Molecular Phylogenetics and Evolution: Gregory Thom, Alexandre Aleixo

Molecular Phylogenetics and Evolution 82 (2015) 95110
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev
Cryptic speciation in the white-shouldered antshrike (Thamnophilus

aethiops, Aves Thamnophilidae): The tale of a transcontinental
radiation across rivers in lowland Amazonia and the northeastern
Atlantic Forest
Gregory Thom a, Alexandre Aleixo b,
a
b
Curso de Ps-graduao em Zoologia, Universidade Federal do Par/Museu Paraense Emlio Goeldi, Caixa Postal 399, CEP 66040-170 Belm, PA, Brazil
Coordenao de Zoologia, Museu Paraense Emlio Goeldi, Caixa Postal 399, CEP 66040-170 Belm, PA, Brazil
a r t i c l e
i n f o
Article history:
Received 23 June 2014
Revised 20 September 2014
Accepted 26 September 2014
Available online 5 October 2014
Keywords:
Diversication hypotheses
Historical biogeography
Phylogeography
Population genetics
Refuges
Taxonomy
a b s t r a c t
The growing knowledge on paleogeography and the recent applications of molecular biology and phylogeography to the study of the Amazonian biota have provided a framework for testing competing hypotheses of biotic diversication in this region. Here, we reconstruct the spatio-temporal context of
diversication of a widespread understory polytypic Amazonian bird species (Thamnophilus aethiops)
and contrast it with different hypotheses of diversication and the taxonomy currently practiced in the
group. Sequences of mtDNA (cytochrome b and ND2) and nuclear (b-brinogen introns 5 and 7 and the
Z-liked Musk4) genes, adding up to 4093 bp of 89 individuals covering the Amazonian, Andean, and Atlantic Forest populations of T. aethiops were analyzed. Phylogenetic and population genetics analyses
revealed ten reciprocally monophyletic and genetically isolated or nearly-isolated lineages in T. aethiops,
highlighting several inconsistencies between taxonomy and evolutionary history in this group. Our data
suggest that the diversication of T. aethiops started in the Andean highlands, and then proceeded into
the Amazonian lowlands probably after the consolidation of the modern Amazonian drainage. The main
cladogenetic events in T. aethiops may be related to the formation and structuring of large Amazonian rivers during the Late MioceneEarly Pleistocene, coinciding with the dates proposed for other lineages of
Amazonian organisms. Population genetics data do not support climatic uctuations as a major source
of diversication in T. aethiops. Even though not entirely concordant with paleobiogeographic models
derived from phylogenies of other vertebrate lineages, our results support a prominent role for rivers as
major drivers of diversication in Amazonia, while underscoring that different diversication scenarios
are probably related to the distinct evolutionary origins of groups being compared.
2014 Elsevier Inc. All rights reserved.
1. Introduction
Since the 19th century, the high species diversity of the Amazon
allied to multifaceted patterns of geographical distributions instigated naturalists and researchers to explain such complexity
(Wallace, 1852; Haffer, 1969; Bates, 2001).
Despite the large number of distinct hypotheses to explain
Amazonian biodiversity (e.g., refuge hypothesis, Haffer, 1969; riverine hypothesis, Wallace, 1852; Ayres and Clutton-Brock, 1992;
riverine-refuge hypothesis, Ayres and Clutton-Brock, 1992;
Haffer, 1993, 2001; ecological gradients hypothesis, Endler, 1977;
museum hypothesis, Roy et al., 1997; and marine incursions,
Corresponding author.
E-mail address: aleixo@museu-goeldi.br (A. Aleixo).
http://dx.doi.org/10.1016/j.ympev.2014.09.023
1055-7903/ 2014 Elsevier Inc. All rights reserved.
Bates, 2001), and the common sense that many causations have
operated for the formation of such diversity (Bush, 1994; Haffer,
2001; Miller et al., 2008), recent phylogeographic and paleobiogeographic reconstructions (Aleixo and Rossetti 2007; Patel et al.,
2011; Weir and Price, 2011; Ribas et al., 2012) have postulated
the formation of the current Amazonian physical landscape as
the main source of cladogenetic events among the studied lineages.
The formation of the Amazon basin was ultimately driven by
the Andean uplift and related arches, which resulted in the formation of a large uvio-lacustrine system in western Amazonia, during the early Miocene (Espurt et al., 2010; Mora et al., 2010). With
the continuous rise of the Andes in the middle Miocene, this system probably expanded to the Purus Arch, a tectonic structure
located 300 km west of Manaus, and which separates the Solimes
and Amazonas sedimentary basins (Figueiredo et al., 2009; Hoorn
96
G. Thom, A. Aleixo / Molecular Phylogenetics and Evolution 82 (2015) 95110
et al., 2010). The period when the Purus Arch was breached probably marked the onset of the development of the transcontinental
Amazon River, draining eastward the western uvio-lacustrine
system, and enabling the formation of upland terra-rme forests
in the western Amazonia; however, the timing of this event is still
a matter of controversy, with two main proposed periods based on
different sources of evidence: late Miocene, between 6.5 and 10 Ma
(Hoorn et al., 2010; Latrubesse et al., 2010) and Pliocene (approximately 2.5 Ma; Campbell et al., 2006). Despite the fact that major
interuves of the Amazonian region delineate the geographical
distribution of most endemic vertebrate taxa and that this has provided the basis for the recognition of the so called areas of endemism (Cracraft, 1985; Silva et al., 2005), the process of river
dynamics driving population divergence is still poorly understood,
with multiple area-relationships patterns documented so far
(Aleixo, 2004; Fernandes et al., 2012; Ribas et al., 2012; dHorta
et al., 2013). This suggests that although rivers have been barriers
to gene ow, other historical events probably contributed to the
overall diversication pattern. However, this knowledge is still
meager when compared with the rich and complex biodiversity
of Amazonia, preventing the recognition of a general model or sets
of heuristic models of diversication, corroborated by several
lineages.
The polytypic species Thamnophilus aethiops (Aves: Thamnophilidae) is a good model to study the paleobiogeography of the
Amazon basin due the following reasons: (1) it is widely distributed in Amazonia and neighboring areas such as the Andes and
the Atlantic Forest in eastern Brazil, two areas known to be historically connected to the development of modern Amazonia; (2) it is
restricted to the understory of upland terra-rme forest, rapidly
responding to environmental change (Stotz et al., 1996); and (3)
subspecies distributions are mainly bounded by the major tributaries of the Amazon River, with some important exceptions that can
provide insights into the circumstances whereby a single lineage
does and does not respond to rivers as barriers. Thus, we estimated
the spatio-temporal scenario of diversication of the polytypic T.
aethiops to address the following questions: (1) what are the evolutionary relationships among the populations/subspecies of T.
aethiops? (2) what are the relationships between the events that
led to diversication in T. aethiops and the paleogeographic models
proposed for the formation of Amazonia? (3) do the demographic
history of the studied populations support any particular diversication hypothesis?
(Hilden, Germany) extraction kit. We amplied the mitochondrial

genes cytochrome b (cyt b, 992 bp, n = 88) and NADH dehydrogenase subunit 2 (ND2, 1041 bp, n = 79), as well as the nuclear genes
b-brinogen intron 7 (Bf7, 963 bp, n = 51), b-brinogen intron 5
(Bf5, 532 bp, n = 63), and the intron 4 of the skeletal muscle receptor of tyrosine kinase (Musk4, 565 bp, n = 64) linked to the Z chromosome (see Table 2 for the primers used). Polymerase chain
reaction (PCR) was performed with 25 ll of nal volume, and
approximately 50 ng of genomic DNA, 1.52.5 mM of MgCl2,
200 mM of dNTPs and 0.1 U of Taq DNA polymerase Promega
(Madison, WI, USA). Reactions started with a denaturation step at
94 C for 5 min, followed by 35 cycles of three steps: (1) 94 C for
1 min; (2) annealing temperatures ranging from 50 C (cyt b and
Bf5) and 52 C (Musk4) to 59 C (ND2, Bf7) for 1 min; and (3)
72 C for 1 min. The last step, for the extension, was at 72 C for
5 min. PCR products were puried with the Polyethylene glycol
protocol (PEG). Sequencing was carried out on an ABI 3130 automated capillary sequencer (Applied Biosystems, Foster City, California, USA) with the ABI Prism Big Dye terminator Kit. To
conrm observed mutations, both strands of each sample were
sequenced. The DNA strands were edited and aligned manually
in BIOEDIT 7.0.5 (Hall, 1999). Saturation of the nucleotide substitutions in the mitochondrial DNA were evaluated using the software
DAMBE (Xia and Xie, 2001). For nuclear loci, heterozygous nucleotide positions were inferred by the presence of double peaks of the
same size in the electropherogram. To obtain the gametic phase of
haplotypes of the heterozygous individuals, we used a Bayesian
approach as implemented in PHASE 2.1 1 (Stephens et al., 2001;
Stephens and Donnelly, 2003; Stephens and Scheet, 2005). The
threshold of 70% of posterior probability was assumed as a condence value for the analyzed haplotypes (see Harrigan et al.,
2008). Lower values were regarded as ambiguous. To obtain the
gametic phase for individuals heterozygous in size (presence of
indels in one of the strands), we used the program CHAMPURU
v1.0 (Flot et al., 2006; Flot, 2007; available online at http://
www.mnhn.fr/jfot/champuru/). Females did not have the gametic
phase estimated for Musk4, since this gene is linked to de Z chromosome. To test the hypothesis of neutral evolution among independent loci we performed the Tajimas D test (Tajima, 1989)
using DNASP v.4.10.9 (Rozas et al., 2003). The signicance for these
tests was assessed through 10.000 coalescence simulations. To
check for possible recombination among the nuclear loci we used
the phi test as implemented in the SPLITSTREE 4.10 software
(Huson and Bryant, 2006).
2. Material and methods
2.2. Phylogenetic analyses
2.1. Samples, laboratory procedures, and data analyses
2.2.1. Concatenated genes and gene trees

