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The evolution of sex chromosomes: similarities and differences between plants and animals Deborah Charlesworth Institute of Evolutionary Biology, University of Edinburgh Male Silene dioica male Female Papaya female Silene latifolia female Classical sex chromosomes Humans Y is ~ 1/3 of the size of the X X ~ 1,098 genes Y 24 genes Y X Male-specific Y region PseudoMSY autosomal region without recombination PAR In Drosophila, the Y is about the same size as the X, but the X has several thousand genes, while the Y has around 20. No X gene has a Y homologue. Sex chromosomes have been known to geneticists for a long time, but many important things have only become clear very recently, and great progress is occurring • Muller (1914): reviewed evidence for X-Y pairing (indicating their homology) and Y genetic degeneration (suggested by C.W. Metz) and discussed recessive loss of function mutations as the cause of degeneration • Haldane (1922, p. 107): “If sex were determined by a single factor, it is very difficult to see what advantage there could be in its being linked with other factors)” – Nei (1969, 1970): models for lack of recombination and consequent accumulation of detrimental mutations leading to degeneration – BC, DC (1978) : evolution of sex-determining region with two loci (driving selection for less recombination) • An excellent review of the classical work is JJ Bull!s 1983 book “Evolution of Sex Determining Mechanisms” • Lahn and Page (1999): human genome sequence reveals sequences of genes shared between X and Y, often highly diverged. Carvalho (2001): Drosophila Y-linked genes • Recent data are starting to help us understand why and how recombination gets stopped between the X and Y (and what the consequences are) • 1. WHAT are sex chromosomes? – and what are NOT sex chromosomes • 2. WHY do sex chromosomes evolve loss of recombination? • 3. WHEN did sex chromosomes of some important species evolve? – and when did recombination stop? • and 4. HOW did recombination stop? • 5. WHERE are the sex-determining loci in relation to the regions where recombination is absent? • 6. WHAT are the consequences for sex chromosomes of stopping recombination? 1. What are sex chromosomes? • “Classical” sex chromosomes – Non-recombining over a large genome region, with small “pseudo-autosomal region(s)” • Mammal and Drosophila X and Y, bird and Lepidopteran Z and W – Genetically degenerated • loss of genes relative to the X (and lower function, see later) – Rearranged relative to the X • BUT sex chromosomes are more diverse than this • I shall take a (molecular) evolutionary perspective The human MSY region genes Many Human Y genes have male functions Genes on Y only Y genes with male functions can be kept on the Y because the sex chromosomes don’t recombine across much of their length. These genes are probably prevented from degenerating Genes on X and Y Heterochromatin There are a few X-Y gene pairs (X homologous genes), even in the nonrecombining regions (NRY) Mammalian Y chromosomes have many fewer genes than the X, and are more rearranged X There are complex genome rearrangements in the human Y due to duplications in a a 4.5-Mb functional region close to the heterochromatic region that includes AZFc Kuroda-Kawaguchi et al. 2001 Nature Genetics: 29, 279 286 Y Heterochromatin (no functional genes) Diversity of sex chromosome types (see Bull’s 1983 book) • CLASSICAL – Non-recombining over a large genome region, with small “pseudoautosomal region(s)” – Genetically degenerated • loss of genes relative to the X (or lower function — see later) • The Y can sometimes be totally lost (X0 systems) – Y is enriched in male-function genes, and is rearranged relative to the X • “LOCAL SEX-DETERMINING REGION” – Chromosome is largely pseudo-autosomal – The same properties as classical sex chromosomes, in a restricted region of genome • HAPLOID – Haploid male genotype is Y and female is X • NOT SEX CHROMOSOMES – fungal and algal incompatibility regions (but have some similar properties) – inversions The genus Silene Dioecious (independent evolution) Gynodioecious Hermaphrodite Female PAR Y Dioecious X Silene latifolia has classical sex chromosomes Hermaphrodite Estimated from ITS sequences by Desfeux, C., et al. 1996. Proc. Roy. Soc. Lond. B. 263:409-414 Recent work with more nuclear genes supports these phylogenetic relationships MSY region Some other plants have small sex-determining regions Several plant ‘sex chromosomes’ have the sexdetermining genes located within a small region (blue; only 10% of chromosome 1 of papaya) where recombination does not occur (Liu et al. 2004) – Some fish sex chromosomes may be similar – Does small size mean young, or primitive? Other plants have heteromorphic sex chromosomes like those of humans and Drosophila, or neo-sex chromosomes neo-sex chromosomes also occur in plants Liu et al. 2004. Nature 427:348-352 Haploid sex chromosomes in a bryophyte with separate sexes (Ceratodon purpureus) Meiosis 2n ! n 288 spores genotyped 59% male Y 41% female X Haploid Diploid sporophyte XY AA NOTE No XX Fertilization n ! 2n YA Male gametophyte XA Female gametophyte McDaniel et al. 2007 Genetics 176:2489-2500 C. purpureus genetic map showing 15 linkage groups, including the XY pair of sex chromosomes 124 markers (121 AFLP, 3 genic) This species has a small sexdetermining region MSY Another bryophyte, Marchantia polymorpha (haploid) has highly heteromorphic sex chromosomes Yamato et al. (2007) Proc. Natl. Acad. Sci. USA 104, 6472-6477 X Y Fusions/translocations, neo-sex chromosomes X Y Autosome Y-autosome fusion X-autosome fusion neo-X Y1 X1 neo-Y or Y2 neo-X or X2 neo-Y Chromosome(s) transmitted to female progeny Chromosome(s) transmitted to male progeny In Drosophila, there is no recombination in males Thus, both kinds of fusion create non-recombining neo-sex chromosomes Chromosome fusions can lead to heteromorphism Fusion can also occur in X0 systems Neo sex chromosomes in the genus Drosophila Complete degeneration of the ancestral Y Carvalho, A. B., and A. G. Clark. 