High Density of Long Dinucleotide Microsatellites in Drosophila subobscura

Marta Pascual3,*{dagger}, Malcolm D. Schug1,* and Charles F. Aquadro*

*Department of Molecular Biology and Genetics, Cornell University; and
{dagger}Departament de Genètica,Universitat de Barcelona, Barcelona, Spain

Abstract

We isolated 96 dinucleotide repeats with five or more tandemly repeated units from a subgenomic Drosophila subobscura library. The mean repeat unit length of microsatellite clones in D. subobscura is 15, higher than that observed in other Drosophila species. Population variation was assayed in 32–40 chromosomes from Barcelona, Spain, using 18 randomly chosen microsatellite loci. Positive correlation between measures of variation and perfect repeat length measures (mean size, most common, and longest allele) is consistent with a higher mutation rate in loci with longer repeat units. Levels of microsatellite variation measured as variance in repeat number and heterozygosity in D. subobscura were similar to those of Drosophila pseudoobscura and higher than those of Drosophila melanogaster and Drosophila simulans. Our data suggest that higher levels of microsatellite variation, and possibly density, in D. subobscura compared with D. melanogaster are due to both a higher average effective population and a higher intrinsic slippage rate in the former species.

Introduction

There has been significant recent interest in microsatellites in Drosophila, including Drosophila melanogaster (Goldstein and Clark 1995Citation ; Schlötterer, Vogl, and Tautz 1997Citation ; Schug, Mackay, and Aquadro 1997Citation ; Schug et al. 1998a, 1998b, 1998cCitation ; Bachtrog et al. 1999Citation ), Drosophila simulans (Hutter, Schug, and Aquadro 1998Citation ; Irvin et al. 1998Citation ), and, recently, Drosophila pseudoobscura and Drosophila persimilis (Noor, Schug, and Aquadro 2000Citation ). These studies have revealed that microsatellites in Drosophila are generally shorter than those in mammals and teleosts, although not all insects have short microsatellites (see review in Schug et al. 1998cCitation ). In addition, the mutation rate for microsatellite length in D. melanogaster is lower than that in humans and other mammals studied to date (Schug, Mackay, and Aquadro 1997Citation ; Schlötterer et al. 1998Citation ; Schug et al. 1998aCitation ).

Drosophila subobscura has been widely used in studies of population genetics, ecology, insect physiology, behavior, and biology in general (e.g., Krimbas 1993Citation ). This polyphagous species is widely distributed in the Palearctic region and appears to have colonized South and North America within the last several decades (Ayala, Serra, and Prevosti 1989Citation ). It has a high frequency of chromosomal rearrangements, some of them presenting latitudinal clines which are likely due to selection since they reestablished after the recent colonization of South and North America (Prevosti et al. 1988Citation ). Dispersal in this species is high. Mark-recapture experiments estimated the mean distance traveled during one activity period as 160 m (Serra, Pegueroles, and Mestres 1987Citation ). The colonization of South America has proved that dispersal can be very fast, since in less than a year it occupied a 2,000-km transect in the north-south direction (Prevosti et al. 1988Citation ). While chromosomal arrangements present latitudinal clines in the Old World, allozymes do not, unless in linkage disequilibrium with inversions, meaning that gene flow is high. Being a generalist species and having a high dispersal rate would be consistent with a large effective population size. We are interested in identifying highly variable markers distributed throughout the genome to study the genetics and evolutionary ecology of this species.

In this study, we report the identification and characterization of microsatellite markers in D. subobscura. We assay variation in a natural population to assess whether microsatellite markers can be informative for future studies in this species. We also compare and contrast microsatellite length, heterozygosity, and variance in repeat number between different Drosophila species and explore the relative influence of mutation rate and effective population size on differences in levels of genetic variation among several Drosophila species.

