Clonal and genetic variation in three collembolan species revealed by isozymes and randomly amplified polymorphic DNA

Pedobiologia 45, 161–173 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/pedo

Clonal and genetic variation in three collembolan species revealed by isozymes and randomly amplified polymorphic DNA Vibeke Simonsen* and Pia Grothe Christensen National Environmental Research Institute, Department of Terrestrial Ecology, Vejlsoevej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark Submitted: 21. June 2000 Accepted: 1. November 2000

Summary Four strains of the collembolan Folsomia candida and one strain of F. fimetaria were analysed for genetic variation revealed by isozymes and randomly amplified polymorphic DNA (RAPD-PCR). Genetic variation for a strain of Hypogastrura assimilis was revealed by isozymes. Both analytical methods are applicable for separating strains of F. candida, and no genetic variation within the strains of this parthenogenetic species was observed by either method. This observation strongly supported the applicability of F. candida as bioindicator like Daphnia magna. As expected for a sexually reproducing species F. fimetaria revealed genetic variation when applying either analytical method and so did H. assimilis when analysing isozymes. The four strains of F. candida were clustering together in a taxon well separated from the two other collembolan species F. fimetaria and H. assimilis based on eight isozymes, representing nine loci. Key words: Collembola, clonal divergence, genetic variation, isozymes, RAPD-PCR

*E-mail corresponding author: [email protected]

0031–4056/01/45/02–161 $ 15.00/0

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Introduction The three collembolan species, Folsomia candida, Folsomia fimetaria and Hypogastrura assimilis are regarded as simple or primitive organisms. Their ability to react to various pollutants has increased their potential as bioindicators (e.g. Kopeszki 1993; Folker-Hansen et al. 1996). The collembolan species, F. candida and F. fimetaria, are under consideration as test organisms for toxicity of chemicals (Hopkin 1997a & b; Wiles & Krogh 1998). F. candida may be used as terrestrial „daphnia”, due to its parthenogenetic reproduction and easiness of cultivation under laboratory conditions. The sensitivity of F. candida to cadmium has been demonstrated (Crommentuijn et al. 1993) by studying various life-history characteristics. Responses of F. candida to cadmium, the organophosphate chlorpyrifos and triphenyltin hydroxide depends on the clone. The interclonal differences were not so large, that the species was not suited for soil ecotoxicological evaluations (Crommentuijn et al. 1995). The advantage of using a parthenogenetic organism for toxicity tests is that the genetic variation is reduced and thereby the genetic impact on the response. However, it has been demonstrated that strains with different genetic composition may react differently when exposed to various chemicals. The different responses complicate the applicability of organisms such as Daphnia magna (Baird et al. 1989, 1991) and F. candida as bioindicator. Several analytical methods have been applied for detecting genetic composition of strains, populations and species. Chromosome number and morphology have been used successfully as genetic markers (e.g. Brummer-Korvenkontio & Saure 1969; Kiauta 1970; Hemmer 1990). The discovery of polytene chromosomes in the salivary glands of the Neanuridae family was extremely useful for studying chromosomal variation (Dallai et al. 1983; Deharveng & Lee 1984). Molecular markers such as proteins were already used about 20 years ago as a versatile tool when studying taxonomy of collembolans (e.g. Hale & Rowland 1977; Hart & Allamong 1979; Prabhoo 1987). Isozymes have been utilised for studying intra- and interspecific variation among populations (Dallai et al. 1983, 1986; Fanciulli et al. 1985, 1991, 1995; Lee & Park 1991; Frati et al. 1992a, 1994, 1997a; Carapelli et al. 1995). Recently, genetic differentiation by analysing ribosomal DNA from two collembolan species (Hwang et al. 1995) and from six sympatric species of the genus Isotomurus (Carapelli et al. 1995) was revealed. Investigation on morphological traits and molecular markers have been demonstrated as useful in taxonomic studies of the genera Isotomurus and Isotoma (Frati et al. 1995; Simonsen et al. 1999). Analyses of the mitochondrial cytochrome oxidase II gene and isozymes (Frati et al. 1997b) revealed evidence for a cryptic species within I. palustris. The impact of metal pollution on isozymes of collembolans revealed a correlation between metal tolerance and the allozyme glutamic oxaloacetic acid transaminase within the species Orchesella cincta (Frati et al. 1992b). However, no impact of soil metal pollution was observed on allelic frequencies of phosphoglucomutase or glucosephosphate isomerase in O. bifasciata (Tranvik et al. 1994), despite the fact that this was observed for several other animals. The aim of the present study was to detect genetic variation within and among four laboratory strains of F. candida, which could support the different ecotoxicological responses as found by Crommentuijn et al. (1995). Two analytical techniques, i.e. isozyme electrophoresis and randomly amplified polymorphic DNA (RAPD-PCR) were