2005. Y chromosome of D. pseudoobscura is not homologous to the ancestral Drosophila Y. Science 307:108-110. • The Y can be lost entirely if genes required for male fertility can move to a different chromosome • Some species have X/0 male genotype, but males are still fertile. – e.g. Drosophila affinis • In D. pseudo-obscura, the sex chromosomes have been fused to an autosome, and the Y has lost all male fertility genes – So, even if the Y chromosome degenerates, we do not need to worry about a future without males • Clearly, the Y cannot be lost unless the sex-determination function is replaced by a new gene (or the Y gene moves to another chromosome) – Such changes are theoretically possible: e.g. DOORN, G. V., and M. KIRKPATRICK, 2007 Turnover of sex chromosomes induced by sexual conflict. Nature 449: 909-912. Neo-sex chromosomes occur in many species Rowell, D. (1985). Complex sex-linked fusion heterozygosity in the Australian huntsman spider Delena cancerides (Araneae: Sparassidae). Chromosoma 93, 169-176. RENS et al., 2004 PNAS 101: 16257-16261 GRÜZNER et al., 2004 Nature 432: 913-917. 2. WHY does recombination stop on sex chromosomes? • Haldane (1922, p. 107): “If sex were determined by a single factor, it is very difficult to see what advantage there could be in its being linked with other factors)” (Sex ratio and unisexual sterility in hybrid animals. J. Genetics 12:101-109) – Nei (1969, 1970): models for lack of recombination and consequent accumulation of detrimental mutations leading to degeneration – Nei, M. 1969. Linkage modification and sex difference in recombination. Genetics 63:681-699; 1970. Accumulation of nonfunctional genes on sheltered chromosomes. American Naturalist 104:311-322. • Many modern authors are much less clear e.g. “heteromorphic sex chromosomes have evolved ….when one autosome develops a dominant sex-determining mutation” – Itoh et al. 2007. Molecular cloning of zebra finch W chromosome repetitive sequences: evolution of the avian W chromosome. Chromosoma 117:111-121. • There is no selection to reduce recombination unless at least 2 genes interact • Qvarnstrom & Bailey. 2009. Heredity 102:4-15 are completely wrong! – The evolution of identifiable heteromorphic sex chromosomes is initiated by the spread of a sex-determining gene (SDG). This occurs when a new mutation at a locus leads all its carriers to become the same (subsequently heterogametic) sex, with the chromosome carrying this mutation becoming the Y/W chromosome (see main text). In eutherian mammals, for example, the development of males is controlled by the SRY gene found only on the Y chromosome • To understand why the sex chromosomes don’t recombine, we need to understand WHY interacting genes are involved, which requires understanding HOW separate sexes evolved, and what kinds of genes were involved – BC, DC (1978) : evolution of sex-determining region with two loci (driving selection for less recombination) • A model for the evolution of dioecy and gynodioecy. Amer. Nat. 112:975-997 • This is a separate question from: how is recombination lost? – i.e. questions like • whether it was it in a single step when the sex chromosomes orginated or a gradual process, with several successive steps? • and were inversions involved? • The question of what gene interactions have selected for loss of recombination is still not fully answered The argument for two or more evolutionary steps 1 M f M f cosexual (hermaphrodite or monoecious) or environmental sex det Female-sterility mutation f !"F Male-sterility mutation M !"m Hermaphrodites and males (androdioecy, very rare) Hermaphrodites and females (gynodioecy, present in 5% of angiosperms) 2 Dominant femalesuppressing mutation(s) f !"F proto-Y proto-X Gynodioecy in Silene vulgaris Males M F m f Females m f m f proto-X proto-X Hermaphrodite Female In S. latifolia, sex-determination is genetically simple: Males and females are simply hermaphrodites with parts missing Mutants support the hypothesis that at least 2 genes are involved 1: loss of stamen promoting factor, SPF, in females X (M! recessive m) 2: suppression of female functions by proto-Y-linked GSF (Suf ! dominant SuF) Genotype at second locus Suf/Suf (no female suppressio n ) Hermaphrodite Genotype at locus that mutates first M/M or M/m Loss of stamen promoting factor (SPF or M) creates females Hermaphrodite Gynoecium suppressing factor (GSF or SuFemale) reduces female functions SuFemale/Suf m /m " ! Female " ! Neuter Male (male fertility increase d ) Picture from Shigeyuki Kawano The simple 2 gene evolutionary model actually suggests that sex determining loci must initially be linked for separate sexes to evolve Once females are present, hermaphrodites are selected to re-allocate more to male and less to female functions 1 2 M SuFemale “proto-Y” “proto-X” m f Selection should then act to reduce recombination between the initial 2 genes slightly older Female M2 M Su proto-Y, with MSY region 3. WHEN did sex chromosome systems evolve? • Some classical sex chromosomes are probably old – We don’t yet know how long it takes for the full set of features to evolve • It is often assumed that all other systems are young CLASSICAL (heteromorphic) SMALL SEX-DETERMINING REGIONS (no heteromorphism) NEO SEX CHROMOSOMES Old Young, Maybe some are old Young • but we need data. It is now possible to get evidence, using DNA sequences, estimating divergence between homologous X and Y sequences, and assuming a molecular clock – heteromorphism can evolve rapidly, e.g. by chromosome fusions • For most species, it is