Revisiting the relationships among howler monkeys through molecular phylogenetic analysis (Primates; Atelidae; Alouatta)

The howler monkeys (Alouatta) are part of the Platyrrhini infraorder, a group of Neotropical primates with the widest geographical distribution, occurring from southern Mexico to northern Argentina. Previous phylogenetic studies based on cytogenetic, morphological, and molecular data have shown inconsistent results, requiring new approaches to clarify relationships within the genus. In this study, we analyzed nine species of Alouatta using 24 molecular markers (2 mitochondrial and 22 nuclear genes). Through Bayesian inference, Maximum Likelihood, and divergence time analyses, we inferred the phylogeny and estimated the timing of speciation events. Our results recovered two major clades within Alouatta, corresponding to Mesoamerican (Trans-Andean) and South American (Cis-Andean) lineages. Most diversification events occurred during the Pliocene. Within the South American clade, we identified two well-supported groups: one composed of species from the Atlantic Forest and eastern Amazon (A. guariba, A. belzebul, and A. discolor), likely shaped by the formation of the South American dry diagonal; and another formed by A. caraya and the A. seniculus complex. Notably, our results confirmed a close phylogenetic relationship between A. discolor and A. belzebul, and between A. macconnelli and A. nigerrima, which occur on opposite sides of the Amazon River.These relationships, along with the confirmation of A. discolor and A. nigerrima as distinct species based on multilocus evidence from known localities, represent advances over previous studies and contribute to a more resolved understanding of Alouatta diversification.