Main findings

The goal of my doctoral thesis was to employ molecular data to provide insight into the evolutionary history and biogeography of Callicebinae, one of the most strikingly poorly studied and among the most species-rich groups of primates. To achieve this goal, large sequence data matrices were generated using multi-locus Sanger sequencing (20 nuclear and 2 mitochondrial loci) and reduced representation, genome-wide double-digest restriction-associated DNA sequencing (ddRADseq). In Chapter 2, we inferred phylogeny and diversification times using the multi-locus data and revised the taxonomy of Callicebinae based on molecular, morphological and biogeographic evidence. In Chapter 3, we employed a statistical biogeographical approach to perform ancestral-area estimations across the phylogeny of Callicebinae

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based on time-calibrated trees inferred using the multi-locus data. In Chapter 4, we conducted phylogenetic analyses, assessed genetic structure and tested for interspecific gene flow using genome-wide ddRADseq data matrices, as well as performed coalescent-based species-tree estimation using the multi-locus sequences.

In Chapter 2, we assembled one of the largest multi-locus Sanger sequenced molecular datasets for any group of platyrrhine primates (22 loci, >14K bp in length; 15 titi species, 59 specimens), reconstructed the first comprehensive species-level molecular phylogeny for Callicebinae, and provided the first molecular review of the subfamily. Our phylogenetic analyses (based on concatenated data matrices) clarified a number of issues on the taxonomic and phylogenetic relationships among the species. We provided evidence for an early divergence (late Miocene) of three major Callicebinae lineages, reconstructed a timeline for Callicebinae diversification, and inferred a well-supported phylogeny for all species included, with the exception of P. miltoni and P. cinerascens, which required further investigation. Based on new molecular evidence and well-established differences in morphology, karyology, and biogeography, we proposed a new genus-level taxonomy for titi monkeys:

Cheracebus (Byrne et al., 2016) in the Orinoco, Negro and upper Amazon basins,

Callicebus Thomas, 1903, in the Atlantic Forest and neighbouring Caatinga regions,

and Plecturocebus (Byrne et al., 2016) in the Amazon basin and Chaco region. We

also reviewed the taxonomic history for Callicebinae, suggested the reintegration of cupreus group species (sensu Kobayashi, 1995) into the moloch group, and questioned the designation of P. dubius (Hershkovitz, 1988) as a valid species. This work illustrates the value of considering molecular evidence in taxonomic classification, provides a basis for future studies on the evolutionary history and taxonomy of titis, and has opened a dialogue on other taxonomic issues that had been left unattended for quite some time. The new taxonomic proposal for Callicebinae brings concordance to the classification of genera across New World primates and better describes the great diversity of this poorly studied group.

In Chapter 3, we provided the first known statistical biogeographical approach applied to reconstruct the biogeography of Callicebinae in an explicit phylogenetic framework. We recovered evidence for the divergence of titi monkey genera in the late Miocene via the fragmentation of a widespread ancestor distributed across the modern-day northwestern Amazon (Cheracebus), wet and dry savanna ecosystems (Plecturocebus), and Atlantic Forest (Callicebus). Our reconstruction

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indicated that species-level diversification among the Amazonian clades initiated from a narrow area of origin (Napo, Cheracebus; Rondônia, P. moloch group), and was characterised by a sequential, long-distance dispersal model of speciation by “island-hopping” across pre-existing river barriers. These founder-events (jump dispersal) were sufficiently rare to allow diversification in isolation after dispersal, emphasising the role of major Amazonian rivers as strong barriers to gene flow among allopatric species, with notable comparisons to island biogeography. We uncovered a complex pattern of diversification among species of the P. moloch group, with a non-monophyletic assemblage of taxa endemic to Rondônia (area of endemism), suggesting that a complex history of river system evolution may have played an important role in driving historical distributions in this region. Our results are taken to suggest that the evolution of the Pebas system in the western Amazon may have influenced the diversification and distribution of extant Callicebinae lineages, which were absent from the western Amazon until the recession of these wetlands and the establishment of suitable forest habitat in the Pleistocene (“Young Amazon” model). This work comprises one of the first investigations of the evolutionary history of titi monkeys in the context of Amazonian and South American historical biogeography based on an explicit phylogenetic hypothesis, and sheds light on the processes that generated the great diversity found among Callicebinae taxa. Although this research provides only a large-scale reconstruction of callicebine biogeography and should be interpreted with caution, it represents a critical starting point for future research that aims to understand diversification within this subfamily.

In Chapter 4, we propelled research on titi monkey evolutionary history into the phylogenomics era using double digest restriction-site associated DNA sequencing (ddRADseq) to generate reduced representation genome-wide molecular data for 12 Callicebinae species (45 specimens). This work is among the first phylogenomic analyses employing genome-wide data for any New World primate group. Here, we began to address more difficult questions regarding the phylogenetic relationships among the lineages, particularly among the species of the P. moloch group. The ddRADseq data matrices contained up to ~ 3K loci, with a maximum concatenated length of ~ 930K bp and up to ~ 15K parsimony informative sites; a massive leap in the quantity of data in comparison to all previous molecular studies on Callicebinae. Our ddRADseq phylogenetic analyses recovered a strongly supported topology (perfect support at all nodes in some analyses) for Callicebinae

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with species of the P. moloch group divided into three major clades (Aripuanã- Tapajós, Eastern, and Western clades). Although we advocate that the ddRADseq topology must be considered provisional until assessed using multispecies coalescent- based analyses with genome-wide data, we consider it the most reliable estimate of the P. moloch group species relationships currently available. We provide evidence of introgressive hybridisation using D-statistic tests that is supported by mitochondrial introgression. Collectively, our results indicate that P. cinerascens individuals from the left bank of upstream Rio Roosevelt, Rondônia, are admixed with P. bernhardi, a significant discovery of broad interest to primatologists, specifically for research on New World primates given the lack of natural hybridising study systems. This research represents the first known application of the D-statistic test among New World primates, and one of the only known cases where genetic evidence has been presented regarding admixture between any New World primates other than howler monkeys. Finally, we discuss putative sources of topological incongruence across loci and across previous studies (analytical and biological, e.g., interspecific gene flow, incomplete lineage sorting), and the complicated nature of lineage diversification across the P. moloch group.

Collectively, the chapters of this thesis provide a detailed picture of the evolutionary history of titi monkeys and add to our understanding of what makes the subfamily Callicebinae, the genera, and the many titi species unique. Within a few years, our knowledge of the evolutionary relationships among Callicebinae taxa has progressed rapidly, perhaps in a relatively manner unprecedented among New World primates; from species relationships based almost entirely upon Kobayashi’s (1995) morphological phylogeny, to phylogenetic hypotheses derived from multi-locus or genome-wide molecular datasets that are among the largest known for any New World primate group.