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`23. Thompson,J. D., Higgins, G. D. & Gibson,T.J. CLUSTAL W:Improvingthesensitivity ofprogressive
`multiple sequencealignment through sequence weighting, position-specific gap penalties and weight
`matrix choice. Nucleic Acids Res. 22, 4673-4680 (1994).
`24. Swofford, D. L., Olsen, G.P., Waddell,P. J. & Hillis, D. M. in Molecular Systematics (eds Hillis, D. M.,
`Moritz, C. & Mable, B. K.) 407-492 (Sinauer, Sunderland, Massachusetts, 1996).
`25. Krajewski, C., Blacket, M., Buckley, L. & Westerman, M. A multigene assessment of phylogenetic
`relationships within the dasyurid marsupial subfamily Sminthopsinae. Mol. Phylogenet. Evol. 8, 236—
`248 (1997).
`26. Swofford, D. L. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods)Version 4,
`(Sinauer, Sunderland, Massachusetts, 1998).
`27. Rambaut, A. & Grassly, N. C. Seq-Gen: An application for the Monte Carlo simulation of DNA
`sequenceevolution along phylogenetic trees. Comput. Appl. Biosci. 13, 303-306 (1997).
`28. Rambaut, A. & Bromham,L.Estimating divergence dates from molecular sequences. Mol. Biol. Evol.
`15, 442-448 (1998).
`
`Supplementary informationis available on Nature’s World-Wide Website
`(http://www.nature.com) oras paper copy from the London editorial office of Nature.
`
`Acknowledgements
`Wethank F. Catzeflis for tissue samples. This work was supported by the NSF (M.S.S.) and
`the TMRprogram of the European Commission (W.W.d.J.; M.J.S.).
`
`Correspondence and requests for materials should be addressed to M.S.S.
`(e-mail: mark.springer@ucr.edu).
`
`acid repeatregion ofthe A2AB gene and a 21-bpregion of the BRCAIgenethatis repeated
`up to four times. The aligned data sets were the following lengths: A2AB (1,164 bp); IRBP
`(1,292 bp); vWF (1,251 bp); 12S rRNA/tRNAvaline-16S rRNA (2,001 bp); and BRCA1
`(2,947 bp). Alignmentsfor the concatenated (A2AB + IRBP + vWF + rRNA) and BRCA1
`datasets are available in the Supplementary Information. Phylogenetic analyses included
`unweighted andtransversion parsimony, minimum evolution (5,708-bp data set) or
`neighbour joining (BRCA1data set) with logdet and maximumlikelihood-GTR™
`distances, neighbour joining with weighted average (WAVE) maximum likelihood
`distances”, and maximumlikelihood under the HKY85(ref. 24) model of sequence
`evolution. Gaps were coded as missing in parsimonyanalyses. Maximum-likelihood
`estimates ofrelative rates and transition to transversion ratios were obtained from
`maximumparsimonytrees and used in subsequent maximum-likelihood analyses and in
`calculating weighted-average maximum-likelihood distances. Bootstrap support values
`are based on 500replications except for maximumlikelihood (100 replications).
`Maximum-likelihood bootstrap analyses with the BRCA1 data set used the following
`backboneconstraint, where taxon numbers correspondto the ordering of taxa (top to
`bottom)in Fig. 1B: (((1—4), (5, 6), (7, 8)), ((9, 10), 11-14), (15, 16), (17, 18), 19-21,
`((((22—24), 25), 26), 27), 28, (29, 30), 31-35, ((36, 37), (38, 39), (40, 41)), ((42, 43), 44),
`(45, 46), 47, ((48, 49), 50, 51)). Kishino and Hasegawatests™ were used to examinea priori
`hypotheses and to examinestatistically acceptable root locations.In the latter case, we
`obtained thebest unrootedlikelihood tree for each data set and then evaluated all possible
`root positions. All phylogenetic analysesandstatistical tests were performed with PAUP
`4.0b2 (ref. 26), except for neighbour-joining with weighted average distances, where
`analyses were performed with PHYLIP 3.572 (J. Felsenstein) and WAVEBOOT(D.King
`and C. Krajewski). Maximum-likelihood analyses with rate partitions allowed the
`following eight rate partitions with the 5,708-bpdataset: third positions of each nuclear
`gene; first + second positions of each nuclear gene; RNA stems; and RNAloops. Tworate
`partitions, correspondingtofirst + second andthird codonpositions,respectively, were
`used with the BRCA1data set. NJ-WAVEanalyses used a weighted-average distance
`approach”with the eight partitions indicated above for the 5,708-bp data set and two
`partitions (first + second codonpositions; third codonpositions) for the 2,947-bp data
`set; each partition was allowed its own rate, base composition, andtransition to
`transversion ratio. Monte Carlo simulations were performed with Seq-Gen 1.1 (ref. 27)
`(Supplementary Information). Molecular dates were estimated using QDATE*
`(Supplementary Information).
