Origin and Evolution of Y Chromosomes: Drosophila Tales

Home , Autosome, B chromosome, Y chromosome

Review Origin and evolution of Y chromosomes: Drosophila tales

A. Bernardo Carvalho1, Leonardo B. Koerich1 and Andrew G. Clark2

1 Departamento de Gene´ tica, Universidade Federal do Rio de Janeiro, Caixa Postal 68011, CEP 21944-970, Rio de Janeiro, Brazil 2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

Classically, Y chromosomes are thought to originate somes arise through this path (or by the related neo-Y from X chromosomes through a process of degeneration pathway; Box 1), invariably being homologous to X chromo- and gene loss. Now, the availability of 12 Drosophila somes. genomes provides an opportunity to study the origin Before 2000, an extensive identification of Y-linked and evolution of Y chromosomes in an informative phy- genes had been performed only for humans [7]. The genome logenetic context. Surprisingly, the majority of Droso- sequencing of Drosophila melanogaster in 2000, followed phila Y-linked genes are recent acquisitions from by Drosophila pseudoobscura (2003) and now by ten autosomes and Y chromosome gene gains are more additional Drosophila genomes (2007), uncovered several frequent than gene losses. Moreover, the Drosophila unexpected phenomena, including the lack of X–Y shared pseudoobscura Y chromosome lacks homology with genes, a wholesale replacement of the Y chromosome in the the Y of most Drosophila species. Thus, the Drosophila D. pseudoobscura lineage, and the preponderance of gene Y has a different evolutionary history from canonical Y gains (compared with gene losses) in the 12 sequenced chromosomes (such as the mammalian Y) and it also Drosophila species. Here, we review these data and explore might have a different origin. their consequences in the broader context of Diptera sex- chromosome evolution. We concentrate on single-copy Sex-chromosome origins genes on the Drosophila Y, with an emphasis on results Sex-chromosome evolution provides a particularly coher- [8,9] appearing since the last review on this subject [10]. ent and satisfying blend of empirical data and theory. Readers interested in the role of repetitive DNA in the According to canonical theory, X and Y chromosomes (for evolution of the Drosophila Y chromosome and on evol- the sake of simplicity we refer to the ‘W’ chromosome of utionary models of Y-degeneration should consult Refs birds and butterflies as ‘Y’ [1]) originate from an autosomal [4,11–13]. pair via a three-step process that begins with the acquisition of one or more strong sex-determining genes by one D. melanogaster Y chromosome: a lack of X–Y autosome, giving rise to nascent X and Y chromosomes. homology Natural selection then favors the suppression of recombi- The D. melanogaster Y chromosome comprises mainly nation between the two chromosomes. The lack of recom- repetitive DNA and is heterochromatic. It does not deter- bination, together with the joint effects of mutation, mine sex; instead, sex determination in Drosophila is natural selection and genetic drift, then leads to progress- basically accomplished by a count of the number of X ive degeneration and loss of Y chromosome genes until only chromosomes [14,15]. However, males devoid of Y chromo- the sex-determining gene(s) and a few relic genes survive somes (‘X0 males’) are sterile [16] and formal genetic [1–5]. As the X chromosome become progressively haploid studies identified six genes that are essential for male in males (hemizygous), natural selection favors increased fertility in the D. melanogaster Y [17–20]. Now, genome transcription of X-linked genes in males through several sequencing and the development of proper bioinformatics dosage-compensation mechanisms [1,2]. In the later methods enables a thorough molecular identification of the stages, the Y usually becomes heterochromatic, accumu- Y chromosome gene content [21–23]. Despite its large size lating large amounts of repetitive DNA. It also frequently (40 Mbp), it contains few single-copy protein-coding acquires male-specific genes from the autosomes [6,7] (or genes: 12 are currently known and indirect evidence female-specific genes in the case of the W chromosome, suggests an upper bound of 20 genes [21–24]. These genes where ZW is female and ZZ is male). Empirical data in a are unusually large, owing to Mbp-sized introns compris- variety of organisms including plants and birds support ing repetitive DNA [25]. D. melanogaster Y-linked genes this scenario, the evidence being particularly clear in have two additional important features; many (and prob- mammals. The most compelling evidence is provided by ably all) have male-related functions (e.g. encoding sperm the observation that, among the 27 different proteins flagella motor proteins) and all arose by duplication from encoded by the human Y, 18 have an ancestral, close autosomal genes [9,21–24]. The initial step of their origin counterpart on the X chromosome [6]. This generality, must have been a gene duplication that created a mutant Y coupled with the beautiful fit between theory and data, carrying a copy of an autosomal gene. Then, in some cases, has led to the widespread assumption that all Y chromo- the mutant Y became fixed in the population, either by Corresponding author: Bernardo Carvalho, A. ([email protected]). genetic drift or because it conferred a selective advantage

