Chromosome Research 10: 55^61, 2002. # 2002 Kluwer Academic Publishers. Printed in the Netherlands
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Direct evidence for the Homo^Pan clade Rainer Wimmer1y , Stefan Kirsch2y , Gudrun A. Rappold2 & Werner Schempp1 * 1 Institute of Human Genetics and Anthropology, University of Freiburg, Breisacher Str. 33, 79106 Freiburg, Germany; Tel: +49 761 270 7062; Fax: +49 761 270 7041; E-mail:
[email protected] 2 Institute of Human Genetics, University of Heidelberg, INF 328, 69120 Heidelberg, Germany *Correspondence y These authors have contributed equally to this work Received 13 July 2001; received in revised form and accepted for publication by M. Schmid 27 September 2001
Key words: comparative mapping, evolutionary breakpoints, in-situ hybridization, phylogeny, primates, Y chromosome
Abstract For a long time, the evolutionary relationship between human and African apes, the `trichotomy problem', has been debated with strong differences in opinion and interpretation. Statistical analyses of different molecular DNA data sets have been carried out and have primarily supported a Homo^Pan clade. An alternative way to address this question is by the comparison of evolutionarily relevant chromosomal breakpoints. Here, we made use of a P1-derived arti¢cial chromosome (PAC)/bacterial arti¢cial chromosome (BAC) contig spanning approximately 2.8 Mb on the long arm of the human Y chromosome, to comparatively map individual PAC clones to chromosomes from great apes, gibbons, and two species of Old World monkeys by £uorescence in-situ hybridization. During our search for evolutionary breakpoints on the Y chromosome, it transpired that a transposition of an approximately 100-kb DNA fragment from chromosome 1 onto the Y chromosome must have occurred in a common ancestor of human, chimpanzee and bonobo. Only the Y chromosomes of these three species contain the chromosome-1-derived fragment; it could not be detected on the Y chromosomes of gorillas or the other primates examined. Thus, this shared derived (synapomorphic) trait provides clear evidence for a Homo^Pan clade independent of DNA sequence analysis.
Introduction In his article `Evidence as to Man's Place in Nature', Thomas H. Huxley concluded that `it is quite certain that the Ape which most nearly approaches man, in the totality of his organization, is either the chimpanzee or the gorilla' (Huxley 1863). Ever since, the evolutionary relationship between humans and African great
apes, the `trichotomy problem', has received considerable attention. Great expectations were raised by molecular methods, when these were ¢rst introduced, regarding their presumed power to resolve the human^chimpanzee^gorilla trichotomy (Sarich & Wilson 1967, Ferris et al. 1981, Sibley & Alquist 1984, 1987, Goldman et al. 1987, Djian & Green 1989, Caccone & Powell 1989, Sibley et al. 1990, Kawamura et al. 1991, Horai et al.
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Direct evidence for the Homo^Pan clade 1992, Rogers 1994, Goodman et al. 1998). However, statistical analyses of DNA sequence data sets, both mitochondrial and nuclear, have provided equivocal solutions depending on the genomic sites analyzed, and, until recently, the relationship between Homo, Pan, and Gorilla has been unresolved because of their close molecular similarity. Ruvolo (1997) applied multiple independent DNA sequence datasets for a statistical analysis, and, of 14 independent data sets, 11 supported a human^chimpanzee clade, two a chimpanzee^gorilla clade, and one a human^ gorilla clade. Her study has recently been expanded by Satta et al. (2000) and by Chen & Li (2001). Both studies conclude that there is strong support for the Homo^Pan clade. Despite the support from morphological features for a chimpanzee^gorilla relationship (Andrews 1992), Gibbs et al. (2000) showed that an extensive morphological data set of soft-tissue characters yielded robust phylogenetic hypotheses that are also compatible with this molecular phylogeny. Here, we report additional evidence for the Homo^Pan clade using a different experimental approach. We used a P1-derived arti¢cial chromosome (PAC)/bacterial arti¢cial chromosome (BAC) contig spanning approximately 2.8 Mb on the long arm of the human Y chromosome to comparatively map PAC clones to metaphase chromosomes from great apes, gibbons, and
57 two species of Old World monkeys by £uorescence in-situ hybridization. We show that the transposition of a small DNA fragment of approximately 100 kb from chromosome 1 onto the Y chromosome must have occurred in a common ancestor of human, chimpanzee and bonobo.
