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Molecular and Genomic Data Identify the Closest Living Relative of Science Jan E. Janecka, etal. Sc/ence 318, 792(2007); DOI: 10.1126/science.1147555

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Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2007 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. I REPORTS 14. M. S. Grinolds, V. A. Lobastov, ]. Weissenrieder, 30. ]. B. Goodenough, Phys. Rev. 120, 67 (1960). 38. From the reflectivity of 0.28 and the absorption depth A. H. Zewail, Proc. Notl. Acod. Sei. U.S.A. 103, 18427 31. T. C. Koethe et al.. Phys. Rev. Lett. 97, 116402 of 100 nm at 800 nm (20), the threshold fluence corresponds (2006). (2006). to 450 m]/mm^ at the surface. With the unit cell volume 15. V. A. Lobastov, ]. Weissenrieder, ]. Tang, A. H. Zewail, 32. Given the x-ray wavelength (1.54 A) and diffraction angle of 118 A^ {21), which contains four vanadium atoms, Nono Lett. 7, 2552 (2007). (13.9°) reported (22), the Bragg peak {22) should be this energy density gives -0.05 photon per vanadium. 16. H. S. Park, ]. S. Baskin, O.-H. Kwon, A. H. Zewail, Nano indexed as (Oil) of the monochnic phase. It was assigned 39. G. Ertl, Adv. CotoL 45, 1 (2000). Lett. 7, 2545 (2007). as (110) of the monoclinic phase, which in fact becomes 40. In to evaluate the maximum range of the intensity 17. K. ]. Caspersen, E. A. Carter, Proc. Notl. Acod. Sei. U.S.A. (110) of the rutile phase upon transformation. decay, we also considered a convoluted step function 102, 6738 (2005). 33. D. Maurer, A. Leue, R. Heichele, V. Müller, Phys. Rev. B instead of a decay process. This distinction becomes 18. D. R. Trinkle et oL. Phys. Rev. Lett. 91, 025701 (2003). 60, 13249 (1999). significant depending on the physics of the process 19. F. ]. Morin, Phys. Rev. Lett. 3, 34 (1959). 34. D. Kucharczyk, T. Niklewski,;. AppL Crystallogr. 12, 370 involved. For a step function, we obtained Ai^ = 760 fs. 20. C. N. Berglund, H. ]. Guggenheim, Phys. Rev. 185, 1022 (1979). The overall fit of the transient was repeated 1000 times to (1969). 35. ]. M. Thomas, Chem. Br. 6, 60 (1970), and references estimate the error in the Af^ range, which was found 21. K. D. Rogers, Powder Diffr. 8, 240 (1993). therein. to be ±80 fs. We note that changes in intensity occur in 22. A. Cavalleri et oL, Phys. Rev. Lett. 87, 237401 (2001). 36. Excess electrons (carriers), which are not bound by strong a step of 250 fs. For the ps component, the range AÍ2 of 23. G. I. Petrov, V. V. Yakovlev, ]. A. Squier, Opt. Lett. 27, correlation with the lattice, may redistribute within the 15 ps is evident from the figure. 655 (2002). excited metallic region but are impeded by the insulating 41. We are grateful to ]. Weissenrieder for helpful 24. A. Cavalleri et oL, Phys. Rev. Lett. 95, 067405 (2005). surrounding. Electron diffusion from the probed layer discussions, L. H. Tjeng for generously providing some 25. A. Cavalleri, M. Rini, R. W. Schoenlein, ]. Phys. Soc. ]pn. (-10 nm) to the metallic region (-100 nm) occurs in crystals, G. R. Rossman for the crystal-cutting equipment, 75, 011004 (2006), and references therein. -100 ps, as calculated from the electron mobility of 1 to and L M. Henling for help with the x-ray measurements. 26. H.-T. Kim et oL. Phys. Rev. Lett. 97, 266401 (2006). 10 cm^/(V-s) for metallic vanadium dioxide (20). The This work was supported by the NSF, by the Air Force 27. P. Baum, A. H. Zewail, Proc. Noti Acad. Sei. U.S.A. 103, diffusion of such electrons into the deeper regions may Office of Scientific Research, and by the Gordon and 16105 (2006). contribute to generation of shear. Betty Moore Center for Physical Biology at Caltech. 28. D.