Multi-Locus Phylogenetic Inference of the Howler Monkey (Alouatta) Radiation in South America

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Multi-locus phylogenetic inference of the howler monkey (Alouatta) radiation in South America.

Esmeralda Ferreira CUNY City College

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Multi-locus phylogenetic inference of the howler monkey (Alouatta) radiation in South America.

Esmeralda Ferreira

Department of Biology, City College of New York,

New York, NY 10031, USA.

Masters Thesis Committee:

Ana Carnaval, PhD. (advisor) ______

Eugene Harris, PhD. (co-advisor) ______

Amy Berkov, PhD. ______

17 May 2019

Contents

Abstract ………………………………………………………………………………….. 3

Introduction ……………………………………………………………………………... 4

Material and Methods …………………………………………………………………... 7

Results …………………………………………………………………………………… 10

Discussion ……………………………………………………………………………….. 13

Acknowledgements ……………………………………………………………………... 17

Cited Literature …………………………………………………………………………17

Appendix ………………………………………………………………………………… 23

Abstract

Howler monkeys (Alouatta) are the most widely distributed New World primates, ranging from southern Mexico to northern Argentina. They occur in tropical rain forests, flooded and gallery forests, and deciduous and semi-deciduous environments. Despite their importance as seed dispersers, howlers have also been known to be ecological indicators. Available phylogenetic hypotheses for this genus have used chromosomal characters, morphological characteristics, and a limited number of molecular markers and specimens. In spite of these analyses, branching patterns among howler species lineages conflict between studies or remain unresolved. Using 14 unlinked non-coding intergenic nuclear regions under both a concatenated Bayesian approach (MrBayes) and a coalescent-based species tree method (*BEAST). I perform a new phylogenetic study of Alouatta, including five of the seven South American species (A. caraya, A. belzebul, A. guariba, A. seniculus, A. sara) and the two recognized species from Mesoamerica (A. pigra, A. palliata). Contrary to previous studies, I find little to no support for a sister relationship between A. guariba (the only howler species endemic to the Atlantic Coastal Forest) and A. belzebul, irrespective of the method utilized. Instead, the Atlantic forest-based A. guariba is recovered as sister to the remaining South American species, with high support both in the Bayesian and the species tree. Relationships among the remaining South American howlers are not as clearly supported, but a sister-species relationship is recovered between A. sara and A. caraya. Although the phylogenetic relationships inferred through the methods utilized here differ from those proposed in previous studies, the divergence time estimation analyses recovered similar dates to other investigations. The age of the genus Alouatta was estimated at ca. 6.1 Mya (95% Highest Posterior Density, or HPD = 5.2 - 6.9 Mya). The Meso-American clade containing Alouatta pigra and A. palliata was dated back to ca. 1.3 Mya (HPD 0.9 – 1.9 Mya), while the South American taxa coalesce at a node dated to ca. 5.1 Mya (HPD 4.3 – 6.0 Mya). While this study advances our knowledge of evolutionary relationships in howler monkeys, several standing questions remain. Tackling them will require further taxonomic, geographic, and genomic sampling in Alouatta.

Keywords: Alouatta, phylogenetics, Brazil, multi-locus.

Introduction

The howler monkeys (genus Alouatta Lacépède 1799, family Atelidae) are the most widely distributed New World primates. These species occur in tropical rain forests, flooded and gallery forests, and deciduous and semi-deciduous seasonal environments from southern Mexico to northern Argentina (Crockett and Eisenberg, 1986; Zunino et al., 2001). Despite having an important ecological role in Neotropical forested biomes (de A. Moura and McConkey, 2007), many species are threatened by habitat loss and habitat alteration according to the IUCN 2016. Knowing that howlers have been observed to adapt well in forest fragments and human altered areas, when absent, they are indicators of high levels of disturbance (Crockett, 1998).

The taxonomy and the phylogenetic relationships among Alouatta species have been the focus of several morphometric, cytogenetics, and molecular analyses. Despite this, the number of species that compose the genus remains unconfirmed (ranging from nine to 14), as well as the evolutionary relationships among them, and the processes underlying diversification (Cortés- Ortiz et al., 2003; Gregorin, 2006; Rylands and Mittermeier, 2009). Linnaeus (1766) was the first one to describe the South America howler monkeys, under the genus Simia (S. belzebul and S. seniculus). In 1799 Lacépède created the name Alouatta, and the taxonomy of the genus has been changing ever since. The South American howlers alone have been referred to using 48 different species-names, which partially explains the difficulty in identifying and classifying these species (Cortés-Ortiz et al., 2015).

