Identifying homomorphic sex chromosomes from wild‐caught adults with limited genomic resources

We demonstrate a genotyping‐by‐sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild‐caught individuals, in the moor frog Rana arvalis. Double‐digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex‐limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex‐linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co‐opted for sex determination among amphibians. The most efficient mapping strategy was a three‐step hierarchical approach, where R. arvalis reads were first mapped to a low‐coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex‐diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.     

Dmrt1 polymorphism and sex‐chromosome differentiation in Rana temporaria

Sex‐determination mechanisms vary both within and among populations of common frogs, opening opportunities to investigate the molecular pathways and ultimate causes shaping their evolution. We investigated the association between sex‐chromosome differentiation (as assayed from microsatellites) and polymorphism at the candidate sex‐determining gene Dmrt1 in two Alpine populations. Both populations harboured a diversity of X‐linked and Y‐linked Dmrt1 haplotypes. Some males had fixed male‐specific alleles at all markers (“differentiated” Y chromosomes), others only at Dmrt1 (“proto‐” Y chromosomes), while still others were genetically indistinguishable from females (undifferentiated X chromosomes). Besides these XX males, we also found rare XY females. The several Dmrt1 Y haplotypes differed in the probability of association with a differentiated Y chromosome, which we interpret as a result of differences in the masculinizing effects of alleles at the sex‐determining locus. From our results, the polymorphism in sex‐chromosome differentiation and its association with Dmrt1, previously inferred from Swedish populations, are not just idiosyncratic features of peripheral populations, but also characterize highly diverged populations in the central range. This implies that an apparently unstable pattern has been maintained over long evolutionary times.     

ANOLIS SEX CHROMOSOMES ARE DERIVED FROM A SINGLE ANCESTRAL PAIR

To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex‐determining mechanisms. We used model‐based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X‐linked bacterial artificial chromosome (BAC) and quantitative PCR of X‐linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long‐term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

Dmrt1 polymorphism covaries with sex‐determination patterns in Rana temporaria

Patterns of sex‐chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern‐boreal population of Ammarnäs displayed well‐differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y‐specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female‐biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co‐option of small genomic regions that harbor key genes from the sex‐determination pathway.     

No evidence that Y‐chromosome differentiation affects male fitness in a Swiss population of common frogs

The canonical model of sex‐chromosome evolution assigns a key role to sexually antagonistic (SA) genes on the arrest of recombination and ensuing degeneration of Y chromosomes. This assumption cannot be tested in organisms with highly differentiated sex chromosomes, such as mammals or birds, owing to the lack of polymorphism. Fixation of SA alleles, furthermore, might be the consequence rather than the cause of recombination arrest. Here we focus on a population of common frogs (Rana temporaria) where XY males with genetically differentiated Y chromosomes (nonrecombinant Y haplotypes) coexist with both XY° males with proto‐Y chromosomes (only differentiated from X chromosomes in the immediate vicinity of the candidate sex‐determining locus Dmrt1) and XX males with undifferentiated sex chromosomes (genetically identical to XX females). Our study finds no effect of sex‐chromosome differentiation on male phenotype, mating success or fathering success. Our conclusions rejoin genomic studies that found no differences in gene expression between XY, XY° and XX males. Sexual dimorphism in common frogs might result more from the differential expression of autosomal genes than from sex‐linked SA genes. Among‐male variance in sex‐chromosome differentiation seems better explained by a polymorphism in the penetrance of alleles at the sex locus, resulting in variable levels of sex reversal (and thus of X‐Y recombination in XY females), independent of sex‐linked SA genes.     

High‐density linkage maps fail to detect any genetic component to sex determination in a Rana temporaria family

Sex chromosome differentiation in Rana temporaria varies strikingly among populations or families: whereas some males display well‐differentiated Y haplotypes at microsatellite markers on linkage group 2 (LG₂), others are genetically undistinguishable from females. We analysed with RADseq markers one family from a Swiss lowland population with no differentiated sex chromosomes, and where sibship analyses had failed to detect any association between the phenotypic sex of progeny and parental haplotypes. Offspring were reared in a common tank in outdoor conditions and sexed at the froglet stage. We could map a total of 2177 SNPs (1123 in the mother, 1054 in the father), recovering in both adults 13 linkage groups (= chromosome pairs) that were strongly syntenic to Xenopus tropicalis despite > 200 My divergence. Sexes differed strikingly in the localization of crossovers, which were uniformly distributed in the female but limited to chromosome ends in the male. None of the 2177 markers showed significant association with offspring sex. Considering the very high power of our analysis, we conclude that sex determination was not genetic in this family; which factors determined sex remain to be investigated.     

