The Evolutionary History of Human and Chimpanzee Y-Chromosome Gene Loss George H. Perry,* à Raul Y. Tito,* and Brian C. Verrelli* *Center for Evolutionary Functional Genomics, The Biodesign Institute, Arizona State University, Tempe; School of Life Sciences, Arizona State University, Tempe; and àSchool of Human Evolution and Social Change, Arizona State University, Tempe 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 dis- proportionate 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 chimpan- zee–bonobo divergence at ;1.8 MYA, which indicates significant Y-chromosome change in the chimpanzee lineage Downloaded from https://academic.oup.com/mbe/article/24/3/853/1246230 by guest on 23 September 2021 relatively early in the evolutionary divergence of humans and chimpanzees. Introduction The initial comparisons of human and chimpanzee Comparative analyses of single-nucleotide differences (Pan troglodytes) Y-chromosome sequences revealed that between human and chimpanzee genomes typically show although there are no lineage-specific gene-disruptive mu- estimates of approximately 1–2% divergence (Watanabe tations in the X-degenerate portion of the Y-chromosome et al. 2004; Chimpanzee Sequencing and Analysis Consor- fixed within humans, surprisingly, 4 genes, CYorf15B, tium 2005). Surprisingly, more focused comparative geno- TBL1Y, TMSB4Y, and USP9Y, are disrupted by one or more mic analyses have identified greater than 100 genes with splice site or premature stop codon mutations in chimpan- lineage-specific coding sequence disruptions in the form zees (Hughes et al. 2005; Kuroki et al. 2006; Tyler-Smith of stop codon or splice site mutations, frameshift inser- et al. 2006). Given that levels of sperm competition are tion/deletions, and gene deletions (Watanabe et al. 2004; likely greater in chimpanzees than in humans (Harcourt Chimpanzee Sequencing and Analysis Consortium 2005; et al. 1981; Dorus et al. 2004) and that the Y-chromosome Newman et al. 2005; Varki and Altheide 2005; Hahn is highly enriched for genes associated with spermatogen- and Lee 2006; Wang et al. 2006). This observation is a rad- esis, the contrast between rates of human and chimpanzee ical change from previous beliefs of the types of genetic Y-chromosome gene disruption was unanticipated. Al- changes that predominantly accompanied the divergence though the specific functions of CYorf15b, TBL1Y, and of human and chimpanzee lineages and strongly implicates TMSB4Y are not well understood (Skaletsky et al. 2003; gene structure and reorganization as important means by Yan et al. 2005), USP9Y is critical for spermatogenesis which our 2 lineages have become genetically and pheno- in humans, with gene-disruptive mutations at this locus re- typically distinct. sulting in azoospermia or the absence of sperm in semen Gene loss at any particular region of the genome can (Sun et al. 1999; Blagosklonova et al. 2000). Thus, the result in many unpredicted changes in phenotype; however, potential loss of this specific gene in the chimpanzee line- lineage-specific gene loss on the Y-chromosome is of par- age is especially puzzling. ticular interest because this chromosome is highly enriched In order to better understand the evolutionary history for genes involved in spermatogenesis (Lahn and Page 1997; and significance of chimpanzee Y-chromosome gene loss, Skaletsky et al. 2003). Therefore, studies of Y-chromosome we have characterized the nucleotide sequences involving gene loss can potentially reveal the history of evolutionary the gene-disruptive mutations in each of the CYorf15B, change between human and chimpanzee mating and fertility TBL1Y, TMSB4Y, and USP9Y genes in the gorilla (Gorilla systems. Furthermore, the Y-chromosome seems to be par- gorilla), the bonobo (Pan paniscus), and in wild-born in- ticularly prone to gene loss; most of the Y-chromosome does dividuals from several chimpanzee subspecies. This analy- not undergo meiotic recombination (Tilford et al. 2001), sis enables us to make inferences about the origin and meaning that positive or negative natural selection can have timing of these gene-disruptive mutations as well as eval- very important implications for ‘‘linked’’ variation com- uate the impact of these potentially important events that pared with that of other chromosomes. As a result, there distinguish the evolutionary histories of human and chim- has been a general trend of