Materials and Methods

Genomic DNA Library Screen
Isolation and characterization of D. subobscura microsatellites followed the protocol described by Schug et al. (1998cCitation ) and Hutter, Schug, and Aquadro (1998)Citation . Genomic DNA of D. subobscura was extracted from a mixture of 28 isofemale lines from Barcelona, Spain, using the Puregene DNA isolation kit (Gentra), and partially digested with Sau3AI. DNA fragments between 400 and 600 bp in length were excised and extracted from a 1% agarose gel using glass beads (Quiaex II, Qiagen), dephosphorylated with calf intestine alkaline phosphatase (Boehringer Mannheim) as in Hutter, Schug, and Aquadro (1998)Citation , and cleaned with the QIAquick PCR purification kit (Qiagen). pUC18 was digested with BamHI, excised from an agarose gel, and purified with glass beads prior cloning. Ligations were carried out using a rapid DNA ligation kit (Boehringer Mannheim), with a ratio of 1 ng vector to 2 ng insert (1:18 pmol ratio). Ligations were purified using the QIAquick purification kit and transformed by electroporation into DH10B electrocompetent Escherichia coli cells (Gibco BRL). Approximately 17,600 white colonies were plated on LB/ampicillin/2% X-gal 150-mm petri dishes (22 plates with approximately 800 white colonies per plate) and lifted onto nylon membranes (Magna lift, MSI).

Denaturation of plasmid DNA and TEMAC hybridization were carried out as described by Schug et al. (1998cCitation ) with 100 ng each of (AC)15 and (AG)15 oligonucleotides end-labeled with [{gamma}-32P] ATP in a 15-µl reaction with 30 U of T4 polynucleotide kinase (USB). Washes prior to prehybridization were as described by Duby (1988)Citation , with a single high-temperature wash at 64°C. Following Schug et al. (1998c)Citation , prehybridizations were performed for 2 h at 64°C, and hybridizations with TEMAC were performed overnight (20 h) at the same temperature. Washes were as described by Jacobs and Celeste (1988)Citation . Nylon membranes were exposed to X-Omat AR film (Kodak) overnight at -80°C. Positive colonies in a secondary screening were sequenced on an ABI 377. All loci were named dsub#, where "#" refers to the number in the order in which the positive clones were picked. Multiple microsatellites within a single cloned DNA fragment were labeled consecutively by letter. Only dinucleotide repeats of five or more perfect units were used for further analysis.

Microsatellite Assay Condition and Allelic Variation
Primers were identified in the sequences flanking the microsatellites using primer 3 of Rozen and Skaletsky (http://www.genome.wi.mit.edu//cgi-bin/primer/primer3.cgi) so that the PCR products were approximately 110–280 bp in length. After optimization of the annealing temperature, one primer was end-labeled in a 7.5-µl reaction using 0.7 µl of primer (0.5 mM), 0.75 µl of 10 x buffer, 1.5 µl of [{gamma}-33P] ATP, and 0.5 µl of T4 polynucleotide kinase (60 min at 37°C and 10 min at 65°C). PCR was performed in a 10-µl reaction with 0.5 µl of each primer (10 µM), 1 µl dNTP's (2 mM), 1 µl 10 x buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, and 15 mM MgCl2), 1 U Taq polymerase, and 1 µl of DNA. A single soak at 95°C for 5 min was followed by 30 cycles of 1 min at 95°C, 30 s at 59–64°C, and 30 s at 72°C. Following PCR, 10 µl of formamide loading dye was added to each reaction, and 2.5 µl was loaded on a 5% acrylamide DNA sequencing gel. A pUC18 DNA sequencing reaction was run adjacent to the PCR products as a size standard. Dried gels were exposed to BIOMAX film (Kodak) for approximately 2 days.

Isofemale lines from Barcelona were surveyed for variation at 18 randomly chosen microsatellite loci using single fly squish preps (Gloor et al. 1993Citation ). For each strain, one individual of the first generation after collection from the wild was used. Analysis of males for most loci allowed these loci to be mapped to autosomes or the X chromosome.

Results

Genomic DNA Library Screen
Of the 17,600 white colonies plated, a total of 380 positive clones were identified, and 88 of them were sequenced. The sequences were visually scored and multiply aligned using the CLUSTAL W program that checks for both orientations (Thompson, Higgins, and Gibson 1994Citation ; available from TRANSFAC) to ascertain whether the clones were different. In three cases, we identified multiple colonies with the same cloned DNA fragments. For two of these cases, the sequence was identical, and for the third case, three alleles of the same locus were cloned and sequenced independently. In this case, we arbitrarily chose the first clone we sequenced for the remaining analysis. We note, however, that the majority of length variation among these sequences was due to differences in the number of microsatellite repeat units, and in all three cases, the number of repeats after an imperfection within the repeated unit was the same.

