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Hybridization in headwater regions, and the role of rivers as drivers of speciation in

Amazonian birds

Jason T. Weir, Maya S. Faccio, Paola Pulido-Santacruz, Alfredo Barrera-Guzmán, and Alexandre Aleixo

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Citation (published version)

Weir, J. T., Faccio, M. S., Pulido‐Santacruz, P., Barrera‐Guzmán, A. O., & Aleixo, A. (2015). Hybridization in headwater regions, and the role of rivers as drivers of speciation in Amazonian birds. Evolution, 69(7), 1823-1834.

Publisher’s Statement This is the peer reviewed version of the following article: Weir, J. T., Faccio, M. S., Pulido‐Santacruz, P., Barrera‐Guzmán, A. O., & Aleixo, A. (2015). Hybridization in headwater regions, and the role of rivers as drivers of speciation in Amazonian birds. Evolution, 69(7), 1823-1834.which has been published in final form at https://doi.org/10.1111/evo.12696. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

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Hybridization in headwater regions, and the role of rivers as

drivers of speciation in Amazonian birds

Journal: Evolution

Manuscript ID: 15-0028.R2

Manuscript Type: Original Article

Date Submitted by the Author: n/a

Complete List of Authors: Weir, Jason; University of Toronto Scarborough, Biological Sciences Faccio, Maya; University of Toronto, Ecology and Evolutionary Biology Pulido-Santacruz, Paola; University of Toronto, Ecology and Evolutionary Biology Barrera-Guzman, Alfredo; University of Toronto, Ecology and Evolutionary Biology Aleixo, Alexandre; Museu Paraense Emílio Goeldi, Dept. of Zoology

Keywords: Hybridization, Gene Flow, Biogeography, Reproductive Isolation

1

Hybridization in headwater regions, and the role of rivers as drivers of speciation in Amazonian birds. 1

Jason T. Weir1,2,*

, Maya S. Faccio2, Paola Pulido-Santacruz

2, Alfredo Barrera-Guzmán

2, Alexandre Aleixo

3 2

1) Department of Biological Sciences, University of Toronto Scarborough, Toronto, Canada3

2) Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada4

3) Department of Zoology, Museu Paraense Emílio Goeldi, Belém, Brazil5

* to whom correspondence should be addressed: jason.weir@utoronto.ca6

Key Words 7

Hybrid Zone, River barriers, gene flow, biogeography 8

9

Running Title: Hybridization in Amazonian headwaters 10

11

Corresponding Author Jason T. Weir 12

Address: Department of Biological Sciences. University of Toronto Scarborough, 1265 Military trail. 13

Toronto, Ontario, Canada. M1C 1A4. Fax: 416-287-7676 E-mail: jason.weir@utoronto.ca 14

15

Word Count: 6444 16

Figure Count: 4 17

Table Count: 0 18

19

Data Archival: Mitochondrial DNA sequences: Genbank accessions in Table S1; SNP datasets deposited 20

in Dryad doi (upon acceptance). Sampling locations in Table S1 21

22

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Abstract 23

Many understory birds and other groups form genetically differentiated subspecies or closely related 24

species on opposite sides of major rivers of Amazonia, but are proposed to come into geographic 25

contact in headwater regions where narrower river widths may present less of a dispersal barrier. 26

Whether such forms hybridize in headwater regions is generally unknown, but has important 27

implications to our understanding of the role of rivers as drivers of speciation. We used a dataset of 28

several thousand SNPs to show that seven taxon pairs which differentiate across a major Amazonian 29

river come into geographic contact and hybridize in headwater regions. All taxon pairs possessed 30

hybrids with low numbers of loci in which alleles were inherited from both parental species, suggesting 31

they are backcrossed with parentals, and indicating gene flow between parental populations. Ongoing 32

gene flow challenges rivers as the sole cause of in situ speciation, but is compatible with the view that 33

the wide river courses in the heart of Amazonia may have driven interfluvial divergence during episodes 34

of wet forest retraction away from headwater regions. Taxa as old as 4 Ma in our Amazonian dataset 35

continue to hybridize at contact zones, suggesting reproductive isolation evolves at a slow pace. 36

37

Introduction 38

Despite its exceptional species richness, little is understood about the historical factors that 39

contributed to species accumulation within the Amazon basin. Factors such as repeated immigration 40

into the Amazon from outside sources, and low levels of extinction within the basin could have 41

promoted species accumulation (see Fjeldså 1994). Nevertheless, in situ speciation within the Amazon 42

basin appears to have contributed importantly to species richness. For example, many groups of 43

Amazonian birds, mammals and other taxa, possess widespread superspecies complexes comprised of 44

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multiple allopatric species or subspecies that occupy different geographic regions within Amazonia (e.g. 45

Haffer 1974). These biogeographic patterns demonstrate that Amazonia is actively producing new taxa 46

(both species and subspecies) which contribute to its species richness. While the important role of in 47

situ speciation is undisputed, the primary driving mechanisms of speciation within Amazonia are poorly 48

understood. 49

Early naturalists like Wallace, Bates and others made the observation that major Amazonian 50

rivers often formed barriers to geographically-replacing taxa, with closely related species or subspecies 51

occupying opposing river banks (Wallace 1853, 1854 1876; Bates 1863). The subsequent accumulation 52

of museum specimen records since the pioneering efforts in the nineteenth century have confirmed 53

these early observations (Hellmayr 1910; Hershkovitz 1977; Ayres and Clutton-Brock 1992; Hayes and 54

Seawall 2004), and have further shown that major Amazonian rivers generally form the geographic 55

divisions between adjacent centers of endemism in which multiple co-distributed superspecies 56

complexes often share similar geographic patterns of replacement of their respective members across 57

the same rivers. For example, in birds, major Amazonian centers of endemism are largely delimited by 58

the Rio Negro, Rio Amazonas, Rio Madeira, Rio Tapajós, Rio Xingu, Rio Tocantins, and their tributaries, 59

with endemic species occurring in the interfluves between these river systems (Fig. 1; Cracraft 1985; 60

Silva et al. 2005). Similar patterns have been found for Amazonian primates (Ayres and Clutton-Brock 61

1992), but not for rodents (Patton et al. 1994; but this study investigated a river not generally thought to 62

present a strong barrier to dispersal). These observations led to the suggestion that rivers themselves 63

(and their floodplains) may act as biogeographic barriers, retarding gene flow and promoting allopatric 64

speciation in Amazonia, either through vicariance of once widespread species following formation of 65

rivers, or subsequent to rare dispersal events across or around river barriers (River-barrier hypothesis 66

hereafter, Sick 1967; Willis 1969; Hershkovitz 1977; Capparella 1988). 67

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Haffer (1969, 1974) and others (e.g. Vamzolini and Willians 1970; Prance 1973, 1982; Brown et 68

al. 1974) challenged the role of river-barriers as the engines of Amazonian speciation. These authors 69

suggested that dry periods, primarily during Pleistocene glacial maxima, resulted in the contraction of 70

