Abstract

Recent studies have suggested that gene gain and loss may contribute significantly to the divergence between humans and chimpanzees. Initial comparisons of the human and chimpanzee Y-chromosomes indicate that chimpanzees have a disproportionate loss of Y-chromosome genes, which may have implications for the adaptive evolution of sex-specific as well as reproductive traits, especially because one of the genes lost in chimpanzees is critically involved in spermatogenesis in humans. Here we have characterized Y-chromosome sequences in gorilla, bonobo, and several chimpanzee subspecies for 7 chimpanzee gene–disruptive mutations. Our analyses show that 6 of these gene-disruptive mutations predate chimpanzee–bonobo divergence at ∼1.8 MYA, which indicates significant Y-chromosome change in the chimpanzee lineage relatively early in the evolutionary divergence of humans and chimpanzees.

Introduction

Comparative analyses of single-nucleotide differences between human and chimpanzee genomes typically show estimates of approximately 1–2% divergence (Watanabe et al. 2004; Chimpanzee Sequencing and Analysis Consortium 2005). Surprisingly, more focused comparative genomic analyses have identified greater than 100 genes with lineage-specific coding sequence disruptions in the form of stop codon or splice site mutations, frameshift insertion/deletions, and gene deletions (Watanabe et al. 2004; Chimpanzee Sequencing and Analysis Consortium 2005; Newman et al. 2005; Varki and Altheide 2005; Hahn and Lee 2006; Wang et al. 2006). This observation is a radical change from previous beliefs of the types of genetic changes that predominantly accompanied the divergence of human and chimpanzee lineages and strongly implicates gene structure and reorganization as important means by which our 2 lineages have become genetically and phenotypically distinct.

Gene loss at any particular region of the genome can result in many unpredicted changes in phenotype; however, lineage-specific gene loss on the Y-chromosome is of particular interest because this chromosome is highly enriched for genes involved in spermatogenesis (Lahn and Page 1997; Skaletsky et al. 2003). Therefore, studies of Y-chromosome gene loss can potentially reveal the history of evolutionary change between human and chimpanzee mating and fertility systems. Furthermore, the Y-chromosome seems to be particularly prone to gene loss; most of the Y-chromosome does not undergo meiotic recombination (Tilford et al. 2001), meaning that positive or negative natural selection can have very important implications for “linked” variation compared with that of other chromosomes. As a result, there has been a general trend of Y-chromosome degradation and gene loss over evolutionary time (e.g., Muller's ratchet), which may sometimes involve the fixation of gene-disruptive mutations on the background of an otherwise adaptive haplotype (Muller 1918; Charlesworth B and Charlesworth D 2000).

The initial comparisons of human and chimpanzee (Pan troglodytes) Y-chromosome sequences revealed that although there are no lineage-specific gene-disruptive mutations in the X-degenerate portion of the Y-chromosome fixed within humans, surprisingly, 4 genes, CYorf15B, TBL1Y, TMSB4Y, and USP9Y, are disrupted by one or more splice site or premature stop codon mutations in chimpanzees (Hughes et al. 2005; Kuroki et al. 2006; Tyler-Smith et al. 2006). Given that levels of sperm competition are likely greater in chimpanzees than in humans (Harcourt et al. 1981; Dorus et al. 2004) and that the Y-chromosome is highly enriched for genes associated with spermatogenesis, the contrast between rates of human and chimpanzee Y-chromosome gene disruption was unanticipated. Although the specific functions of CYorf15b, TBL1Y, and TMSB4Y are not well understood (Skaletsky et al. 2003; Yan et al. 2005), USP9Y is critical for spermatogenesis in humans, with gene-disruptive mutations at this locus resulting in azoospermia or the absence of sperm in semen (Sun et al. 1999; Blagosklonova et al. 2000). Thus, the potential loss of this specific gene in the chimpanzee lineage is especially puzzling.

In order to better understand the evolutionary history and significance of chimpanzee Y-chromosome gene loss, we have characterized the nucleotide sequences involving the gene-disruptive mutations in each of the CYorf15B, TBL1Y, TMSB4Y, and USP9Y genes in the gorilla (Gorilla gorilla), the bonobo (Pan paniscus), and in wild-born individuals from several chimpanzee subspecies. This analysis enables us to make inferences about the origin and timing of these gene-disruptive mutations as well as evaluate the impact of these potentially important events that distinguish the evolutionary histories of human and chimpanzee Y-chromosomes.

Materials and Methods

Although comparison of the exhaustive pseudogene catalogs for humans versus chimpanzees may provide insight into our respective evolutionary and ecological histories in general, the current draft quality of the chimpanzee genome sequence (Build 1.1) precludes the reliable and comprehensive genome-wide identification of chimpanzee pseudogenes without further validations, as discussed by the Chimpanzee Sequencing and Analysis Consortium (2005). However, 2 independent and high-quality chimpanzee Y-chromosome sequences are publicly available (Hughes et al. 2005; Kuroki et al. 2006), offering excellent opportunities for focused analyses on this chromosome.

