Evolutionary History of Caenorhabditis elegans Inferred from Microsatellites: Evidence for Spatial and Temporal Genetic Differentiation and the Occurrence of Outbreeding

Markus Haber, Manuela Schüngel, Annika Putz, Sabine Müller, Barbara Hasert and Hinrich Schulenburg

Department of Evolutionary Biology, Institute for Animal Evolution and Ecology, Westphalian Wilhelms-University, Muenster, Germany

Correspondence: E-mail: hschulen{at}uni-muenster.de.


    Abstract
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Although diverse biological disciplines employ the nematode Caenorhabditis elegans as a highly efficient laboratory model system, little is known about its natural history. We investigated its evolutionary past using 10 polymorphic trinucleotide and tetranucleotide microsatellites, derived from across the whole genome. These microsatellites were analyzed from the 35 previously available natural isolates from different parts of the world and also 23 new strains isolated from northwest Germany. Our results highlight that C. elegans lineages differentiate genetically with respect to geographic distance and, to a lesser extent, differences in the time of strain isolation. The latter indicates some turnover of strain genotypes at specific locations. Our data also demonstrate the coexistence of highly diverse genotypes in the population from northwest Germany, which is best explained by recent migration events. Furthermore, selfing is confirmed as the primary mode of reproduction for this hermaphroditic nematode in nature. Importantly, we also find evidence for the occurrence of occasional outbreeding. Taken together, these results support the previous notion that C. elegans is a colonizer, whereby selfing may permit rapid dispersal within new habitats even in the absence of potential mates, whereas occasional outcrossing may serve to compensate for the disadvantages of inbreeding. Such information about the natural history of C. elegans should be of great value for an in-depth understanding of the complexity of this organism, including its multifaceted developmental, neurological, or molecular genetic pathways.

Key Words: Caenorhabditis elegans • population genetics • AMOVA • inbreeding • microsatellites • recombination


    Introduction
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The soil nematode Caenorhabditis elegans has been extensively and successfully employed under laboratory conditions to delineate the genetics and molecular basis of diverse biological processes (e.g., Riddle et al. 1997). Almost all of these studies were performed with a single C. elegans strain (N2) and derivations thereof. The wealth of information generated in this context contrasts with the paucity of data available on its natural history. Only about 35 natural isolates, which can be unequivocally attributed to C. elegans, are currently available. These strains have been isolated from different places across the temperate regions of the world (Europe, North America, and Australia), usually less than five strains per location. They were mainly found in decomposing material (e.g., compost heaps), where they likely feed on microorganisms (Hodgkin and Doniach 1997). Only a few recent studies were directed at a more detailed characterization of these strains. They provided first evidence of natural variation in body length, offspring production, and reproductive behavior (Hodgkin and Doniach 1997), social behavior (de Bono and Bargmann 1998), aging (Gems and Riddle 2000), sperm size (LaMunyon and Ward 2002), chemosensation (Jovelin, Ajie, and Phillips 2003), and antiparasite defenses (Schulenburg and Müller 2004).

Our study focused on a population genetic analysis of natural C. elegans strains. In this context, the species' reproductive biology is of special importance. Reproduction relies on two genders: hermaphrodites (usually two sex chromosomes, XX) and males (usually X0). Hermaphrodites first produce sperm, which are stored and subsequently used to self-fertilize the later produced eggs. Selfing results in hermaphroditic offspring and occasionally pure males because of nondisjunction of X chromosomes during meiosis. Outcrossing is only possible with the males, whereby male sperm outcompete the hermaphrodite's own sperm (Ward and Carrel 1979; LaMunyon and Ward 1995). Therefore, after successful mating, males father all offspring, resulting in an offspring gender ratio of approximately 1:1 (Hodgkin, Horvitz, and Brenner 1979). However, males appear to be extremely rare in the natural strains, suggesting that selfing represents the primary mode of reproduction in the wild (Hodgkin and Doniach 1997).

Selfing increases homozygosity within individuals, and it may also lead to a decrease in genetic diversity within populations. The latter is predicted for neutral alleles by the neutral theory, because at equilibrium, the effective population size (Ne) is reduced by 50% for strict selfers relative to outbreeders (Pollak 1987), and the level of neutral variability is proportional to the effective population size (Kimura 1971). Genetic diversity may even be reduced further in response to background selection, genetic hitchhiking, or bottleneck effects. Each of these mechanisms is thought to have a much stronger effect in selfers because of the increase in individual homozygosity, the resulting decrease in the effective recombination rate, and the ability of individual selfers to found new populations (Charlesworth, Morgan, and Charlesworth 1993; Nordborg 2000; Charlesworth and Wright 2001; Ingvarsson 2002; Keller and Waller 2002; Cutter and Payseur 2003).

Increased individual homozygosity and decreased population-wide genetic diversity should be disadvantageous. The former is associated with the danger of inbreeding depression; for example, homozygosity of deleterious recessive mutations or homozygosity at specific loci with heterozygote advantage (Keller and Waller 2002). Similarly, low levels of intrapopulational genetic diversity constrains the ability to adapt rapidly to fluctuating environments (Bürger 1999), such as those determined by coevolving parasites (Lively and Howard 1994). At the same time, selfing should lead to increased genetic differentiation between populations. This is a consequence of the above-mentioned forces, which remove genetic variation within populations, because these are unlikely to lead to parallel changes in different populations (Ingvarsson 2002; Keller and Waller 2002). Such an increase in interpopulational differences may intensify genetic incompatibility between populations, potentially leading to reproductive isolation and speciation. Previous studies indicate that the C. elegans isolates from around the world show phenotypic differences but are interfertile and, thus, genetically compatible (Hodgkin and Doniach 1997).

