Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue8-9.Aug2000:

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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 13C/12C and 15N/14N d13C and d15_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, thatendogeic

(primarily soil and organic matter feeders) andepigeic (surface litter feeders, ingesting little or no soil) earthworms would be isotopically distinct and that isotopic values foranecic(surface litter and soil feeders) earthworms would fall between the other two groups based on their feeding strategies, was rejected. Earthworm d13_C _and _d15_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. Average13C-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

Keywords:Anecic; Earthworms; Endogeic; Epigeic; Functional groupings; Stable isotopes

1. Introduction

Several classi®cation schemes have been used to sep-arate earthworm communities into functional group-ings (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

mat-ter; and (iii) anecic: form permanent or

semi-perma-nent 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

earth-worms into two broad groups: humic formers,

compris-ing both epigeic and anecic species that are essentially

plant feeders; and humic feeders, comprising endogeic

species that feed on soil and organic matter. The assig-nation of earthworms into functional groups is proble-matical. 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

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* Corresponding author. Tel.: 562-731; fax: +44-1382-562-426.

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of stable isotopes are eectively an integrated record of assimilated elements such as carbon (C) and nitro-gen (N) (Tieszen et al., 1983; Peterson and Fry, 1987; Hobson and Welch, 1995). Thus, in contrast to analy-sis of gut contents, stable isotope analyanaly-sis 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 13C/12C d13_C _and 15

N/14N d15_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 13C and 15N

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; Mina-gawa 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 13C- and 15N-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 consump-tion by endogeic earthworms, mesofauna such as

fun-givorous nematodes, in theory, become 13C- and 15

N-enriched relative to fungi which, in turn, become 13

C-and 15N-enriched relative to detritus from which fungi

derive nutrition. Similarly, nematophagous amoebae

(Yeates and Foissner, 1995) are likely to be 13C- and

15

N-enriched relative to their nematode prey. Com-pared with epigeic earthworms, endogeic earthworms

potentially consume more 13C- and 15N-enriched

diet-ary material, given that epigeic species feed predomi-nately on plant litter.

Additionally, the whole soil becomes 15N-enriched

with increasing depth in forest soil pro®les (0±45 cm)

by 2.0±8.5- (Shearer et al., 1978; Nadelhoer 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 15N-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

po-tential source of15N-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 earth-worm species are unlikely to be isotopically dierent to either endogeic and epigeic species as they consume material available to both epigeic (surface litter) and endogeic (soil) earthworms.

Utilising d15_N _{data, Schmidt et al. (1997) separated}

earthworms from a single site into three functional groups, epigeic, endogeic and anecic as de®ned by BoucheÂ (1971, 1977). Similarly, Briones et al. (1999)

reported that earthwormd15_N_{was related to their}

eco-logical groupings with endogeic species being more

15

N-enriched than epigeic and epi/anecic species.

How-ever, Martin et al. (1992b) using d13C 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

deter-mine whether the same 15N- and 13C-enrichment of

earthworms relative to vegetation cover and soil exist in dierent habitats; and (ii) to test the hypothesis that endogeic and epigeic earthworms are isotopically dis-tinct 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 1995.

Earthworms were extracted by hand sorting (Boag et al., 1997). At each site, ®ve randomly selected areas

3030 cm were dug to a depth of 30 cm and

earth-worms 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 con-tamination between species. In the laboratory, earth-worms 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 dierent 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 atÿ208_{C prior to}

pro-cessing 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 48_{C until}

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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.

2.2. Isotope analyses

Earthworm, soil and plant samples were analysed as described in Neilson et al. (1998). Isotope natural abundances are reported as:

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 347323 56829'N 3804'W 50 680 Arable

B NN 983133 56818'N 3839'W 70 1145 Arable

C NH 857057 57808'N 3852'W 270 628 Coniferous woodland D NO 484237 56824'N 2850'W 5 647 Coniferous woodland E NO 295506 56839'N 3809'W 80 781 Deciduous woodland F NN 971131 56818'N 3840'W 80 1145 Permanent pasture

Table 2

Meand15_N_{(-) and}_d13_C_{(-) values of individual earthworm species and a weighted mean for each site (study 2)}

