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Riparian soil response to surface nitrogen input: the indicator potential of free-living soil nematode populations
Soil Biology and Biochemistry 31 (1999) 1625±1638
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Riparian soil response to surface nitrogen input: the indicator potential of free-living soil nematode populations Christien H. Ettema a,*, 1, Richard Lowrance b, David C. Coleman a a
Institute of Ecology, University of Georgia, Athens, GA 30602, USA Southeast Watershed Research Lab, US Department of Agriculture, Tifton, GA 31793, USA
b
Accepted 25 April 1999
Abstract Nitrogen saturation of riparian ecosystems may lead to leaching and loss of surface water quality. In the search for early warning signs of N-saturation, we conducted an N-addition experiment to evaluate the indicator potential of free-living nematodes. During 6 months, we measured changes in feeding and life strategy groups, following single high and repeated low inorganic N-additions to a near-stream zone (`zone 1') and an upslope area (`zone 2') within a riparian forest in the southeast Coastal Plain of Georgia. In both zones, N-addition signi®cantly increased the number of bacterivores but not fungivores. Only bacterivores with r-selected life strategies increased. In this group, Rhabditinae multiplied after N-addition in both zones, but Neodiplogasteridae and Myolaimidae responded to N only in zone 1, and Cephalobidae only in zone 2. Microbivore correlations with microbial data, collected concomitantly (Ettema et al., 1999. Riparian soil response to surface nitrogen input: temporal changes in denitri®cation, labile and microbial C and N pools, and bacterial and fungal respiration. Soil Biology & Biochemistry 31, 1609±1624), were considerably stronger in N-amended treatments than in controls, suggesting that N-addition synchronized microbial-microbivorous dynamics. At the end of the experiment, bacterivore abundance returned to control levels, probably partly due to predation, as predator populations had considerably increased several months after the ®rst N-addition. The increase in predator abundance was greater in the single high than repeated low Naddition treatments, as was the case for bacterivore populations. These results suggest that nematodes can be indicators of Nsaturation. However, for practical application, it appears that these indicators could only be meaningful when monitored together with other system characteristics, such denitri®cation rates and litter N. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Riparian zone; Nitrogen saturation; Free-living nematodes; Indicators
1. Introduction Riparian ecosystems are increasingly managed as buers between aquatic bodies and upland agricultural or urban land use, protecting water quality from pollutants such as nitrogen (Gregory et al., 1991; Welsch, * Corresponding author. Tel.: +31-317-482981; fax: +31-317483766. E-mail address: [email protected] (C.H. Ettema) 1 Present address: Wageningen Agricultural University, Soil Biology Group, P.O. Box 8005, 6700 EC Wageningen, The Netherlands.
1991; Fennessy and Cronk, 1997; Lowrance et al., 1997). A major uncertainty in the management of riparian systems is for how long the buers can be sustained when N-loading is continuous (Lowrance and Vellidis, 1995). N-saturation, de®ned as the availability of inorganic N in excess of plant and microbial demand (Aber et al., 1989; Hanson et al., 1994a), may lead to increased nitri®cation and leaching rates, lowered resistance of plants to pests, and plant community changes (Aber et al., 1989), all of which may greatly impair the water quality protection function of the riparian buer zone (e.g., Kadlec and Bevis, 1990). From the management point of view, it is highly desir-
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able to get early warning signs, or `indicators', of impending N-saturation, before riparian systems lose buer capacity. The overall objective of our study was to evaluate the potential of free-living soil nematode assemblages for indicating N-saturation in riparian ecosystems. While it appears easier to measure and interpret biochemical, rather than faunal variables, the advantage of the latter is that they integrate soil N-conditions over a period of time, whereas the former may exhibit a more instantaneous, `snap shot' character. Because of the complementarity of faunal and biochemical indicators, general procedures for monitoring soil quality and disturbance often call for their combined assessment (Linden et al., 1994; Blair et al., 1996; Pankhurst et al., 1997). So far, only biochemical (N-cycling) indicators have been used in the assessment of N-saturation in riparian ecosystems (e.g., Hanson et al., 1994a). The rationale for selecting free-living nematodes was primarily that nematodes were likely to be abundant and diverse along the steep moisture gradient typically found in riparian zones. Secondly, freeliving soil nematodes should be functionally important for riparian soil functioning, by enhancing decomposition and N-mineralization processes (Hunt et al., 1987; Freckman, 1988; Griths, 1994). In many riparian systems, sub-surface ¯ow N, taken up by roots and stored in plant biomass, only becomes available to surface soil denitri®ers when plant litter decomposes (Groman et al., 1992; Hanson et al., 1994b; Lowrance et al., 1997). Thirdly, other studies have shown signi®cant nematode assemblage responses to organic and inorganic N-additions in pastural, agricultural and forest ecosystems (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986; Dmowska and Kozlowska, 1988; Ettema and Bongers, 1993; Griths et al., 1994). In a survey of a riparian wetland zone downslope of a dairy waste application site, Ettema et al. (1998) found high nematode abundances, and a prevalence of bacterial-feeding taxa with r-selected life strategies, at microsites with elevated soil inorganic N-concentration. However, it remained unclear whether these content dierences were causally related to soil Ncontent variation. Thus, the speci®c objective of our N-addition study was to experimentally assess whether free-living riparian soil nematode populations are responsive to riparian soil N changes. To obtain a mechanistic understanding of potential N-induced changes in the nematode populations in this experiment, we examine the links between microbivorous nematode dynamics and concomitantly measured microbial respiration and biomass dynamics (Ettema et al., 1999).
2. Materials and methods 2.1. Site description The experiment was conducted in a mature riparian forest adjoining a second-order stream, located near Tifton, Georgia, USA. The forest had two distinct zones: zone 1, a 5±8 m wide strip bordering the stream, is vegetated by hardwood trees, primarily yellow poplar (Liriodendron tulipifera L.) and swamp tupelo (Nyssa sylvatica var bi¯ora Marsh.). Zone 1 soil is very poorly drained ®ne-loamy sand, with an average water ®lled pore space (WFPS) of 75%, and a soil carbon content of 2±5%. Zone 2, upslope (slope < 1%) from zone 1, is a 30±40 m wide stand of predominantly slash pine (Pinus elliotii Engelm.) and longleaf pine (Pinus palustris Mil.). Zone 2 soil is a poorly drained loamy sand, with an average WFPS of 51%, and a carbon content of 1±3%. 2.2. Experimental design The experiment was set up as a split-plot design, with zone as the main plot eect, and N-addition as the split plot eect. Each N-addition level was replicated in six randomly selected 1.5 by 2.5 m blocks in each zone, and measurements were taken at days 1 (22 Oct. 1996), 10, 22, 42, 78, 134 and 191 (30 April 1997). Ettema et al. (1999) provide details on the experimental design and randomization process. 2.2.1. Nitrogen treatment N-addition treatments were control (C), single high input (H) and repeated low input (L). To prevent Ncontamination by surface runo from the adjacent agricultural ®eld and between treatments, N was added to ®eld soil microcosms instead of larger plots. Microcosms were installed 2 weeks prior to the ®rst Naddition, and were constructed by taking intact littersoil cores to a 20 cm depth, using a corer with a removable inner plastic tube of 25 cm length and 6 cm diameter. Each soil core, contained in a tube, was returned into the sample hole after covering the bottom with nylon mesh (the top remaining uncovered), to allow vertical water ¯ow and nematode migration while facilitating later microcosm retrieval. No plants grew in the microcosms throughout the experiment (sporadically-emerging seedlings were removed by hand). Treatment H was equivalent to 7.5 g N mÿ2, added in a single dose at day 0 (21 Oct. 1996). Treatment L was the same total N input, but was added in ®ve pulses of 1.5 g N mÿ2 each, at days 0, 32, 68, 124 and 181, respectively. N was added in 20 ml Dl water per microcosm, as a 2 : 1 mixture of NH4Cl±N and KNO3±N, mimicking the relative composition of inorganic N ions in surface runo from the
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
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Table 1 Abundances of free-living nematode families in two zones of a riparian forest, subject to repeated low (L), single high (H) or zero nitrogen input (C). Abundances are per 50 g soil (dw), averaged over blocks and time (n 42 for each mean) Zone 1
Zone 2
Trophic group
Life strategy subgroup
Family
C
L
H
C
L
H
Bacterial feeders
Bf 1
Rhabditinaea Neodiplogasteridaeb Bunonematidaec Myolaimidaed Cephalobidaee Plectidaef Monhysteridaeg Rhabdolaimidaeh Prismatolaimidaei Teratocephalidaej Desmodoridaek Chronogasteridael Odontolaimidaem Bastianiidaen Alaimidaeo Bathyodontidaep Aphelenchoididaeq Anguinidaer Aphelenchidaes Diphterophoridaet Leptonchidaeu Aphelenchoididaev Tripylidaew Mononchidaex Belonderidaey Chrysonematidaez Leptonchidaea1 Qudsianematidaeb1 Aporcelaimidaec1
78 11 3 1 208 82 22 103 58 27 4 7 3 1 8 0 351 7 2 5 54 2 3 3 2 0 4 7 3 1059
118 21 9 5 186 67 16 108 57 39 3 4 3 0 8 0 324 4 2 9 77 3 7 4 5 0 3 12 5 1099
149 81 5 14 216 79 14 89 66 29 6 5 3 1 6 1 374 4 0 12 66 1 10 4 2 0 5 5 6 1253
82 6 12 0 223 60 10 31 51 66 15 0 2 0 4 0 163 4 1 24 51 0 4 14 0 0 3 8 5 839
98 4 13 0 304 74 10 29 47 34 9 2 1 0 3 0 208 12 6 22 41 2 4 16 0 0 2 11 6 958
130 6 12 0 315 64 8 28 57 47 8 0 0 0 3 0 221 3 5 27 46 0 9 19 0 1 5 11 6 1031
Bf 2 Bf 3
Bf 4 Fungal feeders
Predators
Omnivores Total a
Ff 2 Ff Ff Pr Pr Pr Pr
3 4 2 3 4 5
Om 4 Om 5
Not identi®ed beyond superfamily level. Pristionchus. c Bunonema. d Myolaimus. e Acrobeloides, Heterocephalobus, Cervidellus, Acrobeles, Acrolobus, Zeldia. f Plectus, Wilsonema, Anaplectus. g Eumonhystera, Monhystrella, Geomonhystera. h Rhabdolaimus. i Prismatolaimus. j Teratocephalus, Metateratocephalus, Euteratocephalus. k Prodesmodora. l Chronogaster. m Odontolaimus. n Bastiania. o Alaimus, Paramphidelus. p Cryptonchus. q Aphelenchoides. r Pseudhalenchus. s Aphelenchus. t Diphtherophora, Tylolaimophorus. u Tylencholaimus, Tylencholaimellus, Basirotyleptus. p Seinura. w Tripyla, Trischistoma. x Prionchulus, Mylonchulus. y Oxydirus, Axonchium. z Chrysonema. a1 Dorylaimoides. b1 Eudorylaimus, Epidorylaimus, Microdorylaimus. c1 Aporcelaimellus, Sectonema. b
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upslope ®eld. Control microcosms received 20 ml Dl water. 2.3. Sample collection and processing On each sampling date, one randomly selected microcosm was retrieved from each treatment in each block. After subsampling for denitri®cation, microcosms were transported to the Soil Ecology Laboratory in Athens GA, and processed within 7 days for measurement of various microbial and nutrient pools as described in Ettema et al. (1999), and the extraction of nematodes. 2.3.1. Nematode extraction Nematodes were extracted from approximately 50 g soil, which was subsampled from the carefully mixed top 10 cm of the microcosm. The sample, which included organic debris, was extracted by decanting and wet sieving (Southey, 1986), including a 5 s blendering step for the litter (cf. Schouten and Arp, 1991). Final separation occurred on a Baermann tray using double cottonwool ®lters (Schouten and Arp, 1991), and extracted nematodes were stored in 5% formaldehyde. First, all nematodes were counted using an inverted microscope. Next, a random subset (specimens occurring in small marked areas randomly distributed over the counting dish grid) was identi®ed to genus using Bongers (1988) and Jairajpuri and Ahmad (1992), until 15% of total abundance in the sample was identi®ed. In the few cases where this was less than 100 individuals, the process proceeded until 100 identi®cations were obtained. Repeated analysis showed that this generally yielded a good estimate of nematode richness and abundances of dominant taxa. Taxa were assigned to feeding groups following Yeates et al. (1993). Within each trophic group, nematodes were ranked by life strategy according to Bongers and Bongers (1998) (Table 1). In this classi®cation, a low number (1±2) indicates a `colonist', r-selected (sensu lato ) life strategy (high reproduction rate, small body size, short life cycle), and a high rating (3±5) signi®es a `persister', K-selected (sensu lato ) life strategy (low reproduction rate, large body size, long life cycle) (Bongers, 1990). 2.3.2. Soil characteristics of fresh soil cores To evaluate the eects of enclosure on nematodes within the microcosms, a fresh soil core was randomly taken from each block, on the ®rst and last sampling date (days 1 and 191, i.e., 14 and 205 days after microcosm installation), using the same soil corer as used for constructing the microcosms. Nematode populations in these cores were compared to the control microcosm data of days 1 and 191.