The phylogenetic analyses were performed using Bayesian
inference (BI) in MrBAYES 3.1.2 (Ronquist and Huelsenbeck,
2003) and Maximum likelihood (ML) in RaxML 7.0.3 (Stamatakis,
2006). The evolutionary models which best explain the evolution
of each dataset were selected in JMODELTEST 0.1.1 (Posada,
2008), using the Bayesian information criterion (BIC) for BI and
the Akaike information criterion (AIC) for ML. For the BI analyses
with the concatenated dataset, a Bayes factor analysis (Brandley
et al., 2005) selected four partitions (mtDNA + one separate partition for each nuclear gene) as the best partitioning scheme. The
evolutionary models for each partition were: (1) mtDNA (GTR + G);
(2) Bf5 (GTR + I + G); (3) Bf7 (GTR + I); and (4) Musk4 (HKY + G).
The BI estimated based on mtDNA only used one model for the
two genes (GTR + G). BI analyses were generated through two
independent runs of 1 107 generations, each with four Markov
chains. The parameters of the chains were sampled every 1000
generations and the rst 1000 trees were discarded as burn-in.
The posterior probabilities for each estimated node were obtained
A total of 89 individuals from 55 localities were sampled

throughout the distribution of T. aethiops in Amazonia, northeastern Atlantic Forest and the Andean foothills, covering nine of ten
described subspecies (Zimmer and Isler, 2003; Fig. 1b, Table 1).
Only T. a. wetmorei, purportedly endemic to the Andean foothills
of Colombia, was not sampled. Nonetheless, we assume here that
that this taxon is closely related to the polionotus subspecies from
northwestern Amazonia, from which it is hardly differentiated
based on plumage characters (Zimmer and Isler, 2003). Subspecic
identication of our samples were made through the inspection of
voucher specimens and followed the most recent taxonomy proposed for T. aethiops (Zimmer and Isler 2003). We used Thamnophilus aroyae, T. unicolor, and T. caerulescens as outgroups due their
close relationship to T. aethiops (Brumeld and Edwards, 2007).
Total DNA was extracted from approximately 20 mg of muscle
tissue following a standard phenol/chloroform protocol
(Sambrook et al., 1989) or with the aid of a DNeasy Quiagen
97
Fig. 1. (a) Phylogenetic relationships among major clades of the polytypic Thamnophilus aethiops and outgroups derived from Bayesian and Maximum likelihood inferences
based on sequences of the mtDNA genes (cyt b and ND2; 2033 bp) and all loci combined (cyt b, ND2, Bf5, Bf7 and Musk4; 4093 bp). Nodal support values correspond to
posterior probabilities (above) and bootstrap pseudo-replicates (below) obtained, respectively, in the mtDNA and concatenated dataset analyses. (b) Map showing the current
distribution and sampled localities of major clades of the polytypic T. aethiops. Sample codes are the same used in Fig. 1a and Table 1. (c) Haplotype network of the ve
sequenced genes estimated through HaploViewer (Salzburger et al., 2011). Clade colors are the same as in Fig. 1a and 1b. Numbers inside circles represent actual number of
individuals having a particular haplotype, with circle sizes being proportional to it, while numbers on connecting branches are nucleotide substitutions higher than one
separating haplotypes. Asterisks beside light blue circles represent the number of individuals from the Pernambuco area of endemism. (d) Bayesian Phylogenetics and
Phylogeography (BP&P) results. Numbers on braches represent the number of times a given node was recovered with a speciation probability P0.95 based on six analyses
performed using two algorithms (0 and 1) and three combinations of priors on ancestral population sizes and divergence times (see methods). (e) Overlapped Bayesian
estimated chronograms derived from alternative dating analyses based on the concatenated mtDNA (cyt b and ND2; 2033 bp; branches and 95% of condence interval in dark
gray) and a multi-locus coalescent species tree (mtDNA, Bf5/Bf7 and Musk4; 4093 bp; branches and 95% condence intervals in Black). Asterisks and crosses represent values
of posterior probability P95% for the species tree and concatenated mtDNA analyses, respectively. (For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.)
98
Table 1
Code (as in Fig. 1a and b), taxon (species or subspecies following the taxonomy in Zimmer and Isler, 2003), lineage (named according to recognized Neotropical areas of endemism as
in Silva et al., 2005, with some modications), N voucher number, Inst. institution (MPEG: Museu Paraense Emlio Goeldi; LSUMZ: Louisiana State University Museum of Natural
Science), and locality information (presented coordinates are plotted in Fig. 1b) associated with each sample of Thamnophilus aethiops and outgroups sequenced in this study.
Code
Taxon
Lineage
Inst.
Locality
Coordinates
T1
T. a. atriceps
Tapajs
63,888
MPEG
Brazil, Par, Altamira, Floresta Nacional de Altamira
T2
T. a. atriceps
Tapajs
70,861
MPEG
Brazil, Par, Itaituba, Miritituba
06S040 1200
55W190 1200
04S260 2400
55W380 2400
T2
T3
T. a. atriceps
T. a. atriceps
Tapajs
Tapajs
70,862
59,159
MPEG
MPEG
Brazil, Par, Itaituba, Miritituba

Brazil, Par, Novo Progresso, 20 km SW
T3
T3
T. a. atriceps
T. a. atriceps
Tapajs
Tapajs
59,160
59,161
MPEG
MPEG

X1
T. a. atriceps
Xingu
57,707
MPEG
Brazil, Par, Senador Jos Porfrio, Rio Xingu
X1
X1
X2
T. a. atriceps
T. a. atriceps
T. a. atriceps
Xingu
Xingu
Xingu
57,708
57,709
70,635
MPEG
MPEG
MPEG

Brazil, Par, Carajs, Serra dos Carajs
X3
T. a. atriceps
Xingu
70,658
MPEG
Brazil, Par, Carajs, Serra dos Carajs, Serra Norte
P1
T. a. distans
Pernambuco
70,454
MPEG
Brazil, Alagoas, Ibateguara, Engenho Coimbra
P1
P1
P2
T. a. distans
T. a. distans
T. a. distans
Pernambuco
Pernambuco
Pernambuco
70,455
70,457
70,459
MPEG
MPEG
MPEG

Brazil, Pernambuco, Barreiros, Cachoeira Linda
P2
P2
T. a. distans
T. a. distans
Pernambuco
Pernambuco
70,460
70,458
MPEG
MPEG

B1
T. a. incertus
Belm
58,647
MPEG
Brazil, Par, Santa Brbara, Parque Ecolgico GUNMA
B2
T. a. incertus
Belm
61,190
MPEG
Brazil, Par, Barcarena
B2
B3
T. a. incertus
T. a. incertus
Belm
Belm
61,192
58,632
MPEG
MPEG
Brazil, Par, Barcarena

Brazil, Par, Tom-A, Quatro Bocas
B4
T. a. incertus
Belm
70,559
MPEG
Brazil, Par, Curua
EI1
T. a. injunctus
Eastern Inambari
68,883
MPEG
Brazil, Amazonas, Careiro, Br 319 km
EI2
T. a. injunctus
Eastern Inambari
71,054
MPEG
Brazil, Amazonas, Humait, Rio Ipixuna
EI2
EI2
EI2
EI3
T.
T.
T.
T.
Eastern
Eastern
Eastern
Eastern
71,055
71,056
71,057
60,637
MPEG
MPEG
MPEG
MPEG
Brazil,
Brazil,
Brazil,
Brazil,
WI1
T. a. kapouni
Western Inambari
58,016
MPEG
Brazil, Acre, Parque Nacional Serra do Divisor
WI2
T. a. kapouni
Western Inambari
59,837
MPEG
Brazil, Acre, Assis Brasil, ESEC Rio Acre
WI3
T. a. kapouni
Western Inambari
60,171
MPEG
Brazil, Amazonas, RDS Cujubim, Rio Juta
WI4
T. a. kapouni
Western Inambari
60,631
MPEG
Brazil, Acre, Tarauac, Br 364 km 40, Rio Liberdade
WI4
WI5
T. a. kapouni
T. a. kapouni
Western Inambari
Western Inambari
60,632
62,071
MPEG
MPEG
Brazil, Acre, Tarauac, Br 364 km 40, Rio Liberdade

Brazil, Acre, Porto Walter, Igarap Cruzeiro do Vale
WI6
T. a. kapouni
Western Inambari
40,722
LSUMZ
Peru, Loreto, 86 km SE Juanjui
WI6
WI7
T. a. kapouni
T. a. kapouni
Western Inambari
Western Inambari
40,661
11,235
LSUMZ
LSUMZ
Peru, Loreto, 86 km SE Juanjui

Peru, Ucayali, SE Cerro Tahuayo, ENE Pucallpa
WI8
T. a. juruanus
Western Inambari
59,966
MPEG
Brazil, Acre, Rio Branco, Fazenda Experimental Catuaba
WI9
T. a. juruanus
Western Inambari
60,634
MPEG
Brazil Acre, Bujari, Floresta Estadual do Antimary
WI9
WI10
T. a. juruanus
T. a. juruanus
Western Inambari
Western Inambari
60,636
60,638
MPEG
MPEG
Brazil Acre, Bujari, Floresta Estadual do Antimary

Brazil, Acre, Plcido de Castro, Ramal Novo Horizonte
WI11
T. a. juruanus
Western Inambari
61,297
MPEG
Brazil, Acre, Rio Branco, Ramal Jarinal
WI11
WI11
T. a. juruanus
T. a. juruanus
Western Inambari
Western Inambari
61,298
61,300
MPEG
MPEG

a.
a.
a.
a.
injunctus
injunctus
injunctus
injunctus
Inambari
Inambari
Inambari
Inambari
Amazonas, Humait, Rio Ipixuna

