Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue8-9.Aug2000:
Soil Biology & Biochemistry 32 (2000) 1053±1061
www.elsevier.com/locate/soilbio
Earthworm d13C and d15N 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 d13 C 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, 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 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. Average 13C-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 15N in earthworms, relative to diet, could be developed as a tool for assessing habitat diversity. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Anecic; Earthworms; Endogeic; Epigeic; Functional groupings; Stable isotopes
1. Introduction
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 mat-
* Corresponding author. Tel.: +44-1382-562-731; fax: +44-1382562-426.
E-mail address: [email protected] (R. Neilson).
ter; 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
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 1 3 - 4
1054
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
of stable isotopes are eectively 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 13C/12C d13 C and 15N/14N
15
d 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; 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 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 13Cenriched 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 13C- and 15Nenriched relative to fungi which, in turn, become 13Cand 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. Compared with epigeic earthworms, endogeic earthworms
potentially consume more 13C- and 15N-enriched dietary material, given that epigeic species feed predominately 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 15Nenriched by c. 2- in the top 35 cm under sugarcane.
Thus, burrowing endogeic species are exposed to a potential source of 15N-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 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
Bouche (1971, 1977). Similarly, Briones et al. (1999)
reported that earthworm d15 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 d13 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 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 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 1995.
Earthworms were extracted by hand sorting (Boag
et al., 1997). At each site, ®ve randomly selected areas
30 30 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 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 at ÿ208C 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
1055
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
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
B
C
D
E
F
NO 347323
NN 983133
NH 857057
NO 484237
NO 295506
NN 971131
56829 'N
56818 'N
57808 'N
56824 'N
56839 'N
56818 'N
3804'W
3839'W
3852'W
2850'W
3809'W
3840'W
50
70
270
5
80
80
680
1145
628
647
781
1145
Arable
Arable
Coniferous woodland
Coniferous woodland
Deciduous woodland
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.
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 2
Mean d15 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)
Aporrectodea caliginosa (End)
Lumbricus castaneus (Epi)
L. rubellus (Epi)
L. terrestris (Ane)
Weighed mean
A. chlorotica (End)
A. rosea (End)
L. terrestris (Ane)
Octolasion cyaneum (End)
Weighed mean
Aporrectodea longa (Ane)
A. rosea (End)
L. castaneus (Epi)
Weighed mean
A. caliginosa (End)
A. chlorotica (End)
A. longa (Ane)
A. rosea (End)
L. castaneus (Epi)
L. rubellus (Epi)
L. terrestris (Ane)
Weighted mean
A. caliginosa (End)
Aporrectodea rosea (End)
Dendrodrilus rubidus (Epi)
L. castaneus (Epi)
L. rubellus (Epi)
L. terrestris (Ane)
Weighted mean
A. caliginosa (End)
A. rosea (End)
L. rubellus (Epi)
L. terrestris (Ane)
Weighted mean
4
27
3
4
3
+8.2
+8.5
+6.5
+7.4
+7.5
+8.1
+10.6
+10.3
+9.0
+10.5
+9.7
+5.5
+4.6
+1.3
+3.8
+8.0
+8.3
+4.5
+7.3
+3.0
+3.1
+4.1
+5.7
+4.4
+3.3
+2.0
+1.3
+0.7
+0.8
+2.2
+5.5
+5.1
+4.5
+4.6
+5.0
0.29
0.20
0.23
2.00
0.53
ÿ25.6
ÿ25.1
ÿ26.8
ÿ25.5
ÿ25.9
ÿ25.4
ÿ25.4
ÿ25.4
ÿ26.2
ÿ26.3
ÿ25.9
ÿ24.4
ÿ24.8
ÿ26.3
ÿ25.2
ÿ25.1
ÿ25.3
ÿ26.5
ÿ25.0
ÿ25.5
ÿ26.0
ÿ26.5
ÿ25.7
ÿ23.9
ÿ23.9
ÿ24.3
ÿ24.7
ÿ25.7
ÿ24.8
ÿ24.4
ÿ25.8
ÿ26.2
ÿ26.7
ÿ26.8
ÿ26.4
0.04
0.09
0.28
0.45
0.20
B
C
D
E
F
a
2
6
10
2
1
4
2
2
1
2
7
1
3
4
1
4
3
4
1
4
8
2
2
10
Codes for ecological groupings: Ane, Anecic; End, Endogeic; Epi, Epigeic.