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used. The variation in F. candida was compared to that revealed in a congeneric species F. fimetaria and in a species from an other genus, represented by Hypogastrura assimilis for confirming the species status of F. candida.

Materials and Methods Strains One of the F. candida strains was a laboratory strain, originating from Berlin, Germany, and was kept in our Department for several years. This strain was designated FCDK. Dr. van Gestel, Vrije Universiteit, Amsterdam kindly provided the three other F. candida strains. The three strains have been in culture for several years and originated from Norwich, England (FCGB), Brunoy, France (FCFR) and Roggebotzand, The Netherlands (FCNL) (Crommentuijn et al. 1995). The F. fimetaria as well as the H. assimilis strain originated from an agricultural field in Denmark and were kept in our Department for several years (Krogh, pers. comm.). The strains were mainly cultivated on plaster at 20°C and with 12 hours of light and 12 hours of dark. However, the strains FCFR and FCNL performed much better when kept in soil (Kjær, pers. comm.). The collembolan specimens utilised in either electrophoretic investigations or RAPD-PCR analyses were stored at - 80°C if not used directly from the culture.

Electrophoresis The electrophoretic methods used were either horizontal starch gel electrophoresis (Harris & Hopkinson 1976) or ultrathin layer isoelectric focusing in agarose gel (Simonsen et al. 1991). Each individual was homogenised in 10 or 5 µl of a 0.1 M Tris-HCl buffer solution, pH 7.0. The solution was applied to the gel by using a small piece of filter paper (Whatman No. 3MM), 4 x 6 mm for the starch gel electrophoresis and 4 x 3 mm for the isoelectric focusing. Up to 30 individuals were analysed per starch gel and up to 15 per isoelectric focusing gel. The staining procedures were similar to those described by Richardson et al. (1986) or by Murphy et al. (1990). All species were compared by a side-by-side analysis for each enzyme. Table 1 lists the enzymes analysed and the methods used for the three species. The abbreviations for the enzymes are shown with capital letters (e.g. ACP) (Table 1) and the corresponding locus are designated with italics (e.g. ACP). If the interpretation of a zymogram for a certain enzyme demanded more than one locus, the zone migrating fastest towards the anode is designated with a hyphen and 1 (e.g. EST-1) and so the corresponding locus with EST-1 and so forth. The alleles were named according to their position on the gel; the allele migrating fastest towards the anode was No. 1 and so forth.

DNA extraction The method for extracting DNA from individual specimens of the two species F. candida and F. fimetaria was performed in the same way as extraction from minute parasitic wasp species (Landry et al. 1993). The method used sodium chloride for denaturing the proteins and iso-propanol for precipitating DNA.

RAPD-PCR analysis The reaction mixture for the PCR-reaction after optimisation was the following: 6.5 µl water, 2.5 µl 100 mM Tris-HCl reaction-buffer (pH = 8.4) with 500 mM KCl (HT Biotechnology, Stratech Scientific Ltd., Luton, UK) and 15 mM MgCl2, 10.0 µl dNTP (2 mM, Pharmacia-Biotech, Uppsala, Sweden), 1.0 µl Taq polymerase (0.25 U/l, HT Biotechnology Ltd.), 2.5 µl pri-

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mer (3 M, Operon) and 2.5 µl DNA extracted as described above. The primers tested were 40 of the following decamer primers (Operon, Alameda, CA, USA): A01-A20 and C01-C20. The PCR reaction was performed by using a thermocycler (Quatro TC-50) for 45 cycles, each consisting of 1 minute at 93 °C, 1 minute at 36 °C and 2 minutes at 72 °C. The PCR products were analysed by submarine 1.4% agarose gel electrophoresis in TBE buffer (89 mM Tris, 89 mM boric acid, 2.5 mM Na2-EDTA), stained with ethidiumbromide and visualized by UV light. Among the 40 primers tested in two experiments, five revealed reproducible banding patterns, i.e. A03, A07, A16, C15 and C19, and these were used for the further analysis. Samples from each strain of F. candida and F. fimetaria were analysed in the same run.