Acre, Senador Guiomard, Ramal Nabor Jnior
07S110 2400
55W300 0000
02S320 2400
51W330 0000
06S000 0000
51W190 4800
06S090 0000
50W240 0000
09S100 1200
35W320 2400
08S480 0000
35W190 1200
01S330 3600
47W390 3600
02S180 0000
48W390 3600
02S320 2400
48W010 4800
01S020 2400
47W270 3600
04S040 4800
60W390 3600
07S310 1200
63W200 2400
09S460 4800
67W190 4800
08S210 0000
73W180 3600
11S030 3600
70W160 1200
04S390 0000
68W190 4800
07S530 2400
71W390 3600
08S330 0000
72W520 1200
07S330 1900
75W540 3200
08S250 3300
74W220 2600
02S190 4800
67W360 0000
09S210 0000
68W060 0000
10S070 4800
67W190 4800
09S540 3600
68W190 1200
99

Table 1 (continued)
Code
Taxon
Lineage
Inst.
Locality
Coordinates
WI12
T. a. juruanus
Western Inambari
62,069
MPEG
Brazil, Acre, Mncio Lima, Estrada do Baro
07S330 0000
72W360 3600
WI12
WI13
T. a. juruanus
T. a. juruanus
Western Inambari
Western Inambari
62,070
59,965
MPEG
MPEG
Brazil, Acre, Mncio Lima, Estrada do Baro

Brazil, Acre, Porto Acre, Reserva Humait
WI14
T. a. juruanus
Western Inambari
63,336
MPEG
Brazil, Acre, Santa Rosa, Rio Purus
WI15
T. a. juruanus
Western Inambari
64,546
MPEG
Brazil, Acre, Senador Guiomard, Ramal Oco do Mundo
WI16
T. a. juruanus
Western Inambari
8984
LSUMZ
Bolivia, Pando, Nicols Suarez, 12 km by road of Cobija
WI17
T. a. juruanus
Western Inambari
1164
LSUMZ
Bolivia, La Paz, 20 km by Rio Beni N. Puerto Linares
EN1
T. a. polionotus
Eastern Negro
59,521
MPEG
Brazil, Amazonas, Barcelos, Rio Arac
EN1
EN1
T. a. polionotus
T. a. polionotus
Eastern Negro
Eastern Negro
59,522
59,523
MPEG
MPEG

WN1
T. a. polionotus
Western Negro
59,524
MPEG
Brazil, Amazonas, Barcelos, Rio Cuiuni
WN2
T. a. polionotus
Western Negro
59,525
MPEG
Brazil, Amazonas, Novo Airo, Igarap-Au
WN3
T. a. polionotus
Western Negro
62,770
MPEG
Brazil, Amazonas, Mara, Lago Cumapi
WN3
T. a. polionotus
Western Negro
62,771
MPEG
Brazil, Amazonas, Mara, Lago Cumapi
NR1
T. a. punctuliger
Northern Rondnia
62,215
MPEG
Brazil, Amazonas, Juruti, Base Capiranga
NR1
NR1
NR2
T. a. punctuliger
T. a. punctuliger
T. a. punctuliger
Northern Rondnia
Northern Rondnia
Northern Rondnia
62,216
58,100
68,944
MPEG
MPEG
MPEG

Brazil, Par, Jacareacanga, Terra do Burandir
NR2
NR3
T. a. punctuliger
T. a. punctuliger
Northern Rondnia
Northern Rondnia
68,965
66,127
MPEG
MPEG
Brazil, Par, Jacareacanga, Terra do Burandir

Brazil, Par, Santarm, Retiro
NR4
T. a. punctuliger
Northern Rondnia
67,048
MPEG
Brazil, Amazonas, Maus, Flona do Pau Rosa
SR1
T. a. punctuliger
Southern Rondnia
54,953
MPEG
Brazil, Rondnia, Guajar-Mirim, REBIO Ouro Preto
SR1
SR2
T. a. punctuliger
T. a. punctuliger
Southern Rondnia
Southern Rondnia
54,955
61,575
MPEG
MPEG
Brazil, Rondnia, Guajar-Mirim, REBIO Ouro Preto

Brazil, Mato Grosso, Querncia, Fazenda Tanguro
SR2
SR2
SR3
T. a. punctuliger
T. a. punctuliger
T. a. punctuliger
Southern Rondnia
Southern Rondnia
Southern Rondnia
61,576
70,226
67,397
MPEG
MPEG
MPEG

Brazil, Mato Grosso, Paranata, Rio Teles Pires
SR3
SR3
SR4
T. a. punctuliger
T. a. punctuliger
T. a. punctuliger
Southern Rondnia
Southern Rondnia
Southern Rondnia
67,396
67,398
67,442
MPEG
MPEG
MPEG

Brazil, Mato Grosso, Paranata, Fazenda Aliana
SR4
SR5
T. a. punctuliger
T. a. punctuliger
Southern Rondnia
Southern Rondnia
67,443
69,215
MPEG
MPEG
Brazil, Mato Grosso, Paranata, Fazenda Aliana

Brazil, Mato Grosso, Paranata, Fazenda Paranata
SR6
T. a. punctuliger
Southern Rondnia
58,704
MPEG
Brazil, Amazonas, Humait, T. Indgena Parintintin
SR6
SR6
SR7
T. a. punctuliger
T. a. punctuliger
T. a. punctuliger
Southern Rondnia
Southern Rondnia
Southern Rondnia
58,705
58,707
14,649
MPEG
MPEG
LSUMZ

Bolivia, Santa Cruz, Serrania de Huanchaca
A1
T. a. aethiops
5500
LSUMZ
Peru, San Martn, 28 km by road NE Tarapoto
T aroyae
T aroyae
T aroyae
38,993
38,985
22,836
LSUMZ
LSUMZ
LSUMZ
Bolivia, Cochabamba, Chapare

Bolivia, Cochabamba, Chapare
Bolivia, La Paz
T.
T.
T.
T.
T.
12,144
6196
7741
32,602
43,515
LSUMZ
LSUMZ
LSUMZ
LSUMZ
LSUMZ
Ecuador, Pichincha, Mindo

Morona-Santiago Province
Ecuador, Caar
Peru, Cajamarca
Peru, San Martn
unicolor
unicolor
unicolor
unicolor
unicolor
09S450 3600
67W400 1200
09S070 1200
69W490 4800
09S500 2400
67W490 1200
11S100 0200
68W260 1800
15S140 0600
67W370 1100
00S250 1200
62W560 2400
00S460 4800
63W160 4800
02S510 0000
60W510 0000
01S430 4800
65W520 4800
02S280 1200
56W000 0000
06S150 0000
57W520 1200
02S240 0000
55W470 2400
03S540 3600
58W240 0000
10S490 4800
64W450 0000
12S530 2400
52W220 1200
09S330 0000
56W340 1200
09S400 4800
56W510 3600
09S340 4800
56W430 1200
07S570 0000
62W220 1200
14S310 4800
60W410 2400
06S260 0600
76W190 0400
100
Table 2
List of primers used in PCR amplications and in DNA cycles sequencing reactions of samples of Thamnophilus aethiops and outgroups sequenced in this study.
Gene
Primer
Sequence 50 30
Source
Cyt b
L14990
H16065
CCA TCC AAC ATC TCA GCA TGA TGA AA

AAC TGC AGT CAT CTC CGG TTT ACA AGA C
Brumeld et al. (2007)

ND2
L5215
H5677
L5758
H6313
TAT CGG GCC CAT ACC CCG AAA AT

DGA DGA RAA DGC YAR RAY YTT DCG
GGN GGN TGR RBH GGN YTD AAY CAR AC
CTC TTA TTT AAG GCT TTG AAG GC
Brumeld
Brumeld
Brumeld
Brumeld
bf7
Fib-B17L
Fib-B17U
TCC CCA GTA GTA TCT GCC ATT AGG GTT

GGA GAA AAC AGG ACA ATG ACA ATT CAC
Prychitko and Moore (1997)

Prychitko and Moore (1997)
bf5
Fib5L
Fib5H
CGC CAT ACA GAG TAT ACT GTG ACA T

GCC ATC CTG GCG ATC TGA A

Musk4
Musk13R
Musk13F
CTCTGAACATTGTGGATCCTCAA
CTTCCATGCACTACAATGGGAAA
Clark and Witt (2)006

Clark and Witt (2006)
through a majority rule consensus of the remaining MCMC samples. The ML analyses were not partitioned and the evolutionary
models selected were GTR + I + G for the concatenated (all loci)
and GTR + G for the mtDNA datasets. Nodal support was estimated
with 1000 bootstrap pseudo-replicates. Both BI and ML analyses
with the concatenated dataset (all loci) were performed using
the nuclear genes with both gametic phases and the mitochondrial
genes duplicated. To visualize genealogical relationships among
individuals of each sequenced gene, haplotype networks were constructed in HaploViewer (Salzburger et al., 2011) based on the
topologies recovered in the species tree analysis. Mean pairwise
p-distances (Nei, 1987) within and among populations were calculated using the mtDNA dataset only in MEGA 4.0 (Tamura et al.,
2007).
2.2.2. Species tree
Recent approaches have shown that multi-locus analyses with
concatenated datasets can result in well-supported topologies that
are incongruent with the true species tree, particularly in recent
scenarios of diversication (Degnan and Rosenberg, 2009;
Kubatko and Degnan, 2007). Thus, we used a Bayesian hierarchical
model implemented in *BEAST 1.6.1 (Drummond and Rambaut,
2007; Heled and Drummond, 2010) to obtain an estimate of the
species tree for the different lineages of T. aethiops. For this
approach, we assumed three independent loci: (1) mtDNA (cyt b
and ND2); (2) Bf5 and Bf7; and (3) Musk4. The evolutionary models
were the same used in the BI for the concatenated dataset, with the
addition of the locus joining the Bf5 and Bf7 genes, for which the
GTR + I + G model was selected. We performed an initial run of
5 107 generations to optimize the analysis operators. Afterwards,
ve independent runs of 5 107 generations were performed,
sampling the parameters of the Markov chain every 1000 generations, until the effective sample sizes (ESS) of all parameters were
equal or higher than 200 (Kuhner 2008). Four independent runs
were performed and combined in a majority rule consensus tree
with posterior probability support. TRACER 1.5 (Rambaut and
Drummond, 2007) was used to access ESS values and verify when
the MCMC reached stable and convergent values, allowing for a
denition of burn-in values. Based on the obtained results and
under a conservative approach, the rst 5000 samples of each
run were discarded as burn-in. The purported species in this analysis were the reciprocally monophyletic populations of T. aethiops
revealed by the BI and ML analyses (Fig. 1a).
2.2.3. Species delimitation
We implemented a coalescent-based bayesian modeling
approach to generate speciation probabilities of closely related
et
et
et
et
al.
al.
al.
al.
(2007)
(2007)
(2007)
(2007)
taxa from multi locus sequence data using the program Bayesian
Phylogenetics and Phylogeography (BP&P v.2.0b; Yang and
Rannala 2010), considered the most accurate coalescent-based
method for species delimitation evaluated by Camargo et al.
(2012). The model implemented in BP&P assumes no gene ow
among species, no recombination, and takes into account gene tree
uncertainty and lineage sorting. BP&P incorporates a model that
include the species divergence times (s), and the population size
parameters h = 4Nl, where N is the effective population size and
l is the substitution rate per site per generation. However, reasonable prior distributions of population parameters can be difcult to
determine, potentially affecting the posterior probability of species
delimitations (Yang and Rannala 2010). Thus, we implemented the
approach of Leach and Fujita (2010), performing analyses using
three combination of priors representing different population sizes
and ages for the root of the species tree as follows: (1) large ancestral population size and deep divergences (h and s gamma priors
G(1, 10) and G(1, 10)); (2) small ancestral population size and shallow divergences among species (h and s gamma priors G(2, 2000)
and G(2, 2000)); and 3) large ancestral population size and shallow
divergences among species (h and s gamma priors G(1, 10) and
G(2, 2000)). We ran the analyses for 1 106 generations, sampling
every ve and discarding the rst 5 104 generations as burn-in,
using the algorithms 0 and 1 with different ne tuning parameters
(e = 5 for algorithm 0 and a = 2 and m = 1 for algorithm 1). We used
the populations and the topology obtained through the species tree
analyses as the guide tree (Fig. 1e). The population occurring disjunctly in the Pernambuco region (Atlantic Forest) was considered
a distinct lineage, sister to that of the Belm area of endemism
population.
2.3. Molecular dating

