Complete Mtdna Genomes Shed Light on The

Molecular Phylogenetics and Evolution 82 (2015) 495–510

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Molecular Phylogenetics and Evolution

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The rise and fall of a genus: Complete mtDNA genomes shed light on the phylogenetic position of yellow-tailed woolly monkeys, Lagothrix ﬂavicauda, and on the evolutionary history of the family Atelidae (Primates: Platyrrhini) ⇑ Anthony Di Fiore a,b, , Paulo B. Chaves a,b,l, Fanny M. Cornejo c,d, Christopher A. Schmitt a,e,f, Sam Shanee g, Liliana Cortés-Ortiz h, Valéria Fagundes i, Christian Roos j, Víctor Pacheco k a Department of Anthropology, New York University, USA b Primate Molecular Ecology and Evolution Laboratory, Department of Anthropology, University of Texas at Austin, USA c Interdepartmental Doctoral Program in Anthropological Sciences, Stony Brook University, USA d Fundación Yunkawasi, Perú e Center for Neurobehavioral Genetics, University of California, Los Angeles, USA f Department of Anthropology, University of Southern California, USA g Neotropical Primate Conservation, UK h Department of Ecology and Evolutionary Biology, University of Michigan, USA i Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Brazil j Gene Bank of Primates and Primate Genetics Laboratory, German Primate Center, Göttingen, Germany k Museo de Historia Natural, Departamento de Mastozoologia, Universidad Nacional Mayor de San Marcos, Perú l New York Consortium in Evolutionary Primatology (NYCEP), USA article info abstract

Article history: Using complete mitochondrial genome sequences, we provide the first molecular analysis of the phylo- Available online 19 April 2014 genetic position of the yellow-tailed woolly monkey, Lagothrix flavicauda (a.k.a. Oreonax flavicauda), a critically endangered neotropical primate endemic to northern Perú. The taxonomic status and phyloge- Keywords: netic position of yellow-tailed woolly monkeys have been debated for many years, but in this study both New World monkeys Bayesian and maximum likelihood phylogenetic reconstructions unequivocally support a monophyletic Oreonax woolly monkey clade that includes L. flavicauda as the basal taxon within the radiation. Bayesian dating Yellow-tailed woolly monkey analyses using several alternative calibrations suggest that the divergence of yellow-tailed woolly mon- Mitogenomics keys from other Lagothrix occurred in the Pleistocene, 2.1 Ma, roughly 6.5 my after the divergence of Dating analysis Phylogenetics woolly monkeys from their sister genus, Brachyteles. Additionally, comparative analysis of the cytochrome oxidase subunit 2 (COX2) gene shows that genetic distances between yellow-tailed woolly monkeys and other Lagothrix from across the genus’ geographic distribution fall well within the range of between-species divergences seen in a large number of other platyrrhine primate genera at the same locus and outside the range of between-genus divergences. Our results thus confirm a position within Lagothrix for the yellow-tailed woolly monkey and strongly suggest that the name Oreonax be formally considered a synonym for this genus. This revision in taxonomic status does not change the dire conservation threats facing the yellow-tailed woolly monkey in Perú, where the remaining wild population is estimated at only 10,000 individuals living in a highly fragmented landscape. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction woolly monkeys distributed throughout the central and western Amazonian lowlands and in montane and submontane regions of The yellow-tailed woolly monkey, Lagothrix (Oreonax) the eastern cordillera of the Andes (Fig. 1). Endemic to northern flavicauda, is one of five currently recognized morphotypes of Perú, the yellow-tailed woolly monkey has long been considered critically endangered (Cornejo et al., 2008; Mittermeier et al., 2009), and its phylogenetic position and taxonomic status within ⇑ Corresponding author. Address: Primate Molecular Ecology and Evolution Laboratory, Department of Anthropology, University of Texas at Austin, 2201 the Atelidae (the neotropical primate family that includes all of Speedway Stop C3200, Austin, TX 78712, USA. the large-bodied, prehensile-tailed platyrrhines) have been contro- E-mail address: anthony.difi[email protected] (A. Di Fiore). versial. While an early comprehensive review (Fooden, 1963) http://dx.doi.org/10.1016/j.ympev.2014.03.028 1055-7903/Ó 2014 Elsevier Inc. All rights reserved. 496 A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510

Fig. 1. Geographic distribution of the five currently recognized morphotypes of woolly monkeys. considered the yellow-tailed woolly monkey as one of two allopat- Bayesian estimation of lineage divergence times. Additionally, we ric species of Lagothrix, a cladistic analysis of craniodental charac- mined NCBI GenBank for mitochondrial cytochrome oxidase sub- ters (Groves, 2001) elevated the taxon to its own monotypic genus, unit II (COX2) gene sequences from all platyrrhine primate genera under the resurrected name Oreonax, and considered it a sister where more than one named species within the genus was repre- taxon to spider monkeys (Ateles) rather than Lagothrix. A more sented. We then used these sequence data to characterize the pair- recent reanalysis, however, argued that the craniodental evidence wise genetic diversity found among species within the same genus is insufficient to warrant assigning this primate to a distinct genus as well as between genera for comparison to the pairwise diversity (Matthews and Rosenberger, 2008; Rosenberger and Matthews, between sequences from the different putative species of woolly 2008), and other morphological studies have confirmed a closer monkeys at the same locus. relationship between L. flavicauda and other woolly monkeys instead of Ateles (e.g., Paredes Esquivel, 2003). To address this thorny taxonomic question as well as other 2. Material and methods unresolved issues concerning atelid evolutionary history, we undertook a new molecular phylogenetic study of the radiation 2.1. Taxon samples and DNA extraction using a large, comparative, mitogenomic dataset. While several previous molecular studies have attempted to resolve relationships Tissue, feces, or extracted DNA were obtained from a variety of within the Atelidae (Canavez et al., 1999; Collins, 2004; Meireles sources and collaborators for each of the five putative forms of et al., 1999; Opazo et al., 2006), none has included all of the puta- woolly monkeys, plus red howler monkeys (Alouatta seniculus) tive genera in the radiation and none has employed a sequence and northern muriquis (Brachyteles hypoxanthus)(Table 1). We dataset of the size we use here. Our study thus had three main extracted DNA from tissue samples using the QIAamp DNA Mini goals: (1) to reconstruct the phylogenetic relationships among Kit (Qiagen, Inc.) following the protocol for DNA Purification from the atelid primates using a large mtDNA dataset encompassing Tissues. The final steps of the protocol were modified slightly in all putative genera, (2) to estimate the timing of major divergences that we heated Buffer AE to 70 °C before applying it to the QIAamp within the atelid clade, and (3) to evaluate whether or not the yel- spin column, and we incubated the column at room temperature low-tailed woolly monkey should be placed in a separate genus for 20–25 min before centrifuging to collect eluted DNA. For etha- from Lagothrix. nol-preserved tissue samples, DNA was eluted from the spin col- To address these objectives, we sequenced the complete mtDNA umn twice using 100 lL of Buffer AE for each eluate, both of genomes of three of the five putative species of woolly monkeys – which were then used as templates for subsequent PCR. For the Lagothrix (Oreonax) flavicauda, Lagothrix poeppigii, and L. lugens – sample of dried tissue from the Field Museum of Natural History plus two other atelid taxa, Alouatta seniculus and Brachyteles collection, we eluted twice with 50 lL of Buffer AE and then com- hypoxanthus. We also sequenced roughly 8 kb of the mtDNA gen- bined eluates. For fecal samples, we extracted DNA from 200 lL omes of the remaining two currently recognized woolly monkey of slurry containing a 1:1 ratio of feces and RNA Later™ nucleic morphotypes, L. cana and L. lagotricha. These were aligned with acid preservation buffer using the QIAamp DNA Stool Mini Kit (Qia- existing mitogenomic data for a large and taxonomically diverse gen, Inc.). Fecal extractions followed the protocol for Isolation of set of primates (Chiou et al., 2011; Finstermeier et al., 2013; DNA from Stool for Human DNA Analysis with the following modifi- Hodgson et al., 2009) and then used for both maximum likelihood cations: (1) after addition of Buffer ASL, we incubated samples on a and Bayesian phylogenetic inference analysis as well as for rocking platform at 56 °C for 30–60 min, (2) following the addition A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510 497

Table 1 Samples newly sequenced for this study.