In the past, the population biology of C. elegans has been analyzed using various molecular markers. However, most of them contained only little variation, including allozymes and DNA sequences of diverse nuclear loci and almost complete mitochondrial genomes (Butler et al. 1981; Thomas and Wilson 1991; Graustein et al. 2002; Denver, Morris, and Thomas 2003). Microsatellites represent a promising alternative because they are usually highly polymorphic, and their interpretation is facilitated because of the availability of well-established models of their evolutionary dynamics (Jarne and Lagoda 1996; Balloux and Lugon-Moulin 2002). One previous study was based on the analysis of dinucleotide microsatellite loci (Sivasundar and Hey 2003). The overall microsatellite variation among strains was found to be surprisingly small. Furthermore, although the data provided some evidence for geographic differentiation, the correlation between geographic and genetic distance was insignificant. These findings were suggested to be the result of background selection to reduce polymorphisms in conjunction with continuous gene flow among populations (Sivasundar and Hey 2003). In our study, we examined the population biology of C. elegans by characterizing 10 polymorphic trinucleptode and tetranucleotide microsatellite loci from across the whole genome. These loci were analyzed for 35 previously available and also 23 new C. elegans strains from northwest Germany.


    Material and Methods
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Isolation of Worms from Nature
Worms were isolated from compost heaps (usually 1-year-old soil) from three locations: (1) Roxel, (2) Mecklenbeck (both suburbs from Münster in Westphalia, northwest Germany; soil collected in summer 2002), and (3) Lingen (Emsland, northwest Germany; soil collected in spring 2002; distance to Münster: approximately 100 km). These locations were at least 5 km distant from scientific institutions, to avoid isolation of worms that escaped from laboratories. Worms were concentrated using the Baermann tray method (Flegg and Hooper 1970). Briefly, a spoon of wet soil was spread onto a 50-µm filter placed on top of a closed water column (autoclaved tap water) inside a plastic funnel. In response to evaporation over a 5-day to 7-day period, worms crawled from the soil into the water column, where they sank to the bottom and from where they were harvested for further analysis. Special care was taken to avoid contamination with existing C. elegans cultures (e.g., the isolation protocol was performed in a specific room where no other worm cultures were maintained) or cross-contamination between soil samples (e.g., equipment was cleaned with boiling, sterile water between separate isolations). The worm concentrate, which included many different nematodes and other organisms, was spread onto large plates (9 cm diameter) with nematode growth medium (NGM) inoculated with Escherichia coli strain OP50 (Stiernagle 1999). Within the next 6 hours, all worms resembling C. elegans were transferred individually onto small NGM plates (diameter: 6 cm), with E. coli OP50 and allowed to reproduce. All worm populations, thus, obtained were considered separate strains. Subsequently, two criteria were employed to identify C. elegans.

Criterion 1
The 18S ribosomal DNA (rDNA) sequences must be completely identical to the published C. elegans gene. Ten offspring per C. elegans candidate were used for DNA isolation. They were transferred to 25 µl PCR-buffer (50 mM KCl; 10 mM Tris-HCl pH 9.0; 0.1% Triton X-100, 2.5 mM MgCl2) and 2 µl proteinase K (10 mg/ml), frozen at –80°C for 2 h to break up tissue, followed by digestion at 60°C for 1 h and proteinase K deactivation at 95°C for 15 min. One microliter of DNA isolate was used for polymerase chain reaction (PCR). For a first PCR screen, we used the primer pair CE1209F (5'-TACTGTCAGTTTCGACTGACTC-3') and CE2250R (5'-ATACGAACCCGAAGATTCGCC-3'), which amplify about 1,000 bp of the 18S rRNA gene of only C. elegans and C. remanei. If no PCR product was obtained, the suitability of the DNA isolate was assessed with a second PCR using primers RHAB1350F (5'-TACAATGGAAGGCAGCAGGC-3') and RHAB1868R (5'-CCTCTGACTTTCGTTCTTGATTAA-3'), which amplify about 500 bp of 18S rDNA of rhabditid nematodes. PCRs were performed in 50 µl reaction volume containing PCR-buffer (see above), 0.8 mM dNTPs, 1 µM primers, 1 U Taq polymerase (Promega Ltd.), and using the following cycling profile: 2 min at 95°C, followed by 35 cycles of 20 s at 95°C, 1 min 55 °C, 1 min 30 s at 72°C (1 min for RHAB1350F X RHAB1868R), and a final extension of 10 min at 72°C. Positive results for the C. elegans–specific PCR (CE1209F X CE2250R) were purified with microcon-50 microconcentrators (Millipore Ltd), followed by DNA sequencing with the primers RHAB1350F and RHAB1868R, which bind within the product of CE1209F and CE2250R, and which encompass a highly variable 18S rDNA region. DNA sequencing reactions were performed with the ABI Prism BigDye Terminator Cycle Sequencing Kit (Applied Biosystems Ltd) including 100 to 500 ng purified PCR product, followed by visualization of results on an ABI PRISM 310 Genetic analyzer (Applied Biosystems Ltd.). At least a 400-bp overlapping sequence was obtained for each candidate and compared with published data using the program WU-Blast2 (http://www.ebi.ac.uk/blast2/).

Criterion 2
The predictions of the biological species concept must be fulfilled (i.e., interfertility of new strains with known C. elegans.) Hermaphroditic offspring from the C. elegans candidates were mated to males from the C. elegans strain PD4790. This strain bears an easily detectable dominant genetic marker: the gene for the green fluorescent protein fused with the C. elegans promoter for pharyngeal myosin, stably integrated into chromosome 3 (Praitis et al. 2001). Both outcrossed F1 and subsequently selfed F2 offspring were examined to ascertain that the new isolates can outcross with known C. elegans and that the resulting offspring are fertile.

Samples from each strain were preserved at –80°C (Stiernagle 1999) and deposited at the Caenorhabditis Genetics Centre (CGC; http://biosci.umn.edu/CGC) with the strain names MY1 to MY23.