Amazonian wet forest into a series of geographically isolated refugia where speciation occurred (Refuge 71

hypothesis). During wetter interglacials, newly diverged species expanded their ranges outwards, with 72

many species expanding until they reached major Amazonian rivers. River barriers were thus considered 73

of secondary importance as speciation had already occurred within refugia. Proponents of the Refuge 74

hypothesis observed that many Amazonian species complexes possess contact zones between 75

geographically-replacing species that do not coincide with river barriers, or that coincide with rivers only 76

along part of the geographic ranges (e.g. Haffer 1997). These patterns were interpreted as evidence that 77

range expansion from forest refugia was not always hindered by rivers. Haffer (1997) also pointed to 78

headwater regions as posing a serious problem for the River-barrier hypothesis. While rivers might be 79

formidable barriers to dispersal along their wide, lower stretches (e.g. the Rio Tapajós can exceed 20km 80

in width), in headwater regions the width of these rivers often narrows to as little as 70m (e.g. 81

headwaters of the Rio Teles Pires; see Fig. 1) where they may no longer function as dispersal barriers. 82

Gene flow through headwater regions could prevent differentiation of species in adjacent river 83

interfluves, and has remained a key argument against the River-barrier hypothesis. 84

There has been limited study of whether gene flow is widespread in headwater regions, and 85

most evidence has been based on morphological patterns hinting at intergradation in these regions for 86

select taxa (see Haffer 1997; Naka et al. 2012). Genetic evidence of gene flow across headwater regions 87

is limited in birds. Based on an analysis of nuclear markers in Glyphorynchus spirurus along a section of 88

the Rio Madeira located towards (but still far from) headwater regions, Fernandes et al. (2013) revealed 89

a single genetically admixed individual between deeply diverged genetic phylogroups that occur on 90

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opposite banks of this river. Similarly, multilocus data indicate small levels of gene flow in headwater 91

regions of the Rio Xingu between deeply diverged forms of the antshrike Thamnophilus aethiops that 92

occur on opposite sides of this river (Thom and Aleixo 2015). A mitochondrial based genetic study of 76 93

taxa pairs separated by the Rio Negro in northern Amazonia found that the Rio Negro or its tributary, 94

the Rio Branco, continued to delineate geographic ranges towards headwater regions in all but 13 pairs 95

(Naka et al. 2012). Those results suggest that rivers may continue to be sufficient barriers promoting 96

divergence for most taxa, even towards the northern periphery of wet forest in Amazonia. In Southern 97

Amazonia, Bates et al. (2004) investigated mitochondrial genetic differentiation across a small section of 98

the Rio Teles Pires that approaches headwater regions (Fig. 1). The Rio Teles Pires is the major tributary 99

flowing into the Rio Tapajós, and together this river system delimits the boundaries between the 100

Rondônia and Tapajós centers of endemism, with many endemic species and subspecies demarcated 101

along much of the length of this river system (Fig. 2). The mitochondrial data demonstrated genetic 102

breaks coinciding with the river for six of the 10 species studied, and highlighted the importance of the 103

river-barrier effect (despite the river being as narrow as 100m at this point) even towards the 104

headwaters. The authors concluded that gene flow in this headwater region seemed unlikely. However, 105

forests capable of supporting many Amazonian wet forest taxa extend another 400 km both to the south 106

and to the east of this Teles Pires study site (an area poorly represented in ornithological collections), 107

and it remains unknown if Amazonian taxa extend into these regions and merge genetically at contact 108

zones (Haffer 1992). 109

What is needed is a clear assessment of whether taxa come into geographic contact in 110

headwater regions and hybridize there as proposed by Haffer (1997). Here we make use of a next 111

generation sequencing approach that samples thousands of single nucleotide polymorphisms (SNPs) to 112

study gene flow in birds along the extreme peripheral edges of Amazonian forest in the headwaters of 113

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the Rio Teles Pires and Rio Xingu. Haffer (1997) highlighted this headwater region as a suture zone 114

where geographic contact was likely between many pairs of avian species or subspecies indicative of the 115

Rondônia and Tapajós centers of endemism (Fig. 1). This headwater region provided a key example to 116

his argument for the homogenizing effect of gene flow through headwater regions. However, the actual 117

contact zones, while inferred to exist based on current knowledge of geographic ranges and the 118

probable absence of intervening barriers to dispersal, have never been demonstrated conclusively (i.e. 119

with local syntopy). By sampling at regular intervals over a 550 km transect of this headwater region we 120

were able to determine the precise locality of geographic contact for five parapatric pairs of species and 121

two pairs of genetically diverged subspecies which are widespread in the Rondônia or Tapajós centers of 122

endemism and whose geographic ranges are largely separated by the Rio Tapajós and its tributaries. We 123

used several analyses of genetic admixture to test for gene flow at contact zones and to determine if 124

genetically admixed individuals represented only F1 hybrids (which could be sterile, in which case gene 125

flow is not demonstrated) or also included later generation hybrids (e.g. backcrossed individuals) 126

indicative of gene flow. The presence of genetically backcrossed individuals in headwater regions would 127

lend support to Haffer’s argument that the River-barrier hypothesis cannot adequately explain the 128

differentiation of taxa in adjacent interfluves. 129

130

Methods 131

Sampling Design: We obtained genetic samples (Table S1) from 17 sites along a 550 km north/south 132

transect in the Brazilian states of Pará and Mato Grosso in 2012, one site in 2014, and one site in 2006 133

and again in 2010 for a total of 19 sites (Fig. 1; Table S2). Sites were spaced every 50 to 60 km or less 134

over most of the transect. In this region, the Rio Teles Pires and Rio Xingu flow in a primarily south to 135

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north direction in parallel to our transect, which lies between the two rivers. Though our transect lies 136

within the Amazonian wet forest biome, the forest in this region has a strong seasonal component, and 137

most of our transect is classified as the “Mato Grosso seasonal forest” by the World Wildlife Fund (Olson 138

et al. 2001). Forest is now heavily degraded along the Rio Teles Pires in this region, but remains intact 139

along most of the Rio Xingu. Most of our sampling sites occurred on privately owned forest contiguous 140

to the pristine forest tracts of Xingu National Park and other protected areas along the Rio Xingu. The 141

transect also included one site (near Nova Mutum) south and west of the Rio Teles Pires along gallery 142

forest and adjacent semi-deciduous forest within the cerrado savannahs that lie to the south of the wet 143

forest biome, and included two sites in wet forest along the east side of the Rio Xingu headwaters. This 144

sampling design was augmented with samples previously collected further afield in the Rondônia and 145

Tapajós Endemism Centers for a total of 144 individuals (Table S1). Our study focusses on taxon pairs 146

believed by Haffer (1992) to come into geographic contact in this region (Xiphorhynchus elegans / X. 147

spixii, Rhegmatorhina hoffmannsi / R. gymnops, Willisornis poecilinotus / W. vidua, and Lepidothrix 148

nattereri / L. iris), as well as subspecies pairs in Dendrocincla fuliginosa, Glyphorynchus spirurus, and the 149

species pair Hypocnemis ochrogyna / H. striata which we found across the transect. 150

151

DNA Sequencing: Whole genomic DNA was extracted from pectoral muscle using the Omega Bio-tek 152

E.Z.N.A DNA Extraction Kit. We sent these samples to the Cornell Institute of Genomic Diversity (IGD) to 153

obtain SNP datasets through a Genotyping by sequencing (GBS) protocol described in Elshire et al. 154

(2011). This protocol uses the PstI enzyme to produce reduced representation single-end libraries which 155

were sequenced on multiple lanes of the Illumina HiSeq 2000 platform with 95 individuals uniquely 156

barcoded and multiplexed for each lane. We processed the resulting 100 base pair (bp) reads through 157

the non-reference genome version of the Universal Network Enabled Analysis Kit (UNEAK) pipeline (Lu 158