We aligned the Hughes et al. (2005) and Kuroki et al. (2006) chimpanzee Y-chromosome sequences and the human Y-chromosome reference sequence (Build 35) for the CYorf15B, TBL1Y, TMSB4Y, and USP9Y genes. This alignment confirmed that the splice site and stop codon mutations described by Hughes et al. (2005) are found in both chimpanzee Y-chromosome sequences (and not in the human sequence), making it highly unlikely that these reflect sequencing or assembly errors. We also identified a 4-bp frameshift deletion in USP9Y exon 36 in the chimpanzee lineage (as inferred by comparison to human and gorilla sequences), which was not originally identified in the study conducted by Hughes et al. (2005). In total, we characterized 7 Y-chromosome gene-disruptive mutations in each primate species (table 1).

Table 1

Summary of Y-Chromosome Gene-Disruptive Mutations

   Similarity to Chimpanzeea 
Gene Exon Type of Mutation Gorilla Bonobo 
CYorf15b Stop codon No Yes 
TBL1Y 12 Splice site mutationb No Yes 
TMSB4Y Splice site mutation No Noc 
USP9Y Splice site deletion (4 bp) No Yes 
 34 Splice site mutation No Yes 
 36 Frameshift deletion (4 bp) No Yes 
 39 Splice site mutation No Yes 
   Similarity to Chimpanzeea 
Gene Exon Type of Mutation Gorilla Bonobo 
CYorf15b Stop codon No Yes 
TBL1Y 12 Splice site mutationb No Yes 
TMSB4Y Splice site mutation No Noc 
USP9Y Splice site deletion (4 bp) No Yes 
 34 Splice site mutation No Yes 
 36 Frameshift deletion (4 bp) No Yes 
 39 Splice site mutation No Yes 
a

Yes: the individual was found to have the gene-disruptive mutation as found in the 2 publicly available chimpanzee Y-chromosome sequences. No: shares the human sequence.

b

Mutations at exon–intron boundaries involved in intron splice site recognition.

c

Because the bonobo did not have this mutation, this gene region was characterized in 1 wild-born individual from each of 4 chimpanzee subspecies, all found to have the identical gene-disruptive mutation as in the chimpanzee reference sequences (see Results and Discussion).

Whole blood samples from wild-born chimpanzees housed at research facilities or zoological institutions were collected during regularly scheduled veterinary examinations. DNA was isolated using a standard phenol/chloroform extraction method (Sambrook and Russell 2001). Chimpanzee subspecies were determined from comparisons of mitochondrial DNA and Y-chromosome sequences to those of wild-born individuals with known capture location, as described by Stone et al. (2002). Bonobo, gorilla, and other chimpanzee DNA samples (PR00107, PR00251, PR00496, and PR00573) were obtained from the Integrated Biomaterials and Information Resource (IPBIR; http://www.ipbir.org). DNA from Clint (S006006), the captive-born chimpanzee who was the donor for the chimpanzee genome sequence project, was obtained from the Coriell Institute for Medical Research (http://www.coriell.org).

To characterize the 7 gene-disruptive mutations (table 1), polymerase chain reaction (PCR) primers were designed based on the human Y-chromosome sequence using the Primer3 computer program (Rozen and Skaletsky 2000) to amplify gene regions encompassing the 7 mutations. Primer sequences were compared first with the chimpanzee Y-chromosome sequences to ensure similarity, and second, with the homologous region on the X-chromosome to ensure Y-chromosome specificity. Primer sequences (all 5′–3′) used were CYorf15B forward: 5′-ACAAGTGTCAGCTGGTTGAGAA-3′ and reverse: 5′-CAGGGGAAAATCTGAATAAAGC-3′ (fragment size 691 bp), TBL1Y forward: 5′-TTCAACAGTTTTCTGCACTTGG-3′ and reverse: 5′-CTCAAGATGGATCAGACATTCG-3′ (917 bp), TMSB4Y forward: 5′-ACAAACCTGGTATGGCTGAGAT-3′ and reverse: 5′-CCTAAACGTTCTGCAAGTGTACC-3′ (733 bp), USP9Y exon 8 forward: 5′-GTTGTGTCCCCATGAACTATGA-3′ and reverse: 5′-TAGCATTGTCCAAATGGTCTGA-3′ (664 bp), USP9Y exon 34 forward: 5′-AAACAATGTGCGTTTCTCCTTT-3′ and reverse: 5′-GGTGGAAACTGAAACCATGAAT-3′ (696 bp), USP9Y exon 36 forward: 5′-CATGAAATTGTTTTAGTTTCTGTTCT-3′ and reverse: 5′-CTGATGGGGTCTTGCAATAGTT-3′ (674 bp), and USP9Y exon 39 forward: 5′-GCAAATAAAAGCTGTTTCTGCAT-3′ and reverse: 5′-GCATTCTAGAGGCACTCAAAAGA-3′ (796 bp). PCR reactions were performed in 25 μL reactions using Platinum Taq (Invitrogen, Carlsbad, California), with the following conditions: 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 s, 59 °C for 30 s, and 70 °C for 30 s.