Data analysis The data obtained by electrophoresis was analysed by the G-stat program (Siegismund 1994). Genetic variation was measured either as P99 equal to the fraction of polymorphic loci (a locus is considered polymorphic when the frequency of the most common allele does not exceed 0.99), as A, the average number of alleles per locus, as Ho, the average observed heterozygosity or as He, the average expected heterozygosity estimated from the observed allelic frequencies assuming Hardy-Weinberg proportions in the population. An unweighted pair-group mean analysis (UPGMA) was performed (e.g. Ferguson 1980) using the genetic identities (Nei 1978) based on isozymes or a similarity index for the RAPD-PCR results (Puterka et al. 1993; Folkertsma et al. 1994).

Results Folsomia candida Ten enzymes were tested, AP revealed very faint or no activity in F. candida so this enzyme was omitted in the further analyses. The zymograms obtained for eight of the enzymes were interpreted as determined by at least one locus, whereas EST was determined by two loci, in all ten loci. No variation within the strains was found for F. candida, see Table 2, as all individuals within a strain showed the same zymogram. However, divergence between the strains was observed (Table 2), and this was reflected by the genetic parameters P99 and A (Table 3). The genetic identities, based on the ten loci, between the strains were estimated (Table 4), and a dendrogram was constructed by applying the UPGMA procedure. Due to the fact, that FCFR and FCNL revealed identical migration rates for all enzymes analysed, the two strains were regarded as one taxon (Fig. 1a). FCDK and FCGB clustered as another taxon. However, the taxon encompassing FCDK and FCGB was difficult to distinguish from the taxon including all four strains, due the very short genetic distance between the two groups. The five primers selected for RAPD-PCR revealed an identical banding patterns within a strain for all the 24 individuals investigated. The number of bands scored was 12, 6, 10, 5 and 10 for the primers A03, A07, A16, C15 and C19, respectively. It was assumed that each band was unique, so in total the data was analysed as 43 different markers. An example is shown in Figure 2. The similarity indices between the strains (Puterka et al. 1993; Folkertsma et al. 1994) are listed in Table 4. A dendrogram based on these similarity indices was constructed by applying the UPGMA procedure (Fig. 1b). The first group consisted of FCFR and FCNL, the second included the first one and FCGB, and FCDK was remotely related to the second group.

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Fig. 1. UPGMA dendrograms of a) F. candida based on ten isozyme loci, b) F. candida based on 43 RAPD-PCR fragments, and c) three collembolan species based on nine isozyme loci. FCDK is F. candida from Denmark, FCGB F. candida from England, FCFR F. candida from France and FCNL F. candida from The Netherlands

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Folsomia fimetaria Among the ten enzymes tested, AP and PGM did not reveal activity, so nine loci were analysed. Four loci, ACP, EST-1, EST-2 and TPI were found to be polymorphic (Table 2). The genotypic distributions for ACP, EST-2 and TPI fitted Hardy-Weinberg expectations. The Hardy-Weinberg expectations for EST-1 was not tested due to the presence of a null allele, designated n, and this locus was not used for the estimation of P99, Ho and He. The genetic variability for the species is listed in Table 3. RAPD-PCR was also applied to this species, using the same five primers as for F. candida. Twenty-four individuals were analysed. Six banding patterns and 4 band-positions were obtained for the primers A03 and A07, 6 patterns and 7 positions for A16, 8 patterns and 9 positions for C15 and 4 patterns and 5 positions for C19. Hypogastrura assimilis Ten loci were revealed when testing the ten enzymes, as PGM expressed nearly no activity. Among these, six were found to be polymorphic (see Table 2). When testing for fit to Hardy-Weinberg expectations only the genotypic distribution for the locus EST2 deviated significantly due to an excess of heterozygotes. The numbers of samples with excess of heterozygotes and with deficiency were three for each category and in accordance with the expected 1:1 ratio. The genetic variability is listed in Table 3.

