SUPPORTING INFORMATION for the description of Dendrocolaptes retentus
Molecular analyzes.— Taxa sequenced: We sequenced all known taxa grouped under the polytypic
Dendrocolaptes certhia in addition to the new taxon retentus described herein (73 specimens; Table 1).
We used sequences of five specimens of D. sanctithomae as the outgroup in our analyzes, the known
outgroup of the D. certhia complex (Derryberry et al. 2011). DNA sequencing: Standard methods were
used to extract, isolate, amplify and sequence two mitochondrial genes. Total genomic DNA was
extracted from frozen or ethanol preserved tissue samples using a a standard phenol/chloroform method
(Hillis et al. 1990). We obtained sequences of the mitochondrial genes cytochrome b [c.a 1016 bp,
primers L14990 and H16064 (Sorenson et al., 1999) and NADH dehydrogenase subunit 2 – ND2 [1041
bp, primers L5215 and H6313 (Johnson and Sorenson 1998, Sorenson et al. 1999)]. All primer numbers
refer to the 3’ base of the published chicken mtDNA sequence (Desjardins and Morais 1990). Fragments
were PCR amplified using standard conditions available upon request. A small aliquot of each
amplification was electrophoresed on agarose gel to check for the correct fragment size and to ensure that
only a single amplification product was obtained. Amplification products were cleaned with a Qiagen
PCR purification kit and cycle-sequenced using a Big Dye Terminator kit (Perkin Elmer, Norwalk,
Connecticut), and all amplification primers listed above. Cycle sequencing reactions were NH4OAC
precipitated, dried, resuspended in formamide EDTA, and run on ABI 3730 and ABI 3130 DNA
Sequencer. We aligned and reconciled sequences from both strands within and between species using
BioEdit version 7.1.7 (Hall 1999). The following measures outlined by Sorenson and Quinn (1998) and
Bates et al. (1999) were taken to ensure that the DNA fragments amplified were accurate and of
mitochondrial origin (not pseudogenes): (1) most sequences were amplified in large fragments (> 1,000
bp); (2) both DNA strands were sequenced; (3) sequences were aligned with the chicken complete
mtDNA sequence, and inspected for insertions, deletions, and stop codons that would result in a
nonfunctional protein; and (4) sequences were expected to exhibit high transition to transversion
substitution ratios characteristic of mitochondrial, not nuclear substitution patterns. Phylogenetic
analyzes: We performed a Bayesian inference (hereafter BI) of phylogeny using the MrBayes software,
version 3.1.2 (Ronquist and Huelsenbeck 2003). Evolutionary models for BI were selected by
MrModeltest 2.3 (Nylander 2004), under the Bayesian information criterion (BIC). Bayes factors
(referred to as BF) were employed to determine the optimal number of partitions to be used in the BI.
Convergence for all parameters was assessed by evaluating whether stationarity was reached by the
Markov chain using the Tracer v1.4.1 software (Rambaut and Drummond 2007). BI were run with two
heated chains for 20 million generations, with resulting trees and parameters being sampled every 1000
generations. We discarded the first 2000 generations as “burn-in” after finding that stationarity was
reached well before this number in each run. To assess the clades’ nodal support, we used the remaining
4001 sampled trees to calculate posterior probabilities.
Morphometric and plumage analyzes.— We examined 378 specimens of all taxa grouped under the
polytypic Dendrocolaptes certhia, except D. c. polyzonus, deposited in the following museums: Museu
Nacional, Rio de Janeiro, Brazil (MN), Museu Paraense Emílio Goeldi, Belém, Brazil (MPEG), and
Museu de Zoologia da Universidade de São Paulo (Appendix). Characters analyzed: Our analyses were
based on morphometric as well as continuous and discrete plumage characters (results available upon
request). For each specimen examined, we measured with a Mitutoyo© digital caliper to the nearest 0.01
mm or counted the following morphometric and continuous plumage characters: 1) wing length; 2) tail
length; 3) tarsus length; 4) bill length from the anterior end of nostril to the tip of the culmen; 5) bill depth
at the anterior end of the nostrils; 6) bill width at the anterior end of the nostrils; 7 - 16) average number
and width of head, throat, back, chest, and belly stripes within a secured area of 10 x10 mm located at the
center of each body part. For each specimen analyzed, the following discrete plumage characters were
also analyzed: 17-20) shape of the head, dorsal, and ventral spots; 21-24) color of the head stripes, belly,
back, and neck. We used Smithe (1975, 1981) as a standard color reference when describing plumage
features. Statistical analyzes: Groupings for statistical analysis of morphological data were based on the
molecular phylogeny obtained, which recognized seven main natural groupings (lineages or populations)
in D. certhia complex. We assessed normality of morphometric data with Kolmogorov-Smirnov tests.
The presence of sexual dimorphism among the populations studied was evaluated by a Hotelling
discriminant analysis. We used Discriminant-Function Analyses (DFA) to test for differences in the
morphometric space among groups. All statistical analyses were performed with software SYSTAT 12
for Windows (Systat Software, San Jose, California, USA - SPSS, 1998). In all tests statistical
significance was accepted at P≤ 0.05.
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