0.18
0.42
0.17
0.01
±
0.35
0.78
0.23
±
0.95
0.18
±
0.30
0.31
±
0.59
0.38
0.29
±
0.35
0.25
1.49
0.81
0.34
0.01
0.11
0.07
0.23
±
0.46
0.06
0.18
±
0.60
0.07
±
0.9
0.18
±
0.30
0.18
0.18
±
0.15
0.15
0.73
0.08
0.19
1056
dsample
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
Rsample ÿ Rstandard
1000Rstandard
where Rsample and Rstandard are the heavy/light isotope
ratios of sample and standard. Analytical precision
was R0.2- for d13 C and R0.4- d15 N:
each site weighted by earthworm abundance. Since no
percentage vegetation cover data were available for the
sampling sites, simple arithmetic mean d15 N and d13 C
values were calculated based on the relevant isotopic
measurement.
2.3. Data analyses
3. Results
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 d15 N and d13 C were calculated for
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 d15 N and d13 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.
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
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, average L. terrestris d15 N ranged from +0.8- (site E) to
+9.0- (site B). This was re¯ected in the weighed
mean earthworm d15 N 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 d15 N and the average d15 N of the
sampled vegetation (Tables 2 and 3). In four of the
®ve sites for which whole soil d15 N values were available, the weighed mean earthworm d15 N also exhibited
a 15N enrichment relative to whole soil d15 N, with the
averaged stepwise increase ranging from 2.8- to 6.3depending 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 d15 N and whole soil d15 N (Fig. 1). When
comparing ecological groupings (BoucheÂ, 1971, 1977),
d15 N 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 3
Mean d15 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
Grasses
Soil
Grasses
Various dicots
Soil
Blaeberry leaves
Birch leaves
Juniper leaves
Soil
Grasses
Soil
Fern
Woodrush
Soil
Grasses
Soil
+5.6
+5.1
N/da
+5.4
+5.1
+5.5
ÿ1.8
ÿ1.6
ÿ2.1
+1.0
+0.5
ÿ0.6
ÿ0.5
ÿ1.7
+2.1
ÿ0.9
+1.7
0.21
0.35
N/d
0.38
0.57
0.06
0.25
0.46
0.38
0.05
0.41
0.04
0.38
0.21
0.04
0.39
0.04
ÿ28.1
ÿ28.2
N/d
ÿ30.7
ÿ29.4
ÿ26.7
ÿ30.9
ÿ29.5
ÿ27.0
ÿ27.9
ÿ30.7
ÿ29.8
ÿ28.3
ÿ26.9
ÿ28.1
ÿ30.7
ÿ28.3
0.04
0.05
N/d
0.37
0.28
0.03
0.09
0.19
0.40
0.02
0.17
0.03
0.21
0.39
0.02
0.09
0.02
B
C
D
E
F
a
N/d = not determined.
