Low Levels of Nucleotide Diversity in Mammalian Y Chromosomes
http://www.100md.com
分子生物学进展 2004年第1期
Department of Evolutionary Biology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
E-mail: hans.ellegren@ebc.uu.se.
Abstract
Sex chromosomes provide a useful context for the study of the relative importance of evolutionary forces affecting genetic diversity. The human Y chromosome shows levels of nucleotide diversity 20% that of autosomes, which is significantly less than expected when differences in effective population size and sex-specific mutation rates are taken into account. To study the generality of low levels of Y chromosome variability in mammalian genomes, we investigated nucleotide diversity in intron sequences of X (1.1–3.0 kb) and Y (0.7–3.5 kb) chromosome genes of five mammals: lynx, wolf, reindeer, cattle, and field vole. For all species, nucleotide diversity was found to be lower on Y than on X, with no segregating site observed in Y-linked sequences of lynx, reindeer, and cattle. For X chromosome sequences, nucleotide diversity was in the range of 1.6 x 10–4 (lynx) to 8.0 x 10–4 (field vole). When differences in effective population size and the extent of the male mutation bias were taken into account, all five species showed evidence of reduced levels of Y chromosome variability. Reduced levels of Y chromosome variability have also been observed in Drosophila and in plants, as well as in the female-specific W chromosome of birds. Among the different factors proposed to explain low levels of genetic variability in the sex-limited chromosome (Y/W), we note that selection is the only factor that is broadly applicable irrespective of mode of reproduction and whether there is male or female heterogamety.
Key Words: sex chromosomes ? effective population size ? male mutation bias ? selective sweeps ? background selection
Introduction
Population genetics theory predicts levels of nucleotide diversity to be determined by a number of factors, including selection, rate of mutation, recombination, and effective population size. Studying the relative importance of these factors in shaping genetic variability is not without difficulties because the factors can be hard to quantify and their effects difficult to disentangle. However, under some circumstances intragenomic comparisons may offer a suitable study design, and one such example concerns studies of the levels of nucleotide diversity on sex chromosomes. In systems with male heterogamety (males XY, females XX), the X and Y chromosomes show distinct characteristics when it comes to the relative magnitude of mutation, recombination, and effective population size. First, while the Y chromosome is exposed to mutations that have arisen in the male germline only, both male and female mutations hit the X chromosome. Given knowledge of m, the male-to-female mutation rate ratio, the relative mutation rate on X and Y can thereby be inferred. Second, as Y is a non-recombining chromosome (with the exception of the pseudoautosomal region), recombination obviously plays different roles on X and Y. Third, under random mating, the effective population size of the Y chromosome is three times smaller than that of the X.
A number of studies have shown that levels of nucleotide diversity on the human Y chromosome are lower than in the rest of the genome (Malaspina et al. 1990; Dorit, Akashi, and Gilbert 1995; Hammer 1995; Whitfield, Sulston, and Goodfellow 1995; Nachman 1998; Anagnostopoulos et al. 1999; Jaruzelska, Zietkiewicz, and Labuda 1999; Shen et al. 2000; The International SNP Map Working Group 2001). This lower incidence of nucleotide diversity may be related to human biology—e.g., sex differences in patterns of migration or biased mating—but may also reflect a general feature of mammalian Y chromosomes or, even more generally, of species with male heterogamety. To address the generality of low Y chromosome variability, we initiated screening for single-nucleotide poplymorphisms (SNPs) in noncoding sequence from the Y chromosome of five mammalian species: lynx (Lynx lynx), wolf (Canis lupus), reindeer (Rangifer tarandus), cattle (Bos taurus), and field vole (Microtus agrestis). Taxonomically, these species are spread in the mammalian phylogenetic tree, and they also differ in terms of population history and mating system. To be able to compare Y chromosome variability with a measure of nucleotide diversity in other parts of the genome from each of the species, which are poorly characterized in terms of intraspecific levels of polymorphism, we also screened for SNPs in noncoding X chromosome DNA sequences. Here we show that lower than expected levels of Y-linked nucleotide diversity are found in all five species, indicating that reduced levels of Y chromosome polymorphism may be a generalized feature of mammalian genomes.