Fig 2. RAPD-PCR fragments of 40 individuals of Folsomia, C19 used as primer. Lane 1-8 F. fimetaria, lane 9-16 FCDK, lane 17-20 FCGB, lane 21-24 FCGB, lane 25-32 FCFR and lane 33-40 FCNL. FCDK is F. candida from Denmark, FCGB F. candida from England, FCFR F. candida from France and FCNL F. candida from The Netherlands

3.1.3.2 3.5.4.4 3.4.11.1 3.1.1.... 5.3.1.9 1.1.1.37 5.3.1.8 1.1.1.44 5.4.2.2 5.3.1.1

PGDH PGM TPI

E.C. number

ACP ADA AP EST GPI MDH MPI

Abbreviation

Tris-morpholine buffer pH = 6.1 (Clayton & Tretiak 1972) B: Tris-citrate buffer pH = 7.0 (Ayala et al. 1972) 3 C: Lithium hydroxide-borate buffer, pH = 8.1 (Ashton & Braden 1961)

2

1 A:

Acid phosphatase Adenosine deaminase Cytosol aminopeptidase Esterase Glucosephosphate isomerase Malate dehydrogenase Mannosephosphate isomerase 6-Phosphogluconate dehydrogenase Phosphoglucomutase Triosephosphate isomerase

Species Enzyme Folsomia fimetaria Hypogastrura assimilis

IEF IEF SGE

IEF SGE SGE IEF IEF IEF SGE pH 4-6 pH 4-6 C

pH 4-6 B2 A1 pH 4-6 pH 4-6 pH 4-6 B IEF IEF SGE

IEF SGE SGE IEF IEF IEF SGE pH 4-6 pH 4-6 A

pH 4-6 B A pH 4-6 pH 4-6 pH 4-6 C3 IEF IEF SGE

IEF SGE SGE IEF IEF IEF SGE

pH 4-6 pH 4-6 C

pH 6-8 B A pH 4-6 pH 4-6 pH 4-6 B

Method Condition Method Condition Method Condition

Folsomia candida

Table 1. List of enzymes analysed for each of three collembolan species. Enzymes are characterised by abbreviations and E.C. numbers. Buffer used for starch gel electrophoresis (SGE) is listed under condition and so is pH range for isoelectric focusing (IEF)

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Table 2. List of loci and alleles, specified by a number, found for Folsomia candida strains from Denmark (FCDK), France (FCFR), United Kingdom (FCGB) and The Netherlands (FCNL), and for F. fimetaria and Hypogastrura assimilis from Denmark. Shared alleles have the same number for all groups. In parentheses the number of individuals analysed is shown Species

Folsomia candida

Folsomia fimetaria

Locus

FCDK

FCFR

FCGB

FCNL

ACP ADA AP EST-1 EST-2 GPI MDH MPI PGDH PGM TPI

5 (30) 3 (55) – 2 (30) 6 (30) 4 (30) 3 (30) 6 (30) 2 (30) 1 (30) 5 (30)

5 (15) 3 (15) – n* (15) 3 (15) 6 (15) 3 (15) 5 (15) 3 (15) 3 (15) 5 (15)

5 (15) 3 (15) – n (15) 3 (15) 5 (15) 2 (15) 6 (15) 2 (15) 2 (15) 5 (15)

5 (15) 3 (15) – n (15) 3 (15) 6 (15) 3 (15) 5 (15) 3 (15) 3 (15) 5 (15)

Hypogastrura assimilis

1, 2, 4 (35) 3, 6 (40) 1 (35) 2, 4 (109) – 1, 2 (89) 3, 4, n (33) 1 (35) 4, 5, 7 (33) 2, 5, 8 (34) 3 (35) 1, 2 (35) 1 (35) 1 (35) 3 (40) 1, 2, 4 (99) 1 (35) 3 (40) – – 1, 3, 4 (104) 2 (49)

* n is a null allele

Comparison of the species A dendrogram based on genetic identity (Table 4) by means of isozymes from the three collembolan species, F. candida encompassing four strains, was depicted (Fig. 1c). The four strains of F. candida clustered together in one taxon. F. fimetaria and H. assimilis clustered remotely with each other and with F. candida, which was expected due to their status as morphologically distinct species. Regarding the RAPD-PCR fragments, all the primers revealed at least one band common for the two species, F. candida and F. fimetaria. However, at least one species-characteristic band was also found for each primer, e.g. for the primer C19 (Fig. 2).