For each PCR fragment amplification, we included DNA from both male and female human, chimpanzee, and bonobo individuals, in order to verify that homologous fragments from the X-chromosome were not amplified. Following amplification, PCR products were purified with Shrimp Alkaline Phosphatase and Exonuclease I (USB Corporation, Cleveland, Ohio), cycle sequenced with BigDye Terminator Cycle Sequencing Kit version 3.1 (Applied Biosystems, Foster City, California), cleaned with isopropanol, and analyzed by electrophoresis on an Applied Biosystems 3730 capillary sequencer. Sequence data were manually aligned and analyzed using the Sequencher version 4.6 computer program (Gene Codes Corporation, Ann Arbor, Michigan). The Y-chromosome sequences generated for this study have been deposited in GenBank with accession numbers EF197918EF197935.

Results and Discussion

Origins of Chimpanzee Y-Chromosome Gene-Disruptive Mutations

Table 1 displays the nucleotide sequence analysis of each of the 7 characterized gene regions. Because the ancestral lineages separating gorillas and the common ancestor of humans and chimpanzees likely diverged over a relatively short period of time, ∼1 Myr or less (see fig. 1), gene genealogies from the nuclear genome do not consistently support the more recent common ancestor for chimpanzees and humans (Chen and Li 2001). This has consequences for determining whether fixation events occurred on the human or chimpanzee lineages using the gorilla as an outgroup sequence. However, given the smaller effective population size and more recent coalescence time of the Y-chromosome (e.g., Stone et al. 2002), the gorilla nucleotide sequence can be appropriately used to polarize human and chimpanzee lineage-specific fixation events on this chromosome. Our analysis of the male gorilla finds that, for each of the 7 gene regions, the nucleotide sequence is identical to the human sequence at the site of each of the 7 gene-disruptive mutations (table 1). From this, we infer that each mutation occurred on the chimpanzee lineage following divergence from the human–chimpanzee common ancestor. This supported the previous conclusions of Hughes et al. (2005) that were based on comparisons of only the human and chimpanzee Y-chromosome sequences to the human X-chromosome sequence.

FIG. 1.—

Timing of Y-chromosome gene losses during chimpanzee evolution. Disruptions to the coding sequences of 3 Y-chromosome genes (CYorf15b, TBL1Y, and USP9Y) are estimated to have an origin in the ancestral chimpanzee–bonobo lineage following divergence from the human lineage, whereas the TMSB4Y coding sequence was disrupted in the chimpanzee lineage following chimpanzee–bonobo Y-chromosome divergence, but prior to the separation of chimpanzee subspecies.

FIG. 1.—

Timing of Y-chromosome gene losses during chimpanzee evolution. Disruptions to the coding sequences of 3 Y-chromosome genes (CYorf15b, TBL1Y, and USP9Y) are estimated to have an origin in the ancestral chimpanzee–bonobo lineage following divergence from the human lineage, whereas the TMSB4Y coding sequence was disrupted in the chimpanzee lineage following chimpanzee–bonobo Y-chromosome divergence, but prior to the separation of chimpanzee subspecies.

In polarizing all 7 mutations to the chimpanzee lineage, this simply purports that they have occurred sometime over the last ∼6 Myr, or since the estimated divergence of Pan and Homo lineages (Kumar et al. 2005). Therefore, in characterizing the male bonobo nucleotide sequence for these 7 gene regions, we can estimate the origin of the gene-disruptive mutations along the chimpanzee lineage. It should be noted here that with our alignment of chimpanzee and human Y-chromosome sequences, we are only identifying mutations that have occurred along these 2 lineages and not necessarily those mutations that may have occurred along the gorilla lineage or the bonobo lineage, following divergence from chimpanzees. Therefore, there may be other gene-disruptive mutations along the Y-chromosome that are fixed between these species, in which case any estimates of gene loss from our analysis would be inaccurate given the ascertainment bias inherent within our sampling design (i.e., gene regions nonrandomly chosen based on the presence of known gene-disruptive mutations). Further nucleotide sequence analysis of the entire gorilla and bonobo Y-chromosome could certainly address this issue at a later date. However, because of the difficulty in large-scale amplification and sequencing of these Y-chromosome regions from genomic DNA (e.g., high X-chromosome homology), this would likely best be accomplished with a bacterial artificial chromosome–based sequencing strategy, similar to those used by Hughes et al. (2005) and Kuroki et al. (2006) to produce their chimpanzee Y-chromosome sequences.

For CYorf15B, TBL1Y, and USP9Y, the same gene-disruptive mutations present in the chimpanzee Y-chromosome sequences were also present in the bonobo sequence (table 1), indicating that these mutations occurred and were fixed in the common ancestor of chimpanzees and bonobos (fig. 1). There are 4 mutations that disrupt the USP9Y coding region in chimpanzees, all of which were observed in the bonobo. Chimpanzee–bonobo Y-chromosome divergence has been estimated to ∼1.8 MYA (Stone et al. 2002). Therefore, the disruptive mutations at these 3 genes likely occurred between ∼6 and ∼1.8 MYA (fig. 1).