Y-chromosome degradation panzee Y-chromosomes. and gene loss over evolutionary time (e.g., Muller’s ratchet), which may sometimes involve the fixation of gene- Materials and Methods disruptive mutations on the background of an otherwise Although comparison of the exhaustive pseudogene adaptive haplotype (Muller 1918; Charlesworth B and catalogs for humans versus chimpanzees may provide in- Charlesworth D 2000). sight into our respective evolutionary and ecological histo- ries in general, the current draft quality of the chimpanzee Key words: pseudogene, Muller’s ratchet, sperm competition, Pan troglodytes, Pan paniscus. genome sequence (Build 1.1) precludes the reliable and comprehensive genome-wide identification of chimpanzee E-mail: [email protected]. pseudogenes without further validations, as discussed by Mol. Biol. Evol. 24(3):853–859. 2007 doi:10.1093/molbev/msm002 the Chimpanzee Sequencing and Analysis Consortium Advance Access publication January 11, 2007 (2005). However, 2 independent and high-quality Ó The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected] 854 Perry et al. Table 1 Y-chromosome sequences to ensure similarity, and second, Summary of Y-Chromosome Gene-Disruptive Mutations with the homologous region on the X-chromosome to en- Similarity to sure Y-chromosome specificity. Primer sequences (all 5#–3#) Chimpanzeea used were CYorf15B forward: 5#-ACAAGTGT- # # Gene Exon Type of Mutation Gorilla Bonobo CAGCTGGTTGAGAA-3 and reverse: 5 -CAGGG- GAAAATCTGAATAAAGC-3# (fragment size 691 bp), CYorf15b 4 Stop codon No Yes # # TBL1Y 12 Splice site mutationb No Yes TBL1Y forward: 5 -TTCAACAGTTTTCTGCACTTGG-3 TMSB4Y 1 Splice site mutation No Noc and reverse: 5#-CTCAAGATGGATCAGACATTCG-3# USP9Y 8 Splice site deletion (4 bp) No Yes (917 bp), TMSB4Y forward: 5#-ACAAACCTGGTAT- 34 Splice site mutation No Yes GGCTGAGAT-3# and reverse: 5#-CCTAAACGTTCTG- 36 Frameshift deletion (4 bp) No Yes CAAGTGTACC-3# (733 bp), USP9Y exon 8 forward: 39 Splice site mutation No Yes 5#-GTTGTGTCCCCATGAACTATGA-3# and reverse: 5#- a Yes: the individual was found to have the gene-disruptive mutation as found in # TAGCATTGTCCAAATGGTCTGA-3 (664 bp), USP9Y Downloaded from https://academic.oup.com/mbe/article/24/3/853/1246230 by guest on 23 September 2021 the 2 publicly available chimpanzee Y-chromosome sequences. No: shares the hu- exon 34 forward: 5#-AAACAATGTGCGTTTCTCCTTT-3# man sequence. # # b Mutations at exon–intron boundaries involved in intron splice site recognition. and reverse: 5 -GGTGGAAACTGAAACCATGAAT-3 c Because the bonobo did not have this mutation, this gene region was charac- (696 bp), USP9Y exon 36 forward: 5#-CATGAAATTGTTT- terized in 1 wild-born individual from each of 4 chimpanzee subspecies, all found to TAGTTTCTGTTCT-3# and reverse: 5#-CTGATGGGG- have the identical gene-disruptive mutation as in the chimpanzee reference sequences TCTTGCAATAGTT-3# (674 bp), and USP9Y exon 39 (see Results and Discussion). forward: 5#-GCAAATAAAAGCTGTTTCTGCAT-3# and reverse: 5#-GCATTCTAGAGGCACTCAAAAGA-3# (796 chimpanzee Y-chromosome sequences are publicly avail- bp). PCR reactions were performed in 25 lL reactions using able (Hughes et al. 2005; Kuroki et al. 2006), offering ex- Platinum Taq (Invitrogen, Carlsbad, California), with the fol- cellent opportunities for focused analyses on this lowing conditions: 94 °Cfor2min,followedby40cyclesof chromosome. 94 °C for 15 s, 59 °C for 30 s, and 70 °C for 30 s. We aligned the Hughes et al. (2005) and Kuroki et al. For each PCR fragment amplification, we included (2006) chimpanzee Y-chromosome sequences and the hu- DNA from both male and female human, chimpanzee, man Y-chromosome reference sequence (Build 35) for the and bonobo individuals, in order to verify that homologous CYorf15B, TBL1Y, TMSB4Y, and USP9Y genes. This align- fragments from the X-chromosome were not amplified. Fol- ment confirmed that the splice site and stop codon muta- lowing amplification, PCR products were purified with tions described by Hughes et al. (2005) are found in Shrimp Alkaline Phosphatase and Exonuclease I (USB Cor- both chimpanzee Y-chromosome sequences (and not in poration, Cleveland, Ohio), cycle sequenced with BigDye the human sequence), making it highly unlikely that these Terminator Cycle Sequencing Kit version 3.1 (Applied reflect sequencing or assembly errors. We also identified Biosystems, Foster City, California), cleaned with isopro- a 4-bp frameshift