Box 1. Neo-Y chromosomes cautiously wrote that ‘the genic content of the Y chromosome would be fluid and may differ among (...) species [of X0/XX sex-chromosome systems are believed to have arisen from an XY/XX system via degeneration and loss of the Y [1].AY the Drosophilidae family]’ [22]. As described in the next chromosome can be ‘regenerated’ in these species when an section, when the 12 Drosophila genomes became avail- autosome fuses to the X chromosome and the new arrangement able, it was found that Y chromosome gene content differs is fixed within a population (Figure 1b). During male meiosis the free even among species of the same group [8,9]. homolog of the fused autosome continues to pair with the newly added portion of the X (termed ‘neo-X’) and moves to the opposite pole of the cell, behaving as a Y chromosome (hence its name, ‘neo- The 12 Drosophila Y chromosomes: a prominent role of Y’). Neo-Y chromosomes are male-restricted and the same evolu- gene gains tionary factors that cause the degeneration of Y chromosomes are Before examining the results of Koerich et al. [9], it is worth expected to act on them. Empirical data confirm that they mentioning a caveat. Ideally, the complete gene set of the Y accumulate repetitive DNA and that their genes degenerate. Indeed Drosophila neo-Y systems are popular models to study Y-chromo- chromosomes in the 12 species should be available before some degeneration [12,48]. There are important differences be- starting a comparative analysis, similar to the approach tween canonical Y and neo-Y evolution [38]; nonetheless, in both used (at least approximately) for analysing the euchro- cases the Y originated through degeneration (of the X or the neo-X). matic portion of the other chromosomes. However, given Fusions between the sex chromosomes (either the X or Y) and the notorious difficulties in sequencing and assembling autosomes also occur in XY/XX species. D. pseudoobscura and its close relatives exemplify the former case: their X chromosomes heterochromatic regions and the Y [27,28], Koerich et al. stem from a fusion between the ancestral X and an autosome [1]. [9] investigated Y-linkage in the Drosophila orthologs of After fixation of the X–autosome fusion the ancestral lineage must the D. melanogaster Y-linked genes. The ensuing ascer- have passed through a ‘X/Y/neo-Y’ stage, in which three chromo- tainment bias should be corrected when estimating gene somes pair during male meiosis (the female meiosis is normal). Not gain and loss rates [9]. The special case of two species (D. surprisingly, such an event causes meiotic problems, and a similar system in another Drosophila species generates 1–3% of sex- pseudoobscura and the closely related D. persimilis)is chromosome aneuploidy [49]. This intermediate stage is usually discussed in the next section, because the changes in their short lived and in a few million years only one Y chromosome Y-chromosome gene content were caused by a wholesale remains; it is believed that the neo-Y is lost or fused to the ancestral replacement of the Y, rather than by individual gains and Y [1]. However, the outcome was surprisingly different in the D. losses of genes [8]. pseudoobscura lineage: the ancestral Y was incorporated into an autosome and was replaced by a new Y chromosome, possibly Koerich et al. [9] found that, in many cases, the orthologs derived from the neo-Y [8]. of the D. melanogaster Y-linked genes were autosomal in other species. This change in chromosomal location can be explained either by an autosome-to-Y transposition in the to males that carried it. Given their redundancy, the D. melanogaster lineage (i.e. a gene gain in the Y) or by a Y- autosomal copy could have pseudogenized and disap- to-autosome transposition in the other lineage (i.e. a gene peared, the new Y-linked copy could have disappeared, loss in the Y). Additional data based on synteny, however, or the two copies could have retained functionality, have provided unambiguous answers (Figure 2). Among possibly by diverging to perform different functions [26]. the 12 known D. melanogaster Y-linked genes, seven were Different Y-linked genes illustrate each of these steps. acquired by the Y in the D. melanogaster lineage <63 Mya Preliminary data suggest that the flagrante delicto Y [9]. This pattern contrasts sharply with the rest of the (FDY) gene is the youngest gene we identified (estimated genome, in which 95% of the genes remained on the same age: 2 million years [My]). It arose from a genomic chromosome arm across the 12 species [29]. However, the duplication of the autosomal gene CG11844 gene to the absolute number of gene movements is similar in the Y and Y, and the flanking sequences remain clearly recognizable in the other chromosomes. So why is the Y-linked gene (http://www.drosophila-conf.org/genetics/gsa/dros/ content so poorly conserved? The most likely explanation is dros2003/; #318C). The autosomal and the Y-linked copies that the other chromosomes had thousands of genes in the are still present and functional. By contrast, the WD40 Y ancestor of the 12 sequenced species, whereas the Y (WDY) gene and the male fertility factor kl5 (kl-5) gene chromosome had a very low number of genes (we know arrived to the Y chromosome 50 million years ago (Mya) of five: male fertility factor kl2 [kl-2], male fertility factor kl3 and, in these cases, the autosomal copies were lost [9,23]. [kl-3], Ppr-Y, Polycystine-related-Y [PRY] and Occludin- However, the autosomal copy does not seem to be lost in all related-Y [ORY]; Figure 2). This condition, coupled with old transpositions: the protein phosphatase 1 regulatory a small and similar number of gene movements in the Y subunit Y (Ppr-Y) gene, for example, arrived on the Y and the other chromosomes, would produce the present chromosome >63 Mya, and this gene and the putative pattern of low conservation of gene content in the Y autosomal parent gene CG13125 have been retained [9,22]. chromosome and high conservation in the other chromo- The D. melanogaster Y chromosome does not share any somes. This pattern dates back to mosquitoes, which single-copy genes with the X. This unusual feature is made diverged from Drosophila 260 Mya; although there are clear when the D. melanogaster and human Y chromo- clear homologies in other chromosomes between these somes are compared (Figure 1a); the ‘X-ancestral’ genes, Diptera [30,31], all orthologs of D. melanogaster Y-linked which comprise the majority of Y-linked genes in mammals genes are autosomal in mosquitoes. Thus, the gene content and are the hallmark of the X–Y shared ancestry, are of the Drosophila Y is younger than the other chromo- absent in D. melanogaster Y. Because Drosophila Y-linked somes. genes came from autosomes, one might expect some inter- Comparisons between gene gains and losses also yield specific variation, but at what time scale? Years ago we interesting clues about the evolution of the Drosophila Y:

271 Review Trends in Genetics Vol.25 No.6

Figure 1. Origins of Y chromosomes. (a) Y-linked genes in (i) D. melanogaster and (ii) humans. Genes ancestrally shared with the X are shown in red and those acquired from autosomes are shown in blue. Genes with unknown origin or that are later additions to both the X and the Y are grouped in the ‘other’ class (green). Genes on the human Y chromosome encode 27 different proteins. The majority of these genes are ancestrally shared with the X chromosome, indicating that these chromosomes are homologous [6,7]. This class of genes is absent among the 12 known single-copy genes in the D. melanogaster Y chromosome, suggesting that X and Y are not homologous [9]. Several human Y-linked genes are multi-copy; we counted them only once. (b) Main paths for the origin of Y chromosomes. Only male karyotypes are shown. (i) The canonical path begins when an autosome acquires a strong male determining gene M, becoming a nascent Y (its homolog became a nascent X). Degeneration of the nascent Y and the evolution of dosage compensation in the X originate mature sex chromosomes [1–5]. (ii) The neo-Y path (shown here in a species with X0/XX sex-determination) begins with an X–autosome fusion, transforming the fused autosome into a neo-X. The free homolog became a neo-Y, which then degenerates [1]. (iii) The non-canonical path begins when a parasitic B chromosome (which usually does not pair) acquires the capacity to pair with the X. Improvement of B–X pairing and the acquisition of male-fertility genes (F) form a chromosome that will be termed ‘Y’ [10,34–36]. However, in contrast with the two other paths, these non- canonical Y chromosomes do not share any homologous region with the X, and are not formed by degeneration. Note that only canonical Y chromosomes are expected to have male-determining function (imparted by the M gene), and that the neo-Y and non-canonical paths can be distinguished because the former reduces the number of autosomes [1]. Color code: blue, autosomes; red, mature sex chromosomes; yellow, extra chromosomes such as a B chromosome. the rate of gene gain is 11 times higher than the rate of male fertility [32], so it presumably acquired one or more gene loss (P = 0.003; 95% confidence interval: 2.3–52.5; [9]). fertility genes from other chromosomes, by the process Thus, the Drosophila Y shows another unusual pattern (for outlined earlier for D. melanogaster (alternatively, some a Y-chromosome): gene gains have a prominent role in its mechanical problem in meiosis might cause sterility in X0 evolution; indeed, its gene content has increased in the last males). We identified 15 genes and pseudogenes in the D. 63 Myr. This finding also suggests that the Drosophila Yis pseudoobscura Y chromosome and none are shared with young because it is gaining genes and yet has few of them. the D. melanogaster Y [8]. Hence, despite their functional and cytogenetic similarities (both are required for male The D. pseudoobscura case: wholesale replacement of fertility, pair with the X, and are heterochromatic), these Y the Y chromosome chromosomes are not homologous. This unexpected finding Given the phylogenetic proximity between D. melanogaster has obvious implications for the origin and evolution of Y and D. pseudoobscura (they belong to the sister groups chromosomes and raises the question of why it happened. melanogaster and obscura), we expected their Y chromo- A possible explanation follows. The incorporation of the somes to be very similar. Instead, they have nothing in ancestral Y into an autosome occurred only in species such common! The ancestral Drosophila Y chromosome became as D. pseudoobscura and D. affinis which also have a part of an autosome in the D. pseudoobscura lineage and known X–autosome fusion, whereas the more distantly was replaced by a new Y chromosome, possibly derived related D. bifasciata and D. guanche (which do not have from a neo-Y [8]. This new Y chromosome is essential for the X–autosome fusion) still carry the ancestral Y [8]. This

