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Maternal ancestry of the Rutilus alburnoides complex (Teleostei, Cyprinidae) as determined by analysis of cytochrome b sequences.

Unisexual lineages consisting predominantly or exclusively of females have been recognized among fishes, amphibians, and reptiles. Essentially all lineages appear to have formed through interspecific hybridization and exhibit reproductive systems that exclude effective genetic recombination (Dawley 1989). These lineages constitute a very small fraction of vertebrate diversity, with approximately only 70 taxa described (Vrijenhoek et al. 1989). Rarity of unisexuality in higher animals has been attributed to low origination rates balanced by high extinction probabilities. Some unisexuals are widely distributed and abundant, but this success has been thought to be restricted to recent evolutionary time, dependent on the opportunity for recruitment of new clones from the genetically variable sexual ancestors (Vrijenhoek 1979, 1984, 1989). In fact, Muller (1964) pointed out that in lineages lacking recombination, females could not produce offspring with fewer mutations than they had, leading to an accumulation of deleterious mutations (i.e., Muller's ratchet) and extinction of clonal organisms within [10.sup.4] to [10.sup.5] generations (Lynch and Gabriel 1990). Recently, however, several studies indicated that some unisexual lineages may have existed for longer than initially suspected (Hedges et al. 1992; Quattro et al. 1992; Spolsky et al. 1992; Schartl et al. 1995a), prompting further examination of the success of unisexual organisms. Substantial insight into the maintenance and evolution of asexual reproduction, and ultimately into the evolutionary significance of sex, can be obtained from understanding how such lineages persist.

An example of a successful unisexual vertebrate is Rutilus alburnoides, a cyprinid fish found in most major basins of the Iberian Peninsula of southwestern Europe [ILLUSTRATION FOR FIGURE 1 OMITTED]. This small fish is one of the most abundant and widespread minnows of Iberian freshwaters, achieving its highest densities in the southern drainages. Chromosomal studies by Collares-Pereira (1985) revealed that most individuals of this complex were triploid (75-80%) with only a small percentage (6.7%) of these triploids males. Diploids were rarer (20-25%) and more localized, exhibiting a normal sex ratio. Collares-Pereira (1985, 1989) used this information to hypothesize that this complex included a bisexual diploid species and a unisexual triploid lineage(s), although she did not know which mechanism(s) of unisexual reproduction was (were) involved. Rare tetraploids have been recently found in three different catchments, including both females and males (Gonzalez-Carmona et al. 1994; Alves et al. 1997; Martins et al., unpubl. data).

Allozyme analysis of R. alburnoides suggested that the entire complex of diploids, triploids, and tetraploids originated by hybridization (Alves et al. 1997). Although the diploid form could represent [F.sub.1] hybrids, some diploid populations exhibited sex ratios skewed toward females, indicating that they may represent a unisexual lineage(s) as well. Comparisons with other minnows from the region implicated members of the genus Leuciscus as one parent involved in production of R. alburnoides, as virtually all individuals exhibited one set of alleles also present in sympatric populations of Leuciscus at each variable locus (Alves et al. 1997), The taxonomy of the genus Leuciscus of the Iberian Peninsula is not well established (Doadrio 1987; Coelho et al. 1995), but recent studies of mtDNA variation (Brito et al. 1997) identified the existence of two allopatric species that coexist with R. alburnoides: L. carolitertii in the more northern Douro and Mondego basins, and L. pyrenaicus in the southern Tejo, Guadiana, and Sado basins. Additional alleles in R. alburnoides presumably contributed by the other parental taxon have been found, but it has not been possible to identify any sympatric species possessing genotype(s) predicted for the missing ancestor (Alves et al. 1997). A low number of R. alburnoides-like diploid males exhibiting the appropriate genotypes were collected in the Tejo and Guadiana basins (Alves et al. 1997; Pires et al., unpubl. data); however, these males may have been reconstituted from the unisexual hybrids (Bogart 1989; Hotz et al. 1992; Goddard and Schultz 1993). Therefore, the identity of the other taxon involved in the putative hybridization event(s) remains unclear.