Code Taxon Provenience Sample type Geographic Locality Approximate Sample origin coordinates contributorw ASE Alouatta seniculus Wild Ethanol Preserved Ecuador Yasuní Biosphere Reserve, Orellana 76.39 W AD Tissue Province 0.68 S BHY Brachyteles Captive DNA extract Brazil Southeastern Brazil Not Available VF hypoxanthus LFL Lagothrix (Oreonax) Wild DNA extract Perú Utcubamba Province, Amazonas Region 78.29 W FMC, LCO, VP ﬂavicauda 5.62 S LPO Lagothrix poeppigii Wild Tissue Ecuador Yasuní Biosphere Reserve, Orellana 76.15 W AD Province 0.64 S LLU Lagothrix lugens Wild Feces Colombia Cueva de los Guacharos National Park, 76.11 W AL, PS Huila Department 1.60 S LLA Lagothrix lagotricha Wild Feces Colombia Caparú, Vaupés Department 69.51 W AL, PS 1.08 S LCA Lagothrix cana Wild Dried Tissue (FMNH Perú Paucartambo Province, Cusco Region 71.59 W BP 170290) 13.10 S wAD = A. Di Fiore, VF = V. Fagundes, FMC = F.M. Cornejo, LCO = L. Cortes-Ortiz, VP = V. Pacheco, AL = A. Link, PS = P. Stevenson, BP = B. Patterson.

of lysis Buffer AL, we incubated samples at 70 °C for 30 min, vor- mtDNA strands in overlapping fragments of 300–600 bp that texing each sample periodically during the incubation period, (3) were assembled into complete mtDNA genome contigs. For direct we heated Buffer AE to 70 °C and allowed it to sit on the spin col- sequencing, we used a large suite of primers that included both umn at room temperature for 20–25 min before elution, and (4) we ones previously designed for sequencing of other platyrrhine eluted one time with 100 lL of Buffer AE rather than 200 lL. mtDNA genomes (Chiou et al., 2011; Hodgson et al., 2009) as well as newly designed primers based on the Ateles belzebuth mitochondrial genome or on other Lagothrix genome sequences generated 2.2. Mitochondrial genome sequencing during this project. Cycle sequencing products were separated via capillary electrophoresis on an ABI 3730 DNA Analyzer system Beginning with published mtDNA genome sequences of other (Applied Biosystems, Inc.) and bases were called using Sequencing New World primates, including one species of atelid (the white- Analysis v5.2 software (Applied Biosystems, Inc.). Base calls were bellied spider monkey, Ateles belzebuth), we designed PCR primers checked by eye, and sequence traces were assembled into contigs to amplify the genomes of the other atelids in two long, overlap- using the software Sequencher v4.7 (Gene Codes Corp.). ping fragments of 9–12 kb (Table 2). These long range PCR ampli- It is important to note that our amplification strategy of initially fications were carried out using two commercially available kits targeting long stretches of mtDNA reduces the chance of inadver- (Toyobo KOD Xtreme™ Hot Start DNA Polymerase kit and Roche tently amplifying nuclear copies of mitochondrial pseudogenes or Expand Long Template PCR System). For the first kit, the reaction ‘numts’ and also allows us to verify that the final contig represents mix consisted of 12.5 lL of 2X Xtreme buffer, 2.0 mM each dNTP, a circular DNA molecule and thus true mtDNA sequence 0.4 lM each of the forward and reverse long range primer, 0.5 (Bensasson et al., 2001; Thalmann et al., 2004). For two woolly units of KOD polymerase, and 2.0 lL of unquantified template monkey taxa (Lagothrix cana and L. lagotricha), we were unable to DNA in a total volume of 25 lL, and the cycling conditions were begin our sequencing from long range templates due to the as follows: 98 °C for 2 min, followed by 40 cycles of denaturing degraded nature of the DNA source material from fecal and at 98 °C for 10 s, primer annealing at 60 °C for 30 s, and extension museum skin samples. Thus, for these two samples, we amplified at 68 °C for 9–11 min, with a final extension at 68 °C for an addi- overlapping shorter fragments (300–600 bp) directly from our tional 9–11 min. For the second kit, the reaction mix comprised initial DNA extractions. As an additional check against the possible 2.5 lL of 10x PCR buffer, 500 lM each dNTP, 0.4 lM forward and inclusion of contaminating ‘numt’ sequences in our dataset, we reverse primer, 0.08 units of Roche polymerase, and 2.0 lLof examined all sequences – both those generated from long range unquantified template DNA in a total reaction volume of 25 lL. amplifications and those generated through targeted sequencing PCR cycling conditions for this mix involved initial denaturation of shorter fragments – for irregularities such as frameshift muta- at 93 °C for 3 min followed by 10 cycles of denaturing at 93 °C tions and premature stop codons falling within coding regions. for 15 s, annealing at 60 °C for 30 s, and extension at 68 °C for 11.5 min. This was followed by an additional 27 cycles of denatur- 2.3. Sequence alignment ation at 93 °C for 15 s, annealing at 55 °C for 30 s, and extension at 68 °C for 11.5 min initially plus an additional 20 s for each cycle Following contig assembly, we aligned our five new complete and a final extension at 68 °C for an additional 11.5 min. atelid genomes and two partial atelid genomes with 41 additional We used the products of these long range amplifications as tem- complete mitochondrial genomes from 28 other primate genera plates for standard cycle-sequencing using the BigDye Terminator available in GenBank, including all mtDNA genomes currently v3.1 chemistry (Applied Biosystems, Inc.) of both heavy and light available for atelids (11 platyrrhines, 11 catarrhines, 4 strepsirrhines, 2 tarsioids) (Table 3). The 12 protein-coding genes located Table 2 Long range amplification primers used for this study. on the mtDNA heavy strand make up the bulk of the mitochondrial genome and are suggested to have similar evolutionary properties Name Sequence Amplicon size (Gissi et al., 2000); thus, for phylogenetic analysis we reduced PlatyF3 50-CAAAAATATTGGTGCAACTCCAAATAAAAG-30 9500 bp our dataset to a concatenated alignment of these 12 gene 0 0 PlatyR3 5 -GAAGGCTCTTGGTCTTATTAACCTAAATTTCT-3 sequences. This resulted in a master alignment of 10,839 bp for a PlatyF4 50-TTCAACGATTAAAGTCTTACGTGATCTGAG-30 11,650 bp PlatyR4 50-ATGGTTTTTCATATCATTAGTCATGGTT-30 total of 33 primate genera, including all five putative atelid genera (Alouatta, Ateles, Brachyteles, Lagothrix, and ‘‘Oreonax’’) as well as Table 3 498 Taxa included in the mtDNA coding sequence alignment.