Materials and Methods
Tissue samples of all five species were extracted with a standard phenol/chloroform protocol. Introns from the Y-linked DBY, SMCY, UBE1Y, UTY, ZFY, and SRY genes (table 1) were amplified using the primers and conditions described in Hellborg and Ellegren (2003). Primers for amplification of introns of the X-linked SMCX, DBX, and ZFX genes (table 2) were designed based on the alignment of available mammalian sequence followed by identification of conserved sites and regions (cf. Lyons et al. 1997). We used a standard polymerase chain reaction (PCR) profile of 95°C for 10 min, followed by 20 cycles of 95°C for 30 s and a touchdown from 65°–55°C, 60°–50°C, or 55°–45°C for 1 min, decreasing 0.5°C per cycle (see table 2), and then 72°C for 1.30 min. These steps were followed by 20 cycles of 95°C for 30 s; 55°C, 50°C, or 45°C for 1 min; and 72°C for 1.30 min, and a final extension step of 72°C for 10 min. Amplification reactions contained 2.5 mM MgCl2, 5 μM of each primer, 2 μM dNTP, and 0.25 U AmpliTaq Gold polymerase (PerkinElmer). Female DNA was used for amplification of X-linked introns. Amplification products of both Y-linked and X-linked introns were screened for polymorphism using either single strand conformation polymorphism (SSCP) analysis (products shorter than 350 bp) or DNA sequencing (in both directions). Different electromorphs identified by SSCP were subsequently sequenced. Amplification products were purified with Qia-Quick PCR purification kit (Qiagen), sequenced using BigDye Terminator Cycle Sequencing chemistry (PerkinElmer), and sequences recorded with an ABI377 semi-automated sequencing instrument (PerkinElmer). Sequences were edited and assembled with AUTOASSEMBLER (Applied Biosystems).
Table 1 Y Chromosome Introns, Primer Sequences Used for Their Amplification, and Size of Amplification Products (in bp) in the Different Species.
Table 2 X Chromosome Introns, Primer Sequences Used for Their Amplification, and Size of Amplification Products (in bp) in the Different Species.
Samples, and their origin, used for polymorphism screening were as follows (numbers refer to the number of males screened; half the number of females were generally used from each geographic region/breed): Lynx: Baltic States (14 animals), Scandinavia (14), Finland (8), and Switzerland (4). Wolf: Russia (14), Scandinavia (10), Baltic States (3), Finland (3), North America (3), Spain (2), and Italy (1). Field vole: Sweden (6), England (3), Denmark (2), Russia (2), Romania (1), Norway (1), and Finland (1). Reindeer: Sweden (6), Alaska (4), Norway (4), Russia (4), and Svalbard (2). Cattle: Swedish Red Polled (2), Swedish Mountain cattle (2), Swedish Red-and-White (2), Swedish Friesian cattle (2), and Hereford (2). Sample size thus varied between species, but the same number of X and Y chromosomes were always screened for each species. Measures of genetic variability, including number of segregating sites per base pair (k) and nucleotide diversity (), and their standard deviations, were calculated in DnaSP (Rozas and Rozas 1999).
Results and Discussion
Levels of Polymorphism in Mammalian X and Y Chromosomes
We surveyed between 0.7 and 3.5 kb of intronic Y chromosome sequence and between 1.1 and 3.0 kb of intronic X chromosome sequence for intraspecific polymorphism in population samples of five mammals. There is evidence for mutation rate variation within mammalian genomes (Matassi et al. 1999; Williams and Hurst 2000; Lercher, Williams, and Hurst 2001; Smith, Webster, and Ellegren 2002), so we chose to study a number of different introns, derived from different genes, rather than a single longer noncoding sequence from each chromosome. Levels of genetic variability were considerably lower on the Y chromosome than on the X chromosome for all five species (tables 3 and 4). In fact, for three of them (lynx, reindeer, cattle), the surveyed Y chromosome sequence was completely monomorphic. For the X chromosome, where 1–7 segregating sites were found per species, the estimated nucleotide diversity (X) ranged from 1.6 x 10–4 (lynx) to 8.0 x 10–4 (field vole). Compared to sequence variation in the human genome, where X is estimated at 4.7 x 10–4 (The International SNP Map Working Group 2001), the lynx shows relatively limited variability (an observation supported by microsatellite data; Hellborg et al. 2002), whereas the opposite situation is suggested for the field vole. For the two species where SNPs were found in Y-linked introns, nucleotide diversity (Y) was estimated at 0.4 x 10–4 (wolf, two segregating sites in 1.6 kb) and 1.7 x 10–4 (field vole, four segregating sites in 3.2 kb), respectively. In humans, Y has been estimated at 1.5 x 10–4 (The International SNP Map Working Group 2001).