To further examine the evolutionary history of R. alburnoides, we have characterized mitochondrial DNA (mtDNA) variation within and among populations of unisexual types and potential progenitors of this complex. Analysis of mtDNA, a maternally transmitted and rapidly evolving molecule, has provided unique insights into the history of unisexual taxa (reviewed by Avise et al. 1992). Accordingly, we characterized sequence variation of the cytochrome (cyt) b gene from the putative parental species L. carolitertii and L. pyrenaicus, representatives of all other endemic "diploid" sympatric minnow genera, and specimens of R. alburnoides representing different ploidy levels and drainage basins from throughout the distribution of the unisexual complex to examine hypotheses regarding the origin and maternal ancestry of this complex.


Collection of Samples. - Collection localities [ILLUSTRATION FOR FIGURE 1 OMITTED] were selected to sample most of the major basins across the range of the unisexual complex (detailed locality data is available from MMC). Multiple localities were sampled from the Tejo and Guadiana basins where R. alburnoides is very abundant. Samples of other cyprinids were obtained from the following sites: L. carolitertii, localities 2 and 3; L. pyrenaicus, localities 5, 6, 8, and 11; Rutilus arcasi, locality 1; Chondrostoma lemmingii, locality 8; and Anaecypris hispanica, locality 9. Samples were collected by electrofishing, killed in the field by exposure to an excess of MS222, and placed on dry ice until storage at -70 [degrees] C.

Ploidy Determination. - Ploidy of unisexuals was determined by flow cytometric measurement of erythrocyte DNA content, following methods described in Goddard and Dawley (1990). Blood samples were drawn from the caudal vein, stabilized in buffer (40 mM citric acid, trisodium salt, 0.25 M sacarose and 5% DMSO) and immediately frozen in liquid nitrogen.

DNA Extraction. - DNA was extracted from liver, heart, gonad or muscle following the equilibrium density gradient method for mtDNA isolation (Dowling et al. 1996) or the [TABULAR DATA FOR TABLE 1 OMITTED] standard SDS-proteinase K/phenol-chloroform procedure for total genomic DNA extraction (Hillis et al. 1996).

Sequencing Analysis. - The entire cyt b gene was sequenced for one diploid or triploid female R. alburnoides from localities 1, 3, 5-11 (n = 9); L. carolitertii from localities 2 and 3 (n = 2); L. pyrenaicus from localities 5, 6, 8, and 11 (n = 4); and one individual each of R. arcasi, C. lemmingii, and A. hispanica. The entire cyt b gene was amplified by polymerase chain reaction (PCR) using two sets of primers modified from those of Schmidt and Gold (1993): LA (5[prime]-GTGACTTGAAAAACCACCGTT-3[prime], position 15248 of Cyprinus carpio; Chang et al. 1994) with HD (5[prime]GGGTTGTTTGATCCTGTTTCG-3[prime], 15894 of C. carpio); and LDIB (5[prime]-ACCCTTGTTCAATGAATCTG-3[prime], 15767 of C. carpio) with HA (CAACGATCTCCGGTTTACAAGAC-3[prime], 16462 of C. carpio). Double-stranded DNA was produced by amplification in a 25 [[micro]liter] reaction volume containing 1.25 units of Taq DNA polymerase and appropriate concentration of supplied reaction buffer, 2.5 [[micro]molar] Mg[Cl.sub.2], each dNTP at 200 [[micro]molar], and each primer at 0.5 [[micro]molar]. Samples were cycled 20 times under the following conditions: 94 [degrees] C denaturation for 1 min, 48 [degrees] C annealing for 1 min, and 72 [degrees] C extension for 2 min. The double-stranded product was diluted 1:100 in sterile, purified [H.sub.2]O, and 5 [[micro]liter] of this dilution was used to initiate an asymmetric amplification with the appropriate L strand primer (i.e., LA or LDIB) diluted 1:100. These reactions were done in 100 [[micro]liter] volumes and followed the conditions given above, except for a higher annealing temperature (52 [degrees] C). Single-stranded amplification products were purified with Millipore Ultrafree-MC (NMWL: 30,000) filters, and sequenced directly by the dideoxynucleotide chain-termination method (Sanger et al. 1977), using the Sequenase kit (Version 2.0, United States Biochemical) and [Alpha]-32P labeling. The labeled products were run on 6% polyacrylamide-5M urea gels and exposed on X-ray film for approximately 14 h. As more sequence became available two internal primers were constructed for sequencing the second half of each amplification product: LCIB (5[prime]-CGAAGCCTACATGGCAATGG-3[prime], 15530 of C. carpio), and LEIB (5[prime]TACTTCTTATTTGCCTACGC-3[prime], 15918 of C. carpio).