Discussion Allozyme polymorphisms revealed by electrophoresis might be affected by the nutritional stage or the age of the organism studied (Ferguson 1980). However, in our study no indication on age-dependent expression, as found for EST (Grimnes 1981), was observed. Grimnes (1986) used polyacrylamide electrophoresis for analysing EST and found more bands than seen in this study. She also observed clonal variation. The divergence of the results for EST between the two studies was probably due to the two different analytical methods applied, but might also be obtained by using different clones. The lack of genetic variation found within F. candida strains might be explained as the result of the cultivation regime, which might select only the genotype best suited

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Table 3. Genetic variability of some collembolan species revealed by isozyme studies. Pops = number of populations, L = number of loci analysed, P99 = fraction of polymorphic loci, A = average number of alleles per locus, Ho = observed heterozygosity, He = expected heterozygosity according to Hardy-Weinberg expectations Species Pops Bilobella aurantiaca 3 B. aurantiaca 3 B. braunerae 1 B. massoudi 1 Dicyrtomina ornata 7 D. saundersi 8 Entomobrya jirisana 1 Folsomia candida 4 F. fimetaria 1 Hypogastrura assimilis 1 Homidia grisea 1 H. koreana (B) 1 H. koreana (J) 1 H. mediaseta 1 H. mundu mundu 1 H. mundu nigra 1 Isotoma anglicana 1 I. riparia 2 I. viridis 1 Isotomurus ghibellinus 1 I. indipendente 1 I. italicus 1 I. maculatus 1 I. palustris 1 I. unifasciatus 1 Orchesella chiantica 3 O. cincta 3 O. dallaii 1 O. ranzii 1 O. sp. from Elba 1 O. villosa 8 Sinella curviseta 1 Tetrodontophora 10 bielanensis Thaumanura ruffoi 3 Tomocerus kinoshitai 1 1P 95

L 5 13 13 13 18 18 14 10 8

P99 0.60 0.39 0.39 0.15 0.241 0.311 0.71 0.70 0.13

A 1.9 1.5 1.5 1.2 1.3 1.4 2.3 1.9 1.3

Ho – 0.10 0.05 0.03 0.09 0.10 – 0.00 0.05

He 0.25 0.11 0.07 0.03 0.09 0.12 0.21 0.00 0.05

Reference Dallai et al. 1983 Dallai et al. 1986 Dallai et al. 1986 Dallai et al. 1986 Fanciulli et al. 1995 Fanciulli et al. 1995 Lee & Park 19912 This study This study

10 14 15 15 15 15 15 11 11 11

0.60 0.93 0.93 0.88 0.80 0.80 0.53 0.82 0.55 0.55

1.8 2.6 2.7 2.3 2.5 2.0 2.1 2.5 1.6 1.7

0.23 – – – – – – 0.15 0.09 0.12

0.22 0.36 0.25 0.18 0.29 0.17 0.19 – – –

This study Lee & Park 19912 Lee & Park 19912 Lee & Park 19912 Lee & Park 19912 Lee & Park 19912 Lee & Park 19912 Simonsen et al. 1999 Simonsen et al. 1999 Simonsen et al. 1999

12 12 12 12 12 12 20 20 20 20 20 20 15 18

0.42 0.33 0.33 0.33 0.50 0.33 0.33 0.25 0.25 0.45 0.40 0.34 0.80 0.13

1.6 1.8 1.8 1.8 1.6 1.8 1.6 1.5 1.5 1.8 1.9 1.6 2.1 1.2

0.09 0.08 0.10 0.14 0.12 0.13 0.08 0.08 0.06 0.12 0.10 0.07 – 0.04

0.13 0.15 0.15 0.19 0.14 0.18 0.11 0.07 0.10 0.14 0.12 0.09 0.28 0.04

15 13

0.33 0.54

1.4 1.9

– –

0.11 0.18

Carapelli et al. 1995 Carapelli et al. 1995 Carapelli et al. 1995 Carapelli et al. 1995 Carapelli et al. 1995 Carapelli et al. 1995 Frati et al. 1994 Frati et al. 1994 Frati et al. 1994 Frati et al. 1994 Frati et al. 1994 Frati et al. 1994 Lee & Park 19912 Fanciulli et al. 1985 and 1991 Dallai et al. 1983 Lee & Park 19912

instead of P99, the proportion of loci for which the frequency of the most common allele does not exceed 0.95 2 Estimated from Table 6 (Lee & Park 1991)