Same sequence used in... Suborder Infraorder Parvorder Superfamily Family Subfamily Taxon GenBank Ref Seq # Common name Original Finstermeier Schrago Perez Chiou Hodgson accession reference et al. (2013) et al. et al. et al. et al. number (2012) (2013) (2011) (2009) Haplorhini Simiiformes Catarrhini Cercopithecoidea Cercopithecidae Cercopithecinae Chlorocebus AY863426 NC_007009 Grivet Raaum et al. XXXX aethiops (2005) Macaca mulatta AY612638 NC_005943 Rhesus monkey Gokey et al. XXXXX (2004) Papio Y18001 NC_001992 Hamadryas Arnason XXXXX hamadryas baboon et al. (1998) Theropithecus FJ85426 NC_019802 Gelada Hodgson XXXX gelada et al. (2009) Colobinae Colobus guereza AY863427 NC_006901 Guereza Raaum et al. XXXXX (2005) 495–510 (2015) 82 Evolution and Phylogenetics Molecular / al. et Fiore Di A. Trachypithecus AY863425 NC_006900 Dusky leaf Raaum et al. XX XX obscurus monkey (2005) Hominoidea Homindae Homininae Gorilla gorilla D38114 NC_001645 Gorilla Horai et al. XXXX (1995) Homo sapiens EF061150 NA Human Friedlaender XX et al. (2007) Pan troglodytes EU095335 NA Chimpanzee Flynn et al. XX (2007) Ponginae Pongo abelii X97707 NC_002083 Sumatran Xu and XX XX orangutan Arnason (1996) Hylobatidae Hylobatinae Hylobates lar X99256 NC_002082 Lar or white- Arnason XXXXX handed gibbon et al.(1996) Platyrrhini Ceboidea Atelidae Atelinae Alouatta caraya KC757384 NA South American Finstermeier X black howler et al. (2013) Redmonkey howler This study w Alouatta monkey Atelesseniculus belzebuth KC757386 NA White-bellied Finstermeier X spider monkey et al. (2013) Ateles belzebuth FJ785422 NC_019800 White-bellied Hodgson XXXX spider monkey et al. (2009) Brachyteles JX262672 NA Southen muriqui Schrago et al. X arachnoides (2012) Brachyteles Northern murIqui This study w hypoxanthus Lagothrix Peruvian yellow- This study (Oreonax) tailed woolly w flavicauda monkey w Lagothrix cana Geoffroy’s woolly This study monkey Lagothrix Humboldt’s or This study w lagotricha common woolly monkey Lagothrix Colombian woolly This study w lugens monkey Lagothrix Poeppig’s or red This study w poeppigii woolly monkey Lagothrix sp. KC757398 NC_021951 Finstermeier X et al. (2013) Cebidae Aotinae Aotus azarai KC757385 NC_021939 Azara’s night or Finstermeier X owl monkey et al. (2013) Aotus FJ785421 NC_019799 Colombian night Hodgson XXXXX lemurinus or owl monkey et al. (2009) Calltrichinae Callithrix KC757388 NC_021941 Geoffroy’s tufted- Finstermeier X geoffroyi ear marmoset et al. (2013) Callithrix AB572419 NA Common Sato et al. X jacchus marmoset (2010) Cebuella KC757389 NC_021942 Pygmy marmoset Finstermeier X (Callithrix) et al. (2013) pygmaea Leontopithecus KC757399 NC_021952 Golden lion Finstermeier X rosalia tamarin et al. (2013) Saguinus FJ785424 NA Cotton-top Hodgson XXXX oedipus tamarin et al. (2009) Saguinus KC757409 NC_021960 Cotton-top Finstermeier X oedipus tamarin et al. (2013) Cebinae Saimiri KC959987 NC_021966 Bolivian squirrel Finstermeier X boliviensis monkey et al. (2013) Saimiri HQ644339 NC_018096 Bolivian squirrel Chiou et al. XX 495–510 (2015) 82 Evolution and Phylogenetics Molecular / al. et Fiore Di A. boliviensis monkey (2011) boliviensis Saimiri HQ644336 NA Central American Chiou et al. XX oerstedii squirrel monkey (2011) citronellus Saimiri sciureusFJ785425 NA Common squirrel Hodgson XXXX sciureus monkey et al. (2009) Cebus albifronsAJ309866 NC_002763 White-fronted Arnason XXXXX capuchin et al. (2000) Sapajus apella JN380205 NC_016666 Tufted or brown Bi et al. XX capuchin (2011) Sapajus KC757410 NC_021961 Yellow-breasted Finstermeier X xanthosternos capuchin et al. (2013) Pitheciidae Callicebinae Callicebus KC959986 NC_021965 Red titi Finstermeier X cupreus et al. (2013) Callicebus FJ785423 NC_019801 Reed titi Hodgson XXXXX donacophilus et al. (2009) Pitheciinae Cacajao calvus KC959985 NC_021967 Bald-headed Finstermeier X uacari et al. (2013) Chiropotes KC757393 NC_021946 White-nosed Finstermeier X albinasus bearded saki et al. (2013) Tarsiformes Tarsioidea Tarsiidae Tarsinae Carlito (Tarsius) AB371090 NC_012774 Phillippine tarsier Matsui et al. XX syrichta (2009) Cephalopachus AF348159 NC_002811 Western tarsier Schmitz et al. XXXXX (Tarsius) (2002) bancanus Strepsirhini Lemuriformes Lemuroidea Lemuridae Lemurinae Eulemur rufus KC757395 NC_021948 Red lemur Finstermeier X et al. (2013) Lemur catta AJ421451 NC_004025 Ring-tailed lemur Arnason XX XX et al. (2002) Lorisiformes Lorisoidea Galagidae Galaginae Galago AB371092 NC_012761 Northern lesser Matsui et al. XXX senegalensis galago (2009) Lorisidae Lorisinae Nycticebus AJ309867 NC_002765 Greater slow loris Arnason X coucang et al. (2000) w Indicates a taxon newly sequenced for this study. Taxonomy, scientific, and common names follow Mitani et al. (2013) . 499 500 A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510 representatives of all currently recognized morphotypes of woolly Yule pure birth process, as appropriate with mainly single monkeys – Lagothrix poeppigii, L. lugens, L. cana, L. lagotricha, and sequences per species. Lagothrix (Oreonax) flavicauda. We ran several divergence time analyses, each using a different set of date estimates to set Bayesian priors for calibrating the 2.4. Phylogenetic inference molecular clock (Table 4). For our first analysis (BEAST1), we employed the same fossil constraints used by Chiou et al. (2011) Phylogenetic trees were reconstructed using both maximum in a recent analysis of the phylogenetic history of squirrel mon- likelihood and Bayesian inference approaches, as implemented keys, and we followed the same method for setting priors for these using the softwares RAxML-HPC2 (version 7.6.6) (Stamatakis, divergences (Table 4). Thus, four of the calibration points were 2006; Stamatakis et al., 2008) and Mr. Bayes (version 3.2.1) implemented by setting priors as lognormal distributions with an (Ronquist and Huelsenbeck, 2003) run on the CIPRES Science Gate- offset, mean, and standard deviation such that 95% of the prior dis- way (Miller et al., 2010). To select the best-fitting model of tribution fell within the boundaries indicated. For an additional sequence evolution for both our likelihood and Bayesian analyses, two calibration points – the divergence of the aotines and the we first used the software jModelTest version 2.1.4 (Darriba et al., cebines and the divergence of Saimiri and Cebus, where the fossils 2012; Guindon and Gascuel, 2003), which identified the general- Aotus dindensis and Neosaimiri with clear affinities to modern Aotus ized time reversible model with gamma-distributed rate variation and Saimiri, respectively, are argued to reflect a hard upper bound and a proportion of invariant sites (GTR + G + I) as the best model for these dates of divergence – we used an exponential distribution under all alternative model selection criteria (i.e., Akaike informa- for the prior with a predefined offset of 12.1 Ma. We initially used tion criterion, Bayesian information criterion, and decision only these six fossil calibrations and allowed the root height of the theoretic frameworks). For both phylogenetic analyses, we con- tree, i.e., the time to the most recent common ancestor (MCRA) of strained the tree space to have a monophyletic ingroup comprising all primate mtDNA genomes, to also be estimated from our data. all of the haplorhine genera in our sample. For our second analysis (BEAST2), we used an alternative set of We ran 25 independent RAxML runs using three data partitions calibration points and priors that were implemented in two other within our alignment (codon positions 1, 2, and 3), with model recent studies that estimated divergence times within the entire parameters estimated independently for each partition. For each primate radiation, one using sequence data from multiple unlinked of these runs, topologic support for the tree with the best likeli- nuclear loci (Perelman et al., 2011) and the other sequence data hood score was estimated with 1000 rapid bootstrap replicates from whole mitochondrial genomes (Finstermeier et al., 2013). using the same GTR + G + I model of evolution as used to generate Both of these studies used a very deep age estimate (90 Ma) for the maximum likelihood tree (Stamatakis, 2006). For our Bayesian the MRCA that was derived from other molecular studies (e.g., phylogenetic inference analysis, we again assumed a GTR + G + I Arnason et al., 2008; reviewed by Tavaré et al., 2002) to set the substitution model and partitioned our alignment by codon posi- prior distribution for the initial divergence of strepsirrhines and tion. Default flat Dirichlet distributions were used for both the sub- haplorhines, though the use of this calibration point is not without stitution rate and stationary nucleotide frequency priors. We ran controversy (Steiper and Seiffert, 2012). four independent Markov Chain Monte Carlo (MCMC) runs, each For our third analysis (BEAST3), we used another set of calibra- with 4 chains, for 1.25 million generations, sampling trees and tion points that were implemented in yet another recent study that the parameter space every 1000 generations. We assessed conver- reconstructed divergence times specifically within the platyrrhines gence in each run visually using the software Tracer v1.5, plotting (Perez et al., 2013). In this study, Perez et al. (2013) did not include the likelihood versus generation and estimating the effective sam- a calibration point for the MRCA of primates, but did include one ple size of all parameters, both within each of the four runs and for the MRCA of haplorhines (tarsiers + anthropoids), based on combined across all runs, after discarding the first 250,000 gener- the fossil taxon Teilhardina, dated to roughly 55.8 Ma. This calibra- ations (20%) of each run as burn-in. Finally, we used TreeAnnotator tion point is not without controversy, however, as it is debated version 1.7.5 (Drummond et al., 2012) to summarize the Bayesian whether this fossil, in fact, represents a stem haplorhine or a stem posterior clade credibility for the topology converged upon within tarsiiform. Perez et al. (2013) also explored the implications of sev- each run and across the set of independent runs. eral different hypotheses about how to set fossil calibration points within the platyrrhines, which are engendered by competing ideas 2.5. Divergence time estimates about the phylogenetic affinities of two platyrrhine fossils, Dolich- ocebus and Tremacebus. We thus ran three different BEAST3 analy- We estimated divergence dates and confidence intervals around ses, corresponding to Perez et al.’s (2013) alternative hypotheses these dates for the monophyletic clades of interest identified in our for calibrations within the Cebidae. phylogenetic analysis (Fig. 2) using a Bayesian MCMC method as The set of calibration point priors used in both of our BEAST2 implemented in the software BEAST version 1.7.5 (Drummond and BEAST3 analyses included deep age estimates for either the et al., 2012) run on the CIPRES Science Gateway (Miller et al., MRCA of primates (Finstermeier et al., 2013; Perelman et al., 2010). For all of our divergence time analyses, we constrained 2011) or the MRCA of haplorhines (Perez et al., 2013), whereas the tree topology based on branching patterns that had >90% boot- our first analysis estimated the age of this root based solely on strap support in our maximum likelihood phylogenetic analyses our sequence data. We thus ran two additional dating analyses and posterior clade credibilities of 1.0 in our Bayesian analyses. using our initial set of fossil calibrations and prior distributions, Thus, we did not constrain a sister grouping of Cebidae + Atelidae but adding one or the other of these early dates as an additional (bootstrap support 56%, posterior clade credibility 0.88), Aoti- calibration point (BEAST4 and BEAST5). Additionally, because nae + Callitrichinae (bootstrap support 96%, posterior clade credi- Perez et al. (2013), also suggested that the shape of the prior distri- bility 0.98), or Leontopithecus + Saimiri (bootstrap support 56%, bution used in conjunction with dates from the La Venta fossils posterior clade credibility 1.0). As in our other analyses we parti- Aotus dindensis and Neosaimiri might influence divergence date tioned the dataset by codon position, allowing for unlinked base estimates, we repeated our BEAST1 analysis using one of their sug- frequencies and variable evolutionary rates across codon positions, gested lognormal rather than exponential priors for the aotine- and we assumed a ‘‘relaxed’’ lognormal molecular clock, allowing cebine and Cebus-Saimiri divergences (BEAST6). evolutionary rates across different branches of the tree to vary Finally, as other researchers have noted, the fact that molecular and be uncorrelated. The prior for lineage branching was set as a data imply an estimated age for crown primates of 30 Ma earlier A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510 501