In contrast to the other 3 genes, the exon–intron splice site mutation in exon 1 of the chimpanzee TMSB4Y gene is not present in our bonobo sequence. Instead, the bonobo splice site sequence is identical to the human sequence (table 1), suggesting that the disruptive mutation at this gene occurred in the chimpanzee lineage following chimpanzee–bonobo divergence. Both of the publicly available chimpanzee Y-chromosome sequences (Hughes et al. 2005; Kuroki et al. 2006) are from the western chimpanzee subspecies (Pan troglodytes verus), and thus, the presence of the mutations in both sequences does not necessarily imply that they are fixed among chimpanzee subspecies. To address this issue, we additionally obtained nucleotide sequence for this TMSB4Y gene region in 4 wild-born male chimpanzees representing each subspecies: 1 western chimpanzee (Pan troglodytes verus), 1 central chimpanzee (Pan troglodytes troglodytes), 1 eastern chimpanzee (Pan troglodytes schweinfurthii), and 1 Nigerian chimpanzee (Pan troglodytes vellerosus). We found that the TMSB4Y gene-disruptive mutation was present in all 4 subspecies (table 1). Although autosome and X-chromosome gene variation is often shared across chimpanzee subspecies because of relatively large effective population sizes for these loci in chimpanzees (e.g., Kaessmann et al. 1999; Fischer et al. 2004; Verrelli et al. 2006), this is not the case for the Y-chromosome (Stone et al. 2002). Therefore, our data suggest that the TMSB4Y mutation is fixed among chimpanzee subspecies and occurred after bonobo–chimpanzee divergence (∼1.8 MYA) but prior to the divergence of chimpanzee Y-chromosome haplogroups (∼0.7 MYA; fig. 1).

Molecular Evidence for Relaxed Functional Constraint

Just as the absence of any of these Y-chromosome gene-disruptive mutations within any lineage does not by itself imply a functional protein, the presence of a disruptive mutation within a coding region does not alone imply pseudogenization. If the gene is still transcribed, the messenger RNA (mRNA) could have regulatory roles and/or may still be translated into a shorter protein that remains functional. Hughes et al. (2005) performed reverse transcriptase–PCR (RT–PCR) experiments for detection of mRNA in various chimpanzee tissues. Although TMSB4Y mRNA was not found, detectable quantities of CYorf15b, TBL1Y, and USP9Y mRNA were present in multiple tissues. The finding of mRNA for these latter 3 genes leaves open the possibility that they are still translated into functional proteins. If this is the case, we may have certain expectations about the molecular evolutionary signature associated with functional constraint acting on protein-coding sequences.

Compared with functional genes that may be subject to purifying or positive selection, pseudogenes are expected to follow a neutral pattern of nucleotide substitution. If a gene region is subject to purifying selection, the ratio of nonsynonymous (amino acid changing) substitutions per nonsynonymous site to synonymous substitutions per synonymous site (dN/dS) is expected to be less than 1, whereas under neutrality dN/dS will approximate 1. To evaluate whether functional constraint may be acting on specific regions of these genes, we aligned the entire human and chimpanzee coding sequences for each of the 4 genes. We computed and compared dN/dS for each gene for the region upstream from the disruptive mutation with the region downstream from the mutation (table 2). If, despite a disruptive mutation, the upstream region of a gene maintains function in chimpanzees, then upstream dN/dS may be different than that of the downstream region, which follows the disruptive mutation and is expected to be free of functional constraint.

Table 2

Human–Chimpanzee dN/dS Comparisons for Disrupted Y-Chromosome Gene Regions

 Upstream of Disruptive Mutation Downstream of Disruptive Mutation 
   
Gene Sites Diffs Sites Diffs dN/dS Sites Diffs Sites Diffs dN/dS 
CYorf15b 112 26 0.0 307 92 0.300 
TBL1Y 680 205 0.301 526 143 0.217 
TMSB4Y 77 19 NA 24 NA 
USP9Y4032 45 1158 14 0.923 1922 14 541 0.985 
 Upstream of Disruptive Mutation Downstream of Disruptive Mutation 
   
Gene Sites Diffs Sites Diffs dN/dS Sites Diffs Sites Diffs dN/dS 
CYorf15b 112 26 0.0 307 92 0.300 
TBL1Y 680 205 0.301 526 143 0.217 
TMSB4Y 77 19 NA 24 NA 
USP9Y4032 45 1158 14 0.923 1922 14 541 0.985 

NOTE.—N, nonsynonymous; S, synonymous. Diffs, Human–chimpanzee differences. NA (not available), could not be calculated due to a zero number of synonymous substitutions in the denominator.

a

The upstream and downstream regions for USP9Y were divided at the point of the exon 34 disruptive mutation (see Results and Discussion).

Using Fisher's exact tests, we find no significant difference between upstream and downstream region dN/dS, for any of the 4 genes with chimpanzee lineage gene-disruptive mutations. For USP9Y, because we are not able to determine which of the 4 disruptive mutations occurred first (i.e., we can infer only that they all occurred prior to chimpanzee–bonobo divergence), we compared upstream with downstream dN/dS in this fashion for each of them, and found no significant difference in any comparison. The results for the USP9Y exon 34 gene-disruptive mutations are shown in table 2 because this provides the most even division (in terms of bp) between upstream and downstream regions. However, it is important to note that the small size of some of these genes (e.g., TMSB4Y; table 2) may prohibit us from detecting differences in functional constraint between upstream and downstream regions.