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et al. 2013) implemented in TASSEL 3.0 (Bradbury et al. 2007). The pipeline was run separately for each 159

taxon pair. The pipeline trims reads to 64bp, merges identical reads into tags within each barcoded 160

individual, and uses pairwise alignment to identify tag pairs with 1 bp mismatch (while probabilistically 161

correcting sequencing errors using the error tolerance rate parameter which we set to 0.03) which are 162

retained as candidate SNPs. For each taxon pair, only loci with 1 SNP are retained. Candidate SNPs 163

present in fewer than 70% of individuals were excluded; then individuals possessing fewer than 10% of 164

candidate SNPs were excluded. Next, a maximum likelihood method (Lynch 2009) was used to 165

reconstruct genotypes based on sequencing coverage using the scripts provided in White et al. 166

(2013)(we used an updated version of the PairDuplicates.pl script provided by T. White which corrected 167

an earlier programming bug which resulted in incorrect genotype calls at some SNPs). This method 168

retains SNPs with an Akaike Information Criterion ≥ 4 units lower than the next best reconstructed 169

genotype. SNPs with observed heterozygosities greater than 0.75 were excluded (this functions to 170

exclude likely paralogs). Other studies have found that varying the heterozygosity threshold cut-off 171

between 0.5 and 1.0 had little effect (Baldassarre et al. 2014), so we used the recommended 0.75 172

threshold. This was followed by a final filtering step in which SNPs present in fewer than 75% to 90% 173

(depending on the taxon pair) of individuals were excluded and individuals with fewer than 40% to 75% 174

(depending on the taxon pair) of SNPs were excluded. 175

For genome-wide SNP datasets, we used FST to determine genetic differentiation between the 176

two taxa in each taxon pair. FST was calculated in the R package Adegenet v1.4-2 (Jombart & Ahmed 177

2011). Hybrid individuals from contact zones (diagnosed using STRUCTURE (Pritchard et al. 2001), see 178

details below) were not included in FST measurements. Because only loci with a single SNP for each 179

taxon pair were retained by the UNEAK pipeline, our estimates of FST are likely to be somewhat 180

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inaccurate, and are used with caution here simply to provide an approximate estimate for genome-wide 181

genetic differentiation. 182

A 970 bp region of the mitochondrial gene cytochrome b was sequenced using standard 183

protocols (Weir and Price 2011a) in order to date when taxon pairs diverged from a common ancestor 184

and to determine whether taxa within taxon pairs are reciprocally monophyletic. GTR-y distances were 185

calculated in PAUP v4.0 (Swofford 2002), and a 2.1% molecular clock applicable to Passerine birds (Weir 186

and Schluter 2008) was used to provide approximate dates of divergence between members of each 187

taxon pair. Bayesian phylogenetic trees were constructed in MrBayes 3.2 (Ronquist and Huelsenbeck 188

2003) using the best fit model of sequence evolution returned by MrModelTest 2 (excluding models 189

which make a correction for invariant sites; Nylander 2004) and were rooted with closely related 190

outgroups (see Table S2). Phylogenies were run for 2 million generations, were sampled every 200 191

generations and majority-rule consensus phylogenies were generated from samples following a 0.5 192

million generation burnin. 193

194

Admixture analysis: We use the term hybrid to describe genetically admixed individuals between pairs 195

of subspecies and species. Principal coordinate analysis (PCoA) in R (cmdscale function; R Core Team 196

2014) and admixture analysis in STRUCTURE 2.3.4 (Pritchard et al. 2001) were used to classify individuals 197

as parental or hybrid. Both analyses were performed on the entire post-filtered SNP datasets for each 198

species pair. Each individual’s genotype at each SNP was coded as 0 or 1 for homozygotes, and 0.5 for 199

heterozygotes for the PCoA analysis. PCoA was performed using Euclidean distances. STRUCTURE was 200

run with the number of populations set at k=2 because, in each case, we are dealing with two taxa that 201

are genetically diverged in both SNPS (with two distinct parental clusters occurring in PCoA space) and in 202

mitochondrial DNA (with deep divergences between, but not within, each cluster in mitochondrial DNA; 203

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see Results). For each taxon pair, eight independent STRUCTURE runs (each with a different starting 204

seed) were performed for 100,000 generations following a 100,000 generation burnin. Post structure 205

results were pooled across runs to determine admixture proportions and their 95% confidence intervals. 206

Individuals highlighted as parental populations in the STRUCTURE and PCoA analyses were then 207

used as reference populations to calculate the hybrid index of each admixed individual using the R 208

package INTROGRESS (Gompert and Buerkle 2010). Parental populations from the Rondônia Endemism 209

Center were set to have a hybrid index of 0, and those from the Tapajós Center were set to an index of 210

1. Interspecific heterozygosity – the proportion of loci in an admixed individual’s genome with alleles 211

inherited from both parental species – was calculated for each admixed individual using INTROGRESS. 212

These values range from 0 (no excess heterozygosity) to 1. Values near 1 are obtained for F1 hybrids, 213

while values less than 1 indicate later generation hybrids that have either backcrossed with the 214

parentals and/or with other hybrids (Fitzpatrick 2012). Triangle plots, in which hybrid index is plotted on 215

the x-axis and interspecific heterozygosity along the y, were used to visualize these results. We used 216

either all SNPs or only SNPs with fixed differences between each of the two parental populations for 217

calculation of the hybrid index and interspecific heterozygosity. Because sample sizes of parental 218

populations were small, SNPs in the fixed SNP dataset were not treated as fixed in the analysis of hybrid 219

index and maximum likelihood methods implemented in INTROGRESS were instead used to estimate 220

parental allele frequencies. Currently available methods for calculating interspecific heterozygosity 221

assume parental allele frequencies are known. Results were similar for almost all admixed individuals 222

when all SNPs or only fixed SNPs were used, and we report results for the fixed SNP dataset only. 223

224

Results 225

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Our dataset was incorporated into multiple Illumina libraries, each of which also had individuals 226

from unrelated projects. Each Illumina library of 95 multiplexed individuals had on average ca. 2.0x108 227

raw reads, thus each individual had on average ca. 2.1x106 raw reads. Our post filtered datasets included 228

between 1,207 and 7,517 SNPS depending on the taxon pair (Fig. 2). A recently published passerine GBS 229

dataset of ca. 68,000 SNPs reported a low Burrow’s composite measure of inter- and intralocus 230

disequilibria, indicating that SNPs are largely independent for datasets of this size (Parchman et al. 231

2013). We lacked reference genomes for our taxon pairs and so have not performed a similar analysis, 232

but given we used far fewer SNPs, we expect most SNPs to likewise be independent. Our post filtering 233

SNP datasets had an average depth of coverage of 20.9 per locus (range of averages: 5.9 to 303.8) and of 234

21.4 per individual (range of averages: 8.9 to 71.1). 235

PCoA analyses of SNPS (Fig. 2) revealed two distinct clusters for each taxon pair that correspond 236

to parental taxa endemic or largely endemic to the Rondônia and Tapajós endemism center. Genome-237

wide Fst values between the two taxa in each taxon pair ranged from 0.15 in Lepidothrix to 0.48 in 238