-S. Yang, N. Gedik, A. H. Zewail, ]. Phys. Chem. C 111, 37. Shear motion leads to a change in principal axes {34). P.B. was partially supported by the Alexander von 4889 (2007). Because not all Bragg spots are equally well in Humboldt Foundation. 29. V. A. Lobastov et al., in Ultrofost Optics IV, F. Krausz, phase with the Ewald sphere at the same time {28), G. Korn, P. Corkum, \. A. Walmsley, Eds., voL 95 of shear motion may enhance or suppress the Bragg Springer Series in Opticol Sciences (Springer, New York, intensities to values above or below the initial intensity, 12 July 2007; accepted 19 September 2007 2004), pp. 419-435. as observed. 10.1126/science.ll47724

Sundatheria (3, 6, 14), although support for this Molecular and Genomic Data hypothesis was lower than for other mammalian interordinal (15). , proposed Identify the Closest Living on morphological grounds (12), has also been in- dicated by some molecular studies (16, 17). Other studies have failed to reject altemative hypotheses, Relative of Primates and analyses of different character subsets support contradictory topologies (18-20). ]an E. Janecka,^ Webb Miller/ Thomas H. Pringle,' Frank Wiens,'' Annette Zitzmann,^ To improve our understanding of early Kristofer l\A. Helgen,* Mark S. Springer/ William ]. Murphy^* euarchontan evolution and determine the closest living relative of primates, we used two inde- A full understanding of morphological and genomic evolution requires the pendent molecular approaches. We first screened identification of their closest living relative. In order to resolve the ancestral relationships among a nonredundant set of 197,522 protein-coding primates and their closest relatives, we searched multispecies genome alignments for exons from the human University of Califor- phylogenetically informative rare genomic changes within the superordinal group , nia Santa Cruz Known Genes track to identify which includes the orders Primates, Dermoptera (), and Scandentia (treeshrews). We also rare genomic changes (exonic indels) that would constructed phylogenetic trees from 14 kilobases of nuclear genes for representatives from most potentially support the three a priori hypotheses. major primate lineages, both extant colugos, and multiple treeshrews, including the pentail We also assembled and analyzed a 14-kb nu- treeshrew, Ptilocercus lowii, the only living member of the family Ptilocercidae. A relaxed clear gene data set of 19 gene fragments in order molecular clock analysis including Ptilocercus suggests that treeshrews arose approximately to estimate a phylogeny and time scale for ex- 63 million ago. Our data show that colugos are the closest living relatives of primates and tant euarchontans. To mitigate against the pos- indicate that their divergence occurred in the . sible effects of long-branch attraction (LBA), which can incorrecfly place rapidly evolving clades The origins of modem primates and their the monophyly of treeshrews, colugos (flying fossil relatives remain a topic of intense ), and primates in the Euarchonta, ^Department of Veterinary Integrative Biosciences, Texas A&M debate (i-i), as there has been an in- with a sister-group relationship to [which University, College Station, TX 77843, USA. ^Center for Com- parative Genomics and Bioinformatics, Pennsylvania State creased focus on identifying adaptive evolution- includes and lagomorphs (3, 6-8)\. In University, University Park, PA 16802, USA. ^Sperling ary changes within primates and the dynamics contrast, the relationships within Euarchonta Foundation, Eugene, OR 97405, USA. "Department of of genome evolution within the primate lineage are not well resolved, most likely because of the Physiology, University of Bayreuth, D-95440 Bayreuth, {4, 5). Resolving higher primate relationships rapid evolution of these groups and inadequate Germany. ^Zoological Institute, Johann Wolfgang Goethe- University, D-60054 Frankfurt/Main, Germany. 