DNA sequences were aligned using MacDNASIS (Vers. 3.0, Hitachi Software), and the identity of the sequenced fragments was confirmed by their alignment to the cyt b sequence of Lythrurus roseipinnis (Schmidt and Gold 1993). Estimates of sequence divergence were calculated by the method of Jukes and Cantor (1969) using the program MEGA (Kumar et al. 1993). Maximum parsimony analyses were conducted using PAUP (Vers. 3.1.1, Swofford 1993), using the North American cyprinid Notemigonus crysoleucas as outgroup (GenBank accession #U01318). Most parsimonious trees were obtained by heuristic search (MULPARS, tree bisection-reconnection method, 50 random addition sequences), weighting transitions and transversions equally. A strict consensus tree was calculated from the shortest length trees. Support for specific nodes was examined by generation of 1000 bootstrap replicates using parameters above, except that simple addition of taxa was used with one tree held per step.

RFLP Analysis. - Sequences of R. alburnoides were searched with MacDNASIS for restriction endonucleases that detected variable sites within the cyt b gene. Five enzymes were selected: BstNI, HaeIII, RsaI, StyI, and TaqI. Ten R. alburnoides were examined from each locality (1, 3-8, 10 and 11), including specimens of both sexes and different ploidy levels. Double-stranded PCR amplifications were performed for each individual in 100-[[micro]liter] reaction volumes and purified as described above; 5 [[micro]liter] of eluted PCR product was digested using 2-3 units of enzyme following the conditions recommended by the suppliers (GibcoBRL, New England Biolabs and Pharmacia Biotech). Restriction fragments were separated through 2% agarose gels, stained with ethidium bromide, and photographed under UV light. Fragment lengths were determined by comparison with a 100-bp molecular weight standard (Pharmacia Biotech) and each fragment profile was analyzed against sequences of R. alburnoides to determine the position of each restriction site change.

Inferred restriction site polymorphisms were used to define unique haplotypes among sampled individuals. Sequence divergence between haplotypes was estimated from the proportion of shared sites using the maximum-likelihood algorithm of Nei and Tajima (1983). Where haplotypes did not share sites for some classes (e.g., 6 bp) of enzymes, a divergence of 0.35 was substituted (McElroy, pers. comm.). Within-sample variation was estimated as haplotype and nucleotide diversity (Nei and Tajima 1981; Nei 1987). Because restriction enzymes were preselected, these estimates are valid only as indicators of relative genetic diversity within the survey presented here. Partitioning of mtDNA variation was accomplished by a hierarchical analysis of molecular variance, as implemented in the program WINAMOVA, (Ver. 1.55, Excoffier et al. 1992). This analysis produces estimates of variance components and analogs to F-statistics (Wright 1951, 1965), designated as [Phi]-statistics, reflecting the correlation of haplotypes at different levels of hierarchical subdivision. Grouping populations by basins, [[Phi].sub.ST] is the correlation of random haplotypes within populations, relative to that of random pairs of haplotypes drawn from the whole species; [[Phi].sub.CT] is the correlation of random haplotypes within populations from the same basin, relative to that of random pairs of haplotypes drawn from the entire species; and [[Phi].sub.SC] is the correlation of random haplotypes within populations, relative to that of random pairs of haplotypes drawn from the basin. The significance of the observed variance components and [Phi]-statistics were tested against the respective null distribution generated by 1000 random permutations.


Sequence Analysis

Sequences obtained in this study can be obtained directly from EMBL-European Bioinformatics Institute under the accession numbers X99421, X99425-X99432 for R. alburnoides, Y10135 and Y10136 for L. carolitertii, Y10131-Y10134 for L. pyrenaicus, X99424 for R. arcasi, X99423 for C. lemmingii, and X99422 for A. hispanica. Two hundred eighty-five variable sites were identified in the sequences of the cyt b gene (1140 bp) from the Iberian taxa, involving 328 murational events. Sequence variation was predominantly due to transitions (261), and no deletions or insertions were observed. Most (246) substitutions occurred in third codon position, with 33 in first codon position and 6 in second codon position. Nineteen amino acid replacement substitutions were observed. Inclusion of the outgroup species, N. crysoleucas, revealed sequence variability at an additional 27 nucleotide positions.