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Table 4. Genetic identities according to Nei (1978) based on isozymes. FCDK, FCFR, FCGB and FCNL represent four different strains of Folsomia candida. Genetic identities were estimated, according to Puterka et al. (1993) based on RAPD-PCR markers obtained by five primers. The number in parentheses are genetic identities according to Folkertsma et al. (1994) Isozymes 10 loci FCDK FCFR FCGB Isozymes 9 loci FCDK FCFR FCGB FCNL F. fimetaria

FCFR 0.39

FCGB 0.49 0.48 0.48

FCNL 0.39 0.97

FCFR 0.43

FCGB 0.54 0.54

FCNL 0.43 0.97 0.54

F. fimetaria H. assimilis 0.00 0.00 0.05 0.12 0.05 0.00 0.05 0.12 0.15

RAPD-PCR 43 markers FCFR FCGB FCNL FCDK 0.28 (0.23) 0.23 (0.21) 0.28 (0.23) FCFR 0.81 (0.76) 1.00 (1.00) FCGB 0.81 (0.76)

to live under laboratory conditions. Another explanation might be that only a single or very few individuals were used as parents for the culture and this combined with the parthenogenetic reproductive mode could cause low or no variation within the strain. Furthermore, a successful transfer of a single very competitive individual from one to the other culture might also cause genetic identity between FCFR and FCNL (Table 2 and Fig. 1). This could not be rejected despite the fact that the strains were kept separately from each other in both laboratories (Van Gestel, pers. comm., Kjær, pers. comm.). It should be emphazised that the analysis of isozymes is only suited for detecting differences and not similarities between individuals. Identical looking zymograms may not represent identical protein compositions. A similar phenomenon might be the case for RAPD-PCR. The genetic identities between the strains of F. candida, based on allozymes, were comparable to those found for Tetrodontophora bielanensis (Fanciulli et al. 1991), for Orchesella cincta, O. chiantica and O. villosa (Frati et al. 1992a & b, 1994), for six sympatric species of Isotomurus (Carapelli et al. 1995) and for three species of Isotoma (Simonsen et al. 1999). This supported the hypothesis that F. candida is a well-defined species. The result obtained by the RAPD-PCR analysis also indicated the species status of F. candida, when compared to F. fimetaria. From the dendrograms obtained by means of isozymes and from RAPD-PCR (Fig. 1a & 1b), a divergence was evident, as FCDK was more remotely related to FCGB, when applying RAPD-PCR markers instead of isozymes. However, this divergence might be caused by the limited number of isozyme loci studied compared to the number of

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fragments. Differences among the strains were also observed when culturing, as FCFR and FCNL did not perform so well on plaster compared to FCDK and FCGB. This observation supported the results obtained by the molecular markers. When using F. candida as a test organism for ecotoxicological tests, the possibility for having different responses to various chemicals by the different strains due to their genetic composition ought to be considered. The study by Crommentuijn et al. (1995) and this study used three of the same strains, i.e. FCFR, FCGB and FCNL, and both studies found that the strain FCGB was different from FCFR and FCNL. The impact of different genetic compositions of the clones might influence the variation obtained by ring-tests as seen for Daphnia magna (Baird et al. 1989). The genetic variability for a number of collembolan species is listed in Table 3, including the three species investigated in the present study. The variability was high in H. assimilis, whereas F. fimetaria was among the species with low genetic variation, which might be due to the establishment of the culture or the culturing regime.

Acknowledgements The Danish Environmental Research Programme supported this work. Dr. C.A.M. van Gestel provided the strains of Folsomia candida from France, The Netherlands and United Kingdom for which we are grateful. We wish to acknowledge Dr. R. B. Jørgensen and B. Andersen for introducing one of us (V. Simonsen) to the RAPD-PCR-method. Dr. C.A.M. van Gestel and Dr. P. Hensbergen are acknowledged for valuable comments on an earlier version of the manuscript. My colleague Dr. P.H. Krogh provided information on the species and collecting sites and A. Christiansen, A. M. Plejdrup, K. Kjær Jacobsen and Z. Gabor provided technical assistance.

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