Spatial association between entomopathogenic and other free-living nematodes and the influence of habitat

Applied Soil Ecology 76 (2014) 1–6

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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Spatial association between entomopathogenic and other free-living nematodes and the influence of habitat Jung-Joon Park a,1 , Ganpati B. Jagdale a,2 , Kijong Cho b , Parwinder S. Grewal a,3 , Casey W. Hoy a,∗ a b

Department of Entomology,The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA Division of Environmental Science and Ecological Engineering, Korea University, Seoul 136-701, South Korea

a r t i c l e

i n f o

Article history: Received 6 June 2013 Received in revised form 26 November 2013 Accepted 7 December 2013 Keywords: Spatial patterns Spatial analysis of distance indices r and K life history traits Nematode community analysis Dispersal

a b s t r a c t Spatial association between entomopathogenic and free-living nematode populations in soil were analyzed at the landscape scale. GPS coordinates were obtained for 479 locations where soil samples were collected to extract nematodes. Habitats sampled included vegetable and agronomic crop fields, grassy borders adjacent to fields, residential lawns, meadow and forested wetlands in a vegetable growing region in northwest Ohio. Free-living nematodes were classified according to trophic level (bacterivores, fungivores, carnivores, and omnivores) and life history characteristics (r-selected colonizing versus K-selected persisting species on a 1–5 scale). Spatial associations based on spatial analysis of distance indices (SADIE) were analyzed and compared among entomopathogenic nematodes and free-living nematode functional guilds defined by the classifications described above. Spatial aggregation indices (Ia ) revealed that each functional guild’s spatial pattern varied among habitats. Considering all data regardless of habitat, spatial aggregation indices showed that functional guilds with K-selected persisting life history traits were less aggregated, whereas those with r-selected colonizer life history traits were more aggregated. The spatial aggregation index of entomopathogenic nematodes was similar to that of the r-selected colonizer type free-living nematodes, which share several life history traits including bacteriophagy, high reproductive rates, insect phoresy, and greater abundance in grassy borders, where spatial associations between entomopathogenic and r-selected colonizing functional guilds of free-living nematodes were particularly strong. The spatial aggregation patterns of entomopathogenic and free-living nematodes, suggest that these species associate over larger areas than previously measured and that the extent of these spatial associations might be predicted by the nematode life history traits. © 2013 Published by Elsevier B.V.

1. Introduction Nematodes are the most abundant metazoa and an evolutionarily successful group of organisms in many ecosystems (Ferris et al., 2001). Nematodes exist as a diverse and highly speciated group in the soil environment. They occupy a central position in the soil food web, occurring at multiple trophic levels (Yeates et al., 1993) including as bacterivores, fungivores, plant parasites, predators and omnivores. Therefore, nematodes can serve as useful ecological indicators and have the potential to provide insights into the

∗ Corresponding author. Tel.: +1 330 263 3611; fax: +1 330 263 3686. E-mail address: [email protected] (C.W. Hoy). 1 Current Address: Department of Applied Biology, Institute of Agricultural and Life Science, Gyeongsang National University, Jinju 600-701, South Korea. 2 Current Address: Department of Plant Pathology, University of Georgia, Athens, GA 30605, USA. 3 Current address: Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, USA. 0929-1393/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.apsoil.2013.12.004

structure and function of the soil food web (Freckman, 1988; Ferris et al., 1999; Ritz and Trudgill, 1999). In addition to their characterization into trophic or feeding groups, the free-living nematodes can also be separated on a colonizer–persister scale (cp scale 1–5) that represents the degree of r (colonizer, cp 1) vs. K (persister, cp 5) life history strategy (Bongers, 1990; Bongers and Bongers, 1998; Ferris et al., 1999, 2001). Colonizer species display a suite of traits that favor rapid population growth and colonization of new habitats, whereas persister species are adapted to competition in saturated habitats (Kokko and Lundberg, 2001) with limited opportunities for niche access or expansion (Ferris et al., 2001; Reznick et al., 2002). Ferris et al. (2001) defined nematode functional guild as a combination of the trophic group and colonizer–persister class. Spatial aggregation and association analyses of nematodes in the soil can contribute new knowledge about soil ecological processes including population dynamics, biological control, and soil food web structure and function. The spatial distributions of freeliving nematodes have been found to vary with genus (McSorley et al., 1985; Ettema et al., 1998; Park et al., 2013), trophic group