Table 3 Summary Statistics for Y Chromosome Variability.
Table 4 Summary Statistics for X Chromosome Variability.
The Effect of Population Size and Sex-Specific Mutation Rates on X and Y Chromosome Variability
As indicated above, estimates of nucleotide diversity on X and Y are not directly comparable. Nucleotide diversity—the normalised measure of heterozygosity—estimates the population genetic parameter theta (), which in a model assuming neutrality, random mating, and a population of constant size can be described by = 4Neμ, Ne being the effective population size and μ the mutation rate. For X, X = 3NeμX and for Y, Y = NeμY, the difference in effective population size reflecting X being hemizygous in males and Y being restricted to the male genome. From this finding, we should expect nucleotide diversity to be three times lower on Y than on X. It is worth emphasising that the expected 3:1 relationship for the effective population size of X and Y is only true under the assumption of random mating and equal numbers of the sexes. However, this may be an invalid assumption if the reproductive success among males is skewed, as may sometimes be the case (see further below). Under such a sex bias in mating, the relative effective population size of Y (compared with X) becomes even smaller.
Although differences in effective population size should thus be expected to lead to low levels of Y chromosome variability, differences in chromosome-specific mutation rates should have the opposite effect, given that μY is typically higher than μX as a result of male-biased mutation (Hurst and Ellegren 1998; Li, Yi, and Makova 2002), and perhaps other factors affecting chromosome-specific mutation rates (Lercher, Williams, and Hurst 2001). Species-specific estimates of the male-to-female mutation rate ratio (m) are not available for the species presented here, but some indication is given by data from other mammals. The most recent estimate of human m is at a factor of 5 (Makova and Li 2002). In Felidae—i.e., including the lynx—Pecon-Slattery and O'Brien (1998) estimated m at 4. For rodents, m is estimated at about 2 (Chang et al. 1994; Chang and Li 1995). The extent of the male bias is likely to be positively correlated with generation time (Chang et al. 1994; Li et al. 1996; Bartosch-H?rlid et al. 2003), and available data would thus suggest that mammalian m ranges approximately from 2 (short-lived species like most rodents) to 5 (humans). Here, we assume m 4 in wolf, cattle, lynx, and reindeer, and 2 in field vole. The corresponding μY/μX ratios are 2.0 and 1.5, respectively [note that μY/μX = m/(1/3m + 2/3)]. Now, combining the effects of differences in effective population size and mutation rate (X/Y = 3μX/μY), we should expect 1.5 times higher nucleotide diversity on X than on Y for wolf, cattle, lynx, and reindeer, and 2.0 times higher for field vole.
Any deviation from the expected difference in nucleotide diversity between X and Y would suggest that other factors are differentially affecting polymorphism levels on the two sex chromosomes. Figure 1 shows the adjusted levels of nucleotide diversity on X when differences in effective population size and mutation rate have been accounted for (i.e., when X is divided by 1.5 for wolf, cattle, lynx, and reindeer, and by 2.0 for field vole). The three species monomorphic for Y—lynx, cattle, and reindeer—obviously remain less variable on Y than on X; for these species the lower 95% confidence interval for X does not include 0. Unfortunately, the absence of observed polymorphism on Y prevents statistical analysis of Y chromosome data. For the two species with observed polymorphism on Y—wolf and field vole—nucleotide diversity also remains lower on Y than on X after compensating for differences in effective population size and mutation rate (fig. 1), although there is overlap in the 95% confidence intervals for X and Y. In summary, our data reveal a general trend of lower than expected levels of nucleotide diversity in mammalian Y chromosomes.
