Received 19 August 1999; received in revised form 8 December 1999; accepted 20 December 1999

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1 Soil Biology & Biochemistry 32 (2000) 1053± Earthworm d 13 C and d 15 N analyses suggest that putative functional classi cations of earthworms are site-speci c and may also indicate habitat diversity Roy Neilson a, *, Brian Boag a, Michael Smith b a Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK b 145 Gloucester Road, Exwick, Exeter, EX4 2EB, UK Received 19 August 1999; received in revised form 8 December 1999; accepted 20 December 1999 Abstract Natural abundances of the stable isotope pairs 13 C/ 12 C and 15 N/ 14 N d 13 C and d 15 N were measured from earthworms sampled from six sites with contrasting habitats (deciduous and coniferous woodland, arable and permanent pasture). Knowledge about the function of earthworms is important to the understanding of their ecology. The hypothesis, that endogeic (primarily soil and organic matter feeders) and epigeic (surface litter feeders, ingesting little or no soil) earthworms would be isotopically distinct and that isotopic values for anecic (surface litter and soil feeders) earthworms would fall between the other two groups based on their feeding strategies, was rejected. Earthworm d 13 C and d 15 N values from six sites indicated that classifying earthworms into the functional groups epigeic, anecic and endogeic is site-dependent. In contrast, d values clearly separated earthworms into humic formers and humic feeders. Average 13 C-enrichment (3.9-) between earthworm and putative dietary source (vegetation) across all sites was larger than the typically reported enrichment (1-) between a single trophic level suggesting that earthworms, as expected, derive nutrition from a number of sources, not just living vegetation. Enrichments of 13 C and 15 N in earthworms, relative to diet, could be developed as a tool for assessing habitat diversity Elsevier Science Ltd. All rights reserved. Keywords: Anecic; Earthworms; Endogeic; Epigeic; Functional groupings; Stable isotopes 1. Introduction * Corresponding author. Tel.: ; fax: address: roy.neilson@scri.sari.ac.uk (R. Neilson). Several classi cation schemes have been used to separate earthworm communities into functional groupings (Perel, 1977; Lavelle, 1979). The most enduring classi cation (BoucheÂ, 1971, 1977) is the separation of earthworms into the following functional groups: (i) epigeic: litter dwellers which feed on decomposing litter with little or no soil ingested; (ii) endogeic: live within the mineral soil horizons and are geophagous, feeding primarily on mineral soil and associated organic matter; and (iii) anecic: form permanent or semi-permanent vertical burrows in soil and feed on surface litter, primarily dead and decaying material (Blair et al., 1995; Edwards and Bohlen, 1996; Fraser and Boag, 1998). Alternatively, Perel (1977) separated earthworms into two broad groups: humic formers, comprising both epigeic and anecic species that are essentially plant feeders; and humic feeders, comprising endogeic species that feed on soil and organic matter. The assignation of earthworms into functional groups is problematical. Conventionally, classi cations are based on techniques that assess only ingested dietary materials, e.g. gut content analyses (Piearce, 1978). However, these provide insights only into short-term rather than long-term dietary preferences. In both plants and invertebrates, natural abundances /00/$ - see front matter Elsevier Science Ltd. All rights reserved. PII: S (00)