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(Robertson and Freckman, 1995; Park et al., 2013), life history strategy (Park et al., 2013), and functional guild (Park et al., 2013). Using Taylor’s power law analysis, Park et al. (2013) showed that bacterivore and plant-parasitic groups were more highly aggregated than omnivorous and predatory nematodes. They also showed that nematodes in cp-classes 1 and 2 tended to be more aggregated than those in higher cp-classes and the functional guilds were generally more highly aggregated than individual genera, suggesting a higher degree of aggregation at the functional guild level. Although functional guild is a basis of many soil nematode community analyses, each functional guild contains species differing in the components of their life-history strategies: reproductive capacity (Ferris et al., 1996); colonizing ability (Bongers, 1990; Ettema and Bongers, 1993); and preference for temperature and moisture conditions (Sohlenius, 1985). These different life history strategies may result in different spatial patterns. Most nematode spatial distribution studies conducted on plantparasitic nematodes show that they are highly aggregated in soil ecosystems (Noe and Campbell, 1985; Ferris et al., 1990), with frequency distributions that are well described by the negative binomial distribution. Such aggregation could influence the net effects of nematodes on ecosystem functioning. For example, the spatial heterogeneity of trophic interactions in soil food webs has been identified as an important determinant of soil trophic dynamics (Parmelee and Alston, 1986; Moore and de Ruiter, 1991). However, little is known of the spatial association between free-living nematodes and entomopathogenic nematodes (EPNs) belonging to Heterorhabditidae and Steinernematidae, which feed on symbiotic bacteria but are obligate insect parasites (Grewal et al., 2005). All available studies on EPN spatial distribution reveal a patchy distribution even in relatively uniform environments (Stuart and Gaugler, 1994; Campbell et al., 1996, 1998; Taylor, 1999). Comparative studies among nematode species reveal that EPNs are associated with soil physical, chemical, and biological characteristics that are most similar to those associated with bacteria-feeding free-living nematodes based on soil sampling, and Baerman funnel extraction and visual identification of nematode species (Hoy et al., 2008) as well as centrifugation and molecular identification by q-PCR (Campos-Herrera et al., 2012). Research to understand the spatial heterogeneity of soil food web structure and function relies on spatial models with realistic properties. Spatial analysis techniques coupled with a variety of statistical tools are currently available to help quantify spatial heterogeneity with specific location information, such as geostatistics and spatial analysis with distance indices (SADIE) (Ettema, 1998; Ettema et al., 1998; Perry and Dixon, 2002; Robertson and Freckman, 1995). Geostatistics uses information from both sample values and sample locations to model spatial dependence as a function of distance among sample points (Cressie, 1993), and can be used to estimate values at points that were not sampled. However, the sampling dimension and measurement scale can affect the performance of geostatistics in describing spatial pattern (Liebhold et al., 1993). In contrast, SADIE (Perry et al., 1999; Perry and Dixon, 2002) allows improved interpretation of the spatial patterns of a single population or the spatial associations between two populations within a given sampling, area because it is designed for data that are distributed in discrete areas with relatively well-defined boundaries, and measures the extent of clustering with subsequent testing for spatial patterns in relationships among sample locations (Korie et al., 2000). The specific objectives of this study were to characterize and quantify the spatial patterns of EPNs and other free-living nematodes, and to test for spatial associations between EPNs and free-living nematodes at the functional guild level, both within various habitats and at landscape scales using SADIE. Our hypothesis was that similarities and differences in life history strategies

between EPN and other guilds would lead to corresponding similarities and differences in spatial structuring of populations, even with sampling taking place over much larger areas than those typical of previous research on spatial patterns of nematodes.

