Successive Radiations, Not Stasis, in the South American Primate Fauna

Home , Common squirrel monkey, Dolichocebus, Morotopithecus, Tremacebus

Successive radiations, not stasis, in the South American primate fauna

Jason A. Hodgsona, Kirstin N. Sternera, Luke J. Matthewsa, Andrew S. Burrella, Rachana A. Jania, Ryan L. Raaumb,c, Caro-Beth Stewartd, and Todd R. Disotella,1

aCenter for the Study of Human Origins, Department of Anthropology, New York University, New York, NY 10003; bDepartment of Anthropology, Lehman College, Bronx, NY 10468; cThe Graduate Center, City University of New York, New York, NY 10016; and dDepartment of Biological Sciences, State University of New York, Albany, NY 12222

Edited by Morris Goodman, Wayne State University, School of Medicine, Detroit, MI, and approved February 18, 2009 (received for review October 16, 2008) The earliest Neotropical primate fossils complete enough for tax- first appearance of their fossil relatives. Here we test the MSH onomic assessment, Dolichocebus, Tremacebus, and Chilecebus, with divergence date estimates and evolutionary rate compari- date to approximately 20 Ma. These have been interpreted as sons inferred from complete heavy-strand protein-coding mito- either closely related to extant forms or as extinct stem lineages. chondrial DNA sequences. The former hypothesis of morphological stasis requires most living Recent molecular studies (9–14) have confirmed monophyly platyrrhine genera to have diverged before 20 Ma. To test this of the 3 major divisions within the platyrrhines: the pitheciids, hypothesis, we collected new complete mitochondrial genomes consisting of the pitheciines and Callicebus; the atelids; and the from Aotus lemurinus, Saimiri sciureus, Saguinus oedipus, Ateles cebids, consisting of the callitrichines, Cebus, Saimiri, and Aotus belzebuth, and Callicebus donacophilus. We combined these with (Table 1). The relationships among these larger groups have published sequences from Cebus albifrons and other primates to proved difficult to resolve (9–14), although a recent study of rare infer the mitochondrial phylogeny. We found support for a cebid/ genomic insertion events supports a basal division of pitheciids atelid clade to the exclusion of the pitheciids. Then, using Bayesian and a sister relationship between atelids and cebids (12). The methods and well-supported fossil calibration constraints, we earliest fossil platyrrhines are known from localities in Argen- estimated that the platyrrhine most recent common ancestor tina, Chile, and Bolivia. Early primate-bearing sediments span