'National has been challenging, making it difficult to sampling within Scandentia and Dermoptera. Museum of Natural History, Smithsonian Institution, Wash- identify character transformations in early pri- Three hypotheses have been proposed: (i) a ington, DC 20013, USA. 'Department of Biology, University mate evolution. An essential part of this chal- sister-group relationship between treeshrews and of California, Riverside, CA 92521, USA. lenge is to determine the closest living relative primates (9-11), (ii) a sister-group relationship *To whom correspondence should be addressed at the to primates, which would provide a broader between colugos and primates [Primatomorpha Department of Veterinary Integrative Biosciences, College of context for understanding primate evolution. (12)], and (iii) both colugos and treeshrews as Veterinary Medicine and Biomédical Sciences, Veterinary Medical Administration Building, Room 107, Texas A&M DNA sequence and morphological studies, sister to the primates [Sundatheria (2, 13)]. Mo- University, College Station, TX 77843-4458, USA. E-mail: and analyses of rare genomic changes, support lecular and morphological studies have favored [email protected]

792 2 NOVEMBER 2007 VOL 318 SCIENCE www.sciencemag.org REPORTS together, we included nuclear DNA sequences plification of candidate indel-containing exons and 102 indels that were treeshrew-specific from both living colugos and the second tree in the and comparison to the treeshrew (potentially informative for Sundatheria). shrew family, Ptilocercidae (21, 22). genome sequence (23). PCR primers were de- After excluding noninformative and hyper- We identified 300 candidate indels in cod- signed for the remaining 196 candidates, of variable indels (23) and the evaluation of ad- ing gene exons within Euarchonta. Of these, which 75% produced a single band in colugo, ditional eutherian genomes (table SI), three 104 were excluded because they lacked flanking distributed in the following categories: 32 indels indels supported the monophyly of Euarchonta sequences that were long or conserved enough that were initially primate-specific (shared by [N4BP2, ZNF12, and CDCA5 (figs. SI to S3)] and for primer design, were determined to be anom- anthropoids and strepsirrhines, potentially in- corroborate the emerging phylogenetic consen- alous misalignments, or were computationally formative for Primatomorpha); 13 indels shared sus that primates, colugos, and treeshrews are a determined to be paralogous gene alignments by primates and treeshrews (potentially in- monophyletic group (3, 8,15,19, 20). No indels (23). The lack of a colugo genome sequence formative for colugos being in a basal position placed treeshrews with rodents and lagomorphs required polymerase chain reaction (PCR) am- or alternatively for euarchontan monophyly); (17) or treeshrews as basal within (24). We identified seven indels that supported colugos as the closest living relative of primates Fig. 1. An example of a coding TEX2 exonA Primatomorpha [Primatomorpha: SPBC25, SMPD3, MTUSl, sequence indel supporting the human SEEKPPAE •-GSEDPKKPPRPQEGTR SH3RF2, NC0A4, TEX2, and SSH2 (Figs. 1 and Primatomorpha hypothesis. A chimp SEEKPPAE •-GSEDPKKPPRPQEGTR 2 and figs. S4 to S10)]. By contrast, no indels three-amino acid deletion in exon orangutan SEEKPPAE •-GSEDPKKPPRPQEGTR supported Sundatheria, despite a larger number 4 of the TEX2 gene is present in all macaque SEEKPPAE •-GSEDPKKPPRPQEGTR of potentially informative