2.4. Data analysis Because of the low frequencies of most genera and families (i.e., many taxa occurred only in a small portion of the microcosms sampled), analysis of variance (ANOVA) was limited to clusters of taxa, grouped according to life-strategies within feeding categories following Bongers and Bongers (1998). Some clusters were merged to further improve frequencies: bacterivores of life strategy classes 3 and 4; predators 2 and 3; predators 4 and 5; and omnivores 4 and 5 (Table 1). Treatment eects were tested using a repeated measures ANOVA in SAS Version 6 (SAS Institute, 1990), according to Ettema et al. (1999). Nematode abundances were log10-transformed prior to statistical testing to better normalize distributions. Data on herbivorous nematodes are not reported here. As no plants grew in the microcosms throughout the experiment (sporadically emerging seedlings were removed by hand), plant feeder dynamics were not directly relevant to this study's objectives. Repeated measures ANOVA was also conducted for the Fisher's a diversity index, a parameter of the logseries distribution (Fisher et al., 1943). This statistic is a measure of richness (in this case, genus richness) that is insensitive to sample size (number of individuals in the sample), and is recommended as the standard diversity index by various authors (Magurran, 1988; Rosenzweig, 1995). It was calculated for all free-living nematodes, and for bacterivores, the most diverse trophic category. Repeated measures analysis was also performed for the maturity index (MI), an index of disturbance based on life strategy group frequencies (Bongers, 1990), and for the ratio of fungal- to bacterial-feeders (nematode F/B ratio). 3. Results The riparian forest soil nematodes were abundant and diverse (Table 1). Throughout the study, 29 freeliving families comprising 49 genera were detected, giving an average total abundance of 1047 individuals (range 334±4277) 50 gÿ1 soil. The mean number of genera occurring together in a single 50 g sample was 17 (range 10±27). Bacterivores were the most abundant and diverse trophic group, representing on average 63% of total abundance and 64% of total genus richness in the microcosms. 3.1. Basic zone dierences Contrasting control (C) microcosms in zones 1 and 2 of the riparian forest indicated that the free-living nematode fauna in zones less than 10 m apart was quite dierent (Tables 1 and 2). Overall, nematodes
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638 Table 2 Comparing nematode abundances and diversities (in 50 g soil dw) between zone 1 and zone 2 soil, using the data from the control (C) microcosms Nematode variablea Bf 1 Bf 2 Bf 3±4 Ff 2 Ff 3 Ff 4 Pr 2±3 Pr 4±5 Om 4±5 (all omnivores) All bacterivores All fungivores All predators Total free-living nematodes Overall genus richness Overall Fisher's a diversity Bacterivorous genus richness Bacterivorous Fisher's a diversity Maturity index Nematode F-to-B ratio
Zone 1b Zone 2b Fc 93 312 211 360 5 54 5 5 14 616 419 10 1059 16.6 5.42 11.4 4.08 2.31 0.71
Table 3 ANOVA of bacterivorous groups.a Abundances (in 50 g soil dw) were log10-transformed before analysis Bf 1
P
100 0.01 0.9153 293 0.07 0.7951 169 5.50 0.0410 168 9.57 0.0114 24 61.67 0.0001 51 0.22 0.6463 4 0.81 0.3887 14 6.57 0.0283 16 0.54 0.4781 562 1.77 0.2132 243 5.69 0.0383 18 11.53 0.0068 839 5.51 0.0408 17.8 9.20 0.0126 6.11 7.69 0.0197 11.0 4.09 0.0706 3.82 2.31 0.1596 2.35 0.93 0.3578 0.48 5.58 0.0398
a
See Table 1 for trophic and life strategy group compositions. Abundances and diversities averaged over blocks and sampling dates (n 42 for each mean). c F-test used log10-transformed abundances, and block within zone as error term (df 1, 10). b
were signi®cantly more numerous in zone 1 than zone 2 soil, averaging 1059 vs. 839 individuals 50 gÿ1 soil, respectively (Table 2). This increase in total abundance was mainly due to signi®cantly higher numbers of rselected fungivores (Ff 2; mainly Aphelenchoididae) and K-selected bacterivores (Bf 3±4; mostly Rhabdolaimidae) in zone 1. Fungivores of life strategy class 3 (Ff 3; only Diphtherophoridae), however, were signi®cantly more abundant in zone 2 control microcosms, as were predators (Pr 4±5: mainly Mononchidae). The maturity index, which is a weighted average of the frequencies of these life strategy groups, was not dierent between zones. However, the F-to-B ratio, which was always smaller than one, was signi®cantly higher in the near-stream zone. Overall genus richness was signi®cantly higher in zone 2, where microcosms contained on an average one genus more than zone 1 enclosures (Table 2). This was mainly due to the higher predator richness in zone 2 samples. Taxa exclusively found in zone 1 were the predators Axonchium and Oxydirus, the bacterivores Bastiania, Cryptonchus, Euteratocephalus and Myolaimus, and the omnivore Epidorylaimus. Taxa limited to zone 2 microcosms were the bacterivorous Cervidellus and fungivorous Tylolaimophorus. Common genera (with frequencies less than 85%, i.e., testable in ANOVA) that were signi®cantly more abundant in zone 1 than in zone 2 control microcosms
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Bf 2
Bf 3±4
All bacterial feeders
Source
SS
F
SS
F
SS
F
SS
F
Zoneb Nc C vs. L, Hd L vs Hd Zone Nc Zone (C vs. L, H)d Zone (L vs. H)d Tieme Time zonee Time Nf Time (C vs. L, H)g Time (L vs. H)g Time N zonef
0.15 3.26 2.64 0.62 0.05 0.04 0.01 4.56 1.03 4.53 2.89 1.64 3.40
0.30 15.8h 25.6h 6.01i 0.25 0.40 0.09 4.54h 1.02 1.78j 2.28i 1.29 1.34
0.54 0.14 0.08 0.06 0.36 0.36 0.00 2.30 1.23 0.38 0.07 0.31 0.45
9.44i 1.89 2.11 1.67 4.66i 9.26k 0.07 7.72h 4.13h 0.79 0.29 1.29 0.94
2.22 0.11 0.07 0.04 0.21 0.11 0.10 0.91 0.81 0.78 0.34 0.44 0.47
13.4h 1.07 1.36 0.79 2.10 2.17 2.04 2.50i 2.21j 1.38 1.21 1.55 0.83
0.05 0.26 0.14 0.12 0.01 0.01 0.00 0.98 0.26 0.44 0.31 0.13 0.20
0.85 4.32i 4.50i 4.13j 0.22 0.44 0.00 4.96i 1.31 1.34 1.85j 0.82 0.61
a
See Table 1 for group composition. F-test (df 1, 10) used Block within Zone [Block(Zone)] as error term. c F-test (df 2, 20) used N Block(Zone) as error term. d F-test (df 1, 20) used Time Block(Zone) as error term. e F-test (df 6, 60) used Time Block(Zone) as error term. f F-test (df 12, 120) used Time N Block(Zone) as error term. g F-test (df 6, 120) used Time N Block(Zone) as error term. h PR0:005;. i PR0:05;. j PR0:10;. k PR0:01. b
Table 4 ANOVA of fungivorous groups.a Abundances (in 50 g soil) were log10-transformed before analysisb Ff 2
Ff 3
Source
SS F
SS
Zone N C vs. L, H L vs. H Zone N Zone (C vs. L, H) Zone (L vs. H) Time Time zone Time N Time (C vs. L, H) Time (L vs. H) Time N zone
2.57 5.85c 0.18 1.23 0.17 2.32 0.01 0.14 0.26 1.77 0.26 3.54d 0.00 0.00 4.54 13.7e 0.32 0.97 0.45 0.93 0.18 0.74 0.27 1.12 0.64 1.34
22.5 0.85 0.84 0.01 1.13 1.12 0.01 2.92 0.71 3.63 1.37 2.26 1.46
a
Ff 4
All fungal feeders
F
SS F
SS F
38.6e 0.87 1.71 0.02 1.15 2.29 0.02 2.07d 0.50 1.01 0.77 1.26 0.41
1.95 1.68 0.10 0.22 0.02 0.07 0.08 0.37 1.19 2.72d 0.44 2.03 0.74 3.41d 2.85 2.58c 2.20 2.00d 0.51 0.31 0.17 0.20 0.35 0.42 1.00 0.60
1.56 3.41d 0.09 0.74 0.09 1.48 0.00 0.01 0.06 0.55 0.05 0.98 0.01 0.01 3.45 16.6e 0.14 0.67 0.21 0.60 0.08 0.43 0.13 0.78 0.36 1.02
See Table 1 for group composition. See Table 3 for explanation of F-tests and dfs. c PR0:05. d PR0:10. e PR0:005. b
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Table 5 ANOVA of predaceous and omnivorous groups.a Abundances (in 50 g soil) were log10-transformed before analysisb Pr 2±3
Pr 4±5
All predators
Om 4±5 (all omnivores)
Source
SS
F
SS
F
SS
F
SS
F
Zone N C vs. L, H L vs. H Zone N Zone (C vs. L, H) Zone (L vs. H) Time Time zone Time N Time (C vs. L, H) Time (L vs. H) Time N zone
0.03 2.33 1.87 0.46 0.60 0.60 0.00 8.14 0.85 15.0 10.9 4.10 2.44
0.05 3.21c 5.14d 1.27 0.83 1.65 0.00 4.60e 0.48 5.23e 7.59e 2.87d 0.85
11.8 1.67 1.31 0.36 0.60 0.03 0.57 3.22 2.01 2.72 0.59 2.13 4.89
10.3f 2.99c 4.68d 1.29 1.07 0.11 2.04 1.47 0.92 1.04 0.45 1.62 1.87d
5.61 1.93 1.93 0.00 0.40 0.21 0.19 3.59 1.14 8.15 4.85 3.30 5.29
8.35d 3.76d 7.52d 0.00 0.77 0.79 0.75 1.82 0.58 2.87e 3.41e 2.48d 1.86d
1.20 0.93 0.93 0.00 0.16 0.06 0.10 1.62 4.01 3.62 1.62 2.00 2.71
2.69 1.75 3.49c 0.00 0.30 0.22 0.37 1.13 2.79d 1.37 1.22 1.52 1.02
a
See Table 1 for group composition. See Table 3 for explanation of F-tests and dfs. c PR0:10;. d PR0:05;. e PR0:005;. f PR0:01. b
were Aphelenchoides, Plectus and Rhabdolaimus. Conversely, Diphtherophora and Teratocephalus were signi®cantly more abundant in zone 2 than in zone 1. 3.2. Eects of N-additions The experimental N-addition signi®cantly changed the abundance of bacterivores but not fungivores, and
in¯uenced the number of predators (Tables 3±5, Figs. 1±3). There were also signi®cant N-eects on maturity and bacterivorous diversity indices (Table 6, Fig. 4). Some of these eects were dependent on zone and time of observation. In many cases, N-eects were greater in single high, than in repeated low, N-addition treatments. Among the bacterivorous taxa, nematodes with r-
Table 6 ANOVA of Fisher's a diversity, maturity index, and fungal to bacterial feeder ratioa Overall Fisher's a diversity
Bacterivore Fisher's a diversity
Maturity index
Nematode F/B ratio
Source
SS
F
SS
F
SS
F
SS
F
Zone N C vs. L, H L vs. H Zone N Zone (C vs. L, H) Zone (L vs. H) Time Time zone Time N Time (C vs. L, H) Time (L vs. H) Time N zone
7.53 9.10 0.58 8.52 4.54 3.85 0.68 26.2 12.4 19.9 10.5 9.43 19.3
2.26 2.47 0.32 4.62b 1.23 2.09 0.37 3.49c 1.65 1.09 1.14 1.03 1.05
16.4 6.25 3.93 2.32 5.30 1.97 3.33 18.4 11.2 14.6 7.06 7.51 14.8
11.3d 4.03b 5.07b 3.00e 3.42e 2.54 4.30e 4.44c 2.70b 1.15 1.12 1.19 1.17
0.01 0.43 0.32 0.11 0.24 0.10 0.14 1.59 0.91 0.18 0.06 0.12 0.46
0.31 7.59c 11.4c 3.81e 4.24b 3.57e 4.91b 7.92c 4.53c 0.55 0.34 0.76 1.40
2.24 0.13 0.02 0.11 0.04 0.04 0.00 3.27 0.63 1.02 0.73 0.29 0.70
4.87e 0.80 0.18 1.42 0.28 0.55 0.01 8.22c 1.59 1.18 1.68 0.68 0.82
a
See Table 3 for explanation of F-tests and dfs. PR0:05;. c PR0:005;. d PR0:01;. e PR0:10. b
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
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Fig. 1. Temporal changes in abundances of bacterial-feeders (Bf) of dierent life strategy categories: (A) Bf 1, (B) Bf 2, and (C) Bf 3±4. Symbols are * for control (C), T for repeated low N input (L) and Q for single high N input (H) microcosms in zone 1, and the same, but open, symbols for zone 2. The marks represent abundances in fresh (unenclosed) soil cores (d 1, 191). Arrows show days at which N-input occurred. Error bars equal two times the pooled standard error for the soil microcosm data.