2. Materials and methods 2.1. Study site and sampling Free living nematodes were extracted from approximately 100 sampling sites selected along transects from each of six habitats in an Ohio muck soil vegetable production area: vegetable crops (‘Vegetables’; 102 sample sites), row crops (corn or soybean; ‘Row crops’; 101 sample sites), residential lawns (‘Lawn’; 101 sample sites), mowed but otherwise unmanaged grass borders of crop fields (‘Grass borders’; 101 sample sites), unmanaged meadow area (‘Meadow’; 104 sample sites) and forested wetlands (‘Forest’; 101 sample sites). Further details on habitats, sites, site selection, sampling, and sample processing can be found in a publication that describes the association of nematode species with soil environmental variables based on these data (Hoy et al., 2008). Soil types were either the same or similar throughout the survey area, consisting primarily of high organic matter “muck” soils. The sites were sampled during September–November of 2004. Sites were marked and a subset of them were georeferenced after sampling was conducted with a global positioning system (GPS) that provided accuracy of within 1 m (Trimble, Sunnyvale, California). Out of the initial 610 sampling locations, sufficiently accurate GPS readings for 479 sampling sites were obtained, the remaining 131 sites were not georeferenced either because the site markers were missing or disturbed, or because we lacked sufficient satellite coverage despite repeated attempts, often due to surrounding trees, foliage or buildings. Distances between sample pairs within the 479 sample sites ranged from 26 m to 7862 m. At each sampling site, ten soil cores were collected (2.0 cm diameter, 15 cm depth), placed in polyethylene bags and kept at 10 ◦ C. EPNs were extracted from these soil samples using a modified insect baiting technique (Fan and Hominick, 1991). The 10 soil cores from each site were mixed and 200 g of the mixed soil was placed in a plastic container (470 ml). Ten last instar wax moth, Galleria melonella L., larvae were released into the soil and the soil samples were incubated in a growth chamber in total darkness, at 25 ◦ C and 88% relative humidity. Three days later, half of the dead larvae were placed in modified White traps (White, 1927) and returned to the growth chambers for an additional 7–10 days to test for emergence of EPN infective juveniles and the other half were dissected to count the numbers of infective juveniles that had penetrated. Emergence of EPNs from modified White traps was verified by inoculating fresh G. melonella larvae with the emerging nematodes. Ten larvae were placed in a Petri dish with moist filter paper, 2 ml of a nematode suspension from the White trap was added, and the dish was incubated under conditions described above for 7 days. Larvae or cadavers showing symptoms typical of EPN infection were individually placed in modified White traps as previously described and observed for the emergence of infective juveniles. The infective juveniles emerging from this second round of infection were identified to species level using methods given in Kaya and Stock (1997) and keys by Stock and Hunt (2005). All EPN nematode cultures were maintained and their species were later confirmed using molecular methods (Maneesakorn et al., 2011). For each sample the presence of infected (verified by the second round of infection) larvae and counts of infective juveniles in the initial infected larvae following dissection (see above) were recorded. The EPN species identified in the sites analyzed for this study were Heterorhabditis bacteriophora (1072 in 31 sites) and Steinernema carpocapsae (127 in 6 sites).

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Free-living nematodes other than EPNs were extracted from another 10 g sample of soil from the composite soil samples from each sampling site within 14 days of collection using the Baerman funnel technique (Flegg and Hooper, 1970). All specimens were identified to the family and/or genus level and counted (see Hoy et al., 2008 for details). Each identified nematode was classified by trophic group according to Yeates et al. (1993) and life history category from colonizer to persister (cp) scale between 1 and 5 according to Bongers (1990). We analyzed the data for only four trophic groups: bacterivores, fungivores, predators, and omnivores, because very few plant-parasitic nematodes were found in vegetable and crop fields. Analyses were performed at the functional group level, for all free-living nematodes, to ensure that sufficient numbers of each group were available for the analysis, and because the question of interest concerned aggregation and association of functional groups more so than individual species within those groups.

2.2. Spatial analysis by distance indices (SADIE) Spatial patterns of EPNs and other free-living nematode groups were analyzed and compared in each habitat using SADIE (Perry, 1998). SADIE enables the spatial characteristics of the observed arrangements to be assessed using indices of clustering for each sample point and a test that compares observed spatial pattern with a random pattern. By permuting the observed counts among sample points the test quantifies the difference between the observed pattern of counts and the alternative random patterns for the same counts. The expected value for the SADIE index of aggregation (Ia , an index of the degree of clustering for the whole sample area) is 1; Ia > 1 indicates that counts are aggregated into a cluster (Perry, 1998). By overlaying cluster maps of the two data groups, SADIE also measures the extent of the spatial association between two populations (Perry and Dixon, 2002), which may be positively associated, negatively associated (dissociated), or occur at random with respect to one another. This measure is based on a comparison of the spatial SADIE clustering indices of the two sets at each sample point rather than the raw data, because the indices have been adjusted to account for the spatial pattern of the data and any local clustering effects (Perry and Dixon, 2002). A global association index, X, was calculated as the mean of local association indices, which is a simple correlation coefficient between clustering indices of EPNs and other free-living nematodes in each habitat: X > 0 for positively associated populations, X ∼ 0 for populations positioned at random with respect to one another, and X < 0 for negatively associated populations (Perry and Dixon, 2002). The indices of aggregation and association were calculated for each of the six sampled habitats and also for the entire study area irrespective of habitat.