The first systematic revision of the genus Alouatta was conducted by Hershkovitz (1949). Using morphological characters based on the hyoid bone, he suggested a division of the genus into three species groups: the seniculus group (A. seniculus, A. belzebul, A. fusca), the palliata group (A. palliata and A. pigra) and the caraya group (A. caraya). Although phylogenetic analyses recovered the Alouatta genus as a monophyletic clade back in 2002 (de Oliveira et al., 2002; Steinberg et al., 2014), it was only in 2006 that Gregorin proposed the first systematic arrangement for the genus, which is still largely followed. Based on the most complete study involving the South American howler monkeys to date, he proposed a systematic of ten species of howlers. More recently, Cortés-Ortiz et al. (2015) reviewed information from previous

5 morphological and genetic data (mtDNA) and proposed the existence of nine species of howlers, distributed in Meso-America and South America (A. palliata, A. pigra, A. seniculus, A. arctoidea, A. sara, A. macconnelli, A. guariba, A. belzebul, A. caraya; Fig 1). Based on molecular data, Cortés-Ortiz et al. (2003) proposed the clade of Meso-American howler monkeys to include five subspecies of A. palliata (A. p. mexicana, A. p. palliata, A. p. coibensis, A. p. trabeata, and A. p. aequatorialis), along with two subspecies of A. pigra (A. p. pigra and A. p. luctuosa). This Meso-American group was shown to be sister to a South American clade, including the remaining seven species. Within the latter, Cortés-Ortiz et al. (2015) recognized three subspecies of A. seniculus (A. s. seniculus, A. s. juara, and A. s. puruensis), and two of A. guariba (A. g. guariba and A. g. clamitans). Cortés-Ortiz et al. (2015) argued that more data were needed to recognize the South American forms A. nigerrima, A. ululata and A. discolor as distinct species, though they have been described as subspecies of A. belzebul.

Figure 1: Geographic distribution of currently recognized species of howler monkeys, genus Alouatta (distributions modified from IUCN 2016. IUCN Red List of Threatened Species,

6 http://www.iucnredlist.org, downloaded on December 24th, 2017): Alouatta pigra (blue), Alouatta palliata (pink), Alouatta seniculus (black) Alouatta macconnelli (orange), Alouatta caraya (red), Alouatta belzebul (purple), Alouatta arctoidea (white), Alouatta guariba (yellow), Alouatta sara (green).

Parallel to the taxonomic instability of the group, phylogenetic reconstructions disagree in regards to the topology of the Alouatta tree (Meireles et al., 1999; Cortés-Ortiz et al., 2003; Villalobos et al., 2004) and suffer from incomplete species sampling. Based on a single nuclear gene (γ1-Globin pseudogene), Meireles et al. (1999) placed A. caraya as sister to a clade containing A. seniculus and its sister clade, which included A. belzebul and A. guariba (Fig. 2, A). A study by Cortes-Ortiz et al. (2003) increased taxonomic sampling by including Alouatta sara, A. macconnelli, A. palliata and A. pigra and analyzed two mitochondrial markers (cytochrome b and ATP-synthase), and two nuclear genes (calmodulin and Recombination Activating Gene 1). In that study, Cortés-Ortiz et al. (2003) inferred a tree in which the South American howlers were divided into two groups: one including A. seniculus, A. sara, A. macconnelli and A. caraya, and another composed of A. belzebul and A. guariba (Fig. 2, B). However, the support for relationships within that South American clade was low. More recently, Villalobos et al. (2004) analyzed mtDNA data (cytochrome b and cytochrome oxidase II) for the South American Alouatta species A. seniculus, A. caraya, A. belzebul, A. guariba and A. sara, finding yet a third topology. While it placed A. sara and A. seniculus as sister taxa, Villalobos et al. (2004) proposed an alternative arrangement for the relationships between A. caraya, A. guariba, and A. belzebul relative to the study of Cortés-Ortiz et al. (2003; Fig. 2, C). It is notable that their results did not find support for a sister-species relationship between A. guariba and A. belzebul, a sister relationship that had been supported, albeit weakly, in previous studies of Meireles et al. (1999) and Cortes-Ortiz et al. (2003).

To clarify the history of the genus, I reconstructed the phylogeny of the South American clade of Alouatta based on DNA sequences from 14 unlinked non-coding nuclear regions (Kiesling et al., 2015), making this the densest genetic sampling of the group to date. Those regions are thought to be useful to search for congruence because they are unlinked; because they are noncoding, they are also less likely to be biased by natural selection, and hence more likely to correctly track evolutionary relationships (Wildman et al., 2009; Kiesling et al., 2015). The data were first

7 concatenated to generate a tree under Bayesian Inference, and then analyzed under a coalescent framework to infer a species tree (Pamilo and Nei, 1988; Edwards, 2009). Using fossil calibrations, I also estimated divergence times for the genus and its species clades.