2 1054 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061 of stable isotopes are e ectively an integrated record of assimilated elements such as carbon (C) and nitrogen (N) (Tieszen et al., 1983; Peterson and Fry, 1987; Hobson and Welch, 1995). Thus, in contrast to analysis of gut contents, stable isotope analysis does not provide a snapshot indication of trophic interactions, but more a representation of the biochemical events and dietary sources of the recent past. Natural abundances of 13 C/ 12 C d 13 C and 15 N/ 14 N d 15 N in animal tissues can be used to directly trace food sources and rank animals into their relative trophic levels, respectively (DeNiro and Epstein, 1978, 1981; Wada et al., 1993). Enrichments of 13 C and 15 N between consumer and diet of c. 1- and 3.4- (for 15 N this can vary from 0±6-), respectively, are typical (DeNiro and Epstein, 1978; Fry et al., 1978a, b; Minagawa and Wada, 1984; Hobson et al., 1993; Wada et al., 1993; Scrimgeour et al., 1995). An organism that feeds on another higher up a food chain will therefore be isotopically more 13 C- and 15 N-enriched than an organism that feeds on another near the base of a food chain. Previous studies, mainly from tropical habitats, have reported that earthworms are 13 C- enriched relative to putative dietary source by more than the expected 1- (Spain et al., 1990; Martin et al., 1992a, 1992b; Spain and Le Feuvre, 1997; Schmidt et al., 1997; Neilson et al., 1998). Whilst ingesting soil, endogeic earthworms also ingest soil-borne micro- and meso-fauna, including protozoa (Miles, 1963; Bonkowski and Schaefer, 1997), bacterivorous nematodes (Yeates, 1981) and fungi (Edwards and Fletcher, 1988). Prior to consumption by endogeic earthworms, mesofauna such as fungivorous nematodes, in theory, become 13 C- and 15 N- enriched relative to fungi which, in turn, become 13 C- and 15 N-enriched relative to detritus from which fungi derive nutrition. Similarly, nematophagous amoebae (Yeates and Foissner, 1995) are likely to be 13 C- and 15 N-enriched relative to their nematode prey. Compared with epigeic earthworms, endogeic earthworms potentially consume more 13 C- and 15 N-enriched dietary material, given that epigeic species feed predominately on plant litter. Additionally, the whole soil becomes 15 N-enriched with increasing depth in forest soil pro les (0±45 cm) by 2.0±8.5- (Shearer et al., 1978; Nadelho er and Fry, 1988; Melillo et al., 1989; Piccolo et al., 1996; Koba et al., 1998). Similarly, Kerley and Jarvis (1997) found that the whole soil became 15 N-enriched by c. 6- and humic material by c. 7- with increasing depth (0±30 cm) under undisturbed grassland. Spain and Le Feuvre (1997) noted that whole soil was 15 N- enriched by c. 2- in the top 35 cm under sugarcane. Thus, burrowing endogeic species are exposed to a potential source of 15 N-enrichment via soil ingestion that is not available to the surface-dwelling epigeic species. It should therefore be possible to separate endogeic and epigeic groups isotopically. However, anecic earthworm species are unlikely to be isotopically di erent to either endogeic and epigeic species as they consume material available to both epigeic (surface litter) and endogeic (soil) earthworms. Utilising d 15 N data, Schmidt et al. (1997) separated earthworms from a single site into three functional groups, epigeic, endogeic and anecic as de ned by Bouche (1971, 1977). Similarly, Briones et al. (1999) reported that earthworm d 15 N was related to their ecological groupings with endogeic species being more 15 N-enriched than epigeic and epi/anecic species. However, Martin et al. (1992b) using d 13 C data, separated both European and tropical earthworm species into only two distinct groups, litter feeders (epigeic and anecic) and soil feeders (endogeic). The aims of this study were two-fold: (i) to determine whether the same 15 N- and 13 C-enrichment of earthworms relative to vegetation cover and soil exist in di erent habitats; and (ii) to test the hypothesis that endogeic and epigeic earthworms are isotopically distinct based on their feeding strategies, and that anecics were isotopically between both epigeic and endogeics. 