Of the 88 clones sequenced, 76 were different and contained one or more perfect dinucleotide repeats with five or more tandemly repeated units: 60 clones contained one repeat, 16 clones contained two repeats, and 2 clones contained three repeats. Thus, a total of 96 dinucleotide repeat units with five or more perfect tandemly repeated units were identified (table 1 ); 65 (67.7%) were AC repeats and 31 (32.3%) were AG repeats. The finding of 96 dinucleotide repeats identified in a total of 76 clones, with the average size of the insert in the sequenced clones being 525 bp (range 393–803 bp), leads to a library-based estimate that there is a dinucleotide repeat of five repeats or more approximately every 21 kb in the genome of D. subobscura. Density of white colonies with microsatellites was corrected for white colonies without insert and for loci appearing more than once. As an aside, nine trinucleotides and four tetranucleotides with five or more perfect repeats were also observed in our sequences. They are reported in table 1 for completeness but are not further analyzed.


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Table 1 Results of the Drosophila subobscura Library Screen

 

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Table 1 Continued

 
In figure 1 , we plotted the numbers and lengths of dinucleotide repeats in all clones identified in the library screen of D. subobscura, D. melanogaster (Schug et al. 1998cCitation ; unpublished results), D. simulans (Hutter, Schug, and Aquadro 1998Citation ), and D. pseudoobscura (Noor, Schug, and Aquadro 2000Citation ). The mean repeat lengths among the four species were significantly different (fig. 1 ; F = 11.44, df = 3, P < 0.001). A post hoc comparison of means using the least significant difference (LSD) test indicates that the difference is primarily due to a longer repeat length in D. subobscura compared with the other species, which were not significantly different from one another.



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Fig. 1.—Frequencies of different repeat length dinucleotide microsatellites in library screens of Drosophila subobscura (present study), Drosophila pseudoobscura (Noor, Schug, and Aquadro 2000Citation ), Drosophila melanogaster (Schug et al. 1998cCitation ), and Drosophila simulans (Hutter, Schug, and Aquadro 1998Citation ). N = number of loci; Mean = average repeat number per locus; Var = variance in repeat number across loci in each library screen

 
Allelic Variation
Allelic variation was surveyed at 18 randomly selected loci for a population sample of D. subobscura from Barcelona, Spain (table 2 ). The average size of the selected loci in the clones was 16.94 repeat units. One locus (dsub19) had primer pairs that amplified two dinucleotide repeats only 6 bp apart in the same PCR product. Repeat number was estimated by comparing the PCR fragment size of natural variants to that of the clone and assuming that all differences in length were due to different numbers of repeats. All changes in length were assumed to reflect differences in the repeat length of the longest perfect repeat. The number of alleles per locus ranges from 3 to 16 (mean = 8.8), the heterozygosity ranges from 0.317 to 0.949 (mean = 0.774), and the variance ranges from 0.225 to 31.554 (mean = 9.152). We tested whether each locus was at Hardy Weinberg equilibrium using GENEPOP, version 1.2 (Raymond and Rousset 1995Citation ). Equilibrium was rejected at five loci: dsub05, dsub04, dsub14, dsub16, and dsub20. An excess of homozygous individuals was found in these loci, possibly indicating the presence of null alleles (Pemberton et al. 1995Citation ).


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Table 2 Dinucleotide Repeat Variation and PCR Conditions

 
To test whether variation among microsatellites is a function of perfect repeat length or whether it is a function of the total length of the repeat regardless of interruption, we examined the correlation between three measures of population variation (variance in repeat number, heterozygosity, and number of alleles) and three measures of repeat length (maximum number of repeats [MAX], mean repeat number [MEAN], and most common allele [MCA]) using a nonparametric Spearman rank order test. We found significant positive association between most measures of variation and the three measures of length of the longest perfect repeat unit (table 3 ). Considering the entire repeat (including interruptions; given in table 2 ), the correlation between measures of variation and total repeat length measures was only significant for the longest allele in the population (tMAX; table 3 ).