difficult to get the genes for such studies • To estimate ages of sex chromosomes, and to study degeneration, we need to find genes and study alignable orthologous X and Y gene pairs. • There are few known mutant phenotypes (as Muller realised) • Molecular methods are needed (Muller realised this too, in 1922) – These methods also allow one to estimate ages and study degeneration • Even with “complete” genome sequences of important “model organisms” there are still great difficulties – The gene content of the Y chromosomes of important “model organisms” have only recently been determined • Drosophila: Carvalho et al. 2001 PNAS 98:13225-13230 • Humans: SKALETSKYet al., 2003 Nature 423: 825 - 837, BHOWMICK et al, 2007 Genome Res. 17: 441-450 • The mouse Y is still not well characterized • and “model organisms” for sex chromosome work are only now starting to be studied – e.g. Dreyer et al. 2007. ESTs and EST-linked polymorphisms for genetic mapping and phylogenetic reconstruction in the guppy, Poecilia reticulata. BMC GENOMICS 8:269. Why is it difficult to sequence Y chromosomes? • Low gene density makes finding genes very difficult. • Rearrangements: one homolog cannot used to help align the other, unlike the autosomes – Y can be sequenced from a single individual • Their intergenic regions and introns contain large amounts of repetitive sequence, so it is difficult to find the different parts of the same gene • Assembly of highly repetitive genomes is very difficult – it requires large sequenced regions, such as BAC clones, but these may be difficult to sequence if they contain repetitive sequences • These are sometimes unstable when cloned, and so cannot be sequenced • They may compete in PCR reactions, so that some copies fail to amplify • If the repetitive sequences are AT-rich, poor strand separation may impede sequencing reactions Human X-Y divergence Stratum 1 Many “X-degenerate” genes still detectable on the Y Stratum 3 Stratum 2 Stratum 4 X-Y divergence, Ks LAHN & PAGE, 1999 Four evolutionary strata on the human X chromosome. Science 286: 964-967. SKALETSKY et al. 2003. Nature 423:825 - 837. Mostly old part of X (all but 2 genes present in marsupial X chromosomes). Few genes on the X are still detectable on the Y Autosomal in marsupials (added to X and Y by transposition. Xp Xq recent transposition PAR1 2 genes transposed very recently to the Y What about plants? I emphasized how helpful it is to identify genes, not just anonymous markers or sequences BUT finding orthologous X and Y gene pairs in non-model species is very difficult, and the S. latifolia genome is awfully big! Maybe one should sequence the genome? Overview of the Marchantia polymorpha YR2 region — so far in this species mainly Y chromosome data, not X and Y. This species is expected to have an old Y chromosome 50 Y-linked housekeeping genes are also found in females (presumably nondegenerated genes, with autosomal or X-linked copies) 14 Y-linked genes are unique to males, and expressed only in reproductive organs G = genes (indicated by arrows ) P = pseudogenes O= organelle sequence T = transposable element How else can one find sex-linked loci? • Testing linkage of known genes in families • MROS3-X and -Y (Dave Guttman, 1998) • Genes involved in flower development – SlAp3-Y (Sachi Matsunaga, 2003) • cDNA probing of micro-dissected Y chromosomes – SlX/Y1 (Delichère et al. , 1999) – SlX/Y4 (Atanassov et al., 2001) – SlX/Y3 (Nicolas et al. 2005 • Genes discovered from cDNA libraries and EST sequences – – – – – SlSS-X/Y SlCyp-X/Y Sl8-Y only Sl6a and b X/Y Sl7X/Y – RB11 and RB18 Dmitry Filatov Roberta Bergero Isomerase, cyclophilin type Roberta Bergero Mono-oxygenase/haem binding protein Roberta Bergero Unknown protein (2 Y and X copies) Roberta Bergero Unknown protein Roberta and Vera Kaiser • Differential display • DD44 (Moore et al., 2003) EST sequences were used to obtain sequences Intron positions of genes at low copy number were determined from the Arabidopsis thaliana and rice genomes PCR primers were designed to cross introns to find length variants to do genetics Parents F1 progeny Y-linked 1830 bp 730 bp maternal X 2072 bp 700 bp 500 bp 510 bp maternal X 590 bp paternal X 590 bp paternal X X- and Y-linkage for locus Sl6 Roberta’s ISVS method Forward primer Intron region Exon A Exon B Reverse primer FAM Incorporation of labeled universal primer after the first PCR cycles FAM For product sizes > 450-500 bp, digest FAM with restriction enzyme MboI FAM MboI HaeIII FAM MboI HaeIII Analysis by capillary electrophoresis Evidence for X/Y linkage of the SlCyp gene Intron 3 variants, showing Y-linkage of 438 bp band Intron 2 variants , showing X-linkage of 259 and 260 bp bands 2 male and 2 female F1 plants Parents 260Xm 257Y 260Xm 447bp 259Xp 257Y 447 bp 438 bp in males only 447 bp 259Xp 260Xm Possible strata in organisms other than humans (X versus Y) Human (Z versus W) (X versus Y) (X versus Y) PAR1 transposition Autosomal in Mostly old part of X marsupials (all but 2 genes (added to X present in and Y by marsupial X transposition) chromosomes) PAR inversion Lawson-Handley et al., 2004 Genetics 167: 367-376 Nam & Ellegren. 