Xiphorhynchus (Fig. 2). All but one of these FST values were higher than the only other genome-wide FST 239

value previously reported for closely related sister species of birds in the Neotropics (Manacus vitellinus 240

and M. candei with an FST of 0.26; Parchman et al. 2013). With the exception of the lower genetic 241

differentiation in Lepidothrix, FST values suggest moderate to strong genetic differentiation between taxa 242

in our taxon pairs. Mitochondrial DNA phylogenies (Fig. 3) and genetic distances (Table S1) showed a 243

similar pattern, with divergence between parental taxa much greater than within each taxa, and with a 244

range of genetic differentiation (Fig. 4). GTR-gamma divergence in mtDNA and genome-wide SNP based 245

FST values were strongly correlated (pearson’s r = 0.48) but not significantly (p = 0.13) correlated. 246

STRUCTURE analysis of the SNP data highlighted between one and six individuals in each taxon 247

pair as being significantly genetically admixed (i.e. 95% confidence intervals did not overlap 0 or 1; Fig 248

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2), and these occurred at intermediate positions along PCo 1 between parental taxa as expected if they 249

were hybrids. All seven taxa pairs studied had at least one individual with more than 25% admixture (Fig. 250

2). In all cases, individuals reconstructed as genetically admixed came from geographic regions precisely 251

at the contact zones between taxa endemic to the Rondônia and Tapajós Endemism Centers as expected 252

if they represent hybrids (Fig. 2). In five of the seven taxa pairs studied, sites with hybrids also had 253

individuals genetically typical of one of the two parental taxa (parental taxa syntopic with hybrids: D. 254

fuliginosa atrirostris and D. f. rufo-olivacea, Willisornis v. nigrigula, Hypocnemis ochrogyna, 255

Rhegmatorhina gymnops, Lepidothrix iris eucephala). However, at no sites did we record both parental 256

taxa syntopically. The geographic regions in which hybridization occurred corresponded closely across 257

most of our contact zones (Fig. 2), all of which appear to occur within a 270 km region, and thus 258

represent a narrow suture zone (sensu Remington 1968). Finer-scaled geographic sampling is necessary 259

to determine the widths of individual hybrid zones, but our preliminary sampling suggests that they are 260

less than 200 km wide for most taxa pairs. 261

The level of missing loci per individual ranged from 0 to 52% for individuals not identified by 262

STRUCTURE as hybrids (mean = 7.6%) and 0 to 61% (mean = 11.4%) for hybrids, and the differences 263

were not significant (t-test; p=0.40). The higher values for the hybrids were due to two individuals with 264

high levels of missing data. One of the two hybrid individuals in Glyphorynchus and the sole Hypocnemis 265

hybrid had 58% and 61% of loci missing respectively. When these two individuals are excluded then the 266

percentage of missing data in the remaining hybrid individuals is similar (mean = 5.4%, range = 0 to 18%) 267

to that for parentals. To be certain that the high level of missing loci were not biasing the admixture 268

proportions for Glyphorynchus and Hypocnemis we filtered datasets for these species so that only SNPs 269

present in the hybrid individual were retained. STRUCTURE analyses were then repeated using identical 270

methods as above. Admixture proportions were almost identical for the two filtering strategies with 271

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admixture proportions for the hybrid individuals differing by less than 1% for the two approaches. This 272

result demonstrates that admixture proportions were not biased by the level of missing loci. 273

We used triangle plots of interspecific heterozygosity versus hybrid index for diagnostic SNPs 274

fixed in our sample from parental populations to determine if hybrids represented F1’s or later 275

generation hybrids. Only 1 individual was a likely F1 hybrid (Willisornis poecilinotus), as evidence by a HI 276

close to 0.5 and an interspecific heterozygosity score close to 1 (Fig. 2). Low values of heterozygosity in 277

the remaining hybrid individuals suggest these individuals represent latter generation hybrids that have 278

crossed with other hybrids and/or with parentals (individuals lying along the diagonal lines in the 279

triangle plots are likely backcrosses with parentals; Fitzpatrick 2012). Results were similar when all SNPs 280

(not just those that are diagnostic) were used, with the exception that the likely F1 hybrid for Willisornis 281

poecilinotus now had a much lower interspecific heterozygosity. 282

Bayesian phylogenetic trees generated from mitochondrial cytochrome b (Fig. 3) indicate that 283

each of the taxon pairs formed two distinct clades which correspond closely to the two groups 284

recovered by the PCoA and STRUCTURE analysis of the SNP datasets (Fig. 2). The SNP and mitochondrial 285

datasets thus support the distinctness of the two named taxa within the taxon pairs. The only lack of 286

correspondence between the mitochondrial and SNP datasets occurred in two individuals of Lepidothrix 287

from population 1 (towards the northern edge of the contact zone) in which the SNP dataset classified 288

them as L. iris eucephala (this classification agrees with the male plumage of this population), but the 289

mitochondrial analysis grouped them with L. nattereri (Fig. 3). However, one of the five individuals 290

sampled from population 1 had a small, but significant signature of admixture in the SNP dataset, thus 291

mitochondrial introgression into some of the individuals of this population is not surprising. 292

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Without fail, individuals with a significant signature of admixture in the SNP dataset all occur at 293

the populations immediately at the contact zones between the taxa in each taxon pair. These admixed 294

individuals group with both parental taxa in Bayesian mitochondrial phylogenies in Dendrocincla, 295

Xiphorhynchus, Lepidothrix and Willisornis, or with just one of the two parental taxa in Hypocnemis (only 296

1 admixed individual detected in this pairs) and Glyphorynchus (Fig. 3). We have not yet successfully 297

sequenced the mitochondrial DNA of the only genetically admixed Rhegmatorhina sample. For two taxa 298

pairs, the population immediately at the contact had individuals grouping with both of the 299

mitochondrial clades (Xiphorhynchus and Willisornis). 300

In each of the two mitochondrial clades detected for each taxon pair, average mitochondrial 301

cytochrome b based GTR-y distances within clades were small (0.0% to 0.7%) compared to between the 302

clades (1.5% to 8.67%; Table S3). Mitochondrial sequence based estimates of gene coalescence times 303

between parental populations from the Rondônia and Tapajós endemism centers ranged from just 0.7 304

Ma in Lepidothrix iris / L. nattereri and Regmatorhina hoffmannsi / R. gymnops to 4.1 Ma in Willisornis 305

poecilinota / W. vidua (Fig. 4). Coalescent dates are estimated to predate population splitting dates on 306

average by only about two to three hundred thousand years in Neotropical birds (see Weir 2006), and 307

the low levels of genetic distance within our taxa support this. 308

309

Discussion 310

Our data provide the first evidence that multiple, co-distributed taxa endemic to adjacent 311

interfluves in Amazonia, and whose geographic ranges are largely separated by river barriers, 312

nevertheless come into geographic contact in the headwater regions where they hybridize. In all seven 313

taxon pairs studied, we found two distinct clades in mitochondrial DNA (Fig. 3), most of which had 314

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moderate to high levels of genome-wide FST, and which correspond closely to two distinct clusters in 315

PCoA plots based on the genome-wide SNP datasets (Fig. 2). These clades demonstrate genetic 316

differentiation between the taxa in each pair largely separated by the Tapajos and Teles Pires rivers. For 317

each pair, PCoA and STRUCTURE analyses detected between one and six genetically admixed individuals 318

from headwater localities which lie precisely at the contact zones between taxa. These results support 319

the key argument of opponents of the River-barrier hypothesis – namely that geographic contact and 320

gene flow in headwater regions should prevent speciation from occurring between adjacent interfluves 321

if river barriers were the only feature driving speciation in these taxa. Rather, other factors – probably in 322

combination with river barriers – must play a role in genetically isolating taxa long enough for speciation 323

to occur. Here we discuss other possible factors, and the implications of ongoing gene flow. 324