Material and Methods

Utilizing previously existing genomic extractions, I amplified and sequenced DNA from 41 individuals of seven species of Alouatta (see Appendix 1). They are A. guariba, A. caraya, A. belzebul (Amazonian site) A. seniculus, A. sara, A. pigra, A. palliata. Samples of A. macconnelli and A. arctoidea. Although A. belzebul has a disjunct distribution, with populations both in Amazonia and the coastal Brazilian forest (Fig. 1), I was only able to include samples of Amazonian A. belzebul for the purposes of this study. Five species were included as outgroups – Ateles paniscus, Ateles belzebuth, Ateles geoffroyi, Lagothrix lagotricha and Brachyteles arachnoides (Kielsling et al., 2015) – and obtained from GenBank (Appendix 2).

Figure 2. Color-coded map of species distributions and existing hypotheses of phylogenetic relationships among the South American howler monkeys. A (top right): maximum parsimony tree based on the γ1-globin pseudogene, including bootstrap values, adapted from Meireles et al. (1999). B (bottom left): neighbor-joining tree based on ATPase 8 and ATPase 6, and Cytb, including bootstrap values, adapted from Cortés-Ortiz et al. (2003). C (bottom right): consensus mtDNA (COII and Cytb) tree, including Bremer Decay Indexes, adapted from Villalobos et al. (2004)

Using the protocols of Kielsling et al. (2015), I established an amplification panel for 16 unlinked non-coding nuclear regions (Appendix 3) using oligonucleotide primers developed by Wildman et al. (2009). Sequence data will be submitted to GenBank upon manuscript acceptance. Polymerase Chain Reaction (PCR) protocols were adapted from Meireles et al. (1997), including an initial denaturation of 3 minutes at 94°C, 30 cycles of denaturation at 94°C (30s), annealing at 55°C (45s), and extension at 72°C (45s), and a final extension of 10 min at 72°C. For a given gene, annealing temperatures varied among species from 42°C to 56C °C. PCR products were visualized through agarose gels electrophoresis (1%), and prepared for sequencing with MacroGen (New York, USA) and Hemocentro USP (Ribeirao Preto, Brazil).

Sequences were edited in Geneious vR6 (http://www.geneious.com, Kearse et al. 2012), combined using Geneious global alignment, examined by eye, and manually adjusted. Nuclear sequences were analyzed using PHASE 2.1.1 (Stephens and Donnelly, 2003) to estimate the

9 haplotypic phase of heterozygotes, after preparation of input files in SeqPHASE (Flot, 2010). PHASE was ran ten independent times, using a 0.90 probability threshold and a parent- independent mutation model. I used Jmodeltest (Posada, 2008) to determine models of nucleotide substitution and best-fit partition schemes, which were used in the phylogenetic analyses.

I performed phylogenetic analyses using two different approaches, and generating three phylogenies. First, I generated a tree under a concatenated approach using Bayesian Inference in MrBayes 3.2.1 (Ronquist et al., 2012). For that, I ran three independent runs and four Markov chains of 20 million generations each, sampling every 1,000 steps. Second, I used a coalescent- based analysis in *BEAST (Heled and Drummond, 2010) to generate a species tree. For that, I set up three independent runs of 100 million generations each, sampling every 10,000 steps. Lastly, I used BEAST 1.8 (Drummond et al., 2012) to estimate a Bayesian tree under concatenation with divergence times by implementing an uncorrelated log-normal relaxed clock (Drummond et al., 2006) under a concatenation approach. This method implemented a uniform prior distribution (interval = 0-1) to the mean rate of the molecular clock (ucld.mean parameter), while using default settings for the parameters that describe substitution rates, nucleotide frequencies, and a Yule tree prior. I ran three independent chains of 100 million steps, sampling every 10,000 steps. I used dates from two extinct primates known from the fossil record - Stirtonia (13.15 Mya) and Solimoea (6.9 Mya) - to calibrate divergence times in BEAST 1.8. Stirtonia tatacoensis was found in Colombia (Flynn et al., 1997) as dental remains in the late 1940s (Stirton, 1951); the species has been widely recognized as related to the Alouatta group (e.g., Szalay and Delson, 1979; Setoguchi et al., 1981; Delson and Rosenberger, 1984; Rosenberger 1992; Hartwig and Meldrum ,2002; but see Hershkovitz, 1970). The other extinct species used, Solimoea acrensis (6.9 Mya), was also described based on a small set of isolated dental elements from Brazil’s Solimões formation (Kay and Cozzuol, 2006).