Fig. 2. Phylogenetic relationships among primates based on analyses of mitogenomic data from 33 genera of primates, including all five putative atelid genera. Topology and branch lengths indicated are based on the combined results of four independent Bayesian MCMC runs of 1.25 million generations each, which were sampled every 1000 generations and yielded a total of 16,000 trees after discarding the first 20% of each run as burn-in. The same topology was recovered in 25 independent maximum likelihood runs. Outside of Lagothrix, all but 4 nodes in the phylogeny (indicated by open diamonds) had both >99% average bootstrap support across likelihood analyses and posterior clade credibilities of 1 in the Bayesian analysis. For two of these nodes (Leontopithecus-Saguinus and Cebus-Sapajus), the Bayesian analysis yielded a posterior clade credibility estimate of 1 despite lower bootstrap support. The pullout indicates inferred phylogenetic relationships within the atelids, where the branching order among different putative species of lowland woolly monkeys could not be confidently resolved. than the appearance of the purported crown primates in the fossil 2.6. COX2 sequence dataset and analyses record (Steiper and Seiffert, 2012; Tavaré et al., 2002) poses chal- lenges for understanding early primate evolutionary history. As a final analysis, we sought to compare the genetic distance Steiper and Seiffert (2012) recently suggested, however, this dis- between the yellow-tailed woolly monkey and other forms of crepancy in dating might be explained by a convergent slowdown woolly monkeys currently included as distinct species within the in rates of molecular evolution across primate phylogeny, associ- genus Lagothrix with that between named species in other primate ated with increasing body and brain size (Steiper and Seiffert, genera. The only mtDNA locus for which sequences from multiple 2012), and they derived a ‘‘corrected’’ molecular estimate for the putative species of Lagothrix have been published is the COX2 gene, age of the MRCA of primates at 60 to 73 Ma. We thus ran a final so we mined GenBank for all available COX2 sequences from differ- dating analysis (BEAST7) using our initial fossil calibrations and ent species within the genus Lagothrix, and from all other currently priors but using as an additional calibration point this early Paleo- recognized genera of platyrrhine primates for which data from cene/late Cretaceous date. more than one putative species within the genus was available. For each of our BEAST dating analyses, we ran 10 independent We combined these sequences with novel COX2 sequences gener- MCMC runs for 12.5 million generations, sampling trees and ated during this study and extracted from other platyrrhine parameter values every 1000 generations. After visual assessment mtDNA genomes. In total, this resulted in a dataset of 320 of convergence and discarding the first 20% of each run as a burn-in sequences from nine different New World monkey genera, includ- period, the final effective sample size (ESS) for all parameter esti- ing three other atelids, five cebids, and one pitheciid. Within each mates for each of our analyses exceeded 175. For visualization of genus and within each family, we aligned the sequences using Clu- divergence date estimates associated with a particular BEAST anal- stalW and adjusted the alignments by eye where necessary. Five ysis, we thinned the set of 100,000 tree sampled across all 10 runs, sequences (two from Ateles fusciceps, one from Cebus capucinus, retaining only those sampled every 10,000 generations (rather one from Aotus azarai, and one from Lagothrix lugens) were than every 1000 generations) using the software LogCombiner ver- removed from the alignment because they were highly divergent sion 1.7.5. The resultant set of 10,000 trees was summarized with and likely represented sequencing errors or inadvertent numt TreeAnnotator version 1.7.5 and visualized with FigTree version amplifications. We then grouped sequences by species, and used 1.4.0 (Drummond et al., 2012). the software MEGA 5.0 (Tamura et al., 2011) to calculate the 502 A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510

Table 4 Calibrations used for divergence dating analyses.

Analysis Node of Interest 2.5% Quantile of 97.5% Quantile of Prior Shape Mean SD Offset References Prior Distribution Prior Distribution BEAST1 MRCA Homo-Pan (Hominini) 5.0 8.0 Lognormal 1.5 0.5 4.5 1, 2 MRCA Ponginae-Homininae (Hominidae) 12.5 18.0 Lognormal 2.725 0.5 11.6 MRCA Theropithecus-Papio 3.5 6.5 Lognormal 1.5 0.5 3.0 MRCA Catarrhini 21.0 30.1 Lognormal 4.5 0.5 19.5 MRCA Aotinae-Cebinae 12.1 NA Exponential 2.5 12.1 MRCA Cebus-Saimiri 12.1 NA Exponential 2.5 12.1 BEAST2 MRCA Galagidae-Lorisidae(Lorisiformes) 34.1 45.9 Normal 40.0 3.0 NA 3, 4 MRCA Anthropoidea (Simiiformes) 34.2 51.8 Normal 43.0 4.5 NA MRCA Catarrhini 17.2 40.8 Normal 29.0 6.0 NA MRCA Platyrrhini 17.6 29.4 Normal 23.5 3.0 NA MRCA Papionini 5.0 9.0 Normal 7.0 1.0 NA MRCA Theropithecus-Papio (Papionini) 3.2 4.8 Normal 4.0 0.4 NA MRCA Hominidae 10.6 20.4 Normal 15.5 2.5 NA MRCA Homo-Pan (Hominini) 4.9 8.1 Normal 6.5 0.8 NA MRCA Primates 78.2 101.8 Normal 90.0 6.0 NA BEAST3 MRCA Haplorhini 56.2 58.9 Lognormal 1.3 0.5 55.8 5 MRCA Catarrhini 24.0 27.0 Lognormal 1.5 0.5 23.5 MRCA Hominini 5.9 6.9 Lognormal 0.5 0.5 5.7 Model 1 MRCA Aotinae-Cebinae 20.4 21.8 Lognormal 0.9 0.4 20.0 MRCA Cebus-Saimiri 20.4 21.8 Lognormal 0.9 0.4 20.0 Model 2 MRCA Aotinae-Cebinae 13.3 16.1 Lognormal 1.8 0.4 12.5 MRCA Cebus-Saimiri 13.3 16.1 Lognormal 1.8 0.4 12.5 Model 3 MRCA Aotinae-Cebinae 20.7 24.7 Lognormal 2.0 0.5 20.0 MRCA Cebus-Saimiri 20.7 24.7 Lognormal 2.0 0.5 20.0 BEAST4 Same as BEAST1 but with MRCA Primates prior set as in BEAST2 BEAST5 Same as BEAST1 but with MRCA Haplorhini set as in BEAST3 BEAST6 Same as BEAST1 but with MRCAs for Aotinae-Cebinae and Cebus-Saimiri set as in BEAST3 Model 2 BEAST7 Same as BEAST1 but with ‘‘corrected’’ MRCA Primates 60.1 72.9 Normal 66.5 3.25 NA 1, 2, 6 set as indicated