`Received 30 August; accepted 10 October 2000.
`1. Novacek, M.J. Mammalian phylogeny: shaking the tree. Nature 356, 121-125 (1992).
`2. Shoshani, J. & McKenna, M.C. Higher taxonomicrelationships among extant mammals based on
`morphology, with selected comparisonsofresults from molecular data. Mol. Phylogenet. Evol. 9, 572—
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`3. de Jong, W. W. Molecules remodel the mammaliantree. Trends Ecol. Evol. 13, 270-275 (1998).
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`endemic African insectivore mammals. Proc. Natl Acad. Sci. USA 95, 9967-9972 (1998).
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`mammalian mtDNA,including amino acid-invariantsites-logdetplussite stripping, to detect
`internal conflicts in the data, with special reference to the positions of hedgehog, armadillo and
`elephant. Syst. Biol. 48, 31-53 (1999).
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`implications from using the logdeterminanttransform for proteins ofdiffering amino acid
`composition. Syst. Biol. 48, 76—93 (1999).
`12. McKenna,M.C.& Bell, S. K. Classification ofMammals Abovethe Species Level (Columbia Univ. Press,
`New York, 1997).
`13. Rainger, R. Agenda for Antiquity: Henry Fairfield Osborn andVertebrate Paleontology at the American
`Museum ofNatural History, 1890-1935 (Univ. AlabamaPress, Tuscaloosa, Alabama, 1991).
`14. Kumar, S. & Hedges, S. B. A molecular timescale for vertebrate evolution. Nature 392, 917-920
`(1998).
`15. Foote, M., Hunter, J. P., Janis, C. M. & Sepkoski,J. J. Jr Evolutionary andpreservationalconstraints on
`originsof biologic groups: divergence timesof eutherian mammals. Science 283, 1310-1314 (1999).
`16. Rich, T. H.et al. A tribosphenic mammal from the Mesozoic of Australia. Science 278, 1438-1442
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`within Eutheria based on analysis of complete mitochondrial sequences. Mol. Biol. Evol. 17, 60-67
`(2000).
`18. Waddell, P. J., Okada, N. & Hasegawa, M. Towards resolvingthe interordinalrelationshipsofplacental
`mammals.Syst. Biol. 48, 1-5 (1999).
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`(Oxford Univ. Press, Oxford, 1989).
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`Hist. 9, 291-567 (1909).
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`mammals. Bull. Am. Mus. Nat. Hist. 183, 1-111 (1986).
`22. Easteal, S. Molecular evidenceforthe early divergence ofplacental mammals. BioEssays 21, 1052-1058
`(1999).
`MSNExhibit 1052 - Page 1 of 5
`6RISN v. Bausch - IPR2023-0001€ © 2001 Macmillan Magazines Ltd
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`Molecular phylogenetics and the
`origins of placental mammals
`
`William J. Murphy*+, Eduardo Eizirik*+{+, Warren E. Johnson*,
`Ya Ping Zhang§,Oliver A. Ryder|l & Stephen J. O’Brien*
`
`* Laboratory of Genomic Diversity, National CancerInstitute, Frederick,
`Maryland 21702, USA
`§ Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of
`Zoology, Chinese Academy of Sciences, Kunming, China
`ll Center for Reproduction ofEndangered Species, Zoological Society ofSan Diego,
`San Diego, California 92112, USA
`£ DepartmentofBiology, University ofMaryland, College Park, Maryland 20742,
`USA
`
`+ These authors contributed equally to this work
`
`Theprecise hierarchy of ancient divergence events that led to the
`present assemblage of modern placental mammals has been an
`area of controversy among morphologists, palaeontologists and
`molecular evolutionists. Here we address the potential weaknesses
`of limited character and taxon sampling in a comprehensive
`molecular phylogenetic analysis of 64 species sampled acrossall
`extant orders of placental mammals. We examined sequence
`variation in 18 homologous gene segments (including nearly
`10,000 base pairs) that were selected for maximal phylogenetic
`informativeness in resolving the hierarchy of early mammalian
`divergence. Phylogenetic analyses identify four primary super-
`ordinal clades:
`(I) Afrotheria (elephants, manatees, hyraxes,
`tenrecs, aardvark and elephant shrews); (II) Xenarthra (sloths,
`anteaters and armadillos); (III) Glires (rodents and lagomorphs),
`as a sister taxon to primates, flying lemurs and tree shrews; and
`(IV)
`the remaining orders of placental mammals (cetaceans,
`artiodactyls, perissodactyls, carnivores, pangolins, bats and core
`insectivores). Our results provide new insight into the pattern of
`the early placental mammal radiation.