Given that we find no clear pattern of differential functional constraint between upstream and downstream regions, we cannot reject the hypothesis that these 4 genes have become pseudogenes in the chimpanzee lineage. However, it is interesting to note that, in general, the whole gene (combined upstream and downstream regions) dN/dS values and levels of nucleotide divergence for these genes are not noticeably different from those observed for other, intact genes in the X-degenerate portion of the Y-chromosome (Hughes et al. 2005; Kuroki et al. 2006). Therefore, despite the relatively ancient age for many of the gene-disrupting mutations, the typical dN/dS values for these genes are inconsistent with expectations of neutrality in the chimpanzee lineage. These seemingly incongruent results could be explained by the tendency for Y-chromosome genes to evolve rapidly in general (Hughes et al. 2005), which may make it difficult to infer subtle differences in functional constraint using comparisons among subsets of these genes. This raises questions about our general expectations of the molecular signature associated with potentially gene-disrupting mutations on the Y-chromosome and whether nonneutral scenarios may explain these patterns.

Once a mutation disrupts a coding region, the gene may continue to degrade through the neutral fixation of additional gene-disruptive mutations, now that functional constraint on the protein-coding sequence has become relaxed. Therefore, we may expect that the age of the original gene-disruptive mutation is correlated with the total number of disruptive mutations for each gene. Among the 4 chimpanzee Y-chromosome genes with disruptive mutations, only USP9Y contains multiple mutations that would disrupt the coding sequence (table 1). In this respect, it is interesting to note that 4 gene-disruptive mutations occurred prior to chimpanzee–bonobo divergence, but that none have become fixed in ∼1.8 Myr. The coding region of USP9Y is considerably larger than the 2 other genes that were disrupted in the chimpanzee–bonobo common ancestor (CYorf15b and TBL1Y; table 2), and the absence of multiple disruptive mutations in these genes may simply reflect gene size rather than functional constraint. Complete coding region sequences for these genes in the bonobo would help to address this issue. For example, 21 bp downstream from the USP9Y exon 36 frameshift deletion that occurred in the chimpanzee–bonobo common ancestor, we coincidentally found a 16-bp frameshift deletion in the bonobo that is not present in any other primate lineage examined here (fig. 2). Therefore, although no gene-disruptive mutations have apparently become fixed in the chimpanzee lineage in the last ∼1.8 Myr, this gene continues to degrade in the bonobo lineage. With additional sampling of the bonobo Y-chromosome sequence, we can better estimate the magnitude of gene-disruptive mutations that may have recently occurred in our close primate relatives.

FIG. 2.—

Continued degradation of the USP9Y coding region in bonobos. The USP9Y exon 36 gene region shown for several primate species with the 4-bp frameshift deletion (A) that occurred in the chimpanzee–bonobo ancestral lineage, by comparison with the gorilla and human sequences. A second example of more recent gene degradation is shown by a 16-bp frameshift deletion (B) that is unique to the bonobo lineage.

FIG. 2.—

Continued degradation of the USP9Y coding region in bonobos. The USP9Y exon 36 gene region shown for several primate species with the 4-bp frameshift deletion (A) that occurred in the chimpanzee–bonobo ancestral lineage, by comparison with the gorilla and human sequences. A second example of more recent gene degradation is shown by a 16-bp frameshift deletion (B) that is unique to the bonobo lineage.

Compared with CYorf15b, TBL1Y, and USP9Y, the coding sequence of TMSB4Y was disrupted more recently (i.e., following chimpanzee–bonobo divergence). However, the absence of TMSB4Y mRNA from any chimpanzee tissue (Hughes et al. 2005) strongly suggests that this gene has become nonfunctional in the chimpanzee lineage. This is an interesting observation; that is, of the 4 genes that have coding sequence–disruptive mutations, the one that produces no detectable mRNA is the one that has occurred much more recently. Similar RT–PCR experiments in bonobo tissue may help to determine whether TMSB4Y transcription was either abolished prior to chimpanzee–bonobo divergence (i.e., following a regulatory region mutation) leading to relaxed functional constraint on the amino acid sequence or, alternatively, if this gene remains functional provided we find no other disruptive mutations in this gene in bonobos.

Mating Systems and Y-Chromosome Evolution

Hughes et al. (2005) originally proposed that CYorf15b, TBL1Y, TMSB4Y, and USP9Y may have been evolutionary “casualties” of strong positive selection elsewhere on the Y-chromosome. By this action, low-frequency disruptive mutations may become fixed because a linked advantageous mutation elsewhere on the Y-chromosome is highly advantageous. Although this is less likely to happen on X-chromosomal and autosomal haplotypes due to recombination, the completely linked nonrecombining nature of the Y-chromosome provides for such a scenario (Rice 1987; Bachtrog 2004). One possible adaptive scenario that could cause differential rates of gene loss on the Y-chromosome may involve the difference in mating systems between humans and chimpanzees.