2008. Genetics 180: 1131 - 1136 PAR Bergero et al., 2007 Genetics 175: 1945-1954 Phylogenetic analysis of bird Z and W chromosomes also suggests that recombination between them stopped at different times Pseudoautosomal end Chicken Z Genes that stopped recombining after split of taxa Genes in region where ZW recombination stopped before split of major bird taxa Some bird taxa probably have small sexdetermining regions from LAWSON-HANDLEY et al., 2004 Genetics 167: 367-376 Gradual evolution of bird sex chromosomes is also evident when different taxa are compared — some taxa have not undergone all the steps that others have taken Giemsa staining C-bands G-bands by BrdU Painting with Locations of chicken Z markers probe Z W Non-recombining region has probably remained small Ostrich Large nonrecombining region Chicken Markers: Z chromosomes of both taxa share several markers Thus they probably had the same ancestral sex chromosome Recombination has been suppressed only in the chicken lineage (including other neognathae), and not in palaeognathous birds Nishida-Umehara et al. 2007 Chromosome Research 15:721-734 Nanda, I et al.. 2008. Cytogenet Genome Res 122:150-156. Gradual evolution of snake sex chromosomes P. molurus (Pythonidae) Females are WZ E. quadrivirgata (Colubridae) Matsubara et al. (2006) PNAS 103: 18190 Many (11/11) genes shared between Z and W (small sexdetermining region) 3/11 genes No genes shared shared between between Z and W Z and W (W has lost most genes) T. flavoviridis (Viperidae) Summary of some of the evolutionary changes that can occur after a small sex determining region evolves Starting state cosexual or environmental sex determination Males M F YA XA (5) Change to ZW system W Z m X Y f Females m f m f (2) Loss of recombination in new region (e.g.mammalian, chicken and viper lineages) (4) New sexdetermining gene arises old Y old X (3) Neo-sex chromosome(s) X XA XA (1) Recombination continues in most of the W (e.g. in ostrich and python lineages) Neo-Y “Y” “X” A change from XY to ZW system in different populations of the same species An example of the use of genes to demonstrate that the XY pair of chromosomes changed into a ZW pair Uno et al. 2008. Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems. Chromosome Research:1217-7 Ks values in 6 X and Y Marchantia polymorpha genes suggest that this sex chromosome system is old (Ks is uncorrected synonymous or silent site divergence) Ks • If we had a good molecular clock, we could translate Ks values into times when X-Y recombination stopped • It is not yet possible to tell whether there are strata in this plant, or if the Y and/or X is degenerated Is papaya (with a small MSY) a young system? Divergence is low between papaya X and Y gene sequences X and Y from hermaphrodite (Yh, YU et al, 2007 Plant Journal 53: 124-132) Ks values in 4 papaya genes from a BAC clone X and Y from male (YU et al, 2008 Tropical Plant Biology 1: 49-57) It is not yet possible to tell whether there are strata in this plant, because only 2 BACs were sequenced (< 150 kb, whereas the size of the MSY is ~ 10 Mb) WHY are there strata? Why don’t organisms stop recombination across the entire sex chromosomes? 2 The simplest 2 gene evolutionary model above suggested that sex determining loci must initially be linked for separate sexes to evolve 1 3 M SuFemale “proto-Y” “proto-X” m f Other genes may be added to the system in a 3rd step (and so on) “proto-Y” sexually antagonistic male-enhancer “Y” M2 M SuFemale Reduced recombination between initial 2 genes Recombination suppression probably eventually evolves across the whole Y chromosome because, once free from the burden of female functions, males keep on becoming better males In hermaphrodites, pressures to increase male and female functions are balanced SuFemale m f m f m f In male-like hermaphrodites (with lowered female function) the balance at other loci changes towards male functions m f In males, only male functions matter • The hypothesis of sexually antagonistic male-enhancers is plausible, but all evidence to date is indirect, and no such genes have yet been identified – without sexual antagonism, there should be no selective pressure converting hermaphrodites into males (the female functions of hermaphrodites could be maintained unchanged while male functions improve). – Mank et al. (2008 American Naturalist 171:35-43) wanted to test for antagonistic genes in chicken and mouse • genes with different male and female expression patterns • many of these will NOT have antagonistic effects, but, as a whole, the set of such genes should include genes with antagonistic effects – They found that this set of are less likely to be expressed in multiple tissues (with the potential for conflicting selection pressures) than the genome average, even after excluding sexlinked genes; however, a difference in tissue-specificity could be explained without sexually antagonistic effects • Drosophila experiments that allowed selection in males only show that female fitness indeed declines • This is consistent with a tradeoff between the sex functions, but it could be due just to stopping selection in females Fertility of female offspring High fertility female parents Low fertility female parents Fertility of male offspring • Reversal in quality of progeny, depending on whether they had high or low fertility parents, is clear evidence for trade-offs, but it does not prove intralocus sexual conflict Low fertility female parents High fertility female parents Low High Fertility of male parents Pischedda & Chippindale (2006, PLoS Biology 4:e356) • The best evidence so far for sexually antagonistic male-enhancers is in the guppy fish, Poecilia reticulata – Guppy males are highly polymorphic for color patterns and their genetics has been studied analysis since 1927 (Winge, Journal of Genetics 18, 1, 1927) – The guppy has 23 pairs of chromosomes — 22 are autosomal and one sex determining. Males are heterogametic (the sex determination mechanism is XX/XY, and the YY genotype is viable) – Almost all the genes determining guppy colour patterns (except for body color) are sex-linked or sex limited (unlike what is found in other teleosts) – though usually not fully sex-linked • Winge, O. 1927. The location of eighteen genes in Lebistes reticulatus. Journal of Genetics 18,. A peculiar mode of inheritance and its cytological explanation. Journal of Genetics 12:137. • With the possibility of using naturally occurring polymorphic sequence variants as genetic