272 Review Trends in Genetics Vol.25 No.6

Figure 2. Gene movements in the Drosophila Y chromosome. Gene gains by the Y chromosome are marked with red arrows and gene losses with blue arrows. The genes assigned with dashed red arrows are probable gains, which have not yet been confirmed owing to lack of close outgroups [9]. The green bar in the D. pseudoobscura–D. persimilis lineage marks the incorporation of the ancestral Y into an autosome, and its replacement by a new Y chromosome [8]. Owing to the experimental design, gene gains can only be detected in the D. melanogaster lineage and gene losses only in the other lineages. After correction of this ascertainment bias, the rate of gene gain is 11- fold higher than the rate of gene loss [9]. Note that the majority of D. melanogaster Y-linked genes (7 out of 12) were acquired after the split between the Drosophila and Sophophora subgenera, which occurred 63 Mya [47]. Figure adapted, with permission, from Ref. [9]. Full gene names: ARY: aldehyde reductase Y; CCY: coiled-coils Y; Pp1-Y1, Pp1-Y2: Y-linked protein phosphatases. association provides a clue: X–autosome fusions cause necessary because the high nucleotide identity (typically meiotic problems in XX/XY species because three chromo- >97%) between these 15 Y-linked genes and their autosomes (the ancestral Y, the X and the neo-Y) must pair (Box somal paralogs might suggest that they are recent acqui- 1). Hence, the incorporation of the ancestral Y into an sitions that occurred after the X–autosome fusion. autosome might have been advantageous because it solved Additional support for the neo-Y hypothesis might come the meiotic problems caused by the X–autosome fusion [8]. from the identification of more Y-linked genes and identi- For a long time, it was thought that the D. pseudoobs- fication of the pairing regions between the X and Y in D. cura Y chromosome was the ancestral Y, and that the neo- pseudoobscura. The bottom line is that the neo-Y hypoth- Y was lost or incorporated into the ancestral Y [1]. Instead, esis provides a simple explanation for the origin of the the genomic data showed that the Y became part of an current Y, but the question remains unsolved. However, autosome. The data also suggest a possible origin for the the data in hand already show that Drosophila Y chromo- current Y: it might be a degenerated neo-Y. The finding somes can be very labile; indeed, chromosomes that seem that ten of the 15 known genes and pseudogenes have to be identical (e.g. those of D. melanogaster and D. pseu- homologs on the neo-X (the autosome that became fused to doobscura) can be non-homologous. These new results also the ancestral X) seems at first sight to confirm this hypoth- hint that X-chromosome pairing and a determining role in esis but, actually, the evidence is at best weak, for two male fertility, which are characteristics that define a Dro- reasons. First, the 15 genes do not represent independent sophila Y chromosome, can evolve rapidly (the current D. events because most of them are adjacent to each other in pseudoobscura Y originated between 2 and 18 Mya [8]) and their original locations. They came from five different provide little information about the origin of the chromo- locations and, among these five, only two are homologous some itself. to the neo-X [8]. Hence, the proper evidence in favor of a How frequent is the phenomenon of wholesale replace- neo-Y origin of the D. pseudoobscura Y is not ten out of 15, ment of the Y? Does it occur in every case of X–autosome but rather two out of five. Second, and more importantly, if fusion? Is it restricted to species with X–autosome fusion, these 15 Y-linked genes are part of the neo-Y chromosome as required by the ‘neo-Y origin’ hypothesis? These three originated by the X-autosome fusion, at least some should questions are amenable to direct experimental investi- be present in the Y chromosome of other species, such as D. gation and the results described in the previous section affinis, that share the same X–autosome fusion. To our have answered one of them. In particular, D. willistoni, knowledge, this has been verified only in D. persimilis (by which has undergone an independent X–autosome fusion, Carlos Machado’s laboratory), which is the closest species carries the ancestral Y chromosome [9]; therefore, Y–auto- to D. pseudoobscura. Testing more distant species is some fusions do not necessarily follow X–autosome fusions.

273 Review Trends in Genetics Vol.25 No.6

The remaining two questions are being addressed by an cannot be completely excluded as a possibility, it might be ongoing study of a large number of Drosophila species. time to consider alternative hypotheses that generate new Preliminary data suggest that, among the 310 tested experimental questions rather than ad hoc explanations. species, 42 had their Y chromosomes replaced, owing to In fact, these discrepancies can be explained parsimo- four independent events (the D. pseudoobscura lineage niously by the hypothesis that the Drosophila Y chromo- case and three additional lineages). None of the three some is not a degenerated X, but rather that it originated additional events (amounting to 34 species) has an X– from the addition of an extra chromosome (that evolved the autosome fusion, so the current Y of these species cannot ability to pair with the X) to an X0/XX sex-chromosome be a degenerated neo-Y. Instead, they must have a differ- system [10,34] (Figure 1b). Although this path might seem ent origin. odd, it happened independently in two Homoptera species [35,36] in which the Y chromosomes originated from ‘B An alternative hypothesis for the origin of the chromosomes’ (supernumerary dispensable chromosomes Drosophila Y that are present in many species [1,37]), which evolved Given the many similarities between the Drosophila Y and accurate pairing with the X-chromosome (Box 2). other Y chromosomes (e.g. X–Y pairing, low gene density, How common are these non-canonical Y chromosomes? heterochromatic state) and the seemingly universal It is common practice to term as Y any chromosome that validity of sex-chromosome evolution theory, it is easy to pairs with the X and is present only in males; moreover, it understand why the Drosophila Y is usually thought to is implicitly assumed to be a degenerated X, even if it does have originated through the same canonical path. How- not have a role in sex-determination (e.g. Drosophila). ever, researchers have repeatedly uncovered contradictory Thus, almost by definition non-canonical Y chromosomes evidence while studying this chromosome in detail. The will be overlooked. The unconventional origin of the Y Drosophila Y lacks a sex-determining gene [16], which, chromosome of the Homoptera Rhinocola aceris was ident- according to the canonical model, should be present. ified owing to the combination of incomplete fixation and Furthermore, Brosseau [17] (see also Ref. [33]) commented high confidence of the ancestral state [35], which probably that ‘the Y has no genetic regions homologous to most of the reflects the fortuitous observation of a recent event. After X(...). This view is diametrically opposed to the notion that the R. aceris study, a careful investigation of Cacopsylla the Y is a degenerate X.’ Finally, the recent genomic data peregrina revealed the same phenomenon [36]. It is likely [8,9,21–23] also do not fit well with the canonical model. that additional cases will be found as researchers become Each of these discrepancies can be accommodated by an ad aware of the possibility of non-canonical origin of Y- hoc assumption: the lack of X–Y shared genes can be chromosomes; tsetse flies, in which the Y chromosome explained by the assumption that all signs of X–Y has clear similarities with B chromosomes, could be an homology were erased. The lack of a male sex-determining example (Box 3). Because the X0/XX sex-chromosome sys- gene can be explained by the assumption that the degener- tem is widespread among insects and is almost certainly ation erased even this gene (or the Drosophila Y being a the ancestral system in several orders [38], there might be neo-Y; but see later). The odd D. pseudoobscura data could many additional cases of Y chromosomes arising from B be rationalized by the assumption that its current Y is a chromosomes. Furthermore, although it is easier to envi- neo-Y (although this explanation ignores the unexpected sage the origin of non-canonical Y chromosomes in species wholesale replacement of the Y). To explain the prominent with X0/XX sex-chromosomes, B chromosomes are also role of gene gains across the 12 species, one could assume able to pair with the sex-chromosomes in XY/XX species that the Drosophila Y arose from the degeneration of the X [39]. chromosome and, hence, only more recently have Will direct testing of the ‘non-canonical origin’ hypoth- gene gains become important. Although these arguments esis be possible in Drosophila? The Homoptera examples