`The panoply of morphological, ecological and genomic diversity
`among extant mammalsoffers considerable potential for studies of
`speciation, adaptation, molecular evolution, genome organization
`and biogeography'™. Studies on morphology and both mitochon-
`drial and nuclear genes have revealed several higher level phylo-
`genetic associations”, but a full resolution ofthe earliest placental
`
`NATURE| VOL409|1 FEBRUARY 2001|www.nature.com
`
`

`

`letters to nature
`
`their sister-grouprelationship with lagomorphs(cohort Glires””’), a
`divergences has yet to be accomplished. We obtained sequences
`groupingthat had been well established on morphological grounds”
`from segments of 15 nuclear and three mitochondrial genes (9,779
`but has been contradicted or unresolved with molecular
`basepairs (bp)) in 64 placental and two marsupial species (Table 1).
`approaches’*!>"®,
`Phylogenetic analyses of the concatenated data set using maximum
`The cohortGliresis, in turn,the sister group of primates, flying
`parsimony, maximum likelihood and distance based (neighbour
`joining) methodsall converged on a nearlyidentical, well supported
`lemurs and tree shrews. Within primates, lemur (Strepsirhini)
`andtarsier (Tarsiiformes) were found to besister taxa (bootstrap
`topology defining four principal eutherian lineages (Fig. 1). The
`results affirm monophyly of traditional placental orders (except
`= 80%) that were separated from anthropoids by a deep divergence.
`This contrasts with the widely held view that tarsiers are more
`Artiodactyla and Insectivora), and also support some previously
`closely related to anthropoids than to Strepsirhini’. Furthermore,
`proposed,as well as new, superordinal clades.
`Ouranalyses provide further support for the superordinal clade
`although maximum parsimony and maximumlikelihood analyses
`Afrotheria*®* (Fig. 1, clade I), and place it as the mostbasal placental
`consistently supported the monophyly of Primates andthesister-
`divergence. Within Afrotheria, our data support a consistently
`group relationship between Dermoptera and Scandentia, distance-
`strong clustering of Paenungulata’(Hyracoidea, Sirenia and Pro-
`based trees suggested primate paraphyly byplacing flying lemurs as
`boscidea) and a nested Tethytheria’’ (Sirenia and Proboscidea). The
`the sister group to anthropoids, and by placing tree shrews basal
`next basal mammalian lineage in our trees was the Neotropical
`among the three orders (Fig. 1). Kishino—Hasegawa (parsimony
`and likelihood) and Templetontests failed to reject either of these
`order Xenarthra (Fig. 1, clade II), whose monophyly wasstrongly
`supported in all analyses. Within Xenarthra, our data consistently
`hypotheses (P>0.1 in all cases), which indicates that further
`sampling within Dermoptera and Scandentia may be required to
`united sloths (Choloepus spp.) and anteaters (Myrmecophaga,
`Tamandua) in the proposed suborder Pilosa®, which was in turn
`fully resolve the deep, contemporaneous divergence among these
`three orders.
`the sister group to armadillos. Although all of our trees showed the
`Seven remaining placental orders—cetaceans,artiodactyls, peris-
`placental root
`to be either between Afrotheria and all other
`placentals
`(neighbour joining and maximum likelihood), or
`sodactyls, carnivores, pangolins, bats, and core insectivores (hedge-
`hogs, shrews and moles)—comprise a fourth superordinal lineage
`within Afrotheria (maximum parsimony; on the branches leading
`to elephant shrewsor the tenrec), we could notstatistically reject a
`(Fig. 1, clade IV), a groupingthat has received support from whole-
`mitochondrial genome sequence analysis (although no pangolin
`scenario with Afrotheria and Xenarthraassister groups, separated
`was sampled)’. Our results affirm the monophyly of Chiroptera
`from all other eutherians by a basal split (P= 0.07 in Kishino—
`Hasegawa (parsimony and likelihood) and Templeton tests).