3. Results 3.1. Spatial pattern of nematode functional groups Total number of georeferenced sample sites, average distance between sample sites and total number of nematode functional groups including EPN in each habitat are summarized in Table 1. In each habitat, BF1, BF2, FF2 and OM4 population densities were relatively high compared with other functional groups. EPN population density was relatively high only in the Grassy borders. Numbers of carnivores (CA3 and CA5) were relatively low in each habitat. CA3 were not present in Grass borders, Row crops or Vegetables. Bacterial feeding genera that were intermediate on the cp scale, BF3, were found infrequently in Row crops and not at all in Vegetables,

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however, their occurrence was similar to that of BF4 in other habitats (Table 1). The SADIE indices of aggregation varied within each habitat among functional groups (Table 2). In the Forest, BF1, BF4 and OM4 were significantly aggregated (p ≤ 0.016), whereas other guilds were not aggregated (p ≥ 0.055). In the Meadow, none of the functional guilds were significantly aggregated (p ≥ 0.080). In the Grass borders, EPN, BF1, BF2, FF2 and OM4 guild types were significantly aggregated (p ≤ 0.016), whereas in the Lawn, each of these were significantly aggregated except EPN, and in addition BF3, and CA5 showed significantly aggregated patterns (p ≤ 0.010). In the Row crops, only the most r-selected bacterial feeders, BF1, were significantly aggregated (p = 0.039). In the Vegetables, however, each of the colonizer groups showed a significantly aggregated pattern (BF1, BF2, FF2; p ≤ 0.016). None of the more K-selected persisting groups showed aggregation in the tilled habitats (BF4, OM4, CA5; p ≥ 0.058). Based on the results within habitats, the colonizing groups were more likely to be significantly aggregated within habitats where any aggregation was observed (all except Meadow), although the persisting groups were occasionally aggregated in relatively undisturbed habitats. When data were pooled across all habitats, the more general pattern observed was significant aggregation in colonizer groups: BF1, BF2, FF2 and EPN (p ≤ 0.021), and no significant aggregation in neutral to persisting life history groups: BF3, CA3, BF4, OM4, and CA5 (p ≥ 0.052) (Table 2). 3.2. Spatial association between EPN and other free-living nematodes Tests of spatial association between EPNs and other free-living nematodes using the SADIE association index, X, were performed for the pooled data regardless of habitat and in each of the habitats except Vegetables, where there were no positive EPN samples (Table 3). EPN’s were by far the most abundant in the Grass borders and results in this habitat were very similar to those for the pooled data: the associations were significant between EPN and the more colonizing groups (BF1, BF2, and FF2, X ≥ 0.344, p ≤ 0.05) but not the other functional groups (X ≤ 0.210, p ≥ 0.068). EPN’s were next most abundant in the Lawn and Forest habitats, where spatial association indices also were significant between EPNs and relatively r-selected bacterial feeders (p ≤ 0.05; BF2 and BF3 in Lawn; BF1 and BF2 in Forest), and also between EPN’s and BF4 and OM4 in Forest only. In the Meadow, all functional groups except FF2 and OM4 were significantly and positively associated with EPNs (p ≤ 0.05), but EPN’s were only found in 4 of the sites (Table 1). In the Row crops, the most disturbed sites in which EPN’s were found, no significant positive association between EPNs and other functional groups were found (p ≥ 0.116). Overall, based on the pooled data, spatial associations were between EPNs and other colonizing bacterial and fungal feeding nematode groups, although exceptions to this overall pattern could be found within some of the habitats and we could not test whether the most important factor was the colonizer–persister scale, the feeding guild, or the combination of the two (Table 1) because relatively r-selected carnivores and omnivore were not sufficiently abundant. 4. Discussion Spatial heterogeneity of soil organisms is a multidimensional concept that requires a variety of analytical techniques, each of which capture a particular aspect of spatial diversity. Soil properties and nematode population densities are spatially correlated within specific distances (Ettema, 1998; Ettema et al., 1998) and spatial scales used for sampling can be very important (Ettema et al., 1998; Yeates and Bongers, 1999; Park et al., 2013).

