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Four of the concatenated ddRADseq loci data matrices assembled in pyRAD were used for phylogenetic inference: A85 and A92 (all samples); B85 (select individuals from each taxon); and PD85 (only individuals of the P. moloch group and Cheracebus). These datasets contained between 1129 and 1987 ddRADseq loci, with a total concatenated length of between 345K and 603K bp, and around 11K to 15.4K parsimony informative sites across all samples (Table 4.9). A total of nine phylogenetic trees were reconstructed using the ddRADseq data; maximum- likelihood and Bayesian (MrBayes) trees were inferred for each of the datasets, and phylogeny and diversification times were jointly estimated for the B85 dataset using BEAST. Species relationships recovered across all trees for all datasets are identical, with only minor topological conflict at some intraspecific nodes (e.g., within P. bernhardi and P. caligatus). As most nodes show perfect support (bootstrap percentage, BP = 100%; posterior probability, PP = 1.0) or strong support (e.g., BP > 95%; PP > 0.99) across the analyses (see Table 4.11), only insignificant (BP < 70%; PP < 0.95) or intermediate support values are mentioned below. Note posterior probability values for the B85 BEAST and MrBayes analyses are not included in Table 4.11 because all nodes show perfect support (PP = 1.0).

In agreement with all previous molecular phylogenies (e.g., Chapter 2 + 3, Carneiro et al., 2016), Cheracebus is recovered as the earliest diverging lineage within Callicebinae, and Callicebus (represented by C. personatus) and Plecturocebus are sister clades (Figure 4.3). Despite the extremely low sequencing coverage, the divergence between C. personatus and Plecturocebus is strongly resolved in most analyses (moderate support in A92, BP = 86%). Among taxa of the genus Cheracebus, C. lucifer and C. purinus are sister species, and for C. lugens, individuals from the left and right bank of the Rio Negro (LN and RN) form a clade to the exclusion of the left bank Rio Japurá (LJ) sample, but with low support at the LN/RN node in some ML trees (e.g., A85 + A92, BP = 53%).

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Figure 4.3 Maximum likelihood tree inferred with the ddRADseq PD85 dataset (Plecturocebus and Cheracebus). Unmarked nodes were strongly supported (BP = 100%), nodes marked with a black circle received significant support (BP = 80 – 99%), while the nodes marked with white circles were recovered without significant support (BP < 70%). Support values are listed according to node numbers in Table 4.11.

The Plecturocebus moloch species group is divided into three major clades, as recovered in the BEAST phylogeny based on seven loci in Chapter 3, and the informal clade names suggested are followed here; the Aripuanã-Tapajós clade containing P. hoffmannsi, P. miltoni and P. cinerascens; the Eastern clade containing

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P. bernhardi, P. cf. moloch, and P. moloch; and the Western clade comprised of P. brunneus, P. caligatus, and P. cupreus. The Aripuanã-Tapajós clade is the earliest diverging lineage within the P. moloch group, while the Eastern and Western clades are sister (B85 BP = 91%; PD85 BP = 87%). Among the Aripuanã-Tapajós species, Plecturocebus hoffmannsi is sister to a P. cinerascens + P. miltoni clade, and among Eastern clade taxa, P. bernhardi is sister to a clade containing P. moloch and P. cf. moloch. Three relatively divergent lineages are represented by the four P. bernhardi specimens in the ddRADseq datasets, and most analyses recover the UFRO(413) and CCM(173) individuals as successive sister lineages to the P. bernhardi clade A specimens (note, the CCM173 sample wasn’t included in the B85 dataset), but with low support at the CCM vs. clade A node in the ML trees (A85 BP = 63%; PD85 BP = 65%). An alternative topology, however, suggests that the UFRO and CCM P. bernhardi individuals are sister (A92 BP = 87%; Figure A3.1, A3.2). This is the only notable conflict between the A85 and A92 analyses, and thus, the clustering threshold used to assemble the ddRADseq datasets appears to have had minimal impact on the recovered species relationships.