For all three approaches (MrBayes, BEAST, *BEAST), I assessed stationary and convergence of model parameters in Tracer 1.5 (Rambaut and Drummond, 2009), applied a 10% burn-in, and combined the three runs in LogCombiner 1.8 (with a 10% burn-in). I then summarized a maximum clade credibility tree in TreeAnnotator 1.8 (Drummond et al., 2012). The final trees were visualized in FigTree 1.4. (available from http://tree.bio.ed.ac.uk/software/figtree/).

Results

Phylogenetic Analyses

Of the 16 pairs of primers screened, I was able to successfully amplify and obtain high quality DNA sequences from 14 loci (see Appendix 4 for amplification success per species); two loci were not included in subsequent analyses (M113t7 and M1803), since those resulted in a poor quality sequences. For the set of 14 markers used, the average size of sequenced fragment was 604 base pairs (bp; ranging from 332 bp to 904 bp). The mean number of polymorphic sites per marker was 26 (ranging from 12 to 46, and the mean number of phylogenetically informative sites was 12 (ranging from 6 to 36); analyses were conducted using MEGA version 4 (Tamura, Dudley, Nei, and Kumar, 2007; Appendix 5).

All phylogenetic trees showed maximum support (posterior probability (PP) of 1.0) for a Meso- American clade containing Alouatta pigra and A. paliatta. The South American clade comprising A. guariba, A. belzebul, A. seniculus, A. caraya, and A. sara also gained high support (MrBayes tree PP= 0.91, Fig. 3; *BEAST PP= 0.99 Fig. 4; dated tree 1, values can be seen in Appendix 6).

Both the MrBayes gene tree and the species tree placed the Atlantic forest A. guariba as sister to the remaining South American howlers with high support (MrBayes PP= 0.91, *BEAST PP= 0.89). The dated BEAST analysis placed A. guariba with A. belzebul, separately from the remaining South American species, but this clade was very weakly supported (PP= 0.44, Fig. 5).

There was, however, no agreement regarding the relative positions of A. belzebul and A. seniculus relative to A. sara and A. caraya. Both gene trees (using MrBayes and BEAST) placed A. seniculus as the sister to the clade that contains A. sara and A. caraya (support in the MrBayes tree 0.82, Fig. 3; in the dated BEAST tree 0.98, Appendix 6) . In contrast, the species tree (*BEAST) inferred A. belzebul to be sister to the A. sara - A. caraya clade, albeit with weak support (0.44; Fig. 4).

All inference methods recovered a sister-species relationship between A. sara and A. caraya. However, support for this clade was only low to moderate (PP= 0.5 in the Mr. Bayes tree, Fig. 3; PP= 0.4 in the species tree, Fig 4; in 0.86 in the dated BEAST tree, Fig. 5, value placed on branches in Appendix 6).

Figure 3: Bayesian Inference tree built with Mr. Bayes, based on concatenated sequences. Posterior probabilities values are provided next to nodes, values only between species.

Figure 4: Species tree generated in *BEAST, following a coalescence-based approach. Posterior probability (PP) values next to nodes.

Estimates of Divergence Times

Fossil dating in BEAST placed the age of the genus Alouatta at ca. 6.1 Mya (95% Highest Posterior Density, or HPD = 5.2 - 6.9 Mya). The Meso-American clade containing Alouatta pigra and A. palliata was dated back to ca. 1.3 Mya (HPD 0.9 – 1.9 Mya), while the South American taxa coalesce at a node dated to ca. 5.1 Mya (HPD 4.3 – 6.0 Mya). The clade that contains A. sara and A. caraya was dated back to ca. 2.9 Mya (HPD 2.2-3.6 Mya), see Fig. 5.

Figure 5: Dated Bayesian analysis based on concatenated sequences and two fossils, Stirtonia (13.15 Mya) and Solimoea (6.9 Mya), placed as per arrows. Bars represent the 95% highest posterior densities (HPD), above the bars values of divergence, in ages (My) on nodes discussed in text.

Discussion

All phylogenetic trees recovered here support previous studies that place Alouatta palliata and A. pigra in a Meso-American clade sister to the South American howlers (Villalobos et al., 2004;

Figueredo, 1998; Cortés-Ortiz et al., 2003).