FIG. 1. Nucleotide diversity (±95% confidence intervals) in Y chromosome (filled bars) and X chromosome (open bars) introns. Observed levels of nucleotide diversity for X chromosome sequences have been divided by 1.5 (lynx, wolf, reindeer, and cattle) or 2.0 (field vole) to take differences in effective population size and sex-specific mutation rates into account
Factors Explaining Reduced Levels of Y Chromosome Variability
Factors potentially able to explain low levels of Y chromosome variability include selection, mating system, or migration patterns, or other mechanisms lowering male effective population size. The non-recombining part of Y effectively behaves as a single locus (haplotype) and is thus susceptible to the influence of selective forces acting on any sequence on the chromosome, with the exception of the pseudoautosomal region/s. This contrasts sharply with the situation on autosomes (in particular) and the X chromosome, where recombination releases sequences from association with a locus under selection. This well-recognized phenomenon (reviewed by Charlesworth and Charlesworth 2000) may act either in the direction of positive selection where advantageous mutations sweep through the population (genetic hitchhiking; Rice 1987) or in the direction of purifying (background) selection (Charlesworth, Charlesworth, and Morgan 1995) whereby chromosomes containing deleterious mutations are eliminated from the gene pool. For instance, strong directional selection on male-specific traits encoded by genes on the Y chromosome (like testis-specific genes involved in male reproduction) may introduce selective sweeps, driven by sexual selection (Roldan and Gomendio 1999; Wyckoff, Wang, and Wu 2000).
The effective population size of Y will be lowered by a high variance in male reproductive success (relative to that of females) (Caballero 1995; Nagylaki 1995; Charlesworth 1996). This may occur in polygynous species (Greenwood 1980), where a few males father a disproportionably large fraction of all offspring. One particular situation when this should apply is in the case of domestic animals; for most of these species, a limited number of males is typically used in breeding programs. The low levels of Y chromosome variability that we found in cattle could be attributed, at least in part, to a strong sex bias in breeding. Another mechanism that has been suggested to lower the effective population size of males is deleterious mutations in mitochondrial DNA (mtDNA) (Gemmel and Sin 2002). Because energy demand for spermatogenesis and sperm motility is very high, much higher than for oogenesis, and because the main function of mitochondrion is to produce ATP through oxidative phosphorylation, sperm may be particularly sensitive to mutations in mtDNA. Such sensitivity could lead to impaired sperm function and thereby lowered male reproductive success.
The nucleotide diversity of the human Y chromosome is about 20% of that found in autosomes (Shen et al. 2000; The International SNP Map Working Group 2001). This is less than expected when differences in effective population size and mutation rate are taken into account. [The International SNP Map Working Group (2001) predicts that Y would have a diversity 31% that of the autosomes; however, they use an m estimate of 1.7 based on Bohossian, Skaletsky, and Page (2000), while a more recent study has estimated m at 5 (Makova and Li 2002). Using the latter estimate we should expect nucleotide diversity on Y be 42% that of the autosomes, thus strengthening the interpretation of lower than expected variability on human Y.] Together with the extensive data available from the human genome, and reports of comparatively low levels of Y chromosome variability in apes (Hammer 1995; Burrows amd Ryder 1997; Stone et al. 2002), our data thus indicate that the mammalian Y chromosome may generally have reduced levels of genetic variability. Moreover, similar observations have been made for Y chromosomes of Drosophila (McAllister and Charlesworth 1999; Bachtrog and Charlesworth 2000) and even of plants (Filatov et al. 2000, 2001). Furthermore, in birds, which have a reversed sex chromosome organization characterized by female heterogamety (females ZW, males ZZ), the W chromosome is very low in nucleotide diversity (Montell, Fridolfsson, and Ellegren 2001; Berlin and Ellegren 2001). These combined data would suggest that a reduced level of genetic variability is a common feature of the sex-limited chromosome (Ellegren 2003), in turn suggesting a common mechanism underlying this phenomenon. Of the potential causative factors listed above, we note that selection is the only one that is broadly applicable irrespective of mode of reproduction or whether there is male or female heterogamety. [High variance in male reproductive success cannot explain the low levels of variability in the female-specific W chromosomes of birds, and lowered male effective population size due to mutations in mtDNA is not applicable to avian and plant systems.] We therefore favor the idea that selection is a key factor in the evolution of nucleotide diversity on the sex-limited chromosome. Unravelling the relative importance of, on the one hand, background selection and, on the other, selective sweeps in shaping genetic variability on the sex-limited chromosome will be an important question for future research in this area.
Acknowledgements
We are grateful to Annika Einarsson, Kristin Eliasson, and Frank Hailer for technical assistance, and to Hannah Sundstr?m and Carles Vilà for helpful comments. Financial support was obtained from the Swedish Research Council and the Nilsson-Ehle Foundation. H.E. is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation.