Among Western clade taxa, P. cupreus and P. caligatus are recovered as sister species but with low support in the ML trees (A85 BP = 60%; A92 BP 78%; B85 BP = 61%), while P. brunneus is the earliest diverging lineage within the clade. In the A85 and A92 analyses including all samples, P. c. dubius is paraphyletic, although some of the nodes within the P. caligatus complex are poorly supported (see Figure A3.1 – A3.4). Subsequently, one P. c. dubius (UFRO427) and one P. c. caligatus (CTGAM181) sample were excluded from the PD85 dataset because of low sequencing coverage and, in these analyses, P. c. dubius and P. c. caligatus are recovered as monophyletic sister taxa (note, only one sample from each was included in the B85 analyses). Overall, the topology recovered across the ddRADseq phylogenetic analyses is identical to the BEAST phylogeny based on seven concatenated loci in Chapter 3, except P. caligatus is sister to P. cupreus in the former and P. brunneus in the latter.

Age estimates in the ddRADseq timetree (Figure 4.4, Table 4.11) are generally younger than those recovered in the combined and nuclear dataset analyses in Chapter 2, especially at species-level, but broadly concordant with the results from Chapter 3. The most recent common ancestor (MRCA) of extant Callicebinae taxa is estimated to have diverged in the late Miocene (10.7 Ma; 95% HPD = 7.9 – 13.9),

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giving rise to the progenitor of the genus Cheracebus and of the

Callicebus+Plecturocebus clade. The divergence between Callicebus and

Plecturocebus also occurred in the late Miocene at c. 7.4 Ma (95% HPD = 5.2 – 10.0). All other diversification events among the Callicebinae taxa represented occurred in the Pleistocene. The MRCA of extant Cheracebus taxa is estimated to have lived in the early Pleistocene (1.9 Ma; 95% HPD = 1.3 – 2.4), while C. purinus and C. lucifer diverged at c. 1.4 Ma (95% HPD = 1.0 – 1.9), and the C. lugens lineages diverged at around 0.9 Ma [(95% HPD = 0.6 – 1.2) LJ vs. RN+LN], and 0.7 Ma [(95% HPD = 0.5 – 1.0) RN vs. LN].

The major P. moloch group clades diversified relatively rapidly in the early Pleistocene, with the Aripuanã-Tapajós clade diverging from the ancestor of the Eastern and Western clades at c. 2.0 Ma (95% HPD = 1.5 – 2.6), the Eastern and Western clades diverging at c. 1.8 Ma (95% HPD = 1.3 – 2.3), and P. hoffmannsi diverging from other species of the Aripuanã-Tapajós clade also at c. 1.8 Ma (95% HPD = 1.4 – 2.4). Thus, within around 200K years, four distinct P. moloch group lineages had emerged which likely gave rise to all known extant taxa. The sister taxa of the Aripuanã-Tapajós clade (P. cinerascens and P. miltoni) and the Eastern clade (P. bernhardi and P. moloch/P. cf. moloch) are estimated to have diverged at 1.5 Ma (95% HPD = 1.1 – 2.0) and 1.3 Ma (95% HPD = 1.0 – 1.7), respectively, while the P. cinerascens (clade A and B), P. moloch (vs. P. cf. moloch), and P. bernhardi (UFRO and clade A) lineages all diverged between c. 0.8 – 0.9 Ma. The earliest diverging lineage within the Western clade, P. brunneus, is estimated to have diverged at c. 1.0 Ma (95% HPD = 0.7 – 1.3), followed by the divergence between P. cupreus and P. caligatus at c. 0.9 Ma (95% HPD = 0.6 – 1.1), and subsequently, the P. cupreus lineages (clade A and B) and the P. caligatus subspecies (P. c. caligatus and P. c. dubius) diverged at c. 0.6 Ma (95% HPD = 0.4 – 0.8) and0.7Ma (95% HPD = 0.5 – 0.9), respectively. Individual trees including outgroups and node support values for each MrBayes and RAxML analysis are found in Appendix 3 (Figure A3.1 – A3.8) and the full timetree including outgroups is found in Appendix 3 (Figure A3.9) A summary of node support for all analyses (except the B85 BEAST and MrBayes analyses, PP = 1.00 for all nodes) and divergence date estimates and 95% HPD intervals for the B85 dataset BEAST analysis is presented in Table 4.11.