2. Materials and methods 2.1. Experimental sites and sampling Six sites, with contrasting vegetation types (Table 1), were sampled during the early autumn of Earthworms were extracted by hand sorting (Boag et al., 1997). At each site, ve randomly selected areas cm were dug to a depth of 30 cm and earthworms in this volume of soil removed by hand. In the eld, earthworms were stored in 100 ml at-bottomed glass honey jars (Steele and Brodie, Wormit, Scotland). The jars were embedded in crushed ice within a cool box to reduce earthworm activity. This limited mucus excretion which may be isotopically species-speci c (Neilson, 1999) and a potential source of isotopic contamination between species. In the laboratory, earthworms were washed in distilled water and identi ed to species where possible, using the taxonomic key of Sims and Gerard (1985). Earthworm species were assigned to the di erent ecological groupings based on that described by Fraser and Boag (1998, their Table 2). Thereafter, they were placed in 3 1 cm glass specimen tubes and stored at 208C prior to processing for isotopic analyses. Adjacent to each sampling location, a representative 500 g soil sample comprising ve smaller 100 g samples was taken from the top 10 cm of soil by a hand-trowel. Soil was bagged and stored at 48C until processed for isotopic analysis. Similarly, at each

3 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053± Table 1 Locations and habitat information of sampled sites Site UK national grid reference Latitude Longitude Altitude (m above sea level) Long-term average annual (1951±1980) rainfall (mm) Habitat A NO 'N 3804'W Arable B NN 'N 3839'W Arable C NH 'N 3852'W Coniferous woodland D NO 'N 2850'W Coniferous woodland E NO 'N 3809'W Deciduous woodland F NN 'N 3840'W Permanent pasture sampling location, fallen leaf samples of the dominant (determined visually) vegetation were also collected. The woodland sites (sites C±E), were open and did not have a readily identi able accumulated litter layer that could be separated from the soil Isotope analyses Earthworm, soil and plant samples were analysed as described in Neilson et al. (1998). Isotope natural abundances are reported as: Table 2 Mean d 15 N (-) and d 13 C (-) values of individual earthworm species and a weighted mean for each site (study 2) Site Earthworm species (ecological grouping a ) n d 15 N SE d 13 C SE A Allolobophora chlorotica (End) Aporrectodea caliginosa (End) Lumbricus castaneus (Epi) L. rubellus (Epi) L. terrestris (Ane) Weighed mean B A. chlorotica (End) A. rosea (End) L. terrestris (Ane) Octolasion cyaneum (End) Weighed mean C Aporrectodea longa (Ane) ± 24.4 ± A. rosea (End) L. castaneus (Epi) Weighed mean D A. caliginosa (End) A. chlorotica (End) ± 25.3 ± A. longa (Ane) A. rosea (End) L. castaneus (Epi) ± 25.5 ± L. rubellus (Epi) L. terrestris (Ane) Weighted mean E A. caliginosa (End) ± 23.9 ± Aporrectodea rosea (End) Dendrodrilus rubidus (Epi) L. castaneus (Epi) L. rubellus (Epi) ± 25.7 ± L. terrestris (Ane) Weighted mean F A. caliginosa (End) A. rosea (End) L. rubellus (Epi) L. terrestris (Ane) Weighted mean a Codes for ecological groupings: Ane, Anecic; End, Endogeic; Epi, Epigeic.

4 1056 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061 d sample ˆ Rsample R standard R standard where R sample and R standard are the heavy/light isotope ratios of sample and standard. Analytical precision was R0.2- for d 13 C and R0.4- d 15 N: 2.3. Data analyses A one-way analysis of variance (ANOVA) using Minitab (Minitab, Pennsylvania, USA) was done to separate earthworm ecological groups based on whole body tissue d values. Mean earthworm d 15 N and d 13 C were calculated for each site weighted by earthworm abundance. Since no percentage vegetation cover data were available for the sampling sites, simple arithmetic mean d 15 N and d 13 C values were calculated based on the relevant isotopic measurement. 3. Results Nine earthworm species known to have widespread distributions in Scotland (Boag et al., 1997) were extracted from the six study sites (Table 2). Five of the nine species, Aporrectodea caliginosa, A. rosea, Lumbri- Fig. 1. Mean d 15 N and d 13 C of weighted average Earthworm (closed triangle), Whole Soil (closed squares) and Vegetation (closed diamonds). A: Site A, Arable; B: Site B, Arable; C: Site C, Coniferous woodland; D: Site D, Coniferous woodland; E: Site E, Deciduous woodland; F: Site F, Permanent pasture. In some instances error bars are smaller than the symbols. Soil was not available for isotopic analysis at Site A.