Previously Available C. elegans Isolates
In addition to the new isolates, we included almost all currently available natural C. elegans strains (table 2). These strains were obtained from the CGC and maintained following standard procedures (Stiernagle 1999). The strains CB4858 and CB4555 originated from the same original isolate from Pasadena, California, but were sent to different laboratories, where they diverged in transposon Tc1 RFLP pattern (Hodgkin and Doniach 1997). A difference in the Tc1 pattern was also detected for the strains CB4851 and RW 7000, which originated from a single lineage isolated in Bergerac, France (Hodgkin and Doniach 1997). These four strains were included in the analysis. All other strains listed derive from different natural isolates. Three of these strains were only recently isolated and have not been included in any previous study. They include strain JU258, isolated by Marie-Anne Félix from a fruit and vegetable garden in Ribeiro Frio on Madeira (Portugal), and the strains JU262 and JU263, isolated by Marie-Anne Félix from the same soil sample from a vegetable garden garbage pile in Le Blanc (Indre, France). We could not include the large number of putative C. elegans strains isolated in Québec (Canada) by Abdul Kader and Côté (1996), because these strains were apparently not archived, and as such, they are not available.


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Table 2 Characteristics of Microsatellites Employed

 
Microsatellite Genotyping
We searched the C. elegans genome database (Stein et al. 2001) and the microsatellite list of Katti, Ranjekar, and Gupta (2001) to identify microsatellite loci with trinucleotide or tetranucleotide motifs and more than 12 repeats from the whole genome (i.e., one per chromosome arm). Trinucleotide and tetranucleotide microsatellites were chosen because they provide greater reliability than mononucleotide or dinucleotide microsatellites (e.g., Schlötterer and Tautz 1992; Chakraborty et al. 1997). Primers were designed for the respective flanking regions using the Primer3 version 0.9 program (Rozen and Skaletsky 2000), as accessible via http://www-genome.wi.mit.edu/genome_software/other/primer3.html. For the microsatellite locus II-L, published primers were used (Degtyareva et al. 2002). The microsatellite V-R could not be amplified with the originally designed primers for strains MY2, MY14, MY15, MY16, and MY23. For these strains, we generated the alternative forward primer CD43D7-ACCT-F2 (table 2). In addition, we could not establish a reproducible PCR protocol for microsatellites along two chromosomal regions: the right arm of chromosome I and the left arm of chromosome III. Therefore, our analysis was based on 10 different microsatellite loci (table 2).

For microsatellite analysis, DNA was isolated from about 1,000 worms of a fresh worm population using a modification of a CTAB-based protocol (Schulenburg et al. 2001). Briefly, worms were centrifuged and the resulting worm pellet ground with a pestle in 400 µl CTAB-buffer (2% w/v CTAB, 0.1 M Tris-HCl [pH 8.0], 0.02 M EDTA, 1.4 M NaCl, 0.5% v/v ß-mercaptoethanol). Tissue was further digested by adding 4 µl proteinase K (10 mg/ml) and incubating at 50°C overnight. Thereafter, DNA was extracted by adding 2 volumes of chloroform:isoamylalcohol (24:1) and centrifuging for 5 min at 13,000 rpm. DNA was precipitated by adding 2/3 volume of ice-cold 100% isopropanol, incubating at –20°C for 1 h, and centrifuging for 30 min at 13,000 rpm. The DNA pellet was washed with 70% ethanol and resuspended in 60 µl sterile H2O.

PCRs were performed in 20 µl volumes using the respective microsatellite primer pairs (table 2) and otherwise the same conditions as above. The following cycling profile was used: initial denaturation for 2 min at 95°C, then 35 cycles with 20 s at 95°C, 30 s at 60°C, 30 s at 72°C, and a final extension for 10 min at 72°C. In one case, namely the alternative primers of microsatellite V-R (see above), a touchdown PCR cycle was used. This cycle only differed from the above profile in that the annealing temperature was initially set to 60°C and then lowered by 0.3°C during each cycle and that the elongation time during cycling was 45 s. Some of the microsatellites were jointly amplified in a multiplex reaction: (1) V-R and IV-L and (2) X-R, V-L, and I-L. The remaining loci were always amplified separately. The fragment size of the PCR products was subsequently analyzed with the ABI PRISM 310 genetic analyzer. PCR was performed as above with one of the primers being fluorescently labeled, and 0.5 µl of the PCR products were loaded together with 0.3 µl TAMRA 500 length standard (Applied Biosystems Ltd.). For the microsatellite IV-R, which produces fragments larger than 500 bp, we used the TAMRA 2500 length standard (Applied Biosystems Ltd.). The run temperature was set to 60°C; the injection voltage was 15 kV; the injection time was varied between 5 and 15 s, depending on signal strength; and run voltage was set to 15 kV. Initial data analysis (collection of data, size calling, and assignment of repeat numbers) was performed with the ABI PRISM Data Collection, Genescan, and Genotyper software (Applied Biosystems Ltd.).

Thereafter, the DNA sequence of some microsatellites was determined to resolve the following ambiguities. The microsatellites IV-L and X-L had very short alleles, which could have resulted from changes in the repeat number of the microsatellite motif or from an unrelated deletion. In addition, the size of some long alleles of the microsatellite II-R could not be identified with absolute certainty. Furthermore, the inferred repeat number for strain N2 was different from the repeat number of the published complete-genome sequence of N2. Before sequencing, the PCR products were purified with microcon-50 microconcentrators and cloned into E. coli DH5{alpha} using the pGEM-T Vector System (Promega Ltd.). Thereafter, plasmids were isolated using the Wizard Minipreps DNA Purification System (Promega Ltd.). Approximately 300 ng of plasmid DNA was sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Kit and the standard plasmid primers (pUCF and pUCR [Promega Ltd.]). Sequencing reactions were run on the ABI PRISM 310 genetic analyzer. To take account of possible PCR errors, three independent clones per sample were used to generate consensus sequences. Three clones were considered to be sufficient for this because all C. elegans strains were homozygous for the different microsatellite loci.