All nine individuals of R. alburnoides exhibited different haplotypes, differing by two to 29 substitutions (Table 1). Estimates of sequence divergence varied from 0.18% to 2.61%, with the largest differences found between the specimen from the Sado basin and all others. Levels of divergence between R. alburnoides and L. pyrenaicus haplotypes ranged from 0.09% to 2.69%, with the smallest values found between specimens from the same basin. Comparisons with L. carolitertii haplotypes revealed uniformly higher estimates of sequence divergence (5.12-6.15%), even when sympatric pairs were compared. Sequence divergences between R. alburnoides and the remaining sympatric taxa (R. arcasi, C. lemmingii, and A. hispanica) were high, ranging from 13.28% to 15.85%.

Parsimony analysis using phylogenetically informative characters resolved 10 equally parsimonious trees, requiring a minimum of 377 mutational steps (CI = 0.653, RI = 0.712). In the strict consensus tree (not shown), Leuciscus and R. alburnoides formed a monophyletic group, distinct from R. arcasi, C. lemmingii, and A. hispanica. The close relationship of Leuciscus and R. alburnoides was well supported in the bootstrap analysis [ILLUSTRATION FOR FIGURE 2 OMITTED] as this clade was found in 100% of all replicates. Within the Leuciscus and R. alburnoides clade, two major groups were strongly supported, one containing samples of L. carolitertii, and the second linking L. pyrenaicus and all unisexuals, independent of their ploidy. This second clade was subdivided into two additional groups, one including R. alburnoides and L. pyrenaicus from the Sado basin, and a second containing L. pyrenaicus from the Tejo and Guadiana basins and the remaining unisexuals. Bootstrap support for these latter two clades was high, occurring in 100% and 87% of the replicates. The branching pattern within the latter group exhibited further subdivision, with a sister group relationship for L. pyrenaicus and R. alburnoides from the Tejo basin and the unisexuals from the northern basins (67% of bootstrap replicates). Relationships among specimens from the Guadiana basin were weakly resolved, with most nodes exhibiting bootstrap values less than 50%.

RFLP Analysis

Restriction endonuclease analysis of cyt b from 90 R. alburnoides with five enzymes revealed 16 restriction sites, 12 of which were polymorphic, defining 13 different composite haplotypes (Table 2). Haplotype diversity of each sample was low, with most exhibiting only one haplotype, even where all three ploidy levels were found. Samples from Macas R. (Douro basin) and Degebe R. (Guadiana basin) exhibited three and four haplotypes, respectively, leading to moderately high estimates of haplotype diversity (0.51 and 0.64, respectively).

Most haplotypes from the same sample of R. alburnoides were similar, differing by one to four restriction sites, leading to low intrasample nucleotide diversities. However, one triploid from the Macas R. (Douro basin) differed from the other specimens of the sample by seven to eight restriction sites, displaying the combination of restriction sites indicative of a L. carolitertii mtDNA genome (haplotype III, Table 2). Accordingly, the Macas R. sample exhibited the highest nucleotide diversity (0.020). Sequence from two regions of the cyt b (positions 1-327 and 888-1105) differed from that of L. carolitertii from the Douro basin by only one transition event, indicating this R. alburnoides possessed the mtDNA of that species.

Hierarchical analysis of molecular variability showed substantial subdivision among populations ([[Phi].sub.ST] = 0.877, Table 3), keeping with the observation that many localities exhibited unique haplotypes. Most variance was found among basins (54.4%), but an appreciable amount was distributed among populations within basins (33.3%). Variation within populations accounted for 12.3% of total variance. All three variance components were significant.


Ancestry of the Unisexual Complex

Sequence and/or restriction site analysis of cyt b from specimens representing both sexes and all ploidy levels of the R. alburnoides complex, the putative parental species L. carolitertii and L. pyrenaicus, and members of other sympatric genera, revealed a monophyletic relationship among unisexuals and L. pyrenaicus. Accordingly, the present study clearly identifies this species as the maternal parent in the hybridization event(s) that led to the formation of the unisexual complex, leaving the "missing" ancestor as the paternal parent.