To estimate the divergence times among T. aethiops populations
revealed by the BI and ML phylogeny estimates, a second BI was
carried out using only the mtDNA dataset (cyt b and ND2). After
the selection of the model of molecular evolution for this dataset
with JMODELTEST 0.1.1 (Posada, 2008), a preliminary run of
1 107 generations was performed in BEAST 1.6.1 (Drummond
and Rambaut, 2007) xing the rate of nucleotide mutations at
1.0 per millions years for the cyt b (for which a robust calibration
is available; Weir and Schluter, 2008) and estimating that for the
ND2 under the options relaxed clock and uncorrelated lognormal.
This analysis was performed to evaluate whether both mitochondrial genes were evolving at similar rates and the cyt b calibration
could be applied to ND2. After the conrmation that these two
genes were evolving at similar rates, the overall molecular clock
calibration used was 2.1% of nucleotide substitutions per million

years (Weir and Schluter, 2008), under the relaxed clock and
uncorrelated lognormal options. We performed an initial run of
1 107 generations to optimize the analysis operators. Afterwards,
two independent runs of 1 107 generations were performed,
sampling the parameters of the Markov chain every 1000 generations, until the ESS of all parameters were equal or higher than
200. Based on the obtained results and under a conservative
approach, the rst 800 samples of each run were discarded as
burn-in. Values of posterior probability and divergence times were
estimated through a majority rule consensus of the remaining
samples. For this analysis we used only one individual from each
populations using the Yule process as a speciation prior.
Analyses based on a multi-locus coalescent approach enable a
more accurate estimate of divergence processes, since gene divergence times normally occur before true speciation events
(Maddison and Knowles, 2006; Carstens and Knowles, 2007;
Edwards et al., 2007; Heled and Drummond, 2010; McCormack
et al., 2010). Thus, during the species tree analysis, we set for the
mtDNA dataset (cyt b and ND2 genes) the mutation rate of 2.1%
substitutions per million years (Weir and Schluter, 2008), whereas
those of the nuclear loci (Bf5/Bf7 and Musk4) were estimated in
comparison to that of the mtDNA under a Yule process speciation
prior.
2.4. Population genetics, historical demography, and gene ow
The recognized groups for the population genetics analyses
were the reciprocally monophyletic populations revealed by the
BI, ML, and species tree analyses (Fig. 1a and e). Even though populations of the Belm and Pernambuco areas of endemism did not
show signicant genetic differentiation and have not acquired reciprocal monophyly, they were recognized as independent populations in the population genetics and network analyses, since they
are currently separated by a long stretch of non-forest environments (Cerrado and Caatinga, AbSaber, 1977), without possibility
of current gene ow.
We analyzed sequences of the three sampled loci (mtDNA, Bf5/
Bf7 and Musk4) and populations with a minimum sampling of at
least ve individuals. The nuclear loci were used with both gametic
phases, except females for Musk4. To estimate changes in historical
demography through non-neutral models of evolution, we performed the Tajimas D (Tajima, 1989), Fus Fs (Fu, 1997), and R2
Ramos-Onsins and Rozas (2002) tests. Signicance was evaluated
through 10,000 coalescent simulations. We also calculated nucleotide diversity (p) and haplotype diversity (h) for all populations. All
analyses were performed in DNASP v.4.10.9 (Rozas et al., 2003).
The dynamics of effective population sizes through time of the
recognized populations of T. aethiops was estimated through
Extended Bayesian Skyline Plots (EBSP; Heled and Drummond,
2008), using a linear model in BEAST 1.6.1 (Drummond and
Rambaut, 2007). EBSP estimate changes in effective population
sizes over time under a multi-locus approach by using the times
of coalescent events among gene trees. We used the rate of 2.1%
of nucleotide substitutions per million years (Weir and Schluter,
2008) for the mtDNA with the relaxed clock and uncorrelated lognormal priors. The evolutionary rates of the nuclear loci (Bf5/Bf7
and Musk4) were estimated in comparison to that the mtDNA.
The MCMC parameters were the same used in the species tree
analysis. The evolutionary models for each locus of each population were estimated in JMODELTEST 0.1.1 (Posada, 2008).
To corroborate the obtained results in the phylogenetic analyses
and test for population structuring across recognized geographical
barriers (rivers and non-forest environments) we performed an
analysis of molecular variance (AMOVA) using the mtDNA dataset
in ARLEQUIN 3.1 (Excofer et al., 2006).
101
We applied the isolation with migration model (Hey and

Nielsen, 2004) implemented in the IMa program (Hey and
Nielsen, 2007) to estimate whether gene ow has occurred among
those parapatric populations potentially in contact (Fig. 1b). For
each pair of populations being compared, IMa estimates values of
H = 4Nl (where l is the mutation rate per year per marker) for
populations 1 and 2, and their ancestor, as well as the t time
(where t = tl, the time of population splitting at t generations in
the past) in which they diverged in the presence of gene ow
(where m = m/l, given in the coalescent). We structured the analyses assuming as populations the reciprocal monophyletic lineages
reveled in the phylogenetic and species tree analyses. IMa estimates compared populations potentially in contact, without an
apparent physical barrier separating them and parapatric populations occurring on opposite banks of major Amazonian tributaries
(see Fig. 1b). Our main objective with IMa analyzes was to test if
the observed absence of phylogenetic signal in the nuclear markers
(see Fig. 1c) is better explained by incomplete lineage sorting as a
result of recent population splitting (m = 0) or by the presence of
gene ow (m > 0). We assumed the HasegawaKishinoYano
(HKY) model of evolution for all analyses and markers. Initial runs
in MCMC mode were performed to establish the best priors for
effective population sizes, divergence times, and migration parameters for each pair of populations being compared. After prior
selection, three independent nal runs in MCMC mode were performed changing the seed number to observe the convergence of
parameter values. Every run was performed using 1,000,000 generations of burn-in, sampling 100,000 trees in 5,000,000 generations
with 20 chains with a geometric heating. We performed a likelihood ratio test (LRT, Nielsen and Wakeley, 2001), using the Loadtrees mode of IMa, sampling 100,000 generations and the results
of the nal MCMC runs, to reject or accept scenarios with migration different from 0. If the LRT rejected a scenario of m > 0, we
assumed that the absence of phylogenetic signal in the nuclear
markers was due to incomplete lineage sorting. On the other hand,
if the LRT rejected a scenario with m = 0, we performed another
MCMC run xing m = 0 to evaluate whether a model without
migration adjusted better to the observed data than the full model
with migration. For these comparison we applied the Akaike information Criterion (AIC), where the model that minimized
AIC = 2[log (L) d] was chosen as the best, where d is the number
of parameters (Nielsen and Wakeley, 2001). If the model with
migration had a better t to the data, we assumed that the phylogeographic pattern may have been affected by gene ow. To convert
the model parameter estimations of m in demographic parameters
we applied the rate of 2.1% of nucleotide substitutions per million
years based on the mtDNA calibration adopted (Weir and Schluter,
2008), and a generation time of 1 year.
3. Results
3.1. Description of DNA sequences
Results of Tajimas D was not signicant for the mtDNA and
Musk4 (P > 0.05), but did reject neutrality signicantly for Bf5/Bf7
(P < 0.05; Table 3), suggesting that this locus does not evolve in a
clock-like fashion. We did not detect signs of saturation for the faster
evolving mitochondrial genes (cyt b and ND2). The phis test did not
reject the null hypothesis of a dataset without recombination for the
nuclear genes (Bf5, Bf7 and Musk4; P > 0.1). Were identied 57 distinct haplotypes for cyt b (992pb, n = 82), 38 for the ND2 (1041pb,
n = 72), 42 for Bf5 (532pb, n = 118), 50 for Bf7 (963pb, n = 98), and
30 for Musk4 (565pb, n = 97; Table 3). All sequences were deposited
in Gen Bank under the accession numbers KF685940-KF686022
(cyt b), KF664029-KF664102 (ND2), KF686023-KF686081 (Bf5),
KF686141-KF686186 (Bf7) and KF686081-KF686140 (Musk4).
102
Table 3
Diversity and demographic parameters of lineages of the polytypic T. aethiops for which at least ve individuals were sequenced for mitochondrial (cyt b and ND2) and nuclear
(Bf5/Bf7, and musk4) genes. Lin. = lineages/clades; N = number of phased alleles; n H = number of haplotypes; H = haplotype diversity; p = nucleotide diversity; D = Tajimas D
test (Tajima,1989); Fs = Fus Fs (Fu, 1997); R2 = Ramos-Onsins, Rozas test (2002). Signicance levels for Tajimas D and R2 = *P < 0.05; **P < 0.01. Signicance levels for Fus
Fs = *P < 0.02.
Lin.
Gene
n H
T. a. aethiops
mtDNA
Bf5/Bf7
musk4
72
118
97
64
42
30
0.996
0.935
0.921
0.0261
0.0054
0.0051
0.3502
1.68371*
1.37883
Tapajs
mtDNA
Bf5/Bf7
musk4
6
6
5
6
5
3
1.000
0.933
0.700
0.0018
0.0061
0.0023
Xingu
mtDNA
Bf5/Bf7
musk4
5
6
5
4
4
3
0.900
0.867
0.700
Pernambuco
mtDNA
Bf5/Bf7
musk4
6
8
6
4
3
3
Belm
mtDNA
Bf5/Bf7
musk4
5
12
8
Eastern IInambari
mtDNA
Bf5/Bf7
musk4
Western Inambari
Fs
R2
0.4155
0.7612
0.1747
1.013
1.009
0.0607
0.1446
0.211
0.2848
0.0014
0.0083
0.0033
0.1909
0.1057
0.2735
0.4448
1.0749
0.6436
0.2186
0.2073
0.1936
0.800
0.714
0.600
0.0006
0.0037
0.0032
0.4474
0.5044
0.5617
1.4544
1.7263
0.957
0.1805
0.2143
0.2206
5
6
3
1.000
0.864
0.714
0.0042
0.0028
0.0024
0.4208
0.7877
0.9657
0.5418
1.8982
0.8748
0.1597
0.2027
0.2381
6
6
6
6
6
5
1.000
1.000
0.933
0.0021
0.0077
0.0044
0.6915
0.9902
1.0484
2.4843
2.4199
1.5652
0.1446
0.1023**
0.2386
mtDNA
Bf5/Bf7
musk4
15
32
25
13
14
11
0.981
0.897
0.847
0.0021
0.0034
0.0031
1.5705*
1.6007*
0.6854
7.6834*
8.3201*
5.3341*
0.0700*
0.0582**
0.0958
Northern Rondnia
mtDNA
Bf5/Bf7
musk4
7
14
11
7
8
9
1.000
0.901
0.945
0.0021
0.0040
0.0045
0.3606
0.768
0.9471
3.4573*
2.8844
5.1712*
0.1267*
0.1174
0.1247
Southern Rondnia
mtDNA
Bf5/Bf7
musk4
15
24
20
12
6
7
0.971
0.757
0.800
0.0047
0.0023
0.0036
0.4438
1.2115
0.5208
2.11639
1.1177
0.9804
0.1208
0.1042
0.1166
3.2. Area and phylogenetic relationships