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E-mail: hans.ellegren@ebc.uu.se.
Abstract
Sex chromosomes provide a useful context for the study of the relative importance of evolutionary forces affecting genetic diversity. The human Y chromosome shows levels of nucleotide diversity 20% that of autosomes, which is significantly less than expected when differences in effective population size and sex-specific mutation rates are taken into account. To study the generality of low levels of Y chromosome variability in mammalian genomes, we investigated nucleotide diversity in intron sequences of X (1.1–3.0 kb) and Y (0.7–3.5 kb) chromosome genes of five mammals: lynx, wolf, reindeer, cattle, and field vole. For all species, nucleotide diversity was found to be lower on Y than on X, with no segregating site observed in Y-linked sequences of lynx, reindeer, and cattle. For X chromosome sequences, nucleotide diversity was in the range of 1.6 x 10–4 (lynx) to 8.0 x 10–4 (field vole). When differences in effective population size and the extent of the male mutation bias were taken into account, all five species showed evidence of reduced levels of Y chromosome variability. Reduced levels of Y chromosome variability have also been observed in Drosophila and in plants, as well as in the female-specific W chromosome of birds. Among the different factors proposed to explain low levels of genetic variability in the sex-limited chromosome (Y/W), we note that selection is the only factor that is broadly applicable irrespective of mode of reproduction and whether there is male or female heterogamety.
Key Words: sex chromosomes ? effective population size ? male mutation bias ? selective sweeps ? background selection
Introduction
Population genetics theory predicts levels of nucleotide diversity to be determined by a number of factors, including selection, rate of mutation, recombination, and effective population size. Studying the relative importance of these factors in shaping genetic variability is not without difficulties because the factors can be hard to quantify and their effects difficult to disentangle. However, under some circumstances intragenomic comparisons may offer a suitable study design, and one such example concerns studies of the levels of nucleotide diversity on sex chromosomes. In systems with male heterogamety (males XY, females XX), the X and Y chromosomes show distinct characteristics when it comes to the relative magnitude of mutation, recombination, and effective population size. First, while the Y chromosome is exposed to mutations that have arisen in the male germline only, both male and female mutations hit the X chromosome. Given knowledge of m, the male-to-female mutation rate ratio, the relative mutation rate on X and Y can thereby be inferred. Second, as Y is a non-recombining chromosome (with the exception of the pseudoautosomal region), recombination obviously plays different roles on X and Y. Third, under random mating, the effective population size of the Y chromosome is three times smaller than that of the X.
A number of studies have shown that levels of nucleotide diversity on the human Y chromosome are lower than in the rest of the genome (Malaspina et al. 1990; Dorit, Akashi, and Gilbert 1995; Hammer 1995; Whitfield, Sulston, and Goodfellow 1995; Nachman 1998; Anagnostopoulos et al. 1999; Jaruzelska, Zietkiewicz, and Labuda 1999; Shen et al. 2000; The International SNP Map Working Group 2001). This lower incidence of nucleotide diversity may be related to human biology—e.g., sex differences in patterns of migration or biased mating—but may also reflect a general feature of mammalian Y chromosomes or, even more generally, of species with male heterogamety. To address the generality of low Y chromosome variability, we initiated screening for single-nucleotide poplymorphisms (SNPs) in noncoding sequence from the Y chromosome of five mammalian species: lynx (Lynx lynx), wolf (Canis lupus), reindeer (Rangifer tarandus), cattle (Bos taurus), and field vole (Microtus agrestis). Taxonomically, these species are spread in the mammalian phylogenetic tree, and they also differ in terms of population history and mating system. To be able to compare Y chromosome variability with a measure of nucleotide diversity in other parts of the genome from each of the species, which are poorly characterized in terms of intraspecific levels of polymorphism, we also screened for SNPs in noncoding X chromosome DNA sequences. Here we show that lower than expected levels of Y-linked nucleotide diversity are found in all five species, indicating that reduced levels of Y chromosome polymorphism may be a generalized feature of mammalian genomes.