This unknown ancestor may remain unsampled (cyprinid populations from some small basins from the south of Spain have not been characterized) or may be extinct. There are several fossil taxa of the Iberian Peninsula whose closest relative appears to be R. alburnoides: the early Miocene Rutilus antiquus in the Ebro basin (Cabrera and Gaudant 1985), the middle Miocene Rutilus antunesi in the Tejo basin (Gaudant 1977), and the late Miocene Rutilus pachecoi in the [TABULAR DATA FOR TABLE 2 OMITTED] Douro and Turia basins (Doadrio 1981; Gaudant 1984). The latter two species were initially identified as belonging to the genus Leuciscus, but Doadrio (1981) transferred them to the genus Rutilus because of their uniseriated pharyngeal teeth, a feature shared with R. alburnoides.

Origin and Relative Age of the Unisexual Complex

All nine individuals of R. alburnoides sequenced exhibited different haplotypes, and RFLP analysis of additional specimens revealed seven more. Individuals of different ploidy levels from the same population possessed identical RFLP haplotypes, supporting a single origin for these lineages. Polyploids have most likely arisen through a hybrid intermediate of lower ploidy that produced unreduced ova subsequently fertilized by haploid sperm.

Mitochondrial DNA variability among unisexual populations exhibited geographical structure, as significant variation was found among populations and basins. A difficulty in interpreting genetic differences within R. alburnoides or any other unisexual form involves determining whether variation results from postformational mutation or reflects multiple hybrid origins from crosses involving divergent ancestors, as first pointed by Parker and Selander [TABULAR DATA FOR TABLE 3 OMITTED] (1976). On the other hand, distinction should be drawn between unique mtDNA origin and individual hybridization event. Two different females having the same mtDNA haplotype could hybridize to produce unisexual lineages (Quattro et al. 1991). Therefore, mtDNA provides a conservative test for multiple origins. The topology recovered from the analysis of cyt b variation among populations indicated that R. alburnoides is polyphyletic, sharing more features with L. pyrenaicus from the same drainage than congeners from other drainages. The data are consistent with the hierarchical model of genetic divergence predicted for unisexual taxa (Parker et al. 1989) in that unisexuals are more similar to the maternal parents from the same basin. This is most easily explained by origin of R. alburnoides through multiple, independent hybridization events involving different female ancestors; however, the number of such events is difficult to determine due to the weak phylogenetic resolution within the Tejo and the Guadiana subgroup. A minimum of two origins is strongly supported, one in the Sado basin and the other in the Tejo/ Guadiana basins, as these groups are supported as distinct monophyletic groups by a minimum of 16 and six synapomorphies, respectively. Even though the origin of unisexual lineages by hybridization is restricted by cytological and developmental constraints, multiple origins are possible once these constraints are overcome (Moritz et al. 1989; Vrijenhoek 1989).

Estimation of the relative age of an asexual lineage can be obtained by comparison of molecular characters with the closest sexual relatives (Avise et al. 1992). The low divergence level between the haplotypes of R. alburnoides and L. pyrenaicus from the Sado basin (0.09%) suggests a relatively recent origin for the unisexual complex in this basin. However, most remaining unisexuals show greater sequence divergence when compared with the genetically closest sexual haplotype assayed, suggesting that some unisexual lineages may be older. Alternatively, L. pyrenaicus haplotypes identical to those of R. alburnoides may not have been sampled, resulting in overestimates of relative age of formation (Avise et al. 1992). The small number of L. pyrenaicus analyzed (n = 6) and the high mtDNA diversity within this species found by Brito et al. (1997) make this last hypothesis more likely.

Origin of the Northern Populations

Although much less abundant, R. alburnoides is present outside the range of L. pyrenaicus, suggesting that it may have dispersed from the Tejo drainage into the northern basins. During the last Wurm glaciation, which occurred 16,000 to 18,000 years ago, sea level along the Atlantic Iberian coast was estimated to be 130-140 m below the present, resulting in the interconnection of adjacent rivers (Rodrigues and Dias 1989). Similar phenomena were possibly involved in determining present-day distribution pattern of many endemic fishes in the peri-Mediterranean region (Bianco 1990, 1995); however, there is no paleogeographic evidence indicating such a connection between the lower courses of the Mondego and the Tejo rivers (J. M. A. Dias, pers. comm.). Instability of the Mondego basin has been documented (Daveau 1976; Daveau et al. 1986; Lourenco 1986), therefore, R. alburnoides may have attained its northern distribution by stream capture between headwaters of the adjacent drainages or inversion of flow direction.