Box 2. Y chromosomes that originated from B chromosomes

Nokkala and co-workers showed that the Y chromosomes of two nearly perfectly with the X (in the same way as a bona fide Y Homoptera species (Rhinocola aceris and Cacopsylla peregrina) chromosome). The foreknowledge that the basic sex-chromosome originated from ‘B chromosomes’ [35,36], supernumerary dispen- system is X0/XX in the Psylloidea family [50], together with the sable chromosomes that are present in many species [1,37].B maintenance of the number of autosomes in R. aceris, ruled out the chromosomes frequently cause detrimental fitness effects but, as in possibility of a neo-Y origin. The lack of chiasmata between the X and other cases of selfish genetic elements, their elimination from Y of the two species (which are present in other Psylloidea species populations is prevented by meiotic drive. In some cases, they with neo-Y) provided additional evidence that their Y chromosomes evolve another mechanism that ensures their maintenance in originated from B chromosomes [35,36]. populations: the ability to pair with X chromosomes in X0/XX Because B chromosomes are rare (although not absent) in species [37]. Drosophila [51], perhaps a more likely source of a non-canonical B chromosomes typically are heterochromatic and occur in variable Y chromosome is the small ‘dot’ chromosome (D. melanogaster numbers, both within and among populations; in the same popula- chromosome 4; [34]). Indeed, trisomy for the dot chromosome is tion, individuals might carry 0, 1, 2, 3 or more copies. This well tolerated [52] and it pairs with the X [53,54]. Of course, the characteristic provides the strongest evidence that the R. aceris Y suggestion of a non-canonical origin of the Drosophila Y does not originated from a B chromosome: in one population, males and imply that the canonical evolutionary mechanism could not females carry a variable number of heterochromatic chromosomes operate in Drosophila; neo-Y chromosomes also are known to which pair imperfectly with the X in males (making it clear that they degenerate in these species (e.g. see Refs [12,48]). Therefore, the are typical B chromosomes), whereas in other populations, a similar important question is whether or not the canonical model provides chromosome is present only in males, in a single copy, and pairs the most likely explanation, given the available evidence.

274 Review Trends in Genetics Vol.25 No.6

Box 3. The Y chromosome of tsetse flies Box 4. Cytogenetics and the origin of the Drosophila Y