1. Chiou et al. (2011). 2. Hodgson et al. (2009). 3. Finstermeier et al. (2013). 4. Perelman et al. (2011). 5. Perez et al. (2013). 6. Steiper and Seiffert (2012). average genetic distance (both Maximum Composite Likelihood Our phylogenetic analysis supports other molecular results – and Kimura 2-Parameter distance estimates) between spe- including those based on multiple nuclear loci (Perelman et al., cies within genera and between all pairs of sequences in the 2011; Steiper and Seiffert, 2012), transposable elements alignment. Pairwise distances were then classified as either (Osterholz et al., 2009; Ray et al., 2005), and other mitogenomic ‘‘between species within genus’’ or ‘‘between genus within family’’ datasets (Chiou et al., 2011; Finstermeier et al., 2013; Hodgson for comparison. et al., 2009) – showing the New World monkeys divided into three distinct, well-supported clades now recognized as families, with the Pitheciidae branching off first (although this is the least well 3. Results supported node in our phylogeny), followed by a split between the Cebidae and the Atelidae. 3.1. Phylogenetic reconstruction Focusing in on just the Atelidae (Fig. 2), our phylogenetic analysis confirms Alouatta as the basal atelid, and all genus-level bifur- All of our Bayesian and maximum likelihood analyses recovered cations within the Atelidae are supported by 100% bootstrap the exactly same tree topology for the best inference of phyloge- support and posterior clade probabilities of 1. In all analyses, netic relationships among the genera in our dataset, with the woolly monkeys (Lagothrix plus ‘‘Oreonax’’) form a monophyletic exception of the branching patterns among different putative clade, and the yellow-tailed woolly monkey is identified very species of common woolly monkeys (i.e., Lagothrix apart from the clearly as the basal sister taxon to all other woolly monkey yellow-tailed woolly monkey) (Fig. 2). Support for this topology included in our analysis. Muriquis (Brachyteles) are confirmed as was very strong, with all but four nodes outside of the Lagothrix the sister taxon to woolly monkeys. clade having >99% bootstrap support averaged across all 25 of our independent maximum likelihood runs. In our Bayesian analy- 3.2. Dating analysis sis, both for each run separately and combining results across runs, only six nodes in the inferred topology had posterior probabilities Table 6 summarizes the results of our various Bayesian diver- of less than one, and three of these occurred within the genus gence time analyses using different sets of calibration points and Lagothrix. Only one node outside of that genus – the node linking alternative priors. While the different analyses yield variation in the common ancestor of pitheciids to the common ancestor of divergence date estimates, particularly for deep nodes in the phy- atelids + cebids within the Platyrrhini – had a posterior clade cred- logeny, the key divergences of interest in this study – i.e., for the ibility of less than 0.95. MRCA of the Atelidae and for the crown ages of lineages within this A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510 503 radiation – are less variable. Not surprisingly, the various analyses that included estimates of the age of the MRCA for crown primates EAST1, or for crown haplorhines yielded dates that were older than our 51–11.0) 29–2.87) 50–2.94) BEAST1 analysis, which utilized a set of six fossil calibrations for 62–1.08) divergences within the anthropoid primates that have been used in previous mitogenomic studies (Chiou et al., 2011; Hodgson et al., 2009) but included no calibration dates for deeper nodes in , including a deep age the primate phylogeny. The oldest estimate for the MRCA of the 6.7) 17.2 (14.7–19.9) 1.5) 10.6 (8.34–13.2) 59.3) 62.4 (56.6–68.5) atelids was 17.2 Ma, using a set of calibration points that were also 14.8) 14.3 (11.5–17.3) 7–3.07) 2.41 (1.48–3.49) .1–19.5).4–16.8) 19.7 (16.9–22.9) 16.4 (13.4–19.7)

used in two other recent large scale molecular studies of primate . BEAST6 – Fossil calibrations as in phylogeny (Finstermeier et al., 2013; Perelman et al., 2011), both of which include a very deep age estimate for crown primates (90 Ma) (BEAST2). Other age estimates for most recent common Perelman et al. (2011) ancestor of atelids fall in the middle of the range implied by the BEAST1 and BEAST2 analyses and seem to be affected most signif- and Perez et al. (2013) icantly by (1) the use of a deep calibration point (e.g., for either primates or haplorhines) and (2) choices about priors for divergences within the playrrhines, speciﬁcally about divergences within the Cebidae. Speciﬁcally, Perez et al.’s (2013) Model 1 and Model 3 priors (here, BEAST3 Model 1 and BEAST3 Model 3) result in much corrected molecular estimate for the Primate root. older estimates for the MRCA of atelids (and for playrrhines) than

do most of the other models we explored, as does using the very Finstermeier et al. (2013) deep age estimate of 90 Ma for crown primates (models BEAST2 and BEAST 4). In Fig. 3, we present divergence time estimates from our BEAST7 analysis, which yield an intermediate – and we would argue a conservative – age estimate for the atelid MRCA and other divergences within the Platyrrhini. This model uses our set of fossil calibrations, but incorporates as an additional cali- Steiper and Seiffert’s (2012) bration point a ‘‘corrected’’ molecular estimate for the age of crown primates at 60 to 73 my (Steiper and Seiffert, 2012). This analysis suggests that (1) howler monkeys shared a last common ancestor with the rest of the atelids roughly 14.3 Ma; (2) spider monkeys shared a common ancestor with the remaining atelins roughly 10.6 Ma, (3) muriquis and woolly monkeys diverged from one another roughly 8.5 Ma, and (4) the two currently recognized species of Brachyteles diverged from one another roughly 2 Ma. Most importantly for this study, this analysis suggests that within the woolly monkeys, the basal divergence of the yellow-tailed woolly monkey from the rest of the . BEAST5 – Fossil calibrations as in BEAST1, plus a deep root for haplorhines following genus was also very recent, on the order of 2.1 Ma in the Pleis- tocene, while the diversiﬁcation among the remaining taxa began only about 0.84 Ma. Even using a deeper age estimate to calibrate the root of the primate tree (e.g., BEAST2, BEAST4) or using a deep estimate for the MRCA of haplorhines coupled with old , including a deep age estimate for haplorhines and alternative calibrations for divergences within the Cebidae. BEAST4 – Fossil calibrations as in B

divergences within the Cebidae (e.g., BEAST3 Models 1 and 3) Perelman et al. (2011) does not push the date estimates for these two nodes much fur- and ther back in time (Table 5).