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Table 1 Number of georeferenced sample sites, average distance between sample sites for each soil habitat and total number of nematodes from each guild that were extracted from soils in the survey. Soil habitat

Forest

Meadow

Grass borders

Lawn

Row crops

Vegetables

Pooled data

Number of georeferenced sample sites

70

93

80

99

63

74

479

237.52 (43.24 )

199.14 (30.83)

713.73 (59.89)

634.02 (76.21)

263.81 (32.60)

276.52 (45.27)

1093.68 (52.50)

14 (10) 1257 1608 981 322 36 185 497 5

4 (2) 1872 1443 1507 100 53 147 1476 11

1149 (21) 4919 2643 1269 195 0 95 615 39

21 (4) 2431 1841 1283 34 2 131 1060 15

11 (3) 961 910 1099 6 0 48 255 2

0 3398 2633 2517 0 0 43 134 1

1199 (40) 14838 11078 8656 657 91 649 4037 73

†

Average distance in meters between sample sites EPN BF1 BF2 FF2 BF3 CA3 BF4 OM4 CA5

Number of nematodes (sites positive for EPN) per guild‡

†

Standard error. EPN: Entomopathogenic nematodes; BF: Bacterivore; CA: Carnivore; FF: Fungivore; OM: Omnivore; numbers 1–5 representing points on a colonizer–persister scale with 1 being more r-selected colonizer types and 5 being more K-selected persisting types according to Bongers (1990). ‡

Nematodes can disperse by passive modes via water and arthropod phoresy, suggesting that larger extents than have typically been examined may be relevant scales for evaluating their spatial distributions. Although many studies on EPN phoresy have examined soil dwelling organisms that disperse within soil habitats, it also has been examined with respect to several adult insects that disperse at the scales relevant to this study including Lepidoptera (Timper et al., 1988) and Coleoptera (Lacey et al., 1995; Kruitbos et al., 2009). Rates of passive dispersal in surface water flow or runoff currently are not well described. Shifting to these larger extents could lead to new understanding of the spatial structure of nematode populations, and how it is influenced by underlying conditions such as moisture, soil chemistry, and biotic communities that interact with nematodes. Western and Blöschl (1999) found, for example, that apparent correlation distances for soil moisture always changed with distance between samples and how thoroughly they cover the soil environment. In this study, we focused on a nematode life history grouping developed and refined by several researchers according to a colonizer-persister continuum (Bongers, 1990; Bongers and Bongers, 1998; Ferris et al., 1999, 2001; Martinez, 1992; Wilson, 1999), which represents the degree of r versus K life history strategies (MacArthur and Wilson, 1967). In broad terms, r species display a suite of traits that favor rapid population growth and colonization of new habitats, whereas K species are adapted to competition in saturated habitats (MacArthur and Wilson, 1967). Accordingly, r species may be expected to form aggregations in patchy and ephemeral habitats, whereas K species may be expected to occur in relatively low and uniform population densities in less

disturbed habitats. We found that aggregation patterns of freeliving nematodes varied somewhat among habitats, as did the abundance of colonizer vs. persister groups among habitats, where selection for the associated life history traits varies (Ferris et al., 2001; Reznick et al., 2002). Nonetheless, colonizer species were significantly aggregated at large spatial extents in 5 (BF1) or 3 (BF2 and FF2) habitats out of 6, whereas neutral to persisting taxa were significantly aggregated in 0 (CA3), 1 (BF4 and CA5) or 3 (OM4) of 6 habitats. These results are generally consistent with the suggested differences among taxa, in that more r selected functional groups were more likely to show significant spatial aggregation, particularly in the more disturbed managed habitats. The exception was relatively persisting omnivores, which may have sufficient breadth in food sources to respond numerically to concentrations of these resources in any habitat. EPN’s spatial pattern was significantly aggregated only in the Grass border habitat, where it was most abundant (Tables 1 and 2). We also tested patterns of spatial association between EPN and each of the other functional groups, and found, in general, that the associations were with the more rselected bacterial and fungal feeding taxa. No nematode taxa are considered to be r-selected carnivores or omnivores, however, so no such comparisons among colonizer and persister groups were made for these functional groups. The observed aggregations and associations among the rselected taxa in this study, at spatial scales ranging from meters to kilometers raises an important question regarding the life history traits behind the observed patterns. The observed patterns of aggregation are assumed to arise from a combination of dispersal, reproduction, and underlying spatial patterns of habitat, in