(MRCA) dates to 19.5 Ma, with all major lineages diverging by 14.3 the Oligocene–Miocene boundary from 26 to 20 Ma (15, 16). ANTHROPOLOGY Ma. Next, we estimated catarrhine divergence dates on the basis The oldest fossil taxa with diagnosable morphology, Dolichoce- of platyrrhine divergence scenarios and found that only a platyr- bus, Tremacebus, and Chilecebus, are found in early Miocene rhine MRCA less than 21 Ma is concordant with the catarrhine fossil layers (approximately 20 Ma). Proponents of the MSH consider record. Finally, we calculated that 33% more change in the rate of Dolichocebus a relative of Saimiri (17), Tremacebus of Aotus (5), evolution is required for platyrrhine divergences consistent with and Chilecebus of the cebines, Cebus and Saimiri (16). This the morphologic stasis hypothesis than for a more recent radiation. requires the 3 major crown platyrrhine lineages to have diverged We conclude that Dolichocebus, Tremacebus, and Chilecebus are by 20 Ma. The SRH considers these taxa to be stem platyrrhines likely too old to be crown platyrrhines, suggesting they were part outside the crown radiation (7, 8). Under the SRH the radiation of an extinct early radiation. We note that the crown platyrrhine of extant platyrrhines is more recent, and similarities between radiation was concomitant with the radiation of 2 South American modern taxa and the early Miocene taxa are convergent (7). xenarthran lineages and follows a global temperature peak and Several studies have estimated platyrrhine divergence dates from tectonic activity in the Andes. nucleotide sequences, with findings of the MRCA of living platyrrhines ranging from 20 to 26 Ma (10, 11, 18, 19). Those studies mitochondrial phylogeny ͉ molecular clock ͉ New World monkeys ͉ finding a MRCA of approximately 20 Ma would seem to falsify the primate evolution ͉ platyrrhine origins MSH, but the confidence intervals for these dates are large enough that an MRCA before the Miocene cannot be excluded (10, 18). By outh America existed as an island continent from 80 to 3.5 choosing mitochondrial genomes, we were able to increase resolu- SMa, resulting in a highly endemic mammalian fauna. Pri- tion by using long DNA sequences and multiple well-constrained mates are thought to have joined this fauna sometime between fossil calibration dates (3). In a comprehensive analysis using 50 and 30 Ma (reviewed in ref. 1) by rafting or island hopping multiple fossil calibration schemes, we developed an exploratory across the then-narrower Atlantic Ocean (2). The phylogenetic approach to estimating the evolutionary rates and divergence times relationships and divergence times of the Neotropical primates of the major platyrrhine lineages under both hypotheses. We (Platyrrhini) are not as well understood as those of their closest applied the Bayesian method of Thorne et al. (20, 21) as imple- relatives, the Old World monkeys and apes (Catarrhini) (e.g., mented with multidivtime from the MULTIDISTRIBUTE pack- refs. 3 and 4). It has been suggested that the macroevolutionary age. This method accommodates uncertainty in fossil calibration pattern among platyrrhines is markedly different from that of points, which avoids potentially misleading confidence intervals catarrhines, with platyrrhine evolution characterized by a single associated with fixed calibration points (22). In addition, rates of ancient radiation with long-lived and morphologically conser- evolution are allowed to vary between branches (i.e., no strict vative lineages, whereas catarrhines have experienced significant molecular clock is enforced), and variance and credibility intervals faunal turnover (5, 6). This ‘‘morphological stasis hypothesis’’ (MSH) is based on an interpretation of the platyrrhine fossil Author contributions: C.-B.S. and T.R.D. designed research; K.N.S., L.J.M., A.S.B., R.A.J., and record that places the known fossil taxa, including the earliest, R.L.R. performed research; J.A.H. analyzed data; J.A.H. and T.R.D. contributed new re- within the crown platyrrhine radiation. The ‘‘successive radia- agents/analytic tools; and J.A.H. wrote the paper. tions hypothesis’’ (SRH) suggests that the earliest fossil taxa are The authors declare no conﬂict of interest. outside the crown radiation and part of an earlier, extinct This article is a PNAS Direct Submission. radiation (7, 8), thus making platyrrhine and catarrhine evolu- Data deposition: The sequences reported in this paper have been deposited in the GenBank tion more similar. If the MSH is correct, then the most recent database (accession nos. FJ785421–FJ785426). common ancestor (MRCA) of crown platyrrhines must be 1To whom correspondence should be addressed at: Department of Anthropology, New York ancient, and the major lineages must have diverged before the University, 25 Waverly Place, New York, NY 10003. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810346106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 Table 1. Sequences used in this study Group Taxa Common name Accession no.

Haplorrhine Anthropoid Platyrrhine Cebid Aotus lemurinus* Gray-bellied night monkey FJ785421 Cebus albifrons White-fronted capuchin AJ309866 Saimiri sciureus* Common squirrel monkey FJ785425 Saguinus oedipus* Cottontop tamarin FJ785424 Atelid Ateles belzebuth* White-fronted spider monkey FJ785422 Pitheciid Callicebus donacophilus* White-eared titi monkey FJ785423 Catarrhine Cercopithecoid Chlorocebus sabaeus Green monkey EF597503 Papio hamadryas Hamadryas baboon NC࿝001992 Theropithecus gelada* Gelada FJ785426 Colobus guereza Mantled guereza AY863427 Trachypithecus obscurus Dusky leaf monkey NC࿝006900 Hominoid Homo sapiens Human EF061150 Pan troglodytes Common chimpanzee EU095335 Pongo pygmaeus Bornean orangutan NC࿝001646 Hylobates lar White-handed gibbon NC࿝002082 Tarsioid Tarsius bancanus Western tarsier NC࿝002811 Strepsirhine Lemurid Lemur catta Ring-tailed lemur AJ421451 Lorisid Nycticebus coucang Slow loris NC࿝002765