Site Earthworm species (ecological groupinga) n d15_N _SE _d13_C _SE

A Allolobophora chlorotica(End) 4 +8.2 0.29 ÿ25.6 0.04

Aporrectodea caliginosa(End) 27 +8.5 0.20 ÿ25.1 0.09

Lumbricus castaneus(Epi) 3 +6.5 0.23 ÿ26.8 0.28

L. rubellus(Epi) 4 +7.4 2.00 ÿ25.5 0.45

L. terrestris(Ane) 3 +7.5 0.53 ÿ25.9 0.20

Weighed mean +8.1 ÿ25.4

B A. chlorotica(End) 2 +10.6 0.18 ÿ25.4 0.01

A. rosea(End) 6 +10.3 0.42 ÿ25.4 0.11

Octolasion cyaneum(End) 2 +10.5 0.01 ÿ26.3 0.23

C Aporrectodea longa(Ane) 1 +5.5 ± ÿ24.4 ±

A. rosea(End) 4 +4.6 0.35 ÿ24.8 0.46

L. castaneus(Epi) 2 +1.3 0.78 ÿ26.3 0.06

D A. caliginosa(End) 2 +8.0 0.23 ÿ25.1 0.18

A. chlorotica(End) 1 +8.3 ± ÿ25.3 ±

A. longa(Ane) 2 +4.5 0.95 ÿ26.5 0.60

A. rosea(End) 7 +7.3 0.18 ÿ25.0 0.07

L. castaneus(Epi) 1 +3.0 ± ÿ25.5 ±

L. rubellus(Epi) 3 +3.1 0.30 ÿ26.0 0.9

Weighted mean +5.7 ÿ25.7

E A. caliginosa(End) 1 +4.4 ± ÿ23.9 ±

Aporrectodea rosea(End) 4 +3.3 0.59 ÿ23.9 0.30

Dendrodrilus rubidus(Epi) 3 +2.0 0.38 ÿ24.3 0.18

L. castaneus(Epi) 4 +1.3 0.29 ÿ24.7 0.18

L. rubellus(Epi) 1 +0.7 ± ÿ25.7 ±

F A. caliginosa(End) 8 +5.5 0.25 ÿ25.8 0.15

A. rosea(End) 2 +5.1 1.49 ÿ26.2 0.73

L. rubellus(Epi) 2 +4.5 0.81 ÿ26.7 0.08

a

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d_sample RsampleÿRstandard Rstandard

1000

-where Rsample and Rstandard are the heavy/light isotope

ratios of sample and standard. Analytical precision wasR0.2-ford13_C_and_R_0.4_-_d15_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 tissued _values.

Mean earthworm d15_N _and _d13_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 d15N and d13C

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. Meand15_N_and_d13_C_{of weighted average Earthworm (closed triangle), Whole Soil (closed squares) and Vegetation (closed diamonds). A:}

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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 BoucheÂ'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).

3.1.d15_N

Values for earthworm d15_N _{at species level varied}

considerably across sites (Table 2). For example, aver-age L. terrestris d15N ranged from +0.8- (site E) to

+9.0- (site B). This was re¯ected in the weighed

mean earthworm d15N that ranged from +2.2- (site

E) to +9.7- (site B) (Table 2). An average 15N

enrichment of 4.6-across all sites (range +2.8±5.9-)

(Fig. 1) was recorded between the calculated weighted

average earthworm d15N and the average d15N of the

sampled vegetation (Tables 2 and 3). In four of the

®ve sites for which whole soil d15_N _{values were}

avail-able, the weighed mean earthwormd15_N_{also exhibited}

a 15N enrichment relative to whole soil d15_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 dierence between the weighed average

earthworm d15N and whole soil d15N (Fig. 1). When

comparing ecological groupings (BoucheÂ, 1971, 1977),

d15N values varied between the sites (Table 4). Epigeic

earthworm species were signi®cantly less 15N-enriched

relative to endogeic species from all the sites except F

(Table 5). All the three ecological groupings were sig-ni®cantly dierent from each other at site D, with

endogeic earthworms being more 15N-enriched than

anecics which in turn were more 15N-enriched than

epigeics (Table 5). In contrast, no dierences were found between anecics and either epigeics or endogeics at sites A, E and F (Table 5).

d15_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).

Dierences in d15_N _{values between humic feeders}

and humic formers were greatest (>3.5-) at sites C

and D (both coniferous woodlands).

3.2.d13C

Earthworm d13C values at species level were less

variable across the sites than d15N(Table 2). This pat-tern was re¯ected in the calculated weighed mean

earthworm d13C value that diered 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 d13_C _{and the mean} _d13_C _{of the}

sampled vegetation (Tables 2 and 3). Similarly, at all

sites, the weighed average earthworm d13_C _{exhibited a}

13

C enrichment relative to whole soil d13_C_{, with a site}

average stepwise increase ranging from 0.8-to 4.1-.