Our results could be interpreted as being consistent with (though not proof of) the Refuge 325

hypothesis that forest refugia, rather than river barriers, drove Amazonian diversification. However, 326

both paleopollen data and molecular dating of phylogenetic splitting events generally argue against the 327

Refuge hypothesis as a general explanation of Amazonian speciation. While paleopollen data has 328

demonstrated the expansion of drier forest types along the northern and southern periphery of 329

Amazonia during cooler/dryer periods (e.g. Mayle et al. 2004), it has not supported the widespread 330

fragmentation of Amazonian forest into a large series of refugia as previously proposed by the Refuge 331

hypothesis (Bush and de Oliveira 2006). Likewise, dating of phylogenetic splitting events in the Amazon 332

has shown evidence that many (but not all) Amazonian species predate the Pleistocene glacial cycles, 333

especially the major cycles of the past one million when forest refugia where deemed most likely (e.g. 334

Moritz et al. 2000; Weir 2006; Rull 2008, 2011; Hoorn et al. 2010; Ribas et al. 2012). The seven taxon 335

pairs studied here have coalescent dates ranging between 0.7 and 4.1 Ma, with three pairs predating 336

the Pleistocene glacial periods altogether, and most pairs predating the severe glacial cycles of the last 337

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one million years of the Pleistocene (Fig. 4). While we acknowledge the imprecision of phylogenetic 338

dating, these results nevertheless suggest that at least some of the taxon pairs studied predate periods 339

when forest refugia seemed likely to have initiated speciation. The span of dates also argues against a 340

single biogeographic event, such as the initial formation of the Rio Tapajós river system, as causing the 341

simultaneous origin of taxon pairs endemic to the Rondônian and Tapajós endemism centers (see also 342

Smith et al. 2014 who argue more generally against river based vicariance as a key driver of Amazonian 343

diversification). 344

The lack of evidence for the Refuge hypothesis and the apparent failings of the River hypothesis 345

given gene flow in headwater regions are addressed by a hybrid of these two known as the River-refuge 346

hypothesis (Capparella 1991; Ayres and Clutton-brock 1992; Haffer 1992). This hypothesis acknowledges 347

that rivers in headwater regions fail to provide barriers sufficient to promote speciation, but builds on 348

paleopollen records that do support periods of past retraction of wet forest at the edges of Amazonia 349

towards its center (reviewed in Mayle et al. 2004). Rivers are wider towards the center of Amazonia and, 350

in combination with the retraction of wet forest out of headwater regions, are believed to provide 351

sufficient barriers to promote speciation under this model. For example, given that the Rio Teles Pires 352

delimits taxa boundaries just 170 km to the west of the northern part of our transect (Haffer 1997; Bates 353

et al. 2004), it seems likely that a retraction of wet forest by just two or three hundred kilometers in the 354

Rio Teles Pires area would probably be sufficient to isolate many populations on opposite sides of this 355

river, leading to their divergence. Though paleopollen records from the Rio Teles Pires headwaters have 356

not been studied, those at the edge of Amazonian wet forest to the west in Bolivia (Mayle et al. 2000; 357

Burbridge et al. 2004) and to the east in the Brazilian state of Pará (Sifeddine et al. 2001) support 358

expansion of dry forest into these regions during the last glacial maxima, and climatic models likewise 359

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17

support dry forest expansion along the entire southern periphery of the Amazon, but without 360

necessarily bisecting Amazonian wet forest (Mayle et al. 2004). 361

Other alternatives to the River-refuge hypothesis could also explain in situ Amazonian speciation 362

despite gene flow in headwater regions. First, speciation might occur despite continual parapatric 363

contact through headwater regions if populations in each interfluve became differentially adapted to 364

local ecological conditions, and headwater regions represented a strong ecotone (Endler 1977, 1982). 365

Selection against hybrid individuals outside of ecotones could be strong and sufficient to maintain 366

species differences despite local genetic admixture at the ecotone. We have not performed a formal 367

assessment of habitat or climatic differentiation between our taxa pairs endemic to the Rondônia and 368

Tapajós endemism centers. Nevertheless, we feel that species in these centers are unlikely to be 369

adapted to greatly different environments given that their ranges are separated only by a river barrier, 370

with presumably similar forest types, climates, and ecological communities on either bank (see Lees et 371

al. 2013 for a comparison of bird communities across the Rio Teles Pires). As such, parapatric speciation 372

via differentiation across an ecological gradient seems unlikely for our birds, though it has been 373

suggested for Amazonian fish across gradients of water chemistry (Cooke et al. 2014). 374

Second, headwater regions may possess sink populations, which, despite gene flow into them, 375

fail to prevent sister taxa from diverging in adjacent interfluves. Our transect occurs at the very southern 376

edge of Amazonian wet forest extent. Much of this region represents a mosaic of interdigitating wet 377

forest types (e.g. primarily along streams and rivers) and semideciduous drier forest (e.g. away from 378

water courses) before finally transitioning to the Chiquitano dry forests and Cerrado savannahs that lie 379

to the south (Fig. 1). While many of our study species also occur in the semideciduous forest types, 380

some appear to do so in low numbers. Rhegmatorhina gymnops, Dendrocincla fuliginosa, and 381

Glyphorynchus spirurus are generally abundant in typical wet forest habitats towards the center of 382

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Amazonia, but were captured in low numbers across our transect (Rhegmatorhina only in the northern 383

half), while other wet forest taxa were not detected at all (e.g. their ranges have not expanded this far 384

into headwater regions). Clearly, transitional forests at headwater regions may prevent some wet forest 385

species from expanding into these areas, while others occur in low numbers and represent sink 386

populations, which fail to retard divergence outside of headwater regions. Despite the transitional 387

nature of forest at headwater regions, some of our study species were abundant along the entire length 388

of the transect, and commonly occurred away from lush streamside vegetation (Willisornis poecilinotus / 389

vidua, Lepidothrix nattereri, Xiphorhynchus elegans/spixii). These latter examples suggest that sink 390

dynamics in headwater regions alone cannot explain the ongoing divergence between interfluves in 391

these groups, and we feel that the River-refuge hypothesis – consistent with paleopollen data – may 392

provide the best explanation in such examples. 393

Regardless of the precise dynamics under which rivers and forests interacted to promote 394

interfluvial differentiation, our data demonstrate that hybridization continues to occur despite one to 395

four million years of divergence since taxa pairs split from a common ancestor (Fig. 4). If the River-396

refuge hypothesis is correct, these results demonstrate that a single vicariant event driven by wet forest 397

retraction along the periphery of Amazonia was not sufficient to generate enough reproductive isolation 398

to prevent gene flow between parental species. Multiple episodes of retraction and divergence across 399

the wider river courses that characterize the heart of the Amazon basin, followed by expansion and 400

contact in headwater regions seems likely given the ages of these taxon pairs. In this respect, these 401

results for the Amazon basin mirror those in the boreal zone of North America, where, despite multiple 402

episodes of boreal forest retraction into refugia during glacials, followed by expansion during 403

interglacials, many boreal birds as old as 1 to 1.5 million years (Fig. 4), as well as mammals, continue to 404

form hybrid zones where their geographic ranges currently come into contact (Arbogast and Kenagy 405

