Rivers acting as barriers for bird dispersal in the Amazon

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/272179867 Rivers acting as barriers for bird dispersal in the Amazon Article in Revista Brasileira de Ornitologia · December 2014 CITATIONS READS 2 76 4 authors: Alexandre M Fernandes Mario Cohn-Haft 28 PUBLICATIONS 221 CITATIONS 44 PUBLICATIONS 553 CITATIONS Universidade Federal Rural de Pernambuco SEE PROFILE Instituto Nacional de Pesquisas da Amazônia SEE PROFILE Tomas Hrbek Izeni Pires Farias 321 PUBLICATIONS 1,603 CITATIONS 232 PUBLICATIONS 1,814 CITATIONS Federal University of Amazonas SEE PROFILE Federal University of Amazonas SEE PROFILE Some of the authors of this publication are also working on these related projects: Genetic variability and population structure in silver arowana Osteoglossum bicirrhosum (Cuvier 1829) from the Amazon River Basin View project Phylogeography and population genetics of Cuvier’s Dwarf caiman (Paleosuchus palpebrosus) in the Amazon: SNPs by next-generation sequencing. View project All content following this page was uploaded by Alexandre M Fernandes on 13 February 2015. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. Revista Brasileira de Ornitologia, 22(4), 361-371 December 2014 article rivers acting as barriers for bird dispersal in the amazon alexandre M. Fernandes1,5, Mario cohn-Haft2, tomas Hrbek3,4 and izeni Pires Farias3 1 2 3 4 5 Universidade Federal Rural de Pernambuco, UFRPE, Unidade Acadêmica de Serra Talhada, Serra Talhada, PE, Brazil. Instituto Nacional de Pesquisas da Amazonia, INPA, Manaus, AM, Brazil. Universidade Federal do Amazonas, UFAM, Manaus, AM, Brazil. University of Puerto Rico, Rio Piedras, UPR-RP, Puerto Rico, USA. Corresponding author: [email protected] Received on 19 November 2014. Accepted on 10 December 2014. aBStract: Morphological, vocal and genetic studies have shown that the Madeira River and its right bank tributaries delimit populations of primates and birds. We sequenced the cytochrome b gene (approx. 950 bp) for individuals of three suboscine passerine bird species, Glyphorynchus spirurus (Furnariidae), Willisornis poecilinotus (hamnophilidae) and Schifornis turdina (Tityridae), on opposite banks of the Madeira River and two of its right-bank tributaries, the Aripuanã and Jiparaná rivers. Phylogenetic hypotheses (parsimony, maximum likelihood and Bayesian analysis) revealed clades that have over 3.1% genetic diferentiation on opposite banks of the Madeira River for G. spirurus, W. poecilinotus and S. turdina, suggesting that this river restricts gene low among populations of these three species. he Jiparaná and Aripuanã rivers apparently separate distinct populations of G. spirurus, the smallest species we examined, but not those of the other two heavier bodied species, W. poecilinotus and S. turdina. In G. spirurus four clades with high levels of genetic diferentiation (3.2–5.5%) were found to be delimited by the three rivers evaluated, whereas in W. poecilinotus and S. turdina no genetic structure across the Jiparaná and Aripuanã rivers was detected. In general, birds that are known to show population structure across the Madeira tributaries (Glyphorynchus spirurus, Hemitriccus minor, Hypocnemis rondoni, Herpsilochmus stotzi, and Hylophylax naevius) have body masses smaller than those of both Willisornis poecilinotus and Schifornis turdina, but some exceptions are discussed. Future studies controlling for several variables are necessary to determine the extent to which body mass is a useful predictor of genetic population structure in understory suboscine passerines. KeY-WordS: Areas of endemism, body mass, comparative phylogeography, conservation, dispersal rate, suboscine birds. introduction Avian distribution patterns are reasonably well known and inluential in studies of evolutionary processes. Jürgen Hafer was one of the irst authors to compile bird distribution data to describe biogeographic patterns in South America (Hafer 1974). His work made a great contribution to evolutionary studies and to the formulation of speciation hypotheses in Amazonia. he “centres of species endemism” he described remain largely unchanged in analyses of distribution patterns