*Produced for this study.

of node times and branch rates are calculated. In total more than (12). It remains unclear whether the inferred mitochondrial phy- 665 multidivtime analyses were performed. logeny within the cebids is reflective of the true population history of divergence. This is not problematic for our purpose. Even if the Results and Discussion Phylogenetic Inference. We sequenced a complete mitochondrial genome from Aotus lemurinus, Saimiri sciureus, Saguinus oedipus, Ateles belzebuth, and Callicebus donacophilus and added them to a

mitochondrial genome of Cebus albifrons taken from GenBank, Saguinus Saimiri thus representing each of the major platyrrhine lineages. We also Papio

sequenced a mitochondrial genome of Theropithecus gelada. This Cebus Theropithecus Chlorocebus Colobus Trachypithecus

taxon was chosen because the fossil record of Theropithecus is well Callicebus

known, and therefore the timing of the Papio/Theropithecus split is Aotus Ateles Pongo

well constrained. To these sequences we added a selection of Homo Pan available mitochondrial genomes from GenBank chosen to provide 100 1.00 Hylobates nodes temporally constrained by a good fossil record, to fill out major branches of the haplorhine tree, or to root the tree (Table 1). 70 1.00 83 1.00 100 1.00 100 1.00 100 1.00 An alignment of the heavy-strand protein-coding genes (10,906 89 1.00 bp total) was used for phylogenetic inference using maximum 63 0.99 likelihood and Bayesian methods as implemented in PAUP* (23) 100 1.00 and MrBayes (24), respectively. In both cases the data were 100 1.00 analyzed with the GTR ϩ I ϩ G model of sequence evolution 100 1.00 (chosen by both Modeltest and MrModeltest). Both methods Tarsius recovered the same topology (Fig. 1). Although the Bayesian clade 100 1.00 credibility values are high across the tree, the maximum likelihood bootstrap values are low for all nodes within the platyrrhines. This 100 1.00 is not surprising given the very short branch lengths inferred for all of the platyrrhine internodes (see branch lengths below and Fig. 1). Two of our inferred relationships are of note. First, we infer a 100 1.00 cebid/atelid clade to the exclusion of the pitheciids. No sequence- Scale based study, including this one, has resolved the platyrrhine family substitutions per = 0.05 nucleotide site relationships convincingly (9–11, 13, 14). Our findings agree with the strong evidence provided by 5 shared Alu insertion events 93 1.00 between cebids and atelids (12). Second, within the cebids, Aotus was found to be sister to Saguinus. Previous sequence-based studies Fig. 1. Topology and branch lengths used for divergence time estimates. Phylogeny was inferred through maximum likelihood (PAUP*) and Bayesian have inferred every possible relationship between Aotus, the cal- (MrBayes) analysis with Lemur and Nycticebus as outgroups. Maximum likelihood litrichines, and the cebines with various levels of support (9–11, 13, bootstrap percentages are left of the branches, and Bayesian clade credibility 14). Our results differ from the Alu insertion data showing one proportions are right. Maximum likelihood branch lengths were estimated using shared event between Aotus and Saimiri to the exclusion of Saguinus the F84 ϩ G model of evolution (estbranches).

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810346106 Hodgson et al. Downloaded by guest on September 26, 2021 Table 2. Evolutionary rate calibration constraints Constraint Divergence Upper (Ma) Lower (Ma) Fossil (reference) Age (Ma)