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19

2001; Weir and Schluter 2004; Toews et al 2007; Irwin et al. 2009; Seneviratne et al 2012). The 406

comparison suggests that differentiation seems possible despite the slow rate at which reproductive 407

isolation accumulates (about a million years at high latitudes and considerably longer in the tropics for 408

birds; Weir and Price 2011b; this study) and despite repeated episodes of gene flow during periods of 409

contact. One possibility is that genetically introgressed populations in contact zone regions go extinct 410

with every repetition of forest expansion and retraction without ever having sufficient time to cause 411

diverging populations to become homogenized genetically (Weir and Schluter 2004). In this way, 412

ongoing divergence is possible despite repeated episodes of parapatric contact. For example, model 413

based estimates of migration rates are zero between parental populations of genetically diverged forms 414

of Thamnophilus aethiops from the Rondônia and Tapajós centers of endemism (Thom and Aleixo 2015), 415

suggesting that away from the vicinity of contact zones in the headwaters, gene flow has limited effect. 416

Hybridization does not necessarily imply a lack of complete reproductive isolation (e.g. Coyne 417

and Orr 2004; Price 2008). For example, hybrids might be sterile, in which case contact regions should 418

possess only F1 progeny (i.e. offspring from direct mating of parentals of each species) and parentals 419

would be fully reproductively isolated. However, low levels of interspecific heterozygosity in all but one 420

of our hybrid individuals suggest that genetically admixed individuals represent later generation hybrids 421

which have crossed with each other and/or with parentals. Admixed individuals falling along the 422

diagonal lines in the triangle plots of heterozygosity and hybrid index (e.g. Dendrocincla fuliginosa, 423

Xiphorhynchus spixii/elegans, Willisornis poecilinotus / vidua, and Lepidothrix nattereri / iris) are 424

expected to result from the direct crossing of a hybrid with a parental (Fitzpatrick 2012). Such individuals 425

suggest gene flow into parental populations, and a lack of complete reproductive isolation. These results 426

contrast with interspecific heterozygosity for hybrids between recently diverged sibling species of 427

flycatcher (Ficedula albicollis and F. hypoleuca) from high latitude regions in Europe where all hybrids 428

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tested had F1 genotypes and suggest strong selection against hybrid offspring (Kawakami et al. 2014). 429

While our data demonstrates that reproductive isolation is incomplete, small sample size limits the 430

scope of our inferences regarding the width of hybrid zones and levels of selection against admixed 431

individuals. Some of our taxon pairs are very old (e.g. 4 Ma; Fig 4.), and are noticeably differentiated in 432

vocalizations (Willisornis poecilinotus / vidua, Xiphorhynchus spixii/elegans), suggesting partial 433

reproductive isolation might be in place despite hybridization. However, other taxon pairs do not show 434

marked differentiation in plumage or song (Glyphorynchus spirurus, Dendrocincla fuliginosa; the latter of 435

which we could not differentiate even in the hand) despite having diverged from a common ancestor 436

approximately 3 Ma. Ironically, the taxon pairs with the most clearly differentiated plumage patterns, 437

and which have always been recognized as specifically distinct (Rhegmatorhina hoffmannsi / R. 438

gymnops; Lepidothrix nattereri / L. iris), are also the youngest (< 1 Ma) in our sample. These 439

observations suggest that plumage differentiation evolves at different rates in different species in our 440

sample. 441

The ages of our Amazonian taxon pairs are similar to those reported for other species with 442

hybrid zones from across the New World tropics (Weir and Price 2011b), with Willisornis representing 443

the oldest taxon pair (ca. 4 Ma) with a hybrid zone that we are aware of for birds. The average and 444

maximum age of taxa pairs with hybrid zones diminishes with increasing latitude in the New World (Fig. 445

4). At boreal latitudes above 45° N, all taxon pairs with hybrid zones diverged from a common ancestor 446

less than 1.5 Ma, suggesting that complete reproductive isolation is in place by one to two million years. 447

Indeed, high latitude species generally have achieved sufficient differentiation, that by about 1.7 million 448

years on average, they are capable of expanding their geographic ranges into sympatry (Weir and Price 449

2011b). Our results for the Amazon suggest that some taxa pairs as old as 3 to 4 million years have still 450

experienced insufficient time to achieve complete reproductive isolation, and continue to exclude each 451

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other geographically across hybrid zones. Tropical birds also are reported to evolve more slowly than 452

high latitude species in song, plumage colouration, body mass and climatic niche (Martin et al. 2010; 453

Weir and Wheatcroft 2011; Weir et al. 2012; Lawson and Weir 2014) – traits important for reproductive 454

isolation and ecological differentiation. Together, these results suggest a slower pace for differentiation 455

in lowland regions of the tropics, despite their high species richness. In the case of Amazonian taxa 456

differentiating on opposite sides of rivers, the apparent lack of strong ecological differences on adjacent 457

river banks is likely to result in lower rates of divergent natural selection between them, when 458

compared to high latitude species (Lawson and Weir 2014). 459

In conclusion, our results confirm the presence of gene flow across headwater regions of 460

Amazonia, despite the very old ages of some of the taxon pairs involved. Our current level of sampling is 461

insufficient to measure hybrid zone width or test for levels of selection against hybrids, and further 462

sampling is necessary to address hybrid zone dynamics in each of these taxon pairs in further detail. 463

Nevertheless, our data do demonstrate low levels of interspecific heterozygosity and suggest the 464

presence of hybrid individuals that have backcrossed with parental populations. These results 465

demonstrate incomplete reproductive isolation and challenge the generality of the River-barrier 466

hypothesis as a driver of in situ speciation in the Amazon. 467

468

Acknowledgments 469

A special thanks to the many landowners who kindly permitted us to sample on their property. The 470

Museu Paraense Emílio Goeldi and the Field Museum of Natural History donated tissue samples from 471

outside our transect. The Brazilian National Research Council (CNPq) granted permits necessary to 472

conduct our research ("Portaria MCTI # 659/2011 - Projeto de Expedição Científica # 00813/2011-7"). 473

Page 21 of 42

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The following funding sources are acknowledged: a Discovery Grant from the Natural Sciences and 474

Engineering Research Council of Canada (402013-2011) and a UTSC VPR-Research Competitiveness Fund 475

(JTW), a Mitacs Student Fellowship (MF), a FAPESPA (ICAAF 23/2011), and several CNPq grants to AA 476

(‘‘INCT em Biodiversidade e Uso da Terra da Amazônia’’ # 574008/2008-0; # 471342/ 2011-4; and # 477

310880/2012-2). 478

479

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ages of hybridizing species along a latitudinal gradient. Am. Nat. 177: 462-469. 629

Weir, J.T., D. L. Wheatcroft, and T. D. Price. 2012. The role of ecological constraint in driving the 630

evolution of avian song pitch across a latitudinal gradient. Evolution. 66: 2773-2783. 631

White, T. A., S. E. Perkins, G. Heckel, and J. B. Searle. 2013. Adaptive evolution during an ongoing range 632

expansion: the invasive bank vole (Myodes glareolus) in Ireland. Mol. Ecol. 22: 2971-2985. 633

Willis, E.O. 1969. On the behavior of five species of Rhegmatorhina, ant-following antbirds of the 634

