Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2607-2611, March 1995 Evolution Fate of a redundant y-globin gene in the atelid clade of New World monkeys: Implications concerning fetal globin gene expression CARLA M. M. MEIRELES*t, MARIA P. C. SCHNEIDER*t, MARIA I. C. SAMPAIO*t, HoRAcIo SCHNEIDER*t, JERRY L. SLIGHTOM4, CHI-HUA CHIUt§, KATHY NEISWANGERT, DEBORAH L. GuMucIoll, JOHN CZELUSNLAKt, AND MORRIS GOODMANt** *Departamento de Genetica, Universidade Federal do Para, Belem, Para, Brazil; Departments of tAnatomy and Cell Biology and §Molecular Biology and Genetics, Wayne State University School of Medicine, Detroit, MI 48201; tMolecular Biology Unit 7242, The Upjohn Company, Kalamazoo, MI 49007; 1Westem Psychiatric Institute and Clinic, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2593; and IlDepartment of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616 Communicated by Roy J. Britten, California Institute of Technology, Corona Del Mar, CA, December 19, 1994 (received for review August 19, 1994) ABSTRACT Conclusive evidence was provided that y', purifying selection. One outcome was that a mutation that the upstream of the two linked simian y-globin loci (5'-y'- made the qr locus a pseudogene was fixed -65 MYA in the 'y2-3'), is a pseudogene in a major group of New World eutherian lineage that evolved into the first true primates (4, monkeys. Sequence analysis of PCR-amplified genomic frag- 8). A later outcome, most likely favored by positive selection, ments of predicted sizes revealed that all extant genera of the was that embryonically expressed -y-globin genes became platyrrhine family Atelidae [Lagothrix (woolly monkeys), fetally expressed in the primate lineage out of which platyr- Brachyteles (woolly spider monkeys), Ateles (spider monkeys), rhines and catarrhines descended (1-3,9, 10). During this same and Alouafta (howler monkeys)] share a large deletion that evolutionary period, a tandem duplication produced the linked removed most of exon 2, all of intron 2 and exon 3, and much yl and y2 genes (1-3). of the 3' flanking sequence of y. The fact that two functional The tandem duplication of the ancestral anthropoid y-glo- 'y-globin genes were not present in early ancestors of the bin gene resulted from an unequal crossover between two Atelidae (and that y1 was the dispensible gene) suggests that homologous Li repetitive elements, one (Lla) upstream of the for much or even all of their evolution, platyrrhines have had single -y gene and the other (Llb) downstream of this gene (1). ly2as the primary fetally expressed 'y-globin gene, in contrast The crossover produced the tandem duplicate 5'-Lla-,y'- to catarrhines (e.g., humans and chimpanzees) that have 'y' as Llba-,y2-Llb-3'. The linked -y1 and ry2 loci and the Li ele- the primary fetally expressed y-globin gene. Results from ments that border them are present in all simian primate promoter sequences further suggest that all three platyrrhine f3-globin gene clusters that have been sequenced across the full families (Atelidae, Cebidae, and Pitheciidae) have y2 rather extent of the two -y loci, including six catarrhines [human (11, than y' as their primary fetally expressed y-globin gene. The 12), chimpanzee (12), gorilla (12), orangutan (12), gibbon (1), implications of this suggestion were explored in terms of how and rhesus monkey (1)] and two platyrrhines [spider monkey gene redundancy, regulatory mutations, and distance of each (1, 2) and capuchin monkey (3)]. In contrast, non-simian- 'y-globin gene from the locus control region were possibly primate B3-globin gene clusters that have been sequenced involved in the acquisition and maintenance of fetal, rather across the full extent of the orthologous y region have only a than embryonic, expression. single -y locus, which is not bordered by the 5'-Lla and 3'-Llb elements (3, 13). These non-simian sequence data have been The simian primates of the two branches of Anthropoidea, obtained from galago (13) and tarsier (3), the former repre- Platyrrhini (New World monkeys) and Catarrhini (Old World senting the primate suborder Strepsirhini and the latter rep- monkeys, apes, and humans), have two linked nonallelic resenting, as do the simians, the other primate suborder ,y-globin genes (1-3) that are found in a genomic domain called Haplorhini (14). Thus, two events, the insertion of Lla and Llb the 3-globin gene cluster. The genes in this cluster are arranged elements and the subsequent duplication of the -y locus, both in the linkage order 5'-s-y'-y_2_-'Iri-S--1-3' (4). These 13-globin occurred in the ancestral simian lineage after it separated from cluster genes, shared by all mammalian orders, descended the tarsier lineage but before it diverged into platyrrhines and from a single ancestral 3-globin gene that tandemly duplicated catarrhines (55-35 MYA). 