1 Homo/Pan 5.0 8.0 Ardipithecus (28) 5.2 Orrorin (29) 6.0 Sahelanthropus (30, 31) 6.0–7.0 2 Homo/Pongo 12.5 18.0 Sivapithecus (32) Ϸ12.5 3 Papio/Theropithecus 3.5 6.5 Theropithecus (33) Ϸ3.5 4 Hominoid/cercopithecoid 21.0 30.0 Morotopithecus (34) Ͼ20.6 Victoriapithecus (35) Ϸ19.0 5 Platyrrhine/catarrhine 31.5 NA Fayum catarrhines (36, 37) 31.5 6 Aotus/Saguinus 12.5 NA Aotus dindensis (38) Ϸ12.5 7 Cebus/Saimiri 12.5 NA Neosaimiri (39) Ϸ12.5

mitochondrial history differs from the population history, it must be We set calibration constraints on 5 nodes within the catarrhines. the case that the populations diverged after the mitochondrial The only constraints used within the platyrrhines were that the lineages diverged. Cebus/Saimiri and the Aotus/Saguinus splits must have occurred by 12.5 Ma on the basis of the La Venta fauna (Table 2). The lack of Branch Lengths. Because of uncertainty in tree topology we have lower bounds within the platyrrhines fully allows for the data to used 4 alternate hypotheses for molecular dating: (i) the best support the MSH. However, the estimated divergence dates for the inferred topology (Fig. 1), (ii) pitheciids sister to atelids, (iii) Aotus, best topology (Fig. 2) are clearly inconsistent with the MSH. The Saguinus, Cebus/Saimiri trichotomy, and (iv) Aotus sister to Cebus/ MRCA of the living platyrrhines is estimated to have lived 19.5 Ma Saimiri. The latter were constructed by maintaining all relationships (95% credibility interval 16.8–23.4 Ma), and the Aotus/Saguinus shown in Fig. 1 except for those indicated. In all cases only Lemur and Cebus/Saimiri splits are estimated at 14.3 (95% credibility was used to root the trees. First, maximum likelihood parameters interval 12.6–17.4) and 14.3 (95% credibility interval 12.6–17.5) under the F84 model of evolution with 5 discrete ␥ categories were Ma, respectively. Although the credibility interval of the platyrrhine calculated using baseml from the PAML package (25) for all 4 MRCA encompasses the early Miocene fossils, the Cebus/Saimiri ANTHROPOLOGY topologies. These parameters were then used to estimate branch and Aotus/Saguinus splits are both far younger than the early lengths and their variance–covariance matrix using the estbranches Miocene fossils, suggesting that Dolichocebus and Tremacebus program from the MULTIDISTRIBUTE package. cannot be close relatives of Saimiri and Aotus, respectively, as The estimated branch lengths for the best topology are depicted proponents of the MSH purport (5, 17). The lower bound of in Fig. 1. Of note is the long branch leading from the anthropoid credibility for the split between the cebids is 19.3 Ma, also making MRCA to the platyrrhine MRCA. This branch is 64% longer than it unlikely that Chilecebus is a cebid (16). Overall, these divergence the branch leading to the catarrhine MRCA, suggesting that estimates suggest that if Dolichocebus, Tremacebus,orChilecebus platyrrhines either diversified much more recently or that the fall within extant diversity, they can only be pitheciids or stem ancestral rate of sequence evolution was faster leading to the members of the atelid/cebid clade. platyrrhine MRCA than to the catarrhine MRCA. The mean To test the effects of topology on the divergence dates, we branch from the anthropoid MRCA to tip amongst platyrrhines has performed the same analysis as above for each of the 3 alternative 0.64 substitutions per site, compared with 0.60 substitutions per site topologies. In no case did the lower bounds of the credibility amongst catarrhines, suggesting that platyrrhines have on average intervals accommodate the MSH. Making pitheciids and atelids evolved faster than catarrhines. However, it has long been recog- sister reduces the age estimate of the platyrrhine MRCA to 17.7 Ma nized that hominoids have evolved at a slower rate than cerco- (95% credibility interval 15.3–21.6), with the lower credibility pithecoids (3, 26, 27). The mean branch from the anthropoid bounds of the Aotus/Saguinus and Cebus/Saimiri splits being no MRCA to tip amongst hominoids has 0.54 substitutions per site, and older than 18.2 Ma. Moving Aotus within the cebids (Aotus, among cercopithecoids has 0.64 substitutions per site. Thus, platyr- Saguinus, Cebus/Saimiri trichotomy and Aotus sister to Cebus/ rhines have, on average, undergone an equivalent amount of Saimiri) results in ages slightly older than those for the best sequence evolution as have cercopithecoids, whereas hominoids topology; however, in no case are the lower-bounds of the cebid have experienced less. This suggests that it is indeed a ‘‘hominoid divergences old enough to accommodate Dolichocebus, Tremace- slowdown’’ and not a cercopithecoid speed-up. Also of note are the bus,orChilecebus within them. Although topology can be critical to extremely short internodes within platyrrhines. The 4 platyrrhine molecular date estimates, the extremely short platyrrhine inter- internodes represent the 4 shortest branches in the tree. This implies nodes mean that any reasonable rearrangement adds little length to that the diversification of the living platyrrhines must represent a the tree and consequently does not effect the dates greatly. In all rapid radiation. subsequent analyses we use the best topology (Fig. 1).