In disagreement with the trees of Meireles et al. (1999) and Cortés-Ortiz et al. (2003), however, I found little to no support for recognizing A. belzebul and A. guariba as sister species. A sister relationship between these two species was only detected in the dated BEAST tree (Fig. 5), albeit with very low support (PP= 0.44). Instead, both the MrBayes (Fig. 3) and *BEAST analyses (Fig. 4) place the Atlantic forest-based A. guariba as the sister lineage to all remaining South American species. This differs from any topology previously published for this group, and suggests a biogeographical history for the genus in which the easternmost Atlantic Forest species is inferred to have split from the ancestor of all species that today occupy the Cerrado and Amazonia. In breaking up the close sister-species relationship previously hypothesized for A. guariba and A. belzebul, the results here resemble that of Villalobos et al. (2004). As such, their close relationship can no longer be assumed and should be further analyzed.

The relationships among the remaining South American species, however, are less clear – particularly regarding the placement of A. seniculus and A. belzebul. The concatenated Mr.Bayes gene tree placed A. belzebul as sister to a clade that includes A. sara, A. caraya and A. seniculus, with moderate support. In placing A. belzebul as external to a clade that contains A. seniculus, the tree recovered by MrBayes (Fig. 3) resembles that of Villalobos et al. (2004) and Cortés- Ortiz et al. (2003). In contrast, the species tree, placed A. seniculus as the sister species to a clade that includes A. sara, A. caraya and A. belzebul, but support for this arrangement was low. In addition, although A. caraya and A. sara were recovered as sister species in all analyses performed here, this relationship consistently had low support. This clade arrangement is a novel result relative to both Cortés-Ortiz et al. (2003) and Villalobos et al. (2004), which recovered A. sara as more closely related to A. seniculus, with A. caraya being place more externally on the tree.

The lack of resolution among the Amazonian and Cerrado-based howlers does not appear surprising to me, and may be related with the age of the species. I propose that, as the first one to diverge, A. guariba may have had more time to undergo allele sorting relative to the remaining South American howlers, whereas the more recently derived species may still be showing incomplete lineage sorting. It is well known that the latter can lead to difficulties in phylogenetic

15 reconstruction, even when coalescence methods are used (Pamilo and Nei, 1988; Maddison and Knowles, 2006; Edwards et al., 2016). However, the inclusion of samples of A. belzebul from the northern Atlantic Forest – thus in closer proximity with the range of the coastal A. guariba – may shed light on this issue. All A. belzebul specimens used in this analysis were collected in the Amazon region.

Evident from my analysis are the observed differences in the topologies inferred from the distinct methods used. Although a combination of gene tree and species tree approaches has been successfully used to infer the phylogeny of African primates based on multiple loci and or mitochondrial genome (Springer et al., 2012; Pozzi et al., 2014; Guevara and Steiper, 2014), this does not seem to be the case for howler monkeys. One potential reason for the differences observed across previous studies, and the difficulty in obtaining well-resolved phylogenies in the present investigation, may be caused by the difficult access to Alouatta DNA samples. For instance, this study does not include samples of Alouatta arctoidea, which was inferred to belong to the South American clade by Cortés-Ortiz et al. (2015). I also was unable to sample A. nigerrima, which is a species recognized by Groves (2005) and Gregorin (2006) based on morphometric data analyses. Another source of potential differences between this and previous studies concerns the methods of inference used. Species trees (*BEAST) and gene trees (used in the Mr. Bayes and BEAST trees) can disagree (Lambert et al., 2015; Edwards, 2009; and Heled and Drummond, 2010). Large differences between these methods can stem from how they are affected by patterns of missing data across species (Lambert et al., 2015; Xi and Davis, 2015). Moreover, it has been proposed that the absence of a full dataset can interfere with the ability of *BEAST to infer the species tree (Heled and Drummond, 2010). This was the case of this study: I was unable to obtain DNA sequences for all the species across markers. (Appendix 4). It seems that additional sampling is needed to elucidate the relationships within this group.

In spite of these differences, the divergence time estimation analyses recovered similar dates to previous studies. Specifically, my estimates agreed with those based on mitochondrial DNA sequences by Cortés-Ortiz et al. (2003). For instance, those authors had timed the split between the Mesoamerican and the Brazilian groups of howlers at about 6.8 Mya. My analysis, which was solely based on nuclear markers, estimated nearly the same divergence time, at 6.11 Mya

(HPD = 5.2 - 6.9 Mya. Cortés-Ortiz et al. (2003) also estimated the divergence between Alouatta guariba and Alouatta belzebul at 4.0 Mya, whereas the present analysis recovered a split dating back to 4.3 Mya (HPD = 3.6 – 5.2 Mya). Other relationships within the South American group differed between my analysis and that of Cortés-Ortiz et al. (2003) and are not discussed here. However, the present analysis differed somewhat from that of Cortés-Ortiz et al. (2003) in the dating of the divergence between the two Mesoamerican species sampled. While I recovered a very recent divergence time for Alouatta pigra and A. palliata, (1.34 Mya; HPD: 0.9 – 1.9), Cortés-Ortiz et al. (2003) estimated it at 2.4 Mya. This relatively small difference, however, is not surprising - for my methods are very distinct from those used from Cortés-Ortiz et al. (2003). Not only the markers used in this study were different, but also the fossils used to calibrate the tree. While I used two fossils related with the Alouatta group, Stirtonia (13.15 Mya) and Solimoea (6.9 Mya), Cortés-Ortiz et al. (2003) used the chimpanzee-human split separation under Sanderson’s (1997) nonparametric rate smoothing approach.