selected life strategies (Bf 1 and Bf 2) increased in number following N-addition, while K-selected bacterivorous populations (Bf 3±4) were unaected (Table 3, Fig. 1). The increase of Bf 1 was most pronounced in zone 1 microcosms, where, 42 days after N-addition, populations in the single high (H) and repeated low (L) addition treatments had increased 3-fold compared to control (C) populations (Fig. 1a). On day 78, numbers in H had further increased, and were on average 2-fold higher than in L, and 6-fold higher than in C. Only at the last date of the experiment, numbers of Bf 1 in L and H were back to control levels. In zone 2, N-eects on Bf 1 were smaller and more variable between dates, but elevated numbers were observed at several times, including the ®nal date. Of the taxa comprising the Bf 1 group (Table 1), the positive response of Rhabditinae to N was comparable among zones, but Neodiplogasteridae and Myolaimidae increased only in zone 1. For the bacterivores of life strategy group 2 (Bf 2), numbers in L and H were signi®cantly higher than in C in zone 2 only (Table 3,
Fig. 1b). This increase appeared to be mainly caused by a rise in Cephalobidae in this zone (Table 1). No signi®cant dierences were found between L and H. Like Bf 1, the increase in Bf 2 populations of L and H emerged 22±42 days after the ®rst N-addition, but, unlike Bf 1, the largest dierence between Bf 2 population sizes in control versus N-addition (L, H) treatments never exceeded 2-fold. The dynamics of fungivorous nematodes were hardly aected by N-addition, except for a small increase (P<0:10) of r-selected fungivores (Ff 2) in N-addition microcosms of zone 2 only (Table 4, Fig. 2a). A slightly signi®cant (P<0:10) zone by N interaction was observed for K-selected fungivores (Ff 4), with L numbers higher than H abundances in zone 1, and vice versa in zone 2 (Table 4, Fig. 2c). However, the F-test contrasting controls with N-addition (L, H) populations was not signi®cant. Unlike the relatively rapid response of bacterivores to N-addition, which declined at the end of the experiment, the N-induced increase of predators occurred
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Fig. 2. Temporal changes in abundances of fungal-feeders (Ff) of dierent life strategy categories: (A) Ff 2, (B) Ff 3, and (C) Ff 4. Symbols and error bars as for Fig. 1.
generally later and was still highly signi®cant 191 days after the ®rst addition, notably for predators of life strategy categories 2 and 3 (Pr 2±3; Table 5, Fig. 3a). This group, which was dominated by Tripylidae (Table 1), behaved remarkably similar in both zones, with a ®nal (day 191) average population size of 30 predators 50 gÿ1 soil in H and 17 predators in L, compared to two predators in control samples. The increase in H emerged 78 days after the single high addition, and remained generally higher than L populations. The Neect on predators of life strategy categories 4 and 5 (Pr 4±5), dominated by Mononchidae, was not as pronounced, but overall populations in addition treatments (L, H) were signi®cantly higher than in controls (Table 5, Fig. 3b). As indicated by the signi®cant zone N time interaction, their dynamics were erratic. There was a weak N-eect on omnivores, with overall higher abundances in N-addition (L and H) treatments than in controls (P<0:10; Table 5, Fig. 3c). Of the community indices tested, the MI was most signi®cantly reduced by N-addition, though this eect
was mostly limited to zone 1, H microcosms. There, the MI was 010% lowered compared to controls during days 42±134, but recovered to control values at day 191 (Table 6, Fig. 4c). Overall genus richness, as estimated by Fisher's a, was on average higher in L than in H, but due to variation by zone and time, no clear N-eect on genus richness emerged (Table 6, Fig. 4a). Re-calculating this statistic with only bacterivore data yielded more signi®cant eects. Overall, bacterivorous genus richness, as estimated by Fisher's a, was signi®cantly reduced in N-addition treatments compared to control microcosms, and this eect was greater (P<0:10) in H than in L. However, examination of the time series (Fig. 4b) indicates that this occurred only in zone 2, whereas in zone 1, during the second half of the experiment, bacterivorous richness in H was higher than in L (but not dierent from C). Although trends indicated lower nematode F-to-B ratios in N-addition treatments compared to control microcosms, N-eects were not signi®cant (Table 6, Fig. 4d).
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Fig. 3. Temporal changes in abundances of predators (Pr) and omnivores (Om) of dierent life strategy categories: (A) Pr 2±3, (B) Pr 4±5, and (C) Om 4±5. Symbols and error bars as for Fig. 1.
3.3. Eects of enclosures There were signi®cant enclosure (microcosm) eects on abundances of individual nematode groups, which often varied by zone and sample date (Table 7; Figs. 1±4). For instance, on day 1, group Bf 1 was signi®cantly more abundant in control microcosms than in fresh (unenclosed) soil cores from the same plots, but day 191 abundances were similar. A similar eect was observed for Bf 2 in zone 1, with a reverse trend occurring in zone 2. Group Bf 3±4 was not aected by the enclosure in zone 1, but it was signi®cantly more abundant in zone 2, at day 1 but not day 191, compared to fresh soil core populations. In contrast to bacterivores, fungivore populations (mostly Ff 2 and Ff 4) generally decreased in microcosms relative to unenclosed soil, particularly at day 191. In contrast, at day 1 predator group Pr 2±3 was more abundant in the microcosms, but not dierent from unenclosed populations on day 191. Abundances of predator
group Pr 4±5, omnivores, and total free-living nematodes were largely unaected by the enclosures, as were indices of genus richness and maturity. The nematode F-to-B ratio was signi®cantly lowered by enclosure, at all times and zones. 4. Discussion 4.1. Riparian nematode assemblages The assumption that free-living nematodes would be abundant and diverse in riparian forests, even in the often water-logged zone 1 soil, was con®rmed by our data. The total nematode population in zone 1 soil was in fact higher than in zone 2, though slightly less diverse (Table 2). The total richness of 49 genera in the riparian forest, and the average total abundance of 1047 free-living nematodes 50 gÿ1 soil, were considerably higher than found in other sites in the coastal
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Fig. 4. Temporal changes in assemblage indices: (A) overall Fisher's a diversity index, (B) bacterivorous Fishers a diversity index, (C) maturity index, and (D) Nematode F-to-B ratio. Symbols and error bars as for Fig. 1.
plain of Georgia and Florida, such as an acidic wetland (Cox and Smart, 1994), and a pine forest on a sandy soil (McSorley, 1993), but were comparable to numbers for a close-by riparian wetland on the same soil type as the forest of the present study (Ettema et al., 1998). The distinct soil dierences between zones less than 10 m apart (Ettema et al., 1999), which are typical for the strong gradient found in riparian zones (e.g., Gregory et al., 1991; Hedin et al., 1998), in¯uenced the composition of the nematode assemblages in these sites (Tables 1 and 2). Total abundance was signi®cantly higher in zone 1 and positively correlated to organic N (rs 0:41, P 0:008, n 42 [zone 1, control microcosms of all dates]), labile C (rs 0:48, P 0:001) and basal respiration rates (rs 0:34, P 0:03). In addition, the near stream zone had higher abundances of taxa that are typically found in aquatic habitats (Bongers, 1988), such as Rhabdolaimus, Euteratocephalus, Monhysteridae, Chronogaster,
Bastiania, Cryptonchus and Paramphidelus (Table 1). In zone 2, higher total abundances were associated with basal respiration rate only (rs 0:34, P 0:03, n 42 [zone 2, control-microcosms of all dates]). In both zones, the microbivorous nematode fauna was dominated by bacterial feeders, but in zone 1 bacterivore dominance was reduced by an increased fungivore population, in concert with the higher amounts of fungal respiration found in this zone (Ettema et al., 1999). Although bacterial respiration was also elevated in zone 1, this did not result in a signi®cant increase of bacterivorous nematodes (Table 1). Within zones, the abundances of bacterivores and fungivores in controls were not signi®cantly correlated to bacterial and fungal respiration, nor to microbial biomass (Table 8). However, in both zones bacterivores were related to basal respiration rates, and in zone 2 fungivore abundance was associated with basal and substrate induced respiration (Table 8). Lack of simple correlation between microbivorous nematodes and their food
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638 Table 7 ANOVA of enclosure (microcosm) eects on nematode groupsa Source Enclosure
Enclosure zone
Fb
Variable
SS
Bf 1 Bf 2 Bf 3±4 Ff 2 Ff 3 Ff 4 Pr 2±3 Pr 4±5 Om4-5 Overall Fisher's a Bacterivore Fisher's a Maturity index Nematode F-to-B ratio
1.10 2.82 0.21 13.2d 0.23 6.60e 0.16 10.9f 0.16 0.53 0.14 3.06 0.28 1.22 0.45 2.49 0.60 1.88 0.00 0.00 1.47 1.27 0.06 3.20g 2.09 17.2d
Enclosure day
SS
Fb
SS
Fc
0.27 0.01 0.31 0.01 0.02 0.10 0.34 0.00 0.14 0.02 0.80 0.02 0.02
0.69 0.20 8.90e 0.39 0.07 2.19 1.48 0.00 0.45 0.01 0.69 1.32 0.18
1.48 4.98e 0.00 0.01 0.08 1.08 0.04 1.13 1.33 1.34 1.18 10.4f 1.17 7.12e 0.06 0.63 0.43 1.05 3.25 1.81 1.46 0.89 0.07 2.47 0.01 0.08
a
Measurements taken on days 1 and 191, on control (C) microcosms and fresh soil cores, in both zones (see Section 2). Abundances were log10-transformed prior to analysis. b F-test (df 1, 10) used Enclosure [Block(Zone)] as error term. c F-test (df 1, 10) used Day Enclosure [Block(Zone)] as error term. d PR0:005;. e PR0:05;. f PR0:01;. g PR0:10.