BI and ML analyses based on the mtDNA and the concatenated
dataset (total of four analyses; Fig. 1a) recovered identical topologies showing with high statistical support the reciprocal monophyly of 10 populations of T. aethiops, with T. aroyae grouping as
the sister species. Two main clades were recovered in T. aethiops,
splitting apparently parapatric and attitudinally segregated
Andean foothill from lowland Amazonian populations, which
diverge by about 5% (uncorrected p genetic distance-value;
Table 4). A subsequent major split involved lowland populations
separated by the Tapajs River in south-central Amazonia, which
diverge by roughly 4% and are hereafter referred to as the eastern
and western clades (populations found to the east and west of the
Tapajs River), respectively. The eastern clade grouped reciprocally
monophyletic populations endemic to the Tapajs, Xingu, and
Belm Amazonian areas of endemism (Silva et al., 2005), as well

as the isolated Pernambuco population in the northern sector of
the Atlantic Forest biome in northeastern Brazil (Fig. 1a and b).
The latter population was nested within the Belm population
despite these two being located in different biomes separated by
more than 1000 km of inhospitable habitat to T. aethiops (extensive
non-forest vegetation). The western clade grouped populations
from the Madeira, Inambari, Napo, Imeri, and Guiana areas of endemism (Silva et al., 2005), but only the Napo/Imeri (west of the
Negro River) and Guiana (east of the Negro River to west Branco
River) areas were occupied by reciprocally monophyletic populations (Fig. 1a and b). Although we did not sample populations from
the Napo area of endemism (Silva et al., 2005), we tentatively
include them in the Imeri population based on their morphological
similarity and placement in the same subspecies polionotus
(Zimmer and Isler, 2003). In contrast, the Inambari and Rondnia
Table 4
Average pairwise uncorrected p-distances (mtDNA) between and within major clades of the polytypic Thamnophilus aethiops and outgroups recovered by Bayesian and Maximum
Likelihood phylogenies.
Clade (average pairwise p-distances within clades)
10
11
1 Western Negro (0.1)

2 Eastern Negro (0)
3 Western Inambari (0.2)
4 Southern Rondnia (0.6)
5 Eastern Inambari (0.2)
6 Northern Rondnia (0.2)
7 Tapajs (0.1)
8 Xingu (0.2)
9 Belm (0.4)
10 Pernambuco (0.1)
11 T. a. aethiops
12 T. aroyae
0.9
1.6
2.2
2.2
2.7
3.6
3.4
3.1
3.1
5.0
5.2
1.4
2.1
2.3
2.8
3.7
3.4
3.1
3.2
5.1
5.5
2.7
2.9
3.1
4.0
3.6
3.5
3.5
5.7
6.2
1.7
3.0
3.8
3.6
3.2
3.3
5.2
5.2
3.4
3.9
3.7
3.2
3.4
5.3
5.5
3.9
3.8
3.4
3.3
6.0
5.5
1.9
1.9
1.8
4.3
5.2
0.7
0.6
4.8
5.8
0.3
4.5
5.5
4.7
5.6
6.0
areas of endemism were inhabited by four distinct non-sister populations with the following distributions: (1) northern portion of
the Rondnia area of endemism in the TapajsMadeira interuve,
probably limited to the south by the Aripuan River; (2) southern
part of the Rondnia area of endemism, probably limited to the
north by the Aripuan River, crossing the TapajsMadeira interuve eastwards into the headwaters of the Tapajs and Xingu Rivers, and hence reaching the southernmost parts of the Tapajs and
Xingu areas of endemism; (3) eastern Inambari area of endemism
in the lowercentral portion of the PurusMadeira interuve; and
(4) western Inambari area of endemism west of the Purus River
and south of the Maraon/Huallaga/Ucayali/Solimes River in
Peru, eastward reaching the headwaters of the PurusMadeira
interuve into northeastern Bolivia (Pando and La Paz Departments). Uncorrected genetic p-distances among populations of T.
aethiops ranged from 0.6% (between Belm/Pernambuco and Xingu
populations) to 6.0% between Andean foothill and northern Rondnia area of endemism populations, with the average p-distances
between reciprocally monophyletic sisters populations around
1.6%.
The coallescent species tree recovered an identical topology to
those of BI and ML analyses based on the concatenated dataset
(Fig. 1e). The species tree also recovered with high statistical support the monophyly of the polytypic T. aethiops, as well as that of
the Amazonian populations, the eastern and western clades, and
the internal relationships within them. Only the sister relationship
between the Western Inambari and the Northern Solimes River
populations was not statistically supported (posterior probability
less than 0.95; Fig. 1e).
The individual topologies of each nuclear gene (Bf5, Bf7 and
Musk4, results not shown) do not support any particular wellresolved relationship among individuals of T. aethiops, except for
Musk4, which supported the monophyly of lowland Amazonian
and Atlantic Forest populations.
3.3. Species delimitation
We obtained equal results among all six BP&P analyses, using
both algorithms and the three different combinations of s and h
values, assuming a cutoff of 95% of posterior probability. Species
delimitations results were robust to the choice of prior distributions, supporting most of the reciprocally monophyletic populations recovered by the BI, ML, and species tree estimates as
distinct species, except for the Belm/Pernambuco and Eastern/
Western Negro sister population pairs (Fig. 1d). The purported speciation event between Belm and Pernambuco populations had
higher speciation probabilities when we used priors representing
large ancestral populations and deep divergences (PP = 0.90) or
shallow divergences (PP = 0.89), but very low values with priors
representing small ancestral populations and shallow divergences
(PP = 0.10), using algorithm 0 with the ne-tuning parameter set
to 5. The split between Eastern and Western Negro populations
had very low speciation probabilities in all analyses (PP 6 0.20).
3.4. Molecular dating
According to the molecular dating based on the concatenated
mtDNA dataset (Fig. 1e), the split between T. aethiops and T. aroyae
took place during the Pliocene between 4.07 and 2.85 Mya (mean
3.45 Mya). The divergence between the Andean (T. a. aethiops) and
the ancestor of the Amazonian and Atlantic Forest populations took
place between 3.54 and 2.44 Mya (mean 2.97 Mya).
The earliest split within Amazonian populations occurred in the
Late PlioceneEarly Pleistocene (2.701.86 Mya, mean 2.27 Mya),
with separation of the eastern and western clades across the
Tapajs River. The rst split in the western clade separated
103
populations from the northern part of the Rondnia area of endemism (east of Aripuan River) from the remaining ones found to
the west of this river and across the Madeira, Solimes, and Negro
rivers, during the EarlyMiddle Pleistocene (1.841.19 Mya, mean
1.50 Mya). Other cladogenetic events in the western clade
stretched from the Middle to Late Pleistocene between 1.40.29
Mya (mean 0.84 Mya), with one of them (involving southern
Rondnia + eastern Inambari versus western Inambari + northern
Solimes populations) coinciding to some extent with the modern
course of the Purus River (Fig. 1b and e). The splits in the eastern
clade occurred between the Middle (1.270.66 Mya, mean 0.95
Mya) and Late Pleistocene (0.490.15 Mya, mean 0.31 Mya) and
separated populations of the Tapajs, Xingu, and Belm/Pernambuco areas of endemism across the Xingu and Tocantins rivers.
The splitting dates estimated by the multi-locus coalescent
approach (species tree) were in general more recent than those
obtained by the single locus mtDNA dataset, particularly for those
younger than 1.0 Mya. However, both coalescent and concatenated
mtDNA timing estimates overlapped to various degrees in their
respective 95% condence intervals, and thus cannot be considered
statistically different.
3.5. Population genetics and historical demography
Haplotype networks recovered for cyt b and ND2 mirrored the
phylogenetic analyses based on BI and ML (Fig. 1a and c). For cyt
b, populations with the highest number of sequenced individuals
differed dramatically in structure: the western Inambari population presented a dominant haplotype shared by six individuals
and 12 other haplotypes represented by one or two individuals,
generally differing from the commonest haplotype by a single
mutation. On the other hand, in the southern Rondnia population,
10 different haplotypes distributed in three groups separated by
two to eight mutations were recovered, with only three haplotypes
present in more than one individual (Fig. 1c). Networks of the
nuclear makers recovered the absence of phylogeographic structure and haplotype sharing among the recognized populations
(Fig. 1c).
Haplotype diversity was generally high and similar among the
sampled Amazonian populations (Table 3). Estimated nucleotide
diversity varied widely among populations depending on the locus
considered, while within a single locus values obtained for the different populations were similar (Table 3), suggesting an overall
demographic stability. The Tajimas D test (1989) showed negative
and signicant values for the mtDNA and Bf5/Bf7 loci in the western Inambari population, consistent with demographic uctuations (Table 3). The Fus Fs (1997) showed signicant and
negative values for the mtDNA and musk4 loci in the northern
Rondnia population and in all three sampled loci in the western
Inambari, also supporting demographic instability. The R2 (2002)
test results showed low and signicant values for the same populations as the Fus Fs (1997), suggesting a similar demographic scenario regardless of the test considered.
The EBSPs (Fig. 2) were in part congruent with the neutrality
tests, inferring tendencies of recent demographic expansions in
both populations of the Inambari and the northern Rondnia areas
of endemism during the Late Pleistocene and Holocene. In contrast,
EBSPs favored scenarios of demographic stability in the Belm,
Pernambuco, Xingu, Tapajs, and southern Rondnia populations
(Fig. 2).
An analysis of molecular variation (AMOVA; Table 5) conrmed
that most of the genetic variation in the Amazonian and Atlantic
Forest populations of T. aethiops is partitioned among the geographically structured clades recovered by the phylogenetic analyses, and not within these groups. Similarly, these results
corroborate that the large tributaries of the Amazon River are at
104
Fig. 2. Demographic histories of major clades of Thamnophilus aethiops for which at least ve individuals were sampled, inferred through Extended Bayesian Skyline Plots
based on mtDNA (cyt b and ND2), Bf5/Bf7, and Musk4 sequences. Black solid lines represent median values, while the dashed lines corresponds to 95% condence intervals.
The X axis corresponds to time in million years before present. The Y axis represents Nes in a logarithmic scale.
least contemporary barriers to gene ow between populations