Materials and methods Chromosome preparation Chromosome spreads were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes taken from human (Homo sapiens), chimpanzee (Pan troglodytes ), pygmy chimpanzee or bonobo (Pan paniscus), western lowland gorilla (Gorilla gorilla gorilla), eastern lowland gorilla (Gorilla gorilla graueri), Sumatran orangutan (Pongo pygmaeus abelii), Bornean orangutan (Pongo pygmaeus pygmaeus), white-handed gibbon (Hylobates lar), pig-tailed macaque (Macaca nemestrina), and proboscis monkey (Nasalis larvatus). Standard methods were applied for all chromosome preparations. Some of the slide preparations were stained with acridine orange (50 mg/ml) and checked for a well-spreading and plasma-free preparation of metaphase plates. Only slides from batches that were con-
Figure 1. Genomic organization of a distinct subinterval of the long arm pericentromeric region of the human Y chromosome £anked by the centromere and the AZFa region. (A) Organization of the Yq11 pericentromeric region and coverage in PAC/BAC clones. (B) Enlarged view of a 650-kb subinterval and precise positioning of PAC and BAC clones mapping to this region. Pre¢xes RP1, 4, and 5 indicate PAC clones and pre¢x RP11 BAC clones. Clones marked in green were used in (C) FISH experiments on human metaphase chromosomal spreads. PAC RP1-76F02 (middle) identi¢ed 3 different cytogenetic locations: a strong signal on Yq11.21, a signal cluster on 1q43 and a weak signal on distal Yq11.23. PAC RP4-532I07 (right) detected strong signals on Yq11.21 and Xp22.3. BAC RP11-322K23 (left) identi¢ed multiple cytogenetic localisations, among others strong signals on the Y-pericentromeric region including Yq11.21, indicating the presence of repetitive sequences. Note that this clone was hybridized without suppression, otherwise no signals would have been obtained. As control, the SHOX gene containing cosmid LLOYNC03`M'34F05 from the pseudoautosomal region 1 on Xpter/Ypter was used. (D) Schematic illustration of the genomic environment of the junction regions. Sequences derived from BAC and PAC clone end-fragments were used to precisely position the corresponding clones on the sequenced BAC clones from the Human Genome Project. Large-scale sequence comparison between the Y-derived BAC clones (Accession Nos. AC011293, AC012502 and AC007247), and clones from the homologous regions (Accession Nos. AF 317888 from Xp22, AL354875 and AL445675 from 1q43) were used to determine the organization of the junction regions at the sequence level. In the process of sex chromosome evolution, a Y-chromosomal rearrangement (Gla«ser et al. 1999; Lahn & Page 1999) excluded the 6.9-kb fragment as part of an X/Y-homology block from recombination on a progenitor Y chromosome. Subsequently, breakage of this X/Y-homology block led to a duplication, and the shifting of one 6.9-kb fragment to human Yp11, with the other 6.9-kb fragment remaining on Yq11 (sequence identities are 88% between the Yq and Xp copy, and 97% between the Yq and Yp copy). The sequence of both junction regions in Yq11 as well as surrounding repeat families consists of: HSATII, MER1, Alu, MER2, AGGAA, ERVL, L1; indicates non-repetitive sequences. As a reference for MER1 and MER2 see Smit & Riggs (1996).