1057
(Table 5). All the three ecological groupings were signi®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. d13 C
Earthworm d13 C values at species level were less
variable across the sites than d15 N (Table 2). This pattern was re¯ected in the calculated weighed mean
earthworm d13 C 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 with d15 N, d13 C of the dierent ecological groupings varied by site (Table 4). With one exception, both
epigeic and anecic species were signi®cantly more 13Cdepleted (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 more 13C-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 soil d15 N data,
earthworms were 15N-enriched relative to soil by
>2.5-, greater than the 1- relative to dietary sources may result from a stepwise 13C-enrichment
along a microbial food web. Alternatively, it may
Table 5
Levels of signi®cance between dierent ecological classi®cations (BoucheÂ, 1971; 1977; Perel, 1977) for d15 N (A) and d13 C (B)
Site
(A) d15 N -
A
D
E
F
(B) d13 C (-)
A
D
E
F
a
Epigeic vs. Endogeic
Epigeic vs. Anecic
Endogeic vs. Anecic
Humic former vs. Humic feeder
0.022
R 0.001
R 0.001
ns
ns
0.011
ns
nsa
ns
R 0.001
ns
ns
0.015
R 0.001
0.011
0.031
0.001
R 0.001
0.021
0.042
ns
ns
ns
ns
0.011
R 0.001
ns
0.001
R 0.001
R 0.001
0.017
R 0.001
ns = not signi®cant at p > 0.05.
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
re¯ect the ingestion of 13C-enriched micro- and mesofauna from a variety of trophic groups and the subsequent assimilation of available 13C 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
d13 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 d15 N (Cabana and Rasmussen, 1994). On this basis,
d15 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 diering 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 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 pattern of earthworm 15N-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 15N-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.
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/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 anecic
> epigeic/anecic Lumbricus spp. In this study, only
data from site D agreed with those ®ndings. Briones et
al. (1999) reported that earthworm 15N was not related
to cropping treatment (maize versus permanent pasture) but was related to ecological grouping with endogeic 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. 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 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 derived 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, 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, JeÂgou 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 d15 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).
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 dierent ecological groupings.
d15 N and d13 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 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 organic matter. Before feeding by humic feeders, soil organic matter may undergo isotopic fractionation,
preferentially removing the 14N fraction, leaving the
residual organic matter 15N-enriched. This is analogous to that reported during sedimentation and microbial transformation of organic N in well-mixed
marine environments (SchaÈfer et al., 1998).
4.3. Conclusions
Earthworm d13 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 possible to separate the dierent ecological groupings isotopically. This supports the conclusion of Edwards
and Bohlen (1996) that it is dicult 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 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 abundances 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 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 Aairs Department.
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www.elsevier.com/locate/soilbio
Earthworm d13C and d15N 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 d13 C 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, 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 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. Average 13C-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 15N in earthworms, relative to diet, could be developed as a tool for assessing habitat diversity. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Anecic; Earthworms; Endogeic; Epigeic; Functional groupings; Stable isotopes
1. Introduction
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 mat-
* Corresponding author. Tel.: +44-1382-562-731; fax: +44-1382562-426.
E-mail address: [email protected] (R. Neilson).
ter; 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
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 1 3 - 4
1054
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
of stable isotopes are eectively 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 13C/12C d13 C and 15N/14N
15
d 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; 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 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 13Cenriched 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 13C- and 15Nenriched relative to fungi which, in turn, become 13Cand 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. Compared with epigeic earthworms, endogeic earthworms
potentially consume more 13C- and 15N-enriched dietary material, given that epigeic species feed predominately 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 15Nenriched by c. 2- in the top 35 cm under sugarcane.
Thus, burrowing endogeic species are exposed to a potential source of 15N-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 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
Bouche (1971, 1977). Similarly, Briones et al. (1999)
reported that earthworm d15 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 d13 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 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 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 1995.
Earthworms were extracted by hand sorting (Boag
et al., 1997). At each site, ®ve randomly selected areas
30 30 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 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 at ÿ208C 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
1055
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
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
B
C
D
E
F
NO 347323
NN 983133
NH 857057
NO 484237
NO 295506
NN 971131
56829 'N
56818 'N
57808 'N
56824 'N
56839 'N
56818 'N
3804'W
3839'W
3852'W
2850'W
3809'W
3840'W
50
70
270
5
80
80
680
1145
628
647
781
1145
Arable
Arable
Coniferous woodland
Coniferous woodland
Deciduous woodland
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.