Table 2 SADIE indices of aggregation (Ia ) for entomopathogenic nematode and free-living nematode functional guilds in each soil habitat. Guild†

Forest Ia

EPN BF1 BF2 FF2 BF3 CA3 BF4 OM4 CA5

1.057 2.075 1.749 0.941 1.154 0.944 1.961 3.182 1.113

P{Ia = 1}‡ 0.346 0.011 0.055 0.522 0.253 0.510 0.016 0.002 0.357

Meadow Ia 0.697 1.699 1.401 2.109 1.114 1.576 0.947 1.382 1.251

P{Ia = 1}‡ 0.750 0.104 0.144 0.074 0.319 0.080 0.487 0.159 0.157

Grass borders Ia 1.525 2.163 2.742 1.914 1.134 – 1.260 1.859 0.920

P{Ia = 1}‡ 0.016 0.002 0.002 0.002 0.231 – 0.103 0.002 0.571

Lawn Ia 1.026 2.287 2.242 1.843 1.977 1.337 0.880 3.006 1.682

P{Ia = 1}‡ 0.378 0.002 0.002 0.010 0.008 0.155 0.604 0.002 0.021

Row crops Ia 1.586 1.709 1.189 1.309 1.558 – 1.317 1.631 –

P{Ia = 1}‡ 0.058 0.039 0.251 0.165 0.067 – 0.175 0.058 –

Vegetables Ia §

– 3.111 3.430 2.059 – – 1.266 1.432 –

Pooled data

P{Ia = 1}‡

Ia

P{Ia = 1}‡

– 0.0016 0.0016 0.0160 – – 0.2035 0.1538 –

2.524 2.697 2.393 1.739 1.826 1.710 1.731 1.416 0.888

0.008 0.002 0.002 0.021 0.056 0.052 0.052 0.102 0.619

† EPN: Entomopathogenic nematodes; BF: Bacterivore; CA: Carnivore; FF: Fungivore; OM: Omnivore; numbers 1–5 representing points on a colonizer–persister scale with 1 being more r-selected colonizer types and 5 being more K-selected persisting types according to Bongers (1990). ‡ The probability that the observed data are no more aggregated than expected from a random permutation of the nematode density, for which Ia ≈ 1. § Not enough data points to run a meaningful SADIE analysis (less than 5).

† EPNs: Entomopathogenic nematodes; BF: Bacterivore; CA: Carnivore; FF: Fungivore; OM; Omnivore; numbers 1–5 representing points on a colonizer–persister scale with 1 being more r-selected colonizer types and 5 being more K-selected persisting types according to Bongers (1990). ‡ Standard Error. § The probability that two populations have neither positive nor negative spatial association, in which case X ≈ 0. # SADIE association index was not feasible because EPNs or other functional guild were not present in any of the samples.

<0.001 <0.001 0.076 0.795 0.982 >0.999 0.471 BF2 FF2 BF3 CA3 BF4 OM4 CA5

0.311 (0.087) –0.277 (0.084) –0.139 (0.035) –0.447 (0.098) 0.304 (0.088) 0.252 (0.085) –0.617 (0.114)

0.013 0.983 0.529 0.999 0.016 0.021 >0.999

0.393 (0.096) –0.340 (0.145) 0.328 (0.102) 0.401 (0.127) 0.451 (0.091) –0.310 (0.080) 0.318 (0.088)

<0.001 0.932 0.015 0.024 <0.001 0.995 0.009

0.387 (0.088) 0.344 (0.091) 0.210 (0.079) – 0.102 (0.052) 0.052 (0.027) –0.174 (0.091)

<0.001 0.004 0.068 – 0.112 0.442 0.883

0.328 (0.078) –0.205 (0.098) 0.527 (0.172) –0.008 (0.006) –0.285 (0.073) –0.247 (0.091) –0.660 (0.131)