`(megabats plus microbats), which has been a topic ofdebate for over
`a decade'*. These dataalso reject (P< 0.0001 forall tests) a direct
`Furthermore, we could notstatistically reject a scenario showing
`Xenarthra basal
`to all other placentals (P=0.50 in Kishino-
`relationship of bats with primates, flying lemurs and tree shrews,
`which wereclassically proposed to form the cohort Archonta". Our
`Hasegawa (parsimony andlikelihood) and Templetontests), sug-
`data support the polyphyly of Insectivora***, with shrews, moles
`gesting that additional data collection will be required to resolve the
`placental root with a highlevel of confidence.
`and hedgehogs forming a monophyletic group provisionally termed
`‘Eulipotyphla’”’, whereas tenrecs clearly cluster within the super-
`After these two basal divergences are two internal superordinal
`clades of eutherians(Fig. 1, clades III and IV ), which were found as
`ordinal clade Afrotheria. We find strong supportfor the traditional
`view of placing hedgehogsin a clade with shrews and moles (boot-
`sister groupsin all of our analyses. CladeIII represents a novel group
`strap = 99%), which is in contrast to the basal position within
`uniting rodents, lagomorphs, primates, tree shrews (Scandentia)
`eutherians observed with mitochondrial DNA (mtDNA) genomes”.
`and flying lemurs (Dermoptera). This is in contrast to molecular
`studies that show support for rodents being basal eutherians”""~”’,
`The inter-ordinal relationships within clade IV were not fully
`which waspossibly influenced by incomplete taxon sampling and
`resolved with high confidence, which is possibly due to the very
`rapid diversification of this group. However, our parsimony and
`extremerate accelerationin this group. Although bootstrap support
`maximum likelihood analyses agree with recent mtDNA data’,
`for this clade is not as high as for the other principal clades
`suggesting a sister-group relationship between Chiroptera and
`(neighbourjoining = 93%, maximumlikelihood = 85%, maximum
`Eulipotyphlaat the base of this principal clade (see Supplementary
`parsimony = 64%andweighted parsimony = 73%), it is consistently
`resolved in all of our analyses. Our increased taxon sampling
`Information). The pangolin (Pholidota) was the sister group to
`carnivores in combined (and manyofthe single-gene) analyses, a
`provides robust support for the monophyly of rodents, as well as
`
`Table 1 Characteristics of nuclear and mitochondrial gene segments
`Gene
`Nucleotides
`Seq.div.
`Ident. mouse
`CI-MP
`No. of species
`Amino acids
`range (%)
`vs human (%)
`amplified
`
`No.ofres.
`110
`278
`-
`230
`186
`-
`334
`-
`326
`-
`111
`258
`148
`142
`68
`-
`2,416
`
`No.of var. sites
`80
`97
`-
`167
`73
`-
`55
`-
`98
`-
`69
`66
`86
`83
`14
`-
`963
`
`No. ofPl
`62
`63
`-
`129
`38
`-
`24
`-
`62
`-
`62
`47
`54
`67
`5
`
`-
`655
`
`No.of Pl
`191
`313
`299
`375
`179
`78
`332
`227
`365
`249
`173
`303
`203
`224
`62
`731
`4,304
`
`0.31-37.08
`0.36-18.06
`0.32-31.16
`0-41.66
`0.54-27.52
`0-16.78
`0.41-27.25
`0-28.53
`0.10-19.26
`0.33-70.40
`0-42.55
`0.78-23.98
`0.45-23.36
`0-34.66
`0-22.99
`0-31.69
`
`78.5
`86.4
`90.5
`87.1
`90.7
`93.4
`89.6
`85.1
`88.5
`78.9
`82.5
`87.1
`89.2
`85.9
`93.6
`81.7
`
`0.34
`0.35
`0.51
`0.41
`0.36
`0.65
`0.30
`0.46
`0.35
`0.44
`0.43
`0.32
`0.38
`0.36
`0.41
`0.25
`
`63
`60
`63
`64
`63
`56
`62
`65
`58
`65
`56
`56
`62
`52
`57
`66
`
`NATURE] VOL409| 1 FEBRUARY 2001| www.nature.com
`
`No.of var. sites
`227
`392
`454
`468
`250
`175
`398
`302
`458
`290
`218
`353
`255
`258
`79
`929
`5,506
`
`No. of bp
`330
`ADORA3
`834
`ADRB2*
`690
`APP-3'UTR
`690
`ATP7A
`558
`BDNF
`340
`BMI1-3'’UTR
`1,002
`CNR1
`422
`CREM-3'UTR
`978
`EDG1
`337
`PLCB4-3'UTR
`333
`PNOC*
`774
`RAG1
`444
`RAG2*
`426
`TYR
`204
`ZFX
`1,417
`mtDNA
`9,779
`Total
`*Nooutgroup included in comparisons.