150-200 million years ago (MYA), producing a 5'-proto-s The first platyrrhine from which an intact y-globin gene was locus and a 3'-proto-P3 locus (4). By the time of the last cloned and sequenced was a spider monkey belonging to the common ancestor of Metatheria (marsupials) and Eutheria species Ateles geoffroyi (Age) (15). This intact gene with (placental mammals) at "125 MYA, the two loci had already conserved coding and promoter sequences was subsequently differentiated into an embryonically expressed s-type locus shown to be the downstream or -y2 locus. In contrast, the upstream yl locus contained a 1.8-kb deletion that removed and a postembryonically expressed {3-type locus (5, 6). This kb of the two gene cluster has persisted in marsupials, as demonstrated most of exon 2, all of intron 2 and exon 3, and >0.6 an and an Australian 3' flanking sequence of the -y' gene (1). Although this finding by American opossum (5) dasyurid locus is a marsupial (6). However, in the early eutherians at 100-80 suggested that in spider monkeys the 'yl-globin locus pseudogene, additional individuals fromAge or closely related MYA, tandem duplications of the embryonic --type not rule out and a tandem species had not been examined. Thus, the data did resulted in E, y, and q loci, similarly, duplication was the result of a of the 13-type locus resulted in 8 and j3 loci (4, 7, 8). This the possibilities that the deletion cloning redundancy of embryonic and postembryonic genes provided opportunities for individual genes to escape the constraints of Abbreviations: MYA, million year(s) ago; Age, Ateles geoffroyi; Apa, Atelespaniscus; Lla, Lagothrix lagotricha; Bar, Brachyteles arachnoides; Abe, Alouatta belzebul; Aca, Alouatta caraya; Ase, Alouatta seniculus; The publication costs of this article were defrayed in part by page charge Cal, Cebus alibifrons; Mmu, Macaca mulatta; Hsa, Homo sapiens; LCR, payment. This article must therefore be hereby marked "advertisement" in locus control region. accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 2607 Downloaded by guest on September 24, 2021 2608 Evolution: Meireles et al. Proc. Nati Acad. Sc. USA 92 (1995) artifact or a mutation in that individual monkey; if either of were included when they greatly increased sequence identity these possibilities were proven to be true, then this deletion among the aligned sequences. would have no evolutionary significance. However, if this yl pseudogene locus resulted from an ancient deletion in the 'y1 RESULTS gene in early ancestors of a major group of New World monkeys, then at least some catarrhine and platyrrhine species The 16 extant genera of the infraorder Platyrrhini (superfam- differ in their predominant fetal y gene expression patterns- ily Ceboidea) belong to seven clades that diverged from i.e., y' is the preferred fetally expressed ry gene in two common ancestors 23-17 MYA (22-26). Moreover, the DNA catarrhines, humans (16) and chimpanzees (17), while y2 sequence evidence from our laboratory (ref. 23 and unpub- predominates in those platyrrhine species that contain the y1 lished results) groups these seven clades into three families pseudogene. Here, we present conclusive evidence that the (Atelidae, Pitheciidae, and Cebidae) and, in agreement with deletion first noted in the yl locus of a single spider monkey paleontological evidence, places the divergence node for the (1) is indeed an ancient deletion and that the y1 locus is a common ancestor of the extant genera of the atelid clade at pseudogene in all genera of the platyrrhine family Atelidae 13 MYA (Fig. 1). [Lagothrix (woolly monkey), Brachyteles (woolly spider mon- key), Ateles (spider monkey), and Alouatta (howler monkey)]. MATERIALS AND METHODS Other Mammals Primates Samples and DNA Extraction. The animal sources of the DNA sequences determined in this study were eight captive monkeys representing the four genera of the family Atelidae: threeAteles-twoAge [the original one (Agel) from the zoo in Madison, WI (15) and the second (Age2) from the zoo in Santa Strepsirhini Haplorhini Ana, CA] and one Ateles paniscus (Apa) from the Centro galago y duplication Nacional de Primatas (CNP), Para, Brazil; one Lagothrix lemur 3555MYA ¶ N\ lagotricha (Lla) from CNP; one Brachyteles arachnoides (Bar) Anthropoidea Tarsier from the Centro de Primatologia, Rio de Janeiro, Brazil; and three Alouatta-one Alouatta belzebul (Abe) from CNP, one Alouatta caraya (Aca), and one Alouatta seniculus (Ase) (the latter two animals from the zoo in Los Angeles, CA). The Platyrrhini Catarrhini DNA sources for the sequences determined in this study were Old World M A clone AgeCh35-19-2 (15) for Agel and genomic DNAs ex- 1 16genera/7clades Apes tracted from peripheral blood cells as described by Bell et al. Ceboidea in 3 families Human (18) forAge2,Aca, andAse and by Sambrooketal. (19) forApa, Radiation 17-23MYA Lla, Bar, and Abe.