I conducted this study motivated by the many different hypotheses previously proposed for Alouatta. My goal was to use multiple nuclear markers to find a well-supported topology for the group, to guide future discussions of the biogeography and history of these important Neotropical monkeys. By using numerous non-coding nuclear markers, which were thought to potentially resolve phylogenetic branching events left ambiguous by coding nuclear markers (Wildman et al., 2009), I sought to contribute to a discussion that was previously solely based on mitochondrial DNA sequences. Nevertheless, that was only partially accomplished. My results differ from all other published hypotheses – and I also find uncertainty in topology estimation that may be tied to the methods utilized here, and sampling difficulties. However, it supports a new hypothesis of early divergence of the Atlantic Forest-based A. guariba relative to the remaining South American howlers, and increases the certainly of divergence times previously proposed for the group. While my study advances our knowledge of evolutionary relationships in howler monkeys, some standing questions remain. Tackling these questions will rely on improved taxonomic, geographic, and genomic sampling in Alouatta.

Acknowledgements

I thank CCNY and NSF for funding the project and my collaborators, Dr. Ivan Prates during analyses in NYC, Dr. Wilson Araújo da Silva (USP/Hemocentro Ribeirao Preto), for lab support in Brazil. Samples were provided by Dr. Celia Koiffmann (Universidade de São Paulo), Dr. Iracilda Sampaio (Universidade Federal do Pará), Dr. Derek Wildman (University of Illinois), and Dr. Liliana Cortés-Ortiz (University of Michigan). A PSC-CUNY Award 66309-0044 to Eugene E. Harris helped support this project.

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Appendix 1: Sampled species and localities.

Species Locality Latitude Longitude 306 Alouatta guariba Rio de Janeiro/RJ - 22.9068467 - 43.1728965 479 Alouatta guariba Tremembé/SP - 23.4598631 - 46.6254738 478 Alouatta guariba Mairiporã/SP - 23.3195045 - 46.5902794 SC2 Alouatta guariba Blumenau/SC - 26.9165792 - 49.0717331 116 Alouatta caraya Presidente Epitácio/SP - 21.7668575 - 52.1096427 118 Alouatta caraya Presidente Epitácio/SP - 21.7668575 - 52.1096427 120 Alouatta caraya Presidente Epitácio/SP - 21.7668575 - 52.1096427 8 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 9 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 5 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 37 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 38 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 66 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 38 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 71 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 74 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 85 Alouatta caraya Hydroelectric Manso, Mato Grosso - 14.870668 - 55.785335 Left side Tocantins River, UHE 821 Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 658 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 493 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 585 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 602 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 656 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 319 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 318 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 809 Left side Tocantins River, UHE Alouatta belzebul Tucuruí, Pará - 3.8321465 - 49.6427149 3044 Alouatta seniculus Trombetas River, Pará - 0.8641523 - 56.9774343 3087 Alouatta seniculus Trombetas River, Pará - 0.8641523 - 56.9774343 2501 Left side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917973 - 59.475864 2542 Left side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917222 - 59.475864 2521 Right side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917222 - 59.473611

2537 Right side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917222 - 59.473611 2559 Right side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917222 - 59.473611 2541 Right side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917222 - 59.473611 2551 Right side Uatumã River, UHE Balbina, Alouatta seniculus Amazonas - 1.917222 - 59.473611 208 Alouatta palliata Cascajal, Veracruz -17.6391699 -94.15111 175 Alouatta palliata xtal, Veracruz -19.1599498 -96.133607 526 Alouatta pigra El Tormento, Campeche -18.6133333 -90.796944 193 Alouatta pigra Pac Chen, Quintana Roo -20.6509367 -87.636137 12 Alouatta seniculus No location No location No location 121 Alouatta sara No location No location No location

Appendix 2: Species and sequences used from GenBank.