markers, it is now possible to make a more detailed genetic linkage map and find out if the Y has an excess of male attractiveness factors • Molecular markers have now been found on the Y chromosome, closely (but none fully) linked to the sex-determining region, SdR. – Shen et al (2007, Aquaculture 271:178-187) mapped the sex locus and nine AFLP markers and four microsatellite DNA markers • Overall, the results suggest that the non-recombining MSY region may not be very large, and that the colour variants may be controlled by polymorphic genes in the PAR 4. HOW did recombination stop, how do MSY regions expand? BUT the known inversions on the Y occurred relatively recently, and these cannot be involved in stopping recombination (since, in most of the Y, it stopped long ago) SRY/ SOX3 Translocation • "..there is little evidence demonstrating the importance of [chromosome rearrangements versus genes modifying recombination] in the evolution of X-Y crossover suppression! (Bull 1983) • There are many inversions on the mammalian Y Lahn & Page’s suggested evolutionary history of the mammalian sex-chromosomes Heterochromatic region X chromosomes Human Mammalian X chromosome gene arrangements are stable, while Y chromosomes are highly rearranged PAR1 Yp BUT inversions occurred since humans (or split from chimpanzees PSA) and modifier genes can also change recombination rates during evolution NRY regions recently transposed from the X degenerated copies of X genes “Ampliconic” (duplications) degenerated X genes inverted PSA2 Yq inverted Chimpanzee The most recent strata in the human MSY already has several inversions • Stratum 5 may involve an inversion, but stratum 4 includes several inversions ( ) • The AMEL locus may span the ancestral boundary between human strata 3 and 4 , but the X and Y genes are intact (Marais & Galtier. 2003. Curr. Biol. 13:R641-643) • Rearrangements may thus be a consequence of lack of recombination Stratum 5 PAR1 Ross et al. 2005. Nature 434:325-337 Stratum 4 Stratum 3 • The papaya MSY regions have already been rearranged, even in just the two BACs so far studied • It is not known whether these inversions caused recombination to stop, but the region is only a small part of the MSY • The rearrangement in this region is shared by the Y and Yh, and differentiates both of them from the X • Thus it pre-dates the evolution of hermaphrodites X from hermaphrodite Y from hermaphrodite (Yh) 6.5 Mb Y from male Y from hermaphrodite (Yh) 6.5 Mb X from male Y from male X-Y divergence (%) 13 - 19 2-4 10 - 13 Yu et al. 2008. Tropical Plant Biology 1: 49-57 Did X-Y recombination stop in S. latifolia due to inversions? Y chromosome deletion map of the Y, based on 3 parental plants A C B In one parent plant, the Y chromosomes gene SlY1 is in a different location In two parent plants the Y chromosomes gene SlY6b is absent Possible rearrangements in the Y, relative to the X Present X gene order Pseudoautosomal Proto-Y1 Proto-Y2 Present Y gene order p arm M M m Suf q arm SuFemale SuFemale Paracentric inversion Pericentric inversion These results show that inversions happened after X-Y recombination stopped 6. Why does stopping recombination lead to sex chromosome degeneration? • It has been known since 1918 that classical Y chromosomes are degenerated chromosomes – Muller, H. J. 1918. Genetic variability, twin hybrids and constant hybrids, in a case of balanced lethal factors. Genetics 3:422-499. • “It is probably needless to point out that the W and especially the Y chromosome ….. show the expected evidences of …. degeneration and differentiation from their homologues, both genetically and cytologically. The evidences are now as follows: – X-linked mutations affecting visible phenotypes are manifested in XY males • therefore the Y does not carry alleles that can cover up mutations – Infrequent dominant Y (and W) linked mutations” – “Great variations in their own size and shape even in closely related species” – Synaptic attraction between them and their homologues • “but the sex chromosomes in the heterozygous sex tend to remain condensed during the growth period, while the autosomes are spinning out for intimate conjugation, and there is frequently delayed synapsis” • “also lack of crossing over between them and their homologues, even …. where other chromosomes are undergoing crossing over” Y chromosome degeneration • Loss of genes – Well illustrated by classical sex chromosomes • for example, the human X region that has been non-recombining longest has the lowest proportion of intact genes on the Y (at most, 5), whereas the probable number carried on the X chromosome is 734 (based on a count done by Gabriel Marais, using Ensembl version 47) • Worse gene function – amino acid substitutions that reduce functioning – less use of optimal codons – expression levels changed relative to X (presumably wrong levels) • Transposable element insertion is often included as an aspect of Y degeneration, and degeneration may indeed be caused partly by transposable element insertions, but we don’t actually know this – it is possible that these insertions are neutral – they could insert after genes or the sequences controlling their expression have degenerated DNA content (Mb) Species Human Total 3,286 X 164 (5%) Y 59 (1.8%) Euchromatic (Mb) genes > 3,000 150 ~ 78 40,000 1148 21 11 7 0.35 114 76 for euchromatin 118 1-2 Drosophila Total 180 > 120 13,600 X Y 33 20 22 0 ~ 2600 20 ??? Estimated number of genes/Mb CpG islands/Mb Region and stratum Humans: we can now compare homologous X and Y gene regions 6-43; mean 10.5 6 2.9 Numbers of functional genes (numbers ampliconic) X and Y copies (X-degenerate) 1 (old X) 5 (4) Genetic degeneration of Y chromosomes Pseudogene numbers Numbers with male function Numbers with ubiquitous expression 0 4 (3 ampliconic) 1 (nonampliconic) 1 