candidates for this major primate lineages and both marmoset SEEKPSAE •-GSEDPKKPPRPQEGTR hypothesis having been screened. One indel SEEKPPPE •-GSEDPQKPPPPQEGTR colugo genera (shaded gray) but is tarsier (ADD2) supported a sister-group relationship be- bushbaby LEEKLPAE •-GSEDPKKPPHPQEGTR absent in all treeshrew lineages and tween treeshrews and primates (fig. Sll). Taken mouse LEEKLPVE •-GSEDPKKPPHPQEGAR eutherian outgroup representatives. together, an analysis of these last eight indels See figs. SI to SID for full align- Phii. flying lemur VEEKLPAE •-GSEDPKKPPVPQEGTR by means of a statistical framework (7) pro- ments and descriptions of additional SEDKPPAERELGSEDPKKPPHSQEGTR vides significant support for Primatomorpha supporting indels for Euarchonta pentail treeshrew SEEKPPAEREPGSEDPKKPPHSQEG-R and Primatomorpha. mouse TEEKPPPEKELPSEDLKKPPQPQEGTK [P < 0.025 (23)1 guinea pig SEEKPPAEKELGSEDPKKPSHPQEGTR The monophyly of Primatomorpha was inde- rabbit SEEKPPAERELASEDPKKPPQPQEGTR pendently confirmed by phylogenetic recon- dog SEEKPPAERELGGEDPKKPPHPQEGTR struction fiüm a 14-kb data set consisting of 19 horse SEEKPPTEKEQGVEDPKKPSPPQEGTR nuclear gene segments with maximum likelihood brown SEEKPPAERDLGVEDPKKPPHPQEGTR [(ML) 90% bootstrap support] and Bayesian cow CEEKPPAERELGGEDPKKPPHPQEGTR (1.00 posterior probability) algorithms (Fig. 2 shrew SEEKLPAEKELGAEDPKKPAHPQEGTR and fig. S12). Previously, the hierarchical order armadillo SEEKPSAERELGSEDSKKPPHSQEGTR at the base of the Euarchonta was difficult to elephant SEEKPPAERELAGEDPKKPP--LEGTR resolve with confidence because of contempo-

Fig. 2. A maximum-likelihood phy- Primatomorpha logeny of the superorder Euarchonta, ML BS: 90% with and lagomorph lineages BAYPP 1.00 as outgroups. Branch lengths were Tamias Indels: estimated under an F84 model of 100/1.0 G 2 aa del. SPBC25 Castor Rodentla L sequence evolution and the re- 2 aa del. SMPD3 1 Pedetes laxed molecular clock approach, 4 aa del. MTUSl R implemented in the program Sylvilagus 1 E 3 aa del. SH3RF2 1 S MULTIDIVTIME (23). Bootstrap (BS) 4 aa del. NC0A4 Ochotona 1 values and Bayesian posterior 3 aa del. TEX2 Galeopterus probabilities (BPPs) are shown on 1 aa del. SSH2 Dermoptera branches for which these values are Cynocephalus 100% and 1.0, respectively. Amino Lemur acid (aa) indels (ins, insertion; del, deletion) supporting the monophyly Microcebus of Euarchonta and Primatomorpha Otolemur are listed in boxes to the left, along Primates Áteles with respective BS and BPP values. 100/1.0 A molecular time scale is presented 100/1.0 Macaca Euarchonta below the tree (23). The 95% cred- Homo ibility intervals (CIs) are shown as ML BS: 92% Tupaia gray bars spanning each node. The BAY PP: 1.00 100/1.0 Indels: 100/1.0 1 point estimates and 95% CIs for all Urogale Scandentia 4 aa del, N4BP2 ; nodes are presented in table S4. 3aains., ZNF12 Ptiiocercus 1 aa del., CDCA5

Cretaceous Paleo Eocene Oligo Miocene Pli

90 70 60 50 40 30 20 10 0 Millions of years

www.sciencemag.org SCIENCE VOL 318 2 NOVEMBER 2007 793 I REPORTS raneous divergence of ancestral lineages dur- ysis supporting Sundatheria placed extinct 17. P. ]. Waddell, S. Shelley, Mol. Phylogenet. Evol. 28, 197 ing the Cretaceous, LBA, and limited taxon plesiadapiforms in a monophyletic clade with (2003). 18. ]. Schmitz, M. Ohme, B. Suryobroto, H. Zischler, and gene sampling (25). The results from our Primates (i), in contrast to Beard {12), who Mol. Biol. Evol. 19, 2308 (2002). expanded data set (table S2) contrast with pre- identified plesiadapiforms as members of Der- 19. 