Chimpanzees and bonobos both have multi-male/multi-female mating systems (Nishida 1968; Kano 1982; Goodall 1986) with presumably high levels of sperm competition relative to humans. Evolutionary consequences of this difference may include a relatively greater testis to body size ratio in chimpanzees than humans (Harcourt et al. 1981) and significantly rapid evolution for genes involved in sperm development and function in the chimpanzee and bonobo lineages (e.g., Dorus et al. 2004). Therefore, given the enrichment for genes involved in spermatogenesis on the Y-chromosome, it seems reasonable that there may have been many selective sweeps during the evolution of the chimpanzee Y-chromosome as a result of strong sexual selection. It is also possible that some of these selective episodes led to the fixation of gene-disruptive mutations elsewhere on the Y-chromosome. In this study, we have shown that 3 of the 4 analyzed Y-chromosome genes were disrupted prior to the divergence of chimpanzees and bonobos, implying that many sex-specific (i.e., Y-chromosomal) fixation events in the chimpanzee lineage were relatively ancient. These initial interspecific analyses shed light on the historical impact that gene-disruptive mutations may have on fixation rates (i.e., dN/dS analyses), whereas population genetic analyses can eventually be used to test hypotheses about more recent events including selective sweeps on the Y chromosome (e.g., Filatov et al. 2000; Bachtrog 2004).

It is also possible that one or more of the gene-disruptive mutations themselves may have been adaptive. Olson (1999) has proposed the “less-is-more” hypothesis, which states that losses of gene function during hominin evolution may in some cases have conferred a fitness benefit. However, as of yet, few examples conclusively support this hypothesis. For example, although Stedman et al. (2004) proposed that a frameshift deletion in the myosin gene MYH16 led to masticatory gracilization and brain-size expansion in the genus Homo, this interpretation has subsequently been called into question (Perry et al. 2005; McCollum et al. 2006). More recently, 2 studies have shown that a premature stop codon mutation in the human CASPASE12 gene was likely swept toward fixation by positive selection (Wang et al. 2006; Xue et al. 2006), possibly because loss of CASPASE12 gene function increases resistance to the system-wide response to infection, or sepsis (Saleh et al. 2004, 2006). Therefore, it will be interesting to determine how many examples of gene loss in fact fit a picture of adaptive evolution when examining differences between humans and other primates.

For example, given the high levels of sperm competition in chimpanzees, it is difficult to reconcile how chimpanzee USP9Y-disruptive mutations could have been neutral because loss of function of this gene in humans leads to the absence of sperm in semen (Sun et al. 1999; Blagosklonova et al. 2000). Interestingly, Gerrard and Filatov (2005) identified 2 different disruptive mutations in a small segment of the USP9Y coding region from the black spider monkey (Ateles geoffroyi). Spider monkeys, like chimpanzees and bonobos, have a multi-male/multi-female mating system (Eisenberger 1973; Cant 1978; Chapman et al. 1993, 1995), raising the possibility that knocking out USP9Y gene function may have been advantageous for primates with high levels of sperm competition. However, we cannot exclude the possibility that other genes compensate for the loss of USP9Y function in nonhuman primates but not in humans, or that USP9Y gained new function in the human lineage, such that its disruption is less deleterious in nonhuman primates. To more fully test these hypotheses will require Y- chromosome sequences from additional primate species, including multiple examples of each mating system.

Conclusion

The discussion of gene gain and loss has been of great interest and debate in understanding how humans and our primate relatives diverged (e.g., Olson 1999; Gilad et al. 2003; Fortna et al. 2004; Hurles 2004; Wang et al. 2006). Although we find that many of the Y-chromosome gene disruptions in the chimpanzee lineage are relatively ancient in origin, others have found that 3 of these genes are still transcribed in chimpanzees, and in general we find little evidence for relaxed functional constraint relative to other Y-chromosome genes. Therefore, the full pseudogene status of these genes warrants additional scrutiny. With a greater sampling of gene disruption events throughout the human and chimpanzee genomes, it will be possible to determine whether the higher rate of gene disruption in chimpanzees is unique in comparison to other chromosomes. In light of the results found here, it will also be of interest to determine whether genes on other chromosomes that play a role in spermatogenesis or fertility also show different patterns of gene disruption between the human and chimpanzee genomes.

We thank Phil Hedrick for encouraging conversation and Anne Stone, the New Iberia Research Center, Primate Foundation of Arizona, IPBIR, and the Coriell Institute for Medical Research for the primate samples used in this study. This work was supported by funding to B.C.V. from the Center for Evolutionary Functional Genomics in The Biodesign Institute at the Arizona State University.