1 (ampliconic) 1 (ampliconic) 2 6 2 Regions added to X about 120 MYA (p arm) Stratum 2 3 (1) 0 Stratum 3 7 (1) 3 Stratum 4 7 6 (2) Recently X-transposed genes — 3 (0) 0 1 0 Other genes on the Y but not the X — 3 (3) 0 3 0 • NOTE that most of the genes present on the Y are found in the youngest stratum of the X (strata 3 and 4 in the initial paper on strata) • This indicates that the older strata are genetically degenerated and have lost most of the genes that were once on the Y • Notice how helpful it is to have identified genes, not just anonymous markers or sequences LAHN & PAGE, 1999 Four evolutionary strata on the human X chromosome. Science 286: 964-967 G C T A T Leaf X/Y4 Flower Leaf X/Y3 X/Y7 Leaf CypX/Y DD44 Gene X/Y1 Leaf Flower Differences between X and Y homologues, estimated using PCR with primers recognising the same sequence in X and Y alleles, and flanking an intron Pyrosequencing Expression studies in S. latifolia give some direct evidence of low Y function C A pyrosequencing primer SlCypY AATTTGCACACCAACAAAGCATCACG SlCypX AATTTGCACACCAACAAAGTATCACG Work of Michael Nicolas and Roberta Bergero TE insertions do NOT necessarily cause loss of function Two Silene latifolia Y genes DD44 Y S. latifolia X Work of Gabriel Marais Blastn Y3 / Y3 Blastn Genbank RepeatMasker (Repbase) Blastx prot TEs Arabidopsis S. vulgaris (not sex-linked) SlXY3 Y X Introns LTR retrotransposon Exons Non-LTR retrotransposon DNA transposon Inverted repeats Direct repeats Another sign of low effectiveness of selection is accumulation of repetitive DNA on Y chromosomes Autosomes Accumulation of retrotransposons on the Drosophila miranda neo-Y chromosome neo-Y chromosome BACHTROG, D., 2003. Mol. Biol. Evol. 20: 173-181. Drosophila miranda Transposable elements Genes The neo-Y is turning into heterochromatin Active MITE insertions were detected by searching for polymorphic inserts in introns of Silene latifolia genes EITRI: 11-bp terminal inverted repeats (5'-CTAGGTAGCAC-3') and 8-bp target site duplications (TSDs, like hAT or P elemenst) A Tourist-like element M Roberta Bergero & DC, in press in Genetics GAAATTCTTT//Sl-To1//TAGTTTC GAAATT--------------TAGTTTC GAACTTCTTC-----------AGTTTC GAACTTCTTC-----------AGTTTC GAACTTCTTC-----------AGTTTC GAACTTCTTC-----------AGTTTC Silene latifolia Not Y-linked (none fixed) Genetic mapping allows us to find ones that are Y-linked As predicted from population genetics theory, MITE insertions are generally at low frequencies, but on the Y chromosome they reach high frequencies Y-linked (3/25 fixed) • Accumulation of transposable element on Y chromosomes may promote rearrangements • Loss of a gene in primates: Nakayama & Ishida. 2006 Genome Res. 16:485-490. • Rearrangements may make it difficult to Cytogenetic maps of the threespine stickleback X and Y chromosomes, based on FISH with genes X Y detect X-Y heteromorphism • Gene conversion between paralogs in the human Y: Bosch et al. 2004. Genome Res. 14:835-844. inversion X Y X Y Heteromorphic X-Y pair deletion of part of Y inversion on Y and/or insertion, making heteromorphism hard to detect deletion Ross and Peichel. Genetics 2008;179:2173-2182 6. WHY are Y chromosomes degenerated? I. The ‘sheltering hypothesis’ (the Y is always heterozygous with an X) • “The reason for this rapid decay of things Y-chromosomal is thought to be quite simple: once the Y chromosome became sex-determining, its presence was limited to the heterogametic sex (in our case, males). Because the Y chromosome was never found in the absence of an X chromosome, there was presumably little selection against the mutational inactivation of those genes on the Y chromosome that were also present on the X chromosome. Thus, over evolutionary time,the Y chromosome gradually lost most of its functional genes by the accumulation of deleterious mutations, resulting in that little dab of male-determining chromatin that we have today.” – HAWLEY, R., 2003 The human Y chromosome: Rumors of its death been greatly exaggerated. Cell 113: 825-828. • This is wrong — it ignores the central importance of the lack of recombination have • It is a challenge to evolutionary biologists that a common observation such as degenerate Y chromosomes is still so far from being understood! (Bull 1983, p. 258) • Genetic degeneration is probably NOT caused by the fact that Y chromosomes are always heterozygous (as Hawley assumed) – Allowing recessive deleterious mutations to arise without major effects on fitness • but, more likely, by lack of X-Y recombination in a large genome region • To answer the important questions – Why does lack of recombination lead to degeneration? – How long does it take? • we need to model non-recombining genomes, and such models have largely answered Bull’s challenge Evidence from modelling: the importance of loss of recombination There are now several other theories for degeneration when there is no recombination Selection for advantageous mutations causes fixation of deleterious mutations , which reduces the effective population size Many different sequences, one carrying the deleterious mutation Only a single sequence, i.e. all carry the deleterious mutation several generations Deleterious mutations prevent spread of advantageous mutations unless their selective advantage is large several generations Muller’s ratchet (probably less important) 10 different sequences Genotype number 1 2 3 4 5 6 7 8 9 Selection against mutations reduces the effective size After many generations with stable numbers of deleterious mutations 4 ancestral sequences Ancestral genotype number 1 4 7 Loss of mutant-free class 8 Data from non-degenerated genes can provide indirect evidence that degeneration is occurring • Degeneration is thought to be caused by lack of recombination – This changes evolutionary processes in several ways – The different