A reciprocal translocation radically reshapes sex‐linked inheritance in the common frog

X and Y chromosomes can diverge when rearrangements block recombination between them. Here we present the first genomic view of a reciprocal translocation that causes two physically unconnected pairs of chromosomes to be coinherited as sex chromosomes. In a population of the common frog (Rana temporaria), both pairs of X and Y chromosomes show extensive sequence differentiation, but not degeneration of the Y chromosomes. A new method based on gene trees shows both chromosomes are sex‐linked. Furthermore, the gene trees from the two Y chromosomes have identical topologies, showing they have been coinherited since the reciprocal translocation occurred. Reciprocal translocations can thus reshape sex linkage on a much greater scale compared with inversions, the type of rearrangement that is much better known in sex chromosome evolution, and they can greatly amplify the power of sexually antagonistic selection to drive genomic rearrangement. Two more populations show evidence of other rearrangements, suggesting that this species has unprecedented structural polymorphism in its sex chromosomes.     

Genomic conflict drives patterns of X‐linked population structure in Drosophila neotestacea

Intragenomic conflict has the potential to cause widespread changes in patterns of genetic diversity and genome evolution. In this study, we investigate the consequences of sex‐ratio (SR) drive on the population genetic patterns of the X‐chromosome in Drosophila neotestacea. An SR X‐chromosome prevents the maturation of Y‐bearing sperm during male spermatogenesis and thus is transmitted to ~100% of the offspring, nearly all of which are daughters. Selection on the rest of the genome to suppress SR can be strong, and the resulting conflict over the offspring sex ratio can result in the accumulation of multiple loci on the X‐chromosome that are necessary for the expression of drive. We surveyed variation at 12 random X‐linked microsatellites across 16 populations of D. neotestacea that range in SR frequency from 0% to 30%. First, every locus was differentiated between SR and wild‐type chromosomes, and this drives genetic structure at the X‐chromosome. Once the association with SR is accounted for, the patterns of differentiation among populations are similar to the autosomes. Second, within wild‐type chromosomes, the relative heterozygosity is reduced in populations with an increased prevalence of drive, and the heterozygosity of SR chromosomes is higher than expected based on its prevalence. The combination of the relatively high prevalence of SR drive and the structuring of polymorphism between the SR and wild‐type chromosomes suggests that genetic conflict because of SR drive has had significant consequences on the patterns of X‐linked polymorphism and thus also probably affects the tempo of X‐chromosome evolution in D. neotestacea.     

RECENT GENE‐CAPTURE ON THE UV SEX CHROMOSOMES OF THE MOSS CERATODON PURPUREUS

Sex chromosomes evolve from ordinary autosomes through the expansion and subsequent degeneration of a region of suppressed recombination that is inherited through one sex. Here we investigate the relative timing of these processes in the UV sex chromosomes of the moss Ceratodon purpureus using molecular population genetic analyses of eight newly discovered sex‐linked loci. In this system, recombination is suppressed on both the female‐transmitted (U) sex chromosome and the male‐transmitted (V) chromosome. Genes on both chromosomes therefore should show the deleterious effects of suppressed recombination and sex‐limited transmission, while purifying selection should maintain homologs of genes essential for both sexes on both sex chromosomes. Based on analyses of eight sex‐linked loci, we show that the nonrecombining portions of the U and V chromosomes expanded in at least two events (～0.6–1.3 MYA and ～2.8–3.5 MYA), after the divergence of C. purpureus from its dioecious sister species, Trichodon cylindricus and Cheilothela chloropus. Both U‐ and V‐linked copies showed reduced nucleotide diversity and limited population structure, compared to autosomal loci, suggesting that the sex chromosomes experienced more recent selective sweeps that the autosomes. Collectively these results highlight the dynamic nature of gene composition and molecular evolution on nonrecombining portions of the U and V sex chromosomes.     

Recent Y chromosome divergence despite ancient origin of dioecy in poplars (Populus)

All species of the genus Populus (poplar, aspen) are dioecious, suggesting an ancient origin of this trait. Despite some empirical counter examples, theory suggests that nonrecombining sex‐linked regions should quickly spread, eventually becoming heteromorphic chromosomes. In contrast, we show using whole‐genome scans that the sex‐associated region in Populus trichocarpa is small and much younger than the age of the genus. This indicates that sex determination is highly labile in poplar, consistent with recent evidence of ‘turnover’ of sex‐determination regions in animals. We performed whole‐genome resequencing of 52 P. trichocarpa (black cottonwood) and 34 Populus balsamifera (balsam poplar) individuals of known sex. Genomewide association studies in these unstructured populations identified 650 SNPs significantly associated with sex. We estimate the size of the sex‐linked region to be ~100 kbp. All SNPs significantly associated with sex were in strong linkage disequilibrium despite the fact that they were mapped to six different chromosomes (plus 3 unmapped scaffolds) in version 2.2 of the reference genome. We show that this is likely due to genome misassembly. The segregation pattern of sex‐associated SNPs revealed this to be an XY sex‐determining system. Estimated divergence times of X and Y haplotype sequences (6–7 Ma) are much more recent than the divergence of P. trichocarpa (poplar) and Populus tremuloides (aspen). Consistent with this, in P. tremuloides, we found no XY haplotype divergence within the P. trichocarpa sex‐determining region. These two species therefore have a different genomic architecture of sex, suggestive of at least one turnover event in the recent past.     