Tsetse flies (genus Glossina) are considered to have an XY/XX sex- Chromosome studies have provided important evidence for the origin chromosome system, but their Y chromosome is not involved in sex of Y chromosomes in many species and they are especially effective determination and shows irregular segregation with the X [55]. when the ancestral state (i.e. before the origin of the Y) is known Indeed, aneuploidy is common in natural populations (e.g. X0, XY [1,35,36]. The majority of Drosophila species have an XY/XX sex- and XYY males co-exist [55]). Furthermore, the Y seems to cause chromosome system [59], so we must infer the ancestral state by meiotic drive in XXY females because they produce twice as many looking outside the genus. The best outgroups for this purpose are XY as X eggs [56]. Variable numbers and meiotic drive are hallmarks the Steganinae subfamily (which belongs to the same Drosophilidae of B chromosomes [1,37] and, if not for the sterility of X0 males [56], family) and sister families such as Ephydridae and Curtonotidae. Little the Glossina Y would perfectly fit the definition of a B chromosome. is known about their chromosomes and the limited data from more Indeed Glossina is known to have B chromosomes [57] and the distant Acalyptrata families are contradictory, with one study close similarity between their B and Y chromosomes in meiotic suggesting that the Drosophilidae karyotype is highly derived [60] behavior and C-band pattern led Amos and Dover [58] to propose and another suggesting that it is ancestral [61]. An investigation of the that their B chromosomes originated from the Y (but note that the karyotype of outgroups such as Ephydridae is clearly needed. This opposite relationship – Y originated from B – additionally explains investigation should eliminate at least one mechanism from the list of the irregular X–Y segregation). The Glossina genome is currently possible origins of the Drosophila Y (i.e. X-degeneration, neo-Y and being sequenced. The genomic data, together with studies aimed at non-canonical). For example, an X0/XX ancestral state would rule out uncovering the ancestral state of sex-chromosomes in related the X-degeneration hypothesis. Neo-Y formation by X–autosome families, might show whether or not the Glossina Y chromosome fusions can be detected by the reduction in the number of autosomes is actually a specialized B chromosome that acquired male-fertility and (in many cases) by the transformation of a single-armed into a genes. two-armed X [1]. The application of these ideas to the study of the origin of the Drosophila Y is hampered by the lack of data from outgroups. The proposed cytogenetic approach also has some caveats. Neo-Y detection is straightforward in evolutionarily recent described in Box 2 might provide some guidance. These events with a known ancestral state [1,50,62], but the Drosophila Y cases were uncovered using cytogenetics (optical micro- originated >63 Mya [9]; pending the rate of chromosomal evolution, scopy of chromosomes), and analogous work in the Droso- further changes that alter chromosome number and structure (such philidae sister families (e.g. Ephydridae) is very much as chromosome fusions, fissions, and inversions) might have blurred needed (Box 4). Interestingly, the only Ephydridae species the signs of the origin of the Y. Another limitation is that Y chromosomes that are non-homologous (e.g. D. melanogaster and investigated so far has X0/XX sex chromosomes [40].As D. pseudoobscura; [8]) can look nearly identical when using simple seen in R. aceris, uncovering recent events is crucial cytogenetics methods (e.g. orcein staining). However, the use of more because the mechanisms of Y origination have not yet been detailed cytogenetic methods, such as fluorescence in situ hybridiza- blurred by additional chromosomal changes. This forms tion (FISH) and chromosome painting (e.g. see Ref. [63]), can alleviate part of the rationale for studying Y-linked gene content in these concerns. 300 Drosophila species; conﬁrmation of the suspected cases of non-canonical Y origin in several Drosophila homologous to the Drosophila X: they are completely het- lineages would support a similar origin for the ancestral erochromatic, carry very few genes, and the genes ortho- Drosophila Y. Finally, as the cost of genome sequencing logous to Drosophila X-linked genes that have been continues to fall, it is certain that many Diptera genome mapped are autosomal in these species [42,43]. Thus, in sequences will become available during the next decade, some Diptera families the X chromosome was probably shedding light on the origin and evolution of their sex replaced; such a possibility creates an interesting problem chromosomes. For example, species in which the Y chromo- with the pre-existing dosage-compensation mechanism. some originated from a neo-Y should have a large block of Namely, the former X chromosome (now an autosome) is additional genes (derived from the fused autosome) on the present in equal dose in males and females in these Dip- X chromosome when compared to proper outgroups, even if tera families and hence its hyper-transcription in males the fusion is ancient. Some glimpses of this approach are should have been turned off [44]. This prediction might also already possible. be tested in Aedes, which seems to have evolved into a Mosquito (Anopheles and Aedes) and Drosophila X system with very little differentiation between the X and chromosomes are homologous [30,31]; therefore, the gen- the Y [41]. esis of the X (and presumably of a canonical Y) occurred >260 Mya, and X chromosome identity is conserved across Concluding remarks very distant Diptera families. This ancestral X is the sole What is a Y chromosome? A good deﬁnition is that it is a major contributor to the Drosophila X [30,31], with no chromosome that regularly segregates from the X chromo- evidence of X–autosome fusions, which strongly suggests somes, irrespective of its origin. Many Y chromosomes will that the ancestral Drosophila Y is not a neo-Y. By contrast, be degenerated Xs or neo-Ys, and a currently unknown half of the Aedes X chromosome is homologous to the number (particularly among insects) will be traced to a Drosophila and Anopheles X, but the other half is homolo- non-canonical origin. The canonical theory of sex-chromo- gous to a single autosome in each of these species [30], some evolution, with its emphasis on X–Y homology, slow presumably owing to an X–autosome fusion in the Aedes degeneration and gene loss, provides an incomplete con- lineage. However, the Aedes Y is not a neo-Y; it is nearly ceptual framework to study the Drosophila Y chromosome, identical to the X (except for the presence of the male- in which there is no sign of X–Y homology, several cases of determining gene [41]), so additional changes must have wholesale replacement seem to have occurred, and gene occurred. In contrast to mosquitoes, the X chromosomes of gains have a prominent role. By considering the possibility the much more closely related families Tephritidae (Cer- of a non-canonical origin, the focus shifts to questions such atitis capitata) and Muscidae (Musca domestica) are not as the origin of the Drosophila Y, its evolutionary lability,