Statistical Analysis
For statistical analysis, seven differently structured data sets were employed (table 3). Data set 1 included all strains without any substructure. Data set 2 was identical to data set 1 except for the exclusion of strain CB4852, for which the exact place of origin was unknown. Data sets 3 and 4 contained all strains from Europe and North America (see table 1), except that data set 4 lacked CB4852 (see above) and that data set 3 was subdivided according to geographic origin (Europe versus North America). Data set 5 only contained strains from northwest Germany, isolated in the current study. Data set 6 included only the strains from Europe, and data set 7 included only those from North America. They were both subdivided into two-strain isolation periods, whereby the cutoff point was chosen such that the two subsets were approximately the same size. Therefore, for the North American population, the cutoff date was set to 1980, resulting in one group with 12 strains isolated before and another group with nine strains isolated after 1980 (see also table 1). The European population contained few strains that were isolated before 2001. Hence, we set the cutoff date at 2001, resulting in one group of six strains being isolated before this date. Of the remaining 26 strains, only 14 were used to obtain a comparatively balanced data set. These 14 were chosen in consideration of the frequency of the underlying genotypes, and, thus, they included five of the most common genotypes from northwest Germany (MY3, MY4, MY5, MY7, and MY8), two of the two next common genotypes (MY15 and MY23; MY21 and MY22), one of each remaining genotype from northwest Germany, and the two strains from Le Blanc, France.


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Table 3 Composition of Data Sets Used for Analysis

 

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Table 1 Genotype and Origin of the Natural C. elegans Isolates

 
For a general characterization of the data, we calculated for each locus the number of different alleles, and for each data subset, the number of alleles, genotypes, polymorphic loci, and also the genetic diversity, the latter computed according to Nei (1987). In addition, we reconstructed a minimum-spanning network from data set 1 to illustrate the general relationships between the C. elegans genotypes. The relationships were inferred from RST (Slatkin 1995), which should produce more reliable results than the alternative FST (Wright 1951,1965), if a stepwise mutation model (SMM) applies (Balloux and Lugon-Moulin 2002). In general, this seems to be true for microsatellites (reviewed in Ellegren [2000] and Balloux and Lugon-Moulin [2002]), including those for which mutation patterns were directly estimated from C. elegans (Degtyareva et al. 2002).

We also assessed the ability of two population-genetic models to describe the pattern of microsatellite evolution in our data. These included the infinite alleles model (IAM) and the SMM (Balloux and Lugon-Moulin 2002). These models differ in their prediction of the expected microsatellite diversity (Estoup et al. 1995), which was calculated as the number of alleles per locus for each model, as previously described (Estoup et al. 1995; Sivasundar and Hey 2003), using data sets 1 (all strains) and 5 (only strains from northwest Germany). The difference between observed and expected diversity was then compared with the Wilcoxon signed rank test using the program SPSS version 11.0.1 (SPSS Inc., Chicago, Ill.).

Analysis of Genetic Differentiation
The genetic differentiation between data subsets (i.e., populations or strains isolated in different time periods) was investigated using an analysis of molecular variance (AMOVA) (Excoffier, Smouse, and Quattro 1992), as implemented in Arlequin version 2.0 (Schneider, Roessli, and Excoffier 2000), which takes into account both the frequency of and the genealogical relationship between genotypes. The fixation index was calculated as RST, defined by Slatkin (1995), following the argumentation above. The significance of genetic differentiation was assessed with a nonparametric permutation test, where haplotypes are randomly swapped between data subsets (i.e., populations), in this case using 1,000 permutations per test (Excoffier, Smouse, and Quattro 1992; Schneider, Roessli, and Excoffier 2000). The genetic differentiation between populations was assessed with data set 3, which excludes all populations for which only few and possibly nonrepresentative strains were available (Hawaii, Australia, and Madeira). The effect of sampling time on the genetic structure in the data was assessed with data sets 6 and 7, where both the North American and the European population were split into two groups (see above; table 3).

We also employed correlation analysis to assess the presence of geographic and temporal genetic differentiation, using the Mantel test (Mantle 1967; Smouse, Long, and Sokal 1986), as implemented in Arlequin version 2.0 (Schneider, Roessli, and Excoffier 2000). Data sets 2 and 4 were analyzed, which either included all strains or only those from Europe and North America, respectively. Data set 4 should produce more reliable results as it excludes the populations for which only single strains are available (Hawaii, Australia, and Madeira). For each data set, RST was used as the genetic distances, calculated from pairwise compared strains. Geographic distances between sampling sites were calculated with the program Koordinaten version 3.5 (http://www.koordinaten.de) and temporal distances from the known dates of isolation (table 1). For the estimation of the null distribution, one matrix was kept constant while rows and columns of the others were permuted. The number of permutation steps was set to 10,000. Full correlations were assessed for comparison of two data matrices, and partial correlations were assessed for three.

Assessment of the Presence of Recombination
The absence of linkage disequilibrium between loci was taken as a first indication for the occurrence of recombination. Linkage disequilibrium was assessed with an extension of the Fisher exact test for contingency tables (Slatkin 1994). As this test is only meaningful for intrapopulational comparisons, we used it for data set 5. The test requires that the haplotypic composition of the sample is known. This condition was fulfilled for the C. elegans strains because all loci studied were homozygous (see Results). For this test, we used a Markov chain with 100,000 steps, including 1,000 initial dememorization steps to start the chain at a random point. All above calculations were performed with the Arlequin version 2.0 program (Schneider, Roessli, and Excoffier 2000).