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Table 3 Spearman Rank Order Correlations Between Measures of Variation and Measures of Population for Perfect Repeat Length and Total Repeat Length

 
Population Variation and Dinucleotide Repeat Lengths Among Drosophila Species
Our comparison of microsatellites in different Drosophila species indicates that D. subobscura has a longer mean repeat length in the clones screened from our microsatellite library than D. melanogaster, D. simulans, and D. pseudoobscura. To determine if these differences are characteristic of alleles found in natural populations, we compared mean allele size, variance in repeat number, and heterozygosity for 18 D. subobscura loci, 28 D. melanogaster loci (Schug et al. 1998bCitation ), 18 D. simulans loci (Hutter, Schug, and Aquadro 1998Citation ), and 10 D. pseudoobscura loci (Noor, Schug, and Aquadro 2000Citation ) with an ANOVA (table 4 ). We chose loci for which information on the repeat unit length from the library clone was available. The populations of D. melanogaster and D. simulans were from Zimbabwe, that of D. pseudoobscura was from Flagstaff, Ariz., and that of D. subobscura was from Barcelona, Spain. These population samples represented the ancestral range of each species. Since different numbers of individuals were used in the three studies and differences in variance were affected by sample size, we limited our D. subobscura samples to approximately 20 chromosomes, similar to the sample size in the studies of the other three species. Heterozygosity was arcsine-transformed, and variance in repeat number was log-transformed.


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Table 4 ANOVA Differences in Repeat Length and Population Variation Measures Between Drosophila subobscura, Drosophila pseudoobscura, Drosophila melanogaster, and Drosophila simulans

 
Differences among species in both heterozygosity and variance in repeat number were significant (table 4 ). Post hoc tests (LSD, Scheffe, and Newman-Keuls, implemented in STATISTICA, StatSoft Inc.) indicated that the strongest differences were for D. subobscura and D. pseudoobscura compared with D. melanogaster and D. simulans. There were no significant differences in mean repeat number among species when population variation was considered (fig. 2 and table 4 ). However, the mode of the repeat number is higher in D. subobscura than in the other three species.



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Fig. 2.—Frequencies of mean allele size for each locus from population surveys of Drosophila subobscura (present study), Drosophila pseudoobscura (Noor, Schug, and Aquadro 2000Citation ), Drosophila melanogaster (Schug et al. 1998bCitation ), and Drosophila simulans (Hutter, Schug, and Aquadro 1998Citation ). N = number of loci; Mean = average repeat number; Mode = most frequent repeat class

 
Discussion

Microsatellite Density Inferred from Library Screens
Dinucleotide repeats (five or more repeat units) appear to be more abundant on average in D. subobscura (one every 21 kb) than in D. melanogaster (one every 60 kb; Schug et al. 1998cCitation ) or D. simulans (one every 291 kb; Hutter, Schug, and Aquadro 1998Citation ). Density data are not available for D. pseudoobscura (Noor, Schug, and Aquadro 2000Citation ). The estimated density of dinucleotide repeats in D. subobscura is similar to that for the honeybee, the brown trout (Estoup et al. 1993a, 1993bCitation ), the rat, and the human (Stallings et al. 1991Citation ). It is unlikely that the differences in density among Drosophila species is due to different stringencies in DNA library screening procedures, since the subgenomic DNA libraries were constructed and screened using similar protocols in our lab. We do note, though, that we did not screen all of the species simultaneously. However, we do not believe that minor differences in the protocols account for our results. Hutter, Schug, and Aquadro (1998)Citation simultaneously screened a D. simulans and D. melanogaster subgenomic library slightly varying the protocol, as we have done, and found that the density of microsatellites in D. melanogaster was the same as that previously found by Schug et al. (1998c)Citation . This observation suggests that no systematic bias exists that would lead us to overestimate the density of microsatellites in D. subobscura.