Materials and Methods
Tissue samples of all five species were extracted with a standard phenol/chloroform protocol. Introns from the Y-linked DBY, SMCY, UBE1Y, UTY, ZFY, and SRY genes (table 1) were amplified using the primers and conditions described in Hellborg and Ellegren (2003). Primers for amplification of introns of the X-linked SMCX, DBX, and ZFX genes (table 2) were designed based on the alignment of available mammalian sequence followed by identification of conserved sites and regions (cf. Lyons et al. 1997). We used a standard polymerase chain reaction (PCR) profile of 95°C for 10 min, followed by 20 cycles of 95°C for 30 s and a touchdown from 65°–55°C, 60°–50°C, or 55°–45°C for 1 min, decreasing 0.5°C per cycle (see table 2), and then 72°C for 1.30 min. These steps were followed by 20 cycles of 95°C for 30 s; 55°C, 50°C, or 45°C for 1 min; and 72°C for 1.30 min, and a final extension step of 72°C for 10 min. Amplification reactions contained 2.5 mM MgCl2, 5 μM of each primer, 2 μM dNTP, and 0.25 U AmpliTaq Gold polymerase (PerkinElmer). Female DNA was used for amplification of X-linked introns. Amplification products of both Y-linked and X-linked introns were screened for polymorphism using either single strand conformation polymorphism (SSCP) analysis (products shorter than 350 bp) or DNA sequencing (in both directions). Different electromorphs identified by SSCP were subsequently sequenced. Amplification products were purified with Qia-Quick PCR purification kit (Qiagen), sequenced using BigDye Terminator Cycle Sequencing chemistry (PerkinElmer), and sequences recorded with an ABI377 semi-automated sequencing instrument (PerkinElmer). Sequences were edited and assembled with AUTOASSEMBLER (Applied Biosystems).
Table 1 Y Chromosome Introns, Primer Sequences Used for Their Amplification, and Size of Amplification Products (in bp) in the Different Species.
Table 2 X Chromosome Introns, Primer Sequences Used for Their Amplification, and Size of Amplification Products (in bp) in the Different Species.
Samples, and their origin, used for polymorphism screening were as follows (numbers refer to the number of males screened; half the number of females were generally used from each geographic region/breed): Lynx: Baltic States (14 animals), Scandinavia (14), Finland (8), and Switzerland (4). Wolf: Russia (14), Scandinavia (10), Baltic States (3), Finland (3), North America (3), Spain (2), and Italy (1). Field vole: Sweden (6), England (3), Denmark (2), Russia (2), Romania (1), Norway (1), and Finland (1). Reindeer: Sweden (6), Alaska (4), Norway (4), Russia (4), and Svalbard (2). Cattle: Swedish Red Polled (2), Swedish Mountain cattle (2), Swedish Red-and-White (2), Swedish Friesian cattle (2), and Hereford (2). Sample size thus varied between species, but the same number of X and Y chromosomes were always screened for each species. Measures of genetic variability, including number of segregating sites per base pair (k) and nucleotide diversity (), and their standard deviations, were calculated in DnaSP (Rozas and Rozas 1999).
Results and Discussion
Levels of Polymorphism in Mammalian X and Y Chromosomes
We surveyed between 0.7 and 3.5 kb of intronic Y chromosome sequence and between 1.1 and 3.0 kb of intronic X chromosome sequence for intraspecific polymorphism in population samples of five mammals. There is evidence for mutation rate variation within mammalian genomes (Matassi et al. 1999; Williams and Hurst 2000; Lercher, Williams, and Hurst 2001; Smith, Webster, and Ellegren 2002), so we chose to study a number of different introns, derived from different genes, rather than a single longer noncoding sequence from each chromosome. Levels of genetic variability were considerably lower on the Y chromosome than on the X chromosome for all five species (tables 3 and 4). In fact, for three of them (lynx, reindeer, cattle), the surveyed Y chromosome sequence was completely monomorphic. For the X chromosome, where 1–7 segregating sites were found per species, the estimated nucleotide diversity (X) ranged from 1.6 x 10–4 (lynx) to 8.0 x 10–4 (field vole). Compared to sequence variation in the human genome, where X is estimated at 4.7 x 10–4 (The International SNP Map Working Group 2001), the lynx shows relatively limited variability (an observation supported by microsatellite data; Hellborg et al. 2002), whereas the opposite situation is suggested for the field vole. For the two species where SNPs were found in Y-linked introns, nucleotide diversity (Y) was estimated at 0.4 x 10–4 (wolf, two segregating sites in 1.6 kb) and 1.7 x 10–4 (field vole, four segregating sites in 3.2 kb), respectively. In humans, Y has been estimated at 1.5 x 10–4 (The International SNP Map Working Group 2001).