Some details of the allozyme data (Alves et al. 1997) conflict with the hypothesis that northern populations originated in the Tejo basin. Unisexuals from this basin exhibit fixed alternative alleles at the loci sIDPH-[1.sup.*] and sIDPH[2.sup.*], which have not been found in unisexual populations from the Mondego and Douro basins. In a survey of mtDNA variation in Leuciscus, Brito et al. (1997) found one specimen in a sample of four from the Mondego basin that exhibited pyrenaicus-like mtDNA. Therefore, R. alburnoides in the northern basins may be the product of an endemic hybridization event involving local Leuciscus carrying the pyrenaicus mtDNA, possibly even L. pyrenaicus, as the maternal ancestor.

Clonally reproducing fishes and amphibians depend on sperm from sexual hosts for successful reproduction, with males of one of the parental species normally serving as sperm donors (Dawley 1989). In the northern basins, where L. pyrenaicus does not occur and males of R. alburnoides are very rare (Collares-Pereira 1984; Alves et al. 1997), R. alburnoides has probably shifted to dependency on a non-parental taxon, most likely L. carolitertii. The observation of one of 20 northern R. alburnoides exhibiting carolitertii-like mtDNA provides some evidence for this hypothesis, suggesting the occurrence of occasional paternal leakage of mtDNA into a unisexual lineage. Paternal leakage of mtDNA seems to be linked to hybrid lines, due to the breakdown of species recognition systems that block the replication of the paternally inherited mtDNA (Avise 1991), and has been hypothesized by Avise and Vrijenhoek (1987) for the hybridogenetic Rana esculenta complex. Alternatively, an individual like this one (R. alburnoides with carolitertii-like mtDNA) could have resulted from a mating between an L. carolitertii female and an R. alburnoides male.


In the R. alburnoides complex de novo hybrid origins probably cannot occur because the paternal ancestor seems to be extinct or, at least, absent from most range of the unisexual complex. According to Vrijenhoek (1979, 1984, 1989), this would compromise the long-term survival of the unisexual complex as new lineages could not compensate the demise of individual lineages due to Muller's ratchet or the eventual disappearance of clonal subniches. Consequences of the impediment to creation of new lineages would be most striking if extensive environmental changes occurred; even a very large asexual population might not have a lineage with the appropriate genotype and become extinct (Crow 1992). However, recent studies described processes apparently responsible for additional genetic variability in unisexual populations: recombination has been hypothesized for several unisexuals (Bogart 1989; Graf and Polls Pelaz 1989; Parker et al. 1989; Sites et al. 1990), occasional occurrence of sex has been suggested in systems where unisexual males occur (Schmidt 1993), and incorporation of subgenomic amounts of DNA from a bisexual host in a gynogenetic fish has been described (Schartl et al. 1995b). The existence of several features, e.g., multiple ploidy levels, rare production of males, incorporation of paternal genome (Alves et al. 1996) and recombination (Alves et al. 1997) makes R. alburnoides a promising system for understanding how lineages may compensate for the disadvantages of asexuality and improve survival of unisexual populations.


We would like to thank the following people for their help with various aspects of this project: T Pacheco, R. Pires, L. M. Vieira, M. J. Martins, and E. Sousa for fish collecting; I. Prospero for ploidy determination; C. A. Tibbets for laboratory and technical assistance; and G. Naylor for data analysis. L. Excoffier kindly provided the computer program WINAMOVA (Version 1.55). We thank Instituto Florestal for permission to collect specimens. This work was supported by Centro de Biologia Ambiental and by grants from Junta Nacional de Investigacao Cientifica e Tecnologica to MJA (CIENCIA/BD/2185/92-RN and PRAXIS XXI/BD/5735/95) and MMC (PEAM/C/GAG/227/93) and the National Science Foundation to TED (DEB-9220683).


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Author:Alves, M.J.; Coelho, M.M.; Collares-Pereira, M.J.; Dowling, T.E.
Date:Oct 1, 1997
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