Table 8 Correlation coecientsa between microbial variables and microbivorous abundances. Variables were log10-transformed before analysis Microbial measurementsb Zone
N-addition Groupc Mic-C Mic-N BR
Zone 1 C L H Zone 2 C L H a
BF FF BF FF BF FF BF FF BF FF BF FF
0.19 0.21 0.33d 0.47e 0.23 0.40f 0.11 0.21 0.19 0.24 0.13 0.17
0.22 0.20 0.48e 0.39d 0.26g 0.29g 0.06 0.22 0.21 0.15 0.18 0.18
0.35d 0.21 0.30d 0.31d 0.08 0.28g 0.28g 0.34d 0.02 0.29g 0.25 0.37d
SIR BACT FUNG 0.22 0.21 ÿ0.14 0.12 0.09 ÿ0.11 0.40f 0.27g 0.08 0.33d 0.25 0.22 0.35d 0.31e 0.02 0.26g 0.03 ÿ0.11 0.10 ÿ0.11 ÿ0.11 0.33d 0.08 0.19 0.20 0.14 0.02 0.26g 0.02 0.24 0.27g 0.31d ÿ0.06 0.29g 0.33d 0.38d
Pearson's r, estimated with n 42 pairs of points in each test. Data from Ettema et al. (1999) Mic-C, microbial carbon; Mic-N, microbial nitrogen; BR, basal respiration; SIR, substrate induced respiration, BACT, bacterial substrate induced respiration; FUNG, fungal substrate induced respiration. c BF, bacterial-feeders; FF, fungal-feeders. d PR0:05;. e PR0:005;. f PR0:01;. g PR0:10. b
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source has been reported before (e.g., Clarholm et al., 1981; Wardle et al., 1995), and has been attributed to asynchronous dynamics and non-linear trophic interactions, which include indirect eects and positive feed backs (Bengtsson et al., 1996). 4.2. N-induced changes in trophic dynamics Interestingly, the N-addition synchronized microbial and microbivorous dynamics, as evidenced by stronger correlations between microbial variables and microbivorous dynamics in both L and H microcosms (Table 8). Notably, positive correlations between bacterivorous nematodes and bacterial respiration were found in the H treatment in both zones, and in the L treatment (P<0:10) in zone 1. In addition, a signi®cant correlation between fungal-feeders and fungal respiration was found in zone 2, H microcosms. In zone 1, fungivorous and bacterivorous abundances were positively associated with microbial C and N in both L and H treatments. Some of these correlations may be coincidental (05%), but their prevalence contrasts highly with the seemingly out-of-phase microbial-microbivorous dynamics in control microcosms. Anderson (1995) presents a conceptual model where disturbances such as re-wetting events after droughts and seasonal litter inputs improve detectibility of faunal eects on soil processes because these disturbances synchronize soil organism activities. Nitrogen in¯ux may have a comparable `re-set' eect on riparian soil population dynamics. While N-eects on microbial dynamics were hardly detectable (Ettema et al., 1999), a measurable signal of increased microbial growth could have been weakened by increased microbivorous grazing pressure (cf. BaÊaÊth et al., 1978; Clarholm et al., 1981). Indeed, when considered in tandem with the highly positive microbialmicrobivorous correlations in N-amended microcosms, the signi®cant increase of some microbivorous groups several weeks after N-addition (Tables 3 and 4, Figs. 1 and 2) is strong evidence that microbial production was temporally elevated by N-input. In zone 1, the 3to 5-fold increase of Bf 1 in N-amended microcosms indicates a fast conversion of consumed bacterial biomass into nematode ospring, although immigration into the open-bottom microcosms cannot be excluded as a partial, though probably minor, source of the population increase. Fast reproduction in response to increased food supply is typical for the rhabditid and diplogasterid taxa in life strategy group 1 (e.g., Sohlenius, 1973; Anderson et al., 1981; Schiemer, 1983; Ettema and Bongers, 1993). In response to inorganic N-addition, these taxa increased in abundance in several studies (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986), which has been attributed to the stimulating eect of
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N on N-limited bacterial production (BaÊaÊth et al., 1978). Although Rhabditinae showed a similar N-induced increase in zones 1 and 2, the more signi®cant N-response in zone 2 came from bacterivores in group 2 (Bf 2), notably Cephalobidae (Tables 1 and 2, Fig. 1b). We suspect that this zone dierence was due to a combination of stochastic and deterministic factors (cf. Sohlenius, 1993). There were fewer Bf 1 than Bf 2 individuals present in zone 2 to start reproduction (Table 1). As zone 2 soil had a lower labile C content than zone 1 (Ettema et al., 1999), it is possible that Ninduced microbial production in zone 2 was not high enough (because of C-limitation) to meet food requirements for some Bf 1 taxa. In neither zone did K-selected bacterivores (Bf 3±4) respond to N-addition, theoretically because they have much longer generation times. In addition, these taxa may feed on `autochtonous' micro¯ora which perhaps was not stimulated as much by N-additions as `zymogenous' micro¯ora was. Given that data on bacterivorous diet speci®city and microbial species-speci®c dynamics are severely limited, these remain speculations. The lack of signi®cant response of fungivorous nematodes to inorganic N may be explained by the notion that fungi tend to be less limited by N, and more limited by C, than bacteria (BaÊaÊth et al., 1978). In other inorganic N-addition studies, fungal-feeders did not respond to N either (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986). Why was not a much greater bacterivorous response to N observed? First of all, adding inorganic N without a C-source may have limited the response of bacteria and bacterivores alike. Although we found a signi®cant 3±5 fold N-induced increase of Bf 1 taxa, these numbers are not as great as the 55-fold increase found by Ettema and Bongers (1993), who added organic N as dried cow manure, or more than the 70fold increase detected by Dmowska and Kozlowska (1988) in ®elds treated with excessive amounts of semiliquid manure. Microbial studies have shown a much stronger microbial production response to combinations of C and N, than to either nutrient alone (BaÊaÊth et al., 1978; Elliott et al., 1983; Zagal and Persson, 1994). Secondly, predation may have signi®cantly weakened the microbivorous growth signal, just like microbivorous grazing may have reduced the microbial signal. Predaceous nematodes greatly increased in Namended systems (and signi®cantly more in H than in L), in both zones (Table 5, Fig. 3a,b). As predaceous abundance increased, bacterivorous numbers decreased back to control-levels. The very similar increase of Tripylidae in each zone suggests that this is a generalist predator. In soil, predation mostly depends on
chance encounters, and most nematode predators are expected to be generalists (Yeates and Wardle, 1996). Finally, nematode abundance changes due to soil enclosure into microcosms may have weakened the eect of N-additions. In microcosms, bacterivore abundance was stimulated, and fungivore abundance inhibited, compared to populations in fresh soil cores (Table 7). The former eect was most signi®cant at the start of the experiment, when the presence of freshly cut roots in the microcosms possibly stimulated bacterivore populations before N-treatment had begun. In contrast, the (negative) eect on fungivore abundance (and fungal respiration: Fig. 6b in Ettema et al., 1999) was most signi®cant at the end of the experiment. The disconnection of fungi and fungivores in the enclosures from larger hyphal networks may have caused their decrease in microcosms, and lessened their ability to take advantage of the added N. 4.3. Indicators of riparian soil N-saturation The general indicator potential of free-living nematodes is based on their presence in measurable numbers and wide distribution, and their critical role in soil processes (Gupta and Yeates, 1997). Their speci®c potential for indicating riparian soil N-saturation is suggested by our results, which showed that r-selected bacterivores (Bf 1 and Bf 2), and predaceous nematodes, were highly responsive to changing soil N-content. The response of r-selected bacterivores to Naddition corroborates results of numerous other Nenrichment studies in non-riparian soils (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986; Dmowska and Kozlowska, 1988; Ettema and Bongers, 1993; Griths et al., 1994), and could explain the positive correlation between rselected bacterivorous nematode abundance and soil nitrate content found by Ettema et al. (1998) in a riparian wetland subject to N-in¯ux. How did nematode group responses compare to the dynamics of other potential N-saturation indicators measured in this experiment? First, nematode dynamics provided a clearer signal of N-saturation than underlying changes in microbial biomass and respiration (Ettema et al., 1999). Second, the N-eect on nematodes was still observable after inorganic N-concentrations ceased to be dierent between treatments. However, inorganic N-pools showed increases immediately after N-addition, whereas nematodes needed at least a month response time (compare Figs. 1 and 3 to Fig. 2 of Ettema et al., 1999). Third, changes in nematode groups following N-addition were observed in both zones, whereas N-induced changes in denitri®cation were only apparent in zone 1 (Fig. 3b in Ettema et al., 1999). Finally, individual nematode groups (notably Bf 1, Bf 2, Pr 3) showed more signi®cant and
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
consistent dierences between N-treatments, than the ecological indices which averaged all nematode populations into single index values (Figs. 1 and 3 versus Fig. 4). Thus, it appears from our experiment that, at the resolution of life-strategy-within-feeding-groups (Table 1), nematodes could be useful indicators of riparian N-saturation. The question remains how to apply nematode indicators in N-saturation monitoring programs. Since so many factors, both deterministic and stochastic, in¯uence nematode abundance and species composition (Ettema, 1998), it seems quite impossible, outside controled experimental settings, to attribute nematode assemblage changes to single causes, if no other variables are measured to support inferences. For instance, a high abundance of Bf 1 nematodes may be due to soil N-saturation, but could also be due to heavy metal pollution (Weiss and Larink, 1991), strong drying-rewetting cycles in organic soils (this study: Bf 1 abundances were considerable in zone 1 control microcosms and untreated, unenclosed soil), a local concentration of animal feces (Rehfeld and Sudhaus, 1989), or any other disturbance that causes a temporary increase in bacterial activity. Therefore, nematode indicators of N-saturation may only be useful when measured together with other key system characteristics, such as denitri®cation (Hanson et al., 1994a, Hanson et al., 1994b; this study) and litter-N (Aber et al., 1989; Hanson et al., 1994a). Moreover, recognizing that the riparian ecosystem reponse to N-in¯ux is spatially variable (within and between zones) and temporally dynamic (with N moving from one pool to another), the combined assessment of a soil faunal, microbiological and biochemical indicator would be a more reliable and ecologically meaningful approach than the single monitoring of any one of them.
Acknowledgements Many thanks are due to Rex Blanchett for his help in setting up the microcosms. Piet Loof provided taxonomic identi®cation of several nematode specimens gathered during a pilot survey of the riparian site. Steve Rathbun guided the statistical analysis. Luc Boerboom provided precious advice and help in data management. Constructive comments by Diana Wall and two anonymous referees greatly improved the manuscript.
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McSorley, R., 1993. Short-term eects of ®re on the nematode community in a pine forest. Pedobiologia 37, 39±48. Pankhurst, C., Doube, B.M., Gupta, V.V.S.R. (Eds.), 1997. Biological Indicators of Soil Health. CAB International, Wallingford. Rehfeld, K., Sudhaus, W., 1989. Die Sukzession der Nematoden im Kuh¯aden: GesetzmaÈssigkeiten und Wege zu einer Kausanalyse. VerhaÈndlungen Gesellschaft fuÈr OÈkologie (GoÈttingen) 17, 745± 755. Rosenzweig, M.L., 1995. Species Diversity in Space and Time. Cambridge University Press, Cambridge. SAS Institute, 1990. SAS/STAT User's Guide, Version 6. Cary, North Carolina. Schiemer, F., 1983. Comparative aspects of food dependence and energetics of freeliving nematodes. Oikos 41, 32±42. Schouten, A.J., Arp, K.K.M., 1991. A study on the eciency of extraction methods for nematodes from dierent forest litters. Pedobiologia 35, 393±400. Sohlenius, B., 1973. Structure and dynamics of populations of Rhabditis (Nematoda: Rhabditidae) from forest soil. Pedobiologia 13, 368±375. Sohlenius, B., 1993. Chaotic or deterministic development of nematode populations in pine forest humus incubated in the laboratory. Biology and Fertility of Soils 16, 263±268. Sohlenius, B., BostroÈm, S., 1986. Short-term dynamics of nematode communities in arable soil Ð in¯uence of nitrogen fertilization in barley crops. Pedobiologia 29, 183±191. Sohlenius, B., Wasilewska, L., 1984. In¯uence of irrigation and fertilization on the nematode community in a Swedish pine forest soil. Journal of Applied Ecology 21, 327±342. Southey, J.F. (Ed.), 1986. Laboratory Methods for Work with Plant and Soil Nematodes. Ministery of Agriculture, Fisheries and Food, Reference Book 402, London. Wardle, D.A., Yeates, G.W., Watson, R.N., Nicholson, K.S., 1995. The detritus foodweb and the diversity of soil fauna as indicators of disturbance regimes in agroecosystems. Plant and Soil 170, 35± 43. Weiss, B., Larink, O., 1991. In¯uence of sewage sludge and heavy metals on nematodes in an arable soil. Biology and Fertility of Soils 12, 5±9. Welsch, D.W., 1991. Riparian forest buers. Publ. NA-PR-07-91. USDA-Forest Service, Washington, DC. Yeates, G.W., Bongers, T., de Goede, R.G.M., Freckman, D.W., Georgieva, S.S., 1993. Feeding habits in nematode families and genera Ð an outline for soil ecologists. Journal of Nematology 25, 315±331. Yeates, G.W., Wardle, D.A., 1996. Nematodes as predators and prey: relationships to biological control and soil processes. Pedobiologia 40, 43±50. Zagal, E., Persson, J., 1994. Immobilization and remineralization of nitrate during glucose decomposition at four rates of nitrogen addition. Soil Biology and Biochemistry 26, 1313±1321.