58 sidered to present optimal metaphase preparations were considered for £uorescence in-situ hybridization, and were stored at 80 C until use. Fluorescence in-situ hybridization (FISH) Prior to FISH, the slides were treated with RNase followed by pepsin digestion as described by Ried et al. (1992). FISH using LLOYNC03`M'34F05 as a marker for the pseudoautosomal gene SHOX (Rao et al. 1997), as well as the PAC and BAC clones derived from the Y-chromosomal contig (Lahn & Page 1999; Kirsch et al., unpublished) essentially followed the methods as described (Schempp et al. 1995). For comparative chromosome painting with a human chromosome-1speci¢c painting probe (AGS Heidelberg) in white-handed gibbon and proboscis monkey, a standard procedure with minor modi¢cations was applied (Jauch et al. 1992). Fluorescence microscopy and imaging Preparations were evaluated using a Zeiss Axiophot epi£uorescence microscope equipped with single-bandpass ¢lters for excitation of red, green and blue (Chroma Technologies, Brattleboro, VT). During exposures, only excitation ¢lters were changed, allowing for pixelshift-free image recording. Images of high magni¢cation and resolution were obtained using a black-and-white CCD camera (Photometrics Kodak KAF 1400; Kodak, Tucson, AZ) connected to the Axiophot. Camera control and digital image acquisition involved the use of an Apple Macintosh Quadra 950 computer. Results and discussion Phylogenetically, the human Y chromosome is of recent origin and has already been shown to harbor secrets whose revelation may be helpful in shedding light on our evolutionary history (Semino et al. 2000, Shen et al. 2000, Jobling & Tyler-Smith 2000). Comparative mapping of human Y chromosome genes and gene families on chromosomes of higher primates by £uorescence in-situ hybridization (FISH) has shown that Y chromosomal rearrangements are con¢ned
R. Wimmer et al. to the non-recombining Y chromosomal portion outside of the pseudoautosomal and sex-determining region in higher primates (GlÌser et al. 1998, 1999). During our search for evolutionary breakpoints within a de¢ned segment on the long arm of the Y chromosome, we performed comparative FISH mapping of individual clones from a human Y-chromosomal PAC contig, to chromosomes of humans, great apes, gibbons and Old World monkeys (Wimmer et al. in preparation). The PAC contig spans approximately 2.8 Mb on proximal Yq11.21 including the entire AZFa-region (Sargent et al. 1999). Here, we report our comparative FISH results in higher primates using a subset of clones proximal to AZFa (Figure 1). In particular, an approximately 100-kb insert in P1-derived arti¢cial chromosome (PAC) RP176F02 resulted in a striking observation and proved useful in de¢ning the phylogenetic order of Homo, Pan and Gorilla. FISH with this PAC clone on human metaphase plates revealed the expected signals on Yq11.21 as well as a weaker cross-hybridization signal more distal on Yq11.23 (Figure 1C). Surprisingly, we detected strong signals also in the subtelomeric region of the long arm of human chromosome 1, suggesting that sequences within RP1-76F02 are present and ampli¢ed on chromosome 1q43. Likewise, in chimpanzee and bonobo, clear RP1-76F02 signals are seen on their respective Y chromosomes and chromosomes 1 (Figure 2a, b). No RP1-76F02 signals were detectable on the Y chromosome of western lowland gorilla (Figure 2c), nor eastern lowland gorilla (not shown), while the strong signals on chromosome 1 remained in the latter two subspecies. It should be mentioned that a triple split signal is consistently seen in chromosome 1 of the gorillas (see Figure 2c). This may be interpreted in a way that a repetitive sequence organisation tends to duplications/ampli¢cations and chromosomal rearrangements. FISH results for RP1-76F02, also con¢ned to chromosome 1, were obtained for the Sumatran orangutan (Figure 2d) and the Bornean orangutan (not shown), as well as for the white-handed gibbon (not shown), the pig-tailed macaque (Figure 2e) and the proboscis monkey (Figure 2f). Our FISH results obtained with PAC clone RP1-76F02 combined with the results of our painting experiments using a human chromosome-1-speci¢c painting
Direct evidence for the Homo^Pan clade
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Figure 2. FISH of human Y-chromosomal PAC clone RP1-76F02 (biotin^FITC: green) to various higher primates. The human-derived cosmid LLOYNC03`M'34F05 for the pseudoautosomal gene SHOX (digoxigenin^TRITC: red) served as an internal standard for judging the hybridization ef¢ciency of our procedure. Clear RP1-76F02 signals (in the intensity range of the SHOX-signal) are seen on the Y chromosomes, and even stronger signals on chromosomes 1 of chimpanzee (a) and bonobo (b). No RP1-76F02 signals were detectable on the Y chromosomes of western lowland gorilla (c), Sumatran orangutan (d), pig-tailed macaque (e), and proboscis monkey (f), while the strong signals remain on their chromosomes 1, respective chromosome 5, the equivalent to human chromosome 1q in proboscis monkey (see Methods). Centromeres are marked by small bars.