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome‐arm identification,integration of genetic linkage groups and analysis of major repeat family distribution

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North–South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome‐specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S‐5.8S‐25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber‐FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.     

Local dynamics of a fast‐evolving sex‐ratio system in Drosophila simulans

By distorting Mendelian transmission to their own advantage, X‐linked meiotic drive elements can rapidly spread in natural populations, generating a sex‐ratio bias. One expected consequence is the triggering of a co‐evolutionary arms race between the sex chromosome that carries the distorter and suppressors counteracting its effect. Such an arms race has been theoretically and experimentally established and can have many evolutionary consequences. However, its dynamics in contemporary populations is still poorly documented. Here, we investigate the fate of the young X‐linked Paris driver in Drosophila simulans from sub‐Saharan Africa to the Middle East. We provide the first example of the early dynamics of distorters and suppressors: we find consistent evidence that the driving chromosomes have been rising in the Middle East during the last decade. In addition, identical haplotypes are at high frequencies around the two co‐evolving drive loci in remote populations, implying that the driving X chromosomes share a recent common ancestor and suggesting that East Africa could be the cradle of the Paris driver. The segmental duplication associated with drive presents an unusual structure in West Africa, which could reflect a secondary state of the driver. Together with our previous demonstration of driver decline in the Indian Ocean where suppression is complete, these data provide a unique picture of the complex dynamics of a co‐evolutionary arms race currently taking place in natural populations of D. simulans.     

Sequence diversity patterns suggesting balancing selection in partially sex‐linked genes of the plant Silene latifolia are not generated by demographic history or gene flow

DNA sequence diversity in genes in the partially sex‐linked pseudoautosomal region (PAR) of the sex chromosomes of the plant Silene latifolia is higher than expected from within‐species diversity of other genes. This could be the footprint of sexually antagonistic (SA) alleles that are maintained by balancing selection in a PAR gene (or genes) and affect polymorphism in linked genome regions. SA selection is predicted to occur during sex chromosome evolution, but it is important to test whether the unexpectedly high sequence polymorphism could be explained without it, purely by the combined effects of partial linkage with the sex‐determining region and the population's demographic history, including possible introgression from Silene dioica. To test this, we applied approximate Bayesian computation‐based model choice to autosomal sequence diversity data, to find the most plausible scenario for the recent history of S. latifolia and then to estimate the posterior density of the most relevant parameters. We then used these densities to simulate variation to be expected at PAR genes. We conclude that an excess of variants at high frequencies at PAR genes should arise in S. latifolia populations only for genes with strong associations with fully sex‐linked genes, which requires closer linkage with the fully sex‐linked region than that estimated for the PAR genes where apparent deviations from neutrality were observed. These results support the need to invoke selection to explain the S. latifolia PAR gene diversity, and encourage further work to test the possibility of balancing selection due to sexual antagonism.     

Evolution of karyotype,sex chromosomes,and meiosis in mygalomorph spiders (Araneae: Mygalomorphae)

Spider diversity is partitioned into three primary clades, namely Mesothelae, Mygalomorphae, and Araneomorphae. Mygalomorph cytogenetics is largely unknown. Our study revealed a remarkable karyotype diversity of mygalomorphs. Unlike araneomorphs, they show no general trend towards a decrease of 2n, as the chromosome number was reduced in some lineages and increased in others. A biarmed karyotype is a symplesiomorphy of mygalomorphs and araneomorphs. Male meiosis of some mygalomorphs is achiasmatic, or includes the diffuse stage. The sex chromosome system X₁X₂0, which is supposedly ancestral in spiders, is uncommon in mygalomorphs. Many mygalomorphs exhibit more than two (and up to 13) X chromosomes in males. The evolution of X chromosomes proceeded via the duplication of chromosomes, fissions, X–X, and X‐autosome fusions. Spiders also exhibit a homomorphic sex chromosome pair. In the germline of mygalomorph males these chromosomes are often deactivated; their deactivation and pairing is initiated already at spermatogonia. Remarkably, pairing of sex chromosomes in mygalomorph females is also initiated at gonial cells. Some mygalomorphs have two sex chromosome pairs. The second pair presumably arose in early‐diverging mygalomorphs, probably via genome duplication. The unique behaviour of spider sex chromosomes in the germline may promote meiotic pairing of homologous sex chromosomes and structural differentiation of their duplicates, as well as the establishment of polyploid genomes. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 377–408.     