5 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053± cus castaneus, L. rubellus and L. terrestris occurred in at least four of the six sites. Site C was excluded from the statistical analyses comparing Bouche 's (1971, 1977) ecological groupings because only a single individual anecic earthworm (A. longa ) was found (Tables 2 and 5). Similarly, Site B was also excluded from analyses as no epigeic species were extracted (Table 4) d 15 N Values for earthworm d 15 N at species level varied considerably across sites (Table 2). For example, average L. terrestris d 15 N ranged from (site E) to (site B). This was re ected in the weighed mean earthworm d 15 N that ranged from (site E) to (site B) (Table 2). An average 15 N enrichment of 4.6- across all sites (range +2.8±5.9-) (Fig. 1) was recorded between the calculated weighted average earthworm d 15 N and the average d 15 N of the sampled vegetation (Tables 2 and 3). In four of the ve sites for which whole soil d 15 N values were available, the weighed mean earthworm d 15 N also exhibited a 15 N enrichment relative to whole soil d 15 N, with the averaged stepwise increase ranging from 2.8- to 6.3- depending upon the site (Fig. 1). The one exception was site E (deciduous woodland), where there was no signi cant di erence between the weighed average earthworm d 15 N and whole soil d 15 N (Fig. 1). When comparing ecological groupings (BoucheÂ, 1971, 1977), d 15 N values varied between the sites (Table 4). Epigeic earthworm species were signi cantly less 15 N-enriched relative to endogeic species from all the sites except F Table 3 Mean d 15 N (-) and d 13 C (-) values of the dominant above-ground vegetation types at each site Site Vegetation and soil d 15 N SE d 13 C SE A Wheat Grasses Soil N/d a N/d N/d N/d B Grasses Various dicots Soil C Blaeberry leaves Birch leaves Juniper leaves Soil D Grasses Soil E Fern Woodrush Soil F Grasses Soil a N/d = not determined. (Table 5). All the three ecological groupings were signi cantly di erent from each other at site D, with endogeic earthworms being more 15 N-enriched than anecics which in turn were more 15 N-enriched than epigeics (Table 5). In contrast, no di erences were found between anecics and either epigeics or endogeics at sites A, E and F (Table 5). d 15 N for humic feeders and humic formers (Perel, 1977) also varied between the sites (Table 4). At all the six sites, humic feeders were always signi cantly more 15 N-enriched than humic formers (Table 5). Di erences in d 15 N values between humic feeders and humic formers were greatest (>3.5-) at sites C and D (both coniferous woodlands) d 13 C Earthworm d 13 C values at species level were less variable across the sites than d 15 N (Table 2). This pattern was re ected in the calculated weighed mean earthworm d 13 C value that di ered by at the most 2.0- between any two sites (sites E and F). A mean 13 C enrichment across all sites of 3.9- (range 2.7± 5.0-) was recorded between the calculated weighed mean earthworm d 13 C and the mean d 13 C of the sampled vegetation (Tables 2 and 3). Similarly, at all sites, the weighed average earthworm d 13 C exhibited a 13 C enrichment relative to whole soil d 13 C, with a site average stepwise increase ranging from 0.8- to As with d 15 N, d 13 C of the di erent ecological groupings varied by site (Table 4). With one exception, both epigeic and anecic species were signi cantly more 13 C- depleted (c. 1-) compared with endogeic species (Table 5), but were not signi cantly di erent from each other. At all the sites, humic formers were signi cantly more 13 C-depleted compared with humic feeders (Table 5), with the greatest depletion occurring in both coniferous woodland sites (C and D). 4. Discussion 4.1. Isotopic enrichment The mean 15 N enrichment of 4.6- between the weighed average earthworm d 15 N and the average d 15 N of the sampled vegetation, across all sites in this study, is within the previously reported range of 0-±6- for a single trophic level (Minagawa and Wada, 1984; Wada et al., 1993; Scrimgeour et al., 1995). At four of the ve sites in this study with available soil d 15 N data, earthworms were 15 N-enriched relative to soil by >2.5-, greater than the <1- gure reported by Neilson et al. (1998) from a grazed and ungrazed upland pasture. Data from this study suggest that earthworms from

6 1058 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061 Table 4 Mean d 15 N (-)(A) and d 13 C (-)(B) of the di erent ecological earthworm groupings (BoucheÂ, 1971; 1977) at each site a Site Epigeic Endogeic Anecic Humic Former Humic Feeder d 15 N SE d 15 N SE d 15 N SE d 15 N se d 15 N SE (A) d 15 N A B N/a b N/a C ± D E F Site Epigeic Endogeic Anecic Humic Former Humic Feeder d 13 C SE d 13 C SE d 13 C SE d 13 C SE d 13 C SE (B) d 13 C (-) A B N/a N/a C ± D E F a Levels of signi cance between the di erent ecological classi cations are presented in Table 5. b N/a = no epigeic earthworm species present. di erent habitats could be ranked in terms of increasing 15 N-enrichment relative to vegetation as follows: deciduous woodland (least 15 N enrichment) < coniferous woodland < grazed pasture < ungrazed pasture < arable (most 15 N-enrichment) which is similar to that reported by Wishart et al. (1997). The average 13 C-enrichment across all the sites of 3.9- between the weighed average earthworm d 13 C and the average d 13 C of the sampled vegetation is substantially greater than the typically reported enrichment of c. 1- between putative trophic levels (e.g., DeNiro and Epstein, 1978; Fry et al., 1978a, 1978b; Wada et al., 1993). However, the 13 C-enrichment is within the previously reported range of 13 C-enrichment (1.0±4.3-) of earthworm tissue relative to dietary sources (Spain et al., 1990; Martin et al., 1992a, 1992b; Schmidt et al., 1997; Spain and Le Feuvre, 1997; Neilson et al., 1998). A general pattern of earthworm 15 N- and 13 C-enrichment relative to putative dietary vegetation appears to be repeatable across a range of habitats from a number of distinct geographic locations. Such enrichment is not a consequence of a large proportion of endogeic species which, as previously noted, could reasonably be expected to be 13 C- enriched relative to other earthworm species. This relatively large 13 C-enrichment relative to putative dietary source could be due to selective feeding, selective assimilation or isotopic fractionation during respiration. Spain and Le Feuvre (1997) postulated that 13 C- enrichments of earthworm tissue >1- relative to dietary sources may result from a stepwise 13 C-enrichment along a microbial food web. Alternatively, it may Table 5 Levels of signi cance between di erent ecological classi cations (BoucheÂ, 1971; 1977; Perel, 1977) for d 15 N (A) and d 13 C (B) Site Epigeic vs. Endogeic Epigeic vs. Anecic Endogeic vs. Anecic Humic former vs. Humic feeder (A) d 15 N - A ns ns D R R R E R ns ns F ns ns a ns (B) d 13 C (-) A ns R D R ns R R E ns ns F ns R a ns = not signi cant at p > 0.05.

7 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053± re ect the ingestion of 13 C-enriched micro- and mesofauna from a variety of trophic groups and the subsequent assimilation of available 13 C by earthworms. These alternatives cannot be distinguished without relevant information about dietary preferences and/or nutritional metabolism. Curry (1994, p. 138) noted that `complex' habitats, with a greater plant diversity, had a wider range of resources (potential food) and supported more diverse soil invertebrate communities. More available dietary sources are likely to be re ected in a wider range of d 13 C throughout the soil ecosystem, i.e. from producers to top predators. Similarly, more trophic levels are likely to occur with increased invertebrate biodiversity and this is likely to be manifested in a wider range of d 15 N (Cabana and Rasmussen, 1994). On this basis, d 15 N data from this study suggests that the coniferous woodland and ungrazed pasture sites (range 5.7±6.3-) have at least one additional trophic level than either the deciduous or arable sites (range 3.4±4.4-), suggesting di ering habitat complexity. In contrast to this study, Wishart et al. (1997) reported that woodlands (coniferous and deciduous) were more `complex' than pastures, although their sampled habitats were within 200 m of each other and not from distinct geographical locations as in this study. Applying an average 15 N-enrichment of 3.4- (Wada et al., 1993; Minagawa and Wada, 1984) to mean foliar d 15 N data listed in Table 1 of Handley et al. (1999), a similar pattern of earthworm 15 N-enrichment in woodlands can be deduced, i.e. woodland < pasture. However, analysing the globally-derived data from Handley et al. (1999) in detail indicates that the 15 N-enrichment in deciduous woodlands is greater than that for coniferous woodlands which is contrary to that found here. This suggests that within global ecological generalisations, contradictory patterns may occur locally Ecological classi cations Endogeic earthworm species at sites C and D were between 3.2 and 5.3- more 15 N-enriched relative to epigeic/anecic Lumbricus spp. Data from these coniferous woodland sites appear to support Schmidt et al. (1997) who, using data gathered from a single site, suggested that endogeic earthworm species were separated from Lumbricus spp. by a single trophic level. In contrast, at the other four sites, endogeic species were generally <2.0- more 15 N-enriched than Lumbricus spp. This may be, as previously noted, because coniferous woodlands support a more diverse soil invertebrate community. The hypothesis that endogeic and epigeic earthworms are isotopically d 15 N and d 13 C distinct based on their known feeding strategies, and that anecics would isotopically fall between the other two groups, was con rmed at only two sites for d 15 N (A and E) and a single site for d 13 C (E). In contrast, Schmidt et al. (1997) found that signi cant di erences existed between each ecological grouping based on earthworm d 15 N, d 15 N decreasing in the order endogeic > anecic > epigeic/anecic Lumbricus spp. In this study, only data from site D agreed with those ndings. Briones et al. (1999) reported that earthworm 15 N was not related to cropping treatment (maize versus permanent pasture) but was related