Recombination should also produce inconsistencies among the genotype relationships inferred from molecular markers, which differ in their mode of inheritance (e.g., biparentally nuclear microsatellite loci versus maternally transmitted mitochondrial DNA molecules). Therefore, we compared the genotype relationships inferred in the present study from microsatellites to those previously inferred from almost completely sequenced mitochondrial genomes (Denver, Morris, and Thomas 2003). We also took into account other previously analyzed nuclear genomic data sets, including about 44,000 bp of nuclear DNA sequences from 56 loci (Denver, Morris, and Thomas 2003), the nuclear glp-1 gene (Graustein et al. 2002), and 55 single-nucleotide polymorphism (SNP) loci (Koch et al. 2000).


    Results
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
New Natural Isolates from Northwest Germany
Based on the two criteria for identification of C. elegans, we isolated 23 new natural strains: 19 from Roxel, three from Mecklenbeck (both suburbs of Münster, northwest Germany), and one from Lingen (northwest Germany). The new strains yielded six distinct microsatellite genotypes that each differed from those of the previously isolated C. elegans strains (table 1). This clearly excludes the possibility of contamination with one of the previous isolates during the isolation process. In three cases, more than one strain derived from a specific soil sample (soil collected on a particular date), including (1) MY2 to MY13 (soil sample C), (2) MY15 to MY16 (soil sample K), and (3) MY17 to MY20 (soil sample S). The strains from the same soil sample may not be entirely independent because their founder animals could have been direct siblings of a single ancestor worm that reproduced during processing of a particular soil sample. Interestingly, the strains from one soil sample differ in microsatellite genotype. Four genotypes were identified among the 12 strains from soil sample C, two among the two strains from soil sample K, and three among the four strains from soil sample S (table 1). Therefore, strain multiplication during the isolation process may only be important in a very few cases.

Characteristics of Microsatellite Variation
Data were obtained for 58 C. elegans strains. For these, the 10 microsatellite loci were polymorphic (table 4). However, the locus I-L was only different for the strain JU258 from Madeira, with all other worm strains sharing an identical allele. The remaining nine microsatellite loci produced at least five different alleles with a maximum of 11 recorded for locus II-R (table 2).


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Table 4 General Characteristics of Microsatellite Variation for Different Data Subsets

 
Three unusual results were obtained. First, the locus IV-R could not be amplified from strain JU258, despite varying PCR conditions and use of alternative primers. This case was, thus, noted as a null allele. Second, for the standard C. elegans laboratory strain N2, six out of 10 loci produced differences in the number of repeat units from the published genome sequence, which was obtained from exactly the same strain. DNA sequencing revealed that two of these loci indeed showed a difference in repeat number: Locus IV-L had two repeats less than predicted (28 instead of 30), and locus X-L had one more than predicted (18 instead of 17). The DNA sequences of the four other loci did not show any differences from the published genome (neither repeat number nor the DNA sequence). This suggests that the observed deviations in fragment size are caused by some PCR artifact, possibly the fluorescent labeling of one of the primers. Third, the two microsatellite loci IV-L and X-L produced very short alleles for some of the strains. Here, DNA sequencing of cloned fragments from strain MY4 (eight repeats for IV-L) and CB4856 (six repeats for X-L) confirmed that the short alleles are not caused by deletions in the flanking regions or amplification of a different locus, but indeed result from a small number of repeat units.

All in all, 26 different genotypes were identified for the whole data set, with an overall genetic diversity of 0.93 (table 4). The populations from Europe and America were highly similar, including the same number of polymorphic loci, similar median numbers of alleles per locus, similar numbers of different genotypes, and almost identical values for genetic diversity. The latter values measured between 0.83 and 0.85, thus, documenting that the two populations are comparatively diverse. Interestingly, the newly isolated strains from northwest Germany, a clearly restricted geographic area, were still very diverse with nine polymorphic loci, a median allele number of three, six different genotypes, and a genetic diversity of 0.72 (table 4). The high level of diversity within populations is also well illustrated by the minimum-spanning tree, where the strains from either Europe or America or from the new northwest Germany population are distributed across the whole tree (fig. 1). Strains isolated in different time periods (early versus late) in either Europe or North America also showed similar levels of diversity. Recent isolates from Europe were slightly less variable than earlier isolates (from either after or before 2001, respectively). In contrast, in North America, strains isolated before 1979 were less diverse than those isolated after 1979 (table 4).



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FIG. 1.— Minimum-spanning network of the relationships of natural C. elegans isolates inferred with microsatellites. The network was inferred using RST as a measure of genetic distance (Note that netlike structures were absent in the inferred topology). Branches were drawn in proportion to the expected number of mutations (see scale in bottom right corner). Each circle refers to a particular genotype as listed in table 1. Circle size corresponds to the number of strains per genotype. The shading indicates the region of origin of the strains, where black denotes the new strains from northwest Germany (EU1–EU6), dark gray denotes the remaining strains from Europe (EU7–EU12), light gray denotes the strains from North America (NA1–NA8), and white denotes the remaining strains from Australia (AUS1–AUS4), Hawaii (PAC), and Madeira (ATL). The genotype labeled N2 includes the standard C. elegans strain N2 and a number of additional strains from both Europe and North America (table 1).

 
For the complete data set (data set 1, table 3), the observed microsatellite diversity was consistent with the predictions of the infinite allele model (IAM) (Wilcoxon signed rank test, Z = –0.051, N = 10, P = 0.859) but not with those of the stepwise mutation model (SMM) (Wilcoxcon signed rank test, Z = –2.803, N = 10, P = 0.008). A similar result was obtained for the population from northwest Germany (data set 5, table 3), where the IAM was consistent with the data (Wilcoxon signed rank test, Z = –1.599, N = 9, P = 0.110), but the SMM must be rejected (Wilcoxon signed rank test, Z = –2.547, N = 9, P = 0.011).

