Soil Biology & Biochemistry 80 (2015) 209e217

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Soil Biology & Biochemistry

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Nest-mounds of the yellow meadow ( flavus) at the “Alter Gleisberg”, Central Germany: Hot or cold spots in nutrient cycling?

* Peggy Bierbaß a, , Jessica L.M. Gutknecht b, Beate Michalzik a a Soil Science, Institute for Geography, University of Jena, Lobdergraben€ 32, 07743 Jena, Germany b Department of Soil Ecology, Helmholtz-Centre for Environmental Research e UFZ, Theodor-Lieser-Straße 4, 06120 Halle (Saale), Germany article info abstract

Article history: Nests of the (Lasius flavus) occur at high densities in grasslands worldwide. Although Received 19 December 2013 many studies have shown that L. flavus nests influence soil nutrient contents, little is known about their Received in revised form effect on soil nutrient cycling rates. The aim of this study was to examine the role of nest-mounds 22 September 2014 inhabited by L. flavus as potential ‘hot spots’ for soil nutrient cycling. Six pairs of nest-mounds and Accepted 23 September 2014 control soils were selected at a grassland site at the plateau of the Alter Gleisberg (Thuringia, Central Available online 13 October 2014 Germany). L. flavus significantly modified the soil environment within the nest. In comparison to the control soils, nest-mounds were characterized by slightly higher soil temperatures during the summer Keywords: Microclimate months. In addition, we found that nests were related to decreased potential C mineralization rates and Soil organic matter increased potential net N mineralization rates. Nest-mound soil exhibited lower amounts of SOC, hot- Soil chemistry water extractable DOC and DN, and higher concentrations of leachable DOC and DN. Moreover, Soil solution chemistry promoted the enrichment of base cations in the nest. Differences in the soil environment between nests Mineralization and control soils were possibly a result of the burrowing activity of ants, soil mixing, accumulation of honeydew, and decreased plant-derived nutrient inputs into the nest-mound soil. In conclusion, L. flavus nest-mounds had a significant but element dependent effect on the soil nutrient cycling and may represent cold spots for C cycling and hot spots for N cycling. Thus, L. flavus nests increase the spatial heterogeneity of soil properties and create unique micro-sites within grassland ecosystems. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Dauber et al., 2006). Therefore, ants are considered to be “ecosystem engineers” (Jones et al., 1994) that increase the spatial Ants are an important component of terrestrial ecosystems due heterogeneity of soil properties. However, the effect of ants on soil to their abundant occurrence (Folgarait, 1998). Elmes (1991) esti- properties is variable and depends on species specific factors, such mated that there are 5e10 ants per m2 of dry land. The activity of as nest size and density, colony age, feeding and nesting strategy ants has been shown to directly affect chemical and physical (Dauber and Wolters, 2000; Boots et al., 2012), as well as on properties of soil, such as organic matter content, nutrient con- properties of the surrounding soil (Frouz et al., 2003). Hence, nest- centrations, water content, bulk density and particle size distribu- mounds might act as hot or cold spots with disproportionately high tion (Cammeraat et al., 2002; Dostal et al., 2005; Holec and Frouz, or low reaction rates in comparison to the surrounding soil 2006). Ant-mediated soil modifications indirectly affect several (McClain et al., 2003). landscape characteristics including: soil microclimatic conditions European grasslands are dominated by ants of the genera (King, 1977a; Rosengren et al., 1987), nutrient cycling rates (Lenoir Formica, Lasius and Myrmica. The yellow meadow ant (Lasius flavus) et al., 2001; Wu et al., 2013), the abundance and activity of other is especially common in grasslands, where nests can occur at soil biota (Wagner et al., 1997; Boots et al., 2012), as well as plant densities of up to 0.5 nests m 2 forming “antscapes” (Kovar et al., community composition and soil seed banks (King, 1977a,b,c; 2001; Steinmeyer et al., 2012). Nests are constructed below the ground or with visible dome-shaped mounds of mineral soil (Waloff and Blackith, 1962; Schreiber, 1969) and are usually main- tained for over 20 years (Woodell and King, 1991). The species lives * Corresponding author. Chair of Silviculture, Faculty of Environment and Natural primarily underground and forages along tunnels extending up to Resources, University of Freiburg, Tennenbacherstraße 4, 79085 Freiburg im fl Breisgau, Germany. Tel.: þ49 7612038623; fax: þ49 7612033781. 1 m around the nest (Woodell and King, 1991). L. avus feeds on E-mail address: [email protected] (P. Bierbaß). sugar-rich honeydew excreted by root as well as on the http://dx.doi.org/10.1016/j.soilbio.2014.09.020 0038-0717/© 2014 Elsevier Ltd. All rights reserved. 210 P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217 aphids themselves, which they cultivate in their nests (Seifert, soil nutrient contents but higher rates and higher amounts of 2007). Thus, the ants potentially fuel the cycling of labile carbon released nutrients in the soil solution. belowground. fl fl Nests of L. avus are known to in uence the nutrient content of 2. Material and methods soils, but there has been little consensus on the exact nature of that fl fl in uence. In this context, several studies on L. avus nests in 2.1. Study site grasslands found higher concentrations of total nitrogen (TN), K, Na and P in nests compared to the surrounding soil (Frouz et al., 2003; The study was performed at a grassland site situated on the Dostal et al., 2005; Platner, 2006; Wu et al., 2010), whereas others limestone plateau of the Alter Gleisberg at 343 m a.s.l. (505702200N, reported lower concentrations of total carbon (TC) and TN (Dean 11 42011 00E). The mean annual temperature in this area is 8.5 C and et al., 1997; King, 1977a; Dostal et al., 2005; Holec and Frouz, mean annual rainfall amounts to 588 mm (1961e1999) (PIK, 2009). 