275 Review Trends in Genetics Vol.25 No.6 the sex chromosomes of Ephydridae and other Diptera 8 Carvalho, A.B. and Clark, A.G. (2005) Y chromosome of D. families, and to the close relationship between Y and B pseudoobscura is not homologous to the ancestral Drosophila Y. Science 307, 108–110 chromosomes in some species. Throughout this review we 9 Koerich, L.B. et al. (2008) Low conservation of gene content in the have emphasized that these questions are amenable to Drosophila Y chromosome. Nature 456, 949–951 experimental investigation and, therefore, will be 10 Carvalho, A.B. (2002) Origin and evolution of the Drosophila Y answered eventually. As the Glossina case suggests (Box chromosome. Curr. Opin. Genet. Dev. 12, 664–668 3), such a shift in focus might illuminate Y-chromosome 11 Gvozdev, V.A. et al. (2005) The Y chromosome as a target for acquired and amplified genetic material in evolution. Bioessays 27, 1256–1262 evolution far beyond Drosophila. 12 Bachtrog, D. et al. (2008) Genomic degradation of a young Y On the experimental side, we cannot emphasize enough chromosome in Drosophila miranda. Genome Biol. 9, R30 the importance of an exhaustive identification of Y-linked 13 Lemos, B. et al. (2008) Polymorphic Y chromosomes harbor cryptic genes. This is the main obstacle to the study of the Y variation with manifold functional consequences. Science 319, 91–93 chromosome because its richness of repetitive DNA pre- 14 Cline, T.W. (1993) The Drosophila sex determination signal: how do flies count to two? Trends Genet. 9, 385–390 cludes sequence assembly into large and easily studied 15 Erickson, J.W. and Quintero, J.J. (2007) Indirect effects of ploidy scaffolds. Instead, short Y-linked scaffolds (usually be- suggest X chromosome dose, not the X:A ratio, signals sex in tween 1 kb and 100 kb) must be individually identified Drosophila. PLoS Biol. 5, e332 among thousands of unmapped scaffolds that came from 16 Bridges, C.B. (1916) Non-disjunction as proof of the chromosome theory of heredity. Genetics 1, 1–52 other heterochromatic regions, using computational 17 Brosseau, G.E. (1960) Genetic analysis of the male fertility factors on methods followed by experimental verification of Y-linkage the Y chromosome of Drosophila melanogaster. Genetics 45, 257–274 [8,21–23]. The experimental verification is necessary 18 Kennison, J.A. (1981) The genetic and cytological organization of the Y owing to the high rate of false-positives and false-negatives chromosome of Drosophila melanogaster. Genetics 98, 529–548 that occur using the current computational methods; this 19 Hazelrigg, T. et al. (1982) A cytogenetic analysis of X- ray induced male steriles on the Y chromosome of Drosophila melanogaster. step is labor intensive (when applied to hundreds of scaf- Chromosoma 87, 535–559 folds) and in practice is done only in the scaffolds that seem 20 Gatti, M. and Pimpinelli, S. (1983) Cytological and genetic-analysis of to encode genes [8,21–23]. Two recent developments will the Y-chromosome of Drosophila melanogaster. 1. Organization of the make this individual experimental verification unnecess- fertility factors. Chromosoma 88, 349–373 ary and hence are likely to have a deep impact on the 21 Carvalho, A.B. et al. (2000) Y chromosomal fertility factors kl-2 and kl-3 of Drosophila melanogaster encode dynein heavy chain polypeptides. knowledge of Y chromosomes. We developed a method Proc. Natl. Acad. Sci. U. S. A. 97, 13239–13244 based on short-read sequences that enables massive identi- 22 Carvalho, A.B. et al. (2001) Identification of five new genes on the Y fication of Y-linked sequences (http://www.drosophila-conf. chromosome of Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. org/2008/pdf/abstract_platform.pdf; #124). This method is 98, 13225–13230 suited for genomes that have already been sequenced using 23 Vibranovski, M.D. et al. (2008) Two new Y-linked genes in Drosophila melanogaster. Genetics 179, 2325–2327 male–female mixed libraries (e.g. see Ref. [45]). However, 24 Gepner, J. and Hays, T.S. (1993) A fertility region on the Y chromosome as proposed by Krzywinski et al. [46], the simple separation of Drosophila melanogaster encodes a dynein microtubule motor. Proc. of male and female libraries before sequencing provides the Natl. Acad. Sci. U. S. A. 90, 11132–11136 most straightforward and powerful method to identify Y- 25 Reugels, A.M. et al. (2000) Mega-introns in the dynein gene DhDhc7(Y) on the heterochromatic Y chromosome give rise to the giant threads linked sequences, at nearly zero additional cost. Currently, loops in primary spermatocytes of Drosophila hydei. Genetics 154, several Anopheles genomes and the hemipteran Rhodnius 759–769 prolixus are being sequenced in this way. We hope that this 26 Lynch, M. and Katju, V. (2004) The altered evolutionary trajectories of will become standard practice for future genome projects. gene duplicates. Trends Genet. 20, 544–549 27 Hoskins, R.A. et al. (2007) Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science 316, 1625–1628 Acknowledgements 28 Carvalho, A.B. et al. (2003) Y chromosome and other heterochromatic We thank B. Lemos, S. Nokkala, J. Lucchesi, Y. Tao, R. Arguello, C. sequences of the Drosophila melanogaster genome: how far can we go? Machado and two anonymous reviewers for many valuable comments on Genetica 117, 227–237 the manuscript. Our work is supported by Conselho Nacional de 29 Bhutkar, A. et al. (2007) Genome-scale analysis of positionally Desenvolvimento Cientı´fico e Tecnolo´gico-CNPq, Coordenaça˜ ode relocated genes. Genome Res. 17, 1880–1887 Aperfeiçoamento do Pessoal de Ensino Superior-CAPES, FAPERJ, FIC- 30 Nene, V. et al. (2007) Genome sequence of Aedes aegypti, a major NIH grant TW007604–02 (to A.B.C.) and NIH grant GM64590 (to A.G.C. arbovirus vector. Science 316, 1718–1723 and A.B.C.). 31 Zdobnov, E.M. et al. (2002) Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298, References 149–159 1 White, M.J.D. (1973) Animal Cytology and Evolution. University Press 32 Dobzhansky, T. (1937) Further data on the variation of the Y 2 Bull, J.J. (1983) Evolution of Sex Determining Mechanisms. Benjamin/ chromosome in Drosophila pseudoobscura. Genetics 22, 340–346 Cummings Pub. Co., Advanced Book Program 33 Lohe, A.R. et al. (1993) Mapping simple repeated DNA sequences 3 Rice, W.R. (1996) Evolution of the Y sex chromosome in animals. in heterochromatin of Drosophila melanogaster. Genetics 134, 1149– Bioscience 46, 331–343 1174 4 Charlesworth, B. and Charlesworth, D. (2000) The degeneration of Y 34 Hackstein, J.H. et al. (1996) Is the Y chromosome of Drosophila an chromosomes. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 1563–1572 evolved supernumerary chromosome? Bioessays 18, 317–323 5 Graves, J.A. (2006) Sex chromosome specialization and degeneration in 35 Nokkala, S. et al. (2000) Achiasmate segregation of a B chromosome mammals. Cell 124, 901–914 from the X chromosome in two species of psyllids (Psylloidea, 6 Skaletsky, H. et al. (2003) The male-specific region of the human Y Homoptera). Genetica 108, 181–189 chromosome is a mosaic of discrete sequence classes. Nature 423, 825– 36 Nokkala, S. et al. (2003) The origin of the achiasmatic XY sex 837 chromosome system in Cacopsylla peregrina (Frst.) (Psylloidea, 7 Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y Homoptera). Genetica 119, 327–332 chromosome. Science 278, 675–680 37 Camacho, J.P. (2004) Preface. Cytogenet. Genome Res 106, 147–148