processes all cause lower “effective population size” and thus they lead to low genetic variation • Diversity studies can thus detect these processes – If degeneration is happening, we should find lower diversity of Ylinked than X-linked genes – We must take into account that the population size of X-linked genes is 3 time higher than for Y-linked genes This is expected if the effective size of the Y is low More autosomal genes are needed (we cannot yet exclude the possibility that X diversity is unusually high) S. latifolia Y diversity is low compared with homologous X-linked or autosomal genes Nucleotide diversity values (%) To estimate subdivision for Y and X genes within S. latifolia, we sampled plants from 23 European populations, and sequenced Y and X alleles SlXY4 Y-linked X-linked or autosomal DD44 -XY SlXY1 SlXY7 Cyp-XY It is expected that all Y diversity estimates are similar— all nonrecombining parts of the Y have the same recent history Type of site SlAp3 (Y vs.A) Studying Y gene degeneration (ii) Molecular evolutionary comparisons of X, Y and outgroup sequences allows one to test whether the X or Y has changed, again using non-degenerated genes Outgroup species X Y chromosome chromosome G A A A G substitution Ancestral sequence X chromosome nucleotide Y chromosome nucleotide Outgroup nucleotide X or Y changed G A G T T C C T C T C A G G C C T C C G A T A G A T C C G T A T C X X Y X Y Y Y Y Inferring the causes of degeneration Maladaptation inferred from divergence • If the neo-Y genes are acquiring harmful mutations that impair their functionality, we expect to find more changes in functionally significant sequences in the ancestry of the neo-Y copies than the neo-X copies (e.g. more non-synonymous substitutions). The opposite is true if the neo-X genes experience adapt more than the neo-Y We can test this by sequence comparisons (X-Y or along the 2 lineages ) Y X outgroup A consistent pattern in X-Y divergence across many genes in D. miranda is difficult to explain by a higher Ka than Ks due to molecular adaptation of the Y (it would be strange if all Y genes were adapting) Bachtrog, D. 2005. Genome Research 15:1393-1401 Chicken compared with turkey Chicken Z Turkey Z Chicken W Turkey W The W chromosome (restricted to females) has relatively more non-synonymous substitutions (higher Ka/Ks) than the Z Berlin & Ellegren. 2006. Journal of Molecular Evolution 62:66-72 In Silene latifolia genes, Gabriel Marais sees both failure of selection to prevent deleterious substitutions, and favorable changes switching to neutral Gene (Numbers of codons analyzed) Site model indicates purifying selection at all loci Branch model X versus Y dN/dSX dN/dSY Significance Branch-site analysis suggests weak efficacy of selection on Y % of codons suggesting degeneration, and changes in Y versus X and outgroup (to neutral evolution or positive selection) SlX1/Y1 (458) 100% ! <<1 10-4 0.11 X < Y*** 6% No significant switching SlCypX/Y (519) 94% ! <<1 6% w~1 0.14 0.14 X=Y 10% No significant switching SlssX/Y (259) 97% ! <<1 3% ! >1 0.18 0.23 X < Y ns 0 No significant switching 63% ! <<1 36% ! ~1, 1% ! > 1 0.13 0.90 dNX=dNY=dSY (all low values) 5.5% under positive selection (!=14.8) Significant switching to positive selection SlX3/Y3 (318) 95% ! <<1 5% ! ~1 0.04 0.13 X < Y* 4% under positive selection (! =3.5) Significant switching to positive selection SlX7/Y7 (246) 88% ! <<1 12% ! ~1 0.08 0.11 X < Y ns 4% No significant switching SlX4/Y4 (362) 92% ! <<1 6% ! ~ 1, 2% ! > 1 0.11 0.25 X < Y* 14% Significant switching to neutrality DD44X/Y (217) Significance of differences in LR tests: ns = non-significant, * p < 0.05, *** p < 0.0005 Male sterility, m Female fertility Summary of steps in the evolution of the Y Maleness factor, M Female suppressor, SuF Proto-X and Y 1 Addition of male function genes, further recombination suppression, rearrangements Loss of parts of Y M2 small MSY region MSY Evolution of a sexSuF determining region in M Suppressed an ancestral recombination on chromosome pair, part of proto-Y forming a nonTransposable element heteromorphic pair 2 The simplest evolutionary model suggests that males and females evolved from hermaphrodites by loss of functions Newest SuF stratum M Oldest stratum PAR accumulation and Some plants, expanded MSY fish, snakes region 3 4 m M2 Y X Classical (humans and Drosophila) 5 Some consequences of sex chromosome evolution • In species with XY systems – The Y chromosome may acquire genes with male functions – Genes with male functions may also evolve more readily on the X than the autosomes, because the X spends a higher proportion of its evolutionary history in males than females • If degeneration has occurred, then, at most X-linked loci XY males have only one gene copy, compared with XX females’ two copies – except for a few housekeeping genes on both X and Y • • Some species have evolved control of levels of expression so that levels of X-linked gene products are correct, relative to expression of other genes: dosage compensation The X must therefore undergo considerable evolutionary change • The compensation mechanisms are different in different organisms inactivation of one X The simplest solution is overexpression of the X in males 2X 2A X 2A Females Males Mammals low expression of both Xs Dosage compensation mechanisms in different organisms 2X 2A X 2A 2X 2A XO 2A Females Males Drosophila “Females” Males C. elegans The Chimpanzee Sequencing and Analysis Consortium 2005 Nature 437: 69-87. Number of 1 MB windows Human-chimpanzee divergence Over all sites, genes on the mammalian X evolve more slowly than autosomal genes (indicating selective constraints, which are more important on the X because of hemizygosity in males) and the Y evolves unusually fast (because there is a higher mutation rate in males, due to multiple