Analysis of Genetic Differentiation
The results from AMOVA highlight that there is significant genetic differentiation between the North American and European populations; 35.17% of the total genetic variability was the result of dividing the data set according to geographic regions (table 5). Similarly, there were significant differences among the strains isolated before and after 2001 in Europe. Here, 34.75% of the total variation resulted from subdivision into early and late isolation periods. In contrast, strains isolated before and after 1979 in North America did not vary significantly, with only 4.69% of the total variation being the result of subdivision of the data set (table 5).


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Table 5 Results for the Analysis of Molecular Variance (AMOVA)

 
An association of genetic differentiation with geographic distance and temporal differences in isolation dates was corroborated by the results of the Mantel test. All comparisons between geographic and genetic distance produced highly significant correlations, including those where the association was corrected for covariance with differences in the time of isolation (table 6). The correlation between temporal and genetic distance was significant for the reduced data set, which only included strains from Europe and North America (table 6). In two of the three other comparisons, the correlation was almost significant (table 6).


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Table 6 Results of the Mantel Test

 
Assessment of the Presence of Recombination
For the population from northwest Germany, linkage disequilibrium between pairwise compared loci was highly significant (Fisher's exact test, P < 0.001), except in two cases. Here, the probability of linkage disequilibrium was P = 0.0110 among X-L and X-R, or P = 0.5693 among V-R and X-L. The latter association was clearly nonsignificant. In this case, the population showed one common allele combination for these two loci, namely 31 and 15 repeat units for V-R and X-L, respectively. This combination was present in 16 strains. The rare exceptions may, thus, have been produced by recombination events (e.g., in the lineage leading to genotype EU2, EU4, and/or EU6 [fig. 2]).



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FIG. 2.— Allele distribution among the newly isolated strains from northwest Germany. Genotypes (GT) and their frequency (#) are as listed in table 1. The roman numbers refer to the C. elegans chromosomes and the boxes to the employed microsatellites on either the left or the right chromosome arm. For chromosomes I and III, only one microsatellite was used per chromosome; two loci were included for all others. The number of repeat units per locus is specified in the boxes. Note that all strains were homozygous, such that only one number is given per locus. The two cases where pairwise compared loci did not show significant linkage disequilibrium (after Bonferroni correction) are indicated at the bottom, including the exact P values, as inferred with the exact test of linkage disequilibrium. For these loci, the common alleles are given in white on a black background.

 
The comparison between the genotype relationships inferred here (fig. 1) and previously analyzed mtDNA and nuclear markers revealed three main inconsistencies, which may be the product of recombination events. Two of these cases refer to the position of the strains AB1 and CB4852. Here, all nuclear DNA analyses unequivocally showed them to belong to the genotype cluster that included strains N2 and RC301. However, if genotype relationships are inferred from mtDNA, they were found in a different cluster that additionally contained CB4858 and KR314 (fig. 3). In the third case, the strain CB4854 showed exactly the same microsatellite genotype as strain CB4858, thus, falling into a clade that excluded strains N2 or RC301 (table 1 and figs. 1 and 3). A similar pattern was observed for the glp-1 gene, although strains CB4854 and CB4858 were not identical, but still belonged to the same clade. In contrast, analysis of a large number of other nuclear loci, nuclear SNPs, and also mtDNA showed this strain to belong to the genotype cluster that included N2 and RC301 but not CB4858 or KR314 (fig. 3).



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FIG. 3.— Indication of outbreeding among the natural C. elegans strains, as inferred from the assignment of strains to the two main C. elegans clusters (black or gray) among different studies based on nuclear (microsat [this study], glp-1 [Graustein et al. 2002], SNPs [Koch et al. 2000], nDNA [Denver, Morris, and Thomas 2003]) or mitochondrial DNA (mtDNA [Denver, Morris, and Thomas 2003]). The strains N2 and RC301, on the one hand, and CB4858 and KR314, on the other hand, always belong to the same two distinct clusters, irrespective of the molecular marker analyzed. In contrast, strains AB1, CB4852, and CB4854 differ in their position among studies. Crosses indicate that a particular strain was not included in the respective study.

 

    Discussion
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Our study assayed microsatellite variation to obtain more detailed insights into the population genetics of one of the main model organisms. For this purpose, we isolated 23 new C. elegans strains from nature, thus, almost doubling the currently available number of natural isolates (Hodgkin and Doniach 1997). We then compared these new isolates to the previously isolated strains. We used 10 microsatellite loci, which were spread across the whole genome and were all polymorphic. Importantly, they provided sufficient resolution for the distinction of strain genotypes and the inference of their relationships. Assignment of strains to specific genotypes was generally consistent with previous studies, based on transposon polymorphisms (Hodgkin and Doniach 1997), snip-SNPs (Koch et al. 2000), nuclear DNA (Graustein et al. 2002; Denver, Morris, and Thomas 2003), or almost complete mitochondrial DNA sequences (Denver, Morris, and Thomas 2003). For instance, seven strains from North America (CB3191 to CB3195 and TR388 and TR389) are found to share the same genotype as N2; strains CB3198 and CB3199 or strains AB3 and AB4 are also consistently shown in each case to bear identical genotypes. In some cases, our microsatellites allowed distinction of strains previously found to be identical (e.g., AB2 versus AB3 and AB4, or RW7000 versus CB4851). The only cases where these microsatellites provided less resolution than previously studied markers were the three strains with high transposon frequencies (DH424, CB4555, and TR403). These strains shared the same genotype but differed in transposon polymorphism (Hodgkin and Doniach 1997).