As withd15_N_,d13_C_{of the dierent ecological}

group-ings 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 dierent from each other. At all the sites, humic formers were

signi®-cantly more13C-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 15N enrichment of 4.6- between the

weighed average earthworm d15_N_{and the average}_d15_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 soild15Ndata,

earthworms were 15N-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 Table 3

Meand15_N_{(-) and}_d13_C_{(-) values of the dominant above-ground}

vegetation types at each site

Site Vegetation and soil d15_N _SE _d13_C _SE

A Wheat +5.6 0.21 ÿ28.1 0.04 Grasses +5.1 0.35 ÿ28.2 0.05

Soil N/da _N/d _N/d _N/d

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dierent habitats could be ranked in terms of

increas-ing 15N-enrichment relative to vegetation as follows:

deciduous woodland (least 15N enrichment) <

conifer-ous woodland < grazed pasture < ungrazed pasture <

arable (most 15N-enrichment) which is similar to that

reported by Wishart et al. (1997).

The average 13C-enrichment across all the sites of

3.9- between the weighed average earthworm d13_C

and the average d13_C _{of the sampled vegetation is}

sub-stantially greater than the typically reported

enrich-ment of c. 1- between putative trophic levels (e.g.,

DeNiro and Epstein, 1978; Fry et al., 1978a, 1978b;

Wada et al., 1993). However, the 13C-enrichment is

within the previously reported range of13C-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;

Neil-son et al., 1998). A general pattern of earthworm15

N-and 13C-enrichment relative to putative dietary

veg-etation appears to be repeatable across a range of habitats from a number of distinct geographic lo-cations. Such enrichment is not a consequence of a large proportion of endogeic species which, as

pre-viously noted, could reasonably be expected to be 13

C-enriched relative to other earthworm species. This

rela-tively large 13C-enrichment relative to putative dietary

source could be due to selective feeding, selective assimilation or isotopic fractionation during

respir-ation. Spain and Le Feuvre (1997) postulated that13

C-enrichments of earthworm tissue >1- relative to

diet-ary sources may result from a stepwise13C-enrichment

along a microbial food web. Alternatively, it may Table 4

Meand15_N_{(-)(A) and}_d13_C_{(-)(B) of the dierent ecological earthworm groupings (BoucheÂ, 1971; 1977) at each site}a

Site Epigeic Endogeic Anecic Humic Former Humic Feeder

d15_N _SE _d15_N _SE _d15_N _SE _d15_N _se _d15_N _SE

(A)d15_N

A 7.1 0.95 8.4 0.18 7.5 0.43 7.2 0.70 8.4 0.18

B N/ab _N/a _10.4 _0.23 _9.0 _0.17 _9.0 _0.17 _10.4 _0.23

C 1.3 0.55 4.6 0.31 5.5 ± 1.3 0.55 4.9 0.34

D 3.1 0.18 7.5 0.17 4.2 0.25 3.8 0.26 7.5 0.17

E 1.5 0.23 3.6 0.45 2.4 0.75 1.8 0.34 3.5 0.50

F 4.5 0.57 5.4 0.25 4.6 0.32 4.6 0.29 5.4 0.26

Site Epigeic Endogeic Anecic Humic Former Humic Feeder

d13_C _SE _d13_C _SE _d13_C _SE _d13_C _SE _d13_C _SE

(B)d13_C_(-)

A ÿ26.1 0.35 ÿ25.1 0.09 ÿ25.9 0.16 ÿ26.0 0.26 ÿ25.1 0.09 B N/a N/a ÿ25.6 0.13 ÿ26.2 0.07 ÿ26.2 0.07 ÿ25.6 0.13 C ÿ26.3 0.04 ÿ24.8 0.40 ÿ24.4 ± ÿ26.3 0.04 ÿ24.7 0.36 D ÿ25.9 0.26 ÿ25.0 0.05 ÿ26.5 0.15 ÿ26.2 0.17 ÿ25.1 0.06 E ÿ24.7 0.19 ÿ23.9 0.20 ÿ24.6 0.28 ÿ24.7 0.16 ÿ23.9 0.23 F ÿ26.7 0.06 ÿ25.9 0.15 ÿ26.8 0.18 ÿ26.8 0.15 ÿ25.9 0.15

a

Levels of signi®cance between the dierent ecological classi®cations are presented in Table 5.

b_{N/a = no epigeic earthworm species present.}

Table 5

Levels of signi®cance between dierent ecological classi®cations (BoucheÂ, 1971; 1977; Perel, 1977) ford15_N_{(A) and}_d13_C_(B)

Site Epigeic vs. Endogeic Epigeic vs. Anecic Endogeic vs. Anecic Humic former vs. Humic feeder

(A)d15_N_-

A 0.022 ns ns 0.015

D R0.001 0.011 R0.001 R0.001

E R0.001 ns ns 0.011

F ns nsa _ns _0.031

(B)d13_C_(-)

A 0.001 ns 0.011 R0.001

D R0.001 ns R0.001 R0.001

E 0.021 ns ns 0.017

F 0.042 ns 0.001 R0.001

a

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re¯ect the ingestion of 13C-enriched micro- and meso-fauna from a variety of trophic groups and the

sub-sequent assimilation of available 13C by earthworms.