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Table 4.11 Summary of node support for the ddRADseq phylogenetic analyses and age estimates from the B85 dataset BEAST analysis. Node numbers correspond to those on Figure 4.3, 4.4. Bold indicates low support (PP < 0.95; BP < 70%). Note, PP = 1.00 for all ingroup nodes in the B85 BEAST and MrBayes analyses. Node Divergence B85 A85 A92 PD85 Mean age 95% HPD _ML BP MB PP ML BP MB PP ML BP MB PP ML BP Lower Upper

1 CheracebusCallicebus (personatus) vs. + Plecturocebus

10.7 7.86 13.9 100 1.00 100 1.00 100 1.00 100 2 Callicebus (personatus) _{vs. Plecturocebus} 7.41 5.22 9.97 99 1.00 96 1.00 86 NA NA 3 C. purinus + C. lucifer _{vs. C. lugens} 1.85 1.31 2.43 100 1.00 100 1.00 100 1.00 100 4 C. purinus vs. C. lucifer 1.42 0.98 1.94 100 1.00 100 1.00 100 1.00 100 5

C. lugens:L bank Rio Japurá vs.R bank Rio Negro + L bank Rio Negro

0.92 0.64 1.24 100 1.00 100 1.00 100 1.00 100

6 C. lugens:Negro vs. L bank Rio R bank Rio Negro 0.7 0.47 0.97 86 0.95 53 0.99 53 1.00 87 7 Aripuanã-Tapajós clade vs. Eastern + Western P. moloch clades 2.01 1.51 2.6 100 1.00 100 1.00 100 1.00 100 8 P. hoffmannsi _{cinerascens + P. miltoni} vs. P. 1.84 1.35 2.37 97 1.00 98 1.00 97 1.00 100 9 P. cinerascens _miltoni vs. P. 1.49 1.09 1.95 100 1.00 100 1.00 100 1.00 100 10 P. cinerascens: _{vs clade B} clade A 0.85 0.56 1.18 100 1.00 100 1.00 100 1.00 100 11 Eastern vs. Western _{moloch clades} P. 1.8 1.34 2.33 91 1.00 95 1.00 95 1.00 87 12 P. bernhardi_{moloch + P. moloch} vs. P. cf. 1.32 0.97 1.73 100 1.00 100 1.00 100 1.00 100 13 P._moloch cf. moloch vs. P. 0.77 0.52 1.04 100 1.00 100 1.00 100 1.00 100 14 P. bernhardi: _vs.remaining UFRO413 0.91 0.61 1.25 100 1.00 97 NA NA 1.00 100 19 P. bernhardi: _{vs. clade A} CCM173 NA 1 _NA 1 _NA 1 NA

1 1.00 63 NA NA 1.00 65

-- P. bernhardi:_{+ CCM173 vs.} UFRO413 _{clade A} NA 1 _NA 1 _NA 1 NA

1 NA NA 1.00 87 NA NA

15 P. brunneus vs. P. _{cupreus + P. caligatus} 1 0.74 1.31 100 1.00 100 1.00 100 1.00 100 16 P. cupreus _caligatus vs. P. 0.86 0.63 1.14 61 1.00 60 1.00 78 1.00 94 17 P. c. caligatus _dubius vs. P. c. 0.67 0.47 0.9 100 0.692 432 1.00 2 632 1.00 100 18 P. cupreus: _{clade B} clade A vs 0.58 0.39 0.78 1.00 1.00 100 1.00 100 1.00 100 1 _{CCM173 was not included in the BEAST analysis}

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Figure 4.4 A time-calibrated phylogeny for Callicebinae inferred with the ddRADseq B85 dataset. All nodes received full support (PP = 1.00). Node bars indicate the 95% highest posterior density (HPD). Estimated divergence ages and 95% HPDs are listed according to node numbers in Table 4.11. See Figure A3.9 for the full timetree with outgroups. Illustrations by Stephen D. Nash ©Conservation International.