Triploidy in a sexually dimorphic passerine provides new evidence for the effect of the W chromosome on secondary sexual traits in birds

In birds, there are two main models for the determination of sex: the ‘Z Dosage’ model in which the number, or dose, of Z chromosomes determines sex, and the ‘Dominant W’ model which argues that a specific gene in the W chromosome may influence Z gene expression and determine sex. The best evidence for W determination of sex comes from birds with 2 copies of the Z chromosome paired with a single W (e.g. ZZW) which are nonetheless females. Here, we expand the species where such a mechanism may operate by reporting a case of a triploid Neotropical passerine bird with sexually dimorphic plumage, the São Paulo marsh antwren Formicivora paludicola. Evidence from 17 autosomal unlinked microsatellite loci, and CHD1 sex‐linked locus, indicate that this individual is a 3n ZZW triploid with intermediate plumage pattern. This example expands our knowledge of sex determination mechanisms in birds by demonstrating that both the W and the two Z chromosomes affect the expression of morphological secondary sexual traits in a non‐galliform bird.     

Evolution of sex chromosomes prior to speciation in the dioecious Phoenix species

Understanding the driving forces and molecular processes underlying dioecy and sex chromosome evolution, leading from hermaphroditism to the occurrence of male and female individuals, is of considerable interest in fundamental and applied research. The genus Phoenix, belonging to the Arecaceae family, consists uniquely of dioecious species. Phylogenetic data suggest that the genus Phoenix has diverged from a hermaphroditic ancestor which is also shared with its closest relatives. We have investigated the cessation of recombination in the sex‐determination region within the genus Phoenix as a whole by extending the analysis of P. dactylifera SSR sex‐related loci to eight other species within the genus. Phylogenetic analysis of a date palm sex‐linked PdMYB1 gene in these species has revealed that sex‐linked alleles have not clustered in a species‐dependent way but rather in X and Y‐allele clusters. Our data show that sex chromosomes evolved from a common autosomal origin before the diversification of the extant dioecious species.     

The Drosophila simulans Y chromosome interacts with the autosomes to influence male fitness

The Y chromosome should degenerate because it cannot recombine. However, male‐limited transmission increases selection efficiency for male‐benefit alleles on the Y, and therefore, Y chromosomes should contribute significantly to variation in male fitness. This means that although the Drosophila Y chromosome is small and gene‐poor, Y‐linked genes are vital for male fertility in Drosophila melanogaster and the Y chromosome has large male fitness effects. It is unclear whether the same pattern is seen in the closely related Drosophila simulans. We backcrossed Y chromosomes from three geographic locations into five genetic backgrounds and found strong Y and genetic background effects on male fertility. There was a significant Y‐background interaction, indicating substantial epistasis between the Y and autosomal genes affecting male fertility. This supports accumulating evidence that interactions between the Y chromosome and the autosomes are key determinants of male fitness.     

Linkage Analysis Reveals the Independent Origin of Poeciliid Sex Chromosomes and a Case of Atypical Sex Inheritance in the Guppy (Poecilia reticulata)

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.     

Amplification of a long terminal repeat-like element on the Y chromosome of the platyfish, Xiphophorus maculatus

The platyfish (Xiphophorus maculatus), in which sex chromosomes are evident from stable and predictable inheritance of sex, is one of the best-studied lower vertebrates with respect to sex determination. In order to identify the structural equivalent for this in the karyotype, which does not contain heteromorphic pairs of chromosomes, two sex-linked molecular probes were used for fluorescent in situ hybridization analysis. One probe, derived from the melanoma oncogene locus ONC-Xmrk, stained both the X and the Y chromosome. This cytogenetic analysis mapped the sex-determining locus to the subtelomeric region of a medium-sized telocentric chromosome. Another probe, a repetitive element (XIR), specifically labeled the Y chromosome in metaphase spreads and in interphase nuclei. The sex chromosomes of X. maculatus can be considered to be at an early stage of evolution of gonosomes. Expansion of the XIR repeat is obviously one of the earliest of the molecular events that lead to divergence of the Y chromosome and recombinational isolation of the sex-determining locus. Received: 10 December 1999; in revised form: 20 January 2000 / Accepted: 24 January 2000