probe in gibbon and proboscis monkey revealed that the orthologous segments to human 1q43 are found on gibbon chromosome 19 and on proboscis monkey chromosome 5. It should be noted that there is a block of Xp22/Yq11 homology of approximately 500 kb in length distal to RP1-76F02. Accordingly, FISH analysis with clones from this X^Y homologous block reveals signals on human Yq11.21 and Xp22.3 (Figure 1C) and, most importantly, this X^Y signal pattern is evolutionarily conserved in all great apes and both species of Old World
monkeys. Thus, the con¢nement of the target sequence of RP1-76F02 to chromosome 1 in gorilla strongly suggests that this represents the ancestral situation, while the Y chromosomal RP176F02 integration must be interpreted as a shared, derived (synapomorphic) trait in human, chimpanzee and bonobo. In order to con¢rm these results, we performed FISH with a PAC clone derived from human chromosome 1q43 to chromosomes from humans and great apes. Indeed, the same hybridization results as those obtained with the Y-chromosomal PAC clone
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Figure 3. Diagram of the comparative FISH mapping of PAC RP1-76F02 ( boxes) on R-banded ideograms of chromosome Y and autosomes of Old World simian primates. Note that the orthologous segments of human 1q43 are found on gibbon chromosome 19q, and on proboscis monkey chromosome 5q (see Methods). The phylogenetic tree is modi¢ed after Martin (1993). Myr, million years before present.
RP1-76F02 were observed. The repeat structure of the £anking sequences of the chromosome 1-homologous segment within the Y-contig (Figure 1) indicates that the transfer of the approximately 100-kb fragment from chromosome 1 to the Y was mediated by a transposition duplication mechanism (Ji et al. 2000) about 5^6 million years ago. For parsimony reasons, this transposition must have occurred in a common ancestor of human,
chimpanzee and bonobo (Figure 3) and therefore provides clear and convincing evidence for a Homo^Pan clade. The results of this study, clearly showing an evolutionary cytogenetic event, combined with the results of the DNA sequence analysis now provide conclusive evidence for a Homo^Pan clade. These results also once again emphasize the power of Y-chromosomal studies in resolving the evolutionary history of mankind.
Direct evidence for the Homo^Pan clade Acknowledgements We thank Wolfram Rietschel from the zoo `Wilhelma' in Stuttgart for kindly providing blood samples from apes and monkeys, Birgit Weiss for excellent technical assistance, Susanne RÎttger and Roscoe Stanyon for helpful discussion, as well as Deborah Morris-Rosendahl for comments on the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sche 214/7-1; Ra 380/10-2). References Andrews P (1992) Evolution and environment in the hominoidea. Nature 360: 641^646. Caccone A, Powell JR (1989) DNA divergence among hominoids. Evolution 43: 925^942. Chen F-C, Li W-H (2001) Genomic divergences between humans and other hominoids and the effective population size of the common ancestor of humans and chimpanzees. Am J Hum Genet 68: 444^456. Djian P, Green H (1989) Vectorial expansion of the involucrin gene and the relatedness of the hominoids. Proc Natl Acad Sci USA 86: 8447^8451. Ferris SD, Wilson AC, Brown WM (1981) Evolutionary tree for apes and humans based on cleavage maps of mitochondrial DNA. Proc Natl Acad Sci USA 78: 2432^2436. Gibbs S, Collard M, Woods B (2000) Soft-tissue characters in higher primate phylogenetics. Proc Natl Acad Sci USA 97: 11130^11132. GlÌser B, GrÏtzner F, Willmann U et al. (1998) Simian Y chromosomes: species-speci¢c rearrangements of DAZ, RBM, and TSPY versus contiguity of PAR and SRY. Mammalian Genome 9: 226^231. GlÌser B, Myrtek D, Rumpler Y et al. (1999) Transposition of SRY into the ancestral pseudoautosomal region creates a new pseudoautosomal boundary in a progenitor of simian primates. Hum Mol Genet 8: 2071^2078. Goldman D, Rathna Giri P, O'Brien SJ (1987) A molecular phylogeny of the hominoid primates as indicated by two-dimensional protein electrophoresis. Proc Natl Acad Sci USA 84: 3307^3311. Goodman M, Porter CA, Czelusniak J et al. (1998) Toward a phylogenetic classi¢cation of primates based on DNA evidence complemented by fossil evidence. Mol Phylogenet Evol 9: 585^598. Horai S, Satta Y, Hayasaka K et al. (1992) Man's place in hominoidea revealed by mitochondrial DNA genealogy. J Mol Evol 35: 32^43. Huxley TH (1863) Evidence as to Man's Place in Nature. New York: P. Appleton & Co. Jauch A, Wienberg J, Stanyon R et al. (1992) Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc Natl Acad Sci USA 89: 8611^8615.
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