0. Madsen et ai, Nature 409, 610 (2001). vious studies supporting Sundatheria. When moptera, within Primatomorpha. Our reanal- 20. W. ]. Murphy et ai, Nature 409, 614 (2001). Ptilocercus lowii and both colugo genera are in- ysis of the data set from {3) that constrains the 21. M. C. McKenna, S. K. Bell, Classification of Above the Species Level (Columbia Univ. Press, New York, cluded, the ML and Bayesian trees become con- monophyly of Euprimates and Dermoptera 1997). sistent with rare genomic changes. The importance agrees with the placement of plesiadapiforms 22. K. M. Helgen, in Species of the World, of P. lowii was evident when it was removed as the sister group to Euprimates, though this D. E. Wilson, D. M. Reeder, Eds. (John Hopkins Univ. from the data set; ML trees lacked significant result is only weakly supported (3) (fig. S14). Press, Baltimore, MD, 2005), pp. 104-109. bootstrap support for the divergence between Finally, our results indicate that a draft genome 23. See supporting material on Science Online. 24. D. Huchon et al., Proc Nati. Acad Sei. U.S.A. 104, 7495 primates, freeshrews, and colugos (fig. S13). sequence from a colugo is a necessary prerequisite (2007). A Bayesian relaxed molecular clock approach to accurately reconstruct the ancesfral primate 25. M. S. Springer, W. J. Murphy, E. Eizirik, S. ]. O'Brien, with eight fossil constraints estimated the origin genome (5). Proc Nati. Acad Sei. U.S.A. 100, 1056 (2003). of Euarchontoglires at 88.8 million years ago (My), 26. M. E. Steiper, N. M. Young, Mol. Phylogenet. Evol. 41, 384 (2006). Euarchonta at 87.9 My, and Primatomorpha References and Notes 27. H. Nishihara, M. Hasegawa, N. Okada, Proc Nati. Acad. Sei 1. S. lavaré, C. R. Marshall, 0. Will, C. Soligo, R. D. Martin, at 86.2 My (see Fig. 2 and table S4 for 95% U.S.A. 103, 9929 (2006). Nature 416, 726 (2002). 28. W. ]. Murphy, T. H. Pringle, T. Crider, M. S. Springer, credibility intervals). Our divergence dates for 2. E. ]. Sargis, Science 298, 1564 (2002). W. Miller, Genome Res. 17, 413 (2007). Hominoidea/Cercopithicoidea (26.8 My), Anthro- 3. ]. I. Bloch, M. T. Silcox, D. M. Boyer, E. J. Sargis, 29. L. H. Emmons, Tupai: A Field Study of Bornean Tree Proc Nati. Acad. Sei. U.S.A. 104, 1159 (2007). poidea (41.7 My), LemurlMicrocebiis (40.4 My), Shrems (Univ. of California Press, Berkeley, CA, 2000). 4. M. Goodman, L. I. Grossman, D. E. Wildman, Trends Strepsirhini (62.1 My), and Primates (79.6 My) 30. This work was supported in part by NSF (grants EF0629849 Genet. 21, 511 (2005). to W.J.M. and EF0629860 to M.S.S.) and the National were very similar to those estimated from an in- 5. E. Pennisi, Science 316, 218 (2007). Institutes of Health (grant HG02238 to W.M.). We thank dependent 59.7-kb alignment of the CFTR gene 6. W. ]. Murphy et al., Science 294, 2348 (2001). A. Jambhekar, T. Crider, A. Wilkerson, V. David, K. Durkin, 7. P. J. Waddell, H. Kishino, R. Ota, Genome Inform. 12, region (2(5) (table S4). The rapid divergence D. Wilson, L. Grassman Jr., and A. Wilting for technical 141 (2001). across the basal euarchontan nodes explains why, advice and support and the Broad Institute/Massachusetts 8. ]. 0. Kriegs, G. Churakov, J. Jurka, J. Brosius, ]. Schmitz, despite the seven indels and high bootstrap and Institute of Technology, Baylor College of Medicine-Human Trends Genet. 23, 158 (2007). Genome Sequencing Center, and Washington University Bayesian support for Primatomorpha, we were 9. R. D. Martin, Primate Origins and Evolution (Princeton Genome Sequencing Center for access to unpublished not able to reject the Sundatheria hypothesis on Univ. Press, Princeton, N], 1990). sequence data. Sequences from this study have been 10. M. J. Novacek, Nature 356, 121 (1992). the basis of sequence data alone (Shimodaira- deposited in GenBank with accession numbers EU142140- 11. ]. Shoshani, M. C. McKenna, Mol. Phylogenet. Evol. 9, Hasegawa test, P = 0.065) {23). We did reject an EU142251 and EU213052-EU213059. 