Table 3 Summary Statistics for Y Chromosome Variability.
Table 4 Summary Statistics for X Chromosome Variability.
The Effect of Population Size and Sex-Specific Mutation Rates on X and Y Chromosome Variability
As indicated above, estimates of nucleotide diversity on X and Y are not directly comparable. Nucleotide diversity—the normalised measure of heterozygosity—estimates the population genetic parameter theta (), which in a model assuming neutrality, random mating, and a population of constant size can be described by = 4Neμ, Ne being the effective population size and μ the mutation rate. For X, X = 3NeμX and for Y, Y = NeμY, the difference in effective population size reflecting X being hemizygous in males and Y being restricted to the male genome. From this finding, we should expect nucleotide diversity to be three times lower on Y than on X. It is worth emphasising that the expected 3:1 relationship for the effective population size of X and Y is only true under the assumption of random mating and equal numbers of the sexes. However, this may be an invalid assumption if the reproductive success among males is skewed, as may sometimes be the case (see further below). Under such a sex bias in mating, the relative effective population size of Y (compared with X) becomes even smaller.
Although differences in effective population size should thus be expected to lead to low levels of Y chromosome variability, differences in chromosome-specific mutation rates should have the opposite effect, given that μY is typically higher than μX as a result of male-biased mutation (Hurst and Ellegren 1998; Li, Yi, and Makova 2002), and perhaps other factors affecting chromosome-specific mutation rates (Lercher, Williams, and Hurst 2001). Species-specific estimates of the male-to-female mutation rate ratio (m) are not available for the species presented here, but some indication is given by data from other mammals. The most recent estimate of human m is at a factor of 5 (Makova and Li 2002). In Felidae—i.e., including the lynx—Pecon-Slattery and O'Brien (1998) estimated m at 4. For rodents, m is estimated at about 2 (Chang et al. 1994; Chang and Li 1995). The extent of the male bias is likely to be positively correlated with generation time (Chang et al. 1994; Li et al. 1996; Bartosch-H?rlid et al. 2003), and available data would thus suggest that mammalian m ranges approximately from 2 (short-lived species like most rodents) to 5 (humans). Here, we assume m 4 in wolf, cattle, lynx, and reindeer, and 2 in field vole. The corresponding μY/μX ratios are 2.0 and 1.5, respectively [note that μY/μX = m/(1/3m + 2/3)]. Now, combining the effects of differences in effective population size and mutation rate (X/Y = 3μX/μY), we should expect 1.5 times higher nucleotide diversity on X than on Y for wolf, cattle, lynx, and reindeer, and 2.0 times higher for field vole.
Any deviation from the expected difference in nucleotide diversity between X and Y would suggest that other factors are differentially affecting polymorphism levels on the two sex chromosomes. Figure 1 shows the adjusted levels of nucleotide diversity on X when differences in effective population size and mutation rate have been accounted for (i.e., when X is divided by 1.5 for wolf, cattle, lynx, and reindeer, and by 2.0 for field vole). The three species monomorphic for Y—lynx, cattle, and reindeer—obviously remain less variable on Y than on X; for these species the lower 95% confidence interval for X does not include 0. Unfortunately, the absence of observed polymorphism on Y prevents statistical analysis of Y chromosome data. For the two species with observed polymorphism on Y—wolf and field vole—nucleotide diversity also remains lower on Y than on X after compensating for differences in effective population size and mutation rate (fig. 1), although there is overlap in the 95% confidence intervals for X and Y. In summary, our data reveal a general trend of lower than expected levels of nucleotide diversity in mammalian Y chromosomes.