2006). Increased levels of nutrients are commonly attributed to The Alter Gleisberg consists of sedimentary rocks of the Mesozoic the accumulation of food, honeydew and organic waste material, era. The plateau and steep flanks are comprised of limestone of the fi and high numbers of nitrogen- xing bacteria in nests (Frouz et al., Lower Muschelkalk (Lower Wellenkalk). The soils are classified as € 1997 cited by Frouz et al., 2003; Dostal et al., 2005; Kilpelainen shallow and loamy Rendzic Leptosol (IUSS Working Group WRB, et al., 2007), while decreased nutrient contents can be explained 2006; Wenzel et al., 2012). The vegetation of the grassland is by the burrowing activity of ants mixing upper organic with deeper dominated by grasses (Arrhenatherum elatius, Brachypodium pin- mineral soil layers (Frouz et al., 2003; Holec and Frouz, 2006). natum, Bromus sp., Dactylis glomerata, Festuca sp.) and herbs Only few studies have dealt with nutrient cycling rates to (Euphorbia cyparissias, Bupleurum falcatum, Melampyrum arvense, explain varying nutrient contents in soils. It is hypothesized that Origanum vulgare). Shrubs (Cornus sanguinea, Crataegus monogyna, higher temperatures and higher amounts of easily available nutri- Prunus spinosa, Rosa canina) occur along the edge of the grassland. ents in nest-mounds stimulate microbial activity, leading to In the last few years, the grassland was regularly grazed by sheep accelerated decomposition and mineralization of organic matter and mowed in autumn to prevent shrub encroachment (Wenzel (OM), nutrient leaching and uptake by plants, and CO2 emissions to et al., 2012). the atmosphere (Ohashi et al., 2007; Wu et al., 2013). In this Ant nest-mounds are common in this grassland, with an average context, Dauber and Wolters (2000) observed higher C minerali- density of 0.33 nest-mounds m 2. Six pairs of L. flavus nest-mounds fl zation rates in nest-mounds of L. avus than in the surrounding soil, containing living colonies and adjacent control soils located within which they attributed to a higher activity of soil microorganisms in an area of approximately 1600 m2 were selected for this study. To “ nest-mounds, consequently forming a mosaic of microbial hot cover the site variability of the grassland, two pairs were chosen ” fi spots . The results corroborate ndings by Stadler et al. (2006), who along the edge of the grassland (2, 6), two pairs in the center of the tested the effect of wood ants (Formica polyctena) on litter solution grassland (4, 5) and two pairs in the transition between grassland chemistry of Norway spruce and microbial enzyme activities in and shrubs (1, 3) (Fig. 1). A wide range of different nest-mound sizes litter extracts. They observed that the presence of ants resulted in were chosen encompassing mound volumes from 0.007 to fi signi cantly increased concentrations of dissolved organic carbon 0.023 m3 (Table 1). Control soils were approximately 1 m away from and nitrogen (DOC and DON) in litter solutions and higher enzyme each nest-mound to exclude ant effects on soil characteristics. activities in litter extracts. In contrast, Lenoir et al. (2001) and Compared to the controls, nest-mounds were characterized by a Domisch et al. (2008) observed very dry moisture conditions in sparse plant cover with higher numbers of vegetation-free patches. nest-mounds of wood ants ( group and Formica aqui- lonia), resulting in decelerated decomposition rates of OM. Thus, 2.2. Microclimatic conditions the mineralization of C, N and P proceeded more slowly in nest- mounds compared to the surrounding forest floor, consequently The study covered a period of 20 weeks, starting on June 8 and representing “cold spots” for nutrient mineralization. Both authors ending on October 25, 2012. To characterize the microclimatic supposed that the decomposition and mineralization of OM are conditions during the study period, the soil temperature and soil accelerated as soon as nest-mounds are abandoned and their dry- volumetric water content were measured in 10 cm depth from the ness is no longer maintained by ants. Hence, abandoned nest- mound top in the central part of all nest-mounds and control soils. mounds could provide “potential hot spots” for nutrient minerali- The soil temperature was continuously recorded at 1-h intervals zation (Lenoir et al., 2001; Domisch et al., 2008). with a data logger (EL-USB-1, Lascar Electronics, Salisbury, UK). The In this study, we characterize the carbon (C) and nitrogen (N) cycling rates and related microclimatic conditions and soil chem- istry of nest-mounds and adjacent control soils located at a grass- land site at the “Alter Gleisberg” (Thuringia, Central Germany). In detail, the objectives of this study are (1) to compare temperature and moisture conditions of L. flavus nest-mounds and the sur- rounding soil, (2) to compare nutrient cycling rates and related C and N contents between L. flavus nest-mounds and the surrounding soil, and (3) to compare base cation concentrations in soil and soil solution between L. flavus nest-mounds and the surrounding soil. The hypothesis is that nest-mounds represent hot spots for soil nutrient cycling by creating favorable environments for other soil biota due to a surplus of easily decomposable organic material (honeydew and organic wastes) as well as suitable temperature and moisture conditions. This environment enhances the microbial activity and therefore leads to higher rates of nutrient mineraliza- Fig. 1. Schematic drawing of the grassland site on the plateau of the Alter Gleisberg. tion and nutrient leaching in comparison to the surrounding soil. Circles mark the position of the six selected L. flavus nest-mounds and control soils, Thus, it is expected that nest-mounds are characterized by lower rectangles mark the position of the three bulk precipitation collectors. P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217 211