Divergence Dates with Best Available Constraints. We estimated the Concordance with Catarrhine Fossil Record. Next, we assessed con- times of divergence between all lineages using the best constraints cordance between divergence dates on the platyrrhine fossil record available for all nodes in the tree for all 4 topologies. Because the and the better-known catarrhine fossil record. Fossil estimates of test of the hypothesis depends on the Bayesian credibility intervals divergence times are concordant when the divergence times inde- estimated for the platyrrhine nodes, we used the largest reasonable pendently predict each other given the observed branch lengths ranges when assigning constraints to conservatively estimate cred- (40). This happens when the branch length per unit time between ibility intervals. Upper constraints (more recent) are logically fossils is proportional to the branch length per unit time between established by the first appearance of a derived lineage in the fossil each fossil and time 0 at the terminal branches. We first tested for record. Lower constraints cannot be definitively set, so we have concordance among the 4 catarrhine divergences used as con- chosen to place lower bounds at or beyond what most authorities strained calibration nodes in the previous analysis. To determine consider possible. When too little fossil information is available we the effects of the calibration constraints at each node on the others, have not set lower bounds. we varied constraints on each node individually while leaving all

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MRCA catarrhines

Ma) Ma detciderP secnegreviD ( Theropithecus Chlorocebus Papio Trachypithecus Tarsius Callicebus Pan Cebus Saimiri Colobus Homo Hylobates Ateles Pongo Aotus 50 Saguinus Pongo/Homo 0 Pan/Homo 40 F Theropithecus/ F J Papio J 30

G 20 10 H

A B 10 K

C K 2 A I 0 D 2 & 0 62-5 L - E 63 1 2 5 1 B - 13 20 62-52 1- 53 -0 6 earliest 12-02 imia ri 3 6 11-01 platyrrhine 1-51 1 } 1- 6-5 /sS 01 fossils M lpyta ACRMrr be u M enihs C Constraint Ranges (Ma) 30 Fig. 3. Surface plot of predicted catarrhine divergence times given various platyrrhine divergence scenarios. The ﬁgure summarizes the results of 540 multidivtime analyses. The constraints used for each analysis are given on the x and y axes, and the predicted catarrhine divergence times given these constraints are N shown on the z axis. Shaded regions indicate platyrrhine calibrations that predict 40 catarrhine divergences concordant with the catarrhine fossil record.