cell divisions in spermatogenesis) Autosomes X Human-chimpanzee divergence Y X • • The PSA recombines at a much higher frequency than the rest of the X, increasingly so as its size is restricted by evolution of new strata High recombination may also cause a high mutation rate (if recombination causes mutations) The PSA is smaller in humans than other mammals, because the boundary has moved towards the tip, compare with its location in bovine X chromosomes and in mice it is small due to movement of genes off the X Van Laere A. et.al. Genome Res. 2008;18:1884-1895 7. What about other non-recombining systems? • WHY did recombination stop, and what are the consequences, e.g. do these genome regions degenerate? • Some fungal incompatibility systems – Different loci (pheromones and receptors) are involved at some of these “loci”, and are sometimes present in inverted regions, but the selective reason for lack of recombination is unknown • Lee et al. 1999. The mating-type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region. PNAS 96:15026-15031. – Some seem to have undergone degeneration. Allelic forms of the genome region each lack genes found in the other orthologous region – TEs are sometimes abundant (50% in U. hordei, maybe accumulated, but comparisons with other genome regions should be made) • Bakkeren et al. 2006. Fungal Genetics and Biology 43: 655-666 • Angiosperm self-incompatibility systems (SI, S-allele systems) – Alleles at two different loci (encoding pollen and pistil function proteins) must be present in the correct combinations, which would lead to selection against recombination, but it is not yet certain whether these regions do have unusually low recombination – Studies testing whether homozygotes for the same allelic form at these “loci” are disfavoured (suggesting “linked load”) are not yet conclusive Fungal and algal mating type loci are NOT sex chromosomes, but they show some very similar evolutionary behaviour, e.g. the mating-type locus of Cryptococcus fungi FRASER et al., 2004 PLoS Biology 2: 22432255 " type Rearranged regions a type Non-diverged region Regions of high a-" divergence Non-diverged region This is like a neosex chromosome system — a part was added recently NOTE: incorrect use of the word ‘rate’ (they mean ‘divergence’) • In Neurospora tetrasperma, there is a non-recombining region whose function is to link the incompatibility gene region to the centromere, guaranteeing first division segregation and thus compatibility among pairs of meiotic products (a mechanism for self-fertilisation) • This region has expanded – Divergence between genes in the two haplotypes varies in a pattern like the sex chromosome strata • Menkis et al. 2008. The mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma represents a model for early evolution of sex chromosomes. PLoS Genetics 4 – The expansion is due to an inversion and the effects of modifier genes • Jacobson 2005. Blocked recombination along the mating-type chromosomes of Neurospora tetrasperma involves both structural heterozygosity and autosomal genes. Genetics 171:839-843. Frequency-dependent selection in plant selfincompatibility maintains many alleles • Genotypes’ survival or fertility (or both, often, for brevity, called “fitness”) depend on the frequencies in the population • e.g. plant self-incompatibility – When an allele is rare, it will usually land on stigmas of plants that do not have that allele, and will thus be compatible with most of the population. Rare alleles have a fertility advantage • Frequency dependent selection also occurs in the interactions between plant defence genes and pathogens Two linked incompatibility loci (S-genes) are involved Sporophytic system Sloci in Arabidopsis species SRK SCR Physical map after Kusaba et al., 2001 NOTE: Differences in gene copy numbers and arrangement in Arabidopsis (and Brassica) suggest suppressed recombination. Gametophytic system S-loci 0.6 0.5 Silent site diversity #s 0.4 0.3 of diversity between SRK classes Kat 0.8 0.6 0.4 0.2 0 -0.2 0.6 0.5 Proportion of diversity between actual populations, Kst ** ** 0.4 0.3 *** * 0.2 0.1 S8 SRK S12 B160 -0.1 Aly8 0 B80 – Diversity and subdivision between alleles are low (there are no associations between SRK alleles and those at the distant loci) – But subdivision between populations of this species is high (for unlinked reference loci, Kst values between European populations is ~ 0.65) ****** *** Proportion 1 SRK • But far from the SRK locus 0 S4 – diversity within types is low, but overall diversity is high, because subdivision between alleles is high (Kat values), but unusually low between populations 0.1 B70 • Close to SRK 0.2 S2 • Treating SRK alleles as “populations”, we measured diversity within each allele type, and “subdivision” between them B120 • In Arabidopsis lyrata, there is evidence that a region around the Slocus has low recombination Gene Significance tested by K* using randomization • Even if the S-locus region has low recombination, it may be too small (contain too few genes to drive the processes described earlier) to undergo genetic degeneration • The possibilities were reviewed by Uyenoyama, M. K. 2005. Evolution under tight linkage to mating type. New Phytologist 165: 63-70. • Some empirical tests have suggested that individuals identical by descent for S alleles may have low survival • BUT it is difficult to test more than a few alleles – one has to compare homozygotes and heterozygotes, matching their inbreeding coefficients, I.e. ensuring that both sets are noninbred (otherwise inbreeding depression might be the cause of lower survival of homozygotes) – it is also hard to rule out an early effect of the incompatibility (which might slow the growth of the pollen that would generate homozygotes, and might lead the maternal plant to abort those zygotes)