Table 1 the same time as collecting mineralized C, a soda lime trap that Characteristics of the selected L. flavus nest-mounds. The mound base area was allows O2 to pass into the cylinder, but captures CO2, was attached calculated using the formula for an ellipse and the mound volume was calculated to a hole in the cap of the cylinders. Three blank measurements using the formula for half an ellipsoid (Ohashi et al., 2005; Domisch et al., 2006). were made to account for atmospheric CO2 that is adsorbed by soda 2 3 Nest-mound Height [cm] Diameter [cm] Base area [m ] Volume [m ] lime during handling. After incubating 40 days at 25 C, the soda Nest 1 23 65 0.306 0.023 lime was removed from the syringes, dried at 105 C, and weighed. Nest 2 16 44 0.124 0.007 The potential C mineralization was calculated by subtracting the Nest 3 9 75 0.294 0.008 blank soda lime weight gain from the soda lime weight gain. The Nest 4 15 56 0.237 0.011 e Nest 5 9 77 0.284 0.009 results were multiplied by 0.27 to convert from CO2 to CO2 C and Nest 6 9 60 0.236 0.007 by 1.69 to correct for the amount of water that is formed during the reaction of CO2 with soda lime and lost during the drying process (Grogan, 1998). volumetric water content was measured weekly with a soil mois- The potential net N mineralization rate (Nmin) was measured as e e ture meter (PCE-SMM 1, PCE Instruments, Meschede, Germany). a production of inorganic N (NH4 N and NO3 N) in soil incubated Three bulk precipitation collectors of 314 cm3 were established in under moist and aerobic conditions as described by Robertson et al. close proximity to the nest-mounds and control soils to determine (1999). Field-moist soil (equivalent 30 g oven dry weight) was weekly precipitation amounts (Fig. 1). weighed into polyethylene bottles and water content was adjusted to 50% soil moisture by weight with deionized water. Bottles were then capped loosely and incubated in the dark at 25 C for 40 days. 2.3. Soil solution sampling and analysis The water content was kept constant by weighing the bottles weekly and adding deionized water if necessary. 10 g subsamples Soil solution collection was conducted in weekly sampling were removed before and after the 40-day incubation and extrac- campaigns in June and July 2012. Soil solution was collected by 12 ted with 100 ml 1 M KCl on an overhead shaker for 1 h. The sus- borosilicate glass suction plates with a pore size of 1 mm (75 cm2 pensions were filtered through 0.45 mm cellulose acetate ecoTech, Bonn, Germany). Prior to the first use, each suction plate membrane filters (Sartorius, Gottingen,€ Germany) and analyzed for was cleaned with 0.1 M HCl and deionized water. In the field, nitrate (NH eN) and ammonium (NO eN) concentrations using a suction plates were installed with a minimum disturbance to the 4 3 photometer (Spectroquant Multy Colorimeter, Merck, Darmstadt, overlaying soil by excavating soil pits next to the nest-mounds and Germany) with a measurement range of 0.01e3.00 mg l 1 (±0.024) control soils. Suction plates were then placed horizontally inside for NH eN and 0.20e20.00 (±0.19) mg l 1 for NO eN(Michalzik the nest-mounds and control soils at a soil depth of approximately 4 3 and Martin, 2013). The potential net N mineralization was calcu- 10 cm. A mixture of site-derived soil and water was used to fix the lated by subtracting the initial NH eN þ NO eN concentrations plates ensuring optimal hydrological contact between the plates 4 3 from the final NH eN þ NO eN concentrations. and overlaying soil. After installation, a negative pressure of 4 3 600 hPa was applied to the plates. Because of low precipitation and 2.4.2. Total elements low soil water contents, soil solution could not be collected for the Soil pH was measured from a 1:2.5 air-dried soil:deionized last three months of the study period. water solution (WTW Multi 340i with SenTix 41-3 electrode, In the laboratory, soil solution samples were analyzed for pH Weilheim, Germany). The electrical conductivity was determined (WTW Multi 340i with SenTix 41-3 electrode, Weilheim, Germany) from a 1:2 air-dried soil:deionized water solution (WTW Multi 340i and electrical conductivity (EC) (WTW Multi 340i with TetraCon with TetraCon 325 electrode, Weilheim, Germany). For the analysis 325 electrode, Weilheim, Germany). Concentrations of dissolved of major and minor element concentrations, oven-dried aliquots carbon (DC), dissolved inorganic carbon (DIC) and dissolved ni- were ground and passed through a 40 mm sieve. Contents of TC and trogen (DN) were measured using a TOC analyzer (TOC-V /TNM- CPN TN were measured using an elemental analyzer (vario TOC cube, 1, Shimadzu, Duisburg, Germany). DOC was calculated as the dif- Elementar, Hanau, Germany). Soil organic carbon (SOC) was ference between DC and DIC. Concentrations of Ca, K, Mg and Na calculated by the difference between TC and total inorganic carbon were determined with ICP-OES (Varian 725-ES). (TIC) determined by the Scheibler volumetric method. Concentra- tions of Ca, K, Mg and Na were determined with ICP-OES (Varian 2.4. Soil sampling and analysis 725-ES) after a microwave assisted modified aqua regia digestion consisting of 1 ml deionized water, 2 ml 30% HCl and 4 ml 30% Soil sampling was conducted at the end of the study on October HNO3 (Mügler et al., 2010). 25, 2012. A total of 12 soil samples were collected from the central e part of each nest-mound and control soil at 0 10 cm depth. In the 2.4.3. Extractable elements < laboratory, the soil samples were homogenized, sieved ( 2 mm), Hot-water extractions (HWE) were done according to the and ants were removed. The soil samples were then divided into method described by Ghani et al. (2003) to estimate elemental fi three parts; eld-moist, air-dried at 50 C, and oven-dried at 105 C, concentrations in the labile soil fraction. Field-moist soil (equiva- for subsequent analysis. lent 4 g oven dry weight) was weighed into centrifuge tubes and extracted with 40 ml deionized water in a water bath at 80 C for 2.4.1. C and N mineralization 16 h. The tubes were then shaken and centrifuged for 20 min at The potential C mineralization rate (Cmin) was measured using 3500 rpm. The supernatants were filtered through 0.45 mm cellu- € the soda lime method that is based on the adsorption of CO2 by lose acetate membrane filters (Sartorius, Gottingen, Germany). The soda lime (Keith and Wong, 2006). 20 g of pre-oven-dried soda hot-water extracts were analyzed for pH (WTW Multi 340i with lime was filled in perforated syringes and attached to the cap of PVC SenTix 41-3 electrode, Weilheim, Germany) and EC (WTW Multi cylinders containing field-moist soil adjusted to 50% soil moisture 340i with TetraCon 325 electrode, Weilheim, Germany). The DC, by weight (equivalent 200 g oven dry weight). The water content of DIC and DN concentrations in the extracts were measured using a the soil was kept constant by weighing the cylinders weekly and TOC analyzer (TOC-VCPN/TNM-1, Shimadzu, Duisburg, Germany). adding deionized water if necessary. To keep the soil well aerated at DOC was calculated as the difference between DC and DIC. NH4eN 212 P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217