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Riparian soil response to surface nitrogen input: the indicator potential of free-living soil nematode populations Christien H. Ettema a,*, 1, Richard Lowrance b, David C. Coleman a a
Institute of Ecology, University of Georgia, Athens, GA 30602, USA Southeast Watershed Research Lab, US Department of Agriculture, Tifton, GA 31793, USA
b
Accepted 25 April 1999
Abstract Nitrogen saturation of riparian ecosystems may lead to leaching and loss of surface water quality. In the search for early warning signs of N-saturation, we conducted an N-addition experiment to evaluate the indicator potential of free-living nematodes. During 6 months, we measured changes in feeding and life strategy groups, following single high and repeated low inorganic N-additions to a near-stream zone (`zone 1') and an upslope area (`zone 2') within a riparian forest in the southeast Coastal Plain of Georgia. In both zones, N-addition signi®cantly increased the number of bacterivores but not fungivores. Only bacterivores with r-selected life strategies increased. In this group, Rhabditinae multiplied after N-addition in both zones, but Neodiplogasteridae and Myolaimidae responded to N only in zone 1, and Cephalobidae only in zone 2. Microbivore correlations with microbial data, collected concomitantly (Ettema et al., 1999. Riparian soil response to surface nitrogen input: temporal changes in denitri®cation, labile and microbial C and N pools, and bacterial and fungal respiration. Soil Biology & Biochemistry 31, 1609±1624), were considerably stronger in N-amended treatments than in controls, suggesting that N-addition synchronized microbial-microbivorous dynamics. At the end of the experiment, bacterivore abundance returned to control levels, probably partly due to predation, as predator populations had considerably increased several months after the ®rst N-addition. The increase in predator abundance was greater in the single high than repeated low Naddition treatments, as was the case for bacterivore populations. These results suggest that nematodes can be indicators of Nsaturation. However, for practical application, it appears that these indicators could only be meaningful when monitored together with other system characteristics, such denitri®cation rates and litter N. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Riparian zone; Nitrogen saturation; Free-living nematodes; Indicators
1. Introduction Riparian ecosystems are increasingly managed as buers between aquatic bodies and upland agricultural or urban land use, protecting water quality from pollutants such as nitrogen (Gregory et al., 1991; Welsch, * Corresponding author. Tel.: +31-317-482981; fax: +31-317483766. E-mail address: [email protected] (C.H. Ettema) 1 Present address: Wageningen Agricultural University, Soil Biology Group, P.O. Box 8005, 6700 EC Wageningen, The Netherlands.
1991; Fennessy and Cronk, 1997; Lowrance et al., 1997). A major uncertainty in the management of riparian systems is for how long the buers can be sustained when N-loading is continuous (Lowrance and Vellidis, 1995). N-saturation, de®ned as the availability of inorganic N in excess of plant and microbial demand (Aber et al., 1989; Hanson et al., 1994a), may lead to increased nitri®cation and leaching rates, lowered resistance of plants to pests, and plant community changes (Aber et al., 1989), all of which may greatly impair the water quality protection function of the riparian buer zone (e.g., Kadlec and Bevis, 1990). From the management point of view, it is highly desir-
0038-0717/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 0 7 2 - 3
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able to get early warning signs, or `indicators', of impending N-saturation, before riparian systems lose buer capacity. The overall objective of our study was to evaluate the potential of free-living soil nematode assemblages for indicating N-saturation in riparian ecosystems. While it appears easier to measure and interpret biochemical, rather than faunal variables, the advantage of the latter is that they integrate soil N-conditions over a period of time, whereas the former may exhibit a more instantaneous, `snap shot' character. Because of the complementarity of faunal and biochemical indicators, general procedures for monitoring soil quality and disturbance often call for their combined assessment (Linden et al., 1994; Blair et al., 1996; Pankhurst et al., 1997). So far, only biochemical (N-cycling) indicators have been used in the assessment of N-saturation in riparian ecosystems (e.g., Hanson et al., 1994a). The rationale for selecting free-living nematodes was primarily that nematodes were likely to be abundant and diverse along the steep moisture gradient typically found in riparian zones. Secondly, freeliving soil nematodes should be functionally important for riparian soil functioning, by enhancing decomposition and N-mineralization processes (Hunt et al., 1987; Freckman, 1988; Griths, 1994). In many riparian systems, sub-surface ¯ow N, taken up by roots and stored in plant biomass, only becomes available to surface soil denitri®ers when plant litter decomposes (Groman et al., 1992; Hanson et al., 1994b; Lowrance et al., 1997). Thirdly, other studies have shown signi®cant nematode assemblage responses to organic and inorganic N-additions in pastural, agricultural and forest ecosystems (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986; Dmowska and Kozlowska, 1988; Ettema and Bongers, 1993; Griths et al., 1994). In a survey of a riparian wetland zone downslope of a dairy waste application site, Ettema et al. (1998) found high nematode abundances, and a prevalence of bacterial-feeding taxa with r-selected life strategies, at microsites with elevated soil inorganic N-concentration. However, it remained unclear whether these content dierences were causally related to soil Ncontent variation. Thus, the speci®c objective of our N-addition study was to experimentally assess whether free-living riparian soil nematode populations are responsive to riparian soil N changes. To obtain a mechanistic understanding of potential N-induced changes in the nematode populations in this experiment, we examine the links between microbivorous nematode dynamics and concomitantly measured microbial respiration and biomass dynamics (Ettema et al., 1999).
2. Materials and methods 2.1. Site description The experiment was conducted in a mature riparian forest adjoining a second-order stream, located near Tifton, Georgia, USA. The forest had two distinct zones: zone 1, a 5±8 m wide strip bordering the stream, is vegetated by hardwood trees, primarily yellow poplar (Liriodendron tulipifera L.) and swamp tupelo (Nyssa sylvatica var bi¯ora Marsh.). Zone 1 soil is very poorly drained ®ne-loamy sand, with an average water ®lled pore space (WFPS) of 75%, and a soil carbon content of 2±5%. Zone 2, upslope (slope < 1%) from zone 1, is a 30±40 m wide stand of predominantly slash pine (Pinus elliotii Engelm.) and longleaf pine (Pinus palustris Mil.). Zone 2 soil is a poorly drained loamy sand, with an average WFPS of 51%, and a carbon content of 1±3%. 2.2. Experimental design The experiment was set up as a split-plot design, with zone as the main plot eect, and N-addition as the split plot eect. Each N-addition level was replicated in six randomly selected 1.5 by 2.5 m blocks in each zone, and measurements were taken at days 1 (22 Oct. 1996), 10, 22, 42, 78, 134 and 191 (30 April 1997). Ettema et al. (1999) provide details on the experimental design and randomization process. 2.2.1. Nitrogen treatment N-addition treatments were control (C), single high input (H) and repeated low input (L). To prevent Ncontamination by surface runo from the adjacent agricultural ®eld and between treatments, N was added to ®eld soil microcosms instead of larger plots. Microcosms were installed 2 weeks prior to the ®rst Naddition, and were constructed by taking intact littersoil cores to a 20 cm depth, using a corer with a removable inner plastic tube of 25 cm length and 6 cm diameter. Each soil core, contained in a tube, was returned into the sample hole after covering the bottom with nylon mesh (the top remaining uncovered), to allow vertical water ¯ow and nematode migration while facilitating later microcosm retrieval. No plants grew in the microcosms throughout the experiment (sporadically-emerging seedlings were removed by hand). Treatment H was equivalent to 7.5 g N mÿ2, added in a single dose at day 0 (21 Oct. 1996). Treatment L was the same total N input, but was added in ®ve pulses of 1.5 g N mÿ2 each, at days 0, 32, 68, 124 and 181, respectively. N was added in 20 ml Dl water per microcosm, as a 2 : 1 mixture of NH4Cl±N and KNO3±N, mimicking the relative composition of inorganic N ions in surface runo from the
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
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Table 1 Abundances of free-living nematode families in two zones of a riparian forest, subject to repeated low (L), single high (H) or zero nitrogen input (C). Abundances are per 50 g soil (dw), averaged over blocks and time (n 42 for each mean) Zone 1
Zone 2
Trophic group
Life strategy subgroup
Family
C
L
H
C
L
H
Bacterial feeders
Bf 1
Rhabditinaea Neodiplogasteridaeb Bunonematidaec Myolaimidaed Cephalobidaee Plectidaef Monhysteridaeg Rhabdolaimidaeh Prismatolaimidaei Teratocephalidaej Desmodoridaek Chronogasteridael Odontolaimidaem Bastianiidaen Alaimidaeo Bathyodontidaep Aphelenchoididaeq Anguinidaer Aphelenchidaes Diphterophoridaet Leptonchidaeu Aphelenchoididaev Tripylidaew Mononchidaex Belonderidaey Chrysonematidaez Leptonchidaea1 Qudsianematidaeb1 Aporcelaimidaec1
78 11 3 1 208 82 22 103 58 27 4 7 3 1 8 0 351 7 2 5 54 2 3 3 2 0 4 7 3 1059
118 21 9 5 186 67 16 108 57 39 3 4 3 0 8 0 324 4 2 9 77 3 7 4 5 0 3 12 5 1099
149 81 5 14 216 79 14 89 66 29 6 5 3 1 6 1 374 4 0 12 66 1 10 4 2 0 5 5 6 1253
82 6 12 0 223 60 10 31 51 66 15 0 2 0 4 0 163 4 1 24 51 0 4 14 0 0 3 8 5 839
98 4 13 0 304 74 10 29 47 34 9 2 1 0 3 0 208 12 6 22 41 2 4 16 0 0 2 11 6 958
130 6 12 0 315 64 8 28 57 47 8 0 0 0 3 0 221 3 5 27 46 0 9 19 0 1 5 11 6 1031
Bf 2 Bf 3
Bf 4 Fungal feeders
Predators
Omnivores Total a
Ff 2 Ff Ff Pr Pr Pr Pr
3 4 2 3 4 5
Om 4 Om 5
Not identi®ed beyond superfamily level. Pristionchus. c Bunonema. d Myolaimus. e Acrobeloides, Heterocephalobus, Cervidellus, Acrobeles, Acrolobus, Zeldia. f Plectus, Wilsonema, Anaplectus. g Eumonhystera, Monhystrella, Geomonhystera. h Rhabdolaimus. i Prismatolaimus. j Teratocephalus, Metateratocephalus, Euteratocephalus. k Prodesmodora. l Chronogaster. m Odontolaimus. n Bastiania. o Alaimus, Paramphidelus. p Cryptonchus. q Aphelenchoides. r Pseudhalenchus. s Aphelenchus. t Diphtherophora, Tylolaimophorus. u Tylencholaimus, Tylencholaimellus, Basirotyleptus. p Seinura. w Tripyla, Trischistoma. x Prionchulus, Mylonchulus. y Oxydirus, Axonchium. z Chrysonema. a1 Dorylaimoides. b1 Eudorylaimus, Epidorylaimus, Microdorylaimus. c1 Aporcelaimellus, Sectonema. b
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upslope ®eld. Control microcosms received 20 ml Dl water. 2.3. Sample collection and processing On each sampling date, one randomly selected microcosm was retrieved from each treatment in each block. After subsampling for denitri®cation, microcosms were transported to the Soil Ecology Laboratory in Athens GA, and processed within 7 days for measurement of various microbial and nutrient pools as described in Ettema et al. (1999), and the extraction of nematodes. 2.3.1. Nematode extraction Nematodes were extracted from approximately 50 g soil, which was subsampled from the carefully mixed top 10 cm of the microcosm. The sample, which included organic debris, was extracted by decanting and wet sieving (Southey, 1986), including a 5 s blendering step for the litter (cf. Schouten and Arp, 1991). Final separation occurred on a Baermann tray using double cottonwool ®lters (Schouten and Arp, 1991), and extracted nematodes were stored in 5% formaldehyde. First, all nematodes were counted using an inverted microscope. Next, a random subset (specimens occurring in small marked areas randomly distributed over the counting dish grid) was identi®ed to genus using Bongers (1988) and Jairajpuri and Ahmad (1992), until 15% of total abundance in the sample was identi®ed. In the few cases where this was less than 100 individuals, the process proceeded until 100 identi®cations were obtained. Repeated analysis showed that this generally yielded a good estimate of nematode richness and abundances of dominant taxa. Taxa were assigned to feeding groups following Yeates et al. (1993). Within each trophic group, nematodes were ranked by life strategy according to Bongers and Bongers (1998) (Table 1). In this classi®cation, a low number (1±2) indicates a `colonist', r-selected (sensu lato ) life strategy (high reproduction rate, small body size, short life cycle), and a high rating (3±5) signi®es a `persister', K-selected (sensu lato ) life strategy (low reproduction rate, large body size, long life cycle) (Bongers, 1990). 2.3.2. Soil characteristics of fresh soil cores To evaluate the eects of enclosure on nematodes within the microcosms, a fresh soil core was randomly taken from each block, on the ®rst and last sampling date (days 1 and 191, i.e., 14 and 205 days after microcosm installation), using the same soil corer as used for constructing the microcosms. Nematode populations in these cores were compared to the control microcosm data of days 1 and 191.