0.002 0.908 <0.001 0.492 0.997 0.968 >0.999

0.119 (0.079) –0.107 (0.250) –0.389 (0.313) – –0.170 (0.222) 0.323 (0.168) 0.779 (0.580)

0.178 0.576 0.707 – 0.654 0.116 0.216

– – – – – –

– – – – – – –

0.472 (0.081) 0.423 (0.074) 0.128 (0.058) –0.158 (0.117) –0.212 (0.070) –0.322 (0.075) 0.006 (0.058)

P{X = 0}

#

Vegetables

P{X = 0} P{X = 0} P{X = 0}

Lawn X (SE) P {X = 0}

Grass borders

X (SE) P{X = 0} Meadow X (SE) P{X = 0}§ Forest X (SE‡ ) Comparison between EPNs and free-living nematode guild†

Table 3 SADIE association indices (X) between entomopathogenic nematode (EPNs) and other free-living nematode functional guilds in each soil habitat.

X (SE)

Row crops

X (SE)

X (SE)

Pooled data

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particular food sources. Dispersal of nematodes at these scales is likely to be passive, particularly via insect phoresy or in surface water during flooding or irrigation, rather than active dispersal through the soil. Movement in surface water along particular drainage patterns could channel long distance movement of each of these groups, although the same pattern of passive movement might be expected for any free-living nematode species. Wind and human activity are additional potential modes of long distance movement for EPN’s that appear to be sufficient to result in large dispersal distances and may account, along with phoresy and water, for their widespread distribution (Koppenhöfer and Kaya, 1996; Stuart and Gaugler, 1994). Spatial pattern of EPNs may have more to do with arthropod associations than the other forms of passive dispersal, however, because EPNs became obligate parasites of insects (Sudhaus, 2008). A propensity for EPN’s, bacterivores and fungivores toward phoretic movement on the same or similar insect species, and the tendency of these insect species to aggregate or disperse according to similar patterns at landscape scales where their densities are abundant, could result in both aggregations within groups and association among them. Regardless of similarities in passive dispersal patterns, however, rapid reproduction in patches of suitable habitat with available prey could also result in observable patterns of aggregation. EPN’s essentially create their own dense patches of bacteria to feed upon within patchily distributed insect hosts. Bacterial feeding nematode’s food resources also may be aggregated spatially in ephemeral patches of dense bacterial populations (Noe and Campbell, 1985; Ferris et al., 1990). Nunan et al. (2002) showed that the spatial aggregation of free-living soil bacteria varied based on the depth of the soil. They showed a strong spatial aggregation pattern of bacterial populations in topsoil (∼25 cm depth) that decreased with subsoil depth (Nunan et al., 2002). To the extent that such aggregations also are associated with insect hosts of EPN’s, or fungi in the case of fungivorous and r-selected nematodes, nematode reproduction on spatially aggregated prey populations could also result in the observed patterns of aggregation at landscape scales. More thorough examination of free-living nematode and arthropod communities, and other habitat characteristics, are needed to examine the relationships between dispersal, reproduction and spatial patterns within and among nematode taxa Recently, Campos-Herrera et al. (2012) found similar spatial association between EPNs and bacterial feeders using molecular identification methods. They showed positive correlations between Acrobeloides-group nematodes, a cp 2 bacterial feeding group (Bongers and Bongers, 1998), and EPNs, as well as a complex of nematophagous fungi that are natural enemies of EPN’s, in soil samples. In their study, however, SADIE association indices failed to show significant relationships between EPNs and Acrobeloidesgroup, the only other free living nematode guild detected by the molecular methods used. However, Pearson correlation coefficients among qPCR estimated abundances of these guilds were significantly different from zero. Our results are consistent with the correlations demonstrated by Campos-Herrera et al. and provide more detailed evidence based on visual identification of free living nematodes in soil samples that spatial pattern and association are closely related to their life history strategies. Hoy et al. (2008) found that EPNs were most similar to r-selected bacterial feeders in their response to a wide range of soil physical and biological conditions, although the formal tests of spatial association in this paper provide a more direct test of these associations. Furthermore, such patterns of aggregation and association were detected at large spatial extents (m–km) compared with those that have been examined in previous