Our study did not confirm the previous finding of extremely low levels of microsatellite variation by Sivasundar and Hey (2003). Their study assayed 20 dinucleotide-motif microsatellite loci, whereas we only considered trinucleotide-motif and tetranucleotide-motif microsatellites. Sivasundar and Hey (2003) found that for a subset of the strains included in the current study, 10 out of the 20 dinucleotide microsatellites (50%) were monomorphic (Sivasundar and Hey 2003). By contrast, for the same strains, we find that only one out of our 10 microsatellites is invariable (10%). The most likely reason for this discrepancy is that Sivasundar and Hey (2003) randomly selected the 20 dinucleotide microstallites, whereas we specifically selected microsatellites with at least 12 repeats to increase the likelihood that they are variable. This strategy followed the previous finding that the extent of microsatellite variation correlates with the number of repeat units (Ellegren 2000; Schlötterer 2000). In fact, in the study by Sivasundar and Hey (2003), fewer than 12 repeats are found for only one out of the 10 polymorphic but for seven out of the 10 monomorphic dinucleotide microsatellites (allele sizes inferred from N2; for further details see Sivasundar and Hey [2003]).

The variation observed at our microsatellite loci was consistent with the IAM but not the SMM. An identical result was also obtained for the dinucleotide microsatellite loci by Sivasundar and Hey (2003). In contrast, a previous laboratory-based mutation accumulation experiment reported that different C. elegans microsatellites, including one assayed in our study (locus II-L), do evolve according to the SMM (Degtyareva et al. 2002). There are two possible explanations for this difference. On the one hand, microsatellite evolution under laboratory conditions may differ from the pattern in nature. This may be supported by the fact that microsatellite evolution in the laboratory was assessed using a strain mutant in DNA repair (Degtyareva et al. 2002). On the other hand, the large majority of previously studied microsatellites in animals do conform to the SMM (reviewed in Ellegren [2000] and Balloux and Lugon-Moulin [2002]), in consistency with the results of Degtyareva et al. (2002). Therefore, Sivasundar and Hey (2003) and our findings may point to the presence of specific factors that bias the distribution of allele sizes in the natural sample, such that it no longer conforms to the predictions of the SMM. The most likely reason is that strains collected from the same location do not form a genetically homogenous population with a common origin in the recent past. This explanation may account for the surprisingly high genetic diversity among the 23 strains from northwest Germany, of which 22 were isolated almost at the same time from two compost heaps in Muenster (all in summer 2002; 19 strains from one compost heap in Roxel and three strains from the other in Mecklenbeck [table 1]). The combined presence of very diverse and apparently unrelated genotypes in close proximity is most likely the result of immigration events. However, it is as yet unclear how C. elegans can spread. It does possess a specific life-history stage, the dauer stage, which is ideally suited for dispersal because it is long-lived, very mobile, and highly resistant to a diversity of stressors, including starvation, heat, and toxins (Riddle and Albert 1997). It is conceivable that this life stage exploits other organisms (e.g., birds, mammals, terrestrial invertebrates, or field biologists) for long-distance dispersal. Interestingly, an association with terrestrial arthropods was suggested for two natural C. elegans isolates (strains PB303 and PB306; information only available from the CGC [http://biosci.umn.edu/CGC]), and such associations are known for other species of the genus Caenorhabditis (Kiontke 1997; Baird 1999; Kiontke, Hironaka, and Sudhaus 2002).

Otherwise, the deviation from the SMM in the global sample (data set 1) may indicate that the currently available strains do not encompass the whole range of genotypes as produced by single-step mutation events. Such a bias may be caused by the fact that only a limited number of strains were available for the different locations, especially for the more distant places such as Hawaii, Madeira, or Australia (table 1) in combination with considerable population subdivisioning. In addition, a bias may have also resulted from the differences in the time of isolation of strains (table 1), if there is a high turnover of genotypes per location. To further explore the underlying reasons for these observations, we examined the presence of genetic differentiation within our data set in relation to both geographic distance and differences in strain isolation time. Considering that microsatellites generally evolve according to the SMM in animals, including C. elegans (see above), this model was used for the respective analyses. We would like to emphasize that employment of the alternative IAM produced essentially identical results. In fact, the significance of inferred differences was generally more pronounced in the IAM-based than in the SMM-based analyses (results not shown).

The results inferred from AMOVA and the Mantel test convincingly demonstrate the presence of both spatial and temporal genetic differentiation. Here, geography seems to have a stronger impact, as it produces clearly significant results in all analyses. Interestingly, the relationship between geographic and genetic distance was weaker after correcting for the factor time in the Mantel test. In contrast, the association between genetic distance and differences in the time of strain isolation was significant in only some of the analyses. Importantly, this includes the Mantel test on the data set 4, which contains approximately equal numbers of strains from two geographic areas only (Europe and North America) and, thus, should yield more reliable results than the complete data set (data set 2). In this case, the effect was still almost significant after correction for the factor geographic distance. Taken together, these results highlight that the factor time may have an important effect on the inferred genetic differentiation between locations.

We would like to emphasize that these results are still consistent with migration between populations, as indicated above from close inspection of the new samples from northwest Germany (e.g., migration within continents). Both AMOVA and the Mantel test were mainly based on strains from two continents (Europe and North America), such that the inferred spatial genetic differentiation is primarily a consequence of differences between, not within, these continents. For these two tests, we decided against further subdividing the data, because this would have reduced reliability of results because of very small sample sizes and unbalanced groups (i.e., large versus small numbers of strains per geographic region). Small and unbalanced sample sizes may also account for the fact that Sivasundar and Hey (2003) obtained different results as to the significance of genetic geographic differentiation. In particular, Sivasundar and Hey (2003) based their analysis on a subdivided data set with a total of 23 natural strains, in which some populations consisted of only a single strain (e.g., Hawaii or Australia). Using AMOVA, they only found significant differentiation when the analysis was based on the SMM but not the IAM. Their Mantel test was insignificant. In contrast, we attempted to reduce possible biases by specifically focusing on data sets in which populations included a larger number of strains and had approximately the same size (i.e., focus on only the European and North American population in data sets 3 and 4).