2.4. Data analysis Because of the low frequencies of most genera and families (i.e., many taxa occurred only in a small portion of the microcosms sampled), analysis of variance (ANOVA) was limited to clusters of taxa, grouped according to life-strategies within feeding categories following Bongers and Bongers (1998). Some clusters were merged to further improve frequencies: bacterivores of life strategy classes 3 and 4; predators 2 and 3; predators 4 and 5; and omnivores 4 and 5 (Table 1). Treatment eects were tested using a repeated measures ANOVA in SAS Version 6 (SAS Institute, 1990), according to Ettema et al. (1999). Nematode abundances were log10-transformed prior to statistical testing to better normalize distributions. Data on herbivorous nematodes are not reported here. As no plants grew in the microcosms throughout the experiment (sporadically emerging seedlings were removed by hand), plant feeder dynamics were not directly relevant to this study's objectives. Repeated measures ANOVA was also conducted for the Fisher's a diversity index, a parameter of the logseries distribution (Fisher et al., 1943). This statistic is a measure of richness (in this case, genus richness) that is insensitive to sample size (number of individuals in the sample), and is recommended as the standard diversity index by various authors (Magurran, 1988; Rosenzweig, 1995). It was calculated for all free-living nematodes, and for bacterivores, the most diverse trophic category. Repeated measures analysis was also performed for the maturity index (MI), an index of disturbance based on life strategy group frequencies (Bongers, 1990), and for the ratio of fungal- to bacterial-feeders (nematode F/B ratio). 3. Results The riparian forest soil nematodes were abundant and diverse (Table 1). Throughout the study, 29 freeliving families comprising 49 genera were detected, giving an average total abundance of 1047 individuals (range 334±4277) 50 gÿ1 soil. The mean number of genera occurring together in a single 50 g sample was 17 (range 10±27). Bacterivores were the most abundant and diverse trophic group, representing on average 63% of total abundance and 64% of total genus richness in the microcosms. 3.1. Basic zone dierences Contrasting control (C) microcosms in zones 1 and 2 of the riparian forest indicated that the free-living nematode fauna in zones less than 10 m apart was quite dierent (Tables 1 and 2). Overall, nematodes
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638 Table 2 Comparing nematode abundances and diversities (in 50 g soil dw) between zone 1 and zone 2 soil, using the data from the control (C) microcosms Nematode variablea Bf 1 Bf 2 Bf 3±4 Ff 2 Ff 3 Ff 4 Pr 2±3 Pr 4±5 Om 4±5 (all omnivores) All bacterivores All fungivores All predators Total free-living nematodes Overall genus richness Overall Fisher's a diversity Bacterivorous genus richness Bacterivorous Fisher's a diversity Maturity index Nematode F-to-B ratio
Zone 1b Zone 2b Fc 93 312 211 360 5 54 5 5 14 616 419 10 1059 16.6 5.42 11.4 4.08 2.31 0.71
Table 3 ANOVA of bacterivorous groups.a Abundances (in 50 g soil dw) were log10-transformed before analysis Bf 1
P
100 0.01 0.9153 293 0.07 0.7951 169 5.50 0.0410 168 9.57 0.0114 24 61.67 0.0001 51 0.22 0.6463 4 0.81 0.3887 14 6.57 0.0283 16 0.54 0.4781 562 1.77 0.2132 243 5.69 0.0383 18 11.53 0.0068 839 5.51 0.0408 17.8 9.20 0.0126 6.11 7.69 0.0197 11.0 4.09 0.0706 3.82 2.31 0.1596 2.35 0.93 0.3578 0.48 5.58 0.0398
a
See Table 1 for trophic and life strategy group compositions. Abundances and diversities averaged over blocks and sampling dates (n 42 for each mean). c F-test used log10-transformed abundances, and block within zone as error term (df 1, 10). b
were signi®cantly more numerous in zone 1 than zone 2 soil, averaging 1059 vs. 839 individuals 50 gÿ1 soil, respectively (Table 2). This increase in total abundance was mainly due to signi®cantly higher numbers of rselected fungivores (Ff 2; mainly Aphelenchoididae) and K-selected bacterivores (Bf 3±4; mostly Rhabdolaimidae) in zone 1. Fungivores of life strategy class 3 (Ff 3; only Diphtherophoridae), however, were signi®cantly more abundant in zone 2 control microcosms, as were predators (Pr 4±5: mainly Mononchidae). The maturity index, which is a weighted average of the frequencies of these life strategy groups, was not dierent between zones. However, the F-to-B ratio, which was always smaller than one, was signi®cantly higher in the near-stream zone. Overall genus richness was signi®cantly higher in zone 2, where microcosms contained on an average one genus more than zone 1 enclosures (Table 2). This was mainly due to the higher predator richness in zone 2 samples. Taxa exclusively found in zone 1 were the predators Axonchium and Oxydirus, the bacterivores Bastiania, Cryptonchus, Euteratocephalus and Myolaimus, and the omnivore Epidorylaimus. Taxa limited to zone 2 microcosms were the bacterivorous Cervidellus and fungivorous Tylolaimophorus. Common genera (with frequencies less than 85%, i.e., testable in ANOVA) that were signi®cantly more abundant in zone 1 than in zone 2 control microcosms
1629
Bf 2
Bf 3±4
All bacterial feeders
Source
SS
F
SS
F
SS
F
SS
F
Zoneb Nc C vs. L, Hd L vs Hd Zone Nc Zone (C vs. L, H)d Zone (L vs. H)d Tieme Time zonee Time Nf Time (C vs. L, H)g Time (L vs. H)g Time N zonef
0.15 3.26 2.64 0.62 0.05 0.04 0.01 4.56 1.03 4.53 2.89 1.64 3.40
0.30 15.8h 25.6h 6.01i 0.25 0.40 0.09 4.54h 1.02 1.78j 2.28i 1.29 1.34
0.54 0.14 0.08 0.06 0.36 0.36 0.00 2.30 1.23 0.38 0.07 0.31 0.45
9.44i 1.89 2.11 1.67 4.66i 9.26k 0.07 7.72h 4.13h 0.79 0.29 1.29 0.94
2.22 0.11 0.07 0.04 0.21 0.11 0.10 0.91 0.81 0.78 0.34 0.44 0.47
13.4h 1.07 1.36 0.79 2.10 2.17 2.04 2.50i 2.21j 1.38 1.21 1.55 0.83
0.05 0.26 0.14 0.12 0.01 0.01 0.00 0.98 0.26 0.44 0.31 0.13 0.20
0.85 4.32i 4.50i 4.13j 0.22 0.44 0.00 4.96i 1.31 1.34 1.85j 0.82 0.61
a
See Table 1 for group composition. F-test (df 1, 10) used Block within Zone [Block(Zone)] as error term. c F-test (df 2, 20) used N Block(Zone) as error term. d F-test (df 1, 20) used Time Block(Zone) as error term. e F-test (df 6, 60) used Time Block(Zone) as error term. f F-test (df 12, 120) used Time N Block(Zone) as error term. g F-test (df 6, 120) used Time N Block(Zone) as error term. h PR0:005;. i PR0:05;. j PR0:10;. k PR0:01. b
Table 4 ANOVA of fungivorous groups.a Abundances (in 50 g soil) were log10-transformed before analysisb Ff 2
Ff 3
Source
SS F
SS
Zone N C vs. L, H L vs. H Zone N Zone (C vs. L, H) Zone (L vs. H) Time Time zone Time N Time (C vs. L, H) Time (L vs. H) Time N zone
2.57 5.85c 0.18 1.23 0.17 2.32 0.01 0.14 0.26 1.77 0.26 3.54d 0.00 0.00 4.54 13.7e 0.32 0.97 0.45 0.93 0.18 0.74 0.27 1.12 0.64 1.34
22.5 0.85 0.84 0.01 1.13 1.12 0.01 2.92 0.71 3.63 1.37 2.26 1.46
a
Ff 4
All fungal feeders
F
SS F
SS F
38.6e 0.87 1.71 0.02 1.15 2.29 0.02 2.07d 0.50 1.01 0.77 1.26 0.41
1.95 1.68 0.10 0.22 0.02 0.07 0.08 0.37 1.19 2.72d 0.44 2.03 0.74 3.41d 2.85 2.58c 2.20 2.00d 0.51 0.31 0.17 0.20 0.35 0.42 1.00 0.60
1.56 3.41d 0.09 0.74 0.09 1.48 0.00 0.01 0.06 0.55 0.05 0.98 0.01 0.01 3.45 16.6e 0.14 0.67 0.21 0.60 0.08 0.43 0.13 0.78 0.36 1.02
See Table 1 for group composition. See Table 3 for explanation of F-tests and dfs. c PR0:05. d PR0:10. e PR0:005. b
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Table 5 ANOVA of predaceous and omnivorous groups.a Abundances (in 50 g soil) were log10-transformed before analysisb Pr 2±3
Pr 4±5
All predators
Om 4±5 (all omnivores)
Source
SS
F
SS
F
SS
F
SS
F
Zone N C vs. L, H L vs. H Zone N Zone (C vs. L, H) Zone (L vs. H) Time Time zone Time N Time (C vs. L, H) Time (L vs. H) Time N zone
0.03 2.33 1.87 0.46 0.60 0.60 0.00 8.14 0.85 15.0 10.9 4.10 2.44
0.05 3.21c 5.14d 1.27 0.83 1.65 0.00 4.60e 0.48 5.23e 7.59e 2.87d 0.85
11.8 1.67 1.31 0.36 0.60 0.03 0.57 3.22 2.01 2.72 0.59 2.13 4.89
10.3f 2.99c 4.68d 1.29 1.07 0.11 2.04 1.47 0.92 1.04 0.45 1.62 1.87d
5.61 1.93 1.93 0.00 0.40 0.21 0.19 3.59 1.14 8.15 4.85 3.30 5.29
8.35d 3.76d 7.52d 0.00 0.77 0.79 0.75 1.82 0.58 2.87e 3.41e 2.48d 1.86d
1.20 0.93 0.93 0.00 0.16 0.06 0.10 1.62 4.01 3.62 1.62 2.00 2.71
2.69 1.75 3.49c 0.00 0.30 0.22 0.37 1.13 2.79d 1.37 1.22 1.52 1.02
a
See Table 1 for group composition. See Table 3 for explanation of F-tests and dfs. c PR0:10;. d PR0:05;. e PR0:005;. f PR0:01. b
were Aphelenchoides, Plectus and Rhabdolaimus. Conversely, Diphtherophora and Teratocephalus were signi®cantly more abundant in zone 2 than in zone 1. 3.2. Eects of N-additions The experimental N-addition signi®cantly changed the abundance of bacterivores but not fungivores, and
in¯uenced the number of predators (Tables 3±5, Figs. 1±3). There were also signi®cant N-eects on maturity and bacterivorous diversity indices (Table 6, Fig. 4). Some of these eects were dependent on zone and time of observation. In many cases, N-eects were greater in single high, than in repeated low, N-addition treatments. Among the bacterivorous taxa, nematodes with r-
Table 6 ANOVA of Fisher's a diversity, maturity index, and fungal to bacterial feeder ratioa Overall Fisher's a diversity
Bacterivore Fisher's a diversity
Maturity index
Nematode F/B ratio
Source
SS
F
SS
F
SS
F
SS
F
Zone N C vs. L, H L vs. H Zone N Zone (C vs. L, H) Zone (L vs. H) Time Time zone Time N Time (C vs. L, H) Time (L vs. H) Time N zone
7.53 9.10 0.58 8.52 4.54 3.85 0.68 26.2 12.4 19.9 10.5 9.43 19.3
2.26 2.47 0.32 4.62b 1.23 2.09 0.37 3.49c 1.65 1.09 1.14 1.03 1.05
16.4 6.25 3.93 2.32 5.30 1.97 3.33 18.4 11.2 14.6 7.06 7.51 14.8
11.3d 4.03b 5.07b 3.00e 3.42e 2.54 4.30e 4.44c 2.70b 1.15 1.12 1.19 1.17
0.01 0.43 0.32 0.11 0.24 0.10 0.14 1.59 0.91 0.18 0.06 0.12 0.46
0.31 7.59c 11.4c 3.81e 4.24b 3.57e 4.91b 7.92c 4.53c 0.55 0.34 0.76 1.40
2.24 0.13 0.02 0.11 0.04 0.04 0.00 3.27 0.63 1.02 0.73 0.29 0.70
4.87e 0.80 0.18 1.42 0.28 0.55 0.01 8.22c 1.59 1.18 1.68 0.68 0.82
a
See Table 3 for explanation of F-tests and dfs. PR0:05;. c PR0:005;. d PR0:01;. e PR0:10. b
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
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Fig. 1. Temporal changes in abundances of bacterial-feeders (Bf) of dierent life strategy categories: (A) Bf 1, (B) Bf 2, and (C) Bf 3±4. Symbols are * for control (C), T for repeated low N input (L) and Q for single high N input (H) microcosms in zone 1, and the same, but open, symbols for zone 2. The marks represent abundances in fresh (unenclosed) soil cores (d 1, 191). Arrows show days at which N-input occurred. Error bars equal two times the pooled standard error for the soil microcosm data.
