was able to survive in the GSMNP ridge-top soil at temperatures of 12 °C and 25 °C. No survival was observed at temperatures of -5, 5, or 35 °C at any soil moisture level. No survival occurred in 25 °C dry (8% gravimetric water) treatments. Of the conditions tested, maximum survival plus fresh-weight maintenace occured at 12 °C and mid-moisture (24% gravimetric water), but highest activity and effects on litter and soil structure occurred at 25 °C and high soil moisture (57% gravimetric water; field capacity). Soil moisture contributed to the success of A. agrestis at higher temperatures within the tolerance conditions; more moisture increased the survival rate and decreased weight-loss. Introduction Charles Darwin was among the first scientists to recognize the importance of earthworms in soil processes (Feller et al. 2003). Numerous studies have confirmed this importance (Satchell 1983), not only in pedogenesis (Darwin 1881), but also in nutrient cycling (James 1991, Robinson et al. 1992, Wilcox et al. 2002). Exotic soil fauna and plants are capable of altering nitrogen and carbon cycles of entire ecosystems (Ehrenfeld and Scott 2001). Exotic earthworms, specifically, are known to affect soil processes and thus alter entire ecosystems in a “bottom up” manner (Hendrix and Bohlen 2002). Impacts of exotic earthworm invasion in the northern forests of North America, primarily by European Lumbricidae, included changes in nitrogen and carbon cycles, increased rates of erosion, and endangerment or extirpation of native species (Bohlen et al. 2004a,b; Frelich et al. 2006). 1Odum School of Ecology, 1033 East Green Street, University of Georgia, Athens, GA 30602-2202. 2Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602-2202. *Corresponding author - hendrixp@uga.edu. 326 Southeastern Naturalist Vol. 8, No. 2 Amynthas agrestis (Goto and Hatai) is an invasive earthworm originating from southeast Asia, with a current North American distribution in at least 16 states, mostly the eastern United States from Maine to Florida, but also as far west as Oklahoma (Reynolds 1978, Reynolds and Wetzel 2004). Currently, an invasion front is being studied in the Great Smoky Mountains National Park (GSMNP, NC and TN) (Snyder 2008). The leading edge of the front appears to be moving along riparian corridors, but specimens have occasionally been found on xeric ridge tops that are dominated by Pinus strobus L. (White Pine). If A. agrestis is able to survive under these more extreme environmental conditions, its invasion of GSMNP could be more rapid and extensive than previously expected. The lack of knowledge regarding A. agrestis and other Asian species stands in contrast to the relatively better-studied European lumbricids. Little is known of most Amynthas species beyond the physical zoological description (Burtelow et al. 1998). Burtelow et al. (1998) reported that Amynthas gracilis invasion decreased soil organic matter in the O horizon by 36%, increased soil pH by more than 1 pH unit, increased microbial biomass, and increased denitrification rates by more than two-fold. Reynolds (1978) reported that A. agrestis is extremely active. Callaham et al. (2003) contributed that A. agrestis behaves as if it is epigeic, also noting its high activity. Reynolds (1978) noted the wide distribution of A. agrestis in Tennessee, mainly in disturbed sites. Callaham et al. (2003) noted the invasiveness of A. agrestis, as the authors report its occurrence in relatively undisturbed, remote locations. The ability of A. agrestis to invade undisturbed soils is very noteworthy, as previous papers have postulated that non-native earthworms tend to be invasive mostly where a disturbance has occurred (Kalisz and Dotson 1989, Kalisz and Powell 2000, Kalisz and Wood 1995). Due to the importance of earthworms in soil processes, we sought more information about the exotic earthworm A. agrestis to determine the impacts this new species may be having on soil processes and other biota in the GSMNP. Therefore, the aim of this study was to understand the pattern of invasion by A. agrestis, with respect to its ability to survive within a range of soil temperatures and moistures in the ridge-top soil in the GSMNP, and to estimate its potential effects on soil structure and organic-matter dynamics. Field-site Description The A. agrestis invasion front currently being studied is on the western edge of the GSMNP along the Chilhowee Reservoir (35°33.0'N, 83°60.5'W). The topography of the area is rugged, with alternating valleys and ridges. Valleys are dominated by Acer spp. (maples), Quercus spp. (oaks), Liquidambar styracifl ua L. (Sweetgum), and Liriodendron tulipifera L. (Tulip Poplar), while the more xeric ridges are dominated by White Pine. In general, the area is classified as mesic–xeric oak-dominated climax forest (Whittaker 1956). 