`bp, basepairs; var., variable sites; Pl, phylogenetically informative sites; res., amino-acid residues; Seq. div., percentage of nucleotide sequencedivergence (Kimura 2-parameter); Cl, consistency index of
`most parsimonioustree(s).
`MSNExhibit 1052 - Page 2 of 5
`Aé © 2001 Macmillan Magazines LWISN vy. Bausch - IPR2023-00016'5
`
`
`
`

`

`letters to nature
`
`fog
`99
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`66
`oo
`«3 00
`83
`67
`
`108
`100
`
`100
`Qt
`6
`
`100
`100
`
`.
`Cetartiodactyla
`
`
`
`Perissodactyla
`
`.
`Carnivora
`
`Pholidota
`
`Chiroptera
`
`‘Eulipotyphla’
`
`| Microchiroptera
`,
`Megachiroptera
`
`TOO
`108
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`a
`99
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`Equus *
`Felis *
`Leopardus
`p.
`Panthera
`Canis *
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`i
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`Nycteris .
`Pteropus
`Rousettus
`*
`Erinaceus *
`Sorex
`Asioscalops
`Condylura *
`‘
`Cavia *
`Hydrochaerus
`.
`i
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`Cavio-
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`90
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`
`Myocastor
`gnathi
`199
`98
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`88
`Hystrix
`< 50
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`Mus
`Rattus
`Cricetus
`Pedetes
`Castor
`i
`Dipodomys
`Tamias
`Muscardinus
`.
`*
`Sylvilagus
`Ochotona *
`400
`35
`Hylobates
`198 400
`a
`62
`Homo *
`109 400
`85
`<50
`
`
`87 4100 Anthropoidea‘|Primates100 Macaca *
`
`60 £25
`Ateles *
`100
`Callimico
`<50
`<0}
`og
`Cynocephalus */ **
`.
`*
`80
`99
`Lemur
`Lemuriformes
`Tarsius *
`Tarsiiformes
`Tupaia *
`400
`Choloepusdid. *
`490
`‘09
`199
`
`100 99|09 Choloepushof.
`
`400
`Tamandua
`100
`00
`Xenarthra
`100
`Myrmecophaga
`100
`Euphractus *
`Pp
`EM
`Chaetophractus
`a
`100 <50 frenviheria|8Trichechus* Sirenia a
`
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`Loxodonta *
`Proboscidea
`u
`r
`5 b0) 50
`Procavia *
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`gg
`9
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`Echinops *
`Tenrecidae
`<39
`i
`|
`400
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`Tubulidentata
`le
`100
`Macroscelides
`Macroscelidea
`r
`Elephantulus *
`ia
`Didelphis *
`| Marsupialia
`Macropus*
`are labelled by their generic designation, except where multiple members of a genus were
`included (see Methods). Brackets indicate higher level taxonomic groups observedin
`resulting trees (right of tree). A difference between MP/ML and NJ analyses was the
`position of the flying lemur (Cynocephalus, double asterisk). MP and MLdiffered from the
`showntree by supporting primate monophyly and a sister-group relationship between
`Dermoptera and Scandentia. Weighted parsimony analyses using only transversions(Tv),
`removalof third position transitions (Ts), and a Tv:Ts weight of 2:1 produced topologies
`congruentwith the showntree, with differences revolving only around branches depicted
`here with low (< 50%) bootstrap support.
`
`Dermoptera **
`Primates
`Scandentia
`
`100
`400 ,100
`72 100.
`Te
`
`71
`83
`
`99
`95
`
`65
`64
`79
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`50
`=50
`55
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`52
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`84
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`100
`100
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`<50
`<50
`79
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`G
`|
`|
`t
`s
`
`| | |
`
`.