Node Age 95% BCI Because of the strong concordance between the 4 catarrhine nodes, we used these as tests to look for concordant platyrrhine A 14.3 12.6 - 17.4 50 B 14.3 12.6 - 17.5 divergence times. To find such times, we took a similar exploratory C 16.0 14.1 - 19.3 approach to that described above. However, here we varied 1-Myr D 18.0 15.7 - 21.6 constraint intervals on the Cebus/Saimiri split and the platyrrhine E 19.5 16.8 - 23.4 MRCA simultaneously. We varied the Cebus/Saimiri split between F 4.5 3.6 - 6.0 G 10.7 8.6 - 13.4 5 and 30 Ma and the platyrrhine MRCA between 10 and 40 Ma in 60 H 11.6 9.1 - 14.5 1-Myr increments. We ran an analysis with every possible combi- I 17.5 14.6 - 20.4 nation of constraints on these nodes. In total, 540 multidivtime J 6.5 5.2 - 7.9 analyses were performed (Fig. 3). This process was automated with K 15.2 12.9 - 17.6 O a Perl script written for this project. L 19.2 16.3 - 21.9 There were 47 platyrrhine constraint combinations that pre- 70 M 27.3 23.4 - 29.9 N 43.9 36.1 - 52.3 dicted divergence times for the Pan/Homo, Papio/Theropithecus, O 66.7 51.8 - 86.7 Pongo/Homo, and catarrhine MRCA within the conservative ranges inferred from the fossil record and described above (Fig. 3, Fig. 2. Divergence time estimates with Bayesian credibility intervals (BCI) using shaded regions). The oldest the Cebus/Saimiri split can be to predict the best available fossil constraints. Gray bars indicate constraint ranges for the the 4 test nodes within the defined ranges is between 18 and 19 Ma, nodes indicated on them. Arrows indicate that no lower constraint was used. whereas the oldest platyrrhine MRCA is between 20 and 21 Ma. Platyrrhine constraint times consistent with the MSH predict divergence times for the catarrhine nodes that are older than the other nodes unconstrained. We slid lower and upper constraints, fossil record suggests. For example, a Cebus/Saimiri split between separated by 1 million years (Myr), across a wide range of ages at 22 and 23 Ma and a platyrrhine MRCA between 27 and 28 Ma 1-Myr intervals. We varied the Papio/Theropithecus split between 0 predicts the Pan/Homo split at 10.1 Ma, the Papio/Theropithecus and 16 Ma, the Pan/Homo split between 1 and 20 Ma, the split at 6.6 Ma, the Pongo/Homo split at 22.8 Ma, and the catarrhine Pongo/Homo split between 6 and 30 Ma, and the MRCA of MRCA at 39.6 Ma. catarrhines between 10 and 40 Ma. In total 89 multidivtime analyses were performed, and the predicted times of divergence from the Rates of Evolution. Because platyrrhine divergence times consistent unconstrained nodes were recorded from each analysis. We found with the MSH are not concordant with the catarrhine fossil record, very strong agreement between the fossil estimates of divergence if the MSH is correct then rates of evolution must have changed times given the estimated branch lengths (data not shown). Only calibration constraints consistent with fossil estimates for a node through time. We characterized the amount of evolutionary rate change required by various platyrrhine divergence times in 2 ways. realistically predict the remaining 3 nodes. For example, a Pan/ ␯ Homo split between 5 and 6 Ma predicts the Papio/Theropithecus First, we tracked changes in the autocorrelation parameter split at 3.7 Ma, the Pongo/Homo split at 12.9 Ma, and the catarrhine calculated with multidivtime under different platyrrhine divergence MRCA at 23.1 Ma, whereas a Pan/Homo split between 9 and 10 Ma scenarios. This parameter characterizes the amount of rate varia- predicts the Papio/Theropithecus split at 6.2 Ma, the Pongo/Homo tion among adjacent branches, with ␯ ϭ 0 meaning no rate change split at 21.7 Ma, and the catarrhine MRCA at 38.3 Ma. Thus, the (e.g., strict molecular clock) and increasing rate variation as ␯ 4 independent fossil estimations of catarrhine divergence times do increases (20, 21). For this analysis, we applied the constraints used not require much evolutionary rate variation given the observed in the first analysis for the 5 nodes outside platyrrhines. Within the branch lengths. platyrrhines we chose to apply constraints to the MRCA of cebids

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810346106 Hodgson et al. Downloaded by guest on September 26, 2021 0.045 CI 45.6–62.6) and the divergence between tarsiers and anthropoids to 79.7 Ma (95% CI 63.8–106.2). These times are well beyond what

SRH MSH most authorities consider possible. To make the rates comparison 0.040 more realistic we reanalyzed the data by tightly constraining all nodes in the tree except for the platyrrhine nodes. To do so we constrained the nodes outside the platyrrhines to Ϯ1 Myr of the 0.035