2009 D.R. Richardson, B.A. Snyder, and P.F. Hendrix 327 Ridge soils are a complex of moderately deep Junaluska and deep Brasstown soils, fine-loamy, mixed, subactive, mesic Typic Hapludults. Valley soils are a complex of shallow Cataska soils and moderately deep Sylco soils, which are loamy skeltal, mixed, active (Sylco) or semi-active (Cataska), mesic Typic Dystrudepts (USDA NRCS and USDOI NPS 2007). Soil temperature was monitored during 2006 and 2007 (Snyder et al., in press); minimum and maximum temperatures recorded on the ridge were -0.56 °C (December 2006) and 32.4 °C (August 2006), respectively. Soil moisture, measured gravimetrically in October 2007, was highly variable across the field site. Moisture averaged 15.1% (n = 52), with a minimum of 2.6% and a field capacity of 57.0%. Methods Approximately 160 mature A. agrestis individuals were collected during a single day by searching through leaf litter from the low-lying areas of the field site. Soil was collected from five random locations at the top of a nearby uninvaded ridge. Litter was collected from the same ridge in a stand dominated by White Pine. Microcosms were constructed from PVC pipe to be 15 cm in height and 10.2 cm in diameter. Mesh screen was attached to the bottom of the PVC tubes to allow the water to drain from the soil. The microcosms were each covered with a perforated plastic cap to allow air flow. Soil was first prepared by coarse (1-cm) sieving to remove rocks and large aggregates, and then homogenized. A 12-cm depth of soil was added to each microcosm, along with 250 cm3 White Pine litter (≈8 g air-dry weight) on the surface, approximately the amount of surface litter at the study site. Soil moisture was adjusted gravimetrically to desired levels. Earthworm fresh weight was recorded. Two earthworms were placed on the litter surface of each microcosm, and immediately placed into incubators set to desired temperatures as described below. Although maximum field density at the site has been estimated at 40 individuals·m-2, A. agrestis populations often display patchy distributions with much higher localized densities; our use of two earthworms per microcosm represents these higher-density areas where we expect significant impacts of A. agrestis on soil processes. Soil moisture was measured gravimetrically and adjusted daily or every other day as needed. Microcosms were maintained within three moisture regimes (low, medium, and high) and five temperatures (-5, 5, 12, 25, and 35 °C). The temperatures were selected to cover a wide range of temperatures over which A. agrestis might survive. Low moisture was established as 8% gravimetric water (air-dried soil), high moisture was 57% gravimetric water (field capacity), and medium moisture was 24% gravimetric water, to establish a soil moisture intermediate to the two extremes. Control microcosms with no earthworms were constructed for all moisture regimes at expected 328 Southeastern Naturalist Vol. 8, No. 2 optimum temperatures (12 and 25 °C) to assess earthworm effects on surface litter and soil structure. Each treatment was replicated three times. Microcosms were deconstructed immediately following removal from the incubators. Since microcosms were removed upon observation of mortality, the 12 and 25 °C treatments ran for the full four weeks, but the other treatments were removed after three days. Because litter was mixed with soil in several treatments, all litter was removed from the microcosms using a standard effort to reduce the effects of unequal litter removal. Earthworms were removed, brushed gently to remove