from opposite banks.
3.6. Gene ow among populations
IMa analyses favored scenarios of very low levels or absence of
gene ow among all populations of T. aethiops potentially in contact (Table 6). Migration events were not rejected by LRT tests
among most pairwise comparisons, with models predicting migration having better AIC scores than models predicting no gene ow
between the following clades: Xingu/Tapajs, Northern Rondnia/

Southern Rondnia, Northern Rondnia/Eastern Inambari, Southern Rondnia/Western Inambari, and Eastern Inambari/Western
Inambari. In all these instances, the estimated migration rate was
very low in both directions (with condence intervals lower bonds
reaching zero except in one comparison), and involved non-sister
populations (Fig. 1, Table 6). The highest migration rate detected
and the only one not including a lower bond likelihood of zero,
involved the Eastern Inambari and Western Inambari populations
(Maximum likelihood = 0.0015; 90% HPD interval = 0.0010.004).
105
Table 5
Results of an Analysis of Molecular Variation (AMOVA) based exclusively on the mtDNA among major clades of the polytypic Thamnophilus aethiops for which at least ve
individuals were sampled, separated by potential modern barriers (rivers and non-forest habitats). Results regarded as signicant if P < 0.001.
Populations
Potential barrier
Among populations on opposite sides of

the barrier
Within the population in the same side

of the barrier
Ust
pValue
Pernambuco/Belm
Caatinga/Cerrado (non-forest
landscapes)
Tocantins R.
Xingu R.
Unknown
Lower Tapajs R.
Teles Pires R. (part)
Aripuan R. (part)
Lower Madeira R.
18.77
81.23
0.18
0.026
60.42
91.08
84.78
71.25
46.49
86.05
93.39
39.58
8.92
15.22
28.75
53.51
13.95
6.61
0.60
0.91
0.84
0.71
0.46
0.86
0.93
0.004
0.002
0.000
0.000
0.000
0.000
0.000
Middle Madeira R.
73.82
26.18
0.73
0.000
Upper Madeira R.
84.84
15.16
0.84
0.000
Purus R. (part)
92.26
7.74
0.92
0.000
Belm/Xingu
Xingu/Tapajs
Xingu/Southern Rondnia
Tapajs/Northern Rondnia
Tapajs/Southern Rondnia
Northern/Southern Rondnia
Northern Rondnia/Eastern
Inambari
Southern Rondnia/Eastern
Inambari
Southern Rondnia/Western
Inambari
Eastern/Western Inambari
Table 6
Summary of IMa results of migration estimates between parapatric population pairs of T. aethiops potentially in contact (see Fig. 1b). Posterior distribution values of migration
were converted to demographic units, where the values of m1 and m2 represent the average number of migrations per 1000 generations per gene copy on the coalescent
(m1 = rate of migration from population 2 to population 1; m2 = rate of migration from population 1 to population 2). Condence intervals represent HPD90. All maximum values
for migration reached likelihood zero, indicating the posterior distribution ends.
Pairwise analyses
m1
m2
AIC
Belm (5)/Xingu (5)
0 (00.0011)
Forced to be zero
0.0027 (00.0063)
Forced to be zero
9569.71
9548.03
Xingu (5)/Tapajs (6)
0 (00.0033)
Forced to be zero
0 (00.003)
Forced to be zero
9586.18
9613.29
Xingu (5)/Southern Rondnia (15)*
Tapajs (6)/Northern Rondnia (7)*
Tapajs (6)/Southern Rondnia (15)*
Northern Rondnia (7)/Southern Rondnia (15)
0 (00.0012)
Forced to be zero
0 (00.0013)
Forced to be zero
10366.19
10378.84
Northern Rondnia (7)/Eastern Inambari (6)
0 (00.0013)
Forced to be zero
0 (00.002)
Forced to be zero
9996.38
10012.88
Southern Rondnia (15)/Eastern Inambari (6)
0 (00.0016)
Forced to be zero
0.0013 (00.0032)
Forced to be zero
9920,21
9898.74
Southern Rondnia (15)/Western Inambari (9)
0 (00.0019)
Forced to be zero
0 (00.0012)
Forced to be zero
10963.26
10997.83
Eastern Inambari (6)/Western Inambari (9)
0 (00.0032)
Forced to be zero
0.0015 (0.0010.004)
Forced to be zero
10369.91
10395.45
Western Inambari (9)/Northern Solimes (7)*
Asterisks indicate pairwise analyses where the likelihood ratio test rejects a scenario with m > 0. For pairwise comparisons where the likelihood ratio test supports a scenario
with m > 0, models with and without migration were contrasted with the Akaike information criterion (AIC). The model that minimized AIC was chosen as the best scenario
(in bold). Numbers in parenthesis next to population names denote the number of individuals of the respective population used in the comparison. See Table 1 for detailed
specimen and locality information.
4. Discussion
4.1. Species limits and taxonomy
Although our analyses support the monophyly of the polytypic
T. aethiops, of the 10 reciprocally monophyletic populations recovered only two are entirely consistent with subspecies limits currently recognized in this group: nominate aethiops from the
foothills of the Andes and T. a. injunctus, which corresponds to
the eastern Inambari population (Zimmer and Isler, 2003; Fig. 1a,
d and e). In fact, high statistical support for key nodes in the
multi-locus coalescent species tree and high speciation probabilities recovered by the species delimitation analysis (BP&P), support
the recognition of nine reciprocally monophyletic lineages tting
the denition of evolutionary species (see Fujita et al., 2012;
Fig. 1d) in T. aethiops.
These lineages are as follows (taxon names are assigned to

recovered populations based on type locality and nomenclatural
priority following Peters 1951 and Zimmer and Isler 2003): (1)
Thamnophilus aethiops Sclater, 1858 (foothills of the Andes); (2)
Thamnophilus atriceps Todd, 1927 (Tapajs population, distributed
throughout most of the Tapajs area of endemism); (3) Thamnophilus incertus Pelzeln, 1869 (Belm and Pernambuco populations;
given their lack of reciprocal monophyly and low speciation probabilities, the former Thamnophilus aethiops incertus from the Belm
area of endemism and Thamnophilus aethiops distans Pinto 1954
from the Atlantic Forest should be regarded as subspecies of a single species, T. incertus, the name with nomenclatural precedence);
(4) Thamnophilus punctuliger Pelzeln 1869 (northern Rondnia
population distributed east of the Aripuan River in the Madeira
Tapajs interuve); (5) Thamnophilus injunctus Zimmer 1933
(eastern Inambari population, distributed through most of the
106
MadeiraPurus interuve); (6) Thamnophilus juruanus Iheringi