Table 2 solution samples between nest-mounds and control soils. Statisti- Mean temperatures (±standard deviation) and mean temperature fluctuations cal analyses were performed using IBM SPSS Statistics 19. (±standard deviation) of nest-mounds and control soils during the study period (June 8eOctober 25, 2012), summer months (June 8eAugust 31, 2012) and autumn months (September 1eOctober 25, 2012). Temperature fluctuations were calculated 3. Results by subtracting the daily minimum temperature from the daily maximum temperature. 3.1. Microclimatic conditions Nest Control fl Temperature [C] The overall mean temperatures of L. avus nest-mounds and Mean temperature during study period 16.3 (±4.0) 16.0 (±3.4) control soils were 16.3 C and 16.0 C(Table 2). Nest-mounds were Mean temperature in summer months 18.9 (±3.8) 18.2 (±3.2) significantly warmer than control soils (p ¼ 0.023; Fig. 2a). In Mean temperature in autumn months 12.3 (±2.9) 12.5 (±2.5) comparison to the control soils, nest-mounds were on average Temperature fluctuation [ C] Mean temperature fluctuation during study period 4.7 (±2.1) 3.6 (±1.4) 0.7 C warmer during the summer months and on average 0.2 C Mean temperature fluctuation in summer months 5.6 (±2.1) 4.3 (±1.4) cooler during the autumn months. Temperature fluctuations were Mean temperature fluctuation in autumn months 3.6 (±1.4) 2.8 (±1.1) also higher in nest-mounds, especially in the summer months (Table 2). Fig. 2b shows that nest-mounds tended to have lower volu- and NO eN were analyzed using a photometer (Spectroquant 3 metric water contents than the control soils, but differences were Multy Colorimeter, Merck, Darmstadt, Germany) (Michalzik and not significant. The weekly amount of precipitation was positively Martin, 2013). Concentrations of Ca, K, Mg and Na were deter- correlated with the volumetric water content of nest-mounds mined with ICP-OES (Varian 725-ES). (r ¼ 0.736, p < 0.001) and control soils (r ¼ 0.669, p ¼ 0.001).