litter, and the fresh weight was recorded per microcosm. Earthworm mass was not recorded for any dead earthworm in a state of decomposition. Soil was removed in 4-cm increments by careful removal with a steel spoon. Each soil layer was placed in a paper bag, weighed, and oven dried to measure soil moisture and to prepare the soil for wet-sieving. Because of the visually obvious effects of A. agrestis on soil structure, soil from the 25 °C high-moisture treatment was selected for soil aggregate analysis (Six et al. 2000) to separate each layer into water-stable aggregate (WSA) fractions and fine particulate organic matter fractions. WSA fractions from the 25 °C high-moisture treatment were analyzed for total carbon using a Carlo Erba NA1500 CHN Combustion Analyzer (Carlo Erba, Milan, Italy). Optimum soil moisture and temperature conditions were investigated by examining changes in earthworm fresh weight between start and finish of the experiment, average earthworm survival, percent change in litter, and formation of WSA (i.e., castings). Statistical analyses were performed using Axum 7 software (Mathsoft 2001). Survival data were analyzed by two-way analysis of variance on arcsine-square root transformed data. Litter, soil aggregate, and soil-carbon data were analyzed by paireddifference t-tests on relevant temperature and moisture treatments combinations. No significant treatment effects were found for soil carbon, so results are not presented. Results Amynthas agrestis was found to tolerate GSMNP ridge-top soil under certain laboratory conditions (Table 1). 100% mortality was observed in -5, 5, and 35 °C temperature treatments within three days. Survival occurred in Table 1. Mean percent survival (± standard error, n = 3) of Amynthas agrestis individuals after 28 days of incubation. Temperature Moisture -5 °C 5 °C 12 °C 25 °C 35 °C Low (8%) 0 0 43.8 ± 22.9 0 0 Medium (24%) 0 0 79.4 ± 6.6 24.0 ± 24.0 0 High (57%) 0 0 69.3 ± 3.5 51.1 ± 27.8 0 2009 D.R. Richardson, B.A. Snyder, and P.F. Hendrix 329 12 °C treatments at all soil moisture levels; survival was also observed in the 25 °C mid-moisture treatment and 25 °C high-moisture treatment. Temperature significantly affected survival, regardless of moisture conditions (Table 2). The temperature tolerance of A. agrestis appears to be greater than 5 °C and less than 35 °C, with optima possibly between 12 and 25 °C. Interestingly, A. agrestis was unable to survive at low soil moisture levels at 25 °C, yet was able to tolerate low soil moisture levels at 12 °C. Among the microcosms with surviving earthworms, a loss of fresh weight was observed (Fig. 1); however, weight loss was dependent upon temperature (Table 2). Treatments at 12 °C showed less weight loss than 25 °C treatments with corresponding moisture levels. Table 2. Analyis of variance table showing main and interactive effects of temperature and soil moisture on survival and weight loss of Amynthas agrestis in laboratory incubations. Dependent variable Independent variable df Sums of squares F P Survival Temperature 1 0.028 9.99 0.008 Moisture 2 0.011 1.95 0.18 Temp*moisture 2 0.007 1.21 0.33 Error 12 0.034 Weight loss Temperature 1 0.008 7.93 0.015 Moisture 2 0.005 2.52 0.12 Temp*moisture 2 0.0009 0.45 0.64 Error 12 0.011 Figure 1. Mean fresh weight of Amynthas agrestis remaining as a percentage of initial fresh weight after 28 days of incubation. Error bars indicate standard error (n = 3). 330 Southeastern Naturalist Vol. 8, No. 2 A comparison of amounts of litter incorporated into soil (Fig. 2) revealed that earthworm activity was significantly increased in the 25 °C high moisture treatment (t = 5.14, P = 0.007). The formation of WSA in the 25 °C treatment with earthworms was significantly higher in the top 4 cm of soil (t = 2.7, P = 0.03) compared to the 25 °C treatment without earthworms, indicating a high level of A. agrestis activity (Fig. 3). Discussion The aggregate data confirms Callaham et al.’s (2003) observation that A. agrestis has an epigeic ecological strategy, since the only significant effects on soil structure occurred in the top 4 cm. Although maximum survival of A. agrestis occurred at 12 °C, A. agrestis appeared to have had highest activity