`Rodentia
`
`Lagomorpha
`
`a
`t
`a
`
`Figure 1 Phylogenetic relationships among 64 placental mammals and two marsupials
`based on analysis of 9,779 bp from 15 nuclear and three mtDNA genes. Thetree
`represents the minimum evolution topology estimated through neighbourjoining (NJ),
`using maximum likelihood (ML) distances (see Methods). Maximum parsimony (MP) and
`MLanalyses (see Supplementary Information) produced a similar topology (MP:
`TL = 26,422, consistency index Cl = 0.34, retention index RI = 0.47; ML: Ln Likelihood =
`—95086.03) with differences revolving mostly around shortinternodeswith low bootstrap
`support. Numbersindicate per cent bootstrap support from NJ(top), MP (middle) and ML
`(bottom), based on 1,000iterations for NJ and MP, and 100iterations for ML. ML
`analyses were based on a pruneddata set (37 taxa marked with asterisks). Terminal taxa
`
`et MSNExhibit 1052 - Page 3 of 5
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`NATURE|VOL 409]
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`1 FEBRUARY2001 |www.nature.com
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`

`letters to nature
`
`Xenarthra: Choloepus didactylus (Linne’s two-toed sloth), Choloepus hoffmanni
`(Hoffmann’s two-toed sloth), Tamanduatetradactyla (tamandua), Myrmecophaga
`tridactyla (giant anteater), Euphractus sexcinctus (six-banded armadillo), Chaetophractus
`villosus (hairy armadillo); Order Insectivora: Erinaceus concolor (European hedgehog),
`Sorex araneus (European shrew), Asioscalops altaica (Old World mole), Condyluracristata
`(star-nose mole), Echinopstelfairi (tenrec); Order Macroscelidea: Elephantulus rufescens
`(long-eared elephant shrew), Macroscelides proboscideus (short-eared elephant shrew);
`Order Tubulidentata: Orycteropus afer (aardvark); Order Hyracoidea: Procavia capensis
`(rock hyrax); Order Sirenia: Trichechus manatus (West Indian manatee); Order
`Proboscidea: Loxodonta africana (African elephant); Order Rodentia: Tamiasstriatus
`(eastern chipmunk), Castor canadensis (American beaver), Muscardinus avellanarius
`(dormouse), Pedetes capensis (springhare), Mus musculus (mouse), Rattus norvegicus
`(brown rat), Cricetus griseus (hamster), Hystrix brachyurus (Malayan porcupine),
`Erethizon dorsatum (North American porcupine), Dipodomys heermanni (kangaroorat),
`Heterocephalus glaber (naked molerat), Cavia tschudii (guinea-pig), Hydrochaeris
`hydrochaeris (capybara), Myocastor coypus (nutria), Dinomys branickii (pacarana), Agouti
`taczanowskii (mountain paca); Order Lagomorpha: Ochotona hyperborea (pika),
`Sylvilagus cf. floridanus (eastern cottontail); Order Dermoptera: Cynocephalus variegatus
`(Malayanflying lemur); Order Scandentia: Tupaia minor(lesser tree shrew); Order
`Primates: Lemurcatta (ring-tailed lemur), Tarsius bancanus (Western tarsier), Macaca
`mulatta (rhesus macaque), Ateles fusciceps (brown-headed spider monkey), Callimico
`goeldi (Goeldi’s monkey), Hylobates concolor (gibbon), Homo sapiens (human); Order
`Chiroptera: Rousettus lanosus (long-haired rousette), Pteropus giganteus(flying fox),
`Nycteris thebaica (slit-faced bat), Artibeus jamacensis (Jamaicanfruit-eating bat); Order
`Carnivora: Canis familiaris (domestic dog), Ursus arctos (brownbear), Felis catus
`(domestic cat), Leopardus pardalis (ocelot), Panthera onca (jaguar); Order Perissodactyla:
`Ceratotherium simum (white rhinoceros), Equus caballus (horse), Tapirus indicus
`(Malayantapir); Order Artiodactyla: Lama glama(llama), Sus scrofa (pig), Hippopotamus
`amphibus (hippopotamus), Okapia johnstoni (okapi), Tragelaphus euryceros (bongo);
`Order Cetacea: Megaptera novaeangliae (humpback whale), Tursiops truncatus (bottlenose
`dolphin); Order Pholidota: Manis pentadactyla (Chinese pangolin). For outgroup
`comparison weincluded both Australian (Macropus eugenii, Tammar wallaby) and
`American (Didelphis virginianus, opossum) marsupial representatives.