References

Bachtrog
D
Evidence that positive selection drives Y-chromosome degeneration in Drosophila miranda
Nat Genet
 , 
2004
, vol. 
36
 (pg. 
518
-
522
)
Blagosklonova
O
Fellmann
F
Clavequin
MC
Roux
C
Bresson
JL
AZFa deletions in Sertoli cell-only syndrome: a retrospective study
Mol Hum Reprod
 , 
2000
, vol. 
6
 (pg. 
795
-
799
)
Cant
JGH
Population survey of the spider monkey, Ateles geoffroyi, at Tikal, Guatemala
Primates
 , 
1978
, vol. 
19
 (pg. 
525
-
535
)
Chapman
CA
White
FJ
Wrangham
RW
Defining subgroup size in fission-fusion societies
Folia Primatol (Basel)
 , 
1993
, vol. 
61
 (pg. 
31
-
34
)
Chapman
CA
Wrangham
RW
Chapman
LJ
Ecological constraints on group size—an analysis of spider monkey and chimpanzee subgroups
Behav Ecol Sociobiol
 , 
1995
, vol. 
36
 (pg. 
59
-
70
)
Charlesworth
B
Charlesworth
D
The degeneration of Y chromosomes
Philos Trans R Soc Lond B Biol Sci
 , 
2000
, vol. 
355
 (pg. 
1563
-
1572
)
Chen
FC
Li
WH
Genomic divergences between humans and other hominoids and the effective population size of the common ancestor of humans and chimpanzees
Am J Hum Genet
 , 
2001
, vol. 
68
 (pg. 
444
-
456
)
Chimpanzee Sequencing and Analysis Consortium
Initial sequence of the chimpanzee genome and comparison with the human genome
Nature
 , 
2005
, vol. 
437
 (pg. 
69
-
87
)
Dorus
S
Evans
PD
Wyckoff
GJ
Choi
SS
Lahn
BT
Rate of molecular evolution of the seminal protein gene SEMG2 correlates with levels of female promiscuity
Nat Genet
 , 
2004
, vol. 
36
 (pg. 
1326
-
1329
)
Eisenberger
JF
Reproduction in two species of spider monkeys, Ateles fusciceps and Ateles geoffroyi
J Mammal
 , 
1973
, vol. 
54
 (pg. 
955
-
957
)
Filatov
DA
Moneger
F
Negrutiu
I
Charlesworth
D
Low variability in a Y-linked plant gene and its implications for Y-chromosome evolution
Nature
 , 
2000
, vol. 
404
 (pg. 
388
-
390
)
Fischer
A
Wiebe
V
Paabo
S
Przeworski
M
Evidence for a complex demographic history of chimpanzees
Mol Biol Evol
 , 
2004
, vol. 
21
 (pg. 
799
-
808
)
Fortna
A
Kim
Y
MacLaren
E
, et al.  . 
Lineage-specific gene duplication and loss in human and great ape evolution
PLoS Biol
 , 
2004
, vol. 
2
 pg. 
E207
  
(16 co-authors)
Gerrard
DT
Filatov
DA
Positive and negative selection on mammalian Y chromosomes
Mol Biol Evol
 , 
2005
, vol. 
22
 (pg. 
1423
-
1432
)
Gilad
Y
Man
O
Paabo
S
Lancet
D
Human specific loss of olfactory receptor genes
Proc Natl Acad Sci USA
 , 
2003
, vol. 
100
 (pg. 
3324
-
3327
)
Goodall
J
The chimpanzees of Gombe: patterns of behavior
1986
Cambridge
Belknap Press of Harvard University Press
Hahn
Y
Lee
B
Human-specific nonsense mutations identified by genome-sequence comparisons
Hum Genet
 , 
2006
, vol. 
119
 (pg. 
169
-
178
)
Harcourt
AH
Harvey
PH
Larson
SG
Short
RV
Testis weight, body weight and breeding system in primates
Nature
 , 
1981
, vol. 
293
 (pg. 
55
-
57
)
Hughes
JF
Skaletsky
H
Pyntikova
T
Minx
PJ
Graves
T
Rozen
S
Wilson
RK
Page
DC
Conservation of Y-linked genes during human evolution revealed by comparative sequencing in chimpanzee
Nature
 , 
2005
, vol. 
437
 (pg. 
100
-
103
)
Hurles
M
Gene duplication: the genomic trade in spare parts
PLoS Biol
 , 
2004
, vol. 
2
 pg. 
E206
 