The best estimate of microsatellite density will come from the analysis of large segments of DNA sequence from the genome of each species. Sufficient sequences do not exist at present. An alternative is to compare a collection of smaller homologous segments of the genome among species. Unfortunately, the data in GenBank at present are limited. However, there are 12 gene regions for which there is genomic sequence in both D. subobscura and D. melanogaster (only a few of these regions are available for D. simulans or D. pseudoobscura and are thus not compared). These comparisons are presented in table 5 , which shows that D. melanogaster has an average density of dinucleotide repeats of 1 per 5,518 bp (for microsatellites of five or more repeats), with an average length of 5.56 repeats. This estimate is in agreement with that obtained by Kruglyak et al. (1998)Citation for 1 Mb of D. melanogaster sequence (large continuous regions, therefore not biased by gene content) where the density was observed to be one microsatellite every 5,376 bp, with an average length of 6.35 repeats. Table 5 also shows that the density of microsatellites in D. subobscura is higher (on average, one every 3,978 bp), and they are longer on average (7.93 repeats).


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Table 5 Sequence Length and Number of Dinucleotide Repeats (Length of Repeat in Parentheses) to Genes Available in GenBank for Drosophila subobscura and Drosophila melanogaster

 
Returning to our clones from our library screens, the mean length of dinucleotide repeats is 14.94 in D. subobscura, compared with 10.39 for D. melanogaster (Schug et al. 1998cCitation ), 10.84 for D. simulans (Hutter, Schug, and Aquadro 1998Citation ), and 12.11 for D. pseudoobscura (Noor, Schug, and Aquadro 2000Citation ). (AC)n repeats are the most abundant in all of the Drosophila library screen, although the frequency of (AG)n units is higher in D. subobscura (32.3%) than in D. melanogaster (8%; Schug et al. 1998cCitation ), D. simulans (10%; Hutter, Schug, and Aquadro 1998Citation ), and D. pseudoobscura (7%; Noor, Schug, and Aquadro 2000Citation ). However, the longer repeat unit length we find in D. subobscura is not due to differences in repeat unit length among repeat types ((AC)n = 14.2 [range 5–34], (AG)n = 16.45 [range 5–36]).

The comparison of homologous gene regions in D. subobscura and D. melanogaster given in table 5 corroborates this inference of longer microsatellites in D. subobscura compared with D. melanogaster (7.93 vs. 5.56 repeats on average). Thus, while the library approach apparently underestimates density significantly, both library results and GenBank surveys indicate that microsatellites are longer and have a higher density in D. subobscura than in D. melanogaster.

Microsatellite Variability in a Natural Population
Dinucleotide repeat loci show high levels of heterozygosity and variance in repeat number and a large number of alleles in D. subobscura, similar to those found in D. pseudoobscura (Noor, Schug, and Aquadro 2000Citation ) but larger than in D. melanogaster (Wetterstrand 1997Citation ) or D. simulans (Irvin et al. 1998Citation ). There is no significant difference in mean repeat unit length among alleles segregating in populations between these species, although the mode suggests a longer repeat length in D. subobscura (fig. 2 ).

The strong correlation between variance and heterozygosity in our population survey and different perfect repeat length measures (MEAN, MAX, MCA, and number of alleles) suggests that long microsatellites have a higher mutation rate, as previously noted by Goldstein and Clark (1995) and Schug et al. (1998b)Citation for D. melanogaster. Furthermore, Schlötterer et al. (1998)Citation found nine mutations in a long perfect 28-repeat allele of one locus in a screen of 119 D. melanogaster lines maintained for 250 generations. These results are also consistent with in vitro studies of yeast, which indicate that long dinucleotide repeat units are more mutable than shorter dinucleotide repeats (Wierdl, Dominska, and Petes 1997Citation ).

Factors Contributing to Higher Microsatellite Variability in D. subobscura
The high levels of microsatellite variability observed in D. subobscura relative to D. melanogaster and D. simulans could be due to a larger effective population size (Ne) and/or a higher microsatellite mutation rate in D. subobscura. A mutation rate difference is a particular possibility given the tendency toward longer microsatellites in D. subobscura and the positive correlation between allele length and variability (presumably reflecting a length-dependent mutation rate). The challenge is to distinguish effective population size from mutation rate by their effects on variation. One approach is to compare Ne estimated from base pair polymorphism data with that estimated from microsatellites. This comparison assumes that the microsatellite mutation rate in D. subobscura is identical to that observed in D. melanogaster and relies on estimates of base pair mutation rate calculated from divergence data between species.