`Wedesigned primersas part of an ongoingeffort to develop markers with uses in both
`mammalian comparative gene mapping and mammalian phylogenetics. The GenBank
`and UniGene databases (NCBI) were searched for genes with exonsofsufficient length
`(> 200 bp) andvariability (80-95%nucleotide identity between mouse and human),
`thereby providing adequate variation for the purpose of phylogenetic and somaticcell/
`radiation hybrid mapping. We designed primers in conserved stretches of the genes using
`Primer 3.0 (Whitehead Institute). Primer design avoided regions showing BLAST
`similarity to paralogues of the target gene. Primer pairs were selected by pre-screening
`each set in a panel of DNA from 11 individuals, including ten species from different
`eutherian Orders and one marsupial, using a touchdown PCRprotocol with Taq-Gold
`DNApolymerase (PE Biosystems), and either 1.5 or 2.0 mM MgCl). Only those markers
`that showed amplification ofa single, intense productof the predicted size in at least nine
`ofthe ten eutherian orders were chosenforthefinal study. We then chose successful primer
`pairs (see Supplementary Information) for amplification in a 96-well format (2 x 48
`species or 6X 16 species). We amplified remaining species in separate reactions with
`appropriate controls. Amplification products and Big Dye terminator (Applied
`Biosystems) sequencing reactions were purified using the Psiclone 96-well PCR purifi-
`cation system (Princeton Separations), and the 96-well Centri-sep system (Princeton
`Separations), respectively. Sequencingreactions were largely performed in a 96-well
`format using the ABI-3700 capillary sequencing apparatus, whereas smaller numbers of
`reactions were analysed using either an ABI-373 stretch or ABI-377 DNA sequencer.
`Ourinitial data set consisted of > 12,000 bp from 20 nuclear-coding loci. We removed
`five genes (1,672 bp) owingto inconsistent amplification in > 20% ofthe species,failure of
`key taxa, or amplification ofputative paralogousloci in somespecies. Thefinal set of genes
`comprised 10,704 bp of which 925 bp were deleted because of ambiguousalignment,
`resulting in the total data set of 9,779 bp. A summary of the genes used and their
`characteristics is given in Table 1.
`
`relationship that has also been suggested by previous morphological
`and molecular data®”’”’. Although our support for the Carnivora—
`Pholidota sister grouping fails statistical tests against alternative
`hypotheses (Kishino—Hasegawa (parsimony and likelihood) and
`Templeton tests; P=0.345), our data do firmly place Pholidota
`within cladeIV, andreject the view of an alliance between pangolins
`and the Neotropical Xenarthrans’ (Kishino—Hasegawa (parsimony
`and likelihood) and Templeton tests, P<0.0017). Our data also
`consistently
`indicated
`a
` sister-group
`relationship
`between
`Cetartiodactyla’ and Perissodactyla, albeit with only moderate
`bootstrap support (63-72%).
`We also performed phylogenetic analyses with the combined
`amino-acid sequences from the 11 coding nuclear genes, which
`provided medium to high support for the monophyly of most
`orders, as well as some superordinal relationships (for example,
`Afrotheria, Paenungulata, Glires, and a split between Afrotheria
`plus Xenarthraversusother placentals; see Supplementary Informa-
`tion). However, these data lacked sufficient power to resolve the
`otherbasal relationships identified with the nucleotide dataset. This
`is not unexpected, given that the amino-acid data set contains less
`than 20%of the numberof variable and parsimony-informative
`sites present in the nucleotide data set (Table 1).
`Notably, our data set shows consistency of amplification and
`phylogenetic signal associated with the four non-coding nuclear
`segments (APP, BMI1, CREM and PLCB4) all of which were derived
`from 3’ untranslated regions (3’ UTRs). Whereas the average
`percentage of variable sites was expectedly higher for the 3’ UTRs
`(68% of 1,789 bp, n = 4) versus coding regions (51% of 6,573 bp,
`n = 11), the average consistency index was also higher for the
`3’ UTRs(0.51) compared with the codingregions(0.33). Within the
`UTRs, we generally observed conserved blocks of sequence inter-
`rupted by variable (yet usually alignable) indels, which were often
`phylogenetically informative. It is probable that 3’ UTRswill be of
`importancein future phylogenetic studies of mammalian orders,as
`well as higher level phylogeny within other taxonomic groups.