Our results also support the conclusion of previous studies that C. elegans primarily reproduces via selfing in nature (Hodgkin and Doniach 1997). We found significant linkage disequilibrium between almost all loci within the sample from northwest Germany, as expected for selfing populations (Nordborg 2000; Ingvarsson 2002). Moreover, we observed a complete absence of heterozygosity within each of the natural strains. However, the latter result may represent an artifact caused by the isolation procedure and/or subsequent culturing in the laboratory. In both cases, selfing hermaphrodites may be favored over outcrossing conspecifics of the same lineage because they can reproduce faster under laboratory conditions and, thus, show higher population growth rates (e.g., Hodgkin and Barnes 1991; Chasnov and Chow 2002; Stewart and Phillips 2002). In turn, any within-strain heterozygosity present in the initial sample may have been rapidly lost as a consequence of simple stochastic processes in response to inbreeding within this period.

At the same time, our results strongly suggest the occurrence of rare outcrossing. A first indication comes from the absence of significant linkage disequilibrium among two pairs of microsatellite loci within the sample from northwest Germany. However, this result may also be explained by immigration of specific genotypes into northwest Germany. A second and much stronger line of evidence stems from the observation that certain strains are inferred from independent data sets to fall into different C. elegans genotype clusters. This evidence is most convincing for the strains AB1 and CB4852, where the differences are consistenly found between biparentally inherited nuclear DNA and the maternally inherited mtDNA. Such differences suggest that these strains are not the result of clonelike reproduction and, thus, exclusively maternally transmitted genomic material (as expected for selfing lineages) (see also Denver, Morris, and Thomas [2003]). The evidence is less clear for the strain CB4854, where only two out of four nuclear data sets generate different results than the mtDNA data set. Here, the detectable contribution of paternal material may be restricted to only part of the nuclear genome. Interestingly, AB1 and CB4852 were already previously indicated to represent recombinant strains. However, for only one of them, AB1, this supposition was based on the comparison of mitochondrial and nuclear genomes (Denver, Morris, and Thomas 2003). For CB4852, it was tentatively suggested from the comparison of the mtDNA-based phylogeny and the distribution of specific phenotypic traits along the phylogeny (Denver, Morris, and Thomas 2003). CB4854 represents a new case.

The comparison between our data set, which contains the largest number of strains among the nuclear DNA–based data sets, and the mtDNA data set by Denver, Morris, and Thomas (2003) indicates three out of 24 strains with genome mixing (12.5%). This estimate is likely to be conservative, because our approach would have failed to detect recombinant strains if the nuclear genomes of the two parental strains were very similar and fell into the same clade. These results imply that males do contribute genetically to C. elegans evolution and may, thus, have an adaptive value as vehicles for genetic exchange (see also Cutter, Aviles, and Ward [2003]). This clearly contrasts with the previous suggestion that C. elegans males are evolutionary relics (Chasnov and Chow 2002; Stewart and Phillips 2002). An example of the selective advantage of rare outcrossing over inbreeding is the improved ability to adapt to coevolving parasites: Using a theoretical approach, Agrawal and Lively (2001) showed that over almost the whole parameter space, hermaphrodite host species can persist in the presence of parasites if they show at least some outcrossing.

In conclusion, the analysis of polymorphic microsatellite loci and consideration of new natural isolates permits reconstruction of a more comprehensive picture of the evolutionary history of C. elegans. In particular, our data strongly suggest that C. elegans strains differentiate genetically depending on geographic distance and, to a lesser extent, on time. The latter result indicates a moderate but still detectable turnover of strain genotypes over time. Our results also highlight that extremely diverse genotypes coexist at single locations (e.g., in single compost heaps in northwest Germany), which is most likely the result of migration (e.g., within continents). Furthermore, we detected evidence for the occurrence of rare outbreeding. Such information about the worm's population history should be of great value for an in-depth understanding of the complexity of this species. In particular, consideration of the selective pressures encountered by C. elegans in nature may greatly help to understand many details of the morphology, development, physiology, or genetics of this species. Because this species can be efficiently investigated under laboratory conditions, and several natural isolates are available, C. elegans may provide a promising model system for the comprehensive analysis of natural variation in complex traits. For such analysis, our study may here facilitate choice of genetically distinct strains from either the whole world or a single population, such as the one from northwest Germany.

Finally, we would like to emphasize that many aspects of the natural history of this model organism cannot as yet be addressed in full detail, because only a limited number of strains is currently available, which have been isolated at different timepoints using different methods. Differences in the methods employed may potentially lead to a bias in the genotypes obtained. For instance, different periods of maintenance under laboratory conditions after isolation may lead to differences in mutation accumulation among strains. Therefore, in the future, it would be desirable to repeat this study using a larger sample and a more stringent approach. Our results highlight that in this context, it would be very promising to obtain C. elegans strains from single populations from within the same continent, to obtain reliable estimates on migration between populations and outcrossing rates within populations. Our results additionally emphasize that it is essential to collect strains more or less at the same time to avoid biased results caused by a possible turnover of strain genotypes at specific locations. Similarly, identical isolation protocols should be used to ascertain an unbiased recovery of the natural genetic diversity.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We are very grateful to Nico Michiels, Thomas D'Souza, Lukas Schärer, Ruza Bruvo, Stu Field, Martin Hasshoff, Gregor Schulte, and Iris Michiels for support and valuable advice on this project; to a student course in summer 2002 for their enthusiasm during the first worm isolation attempt; to Theresa Stiernagle of the Caenorhabditis Genetics Centre for sending us the previously isolated strains; to Asher Cutter, John Dennehy, Marie-Anne Félix, Jody Hey, Charles Baer, Claus-Peter Stelzer, Thomas D'Souza, and two anonymous reviewers for helpful comments on this manuscript; and to the German Science Foundation for financial support (DFG grant SCHU1415/1–1).


    Footnotes
 
Arndt von Haeseler, Associate Editor


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 Material and Methods
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Accepted for publication September 9, 2004.