2.5. Statistical analysis 3.2. Soil chemistry

The non-parametric Wilcoxon signed-rank test for paired sam- 3.2.1. C and N mineralization ples (p < 0.05) was used to test for significant differences in The potential C and net N mineralization rates were significantly microclimatic conditions, chemical characteristics of soil and soil affected by ants (Fig. 3). On average, nest-mounds had a 1.5 fold

Fig. 2. (a) Variation of daily means of soil temperature of nest-mounds and control soils during the study period, (b) average volumetric water contents of nest-mounds and control soils and average weekly precipitation amounts during the study period. Sampling dates with significant differences (Wilcoxon signed-rank test with Bonferroni correction, p < 0.0025) in soil water contents between nest-mounds and control soils are marked with *. P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217 213 lower potential C mineralization rate (p ¼ 0.028) and a 17.0 fold Table 3 higher potential net N mineralization rate than control soils Chemical characteristics (mean ± standard deviation) of soil samples taken from ¼ nest-mounds and control soils and the results of the Wilcoxon signed-rank test. Bold (p 0.028). Average potential C mineralization rates were fi < numbers indicate differences signi cant at p 0.05. 38.96 mgg 1 d 1 for nest-mounds and 57.77 mgg 1 d 1 for control soils. Average potential net N mineralization rates of nest-mounds Nest Control p-value and control soils were 0.51 mgg1 d 1 and 0.03 mgg1 d 1, Total elements pH (H2O) 7.79 (±0.03) 7.65 (±0.02) 0.027 respectively. EC [mScm 1] 346 (±20) 380 (±19) 0.028 Organic matter [mgg 1] 3.2.2. Total elements SOC 65,331 (±8898) 84,286 (±11,302) 0.028 TN 3319 (±439) 3541 (±476) 0.400 Chemical characteristics of soil samples exhibited significant Element concentration [mgg 1] differences between nest-mounds and control soils (Table 3). Soil Ca 107,267 (±20,727) 96,067 (±22,303) 0.058 pH was neutral to slightly alkaline and increased from control soils K 13,900 (±832) 12,783 (±828) 0.028 to nest-mounds (p ¼ 0.027). EC was significantly lower in nest- Mg 7965 (±408) 7660 (±346) 0.046 mound soils (p ¼ 0.028). Nest-mounds had significantly lower Na 443 (±18) 448 (±13) 0.463 Extractable elements contents of SOC (p ¼ 0.028). Contents of TN did not differ signifi- pH 7.52 (±0.04) 7.39 (±0.05) 0.027 fi cantly across the experiment. Nest-mounds were signi cantly EC [mScm 1] 239 (±22) 280 (±11) 0.028 enriched in K (p ¼ 0.028) and Mg (p ¼ 0.046). Organic matter [mgg 1] DOC 2155 (±339) 2827 (±265) 0.028 DN 244.42 (±45.90) 311.93 (±32.22) 0.028