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 2
Mean d15 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)
Aporrectodea caliginosa (End)
Lumbricus castaneus (Epi)
L. rubellus (Epi)
L. terrestris (Ane)
Weighed mean
A. chlorotica (End)
A. rosea (End)
L. terrestris (Ane)
Octolasion cyaneum (End)
Weighed mean
Aporrectodea longa (Ane)
A. rosea (End)
L. castaneus (Epi)
Weighed mean
A. caliginosa (End)
A. chlorotica (End)
A. longa (Ane)
A. rosea (End)
L. castaneus (Epi)
L. rubellus (Epi)
L. terrestris (Ane)
Weighted mean
A. caliginosa (End)
Aporrectodea rosea (End)
Dendrodrilus rubidus (Epi)
L. castaneus (Epi)
L. rubellus (Epi)
L. terrestris (Ane)
Weighted mean
A. caliginosa (End)
A. rosea (End)
L. rubellus (Epi)
L. terrestris (Ane)
Weighted mean
4
27
3
4
3
+8.2
+8.5
+6.5
+7.4
+7.5
+8.1
+10.6
+10.3
+9.0
+10.5
+9.7
+5.5
+4.6
+1.3
+3.8
+8.0
+8.3
+4.5
+7.3
+3.0
+3.1
+4.1
+5.7
+4.4
+3.3
+2.0
+1.3
+0.7
+0.8
+2.2
+5.5
+5.1
+4.5
+4.6
+5.0
0.29
0.20
0.23
2.00
0.53
ÿ25.6
ÿ25.1
ÿ26.8
ÿ25.5
ÿ25.9
ÿ25.4
ÿ25.4
ÿ25.4
ÿ26.2
ÿ26.3
ÿ25.9
ÿ24.4
ÿ24.8
ÿ26.3
ÿ25.2
ÿ25.1
ÿ25.3
ÿ26.5
ÿ25.0
ÿ25.5
ÿ26.0
ÿ26.5
ÿ25.7
ÿ23.9
ÿ23.9
ÿ24.3
ÿ24.7
ÿ25.7
ÿ24.8
ÿ24.4
ÿ25.8
ÿ26.2
ÿ26.7
ÿ26.8
ÿ26.4
0.04
0.09
0.28
0.45
0.20
B
C
D
E
F
a
2
6
10
2
1
4
2
2
1
2
7
1
3
4
1
4
3
4
1
4
8
2
2
10
Codes for ecological groupings: Ane, Anecic; End, Endogeic; Epi, Epigeic.
0.18
0.42
0.17
0.01
±
0.35
0.78
0.23
±
0.95
0.18
±
0.30
0.31
±
0.59
0.38
0.29
±
0.35
0.25
1.49
0.81
0.34
0.01
0.11
0.07
0.23
±
0.46
0.06
0.18
±
0.60
0.07
±
0.9
0.18
±
0.30
0.18
0.18
±
0.15
0.15
0.73
0.08
0.19
1056
dsample
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
Rsample ÿ Rstandard
1000Rstandard
where Rsample and Rstandard are the heavy/light isotope
ratios of sample and standard. Analytical precision
was R0.2- for d13 C and R0.4- d15 N:
each site weighted by earthworm abundance. Since no
percentage vegetation cover data were available for the
sampling sites, simple arithmetic mean d15 N and d13 C
values were calculated based on the relevant isotopic
measurement.
2.3. Data analyses
3. Results
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 d15 N and d13 C were calculated for
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 d15 N and d13 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.