FIG. 1. Nucleotide diversity (±95% confidence intervals) in Y chromosome (filled bars) and X chromosome (open bars) introns. Observed levels of nucleotide diversity for X chromosome sequences have been divided by 1.5 (lynx, wolf, reindeer, and cattle) or 2.0 (field vole) to take differences in effective population size and sex-specific mutation rates into account
Factors Explaining Reduced Levels of Y Chromosome Variability
Factors potentially able to explain low levels of Y chromosome variability include selection, mating system, or migration patterns, or other mechanisms lowering male effective population size. The non-recombining part of Y effectively behaves as a single locus (haplotype) and is thus susceptible to the influence of selective forces acting on any sequence on the chromosome, with the exception of the pseudoautosomal region/s. This contrasts sharply with the situation on autosomes (in particular) and the X chromosome, where recombination releases sequences from association with a locus under selection. This well-recognized phenomenon (reviewed by Charlesworth and Charlesworth 2000) may act either in the direction of positive selection where advantageous mutations sweep through the population (genetic hitchhiking; Rice 1987) or in the direction of purifying (background) selection (Charlesworth, Charlesworth, and Morgan 1995) whereby chromosomes containing deleterious mutations are eliminated from the gene pool. For instance, strong directional selection on male-specific traits encoded by genes on the Y chromosome (like testis-specific genes involved in male reproduction) may introduce selective sweeps, driven by sexual selection (Roldan and Gomendio 1999; Wyckoff, Wang, and Wu 2000).
The effective population size of Y will be lowered by a high variance in male reproductive success (relative to that of females) (Caballero 1995; Nagylaki 1995; Charlesworth 1996). This may occur in polygynous species (Greenwood 1980), where a few males father a disproportionably large fraction of all offspring. One particular situation when this should apply is in the case of domestic animals; for most of these species, a limited number of males is typically used in breeding programs. The low levels of Y chromosome variability that we found in cattle could be attributed, at least in part, to a strong sex bias in breeding. Another mechanism that has been suggested to lower the effective population size of males is deleterious mutations in mitochondrial DNA (mtDNA) (Gemmel and Sin 2002). Because energy demand for spermatogenesis and sperm motility is very high, much higher than for oogenesis, and because the main function of mitochondrion is to produce ATP through oxidative phosphorylation, sperm may be particularly sensitive to mutations in mtDNA. Such sensitivity could lead to impaired sperm function and thereby lowered male reproductive success.
The nucleotide diversity of the human Y chromosome is about 20% of that found in autosomes (Shen et al. 2000; The International SNP Map Working Group 2001). This is less than expected when differences in effective population size and mutation rate are taken into account. [The International SNP Map Working Group (2001) predicts that Y would have a diversity 31% that of the autosomes; however, they use an m estimate of 1.7 based on Bohossian, Skaletsky, and Page (2000), while a more recent study has estimated m at 5 (Makova and Li 2002). Using the latter estimate we should expect nucleotide diversity on Y be 42% that of the autosomes, thus strengthening the interpretation of lower than expected variability on human Y.] Together with the extensive data available from the human genome, and reports of comparatively low levels of Y chromosome variability in apes (Hammer 1995; Burrows amd Ryder 1997; Stone et al. 2002), our data thus indicate that the mammalian Y chromosome may generally have reduced levels of genetic variability. Moreover, similar observations have been made for Y chromosomes of Drosophila (McAllister and Charlesworth 1999; Bachtrog and Charlesworth 2000) and even of plants (Filatov et al. 2000, 2001). Furthermore, in birds, which have a reversed sex chromosome organization characterized by female heterogamety (females ZW, males ZZ), the W chromosome is very low in nucleotide diversity (Montell, Fridolfsson, and Ellegren 2001; Berlin and Ellegren 2001). These combined data would suggest that a reduced level of genetic variability is a common feature of the sex-limited chromosome (Ellegren 2003), in turn suggesting a common mechanism underlying this phenomenon. Of the potential causative factors listed above, we note that selection is the only one that is broadly applicable irrespective of mode of reproduction or whether there is male or female heterogamety. [High variance in male reproductive success cannot explain the low levels of variability in the female-specific W chromosomes of birds, and lowered male effective population size due to mutations in mtDNA is not applicable to avian and plant systems.] We therefore favor the idea that selection is a key factor in the evolution of nucleotide diversity on the sex-limited chromosome. Unravelling the relative importance of, on the one hand, background selection and, on the other, selective sweeps in shaping genetic variability on the sex-limited chromosome will be an important question for future research in this area.
Acknowledgements
We are grateful to Annika Einarsson, Kristin Eliasson, and Frank Hailer for technical assistance, and to Hannah Sundstr?m and Carles Vilà for helpful comments. Financial support was obtained from the Swedish Research Council and the Nilsson-Ehle Foundation. H.E. is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation.
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