3.2.3. Extractable elements NH4eN 11.18 (±4.45) 7.23 (±4.91) 0.138 e ± ± The hot-water extracts of nest-mounds were characterized by a NO3 N 18.86 ( 4.92) 35.89 ( 4.62) 0.028 m 1 significantly higher pH (p ¼ 0.027) and lower EC (p ¼ 0.028) Element concentration [ gg ] Ca 339.13 (±86.57) 445.77 (±56.69) 0.028 (Table 3). In comparison to the control soils, nest-mounds con- K 72.79 (±40.86) 60.84 (±15.17) 0.917 tained significantly lower concentrations of DOChwe (p ¼ 0.028), Mg 9.99 (±3.35) 16.62 (±1.87) 0.028 DNhwe (p ¼ 0.028) and NO3eNhwe (p ¼ 0.028). Concentrations of Na 22.75 (±7.39) 20.06 (±3.73) 0.225 hot-water extractable Ca (p ¼ 0.028) and Mg (p ¼ 0.028) were

significantly lower in nest-mounds. The hot-water extractable el- ements were generally less than 9% of their respective total soil element.

3.3. Soil solution chemistry

Soil solution EC was significantly higher in nest-mounds compared to control soils, as opposed to the bulk soil where EC was significantly lower in nest-mounds (Table 4). Soil solutions of nest-mounds were also significantly enriched in DOC, DN, Ca, K and Mg (Fig. 4).

4. Discussion

4.1. Effect of L. flavus on microclimatic conditions

Differences in microclimate between L. flavus nests and sur- rounding soil were only modest. Increased nest temperatures during the summer months are attributable to a higher amount of bare soil on the mounds leading to a greater insolation and heating (Dlussky, 1981). Additional heat is produced by the metabolic ac- tivity of the ants and microorganisms inside the nest (Holldobler€

Table 4 Chemical characteristics (mean ± standard deviation) of soil solution from nest- mounds and control soils and the results of the Wilcoxon signed-rank test. Bold numbers indicate differences significant at p < 0.05.

Nest Control p-value

pH 8.17 (±0.06) 7.96 (±0.08) 0.078 EC [mScm 1] 424 (±37) 279 (±36) 0.043 Organic matter [mg l 1] DOC 27.31 (±2.41) 23.09 (±2.68) 0.028 DN 3.95 (±2.84) 2.85 (±2.80) 0.046 Element concentration [mg l 1] Ca 87.20 (±4.40) 59.80 (±6.41) 0.043 Fig. 3. Boxplots showing potential C and net N mineralization rates of nest-mounds K 4.32 (±0.47) 2.55 (±1.69) 0.046 and control soils. The box spans the first to the third quartile; the vertical line in the Mg 2.90 (±0.29) 1.93 (±0.38) 0.043 box shows the median; bars above and below the box represent the minimum and Na 3.55 (±0.96) 2.25 (±1.10) 0.080 maximum values. 214 P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217