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research. Therefore, our results improve understanding of ecological characteristics of EPNs and free-living nematodes in soil ecosystems. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A1A2007061) to J.-J. Park, Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0027429) to K. Cho, the USDA’s National Research Initiative Competitive Grants Program (Grant No. 2004-35302-15004) to C.W Hoy and P.S. Grewal, and state and federal funds appropriated to the Ohio Agricultural Research and Development Center. We thank Nuris Acosta and Mike Dunlap for their technical assistance with sample collection and processing. References Bongers, T., 1990. The maturity index, an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Bongers, T., Bongers, M., 1998. Functional diversity of nematodes. Appl. Soil Ecol. 10, 239–251. Campbell, J.F., Lewis, E., Yoder, F., Gaugler, R., 1996. Entomopathogenic nematode (Heterorhabditidae and Steinernematidae) spatial distribution in turfgrass. Parasitology 113, 473–482. Campbell, J.F., Orza, G., Yoder, F., Lewis, E., Gaugler, R., 1998. Entomopathogenic nematode (Heterorhabditidae and Steinernematidae) spatial distribution in turfgrass. Entomol. Exp. Appl. 86, 1–11. Campos-Herrera, R., El-Borai, F.E., Duncan, L.W., 2012. Wide interguild relationships among entomopathogenic and free-living nematodes in soil as measures by real time qPCR. J. Invertebr. Pathol. 111, 126–135. Cressie, N., 1993. Statistics for Spatial Data. Wiley, New York, NY. Ettema, C.H., 1998. Soil nematode diversity, species coexistence and ecosystem function. J. Nematol. 30, 159–169. Ettema, C.H., Bongers, T., 1993. Characterization of nematode colonization and succession in disturbed soil using the maturity index. Biol. Fertil. Soils 16, 79–85. Ettema, C.H., Coleman, D.C., Vellidis, G., Lowrance, R., Rathbun, S.L., 1998. Spatiotemporal distributions of bacterivorous nematodes and soil resources in a restored riparian wetland. Ecology 79, 2721–2734. Fan, X., Hominick, W.M., 1991. Efficiency of the Galleria (wax moth) baiting technique for recovering infective stages of entomopathogenic rhabditids (Steinernematidae and Heterorhabditidae) from sand and soil. Rev. Nématol. 14, 381–387. Ferris, H., Eyre, M., Venette, R.C., Lau, S.S., 1996. Population energetics of bacterialfeeding nematodes: stage-specific development and fecundity rates. Soil. Biol. Biochem. 28, 217–280. Ferris, H., Bongers, T., de Goede, R.G.M., 1999. Nematode faunal indicators of soil food web condition. J. Nematol. 31, 534–535. Ferris, H., Bongers, T., de Goede, R.G.M., 2001. A framework for soil food web diagnostics, extension of the nematode faunal analysis concept. Appl. Soil Ecol. 18, 13–29. Ferris, H., Mullens, T.A., Foord, K.E., 1990. Stability and characteristics of spatial description parameters for nematode populations. J. Nematol. 22, 427–439. Flegg, J.M., Hooper, D.J., 1970. Extraction of free-living stages from soil. In: Southey, J.F. (Ed.), Laboratory Methods for Work with Plant and Soil Nematodes. Her Majesty’s Stationery Office, London, pp. 5–22. Freckman, D.W., 1988. Bacterivorous nematodes and organic-matter decomposition. Agric. Ecosyst. Environ. 24, 195–217. Grewal, P.S., Ehlers, R.-U., Shapiro-Ilan, D.I. (Eds.), 2005. Nematodes as Biocontrol Agents. CABI Publishing, CAB, International, Oxon, UK. Hoy, C.W., Grewal, P.S., Lawrence, J.L., Jagdale, G., Acosta, N., 2008. Canonical correspondence analysis demonstrates unique soil conditions for entomopathogenic nematode species compared with other free living nematode species. Biol. Control 46, 371–379. Kaya, H.K., Stock, S.P., 1997. Techniques in insect nematology. In: Lacey, L.A. (Ed.), Biological Techniques Manual of Techniques in Insect Pathology. Academic Press, New York, NY, pp. 281–324. Kokko, H., Lundberg, P., 2001. Dispersal, migration, and offspring retention in saturated habitats. Am. Nat. 157, 188–202.

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