to ecological grouping with endogeic species being more 15 N-enriched than epigeic and epi/anecic species. In terms of earthworm d values, it appears that Bouche 's (1971, 1977) ecological classi cations are site-dependent. Reasons for the inconsistencies between our study and that of Schmidt et al. (1997) are not obvious. Generally, these putative site-dependent patterns may support the hypothesis that earthworms were `ecosystemivorous' (Pokarzhevskii et al., 1997), i.e. when earthworms consume soil and micro- and mesofauna, they are in e ect ingesting micro-ecosystems. Therefore, earthworm d values could merely re ect those of their habitat. Alternatively, a soil with a greater faunal biodiversity is likely to comprise more trophic levels. Earthworms ingest a variety of materials when consuming soil and organic matter (Edwards and Bohlen, 1996; Edwards, 1998) and material derived from more trophic levels may produce a greater di erence between earthworm d 15 N and d 13 C and their putative diet (foliage and soil). The isotopic values measured here may indicate habitat biodiversity, earthworm feeding strategy or both. At a ner scale, gut analyses have shown that earthworms can have both, species-speci c diets and diets that pertain to the ecological group to which they have been assigned (Bernier, 1998). Bernier (1998) reported that L. terrestris had feeding attributes similar to both L. castaneus (epigeic) and Aporrectodea icterica (endogeic), whereas, Je gou et al. (1999) noted that the feeding habits of L. terrestris were similar to the epigeic species Eisenia andrei. Bouche (1971) considered L. terrestris to be epigeic/anecic, i.e., mainly anecic when litter quantity decreased during winter through summer but epigeic when litter was abundant in autumn. Since sampling in this study was done in early autumn prior to litter accumulation and that stable isotope analyses is a record of biochemical events/dietary sources of the recent past, L. terrestris was assumed to be exhibiting anecic behaviour at the time of sampling. However, it is not possible to discount that the similarity in d 15 N between epigeic species and anecic species, comprising mainly of L. terrestris, is due to epi/anecic behaviour. Additionally, rates of organic matter assimilation depend on the quality of ingested food (Lavelle et al., 1997) and some endogeic species can digest speci c fractions of soil organic matter (Fragoso et al., 1997).

8 1060 R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061 These factors could in uence the isotopic values of individual earthworms and species which, in turn, would be re ected in the isotopic values of the di erent ecological groupings. d 15 N and d 13 C values separated earthworms into the ecological groupings suggested by Perel (1977). Essentially, humic formers represent those earthworms that feed on plant litter, whereas humic feeders predominately consume 15 N enriched soil (Shearer et al., 1978; Nadelho er and Fry, 1988; Melillo et al., 1989; Piccolo et al., 1996; Kerley and Jarvis, 1997; Spain and Le Feuvre, 1997; Koba et al., 1998) and decomposed organic matter. Before feeding by humic feeders, soil organic matter may undergo isotopic fractionation, preferentially removing the 14 N fraction, leaving the residual organic matter 15 N-enriched. This is analogous to that reported during sedimentation and microbial transformation of organic N in well-mixed marine environments (SchaÈ fer et al., 1998) Conclusions Earthworm d 13 C and d 15 N data presented here from a variety of habitats suggest that earthworm ecological groupings are site speci c and generally it is not possible to separate the di erent ecological groupings isotopically. This supports the conclusion of Edwards and Bohlen (1996) that it is di cult to classify earthworms ecologically in ways that are relevant globally. Blair et al. (1995) questioned the validity of Bouche's ecological classi cation and called for a rede nition. Although it is possible to isotopically distinguish earthworm species from di erent ecological groups, it is unclear whether isotopic natural abundance values would separate species from within the same ecological grouping. Consequently, this questions the validity of the potential of using stable isotope natural abundances in taxonomic studies as suggested by Briones et al. (1999). Further research is required to determine whether earthworm 13 C- and 15 N-enrichment relative to putative dietary source is indicative of habitat complexity. Acknowledgements We are grateful to W. Stein for technical assistance and to D. Robinson and L. Handley for constructive comments on the manuscript. The Scottish Crop Research Institute is grant-aided by the Scottish Executive Rural A airs Department. References Bernier, N., Earthworm feeding activity and development of the humus pro le. Biology and Fertility of Soils 26, 215±223. Blair, J.M., Parmelee, R.W., Lavelle, P., In uences of earthworms on biogeochemistry. In: Hendrix, P.F. (Ed.), Earthworm Ecology and Biogeography in North America. CRC Press, Boca Raton, USA, pp. 127±158. 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