The species Myrmotherula iheringi (Thamnophilidae) is composed of two subspecies occurring in central-western Amazonia: M. i. iheringi and M. i. heteroptera. Morphologically, these taxa are differentiated only by subtle plumage characters of the females. However, recent field studies revealed that M. i. iheringi and M. i. heteroptera also have distinct vocalizations, which led to the suspicion that they are independent evolutionary units. To this end, we assessed the level of evolutionary independence of these taxa based on 1701 bp of the mitochondrial ND2 and citb genes, sequenced for one specimen of M. i. iheringi and 10 specimens of heteroptera. In addition, 39 skins deposited in the collections of the Museu Paraense Emilio Goeldi, Museu de Zoologia da Universidade de São Paulo and the Carnegie Museum of Natural History were analyzed, corresponding to 10 localities, which encompass the entire distribution of M. iheringi. The following characters were used: Wing length, Tail length, Tarsus length, Beak length, Beak height and Beak width; and analyzed using a testt to detect differences between taxa and multivariate analyses (PCA and AFD) to demonstrate segregation between them. Phylogenetic analyses were performed in PAUP 4.0b software using Maximum Parsimony (MP) and Maximum Likelihood (MV) methods.MP and MV trees were congruent (0=0.69; RI=0.90). The t-test was used to show the existence of three main data with strong statistical support: data A, inducing the specimens of M. i. ihering from the Rondônia center of endemism; data B, containing the specimens in the southern portion of the Inambari center of endemism; and data C, containing the specimens from the northern portion of the Inambari center of endemism. According to the t-test, few morphological characters are diagnostic between taxa from data A and B; only the characters Comproda cauda and Altura do beico presented significant differences. However, when compared to data C, all characters showed differences. AACP also corroborated this segregation, demonstrating that tail length and beak measurements account for approximately 67% of the variation. Discriminant function analysis showed that 89% of the specimens considered within data A were correctly grouped; similarly 75% of the specimens considered as belonging to data B and 89% of the specimens considered from data C, were also correctly grouped (Wilks' lambda = 0.213; F= 4.280; P = 0.000). In addition to these differences, data A B and C correspond to the two distinct vocal types of M. iheringi, reinforcing their status as independent species. The data also revealed that populations assigned to M. i. heteropterado east of Acre are closer to M. i. iheringi, which is also corroborated by vocal characters.
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