1904 (western Inambari population, distributed through most of
the Inambari area of endemism west of the Purus River; the taxon
name Thamnophilus aethiops kapouni Seilern 1913 also applies to
this population, but it has no nomenclatural priority, and therefore
should be synonymized with juruanus); (7) Thamnophilus polionotus Pelzeln 1869 (western and eastern Negro populations distributed north of the Amazon in the BrancoSolimes interuve; as
explained above the taxon Thamnophilus aethiops wetmorei de
Schauensee, 1945 is tentatively included in this lineage); and,
nally, two additional unnamed lineages (no known taxon
described previously from their ranges) corresponding respectively
to most of the Xingu area of endemism (assigned currently to T.
atriceps, whose type locality is in the Tapajs area of endemism)
and the southern part of the Rondnia, Tapajs, and Xingu areas
of endemism (assigned currently to T. punctuliger, whose type
locality lies in the northern part of the Rondnia area of
endemism).
By occupying mostly shaded environments in the understory of
tropical forests, the evolution of plumage color states traditionally
used in subspecies delimitations in T. aethiops probably proceeded
at a slower pace than at least one of the sequenced loci (mtDNA).
This process can generate the formation of morphologically cryptic
and sometimes polymorphic lineages, resulting in the taxonomic
inconsistencies cited above (Baker et al., 1995; Irwin et al., 2001;
Fernandes et al., 2012). In contrast, vocal characters are thought
to be directly involved in the reproductive isolation of forest interior suboscine lineages (Carneiro et al., 2012). We do not report on
the vocal variation within the polytypic T. aethiops in this paper,
but will present in a separate publication a detailed taxonomic
overhaul of the T. aethiops complex based on a multi character
approach, where the unnamed evolutionary lineages mentioned
above will be described.
The only sister populations of T. aethiops for which low speciation probabilities were recovered are the parapatric eastern/western Negro populations (T. polionotus) and the allopatric Belm/
Pernambuco populations (T. i. incertus and T. i. distans). Even
though the species tree recovered the former pair as distinct species, the species delimitation analyses favored a scenario of very
shallow divergence between them, not supporting their status as
distinct evolutionary species (Fig. 1d and e). Indeed, the lack of
reciprocal monophyly in the mtDNA between the Belm (T. i. incertus) and Pernambuco (T. i. distans) populations supports a very
recent divergence between them, despite their current separation
by over 1000 km.
The overall lack or very low rate of gene ow estimated among
most parapatric populations of T. aethiops are also indicative of
their status as independent species (Table 6). Interestingly, whenever lack of gene ow (i.e., zero migration rates) could be statistically rejected between any population pair potentially in contact, it
always involved non-sister lineages, implying in secondary contact
rather than primary intergradation (Table 6). Hence, it is possible
that these very low rates of gene ow are explained by a signicant
degree of reproductive isolation already acquired by these populations that have diverged in allopatry for a relatively long time
before coming in contact. Estimates of gene ow between non-sister populations or populations that exchange migrants with a third
unsampled population go against a main assumption of the IMa
model and can generate several biases in the estimation of the
main parameters, although they have little effect when low levels
of gene ow are detected (see Strasburg and Rieseberg 2010), as
documented herein. IMa analyses supported absolute no gene ow
among all populations of the two main Amazonian clades of T.
aethiops separated by the Tapajs River (eastern clade: Xingu and
Tapajs populations; and western clade: northern Rondnia and
southern Rondnia populations), which likely come in contact
across a wide area in the headwaters of the Tapajs, Xingu, and

Tocantins rivers (Fig. 1b, Table 6). Despite sampling limitations,
this study provides the best evidence available so far that interuvial populations of terra-rme avian lineages in contact across the
headwaters of southern Amazonian tributaries can retain their
genetic diagnosabilities, probably as a result of an advanced degree
of reproductive isolation acquired in allopatry before coming in
contact in more recent times, as suggested by several other studies
based solely on mtDNA datasets (Carneiro et al., 2012; Batista
et al., 2013; Rodrigues et al., 2013). In contrast, gene ow was present, yet at a very small rate, between the non-sister Tapajs and
Xingu populations belonging to the same eastern clade, as well
as between the southern Rondnia and western Inambari populations of the western clade across the upper Madeira River (Fig. 1,
Table 6). Therefore, comparative levels of gene ow across the
headwaters of major Amazonian tributaries are likely to be more
correlated with the phylogenetic distance of the lineages/populations in contact, rather than the smaller barrier effect of rivers in
these areas (Haffer 1992), which result in a more random pattern
of gene ow.
4.2. The diversication of the Thamnophilus aethiops complex in CisAndean South America
Populations of T. aethiops distributed in the Amazon and Atlantic Forests are in a clade with several basal Andean taxa typically
occurring above 1000 m (T. unicolor, T. aroyae, and T. a. aethiops),
supporting an ancestor of Andean origin for these lowland forest
populations (see also Brumeld and Edwards, 2007). Diversication events along Andean altitudinal gradients have been proposed
for some avian lineages (Bates and Zink, 1994; Weir, 2006; Isler
et al., 2012; dHorta et al., 2013), and are probably related to allopatric speciation during altitudinal shifts in habitats due to climatic oscillations (Graham et al., 2004). Under this diversication
model, the dispersal of an Andean ancestor into the Amazonian
lowlands would only be possible after the draining of the lacustrine system of the Solimes Basin in western Amazonia (the so
called Pebas and Amazonas lakes), which led to the formation of
the modern Amazon drainage (Hoorn et al., 2010) and the establishment of upland terra-rme forests in this region. Paleogeographic hypotheses differ in the time frame when these events
occurred (Campbell et al., 2006; Figueiredo et al., 2009;
Latrubesse et al., 2010), even though they coincide in postulating
a favorable environment for the establishment of upland terrarme forests in western Amazonia between the late Pliocene and
early Pleistocene, when the diversication of the T. aethiops complex in Amazonia began (Fig. 1e).
The genetically poorly-differentiated yet isolated Atlantic Forest
population (T. i. distans) may have originated from a recent dispersal event of the Belm population (T. i. incertus) or through isolation caused by climatic uctuations leading to the disruption of
the forest corridor linking eastern Amazonia with the northeastern
Atlantic Forest during the Late Quaternary (Batalha-Filho et al.,
2013). In contrast, other Amazonian and Atlantic Forest avian sister
lineages exhibited reciprocal monophyly and high levels of genetic
differentiation estimated to date back to a time frame stretching
from the Late Pliocene to the Early Pleistocene (Aleixo, 2002;
Cabanne et al., 2008; Weir and Price, 2011). These different phylogeographic patterns are consistent with multiple and cyclical connection and isolation episodes between eastern Amazonia and
the northern sector of the Atlantic Forest until at least the Late
Quaternary (Batalha-Filho et al., 2013).
The data presented herein support that distinct events of vicariance related to river formation and reorganization mostly during
the Pleistocene have inuenced the diversication of the T. aethiops
complex, providing another body of evidence that different
modern interuves in Amazonia harbor distinct evolutionary lineages, with little or no gene ow among them (Table 6). In the T.
aethiops complex, all splits involving the recognized lineages coincide with at least part of the course of a major Amazonian river,
indicating that at some point in time these rivers might have acted
as primary diversication barriers. Interestingly, the absence of a
physical barrier separating the eastern and western Inambari
clades where they meet in the middle PurusMadeira interuve
is associated with the highest gene ow rate recovered herein
among lineages of the T. aethiops complex, yet still very low
(0.0015; Table 6).
The phylogeographic data presented herein is consistent with at
least two independent splits across different portions of the modern course of the Madeira River as also suggested for several other
avian lineages (Aleixo 2004; Patan et al., 2009; Fernandes et al.,
2013; Sousa-Neves et al., 2013). Latrubesse (2002) postulated that
the Madeira paleodrainage was broader and more complex than
today until the late Pleistocene, reporting the presence of two
mega-fans related to the Aripuan and Jiparan rivers, both of
which currently ow into the Madeira River. Thus, it is possible
that the initial isolation of the northern Rondnia lineage in the
western clade is related to a former larger barrier (Aripuan
mega-fan), which decreased in size due to a drainage-capture
event (Wilkinson et al., 2010), promoting the secondary contact
between different non-sister lineages that diverged in allopatry,
as postulated for other terra-rme avian lineages (Fernandes
2013). The absence or very low levels of gene ow detected among
populations of the T. aethiops complex suggest a ckle scenario of
migration across Amazonian rivers, which is probably best
explained by an overall inability to cross open water, as demonstrated for other understory Neotropical avian lineages (Moore
et al., 2008). Thus, multiple and independent splitting events along
a single modern river course are more likely explained by the disruption of river drainages, whereby barrier effects move across
neighbouring interuvia.
Likewise, the estimated dates for the splits across the Solimes
and Negro rivers long after the formation of these barriers, suggest
a similar scenario of river lateral change or course capture. The formation of the Solimes River is closely related to the formation of
the transcontinental Amazonian drainage during the Late PlioceneEarly Pleistocene (Campbell et al., 2006; Hoorn et al., 2010;
Latrubesse et al., 2010). Thus, if these time estimates for the establishment of the Solimes River are correct, the relatively recent
split of T. aethiops populations across this river (0.680.26 Mya)
is explained by vicariance following dispersal after the formation
of the current Solimes River. As observed for clades separated
by the Solimes, the formation of the Negro River (see AlmeidaFilho and Miranda 2007) probably predates the cladogenetic event
separating the Imeri/Napo and western Guianan clades of T. polionotus (0.230.02 Mya). Unlike widely reported local instances of
river-channel migration due to sedimentation involving whitewater rivers in western Amazonia (Salo et al., 1986; Patton and
Silva, 1998), some shifts in Amazonian rivers courses take place
over long periods of time and are probably related to drainage capture events mediated by tectonics (Almeida-Filho and Miranda
2007; Rossetti and Valeriano, 2007; Shephard et al., 2010). The
growing body of phylogeographic evidence available now for Amazonian lineages suggest that river courses, particularly those of the
Negro, Madeira, Tapajs and Tocantins, have been stable for long
periods of time until changing part of their courses, and then
remaining stable for an additional time (Ribas et al., 2012;
dHorta et al., 2013; Fernandes et al., 2012, 2013; MaldonadoCoelho et al., 2013; Sousa-Neves et al., 2013). Hence, a dynamic
drainage landscape, over long periods of time, can act like a speciation pump rather than prevent diversication, as suggested previously (Haffer 1993; Colwell 2000; Gascon et al., 2000).
107
When compared to other upland terra-rme avian lineages