`The molecular resolution of placental mammals into four super-
`ordinal clades has been independently corroborated by an accom-
`panying analysis”’. Estimatesfor the timing of molecular divergence
`indicate that the superordinal diversification occurred 64—104 Myr
`ago (median 84 Myr ago”), several millions ofyears prior to the K/T
`boundary which marked the disappearanceofthe dinosaurs”. The
`earliest divergences (Afrotheria and Xenarthra) apparently occurred
`in the Southern Hemisphere, probably associated with the geologi-
`cal separation of the southern supercontinent Gondwanaland,
`followed by dispersal of the ancestors of Clades III and IV into the
`northern supercontinent of Laurasia™“. The placement of several
`primitive insectivorous/generalist taxa near basal positions in each
`of the major clades (for example, Tenrecidae, Eulipotyphla and
`Scandentia; Fig. 1) is consistent with the early eutherian divergences
`preceding their remarkable morphological diversification. Geogra-
`phical isolation of these primitive forms in different regions and the
`appearance of ecological niches vacated by the dinosaurs may
`account for the remarkable parallel adaptive radiations suggested
`by Madsenetal.”
`From a genomics standpoint, these findings suggest that mouse
`and human,the twospecies at the forefront of genomic sequencing,
`are phylogenetically restricted to just one of the four principal
`placental lineages identified in our analyses. A broader knowledge
`of mammalian genomic organization, function and evolutionwill be
`gained by applying large-scale genomeanalysis to other species from
`each of these principal lineages, particularly Afrotheria and Xenar-
`thra. This will achieve a better understanding of the history of our
`
`mammalian ancestors and the uniqueness of our own genome.
`[1]
`
`Data alignment and phylogenetic analysis
`Phylogenetic analysis was performed on datasets aligned using the computer software
`CLUSTAL-X”*. We manually inspected and modified alignmentoutput. Regions of the
`alignments in which determination of homology was ambiguousweredeleted before
`phylogenetic analyses.In thefinal dataset, all taxa were representedbyatleast 11 of the 17
`gene segments (see Supplementary Information). Phylogenetic analyses were performed
`using maximum parsimony, distance-based (neighbourjoining), and maximumlike-
`lihood methods implemented in PAUP* 4.0b2 (ref. 27), PHYLIP 3.57, (DNAMLand
`Protdist, J. Felsenstein, Univ. of Washington) and PUZZLE”. PAML2.0 (Z.Yang,
`University College, London) was usedfor estimating the transition/transversion ratio
`(Ts/Tv = 2.0) and the a-parameter for rate variation amongsites (a = 0.45). Parsimony
`analyses were performed using 50 replicates with random addition oftaxa andtree-
`bisection reconnection branch swapping.
`Weperformed minimum evolution (neighbourjoining) analyses with several distance
`measures (Kimura 2-parameter, Logdet and maximumlikelihood distances with HKY85
`model) to examineeffects on topological stability (see Supplementary Information).
`Maximumlikelihood analyses for the nucleotide data set were performed with DNAML
`(assuming equal substitution rates amongsites) and with PAUP* (using an HKY-y model
`with estimated parameters); maximumlikelihoodtrees for the amino-acid data set were
`constructed using PUZZLE(y-corrected JTT model). Reliability of nodes was assessed
`using 1,000 bootstrap iterations for the nucleotide maximum parsimonyand neighbour-
`MSN Exhibit 1052 - Page 4 of 5
`ZA © 2001 Macmillan MagazinesLtd MSN Vv. Bausch _ IPR2023-00676
`
`Methods
`
`Taxon sampling, amplification and sequencing
`Ourdataset contains representatives from divergentlineagesofall eutherian orders. Order
`
`NATURE] VOL409| 1 FEBRUARY 2001| www.nature.com
`
`
`
`

`

`letters to nature
`
`FPyepr
`
`Horsetails and fems area
`monophyletic group and the
`closest living relatives to seed plants
`
`Kathleen M.Pryer*, Harald Schneider’, Alan R. Smith;,
`Raymond Cranfill{, Paul G. Wolf, Jeffrey S. Hunt* & Sedonia D. Sipes:
`
`* Department ofBotany, The Field Museum ofNatural History,
`1400 S. Lake Shore Drive, Chicago, Illinois 60605, USA
`+ University Herbarium, University of California, 1001 Valley Life Sciences
`Building 2465, Berkeley, California 94720, USA
`£ DepartmentofBiology, 5305 Old Main Hill, Utah State University, Logan,
`Utah 84322, USA
`
`joining trees and the amino-acid maximum parsimonyphylogenies, and 100 replicates for
`the nucleotide maximumlikelihood tree and the amino-acid distance-based analyses
`(Dayhoff PAM matrix) (see Supplementary Information