3.3. COX2 divergences within and between genera Perez et al. (2013)

Estimates of the average genetic distance at the COX2 locus a between pairs of New World monkey species from the same genus, BEAST1 BEAST2 BEAST3 Model 1 BEAST3 Model 2 BEAST3 Model 3 BEAST4 BEAST5 BEAST6 BEAST7 1.74 (1.10–2.51)1.87 (1.32–2.56) 2.33 (1.44–3.47)0.74 (0.54–0.96) 2.50 (1.67–3.51) 2.25 (1.35–3.35) 0.98 (0.71–1.28) 2.37 (1.60–3.36) 1.87 (1.18–2.65) 0.92 (0.68–1.21) 1.97 (1.40–2.66) 2.22 (1.31–3.36) 0.78 (0.59–1.00) 2.38 (1.65–3.31) 2.20 (1.37–3.31) 0.94 (0.68–1.22) 2.38 (1.62–3.29) 1.88 (1.25–2.71) 0.93 (0.67–1.23) 2.03 (1.46–2.72) 1.77 (1.15–2.55) 0.80 (0.60–1.03) 1.87 (1.32–2.53) 1.98 (1. 0.74 (0.55–0.95) 2.12 (1. 0.84 (0. measured as Maximum Composite Likelihood distances, are sum- 7.61 (5.62–9.65) 10.2 (7.42–13.4) 9.97 (7.37–12.9) 7.97 (6.18–9.98) 10.3 (7.59–13.4) 9.42 (6.93–12.3) 8.14 (6.20–10.4) 7.60 (5.79–9.55) 8.58 (6. Model 2 calibrations within the Cebidae. BEAST7 – Fossil calibrations as in BEAST1, plus marized in Table 6 (results using Kimura 2-Parameter distances Finstermeier et al. (2013) are equivalent: data not shown). The mean genetic distance seen between the four lowland morphotypes of Lagothrix (0.019 MCL flavicauda distance) was lower than that seen between species within all of flavicauda the other genus examined except Saimiri, and the same is generally Lagothrix true even when the yellow-tailed woolly monkey is included with and including without the other forms. For example, the average genetic distance Perez et al.’s (2013) between different species of marmosets (genus Callithrix sensu lato) is 0.089, or close to four times that seen among woolly mon- Brachyteles Brachyteles Lagothrix Lagothrix keys, including the yellow-tailed woolly monkey, and even when Model summaries: BEAST1 – Fossil calibrations as in Table 4, with no prior specified for crown Primates or haplorhines. BEAST2 – Priors based on MRCA Primates 50.4 (42.3–59.4) 75.9 (66.9–85.3) 58.6 (56.4–62.8) 57.8 (56.3–60.4) 58.8 (56.4–63.0) 72.7 (61.9–83.7) 57.8 (56.3–60.6) 50.5 (43.1– MRCA PlatyrrhiniMRCA PitheciidaeMRCA CebidaeMRCA AtelidaeMRCA AlouattiniMRCA AteliniMRCA MRCA MRCA 17.4 (15.2–20.2)MRCA 14.5 (12.0–17.5) 23.8 (20.6–27.3) 19.7 (16.1–23.4) 15.2 (13.4–17.6) 24.3 (22.9–25.9) 12.7 (10.2–15.3) 20.1 2.09 (17.0–22.9) (1.34–3.03) 20.9 (17.8–24.2) 18.2 (16.6–20.0) 17.1 (13.6–21.0) 2.76 15.1 (1.61–4.09) (12.9–17.4) 21.8 9.42 (21.1–22.7) (7.37–11.8) 25.0 (23.5–27.0) 17.1 (14.0–20.3) 2.64 20.7 (1.53–3.99) (17.4–23.8) 15.8 12.6 (14.6–17.2) (9.62–16.0) 21.7 (17.8–25.8) 13.2 (11.0–15.4) 2.21 18.0 (1.41–3.19) (14.1–22.0) 22.6 12.4 (21.6–23.8) (9.57–15.4) 18.8 (16.3–21.5) 17.6 (14.4–21.1) 2.69 15.7 (1.56–4.13) (12.9–18.4) 18.8 9.89 (15.4–22.3) (7.90–11.9) 17.6 (16 15.7 (12.3–19.3) 2.64 14.7 (1.65–3.89) (12 16.4 12.8 (14.2–18.7) (9.90–16.0) 13.6 (11.2–16.3) 2.31 (1.48–3.29) 15.4 11.7 (14.3–1 (8.92–14.9) 12.7 (10.6– 2.13 (1.3 10.1 (7.94–12.4) 9.43 (7.50–1 genera of Atlantic Forest and Amazonian marmosets (Callithrix Node of interest Model a Table 5 BEAST1, but using plus a deep root for Primates following estimate for the primate root. BEAST3 – Priors based on and Mico, respectively) are considered separately, the mean Median date estimates and 95% HPD ranges for the different models explored in this study. 504 A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510

Table 6 Genetic distances at the COX2 locus between species of New World monkeys within the same genus.

Genus Species # Sequences Total # species Total # sequences Mean (and Range) in genetic distance References Alouatta caraya 3 3 16 0.072 (0.050–0.090) 1, 2, 3, 4, 5, 6 palliata 2 senciulus 11 Aotus azarai 9 6 30 0.044 (0.018–0.055) 6, 7, 8, 9 grisseimembra 1 lemurinus 5 nancymaae 9 nigriceps 4 vociferans 2 Ateles belzebuth 2 7 34 0.034 (0.017–0.044) 1, 3, 4, 5, 6, 10, 11 chamek 8 fusciceps 5 geoffroyi 5 hybridus 3 marginatus 7 paniscus sp. indet.a 2 Brachyteles arachnoides 1 2 3 0.053 (NA) 1, 10, 12 hypoxanthus 2 Callicebus donacophilus 2 2 2 0.081 (NA) 6, 8 cupreus 1 Callithrix aurita 29 23 Callithrix sensu lato 0.089 (0.012–0.154) 6, 13, 14, 15, 16 geoffroyi 2 Callithrix sensu stricto 0.033 (0.010–0.064) jacchus 4 Mico 0.020 (0.013–0.025) kuhlii 1 pencillata 3 (Mico) argentatus 3 (Mico) emiliae 2 (Mico) humeralifer 2 (Mico) mauesi 2 (Mico) saterei 2 Cebus albifrons 1 2 120 0.036 (NA) 11, 17 capucinus 119 Saimiri boliviensis 5 4 24 0.018 (0.011–0.026) 4, 7, 8, 11, 18, 19, 20 oerstedii 3 peruviensis 1 sciureus 15 Sapajus apella 3 2 4 0.055 (NA) 4, 6, 21 xanthosternos 1 Lagothrix cana 7 4 58 0.019 (0.015–0.022) excluding flavicauda 1, 2, 6, 22, 23 lagotricha 19 lugens 22 poeppigii 8 sp. indet.a 1 flavicauda 1 1 1 0.023 (0.025–0.034) including flavicauda 1

1. This study. 2. Adkins and Honeycutt (1994). 3. Ascunce et al. (2002). 4. Ascunce et al. (2003). 5. Figueiredo et al.(1998). 6. Finstermeier et al. (2013). 7. Ashley and Vaughn (1995). 8. Babb et al. (2011). 9. Suarez et al. (2011). 10. Collins and Dubach (2000). 11. Hodgson et al. (2009). 12. Schrago et al. (2012). 13. Sato et al. (2010). 14. Sena et al. (2002). 15. Yang and Yoder (2003). 16. Yoder and Yang (2004). 17. Ruiz-García et al. (2011b); Note: citation for these sequences in NCBI GenBank appears to be in error as it refers to Aotus COX2; presumably these sequences are those used in Ruiz-García et al. (2011a) on Cebus capucinus. 18. Chiou et al. (2011). 19. Matsui et al. (2009). 20. Ruiz-Garcia et al. (2013). 21. Bi et al. (2011). 22. Botero et al. (2010). 23. Ruiz-García and Pinedo-Castro (2010); Note: citation in NCBI GenBank indicates no journal reference for these sequences, but they presumably are those used in Ruiz- García and Pinedo-Castro, 2011. a Data only included for between genus comparisons. distance among species within each of genus is greater than that woolly monkey and other woolly monkeys (0.034) is in all cases among the four common woolly monkey morphotypes. Moreover, but that of Saimiri, less than or equal to the average distance seen the maximum genetic distance seen between the yellow-tailed among species within a genus for all other platyrrhine genera. A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510 505

Fig. 3. Divergence time estimates for platyrrhines based on the BEAST7 model. Nodes in the phylogeny indicate the median divergence date estimate based on 10,000 trees compiled from 10 BEAST MCMC runs of 12.5 million generations each, with trees sampled every 10,000 generations after discarding an initial burn-in period of 2.5 million generations per run. Shading indicates the 95% highest posterior density (HPD) range around the median divergence date estimate.