which were also shown to have their diversication mediated by
Amazonian rivers (Fernandes et al., 2012, 2013; Ribas et al.,
2012; dHorta et al., 2013; Sousa-Neves et al., 2013), the order
and timing in which different rivers accounted for splits of lineages
of the T. aethiops complex differ signicantly from most of them,
indicating that distinct lineages responded differently to the process of drainage evolution in Amazonia. Perhaps the most similar
cases to that of the T. aethiops complex are those reported for the
Xiphorhynchus spixii/elegans (Dendrocolaptidae; Aleixo 2004) and
Phlegopsis nigromaculata (Thamnophilidae; Aleixo et al., 2009),
whereby the Tapajs River also accounted for the rst split in time.
One hypothesis is that these differences in the relative order and
timing in which different rivers account for splits throughout the
Amazon are related to the geograc origins of the ancestral populations of these widespread lineages (Aleixo and Rossetti 2007). In
contrast to the T. aethiops complex, X. spixii/elegans and Sclerurus
mexicanus (dHorta et al., 2013), whose sister groups in Amazonia
include widespread Andes lineages, a different but pervasive phylogeographic pattern in the Amazon is that where earliest splits
usually involve lineages endemic to the Guianan shield and coincide either with the present course of the lower Amazon or Negro
rivers (Carneiro et al., 2012; Batista et al., 2013; dHorta et al.,
2013; Rodrigues et al., 2013; Sousa-Neves et al., 2013). This alternative pattern imply a Guianan shield origin for these lineages,
whereas those reported for T. aethiops, P. nigromaculata, and X. spixii/elegans (which are absent or nearly absent from the Guianan
shield) support a Brazilian shield origin (see also Aleixo and
Rossetti 2007). Therefore, we hypothesize that the structuring of
the modern Amazon drainage and the drying out of the western
Amazonian sedimentary basins from the late Pliocene to the late
Pleistocene were keystone events to promote concomitantly: (1)
an increase in the dispersal rates of both Brazilian and Guianan
shield lineages across the more recent upland terra-rme forests
of the upper Amazonia (Solimes sedimentary basin); and (2) an
increase in cladogenetic events involving upland terra-rme forest
lineages due to the latest cycle of drainage evolution throughout
Amazonia, which shaped the modern transcontinental river system. Under this scenario, depending on the geographical origins
of any given lineage before this latest cycle of widespread drainage
structuring (late PlioceneEarly Pleistocene), their response to riverine barriers can differ widely, as veried by the phylogeographic
studies conducted so far.
When climate change is evaluated as a possible driver of diversication in the T. aethiops complex, neutrality tests and EBSPs support demographic expansions in lineages distributed in the
Inambari and northern Rondnia areas of endemism since the last
interglacial cycle (approximately 0.13 Mya) to the present (Fig. 2).
Recent demographic expansions have been postulated for other lineages of birds in western Amazonia (Aleixo, 2004; Fernandes et al.,
2012; Ribas et al., 2012), but in all cases there was no evidence that
purported refuges were correlated with cladogenetic events in
these lineages. The dates estimated for the origin of the main reciprocally monophyletic lineages of the T. aethiops complex based on
the multi-locus species tree precede the coalescent time of the
locus used in EBSPs analyzes as well as the estimated time of the
demographic expansions. Thus, if climatic oscillations during the
last 0.2 Mya affected populations of the T. aethiops complex, they
apparently were not strong enough to generate the formation of
new clades, hence not supporting the refuge hypothesis as an
important cause of diversication in the T. aethiops complex
(Haffer, 2001). Furthermore, differences in the historical population
demography among T. aethiops complex lineages (Fig. 1c and 2),
with some lineages exhibiting relatively stable demographic histories, suggest that climate oscillations during the Late Pleistocene
did not impact uniformly all sectors of Amazonia. Even though
108
the sparse sampling of some T. aethiops complex lineages may yield

unreliable results that underestimate the effect of climatic oscillations on ancestral population sizes during the last glacial cycles,
our data is in agreement with that recovered for several other
linages of Amazonian organisms where no signicant changes in
historical population structures were found since the Last Glacial
Maximum (Lessa et al., 2003; Fernandes et al., 2012; Ribas et al.,
2012; Maldonado-Coelho et al., 2013). These results have ignited
an intense debate on the applicability of the refuge hypothesis in
explaining patterns of diversication in Amazonia (Bush and de
Oliveira, 2006), along with paleo-environmental uncertainties concerning the existence and location of refuges (Bush, 1994; van der
Hammen and Hooghiemstra, 2000; Haffer, 2001).
Acknowledgments
We thank the curator and curatorial assistants of the Museum
of Natural Science, Louisiana State University (LSUMZ), for allowing us to sequence tissues and study specimens under their care.
We also grateful to the tenacious effort of many specimen collectors working for or together with the Museu Paraense Emlio Goeldi (MPEG), who along years of intense eld work in the Amazon,
amassed the tissues necessary for the analyzes contained in this
paper, with permits provided by ICMBIO (Instituto Chico Mendes
de Conservao da Biodiversidade). Field and laboratory work
related to this paper were generously funded by the Brazilian
Research Council (CNPq; grants # 310593/2009-3; INCT em Biodiversidade e Uso da Terra da Amaznia # 574008/2008-0; #
563236/2010-8; and # 471342/ 2011-4) and the Par State Funding
Agency (ICAAF 023/2011). Support to GTs graduate research was
provided by a CNPq Masters fellowship (# 131000/2010-1). AA
is supported by a CNPq research productivity fellowship (#
310880/2012-2). We thank an anonymous referee as well as Marcos Maldonado-Coelho, Renato Caparroz, Luciano N. Naka, Camila
Ribas, and Pricles S. Rego for reviewing earlier versions of this
manuscript.
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Molecular Phylogenetics and Evolution 82 (2015) 95110

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

Cryptic speciation in the white-shouldered antshrike (Thamnophilus

G. Thom, A. Aleixo / Molecular Phylogenetics and Evolution 82 (2015) 95110

(Hilden, Germany) extraction kit. We amplied the mitochondrial

2. Material and methods

2.2. Phylogenetic analyses

2.1. Samples, laboratory procedures, and data analyses

2.2.1. Concatenated genes and gene trees

A total of 89 individuals from 55 localities were sampled

G. Thom, A. Aleixo / Molecular Phylogenetics and Evolution 82 (2015) 95110

G. Thom, A. Aleixo / Molecular Phylogenetics and Evolution 82 (2015) 95110

Brazil, Par, Altamira, Floresta Nacional de Altamira

Brazil, Par, Itaituba, Miritituba

Brazil, Par, Itaituba, Miritituba

Brazil, Par, Novo Progresso, 20 km SW

Brazil, Par, Senador Jos Porfrio, Rio Xingu

Brazil, Par, Senador Jos Porfrio, Rio Xingu

Brazil, Par, Carajs, Serra dos Carajs, Serra Norte

Brazil, Alagoas, Ibateguara, Engenho Coimbra

Brazil, Alagoas, Ibateguara, Engenho Coimbra

Brazil, Pernambuco, Barreiros, Cachoeira Linda

Brazil, Par, Santa Brbara, Parque Ecolgico GUNMA

Brazil, Par, Barcarena

Brazil, Par, Barcarena

Brazil, Par, Curua

Brazil, Amazonas, Careiro, Br 319 km

Brazil, Amazonas, Humait, Rio Ipixuna

Brazil, Acre, Parque Nacional Serra do Divisor

Brazil, Acre, Assis Brasil, ESEC Rio Acre

Brazil, Amazonas, RDS Cujubim, Rio Juta

Brazil, Acre, Tarauac, Br 364 km 40, Rio Liberdade

Brazil, Acre, Tarauac, Br 364 km 40, Rio Liberdade

Peru, Loreto, 86 km SE Juanjui

Peru, Loreto, 86 km SE Juanjui

Brazil, Acre, Rio Branco, Fazenda Experimental Catuaba

Brazil Acre, Bujari, Floresta Estadual do Antimary

Brazil Acre, Bujari, Floresta Estadual do Antimary

Brazil, Acre, Rio Branco, Ramal Jarinal

Brazil, Acre, Rio Branco, Ramal Jarinal

Amazonas, Humait, Rio Ipixuna

G. Thom, A. Aleixo / Molecular Phylogenetics and Evolution 82 (2015) 95110

Brazil, Acre, Mncio Lima, Estrada do Baro

Brazil, Acre, Mncio Lima, Estrada do Baro

Brazil, Acre, Santa Rosa, Rio Purus

Brazil, Acre, Senador Guiomard, Ramal Oco do Mundo

Bolivia, Pando, Nicols Suarez, 12 km by road of Cobija

Bolivia, La Paz, 20 km by Rio Beni N. Puerto Linares

Brazil, Amazonas, Barcelos, Rio Arac

Brazil, Amazonas, Barcelos, Rio Arac

Brazil, Amazonas, Barcelos, Rio Cuiuni

Brazil, Amazonas, Novo Airo, Igarap-Au

Brazil, Amazonas, Mara, Lago Cumapi

Brazil, Amazonas, Mara, Lago Cumapi

Brazil, Amazonas, Juruti, Base Capiranga

Brazil, Amazonas, Juruti, Base Capiranga

Brazil, Par, Jacareacanga, Terra do Burandir

Brazil, Amazonas, Maus, Flona do Pau Rosa

Brazil, Rondnia, Guajar-Mirim, REBIO Ouro Preto

Brazil, Rondnia, Guajar-Mirim, REBIO Ouro Preto

Brazil, Mato Grosso, Querncia, Fazenda Tanguro

Brazil, Mato Grosso, Paranata, Rio Teles Pires