selected life strategies (Bf 1 and Bf 2) increased in number following N-addition, while K-selected bacterivorous populations (Bf 3±4) were unaected (Table 3, Fig. 1). The increase of Bf 1 was most pronounced in zone 1 microcosms, where, 42 days after N-addition, populations in the single high (H) and repeated low (L) addition treatments had increased 3-fold compared to control (C) populations (Fig. 1a). On day 78, numbers in H had further increased, and were on average 2-fold higher than in L, and 6-fold higher than in C. Only at the last date of the experiment, numbers of Bf 1 in L and H were back to control levels. In zone 2, N-eects on Bf 1 were smaller and more variable between dates, but elevated numbers were observed at several times, including the ®nal date. Of the taxa comprising the Bf 1 group (Table 1), the positive response of Rhabditinae to N was comparable among zones, but Neodiplogasteridae and Myolaimidae increased only in zone 1. For the bacterivores of life strategy group 2 (Bf 2), numbers in L and H were signi®cantly higher than in C in zone 2 only (Table 3,
Fig. 1b). This increase appeared to be mainly caused by a rise in Cephalobidae in this zone (Table 1). No signi®cant dierences were found between L and H. Like Bf 1, the increase in Bf 2 populations of L and H emerged 22±42 days after the ®rst N-addition, but, unlike Bf 1, the largest dierence between Bf 2 population sizes in control versus N-addition (L, H) treatments never exceeded 2-fold. The dynamics of fungivorous nematodes were hardly aected by N-addition, except for a small increase (P<0:10) of r-selected fungivores (Ff 2) in N-addition microcosms of zone 2 only (Table 4, Fig. 2a). A slightly signi®cant (P<0:10) zone by N interaction was observed for K-selected fungivores (Ff 4), with L numbers higher than H abundances in zone 1, and vice versa in zone 2 (Table 4, Fig. 2c). However, the F-test contrasting controls with N-addition (L, H) populations was not signi®cant. Unlike the relatively rapid response of bacterivores to N-addition, which declined at the end of the experiment, the N-induced increase of predators occurred
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C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
Fig. 2. Temporal changes in abundances of fungal-feeders (Ff) of dierent life strategy categories: (A) Ff 2, (B) Ff 3, and (C) Ff 4. Symbols and error bars as for Fig. 1.
generally later and was still highly signi®cant 191 days after the ®rst addition, notably for predators of life strategy categories 2 and 3 (Pr 2±3; Table 5, Fig. 3a). This group, which was dominated by Tripylidae (Table 1), behaved remarkably similar in both zones, with a ®nal (day 191) average population size of 30 predators 50 gÿ1 soil in H and 17 predators in L, compared to two predators in control samples. The increase in H emerged 78 days after the single high addition, and remained generally higher than L populations. The Neect on predators of life strategy categories 4 and 5 (Pr 4±5), dominated by Mononchidae, was not as pronounced, but overall populations in addition treatments (L, H) were signi®cantly higher than in controls (Table 5, Fig. 3b). As indicated by the signi®cant zone N time interaction, their dynamics were erratic. There was a weak N-eect on omnivores, with overall higher abundances in N-addition (L and H) treatments than in controls (P<0:10; Table 5, Fig. 3c). Of the community indices tested, the MI was most signi®cantly reduced by N-addition, though this eect
was mostly limited to zone 1, H microcosms. There, the MI was 010% lowered compared to controls during days 42±134, but recovered to control values at day 191 (Table 6, Fig. 4c). Overall genus richness, as estimated by Fisher's a, was on average higher in L than in H, but due to variation by zone and time, no clear N-eect on genus richness emerged (Table 6, Fig. 4a). Re-calculating this statistic with only bacterivore data yielded more signi®cant eects. Overall, bacterivorous genus richness, as estimated by Fisher's a, was signi®cantly reduced in N-addition treatments compared to control microcosms, and this eect was greater (P<0:10) in H than in L. However, examination of the time series (Fig. 4b) indicates that this occurred only in zone 2, whereas in zone 1, during the second half of the experiment, bacterivorous richness in H was higher than in L (but not dierent from C). Although trends indicated lower nematode F-to-B ratios in N-addition treatments compared to control microcosms, N-eects were not signi®cant (Table 6, Fig. 4d).
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
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Fig. 3. Temporal changes in abundances of predators (Pr) and omnivores (Om) of dierent life strategy categories: (A) Pr 2±3, (B) Pr 4±5, and (C) Om 4±5. Symbols and error bars as for Fig. 1.
3.3. Eects of enclosures There were signi®cant enclosure (microcosm) eects on abundances of individual nematode groups, which often varied by zone and sample date (Table 7; Figs. 1±4). For instance, on day 1, group Bf 1 was signi®cantly more abundant in control microcosms than in fresh (unenclosed) soil cores from the same plots, but day 191 abundances were similar. A similar eect was observed for Bf 2 in zone 1, with a reverse trend occurring in zone 2. Group Bf 3±4 was not aected by the enclosure in zone 1, but it was signi®cantly more abundant in zone 2, at day 1 but not day 191, compared to fresh soil core populations. In contrast to bacterivores, fungivore populations (mostly Ff 2 and Ff 4) generally decreased in microcosms relative to unenclosed soil, particularly at day 191. In contrast, at day 1 predator group Pr 2±3 was more abundant in the microcosms, but not dierent from unenclosed populations on day 191. Abundances of predator
group Pr 4±5, omnivores, and total free-living nematodes were largely unaected by the enclosures, as were indices of genus richness and maturity. The nematode F-to-B ratio was signi®cantly lowered by enclosure, at all times and zones. 4. Discussion 4.1. Riparian nematode assemblages The assumption that free-living nematodes would be abundant and diverse in riparian forests, even in the often water-logged zone 1 soil, was con®rmed by our data. The total nematode population in zone 1 soil was in fact higher than in zone 2, though slightly less diverse (Table 2). The total richness of 49 genera in the riparian forest, and the average total abundance of 1047 free-living nematodes 50 gÿ1 soil, were considerably higher than found in other sites in the coastal
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Fig. 4. Temporal changes in assemblage indices: (A) overall Fisher's a diversity index, (B) bacterivorous Fishers a diversity index, (C) maturity index, and (D) Nematode F-to-B ratio. Symbols and error bars as for Fig. 1.
plain of Georgia and Florida, such as an acidic wetland (Cox and Smart, 1994), and a pine forest on a sandy soil (McSorley, 1993), but were comparable to numbers for a close-by riparian wetland on the same soil type as the forest of the present study (Ettema et al., 1998). The distinct soil dierences between zones less than 10 m apart (Ettema et al., 1999), which are typical for the strong gradient found in riparian zones (e.g., Gregory et al., 1991; Hedin et al., 1998), in¯uenced the composition of the nematode assemblages in these sites (Tables 1 and 2). Total abundance was signi®cantly higher in zone 1 and positively correlated to organic N (rs 0:41, P 0:008, n 42 [zone 1, control microcosms of all dates]), labile C (rs 0:48, P 0:001) and basal respiration rates (rs 0:34, P 0:03). In addition, the near stream zone had higher abundances of taxa that are typically found in aquatic habitats (Bongers, 1988), such as Rhabdolaimus, Euteratocephalus, Monhysteridae, Chronogaster,
Bastiania, Cryptonchus and Paramphidelus (Table 1). In zone 2, higher total abundances were associated with basal respiration rate only (rs 0:34, P 0:03, n 42 [zone 2, control-microcosms of all dates]). In both zones, the microbivorous nematode fauna was dominated by bacterial feeders, but in zone 1 bacterivore dominance was reduced by an increased fungivore population, in concert with the higher amounts of fungal respiration found in this zone (Ettema et al., 1999). Although bacterial respiration was also elevated in zone 1, this did not result in a signi®cant increase of bacterivorous nematodes (Table 1). Within zones, the abundances of bacterivores and fungivores in controls were not signi®cantly correlated to bacterial and fungal respiration, nor to microbial biomass (Table 8). However, in both zones bacterivores were related to basal respiration rates, and in zone 2 fungivore abundance was associated with basal and substrate induced respiration (Table 8). Lack of simple correlation between microbivorous nematodes and their food
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638 Table 7 ANOVA of enclosure (microcosm) eects on nematode groupsa Source Enclosure
Enclosure zone
Fb
Variable
SS
Bf 1 Bf 2 Bf 3±4 Ff 2 Ff 3 Ff 4 Pr 2±3 Pr 4±5 Om4-5 Overall Fisher's a Bacterivore Fisher's a Maturity index Nematode F-to-B ratio
1.10 2.82 0.21 13.2d 0.23 6.60e 0.16 10.9f 0.16 0.53 0.14 3.06 0.28 1.22 0.45 2.49 0.60 1.88 0.00 0.00 1.47 1.27 0.06 3.20g 2.09 17.2d
Enclosure day
SS
Fb
SS
Fc
0.27 0.01 0.31 0.01 0.02 0.10 0.34 0.00 0.14 0.02 0.80 0.02 0.02
0.69 0.20 8.90e 0.39 0.07 2.19 1.48 0.00 0.45 0.01 0.69 1.32 0.18
1.48 4.98e 0.00 0.01 0.08 1.08 0.04 1.13 1.33 1.34 1.18 10.4f 1.17 7.12e 0.06 0.63 0.43 1.05 3.25 1.81 1.46 0.89 0.07 2.47 0.01 0.08
a
Measurements taken on days 1 and 191, on control (C) microcosms and fresh soil cores, in both zones (see Section 2). Abundances were log10-transformed prior to analysis. b F-test (df 1, 10) used Enclosure [Block(Zone)] as error term. c F-test (df 1, 10) used Day Enclosure [Block(Zone)] as error term. d PR0:005;. e PR0:05;. f PR0:01;. g PR0:10.
