Inferring evolutionary relationships between recently diverged taxa is still challenging, especially taking into account the likely occurrence of incomplete lineage sorting and/or introgression. The Xiphorhynchus pardalotus/ocellatus species complex includes between two to three polytypic species and eight to nine subspecies distributed throughout most of lowland Amazonia and the foothills of the eastern Andes. To understand its historical diversification and address the main unsettled issues of phylogenetic relationships and taxonomy, we apply several approaches using data from two mitochondrial (Cyt b and ND2) and three nuclear genes (β-fibint7, CPZint3 and CRYAAint1) for all described species and most subspecies of this complex. We compared single gene trees with a multilocus concatenated tree and Bayesian species tree inferred under a coalescent framework ((*)BEAST). Our results showed a general pattern of incongruence among gene trees and multilocus trees. Despite of this, the coalescent-based species tree analysis supports the sister-taxa relationship of X. ocellatus and X. chunchotambo, while X. pardalotus comes out as the basal taxon. With exception of the last, our results revealed within both X. ocellatus and X. chunchotambo high levels of genetic differentiation (p-distances 0.5-5.5%) with well-supported lineages. Our phylogenetic analyses showed several incongruences with current subspecies taxonomy, revealing that X. o. ocellatus is paraphyletic relative to X. o. perplexus, and the currently recognized subspecies X. c. napensis corresponds to two distinct evolutionary lineages, which are not supported as sister-lineages. In addition, the deep level of genetic divergence between X. o. beauperthuysii and the extant subspecies of X. ocellatus is more consistent with species-level differences found in this complex. Divergence time estimates were consistent with a historical scenario of intense population subdivision and speciation during the Early-mid Pleistocene. The spatial pattern and timing of diversification overlap broadly with that reported for other Amazonian vertebrate lineages.
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