Species Gene GenBank # Length Alouatta belzebul M002 KC760209.1 318 bp Alouatta belzebul M003 KC760244.1 479 bp Alouatta belzebul M085 KC760970.1 311 bp Alouatta belzebul M093 KC761003.1 445 bp Alouatta belzebul M194 KC761125.1 453 bp Alouatta belzebul M201 KC761158.1 732 bp Alouatta belzebul M252 KC761551.1 680 bp Alouatta belzebul M254 KC761585.1 428 bp Alouatta belzebul M258 KC761614.1 719 bp Alouatta belzebul M265 KC761676.1 836 bp Alouatta belzebul M271 KC761767.1 688 bp Alouatta belzebul M4344 KC760662.1 415 bp Alouatta caraya M002 KC760210.1 319 bp Alouatta caraya M003 KC760245.1 475 bp Alouatta caraya M085 KC760971.1 353 bp Alouatta caraya M093 KC761004.1 445 bp Alouatta caraya M190 KC761096.1 605 bp Alouatta caraya M194 KC761126.1 462 bp Alouatta caraya M201 KC761159.1 732 bp Alouatta caraya M252 KC761552.1 745 bp Alouatta caraya M254 KC761586.1 421 bp Alouatta caraya M258 KC761615.1 660 bp Alouatta caraya M265 KC761677.1 822 bp Alouatta caraya M271 KC761768.1 694 bp Alouatta caraya M1701 KC762029.1 596 bp Alouatta caraya M4344 KC760663.1 888 bp Alouatta palliata M002 KC760211.1 320 bp Alouatta palliata M003 KC760246.1 479 bp

Alouatta palliata M085 KC760972.1 353 bp Alouatta palliata M190 KC761097.1 620 bp Alouatta palliata M194 KC761127.1 461 bp Alouatta palliata M201 KC761160.1 723 bp Alouatta palliata M252 KC761553.1 638 bp Alouatta palliata M258 KC761616.1 708 bp Alouatta palliata M265 KC761678.1 835 bp Alouatta palliata M4344 KC760664.1 807 bp Ateles belzebuth M003 KC760247.1 478 bp Ateles belzebuth M085 KC760973.1 358 bp Ateles belzebuth M093 KC761005.1 444 bp Ateles belzebuth M190 KC761098.1 620 bp Ateles belzebuth M194 KC761128.1 464 bp Ateles belzebuth M201 KC761161.1 727 bp Ateles belzebuth M252 KC761554.1 720 bp Ateles belzebuth M254 KC761587.1 425 bp Ateles belzebuth M258 KC761617.1 719 bp Ateles belzebuth M265 KC761679.1 468 bp Ateles belzebuth M271 KC761769.1 694 bp Ateles belzebuth M1701 KC762031.1 587 bp Ateles belzebuth M4344 KC760665.1 819 bp Ateles belzebuth M002 KC760212.1 320 bp Ateles geoffroyi M002 KC760213.1 320 bp Ateles geoffroyi M003 KC760248.1 478 bp Ateles geoffroyi M093 KC761006.1 435 bp Ateles geoffroyi M190 KC761099.1 620 bp Ateles geoffroyi M194 KC761129.1 464 bp Ateles geoffroyi M201 KC761162.1 727bp Ateles geoffroyi M252 KC761555.1 720 bp Ateles geoffroyi M254 KC761588.1 324 bp Ateles geoffroyi M258 KC761618.1 705 bp Ateles geoffroyi M265 KC761680.1 790 bp Ateles geoffroyi M271 KC761770.1 693 bp Ateles geoffroyi M4344 KC760666.1 887 bp Ateles paniscus M002 KC760214.1 320 bp Ateles paniscus M003 KC760249.1 478 bp Ateles paniscus M085 KC760974.1 313 bp Ateles paniscus M093 KC761007.1 445 bp Ateles paniscus M190 KC761100.1 620 bp Ateles paniscus M194 KC761130.1 464 bp Ateles paniscus M201 KC761163.1 718 bp Ateles paniscus M252 KC761556.1 742 bp Ateles paniscus M254 KC761589.1 421 bp Ateles paniscus M258 KC761619.1 701 bp Ateles paniscus M265 KC761681.1 792 bp Ateles paniscus M271 KC761771.1 691 bp Ateles paniscus M1701 KC762032.1 598 bp

Ateles paniscus M4344 KC760667.1 889 bp Brachyteles arachnoides M002 KC760215.1 319 bp Brachyteles arachnoides M003 KC760250.1 478 bp Brachyteles arachnoides M085 KC760975.1 342 bp Brachyteles arachnoides M093 KC761008.1 445 bp Brachyteles arachnoides M190 KC761101.1 618 bp