potential C mineralization rate than control soils pointing to a decreased microbial activity in nest-mounds. Due to the lower amount of SOC and hot-water extractable DOC in nests it can be suggested that C limitation is the major reason for the diminished C mineralization rate. In contrast, higher rates of plant root- derived labile C inputs into the control soils increase the C avail- ability and thereby promote the growth and activity of soil mi- croorganisms (Blume et al., 2010). According to Amador and Gorres€ (2007), soil mixing is another factor influencing the microbial ac- tivity. Ant bioturbation mixes upper organic soil layers with deeper mineral soil layers that may contain fewer microorganisms with different metabolic capacities. Moreover, ants produce antifungal and antibacterial agents that are secreted by the paired thoracic metapleural glands. The secretions primarily protect the ants against the growth of bacteria and fungal spores (Holldobler€ and Wilson, 2001; Beattie, 2010) and can therefore affect the size and composition of the microbial community in nest-mounds (Amador and Gorres,€ 2007). This is in agreement with findings of Blomqvist et al. (2000), Dauber and Wolters (2000) and Boots et al. (2012) who reported that the composition of the microbial community in L. flavus nests and the surrounding soil were significantly different. Moreover, Holec and Frouz (2006) found lower bacteria counts in nests of L. flavus. Under field conditions, the microclimate of nests may also play an important role in affecting microbial activity. In this study, L. flavus nest-mounds were characterized by higher temperatures during the summer months and slightly lower water contents. High nest temperatures could speed up metabolic processes, whereas low water contents inhibit metabolic processes. The results are consistent with observations by Lenoir et al. (2001) and Domisch et al. (2008) who reported that nests of Formica species were very dry resulting in a lower microbial Fig. 4. Variation in mean DOC and DN concentrations in soil solution of nest-mounds and control soils during the study period. Error bars represent one standard deviation. activity and slow decomposition of OM, which is probably due to the fact that wood ant nests are constructed mainly of organic material. and Wilson, 1990). Temperature fluctuations were also greater in Despite the fact that L. flavus nest-mounds contained signifi- nest-mounds than in the control soils probably due to the lack of a cantly lower amounts of hot-water extractable DN, nest-mounds heat insulating vegetation cover. Thus, the thermal storage of had a considerably higher potential net N mineralization rate in L. flavus nests is relatively short-term (Schreiber, 1969; Frouz and comparison to the control soils. Higher potential net N mineral- Jílkova, 2008). ization rates were also reported by Wagner and Jones (2006) in L. flavus nest-mounds were slightly but not significantly drier harvester ant nests, while Lenoir et al. (2001) found no net N than control soils. Many other studies found that L. flavus nests mineralization in wood ant nests. The enrichment of inorganic N were drier (Dean et al., 1997; King, 1977a; Blomqvist et al., 2000; in nests is possibly a result of a combination of increased N Holec and Frouz, 2006; Wu et al., 2010; Boots et al., 2012). As mineralization and decreased N utilization (Wagner and Jones, with the highly fluctuating nest temperatures, a lower water con- 2006). The environmental conditions in L. flavus nest-mounds tent is possibly a result of the larger amount of bare soil, increasing could favor a specific microbial community associated with the evaporation (Cammeraat and Risch, 2008). Woodell and King (1991 N cycle resulting in increased N mineralization rates. In contrast, cited by Blomqvist et al., 2000) suggested that evaporation is the very low potential net N mineralization rate in control soils further promoted by the burrowing activity of ants and the indicates that available inorganic N might be incorporated by soil resulting loose soil structure with an increased porosity and microorganisms. It is likely that the higher availability of labile C decreased bulk density. Moreover, nest-mounds have been found to to microorganisms and higher C mineralization rates in the allow more water infiltration (Green et al., 1999). control soils increase microbial N demand and the immobiliza- tion of inorganic N by microorganisms (Schaeffer and Evans, 2005). 4.2. Effect of L. flavus on nutrient cycling rates and related C and N contents 4.2.2. Total C and N In agreement with previous studies (Dostal et al., 2005; Holec 4.2.1. C and N mineralization and Frouz, 2006; Platner, 2006; Wu et al., 2010; Boots et al., Several field and laboratory studies reported an increase in CO2 2012), L. flavus nest-mounds were more depleted in SOC than efflux from nests of L. flavus (Dauber and Wolters, 2000; Wu et al., the surrounding soil. In contrast, L. flavus had no effect on TN 2013), wood ants (Ohashi et al., 2005, 2007; Risch et al., 2005; concentrations. Ants may have reduced SOC concentrations in two Domisch et al., 2006), fire ants (Bender and Wood, 2003), and different ways; they either decreased the input of C into the soil or leaf-cutting ants (Sousa-Souto et al., 2012) compared to the sur- they increased the output of C. Frouz et al. (2003) suggested that rounding soil. Only few studies showed a lower CO2 efflux from ant the depletion of C might be a result of a higher microbial activity in nests (Holec and Frouz, 2006; Amador and Gorres,€ 2007). In this nest-mounds accelerating the decomposition, mineralization, study, soils from L. flavus nest-mounds had a 1.5 fold lower leaching and consequently the loss of stored C. Although L. flavus P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217 215 nest-mounds exhibited higher DOC concentrations in the soil so- 4.2.4. Leachable C and N lution indicating increased leaching