276 Review Trends in Genetics Vol.25 No.6

38 Blackman, R.L. (1995) Sex determination in insects. In Insect 52 Lindsley, D.L. and Grell, E.H. (1972) Genetic variations of Drosophila Reproduction (Leather, S.R. and Hardie, J., eds), pp. 224–243, CRC melanogaster. Carnegie Institute press 53 Hughes, S.E. et al. (2009) Heterochromatic threads connect oscillating 39 Nokkala, S. (1986) The meiotic behaviour of B-chromosomes and their chromosomes during prometaphase I in Drosophila oocytes. PLoS effect on the segregation of sex chromosomes in males of Hemerobius Genet. 5, e1000348 marginatus. Hereditas 105, 221–227 54 Sandler, L. and Novitski, E. (1956) Evidence for genetic homology 40 Heitz, E. (1933) Die somatische Heteropyknose bei Drosophila between chromosomes I and IV in Drosophila melanogaster, with a melanogaster und ihre genetische Bedeutung. Cell Tissue Res. 20, proposed explanation for the crowding effect in triploids. Genetics 41, 237–287 189–193 41 Clements, A.N. (1992) The Biology of Mosquitoes. Chapman & Hall 55 Maudlin, I. (1979) Chromosome polymorphism and sex determination 42 Hiroyoshi, T. (1977) Some new mutants and the revised linkage maps in a wild population of tsetse. Nature 277, 300–301 of the housefly, Musca domestica. Jpn. J. Genet. 52, 275–288 56 Southern, D.I. (1980) Chromosome diversity in Tsetse flies. In Insect 43 Stratikopoulos, E.E. et al. (2008) An integrated genetic and cytogenetic Cytogenetics (Blackman, R.L. et al., eds), Blackwell Scientific map for the Mediterranean fruit fly, Ceratitis capitata, based on Publications microsatellite and morphological markers. Genetica 133, 147–157 57 Southern, D.I. and Pell, P.E. (1973) Chromosome relationships and 44 Larsson, J. and Meller, V.H. (2006) Dosage compensation, the origin meiotic mechanisms of certain morsitans group tsetse flies and their and the afterlife of sex chromosomes. Chromosome Res. 14, 417–431 hybrids. Chromosoma 44, 319–334 45 Drosophila 12 Genomes Consortium (2007) Evolution of genes and 58 Amos, A. and Dover, G. (1981) The distribution of repetitive DNAs genomes on the Drosophila phylogeny. Nature 450, 203–218 between regular and supernumerary chromosomes in species of 46 Krzywinski, J. et al. (2004) Isolation and characterization of Y Glossina (Tsetse): a two-step process in the origin of supernumeraries. chromosome sequences from the African malaria mosquito Chromosoma 81, 673–690 Anopheles gambiae. Genetics 166, 1291–1302 59 Clayton, F.E. (1998) Published karyotypes of the Drosophilidae. 47 Tamura, K. et al. (2004) Temporal patterns of fruit fly (Drosophila) Drosoph. Inf. Serv. 81, 5–125 evolution revealed by mutation clocks. Mol. Biol. Evol. 21, 36–44 60 Boves, J.W. et al. (1973) Chromosomes of Richardiidae, Otitidae 48 Steinemann, M. et al. (1993) How Y chromosomes become genetically and Platystomatidae (Diptera: Acalyptratae). Genetica 44, 553– inert. Proc. Natl. Acad. Sci. U. S. A. 90, 5737–5741 571 49 Cooper, K.W. (1946) The mechanism of non-random segregation of 61 Block, K. (1976) Chromosomal variation in Agromyzidae (Diptera). VI. sex chromosomes in male Drosophila miranda. Genetics 31, 181– Comparative chromosome studies. Hereditas 84, 177–212 194 62 Patterson, J.T. and Stone, W.S. (1952) Evolution in the Genus 50 Maryanska-Nadachowska, A. (2002) A review of karyotype variation in Drosophila. The MacMillan Company jumping plant-lice (Psylloidea, Sternorrhyncha, Hemiptera) and 63 Rens, W. et al. (2004) Resolution and evolution of the duck-billed checklist of chromosome numbers. Folia Biol. (Krakow) 50, 135–152 platypus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male 51 Ramachandra, N.B. and Ranganath, H.A. (1985) Suprenumerary sex chromosome constitution. Proc. Natl. Acad. Sci. U. S. A. 101, chromosomes in Drosophila nasuta albomicans. Experientia 41, 680–681 16257–16261

277