Kaessmann
H
Wiebe
V
Paabo
S
Extensive nuclear DNA sequence diversity among chimpanzees
Science
 , 
1999
, vol. 
286
 (pg. 
1159
-
1162
)
Kano
T
The social group of pygmy chimpanzees (Pan paniscus) of Wamba
Primates
 , 
1982
, vol. 
23
 (pg. 
171
-
188
)
Kumar
S
Filipski
A
Swarna
V
Walker
A
Hedges
SB
Placing confidence limits on the molecular age of the human-chimpanzee divergence
Proc Natl Acad Sci USA
 , 
2005
, vol. 
102
 (pg. 
18842
-
18847
)
Kuroki
Y
Toyoda
A
Noguchi
H
, et al.  . 
Comparative analysis of chimpanzee and human Y chromosomes unveils complex evolutionary pathway
Nat Genet
 , 
2006
, vol. 
38
 (pg. 
158
-
167
(19 co-authors)
Lahn
BT
Page
DC
Functional coherence of the human Y chromosome
Science
 , 
1997
, vol. 
278
 (pg. 
675
-
680
)
McCollum
MA
Sherwood
CC
Vinyard
CJ
Lovejoy
CO
Schachat
F
Of muscle-bound crania and human brain evolution: the story behind the MYH16 headlines
J Hum Evol
 , 
2006
, vol. 
50
 (pg. 
232
-
236
)
Muller
HJ
Genetic variability, twin hybrids and constant hybrids, in a case of balanced lethal factors
Genetics
 , 
1918
, vol. 
3
 (pg. 
422
-
499
)
Newman
TL
Tuzun
E
Morrison
VA
Hayden
KE
Ventura
M
McGrath
SD
Rocchi
M
Eichler
EE
A genome-wide survey of structural variation between human and chimpanzee
Genome Res
 , 
2005
, vol. 
15
 (pg. 
1344
-
1356
)
Nishida
T
The social group of wild chimpanzees in the Mahale Mountains
Primates
 , 
1968
, vol. 
9
 (pg. 
167
-
224
)
Olson
MV
When less is more: gene loss as an engine of evolutionary change
Am J Hum Genet
 , 
1999
, vol. 
64
 (pg. 
18
-
23
)
Perry
GH
Verrelli
BC
Stone
AC
Comparative analyses reveal a complex history of molecular evolution for human MYH16
Mol Biol Evol
 , 
2005
, vol. 
22
 (pg. 
379
-
382
)
Rice
WR
Genetic hitchhiking and the evolution of reduced genetic activity of the Y sex chromosome
Genetics
 , 
1987
, vol. 
116
 (pg. 
161
-
167
)
Rozen
S
Skaletsky
HJ
Krawetz
S
Misener
S
Primer3 on the WWW for general users and for biologist programmers
Bioinformatics methods and protocols: methods in molecular biology
2000
Totowa (NJ)
Humana Press
(pg. 
365
-
386
)
Saleh
M
Mathison
JC
Wolinski
MK
Bensinger
SJ
Fitzgerald
P
Droin
N
Ulevitch
RJ
Green
DR
Nicholson
DW
Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice
Nature
 , 
2006
, vol. 
440
 (pg. 
1064
-
1068
)
Saleh
M
Vaillancourt
JP
Graham
RK
, et al.  . 
Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms
Nature
 , 
2004
, vol. 
429
 (pg. 
75
-
79
(16 co-authors)
Sambrook
J
Russell
DW
Molecular cloning: a laboratory manual
2001
Cold Spring Harbor (NY)
Cold Spring Harbor Laboratory Press
Skaletsky
H
Kuroda-Kawaguchi
T
Minx
PJ
, et al.  . 
The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes
Nature
 , 
2003
, vol. 
423
 (pg. 
825
-
837
(40 co-authors)
Stedman
HH
Kozyak
BW
Nelson
A
Thesier
DM
Su
LT
Low
DW
Bridges
CR
Shrager
JB
Minugh-Purvis
N
Mitchell
MA
Myosin gene mutation correlates with anatomical changes in the human lineage
Nature
 , 
2004
, vol. 
428
 (pg. 
415
-
418
)
Stone
AC
Griffiths
RC
Zegura
SL
Hammer
MF
High levels of Y-chromosome nucleotide diversity in the genus Pan
Proc Natl Acad Sci USA
 , 
2002
, vol. 
99
 (pg. 
43
-
48
)
Sun
C
Skaletsky
H
Birren
B
Devon
K
Tang
Z
Silber
S
Oates
R
Page
DC
An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y
Nat Genet
 , 
1999
, vol. 
23
 (pg. 
429
-
432
)
Tilford
CA
Kuroda-Kawaguchi
T
Skaletsky
H
, et al.  . 
A physical map of the human Y chromosome
Nature
 , 
2001
, vol. 
409
 (pg. 
943
-
945
(12 co-authors)
Tyler-Smith
C
Howe
K
Santos
FR
The rise and fall of the ape Y chromosome?
Nat Genet
 , 
2006
, vol. 
38
 (pg. 
141
-
143
)
Varki
A
Altheide
TK
Comparing the human and chimpanzee genomes: searching for needles in a haystack
Genome Res
 , 
2005
, vol. 
15
 (pg. 
1746
-
1758
)
Verrelli
BC
Tishkoff
SA
Stone
AC
Touchman
JW
Contrasting histories of G6PD molecular evolution and malarial resistance in humans and chimpanzees
Mol Biol Evol
 , 
2006
, vol. 
23
 (pg. 
1592
-
1601
)
Wang
X
Grus
WE
Zhang
J
Gene losses during human origins
PLoS Biol
 , 
2006
, vol. 
4
 pg. 
e52
 
Watanabe
H
Fujiyama
A
Hattori
M
, et al.  . 
DNA sequence and comparative analysis of chimpanzee chromosome 22
Nature
 , 
2004
, vol. 
429
 (pg. 
382
-
388
(45 co-authors)
Xue
Y
Daly
A
Yngvadottir
B
, et al.  . 
Spread of an inactive form of caspase-12 in humans is due to recent positive selection
Am J Hum Genet
 , 
2006
, vol. 
78
 (pg. 
659
-
670
(14 co-authors)
Yan
HT
Shinka
T
Kinoshita
K
, et al.  . 
Molecular analysis of TBL1Y, a Y-linked homologue of TBL1X related with X-linked late-onset sensorineural deafness
J Hum Genet
 , 
2005
, vol. 
50
 (pg. 
175
-
181
(11 co-authors)

Author notes

Yoko Satta, Associate Editor