ν ) best estimate of divergence times shown in Fig. 2. To these we added either the MSH or SRH platyrrhine constraints used in the previous

analysis. We found that given the SRH the mean rate change 0.030 between branches is 0.0027 substitutions per site per Myr, compared with 0.0036 substitutions per site per Myr given the MSH. Rate Change (

Thus, given the divergence times for the taxa outside the platyr- 0.025 rhines shown in Fig. 2, the MSH requires on average 33% greater evolutionary rate change between adjacent branches than does the SRH. This difference was found to be significant with a paired t test 0.020 (t ϭ 1.735, df ϭ 29, P ϭ 0.047, one-tailed). The reason for the greater amount of evolutionary rate change required by the MSH is clear from the inferred branch lengths. The 0.015 branch leading from the anthropoid MRCA to the platyrrhine MRCA is much longer than that leading to the catarrhine MRCA 5-6 (Fig. 1). However, the MSH requires the MRCA of platyrrhines be 30-31 10-11 20-21 25-26 35-36 15-16 Cebid MRCA Constraint Range (Ma) as old or older than the MRCA of catarrhines. Consequently, the MSH requires relatively fast evolution in the ancestral platyrrhine Fig. 4. Change in the rate of evolution between branches given various lineage. Because the root-to-tip branch lengths of the platyrrhines platyrrhine divergence scenarios. The ﬁgure gives the estimated autocorrelation ␯ are on average as long as those of cercopithecoids (Fig. 1), the MSH parameter ( ) for 35 multidivtime runs. Five constraint intervals were constant for then requires the rate of evolution to slow independently in many all runs (Table 2, constraints 1–5). The cebid MRCA was varied for each run, and the constraint intervals used are shown on the x axis. The dashed lines indicate the of the terminal platyrrhine lineages. This accounts for the greater La Venta fossils at 12.5 Ma and the disputed early Miocene fossils at 20 Ma and amount of evolutionary rate change seen in the MSH compared ANTHROPOLOGY demarcate the SRH and the MSH. with the SRH. Thus, accepting fossil interpretations consistent with the MSH requires accepting much greater disparity in evolutionary rates among branches (e.g., ref. 41). because Tremacebus, Dolichocebus, and Chilecebus are considered Our analyses suggest that the radiation of crown platyrrhines was to be cebids by the MSH. Using an approach similar to that used rapid and began approximately 20 Ma, long after primates reached in the concordance analysis, we slid lower and upper constraints South America. This suggests that the multiple fossil taxa present separated by 1 Myr between 5 and 40 Ma in 1-Myr intervals. before 20 Ma are outside of the crown radiation and are therefore Thirty-five multidivtime analyses were performed, and ␯ was re- part of an earlier radiation. The cause of faunal turnover in South corded for each (Fig. 4). It was found that a cebid MRCA between American primates is unclear. We note that amongst the South 15 and 16 Ma requires the least amount of evolutionary rate change American-endemic xenarthrans (armadillos, sloths, and anteaters), (␯ ϭ 0.0151). For cebid MRCA divergence times consistent with the both armadillos and sloths independently experienced radiations at SRH, ␯ ranges from 0.0151 to 0.0175, whereas for times consistent approximately 20 Ma (42). The primate and xenarthran radiations with the MSH, the minimum ␯ is 0.0185. in the early Miocene follow a peak in global temperature (43) and Finally, we estimated the average difference in evolutionary rates a major compressional episode in the Chilean Andes caused by an between adjacent branches under the MSH and the SRH. To do so acceleration in the rate at which the Nazca and South American we used multidivtime to estimate evolutionary rates along each plates met (44). It remains to be seen whether climatic or tectonic branch using the best estimates of divergence times consistent with changes played any role in the faunal turnover of South American each hypothesis respectively. We then calculated the average rate primates or the simultaneous radiations among xenarthrans. change as the mean absolute difference in rate between adjacent branches. To calculate rates under the SRH we used the 5 ca- Conclusions tarrhine constraints used in the previous analyses (Table 2) and Using a phylogeny representing all major anthropoid clades, we constrained the Cebus/Saimiri and Aotus/Saguinus divergences be- have demonstrated that the MRCA of platyrrhines likely lived tween 12.5 and 20 Ma. To calculate rates under the MSH we took too recently for the early Miocene South American primate a similar approach but constrained the Cebus/Saimiri and Aotus/ fauna to fall within extant diversity, indicating that Dolichocebus, Saguinus divergences older than 20 Ma. The average evolutionary Tremacebus, and Chilecebus are stem platyrrhines. This suggests rate change between adjacent branches given the SRH constraints that platyrrhine evolution is characterized by an extinct early is 0.0028 substitutions per site per Myr, whereas that for the MSH radiation followed by the radiation of crown platyrrhines. Al- is 0.0031 substitutions per site per Myr. Thus, the MSH requires on though platyrrhine evolution may involve morphological stasis average slightly greater change in evolutionary rates between from the time of La Venta approximately 12.5 Ma, this stasis is adjacent branches; however, a paired t test shows this difference not not likely to extend to the early Miocene. to be significant (t ϭ 0.815, df ϭ 29, P ϭ 0.211, one-tailed). In the previous analysis, given the SRH constraints, the estimated Materials and Methods divergence times are similar to those shown in Fig. 2, whereas the Mitochondrial Genome Sequencing. Total DNA was extracted from tissue, and divergence times given the MSH constraints are very different. To mitochondrial genomes were sequenced with established methods that ensure true mitochondrial sequence and not nuclear insertions (3, 45). Sequencing and minimize the amount or evolutionary rate change in the latter PCR primers are available upon request. experiment, multidivtime pushed the divergence times for the nodes outside the platyrrhines back in time to account for the necessarily Phylogenetic Inference. Heavy-strand protein-coding genes were extracted from older platyrrhine divergences. For example, the divergence be- complete genome sequences according to GenBank annotations. These genes tween platyrrhines and catarrhines is pushed back to 52.8 Ma (95% were individually aligned with ClustalW 2.0.8 (46), and these alignments were