Brachyteles arachnoides M194 KC761131.1 464 bp

Brachyteles arachnoides M201 KC761164.1 683 bp

Brachyteles arachnoides M252 KC761557.1 750 bp

Brachyteles arachnoides M254 KC761590.1 429 bp Brachyteles arachnoides M258 KC761620.1 718 bp Brachyteles arachnoides M265 KC761682.1 838 bp Brachyteles arachnoides M271 KC761772.1 686 bp Brachyteles arachnoides M4344 KC760668.1 888 bp Lagothrix lagotricha M002 KC760216.1 306 bp Lagothrix lagotricha M003 KC760251.1 476 bp Lagothrix lagotricha M085 KC760976.1 353 bp Lagothrix lagotricha M093 KC761009.1 442 bp Lagothrix lagotricha M190 KC761102.1 478 bp Lagothrix lagotricha M194 KC761132.1 464 bp Lagothrix lagotricha M201 KC761165.1 731 bp Lagothrix lagotricha M252 KC761558.1 719 bp

Lagothrix lagotricha M254 KC761591.1 430 bp

Lagothrix lagotricha M265 KC761683.1 837 bp

Lagothrix lagotricha M271 KC761773.1 692 bp

Lagothrix lagotricha M1701 KC762033.1 592 bp Lagothrix lagotricha M4344 KC760669.1 887 bp

Appendix 3: Primes, forwards and reverses sequences.

Marker Strand Primers M002 F CAG CAT CTT AGA CCT GAC CAG TGA R CCA GAT ACT CAT TGT GTC TCA CA M003 F GCC TAA GAT CTA ATC AGC ACA TTG R TCT GTA TCT GCC TTT GTA AGA GG M085 F GCT TCA AAC ACT CAG TGC TC R CAG GCA GYT TCA ATG ATT CTC M093 F TAA CTA GAG TGA ACA TTT GGA CTG R ATA GAG GGC TAC AGA ACG M190 F CAC TGG CCA TYC AGC CTC CTG GT R TGT CTG GTG CTA CAT CCA GAG C M194 F ATT CTA TTC CCT GTG ATG AWA GCA GA R TCT TTT CAC TCA ACA TAT GCC TGG A M201 F GGT AAC CAG TTT GGG CTT CTG R CAT CGT CCT GGT TGT GAA GTG M252 F GAG CAG TCA TGA TAC AAA ATA GGG R CTC AAG TAT TCT TTG TAT CTG GC M254 F ACA TAT GAC TCA GGC CAA ATC R GCA TTG CAG TAG CTA GCA C M258 F CCA AGG CAT AGT GTC TTA ACA R AAC CTG TCC CTG TAT CTA AAA C M265 F TCC ATA GTA CAC CAA GGG ACC R CCA ATA GTA TGC ACT GTG AGC ATG M271 F CTT GAA CAA CAC ATA TTT CCA ACC R GAA AGA TGG CAT CTA CTG GTG A M1701 F GTC CTG GAT TTC CTA TCA CC R GGA GCT GTC TTC CTC TGT AA M4344 F AAG GAA CCA CCA CTA GGA G R GTG CAA CAT TTA TCA TGA CTT C M113T7 F CAT GCT AGC AAG TAA GCT TGT C R GAG ATG GGT CCT TCC TGT CC M1803 F GCA TAC ATG CTT GCA TCC AA R GCA TAG TAG GGG TGC ACA G

Appendix 4: Amplification success across markers and species.

Marker A. caraya A. belzebul A. guariba A. sara A. seniculus M002 YES YES NO YES YES M003 YES YES YES YES YES

M085 YES YES YES YES YES

M093 YES YES YES YES NO M190 YES YES YES NO YES M194 YES YES YES YES YES M201 YES YES NO YES YES

YES M252 YES NO YES YES M254 YES YES YES NO YES M258 YES YES YES YES YES M265 YES YES YES YES YES

M271 YES YES YES YES YES

M1701 YES YES YES YES YES M4344 YES YES YES NO YES M113T7 NO NO NO NO NO M1803 NO NO NO NO NO

Appendix 5. Number of polymorphic sites, number of phylogenetically informative sites, and models of nucleotide substitution per marker.

Marker Polymorphic Phylogenetically Informative Length bp Model selected M002 14 6 332 HKY + I M003 12 11 480 HKY + I M085 19 10 367 HKY M093 14 6 450 HKY M190 28 7 620 HKY + G M194 18 10 472 HKY + G M201 10 6 738 HKY + G M252 46 36 763 HKY M254 29 10 460 HKY M258 27 8 726 HKY + G M265 24 9 843 GTR + G M271 46 19 703 GTR M1701 29 11 600 K80 M4344 43 15 904 HKY

Appendix 6: Dated tree, Posterior probability (PP) values next to nodes between species.