These alternatives cannot be distinguished without rel-evant 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

d13_C _{throughout the soil ecosystem, i.e. from}

produ-cers to top predators. Similarly, more trophic levels are likely to occur with increased invertebrate biodiver-sity and this is likely to be manifested in a wider range

of d15N (Cabana and Rasmussen, 1994). On this basis,

d15N 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 diering habitat complexity. In contrast to this study, Wishart et al. (1997) reported that wood-lands (coniferous and deciduous) were more `complex' than pastures, although their sampled habitats were within 200 m of each other and not from distinct geo-graphical locations as in this study. Applying an

aver-age 15N-enrichment of 3.4- (Wada et al., 1993;

Minagawa and Wada, 1984) to mean foliar d15_N _data

listed in Table 1 of Handley et al. (1999), a similar

pat-tern of earthworm 15N-enrichment in woodlands can

be deduced, i.e. woodland < pasture. However, analys-ing the globally-derived data from Handley et al.

(1999) in detail indicates that the 15N-enrichment in

deciduous woodlands is greater than that for conifer-ous woodlands which is contrary to that found here. This suggests that within global ecological generalis-ations, contradictory patterns may occur locally.

4.2. Ecological classi®cations

Endogeic earthworm species at sites C and D were

between 3.2 and 5.3- more 15N-enriched relative to

epigeic/anecicLumbricus spp. Data from these

conifer-ous woodland sites appear to support Schmidt et al. (1997) who, using data gathered from a single site, suggested that endogeic earthworm species were

separ-ated from Lumbricus spp. by a single trophic level. In

contrast, at the other four sites, endogeic species were

generally <2.0- more 15N-enriched than Lumbricus

spp. This may be, as previously noted, because conifer-ous woodlands support a more diverse soil invertebrate community.

The hypothesis that endogeic and epigeic earth-worms are isotopically d15N and d13C 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 d15N (A and E)

and a single site for d13C (E). In contrast, Schmidt et

al. (1997) found that signi®cant dierences existed between each ecological grouping based on earthworm

d15N, d15N 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 earthworm15N was not related

to cropping treatment (maize versus permanent pas-ture) but was related to ecological grouping with

endo-geic species being more 15N-enriched than epigeic and

epi/anecic species. In terms of earthworm d _{values, it}

appears that BoucheÂ'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. Gen-erally, these putative site-dependent patterns may

sup-port the hypothesis that earthworms were

`ecosystemivorous' (Pokarzhevskii et al., 1997), i.e. when earthworms consume soil and micro- and meso-fauna, they are in eect 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 de-rived from more trophic levels may produce a greater

dierence between earthworm d15_N _and_d13_C _{and their}

putative diet (foliage and soil). The isotopic values measured here may indicate habitat biodiversity, earth-worm feeding strategy or both.

At a ®ner scale, gut analyses have shown that earth-worms 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. terrestrishad feeding attributes similar to both L. castaneus (epigeic) andAporrectodea icterica (endo-geic), whereas, JeÂgou et al. (1999) noted that the

feed-ing habits of L. terrestris were similar to the epigeic

speciesEisenia andrei. BoucheÂ (1971) consideredL. ter-restristo be epigeic/anecic, i.e., mainly anecic when lit-ter quantity decreased during winlit-ter 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 d15N

between epigeic species and anecic species, comprising

mainly of L. terrestris, is due to epi/anecic behaviour.

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These factors could in¯uence the isotopic values of in-dividual earthworms and species which, in turn, would be re¯ected in the isotopic values of the dierent eco-logical groupings.

d15N andd13C values separated earthworms into the

ecological groupings suggested by Perel (1977). Essen-tially, humic formers represent those earthworms that feed on plant litter, whereas humic feeders

predomi-nately consume 15N enriched soil (Shearer et al., 1978;

Nadelhoer 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 ganic matter. Before feeding by humic feeders, soil or-ganic matter may undergo isotopic fractionation,

preferentially removing the 14N fraction, leaving the

residual organic matter 15N-enriched. This is

analo-gous to that reported during sedimentation and mi-crobial transformation of organic N in well-mixed marine environments (SchaÈfer et al., 1998).

4.3. Conclusions

Earthwormd13_C _and_d15_N_{data presented here from}

a variety of habitats suggest that earthworm ecological groupings are site speci®c and generally it is not poss-ible to separate the dierent ecological groupings iso-topically. This supports the conclusion of Edwards and Bohlen (1996) that it is dicult to classify earth-worms 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 earth-worm species from dierent 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 abun-dances in taxonomic studies as suggested by Briones et al. (1999). Further research is required to determine

whether earthworm 13C- and 15N-enrichment relative

to putative dietary source is indicative of habitat com-plexity.

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 Aairs Department.

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