at a combination of 25 °C temperature and soil moisture at field capacity. The amount of A. agrestis activity in the 25 °C high-moisture treatment might have led to a limitation of food resources, which in turn may have been responsible for the higher mortality in otherwise optimal conditions. Greater resolution is needed to determine more precise temperature optima. Because so little is known about ecological aspects of A. agrestis, inferences may be drawn from other Asian earthworm species that are congeners or in closely related genera (i.e., pheretimoid earthworms formerly in the genus Pheretima, which has been split into 10 genera; see Chang and Chen Figure 2. Change in litter mass in temperature and soil moisture treatments in which earthworms survived. Asterisk indicates significance (P < 0.01). Error bars indicate standard error (n = 3). 2009 D.R. Richardson, B.A. Snyder, and P.F. Hendrix 331 Figure 3. Water-stable soil aggregates greater than 2 mm in the 25 °C and high soil moisture treatment. Asterisk indicates significance (P < 0.01). Error bars indicate standard error (n = 3). 2005), and these data may be compared with the findings of this study. El-Duweini and Ghabbour (1965) list soil water content as a key factor to understanding the distribution of Egyptian earthworms including Metaphire californica (Kinberg) and Polypheretima elongata (Perrier). Fragoso et al. (1999) reported that temperature tolerances for A. gracilis were between 15 and 26 °C and those for A. corticis were 13 to 26 °C. Grant (1955a, b) studied the temperature preferences and moisture relationships of A. hupeiensis (Michaelsen). He found a temperature preference for between 15 and 23 °C and a soil moisture preference for the highest soil moisture possible in the experiment (≈30%). In studies of non-pheretimoid, subtropical earthworms, Viljoen and Reinecke (1992) and Hallat et al. (1992) reported results consistent with the conclusions of this study, namely that reproductive success for the epigeic earthworms they studied (Eudrilus eugeniae (Kinberg) and Perionyx excavatus (Perrier), respectively) were optimal at 25 °C and a high moisture level. These studies therefore support our observation that conditions for maximum activity of A. agrestis exist at approximately 25 °C and field capacity of soil. Since the duration and primary aim of our study did not allow us to investigate reproduction, it is unknown how soil temperature and moisture may affect A. agrestis population dynamics. There remains a need to conduct microcosm studies that factor in other biological parameters such 332 Southeastern Naturalist Vol. 8, No. 2 as reproduction. However, by comparing data from the studies mentioned above along with the data from this study, particularly earthworm activity (as indicated by formation of WSA), it appears that optimal conditions for activity are somewhere around soil field capacity and 25 °C. Since few A. agrestis individuals were observed on the pine-dominated, xeric ridge-tops surrounding the invasion front in GSMNP prior to this study, it was hypothesized that an A. agrestis invasion would be hindered at these locations. However, the results of this study suggest otherwise. The likelihood of an extensive invasion in the GSMNP may be higher than we thought, since A. agrestis is able to survive in ridge-top soil during periods where soil temperature and moisture conditions are tolerable. Being able to survive in ridge-top soil when conditions are favorable may allow for migration through the less favorable ridge-top microclimates, and onto other more favorable low-lying patches within the GSMNP. Continuous monitoring of microclimatic conditions along the invasion front at GSMNP might allow us to predict such suitable invasion corridors based upon the results from this study. If further study of the A. agrestis invasion reveals a relationship between actual invasion routes and physiological tolerances, it may be possible to construct predictive models of the extent of earthworm invasion. Acknowledgments A National Science Foundation Research Experience for Undergraduates (REU) Grant under NSF DEB-0236276 funded this research. 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