Amazon basin. Wilson Bull. 81: 363-395. 635

636

Page 29 of 42

30

637

638

639

Figure legends (Colour versions for online) 640

Figure 1 641

Sampling sites (numbered 0 to 18; see Table S1) in the headwaters of the Rio Teles Pires and Rio Xingu 642

of the Brazilian Amazon. This headwater region occurs primarily in seasonal forest (purple) which forms 643

a transition between the wet forests to the north (green) and the Cerrado savannas (yellow) and 644

Chiquitano dry forests (pink) to the south (drawn from Olson et al. 2001). Other savanna areas shown in 645

pale yellow. Major rivers believed to form important dispersal barriers and which delineate endemism 646

regions in adjacent interfluves are shown in blue. Endemism centers are as follows: A) Guiana, B) Napo, 647

C) Inambari, D) Rondônia, E) Tapajós, F) Xingu, and G) Belém. Locations where each river first narrows to 648

<100 m is shown by red star (as measured from each rivers main channel in Google Earth). Width of the 649

Rio Xingu greatly exceeds 100m across most of its length, but has a small region only ca. 200 km from its 650

mouth where it narrows to less than 100m (northern star). The next region of narrowing occurs in the 651

headwaters (southern star). The black triangle indicates location of the Bates et al. (2004) study of river 652

barriers. Each segment of the vertical scale bar indicates 100 km. 653

654

Figure 2 655

Seven taxon pairs largely separated by the Rio Tapajós (red, taxa west of Tapajós; blue, taxa east of 656

Tapajos) and its tributaries, but which hybridize in headwater regions. Shown are the geographic ranges 657

Page 30 of 42

31

and sampling localities, PCoA plots (x-axis PCo 1, y-axis PCo 2), FST values (calculated from all SNPs), 658

STRUCTURE analysis, and triangle plots of interspecific heterozygosity (y-axis) and hybrid index (x-axis) 659

based on genome-wide GBS SNP data. Genetically admixed individuals on distribution maps and PCoA 660

analysis are represented by black stars, and on triangle plots by circles. Asterisks above STRUCTURE 661

plots indicate individuals where 95% confidence intervals on admixture proportions did not overlap 0 or 662

1. The number of SNP loci used in PCoA and STRUCTURE analyses are indicated above the PCoA plots (all 663

SNPs used), and the number of SNPs fixed between parental populations and used in triangle plots are 664

indicated. Numbers along the x-axis of STRUCTURE plots indicate the sampling population corresponding 665

to Fig. 1 and W and E indicate parental populations outside of our transect from west and east of the Rio 666

Tapajós / Rio Teles Pires respectively. The two subspecies of Glyphorynchus spirurus possess multiple 667

phylogroups based on mitochondrial DNA (sensu Fernandes et al. 2013). We show the ranges only for 668

the two phylogroups that meet in our transect. 669

670

Figure 3 671

Bayesian phylogenetic trees generated from 970 bp of mitochondrial DNA for taxon pairs largely 672

separated by the Rio Tapajós (red, taxa west of Tapajós; blue, taxa east of Tapajos) and its tributaries. 673

Individuals highlighted as significantly admixed in genome-wide SNP datasets are shown by black stars. 674

Posterior probabilities are shown at the basal node for each taxon (* indicates > 0.95). Individuals are 675

numbered according to the sampling population corresponding to Fig. 1 and W and E indicate parental 676

populations outside of our transect from west and east of the Rio Tapajós / Rio Teles Pires respectively. 677

All taxon pairs are drawn to scale (scale bar is show under the first taxon pair). 678

679

Page 31 of 42

32

680

Figure 4 681

A comparison of mitochondrial cytochrome b coalescent dates (estimated using a 2.1% molecular clock 682

applied to GTR-y distances; Weir and Schluter 2008) for our Amazonian taxon pairs and for other New 683

World terrestrial birds which possess hybrid zones. Amazonian birds studied here are indicated by black 684

triangles (taxa pairs as in Figure 2: 1 Willisornis, 2 Glyphorynchus, 3 Dendrocincla, 4 Xiphorhynchus, 5 685

Hypocnemis, 6 Rhegmatorhina, 7 Lepidothrix). Boreal birds (i.e. above 45° N) are indicated by gray 686

diamonds, all other hybrid zones are indicated by open circles. The absolute midpoint latitude of each 687

hybrid zone is plotted on the x-axis. Non-Amazonian species and dates taken from Weir and Price 2011. 688

689

Figure legends (Grayscale versions for print) 690

Figure 1 691

Sampling sites (numbered 0 to 18; see Table S1) in the headwaters of the Rio Teles Pires and Rio Xingu 692

of the Brazilian Amazon. This headwater region occurs primarily in seasonal forest (dark gray) which 693

forms a transition between the wet forests to the north (pale gray) and the Cerrado savannas (right 694

sloping diagonal pattern) and Chiquitano dry forests (labelled) to the south (drawn from Olson et al. 695

2001). Other savanna areas shown in pale left sloping diagonal patterning. Major rivers believed to form 696

important dispersal barriers and which delineate endemism regions in adjacent interfluves are 697

illistrated. Endemism centers are as follows: A) Guiana, B) Napo, C) Inambari, D) Rondônia, E) Tapajós, F) 698

Xingu, and G) Belém. Locations where each river first narrows to <100 m is shown by black star (as 699

measured from each rivers main channel in Google Earth). Width of the Rio Xingu greatly exceeds 100m 700

Page 32 of 42

33

across most of its length, but has a small region only ca. 200 km from its mouth where it narrows to less 701

than 100m (northern star). The next region of narrowing occurs in the headwaters (southern star). The 702

black triangle indicates location of the Bates et al. (2004) study of river barriers. Each segment of the 703

vertical scale bar indicates 100 km. 704

705

Figure 2 706

Seven taxon pairs largely separated by the Rio Tapajós (dark gray and circles, taxa west of Tapajós; light 707

gray and triangles, taxa east of Tapajos) and its tributaries, but which hybridize in headwater regions. 708

Shown are the geographic ranges and sampling localities, PCoA plots (x-axis PCo 1, y-axis PCo 2), FST 709

values (calculated from all SNPs), STRUCTURE analysis, and triangle plots of interspecific heterozygosity 710

(y-axis) and hybrid index (x-axis) based on genome-wide GBS SNP data. Genetically admixed individuals 711

on distribution maps and PCoA analysis are represented by black stars, and on triangle plots by circles. 712

Asterisks above STRUCTURE plots indicate individuals where 95% confidence intervals on admixture 713

proportions did not overlap 0 or 1. The number of SNP loci used in PCoA and STRUCTURE analyses are 714

indicated above the PCoA plots (all SNPs used), and the number of SNPs fixed between parental 715

populations and used in triangle plots are indicated. Numbers along the x-axis of STRUCTURE plots 716

indicate the sampling population corresponding to Fig. 1 and W and E indicate parental populations 717

outside of our transect from west and east of the Rio Tapajós / Rio Teles Pires respectively. The two 718

subspecies of Glyphorynchus spirurus possess multiple phylogroups based on mitochondrial DNA (sensu 719

Fernandes et al. 2013). We show the ranges only for the two phylogroups that meet in our transect. 720

721

Figure 3 722

Page 33 of 42

34

Bayesian phylogenetic trees generated from 970 bp of mitochondrial DNA for taxon pairs largely 723

separated by the Rio Tapajós (circles, taxa west of Tapajós; triangles, taxa east of Tapajos) and its 724

tributaries. Individuals highlighted as significantly admixed in genome-wide SNP datasets are shown by 725

black stars. Posterior probabilities are shown at the basal node for each taxon (* indicates > 0.95). 726