Table 8 Correlation coecientsa between microbial variables and microbivorous abundances. Variables were log10-transformed before analysis Microbial measurementsb Zone
N-addition Groupc Mic-C Mic-N BR
Zone 1 C L H Zone 2 C L H a
BF FF BF FF BF FF BF FF BF FF BF FF
0.19 0.21 0.33d 0.47e 0.23 0.40f 0.11 0.21 0.19 0.24 0.13 0.17
0.22 0.20 0.48e 0.39d 0.26g 0.29g 0.06 0.22 0.21 0.15 0.18 0.18
0.35d 0.21 0.30d 0.31d 0.08 0.28g 0.28g 0.34d 0.02 0.29g 0.25 0.37d
SIR BACT FUNG 0.22 0.21 ÿ0.14 0.12 0.09 ÿ0.11 0.40f 0.27g 0.08 0.33d 0.25 0.22 0.35d 0.31e 0.02 0.26g 0.03 ÿ0.11 0.10 ÿ0.11 ÿ0.11 0.33d 0.08 0.19 0.20 0.14 0.02 0.26g 0.02 0.24 0.27g 0.31d ÿ0.06 0.29g 0.33d 0.38d
Pearson's r, estimated with n 42 pairs of points in each test. Data from Ettema et al. (1999) Mic-C, microbial carbon; Mic-N, microbial nitrogen; BR, basal respiration; SIR, substrate induced respiration, BACT, bacterial substrate induced respiration; FUNG, fungal substrate induced respiration. c BF, bacterial-feeders; FF, fungal-feeders. d PR0:05;. e PR0:005;. f PR0:01;. g PR0:10. b
1635
source has been reported before (e.g., Clarholm et al., 1981; Wardle et al., 1995), and has been attributed to asynchronous dynamics and non-linear trophic interactions, which include indirect eects and positive feed backs (Bengtsson et al., 1996). 4.2. N-induced changes in trophic dynamics Interestingly, the N-addition synchronized microbial and microbivorous dynamics, as evidenced by stronger correlations between microbial variables and microbivorous dynamics in both L and H microcosms (Table 8). Notably, positive correlations between bacterivorous nematodes and bacterial respiration were found in the H treatment in both zones, and in the L treatment (P<0:10) in zone 1. In addition, a signi®cant correlation between fungal-feeders and fungal respiration was found in zone 2, H microcosms. In zone 1, fungivorous and bacterivorous abundances were positively associated with microbial C and N in both L and H treatments. Some of these correlations may be coincidental (05%), but their prevalence contrasts highly with the seemingly out-of-phase microbial-microbivorous dynamics in control microcosms. Anderson (1995) presents a conceptual model where disturbances such as re-wetting events after droughts and seasonal litter inputs improve detectibility of faunal eects on soil processes because these disturbances synchronize soil organism activities. Nitrogen in¯ux may have a comparable `re-set' eect on riparian soil population dynamics. While N-eects on microbial dynamics were hardly detectable (Ettema et al., 1999), a measurable signal of increased microbial growth could have been weakened by increased microbivorous grazing pressure (cf. BaÊaÊth et al., 1978; Clarholm et al., 1981). Indeed, when considered in tandem with the highly positive microbialmicrobivorous correlations in N-amended microcosms, the signi®cant increase of some microbivorous groups several weeks after N-addition (Tables 3 and 4, Figs. 1 and 2) is strong evidence that microbial production was temporally elevated by N-input. In zone 1, the 3to 5-fold increase of Bf 1 in N-amended microcosms indicates a fast conversion of consumed bacterial biomass into nematode ospring, although immigration into the open-bottom microcosms cannot be excluded as a partial, though probably minor, source of the population increase. Fast reproduction in response to increased food supply is typical for the rhabditid and diplogasterid taxa in life strategy group 1 (e.g., Sohlenius, 1973; Anderson et al., 1981; Schiemer, 1983; Ettema and Bongers, 1993). In response to inorganic N-addition, these taxa increased in abundance in several studies (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986), which has been attributed to the stimulating eect of
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C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
N on N-limited bacterial production (BaÊaÊth et al., 1978). Although Rhabditinae showed a similar N-induced increase in zones 1 and 2, the more signi®cant N-response in zone 2 came from bacterivores in group 2 (Bf 2), notably Cephalobidae (Tables 1 and 2, Fig. 1b). We suspect that this zone dierence was due to a combination of stochastic and deterministic factors (cf. Sohlenius, 1993). There were fewer Bf 1 than Bf 2 individuals present in zone 2 to start reproduction (Table 1). As zone 2 soil had a lower labile C content than zone 1 (Ettema et al., 1999), it is possible that Ninduced microbial production in zone 2 was not high enough (because of C-limitation) to meet food requirements for some Bf 1 taxa. In neither zone did K-selected bacterivores (Bf 3±4) respond to N-addition, theoretically because they have much longer generation times. In addition, these taxa may feed on `autochtonous' micro¯ora which perhaps was not stimulated as much by N-additions as `zymogenous' micro¯ora was. Given that data on bacterivorous diet speci®city and microbial species-speci®c dynamics are severely limited, these remain speculations. The lack of signi®cant response of fungivorous nematodes to inorganic N may be explained by the notion that fungi tend to be less limited by N, and more limited by C, than bacteria (BaÊaÊth et al., 1978). In other inorganic N-addition studies, fungal-feeders did not respond to N either (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986). Why was not a much greater bacterivorous response to N observed? First of all, adding inorganic N without a C-source may have limited the response of bacteria and bacterivores alike. Although we found a signi®cant 3±5 fold N-induced increase of Bf 1 taxa, these numbers are not as great as the 55-fold increase found by Ettema and Bongers (1993), who added organic N as dried cow manure, or more than the 70fold increase detected by Dmowska and Kozlowska (1988) in ®elds treated with excessive amounts of semiliquid manure. Microbial studies have shown a much stronger microbial production response to combinations of C and N, than to either nutrient alone (BaÊaÊth et al., 1978; Elliott et al., 1983; Zagal and Persson, 1994). Secondly, predation may have signi®cantly weakened the microbivorous growth signal, just like microbivorous grazing may have reduced the microbial signal. Predaceous nematodes greatly increased in Namended systems (and signi®cantly more in H than in L), in both zones (Table 5, Fig. 3a,b). As predaceous abundance increased, bacterivorous numbers decreased back to control-levels. The very similar increase of Tripylidae in each zone suggests that this is a generalist predator. In soil, predation mostly depends on
chance encounters, and most nematode predators are expected to be generalists (Yeates and Wardle, 1996). Finally, nematode abundance changes due to soil enclosure into microcosms may have weakened the eect of N-additions. In microcosms, bacterivore abundance was stimulated, and fungivore abundance inhibited, compared to populations in fresh soil cores (Table 7). The former eect was most signi®cant at the start of the experiment, when the presence of freshly cut roots in the microcosms possibly stimulated bacterivore populations before N-treatment had begun. In contrast, the (negative) eect on fungivore abundance (and fungal respiration: Fig. 6b in Ettema et al., 1999) was most signi®cant at the end of the experiment. The disconnection of fungi and fungivores in the enclosures from larger hyphal networks may have caused their decrease in microcosms, and lessened their ability to take advantage of the added N. 4.3. Indicators of riparian soil N-saturation The general indicator potential of free-living nematodes is based on their presence in measurable numbers and wide distribution, and their critical role in soil processes (Gupta and Yeates, 1997). Their speci®c potential for indicating riparian soil N-saturation is suggested by our results, which showed that r-selected bacterivores (Bf 1 and Bf 2), and predaceous nematodes, were highly responsive to changing soil N-content. The response of r-selected bacterivores to Naddition corroborates results of numerous other Nenrichment studies in non-riparian soils (BaÊaÊth et al., 1978; Sohlenius and Wasilewska, 1984; Sohlenius and BostroÈm, 1986; Dmowska and Kozlowska, 1988; Ettema and Bongers, 1993; Griths et al., 1994), and could explain the positive correlation between rselected bacterivorous nematode abundance and soil nitrate content found by Ettema et al. (1998) in a riparian wetland subject to N-in¯ux. How did nematode group responses compare to the dynamics of other potential N-saturation indicators measured in this experiment? First, nematode dynamics provided a clearer signal of N-saturation than underlying changes in microbial biomass and respiration (Ettema et al., 1999). Second, the N-eect on nematodes was still observable after inorganic N-concentrations ceased to be dierent between treatments. However, inorganic N-pools showed increases immediately after N-addition, whereas nematodes needed at least a month response time (compare Figs. 1 and 3 to Fig. 2 of Ettema et al., 1999). Third, changes in nematode groups following N-addition were observed in both zones, whereas N-induced changes in denitri®cation were only apparent in zone 1 (Fig. 3b in Ettema et al., 1999). Finally, individual nematode groups (notably Bf 1, Bf 2, Pr 3) showed more signi®cant and
C.H. Ettema et al. / Soil Biology and Biochemistry 31 (1999) 1625±1638
consistent dierences between N-treatments, than the ecological indices which averaged all nematode populations into single index values (Figs. 1 and 3 versus Fig. 4). Thus, it appears from our experiment that, at the resolution of life-strategy-within-feeding-groups (Table 1), nematodes could be useful indicators of riparian N-saturation. The question remains how to apply nematode indicators in N-saturation monitoring programs. Since so many factors, both deterministic and stochastic, in¯uence nematode abundance and species composition (Ettema, 1998), it seems quite impossible, outside controled experimental settings, to attribute nematode assemblage changes to single causes, if no other variables are measured to support inferences. For instance, a high abundance of Bf 1 nematodes may be due to soil N-saturation, but could also be due to heavy metal pollution (Weiss and Larink, 1991), strong drying-rewetting cycles in organic soils (this study: Bf 1 abundances were considerable in zone 1 control microcosms and untreated, unenclosed soil), a local concentration of animal feces (Rehfeld and Sudhaus, 1989), or any other disturbance that causes a temporary increase in bacterial activity. Therefore, nematode indicators of N-saturation may only be useful when measured together with other key system characteristics, such as denitri®cation (Hanson et al., 1994a, Hanson et al., 1994b; this study) and litter-N (Aber et al., 1989; Hanson et al., 1994a). Moreover, recognizing that the riparian ecosystem reponse to N-in¯ux is spatially variable (within and between zones) and temporally dynamic (with N moving from one pool to another), the combined assessment of a soil faunal, microbiological and biochemical indicator would be a more reliable and ecologically meaningful approach than the single monitoring of any one of them.
Acknowledgements Many thanks are due to Rex Blanchett for his help in setting up the microcosms. Piet Loof provided taxonomic identi®cation of several nematode specimens gathered during a pilot survey of the riparian site. Steve Rathbun guided the statistical analysis. Luc Boerboom provided precious advice and help in data management. Constructive comments by Diana Wall and two anonymous referees greatly improved the manuscript.
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