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
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, average L. terrestris d15 N ranged from +0.8- (site E) to
+9.0- (site B). This was re¯ected in the weighed
mean earthworm d15 N 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 d15 N and the average d15 N of the
sampled vegetation (Tables 2 and 3). In four of the
®ve sites for which whole soil d15 N values were available, the weighed mean earthworm d15 N also exhibited
a 15N enrichment relative to whole soil d15 N, with the
averaged stepwise increase ranging from 2.8- to 6.3depending 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 d15 N and whole soil d15 N (Fig. 1). When
comparing ecological groupings (BoucheÂ, 1971, 1977),
d15 N 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 3
Mean d15 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
Grasses
Soil
Grasses
Various dicots
Soil
Blaeberry leaves
Birch leaves
Juniper leaves
Soil
Grasses
Soil
Fern
Woodrush
Soil
Grasses
Soil
+5.6
+5.1
N/da
+5.4
+5.1
+5.5
ÿ1.8
ÿ1.6
ÿ2.1
+1.0
+0.5
ÿ0.6
ÿ0.5
ÿ1.7
+2.1
ÿ0.9
+1.7
0.21
0.35
N/d
0.38
0.57
0.06
0.25
0.46
0.38
0.05
0.41
0.04
0.38
0.21
0.04
0.39
0.04
ÿ28.1
ÿ28.2
N/d
ÿ30.7
ÿ29.4
ÿ26.7
ÿ30.9
ÿ29.5
ÿ27.0
ÿ27.9
ÿ30.7
ÿ29.8
ÿ28.3
ÿ26.9
ÿ28.1
ÿ30.7
ÿ28.3
0.04
0.05
N/d
0.37
0.28
0.03
0.09
0.19
0.40
0.02
0.17
0.03
0.21
0.39
0.02
0.09
0.02
B
C
D
E
F
a
N/d = not determined.
1057
(Table 5). All the three ecological groupings were signi®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. d13 C
Earthworm d13 C values at species level were less
variable across the sites than d15 N (Table 2). This pattern was re¯ected in the calculated weighed mean
earthworm d13 C 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 with d15 N, d13 C of the dierent ecological groupings varied by site (Table 4). With one exception, both
epigeic and anecic species were signi®cantly more 13Cdepleted (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 more 13C-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 soil d15 N data,
earthworms were 15N-enriched relative to soil by
>2.5-, greater than the 1- relative to dietary sources may result from a stepwise 13C-enrichment
along a microbial food web. Alternatively, it may
Table 5
Levels of signi®cance between dierent ecological classi®cations (BoucheÂ, 1971; 1977; Perel, 1977) for d15 N (A) and d13 C (B)
Site
(A) d15 N -
A
D
E
F
(B) d13 C (-)
A
D
E
F
a
Epigeic vs. Endogeic
Epigeic vs. Anecic
Endogeic vs. Anecic
Humic former vs. Humic feeder
0.022
R 0.001
R 0.001
ns
ns
0.011
ns
nsa
ns
R 0.001
ns
ns
0.015
R 0.001
0.011
0.031
0.001
R 0.001
0.021
0.042
ns
ns
ns
ns
0.011
R 0.001
ns
0.001
R 0.001
R 0.001
0.017
R 0.001
ns = not signi®cant at p > 0.05.
R. Neilson et al. / Soil Biology & Biochemistry 32 (2000) 1053±1061
re¯ect the ingestion of 13C-enriched micro- and mesofauna from a variety of trophic groups and the subsequent assimilation of available 13C 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
d13 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 d15 N (Cabana and Rasmussen, 1994). On this basis,
d15 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 diering 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 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 pattern of earthworm 15N-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 15N-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.
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/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 anecic
> epigeic/anecic Lumbricus spp. In this study, only
data from site D agreed with those ®ndings. Briones et
al. (1999) reported that earthworm 15N was not related
to cropping treatment (maize versus permanent pasture) but was related to ecological grouping with endogeic 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. 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 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 derived 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, 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, JeÂgou 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 d15 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).
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 dierent ecological groupings.
d15 N and d13 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 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 organic matter. Before feeding by humic feeders, soil organic matter may undergo isotopic fractionation,
preferentially removing the 14N fraction, leaving the
residual organic matter 15N-enriched. This is analogous to that reported during sedimentation and microbial transformation of organic N in well-mixed
marine environments (SchaÈfer et al., 1998).
4.3. Conclusions
Earthworm d13 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 possible to separate the dierent ecological groupings isotopically. This supports the conclusion of Edwards
and Bohlen (1996) that it is dicult 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 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 abundances 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 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 Aairs Department.
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