Finally, when we plot frequency spectra of the pairwise dis- (Chiou et al., 2011; Finstermeier et al., 2013; Hodgson et al., tances among COX2 sequences between species within the same 2009) as well as multiple nuclear loci (Perelman et al., 2011; genus and between genera within the same family, these distribu- Steiper and Seiffert, 2012), transposable elements (Osterholz tions are almost perfectly disjunct (Fig. 4). Few ‘‘between genus- et al., 2009; Ray et al., 2005). As in these other studies, we find within family’’ distances are less than 0.1 (and all of these involve clear, strong support for the existence of three major clades within the pair of sister genera Cebus and Sapajus), while few ‘‘between the platyrrhines: the pitheciids (comprising titis, sakis, and uak- species-within genus’’ distances are greater than 0.1. Moreover, aris); the cebids (comprising capuchins, squirrel monkeys, owl the genetic distances between the yellow-tailed woolly monkey monkeys, and the callitrichines); and the large-bodied, prehensile and each of the other woolly monkey taxa fall squarely inside of tailed atelids. Our analysis also suggests that the pitheciid lineage the ‘‘between species-within genus’’ distribution (Fig. 4). diverged first within the New World monkeys. Though this node has the weakest support in our phylogeny, a basal divergence of the Pitheciidae has also been recovered in many, though not all, 4. Discussion prior molecular studies. Additionally, our study sheds particular light on the evolution- 4.1. Atelid phylogenetic history and divergence dates ary history of the atelid clade, which has been underrepresented in prior phylogenetic analysis. First, our results clarify the relation- Our mitogenomic analysis of platyrrhine evolutionary history, ship among taxa within the atelin subfamily. Woolly monkeys which incorporates new sequence data from seven additional ate- and muriquis are robustly identified as sister lineages that shared lid taxa (including red howler monkeys, northern muriquis, and all a MRCA in the middle Miocene, 8–9 Ma, while spider monkeys five currently recognized morphotypes of woolly monkeys) corrob- diverged from the woolly monkey-muriqui common ancestor ear- orates and fills in other recent molecular reconstructions of New lier, 10–11 Ma. While ours is not the first study to identify a sister World monkey phylogeny, several of which have been based on relationship between Brachyteles and Lagothrix to the exclusion of less extensive mitogenomic datasets for the taxa in question Ateles (Canavez et al., 1999; but see Collins, 2004; e.g., Perelman 506 A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510

Fig. 4. Distribution of genetic distances at the COX2 locus between pairs of platyrrhine sequences. White bars summarize pairwise distances between samples attributed to individuals from different species within the same genus, while grey bars summarize distances among pairs from different genera within the same family. Distances between the yellow-tailed woolly monkey and other forms of woolly monkeys are shown in black. et al., 2011; Schneider et al., 2001), the relationship among these As is clear from the Table, the crown ages estimated in various three genera has remained contentious (Collins, 2004) and this studies using different marker types and employing different study helps resolve that issue. Second, our study provides the first molecular clock assumptions are variable, with studies based on genetic estimate for the divergence date between northern and single mitochondrial genes and/or employing non-Bayesian meth- southern muriquis (Brachyteles hypoxanthus and Brachyteles arach- ods for estimating divergence times yielding dates that are some- noides, respectively), placing this split at 2 Ma. This time depth what older than those based on larger sequence data sets and well exceeds that between sister species within many other platyr- that used Bayesian approaches. Moreover, even those studies uti- rhine genera, and, when coupled with morphological differences lizing Bayesian methods with large datasets – i.e., this one using between the two forms (Lemos de Sá and Glander, 1993; Lemos mitogenomic data, that by Perelman et al. (2011) using multiple de Sá et al., 1993), strengthens the argument for separating these nuclear loci, and that by Springer et al. (2012) using both nuclear taxa at the species level. The International Union for the Conserva- and mtDNA genes – are not entirely comparable given that they tion of Nature (IUCN) has classified the southern muriqui as incorporate somewhat different sets of species within each genus. ‘‘endangered’’ (Mendes et al., 2008a) and the northern muriqui as Additionally, estimated crown divergence times tend to be slightly ‘‘critically endangered’’ (Mendes et al., 2008b), and conservation older in studies that had more taxa represented within a particular efforts should focus on managing these taxa separately. Most genus (e.g., for Saguinus and Ateles). Nonetheless, it is still quite importantly, our study provides the first molecular assessment of clear that within each of the large-scale Bayesian studies, and the phylogenetic position of the yellow-tailed woolly monkey with across the set of studies employing smaller datasets, the minimum respect to other woolly monkeys and to other atelids. We find that crown ages that have been estimated for other platyrrhine genera yellow-tailed and common woolly monkeys diverged from one are often much older than corresponding estimates for the MRCA another very recently – also on the order of 2 Ma, or more than of yellow-tailed and common woolly monkeys and for the MRCA 6 million years after the divergence of the woolly monkey and of the common woolly monkey clade. Put another way, the diver- muriqui lineages. Finally, we estimate that the radiation of com- gence between yellow-tailed and common woollies is much more mon woolly monkeys began much more recently, less than 1 Ma. recent than the deepest species-level divergence between members of a number of other well-accepted platyrrhine genera, and the age of the MRCA of the common woolly monkey clade is even 4.2. Implications for the taxonomy of woolly monkeys more recent. These observations – coupled with the fact that pairwise To put the evolutionary history of woolly monkeys in a compar- genetic distances at the COX2 gene between yellow-tailed woolly ative perspective, Table 7 summarizes estimates of the minimum monkeys and other forms of Lagothrix lie well within the range crown ages of other platyrrhine genera, which we have collated seen between pairs of species within a genus, and well outside of from several other mtDNA studies (which have typically focused the range seen between species from different genera within the on a single gene of relatively short length, e.g., Lavergne et al., same family (Fig. 4) – argue strongly against the idea that yel- 2009; Lynch Alfaro et al., 2012a; Matauschek et al., 2011; Ruiz- low-tailed and common woolly monkeys should be classified in García et al., 2010; Ruiz-García and Pinedo-Castro, 2011) and from different genera. Other recent examinations of the phylogenetic Perelman et al.’s (2011) and Springer et al.’s (2012) recent large position of yellow-tailed woolly monkeys utilizing morphological scale analyses using multiple nuclear loci. These latter studies both data also challenge the idea of recognizing a distinction at the estimated divergence dates using the Bayesian approach we follow genus level between yellow-tailed woolly monkeys and other mor- here, and, as noted above, Perelman et al.’s (2011) study was the photypes. For example, in replicating and revising Groves’ (2001) source for the calibration dates used in our BEAST2 analysis. cladistic analysis of ateline craniodental features that led him to Accordingly, the dates indicated in Table 7 correspond to the deep- elevate the yellow-tailed woolly monkey to its own monotypic est divergence within the indicated genus where two or more dis- genus (Oreonax) as a sister taxon to spider monkeys (Ateles), tinct species were included in the respective dataset. Table 7 Estimated age of the most recent common ancestor (MRCA) for woolly monkeys and other platyrrhine genera.

Family Taxon Date Based on Multiple Date Based on Multiple Date Based on mtDNA Date Based on mtDNA Date From Studies Loci Used Reference for Nuclear Loci (Perelman Nuclear + mtDNA Loci Genomes (This Study Genomes (This Study Using Fewer other mtDNA et al., 2011) (Springer et al., 2012) a BEAST2) BEAST7) mtDNA Loci Studies Pitheciidae Callicebus 9.86 Ma 7.04 Ma 4.76 Ma b 4.12 Ma b –––

Pithecia 4.00 Ma 2.82 Ma – – – – – 495–510 (2015) 82 Evolution and Phylogenetics Molecular / al. et Fiore Di A. Chiropotes 1.88 Ma 1.82 Ma – – – – – Cacajao 3.39 Ma 2.15 Ma – – – – – Cebidae Aotus 5.54 Ma 2.64 Ma 3.48 Mab 2.96 Ma b 12.5 Ma c COX2 1 Saguinus 8.84 Ma 5.59 Ma – – 10.1 Mad Cyt b, HV1 2 Callithrix sensu lato 5.96 Ma 5.0 Ma 6.60 Ma 5.57 Ma – – – (Callithrix + Mico + Cebuella) Callithrix sensu stricto 3.79 Ma 0.94 Mab 0.81 Ma b ––– Mico 2.17 Ma – – – – – Leontopithecus 0.50 Ma 0.4 Ma – – – – – Cebus sensu lato 6.00 Ma 4.4 Ma 6.34 Ma 5.39 Ma 6.2 Ma Cyt b 3 (Cebus + Sapajus) Saimiri 2.24 Ma 1.44 Ma 2.01 Ma 1.69 Ma 4.3 Ma Cyt b 4 b b Atelidae Alouatta 6.03 Ma 2.98 Ma 2.76 Ma 2.41 Ma 6.8 Ma ATP6, ATP8, and Cyt 5 b (+ 2 nuclear regions) Ateles 5.07 Ma 1.97 Ma – – 8.0 Mad NAD5, NAD6, Cyt b 6 Brachyteles 2.92 Ma 1.9 Ma 2.32 Ma 1.98 Ma – – – Lagothrix (including L. – 2.50 Ma 2.12 Ma – – – ﬂavicauda) Lagothrix (without L. 0.64 Ma 0.9 Ma 0.98 Ma 0.84 Ma 2.9 Ma COX2 7 ﬂavicauda)