572 (1998). alliance of treeshrews and primates (P = 0.047), 12. K. C. Beard, in Mammal Phytogeny: Placentats, F. S. Szalay, Supporting Online Material despite the single discrepant indel supporting M. ]. Novacek, M. C. McKenna, Eds. (Springer, New York, www.sciencemag.org/cgi/content/full/318/5851/792/DCl primates + free shrews. This observation is sim- 1993), pp. 129-150. Materials and Methods ilar to other findings of incomplete lineage sorting 13. E. ]. Sargis, Evol. Anthropol. 13, 56 (2004). Figs. SI to S14 in the common ancestor of rapidly diversifying 14. F.-G. R. Liu et ai, Science 291, 1786 (2001). Tables SI to S5 15. M. S. Springer, M. J. Stanhope, 0. Madsen, W. W. de Jong, References eutherian clades {27, 28). Trends Ecol. Evol. 19, 430 (2004). The inclusion of nuclear gene sequences 16. R. M. Adkins, R. L. Honeycutt, Proc Nati. Acad Sd. U.S.A. 9 July 2007; accepted 1 October 2007 from ptilocercid freeshrews allowed us to date 88, 10317 (1991). 10.1126/science.ll47555 the origin of extant freeshrews (Scandentia) to -63.4 My (Fig. 2 and table S4), near the Cretaceous-Tertiary boundary, concomitant with divergence estimates of many eutherian orders and consistent with the long-frise model of eu- A Gene Regulatory Network therian diversification (25). This deep divergence between Ptilocercus and other scandentians Subcircuit Drives a Dynamic complements profound anatomical and behav- ioral distinctions that have been documented be- tween these groups (2, 13, 21, 29) and vindicates Pattern of Gene Expression recent classifications that have separated Ptilo- Joel Smith, Christina Theodoris, Eric H. Davidson* cercus in a unique family, Ptilocercidae {21, 22). As the sole living representative of a eutherian Early specification of endomesodermal territories in the sea urchin embryo depends on a moving torus lineage that diverged in the early Tertiary along of regulatory gene expression. We show how this dynamic patterning function is encoded in a gene with many modem mammalian orders, we sug- regulatory network (GRN) subcircuit that includes the otx, wnt8, and blimpl genes, the cis-regulatory gest that the phylogenetic uniqueness of Ptilo- control systems of which have all been experimentally defined. A cis-regulatory reconstruction cercus, combined with its restriction to lowland experiment revealed that blimpl autorepression accounts for progressive extinction of expression in the forest habitats within a relatively limited global center of the torus, whereas its outward expansion follows reception of the Wnt8 ligand by adjacent range, should render it an important conservation cells. GRN circuitry thus controls not only static spatial assignment in development but also dynamic priority in global context. regulatory patterning. Because our conclusions imply that colugos, rather than treeshrews, are the most appro- priate outgroup for Primates in studying the The genomic regulatory code that confrols work (GRN). The GRN states the interactions of evolution of adaptive traits, these results may the specification of the future skeleto- about 50 genes encoding franscription factors, as affect the placement of euarchontan fossils and genic, gut endoderm, and nonskeletogenic determined in an extensive perturbation analysis our understanding of primate genomic evolution mesodermal components of the sea urchin along with other data {1, 2). The subcircuits of {3-5). For example, a recent morphological anal- embryo is embodied in a gene regulatory net- this network confrol the establishment of fran-

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