Hodgson et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 concatenated. This process was automated with a Perl script written for this Root-to-tip mean was set at 75 Ma, with a 75 Ma standard deviation. The project. To infer the model of nucleotide change that best describes the data set, evolutionary rate at the root was set at 0.12 substitutions per nucleotide site per Modeltest 3.7 (47) and MrModeltest 2.2 were used and evaluated under the Myr, with a standard deviation of 0.12. This was calculated as the median Aikake Information Criterion. Model and parameters chosen by Modeltest 3.7 root-to-tip branch length divided by the root-to-tip mean prior. The autocorre- were used for maximum likelihood phylogenetic inference implemented in lation parameter prior (brownmean) and its standard deviation were set to 0.026, PAUP* 4.0b10 (23). Topologic support was estimated with 660 bootstrap repli- such that brownmean multiplied by the root-to-tip mean prior (75 Ma) equals Ϸ2. cates. The model chosen by MrModeltest 2.2 was used for Bayesian phylogenetic After a burn-in period of 100,000 generations, MCMC chains were sampled every inference as implemented by MrBayes 3.1.2 (24). For the Bayesian analysis, the 100 generations until 10,000 samples were taken. To test the effect of the priors data were partitioned by codon position, and model parameters were estimated on the posteriors, several runs were performed with various prior choices. The independently for each partition. Four Markov Chain Monte Carlo (MCMC) results were extremely robust to changes in priors. simulations were run for 10,000,000 generations sampled every 1,000 generations after a burn-in period of 1,000,000 generations. ACKNOWLEDGMENTS. We thank Alfred Rosenberger and Terry Harrison for helpful comments and suggestions; Heidi Reinholdt and Laura Gaydosh for help Molecular Dating. The various divergence date and evolutionary rate analyses with laboratory work; and Anthony Di Fiore, Cathi Lehn, and Derek Wildman for were performed as described above. Bayesian priors for multidivtime were cho- providing samples. This research was supported by National Institutes of Health sen according to J. Thorne’s recommendations in the multidivtime manual. Grants R01-GM060760 and R24-GM65580 (to C.-B.S. and T.R.D.).

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