rates, the potential C miner- Although nest-mounds contained lower contents of SOC, the alization rate was considerably lower. The decreased SOC content DOC concentration in soil solution was higher compared to the must therefore be a consequence of reduced C inputs into the nest- control soils. In a greenhouse experiment, Stadler et al. (2006) also mound soil. Shoot and root remains, root exudates from living found larger DOC concentrations in Norway spruce litter solutions roots as well as root hairs and fine roots sloughed by root elon- affected by 100 workers of F. polyctena in comparison to unaffected gation represent an important source of C input into the soil litter. However, DOC concentrations in litter solution affected by F. (Kuzyakov and Domanski, 2000). Nest-mounds provide less polyctena (approximately 75 mg l 1) were considerably higher than favorable sites for plant colonization due to the burrowing activity DOC concentrations in soil solution affected by L. flavus (27 mg l 1). of ants (Dostal et al., 2005), high seed mortality (King, 1977c) and Differences in DOC concentrations between nest-mounds and dry soil conditions (Petal, 1978 cited by Lenoir et al., 2001). control soils were probably due to the input of easily decomposable Therefore, nest-mounds are characterized by less plant cover honeydew from root aphids farmed by L. flavus inside the nest- (Dean et al., 1997) or root biomass (Dostal et al., 2005) compared to mound. Honeydew is a sticky liquid that is mainly composed of the surrounding soil. Besides decreased C inputs, the depletion of C carbohydrates (90e95 % of the honeydew dry weight) and contains in nest-mounds can be attributed to the burrowing activity of ants smaller amounts of organic acids, B-vitamins and minerals and the consequent soil mixing (Frouz et al., 2003; Holec and (Holldobler€ and Wilson, 1990). Seifert (2007) estimated that a Frouz, 2006). medium-sized nest with 23,000 L. flavus workers farms approxi- mately 17,000 root aphids. During the ant-active season, L. flavus would be able to harvest 6 ml honeydew per day. 4.2.3. Extractable C and N Hot-water extracts represent a small and relatively labile soil 4.3. Effect of L. flavus on base cations in soil and soil solution fraction. They provide easily available nutrients for soil microor- ganisms and thereby contribute to the soil nutrient cycle (Strosser, Ants promoted the enrichment of base cations in the nest- 2010). It was expected that L. flavus nest-mounds would contain mound as indicated by higher Ca, K and Mg concentrations both higher amounts of extractable soil elements in comparison to the in bulk soil extractions and in the soil solution. This is likely for control soils due to the accumulation of easily decomposable several reasons including the accumulation of food and waste organic material in form of food and waste material. However, nest- material in the nest (Frouz et al., 2003), carbonate weathering mounds exhibited significantly reduced amounts of DOChwe,DNhwe (Blume et al., 2010) and the upward transport of calcareous subsoil and NO3eNhwe, and had no effect on NH4eNhwe. Therefore, it ap- through ant bioturbation (Levan and Stone, 1983). The higher pears that accumulated food and waste material inside nest- content of base cations could also be a reason for the increased pH, mounds play only a minor role in labile C and N supply. Vegeta- that we and others have observed in nest-mounds (Levan and tion seems to play a more important role. According to Kuzyakov Stone, 1983; Frouz et al., 2003; Jílkova et al., 2012). and Domanski (2000), root exudates, root hairs and fine roots are considered to be important sources of labile C and N inputs into the 5. Conclusions: L. flavus nest-mounds as hot or cold spots in soil, because they are easily decomposed by soil microorganisms nutrient cycling? and contain relatively high concentrations of C and N. Due to the decreased vegetation cover on the mounds, there are lower inputs To sum up, L. flavus significantly modified the soil environment of labile C and N in comparison to the control soils. Some authors within the nest. Nest-mounds had decreased potential C mineral- showed that hot-water extractable DOC is strongly correlated with ization rates, but increased potential net N mineralization rates, the microbial biomass-C (Sparling et al., 1998; Ghani et al., 2003) which were related to lower amounts of SOC, hot-water extractable and CO2 evolution (Fischer, 2003 cited by Ghani et al., 2003), which DOC and DN, and higher concentrations of leachable DOC and DN indicates that hot-water extractable DOC is an easily available (Fig. 5). substrate for microbial utilization (Ghani et al., 2003). The C limi- It is impossible to say that L. flavus nest-mounds acted as overall tation in nest-mounds could therefore reduce the microbial activity hot or cold spots in nutrient cycling. Rather a differentiated view is and C mineralization. necessary. L. flavus nest-mounds represented cold spots for the C

Fig. 5. Schematic summary of the effects of L. flavus nest-mounds on microclimatic conditions, soil solution chemistry and soil chemistry. Plus (þ) and minus () show statistically significant increases and decreases in soil properties of nest-mounds in comparison to the control soils. The equal sign (¼) shows non-significant differences in soil properties between nest-mounds and control soils. 216 P. Bierbaß et al. / Soil Biology & Biochemistry 80 (2015) 209e217 cycling because of a lower soil C and labile C enrichment as well as a Folgarait, P.J., 1998. Ant and its relationship to ecosystem functioning: a e lower potential C mineralization rate. However, nest-mounds review. Biodiversity and Conservation 7, 1221 1244. Frouz, J., Holec, M., Kalcík, J., 2003. The effect of Lasius niger (, For- exhibited increased concentrations of leachable DOC. In contrast, micidae) ant nest on selected soil chemical properties. 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