1. Ruiz-García et al. (2011b). 2. Matauschek et al. (2011). 3. Lynch Alfaro et al. (2012a). 4. Lavergne et al. (2009). 5. Cortés-Ortiz et al. (2003). 6. Morales Jimenez et al. (2015). 7. Ruiz-Garcia and Pinedo-Castro (2011). a While this study utilized data from many nuclear and mtDNA genes, many individual platyrrhine species were represented by only a single locus. b This study used data from fewer and less genetically divergent species within the genus indicated than Perelman et al. (2011). c This study inferred a very different branching pattern of species within the genus than Perelman et al. (2011). d This study used a larger number of species within the genus than either Perelman et al. (2011) or this study. 507 508 A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510

Matthews and Rosenberger (2008; Rosenberger and Matthews, monkeys had low bootstrap support in our maximum likelihood 2008) found that using different taxon combinations in the analy- analyses, and the posterior clade credibilities associated with these sis yielded different results about branching patterns within the nodes were less than 0.70. The uncertain branching pattern among clade. While their study did not conclusively resolve the position the common woolly monkeys – along with our recent age estimate of the yellow-tailed woolly monkey within the atelines (nor the for the MRCA of this clade, the small average genetic distances monophyly of Lagothrix, sensu lato), their analysis highlighted between clade members at the COX2 locus, and other recent the fact that no compelling morphological evidence exists to link research suggesting a lack of reciprocal monophyly among the dif- yellow-tailed woolly monkeys more closely to Ateles than to other ferent putative species (Botero, 2010; Botero and Stevenson, in woolly monkeys. Notably, Matthews and Rosenberger (2008; press; Ruiz-Garcia and Pinedo-Castro, 2011) – argues strongly Rosenberger and Matthews, 2008) did not have access to the size- against recognizing these morphotypes as distinct species. Thus, able number of specimens of yellow-tailed woolly monkeys pres- we conclude that the remaining taxa of woolly monkeys are ade- ent in the Museo de Historia Natural at the Universidad Nacional quately accommodated within a single species (Lagothrix lagotri- Mayor de San Marcos in Perú for their analysis. However, using cha) that encompasses several geographically distinct subspecies, these 15 specimens (plus more than 90 from the four other mor- some of which are paraphyletic and may be linked by some level photypes of woolly monkeys and 30 from other atelines), Paredes of gene flow (Botero, 2010; Botero and Stevenson, in press; Ruiz- Esquivel (2003) conducted the most thorough morphology-based Garcia and Pinedo-Castro, 2011) (see also Botero et al., 2015). study yet done on the atelid clade. Her cladistic analysis of 54 morphological characters (including craniodental traits, as well as traits describing pelage color and banding) concluded that yel- 4.3. Biogeographic implications low-tailed woolly monkeys and other forms of Lagothrix indeed form a monophyletic clade within the atelids, and, as in our study, Yellow-tailed woolly monkeys are currently limited in their dis- yellow-tailed woolly monkeys diverged first within this lineage. tribution to montane and submontane forests in northern Perú, In short, we concur with other recent examinations of the ate- while the other samples utilized in this study are either from the line phylogenetics and of the phylogenetic position of yellow- Amazonian lowlands of Ecuador, Perú, or Colombia or from a mon- tailed woolly monkeys based on morphological data (Matthews tane population of woolly monkeys in southern Colombia (Fig. 1). and Rosenberger, 2008; Paredes Esquivel, 2003; Rosenberger and That fact that yellow-tailed woolly monkeys are the basal lineage Matthews, 2008): woolly monkeys constitute a monophyletic to split off within Lagothrix and are estimated to have done so more group, and there is no compelling reason to continue using the than one million years before the MRCA of the other woolly mon- genus name Oreonax. Rather, that genus name should be consid- keys in our sample began diversifying suggests that the woolly ered a synonym for Lagothrix, and the yellow-tailed woolly monkey monkey lineage likely originated from an ancestral population liv- should be referred to as Lagothrix flavicauda,asFooden (1963) con- ing in the upper Amazon basin or Andean foothills in northern Perú cluded in his review of the woolly monkeys over 50 years ago. and from there spread across the western and central Amazon as Based on our results (and the morphometric analysis of Paredes well as into the inter-Andean valleys and montane forests of north- Esquivel, 2003), it does seem likely that yellow-tailed woolly ern Colombia. This suggestion is consistent with a recent phyloge- monkeys comprise a distinct evolutionary lineage from the ographic analysis of a large set of COX2 sequences from all four remaining forms, which cluster together in our analysis as a mono- morphotypes of common woolly monkeys (Ruiz-Garcia and phyletic group with a much more recent common ancestor Pinedo-Castro, 2011). That study found that Lagothrix lagotricha (840,000 years). However, we caution that until sequence data poeppigii – the taxon whose current geographic range is adjacent from additional yellow-tailed woolly monkey individuals are avail- to that of Lagothrix flavicauda – contained the highest haplotype able – as well as sequence data from additional higher altitude diversity and had broadest distribution across the inferred COX2 populations of other Lagothrix – that idea remains a working haplotype network, leading the authors to conclude that the source hypothesis. Moreover, given the paucity of field data on yellow- population for common woolly monkeys was in the upper Amazon tailed woolly monkeys and on high-altitude populations of the region of Perú (Ruiz-Garcia and Pinedo-Castro, 2011). other morphotypes, it still remains to be seen how distinct these Several recent reevaluations of the geological history of the lineages may be in terms of diet, ecology, or behavior. If reciprocal Amazon basin suggest that the modern drainage system developed monophyly were confirmed for yellow-tailed versus other woolly during the Pliocene (Campbell et al., 2006; Latrubesse et al., 2010) monkeys, an argument might be made for considering Oreonax as and would thus have been in place well before 840 kyr, the esti- a subgenus of Lagothrix, and, indeed, there is some precedent for mated age of the MCRA of common woolly monkeys. If this model considering such an option. For example, it was recently recom- for the origin of the modern Amazon River system is correct, then mended that Sapajus (a subgenus of Cebus) be elevated to genus our genetic results also imply either that common woolly monkeys status – a taxonomic revision that we utilize here – based, in part, dispersed rapidly and simultaneously from the same upper Amazon on molecular evidence showing a deep split (5–6 Ma) between source into different biogeographic regions in the lowlands but the ‘‘gracile’’ and ‘‘robust’’ forms of capuchins (Lynch Alfaro et al., have not yet had time to become clearly differentiated, or that riv- 2012b). However, in light of the data at hand, we think that consid- ers and other landscape features in the upper Amazon basin and ering Oreonax as even a subgenus is unnecessary: the split between Andean forelands are only semi-permeable barriers to gene flow yellow-tailed woolly monkeys and other Lagothrix occurred much for Lagothrix and that some genetic exchange between morpho- more recently than the Cebus-Sapajus split and the two lineages types is ongoing. A full understanding of the phylogeography of have an allopatric distribution, where contact and possible gene the genus Lagothrix will require additional sampling within each flow is precluded. By contrast, the distributions of Cebus and Sapa- of the putative common woolly monkey subspecies, particularly jus overlap throughout much of Amazonia, and molecular data from populations in south central Colombia and northeastern confirm an absence of gene flow between these genera even in Ecuador (where the distributions of L.l. lugens, L.l. lagotricha, and the face of widespread sympatry (Lynch Alfaro et al., 2012a). L.l. poeppigii interdigitate) and on opposing banks of the Río Juruá With regard to the other currently recognized morphotypes of (the presumed boundary between L.l. poeppigii and L.l. cana)andof Lagothrix (poeppigii, lugens, lagotricha, and cana), our phylogenetic the Río Amazonas/Solimões between the Ríos Napo and Japurá (the analysis was unable to robustly identify the branching order presumed boundary between eastern populations of L.l. lagotricha among taxa; several nodes within the clade of common woolly and L.l.poeppigii). A. Di Fiore et al. / Molecular Phylogenetics and Evolution 82 (2015) 495–510 509

4.4. Conservation References

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