Individuals are numbered according to the sampling population corresponding to Fig. 1 and W and E 727

indicate parental populations outside of our transect from west and east of the Rio Tapajós / Rio Teles 728

Pires respectively. All taxon pairs are drawn to scale (scale bar is show under the first taxon pair). 729

730

731

Figure 4 732

A comparison of mitochondrial cytochrome b coalescent dates (estimated using a 2.1% molecular clock 733

applied to GTR-y distances; Weir and Schluter 2008) for our Amazonian taxon pairs and for other New 734

World terrestrial birds which possess hybrid zones. Amazonian birds studied here are indicated by black 735

triangles (taxa pairs as in Figure 2: 1 Willisornis, 2 Glyphorynchus, 3 Dendrocincla, 4 Xiphorhynchus, 5 736

Hypocnemis, 6 Rhegmatorhina, 7 Lepidothrix). Boreal birds (i.e. above 45° N) are indicated by gray 737

diamonds, all other hybrid zones are indicated by open circles. The absolute midpoint latitude of each 738

hybrid zone is plotted on the x-axis. Non-Amazonian species and dates taken from Weir and Price 2011. 739

740

Page 34 of 42

1

234

5 6 789

1011

13

12

14

15

16

17

BeniSavanna

Seasonal forest

Cerrado

Wet Forest

ChiquitanoDry Forest

A

B

DE

F

G

Andes

Figure 1

0

18

C

Page 35 of 42

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

-90

-40

10

60

-30 -15 0 15 30

-60

-40

-20

0

20

-60 -30 0 30 60

-60

-30

0

30

60

-60 -30 0 30 60

-40

-20

0

20

40

-30 0 30

0.00

0.25

0.50

0.75

1.00

W W 18

15

14

13

10

8 8 8 6 4 2 1 E E E

0.00

0.25

0.50

0.75

1.00

17

16

15

11

4 3 3 0 1 1 1 1 1 1 1 E E

0.00

0.25

0.50

0.75

1.00

W W 17

16

15

13

12

8 8 6 6 3 3 3 3 2 E E E

0.00

0.25

0.50

0.75

1.00

14

14

13

12

12

8 8 4 4 1 E E

0.00

0.25

0.50

0.75

1.00

W 16

8 7 4 3 3 2 2 2 1 1 1 1 E E

211 loci

951 loci

84 loci

234 loci

41 loci

240 loci

Figure 2

2103 loci

7517 loci

3091 loci

2387 loci

1207 loci

4631 loci

* * *

* * *

* *

*

* *

*

Dendrocincla fuliginosa atritostris / D. f. rufo-olivacea (FST = 0.35)

Xiphorhynchus elegans elegans / X. spixii (FST = 0.48)

Glyphorynchus spirurus inornatus / G. s. paraensis (FST = 0.34)

Hypocnemis ochrogyna / H. striata striata (FST = 0.46)

Rhegmatorhina hoffmannsi / R. gymnops (FST = 0.37)

Willisornis poecilinotus griseiventris / W. vidua nigrigula (FST = 0.45)

22 loci7495 loci****

Lepidothrix nattereri / L. iris eucephala (FST = 0.15)

0.00

0.25

0.50

0.75

1.00

W W W 12

12

E E E

***

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

-20

0

20

40

-30 -15 0 15 300.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

-30

0

30

60

-40 -20 0 20 40

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

-60

-30

0

30

60

-40 -20 0 20 40

0.00

0.25

0.50

0.75

1.00

W W 17

15

12

8 5 4 2 2 2 2 1 1 1 E E E E

Page 36 of 42

0.01

0.89

*

*

0.89

*

**

A) Dendrocincla B) Xiphorhynchus C) Glyphorynchus

D) Hypocnemis E) Rhegmatorhina F) Willisornis G) Lepidothrix

0.94

*

*

*

0.91

*

E

1714

W

15

8

W

W13

8

18

8

E

E6

142

0.71

2

E

3

4

W

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1

14

3

E

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1

1

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E

W

W

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612

15

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2

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1

2

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4

176

WWW

12158

E

E1

EE

4

4

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8

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Figure 3

Page 37 of 42

Absolute midpoint latitude

Coal

esce

nt d

ate

(Ma)

Figure 4

0

1

2

3

4

5

0 10 20 30 40 50 60

1

23

4

5

67

Page 38 of 42

1

234

5 6 789

1011

13

12

14

1517

BeniSavanna

Seasonal forest

Wet Forest

ChiquitanoDry Forest

A

B

DE

F

G

Figure 1

0

18

C

16

Cerrado

Page 39 of 42

-90

-40

10

60

-30 -15 0 15 30

-60

-40

-20

0

20

-60 -30 0 30 60

-60

-30

0

30

60

-60 -30 0 30 60

-40

-20

0

20

40

-30 0 30

-20

0

20

40

-30 -15 0 15 30

-30

0

30

60

-40 -20 0 20 40

-60

-30

0

30

60

-40 -20 0 20 40 0.00

0.25

0.50

0.75

1.00

W W 18 15 14 13 10 8 8 8 6 4 2 1 E E E

0.00

0.25

0.50

0.75

1.00

17 16 15 11 4 3 3 0 1 1 1 1 1 1 1 E E

0.00

0.25

0.50

0.75

1.00

W W 17 16 15 13 12 8 8 6 6 3 3 3 3 2 E E E

0.00

0.25

0.50

0.75

1.00

14 14 13 12 12 8 8 4 4 1 E E

0.00

0.25

0.50

0.75

1.00

W 16 8 7 4 3 3 2 2 2 1 1 1 1 E E

211 loci

951 loci

84 loci

234 loci

41 loci

240 loci

Figure 2

2103 loci

7517 loci

3091 loci

2387 loci

1207 loci

4631 loci

* * *

* * *

* *

*

* *

*

Dendrocincla fuliginosa atritostris / D. f. rufo-olivacea (FST = 0.35)

Xiphorhynchus elegans elegans / X. spixii (FST = 0.48)

Glyphorynchus spirurus inornatus / G. s. paraensis (FST = 0.34)

Hypocnemis ochrogyna / H. striata striata (FST = 0.46)

Rhegmatorhina hoffmannsi / R. gymnops (FST = 0.37)

Willisornis poecilinotus griseiventris / W. vidua nigrigula (FST = 0.45)

22 loci7495 loci****

Lepidothrix nattereri / L. iris eucephala (FST = 0.15)

0.00

0.25

0.50

0.75

1.00

W W W 12 12 E E E

***

0.00

0.25

0.50

0.75

1.00

W W 17 15 12 8 5 4 2 2 2 2 1 1 1 E E E E

Page 40 of 42

0.01

0.89

*

*

0.89

*

**

A) Dendrocincla B) Xiphorhynchus C) Glyphorynchus

D) Hypocnemis E) Rhegmatorhina F) Willisornis G) Lepidothrix

0.94

*

*

*

0.91

*

E

1714

W

15

8

W

W13

8

18

8

E

E6

142

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176

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EE

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Figure 3

Page 41 of 42

Absolute midpoint latitude

Coal

esce

nt d

ate

(Ma)

Figure 4

0

1

2

3

4

5

0 10 20 30 40 50 60

1

23

4

5

67

Page 42 of 42