We estimated Ne for D. subobscura from DNA sequence data of rp49 (Rozas et al. 1999Citation ) and Acp70A (Cirera and Aguadé 1998Citation ) by rearranging the neutral equilibrium expectation {pi} = 4Neµ, since both genes are autosomal. The estimated rate of silent substitution per site per year for D. subobscura Adh is 1.28 x 10-8 (Marfany and González-Duarte 1993Citation ), and that for rp49 is 9.1 x 10-9 to 14.1 x 10-9 (Rozas et al. 1999Citation ). Using these estimates, Ne ranges from 2.5 x 105 to 3.8 x 105 for Acp70A and from 1.8 x 105 to 2.7 x 105 for rp49. We estimated Ne of D. subobscura from microsatellite data using the dinucleotide repeat mutation rate of D. melanogaster (9.3 x 10-6 per locus per generation) empirically determined by Schug et al. (1998bCitation ). The infinite-allele model (IAM; Kimura and Crow 1964Citation ) predicts H = 4Neµ/(1 + 4Neµ). The stepwise mutation model (SMM; Ohta and Kimura 1973Citation ) predicts H = 1 - (1/). A similar calculation can be done using the variance in repeat number (Var); Slatkin (1995)Citation has shown that Var = 4Neµ under a single-step stepwise mutation model (SMM(Var)). By rearranging the equations, solving for Ne at each locus, and averaging across all 18 loci, we obtain estimates for the Barcelona sample of D. subobscura (table 6 ). Our estimates of Ne from microsatellites for D. subobscura with IAM and SMM(Var) are similar to those obtained from single-copy nuclear genes for this species.


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Table 6 Estimates of Effective Population Size from Microsatellites and for Single-Copy Genes for Different Drosophila Species

 
The effective population size obtained from microsatellites for D. subobscura is similar to that of D. pseudoobscura but larger than those of D. melanogaster and D. simulans (table 6 ). A larger population size, and thus a greater range of allele lengths, in the obscura group species compared with the melanogaster group species could explain why the former has longer microsatellite alleles on average. However, effective population size estimated from sequences of single-copy nuclear genes in D. subobscura and D. melanogaster are similar to each other and smaller than D. pseudoobscura. While these data are limited, taken at face value, they suggest that the slippage rate per repeat unit that we used is correct for D. subobscura, but an overestimate for the other species.

Kruglyak et al. (1998)Citation recently presented a model of microsatellite evolution that demonstrates how differences in slippage rate per repeat unit can explain both density and variation in repeat length across different organisms. For example, the mouse has the highest predicted slippage rate, the highest microsatellite density, and the longest repeats, while yeast and D. melanogaster have the lowest predicted slippage rate, a low density, and a shorter average repeat length. Thus, the higher density and longer repeat length of microsatellites in D. subobscura compared with D. melanogaster, could be explained according to the Kruglyak et al. (1998)Citation model by a slightly higher slippage rate in D. subobscura. Our results suggest that the slippage rate per repeat unit might vary not only between distantly related taxa, but also between species belonging to the same genus. The possibility of having a different slippage rate per unit has to be taken into account when using microsatellites to make inferences about effective population size and the timing of demographic events or divergence between species.

Acknowledgements

This work was supported by a Postdoctoral Fellowship to M.P. from Ministerio de Educación y Ciencia, Spain, an NIH National Service Research Fellowship to M.D.S., and NIH grant GM36431 to C.F.A. We thank members of the Aquadro lab, L. Serra and his lab, and Rick Durrett for helpful discussion.

Footnotes

Pierre Capy, Reviewing Editor

1 Present address: Department of Biology, University of North Carolina–Greensboro. Back

2 Keywords: microsatellites Drosophila subobscura, variation mutation rate effective population size Back

3 Address for correspondence and reprints: Marta Pascual, Departament de Genètica, Universitat de Barcelona, Diagonal 645, 08028, Barcelona, Spain. E-mail: mpascual{at}porthos.bio.ub.es Back

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Accepted for publication May 4, 2000.