for many avian groups. hey have been generally accepted in subsequent works, and in the Amazon basin these regions are often delimited by large rivers (Hafer 1974; Cracraft 1985; da Silva & Oren 1996). Bird species are usually separated by the Amazon River and its major tributaries such as the Negro, Madeira, Tapajós and Tocantins rivers (Cohn-Haft 2000; Ribas et al. 2012; D’Horta et al. 2013; Fernandes et al. 2012, 2013, 2014). Similar patterns are also found in other Amazonian vertebrate taxa, including primates and butterlies (Wallace 1852; van Roosmalen et al. 1998; Hall & Harvey 2002), suggesting that rivers are important barriers to dispersal. Recent studies of primates and birds in the Madeira-Tapajós interluvium (M-T), also known as the Rondônia area of endemism (Cracraft 1985), have suggested that smaller rivers also limit the distributions of some taxa, thus forming smaller areas of endemism in what was referred to as “mini-interluvia” (Cohn-Haft et al. 2007). Willis (1969), in a study of birds of the genus Rhegmatorhina, was one of the irst to document complex patterns of bird distributions in this area. He discussed the parapatric occurrence of Rhegmatorhina berlepschi and R. hofmannsi within the M-T and suggested that the Madeira and Tapajós rivers have occasionally changed their courses, resulting in the separation of populations and subsequent speciation. Van Roosmalen and collaborators (1998) described geographic substitutions of species in primates of the genera Callithrix and Callicebus on opposite banks of small rivers within this interluvium and described a new species of marmoset, Callithrix humilis, that occurs only on the west bank of the Aripuanã River. Subsequently, several other bird species in this region have been found to contain vocally, morphologically or genetically distinct populations, with restricted distributions and geographic substitution 362 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias on opposite banks of Madeira tributaries, such as the Aripuanã and Jiparaná (or Machado) rivers (Cohn-Haft et al. 2007; Isler et al. 2007; Tobias et al. 2008; Fernandes et al. 2012, 2013, 2014; Whitney et al. 2013a, b, c, d, e). Similar geographic patterns, with diferent races being separated by small Amazonian rivers, have also been found for butterlies (Hall & Harvey 2002). Sardelli (2005) found genetic diferentiation (cytochrome b, 500 bp) among morphologically indistinguishable populations of the Snethlage’s TodyTyrant (Hemitriccus minor) apparently bounded by the Jiparaná and Aripuanã rivers. his study raised the possibility of the existence of cryptic endemism in miniinterluvia, which was subsequently investigated for the other three species of passerine birds (Myrmeciza hemimelaena, Glyphorynchus spirurus, Hylophylax naevius (Fernandes et al. 2012, 2013, 2014). Fernandes (2013) cited in a review a number of publications corroborating the importance of the mini-interluvia, highlighting that these diversity patterns are a key (and possibly unique) Amazonian feature and that despite the fact that this inescale endemism is well known and recognized among systematists working in the Amazon, it is not taken into account in conservation plans. Fernandes (2013) pointed out that many taxa in this region, including those yet to be given formal scientiic names, may now be endangered or even extinct. hus it is of utmost importance to consider species that present this kind of ine scale diferentiation in future conservation proposals. he objective of our study was threefold: 1) describe phylogeographic patterns for three species of suboscine passerines across the Rondônia area of endemism; 2) compare these patterns to those of other species know to have populations delimited by the Madeira, Aripuanã and Jiparaná rivers; and 3) investigate the relationship between degree of phylogeographic structure and ecological attributes in the light of the riverine barrier hypothesis. rivers along their lower courses are roughly 3.0, 0.8, 0.4 and 0.3 km, respectively. Each collection point had a corresponding point located on the opposite bank and therefore in a diferent interluvium. For purposes of sampling and analyses, we suggest the existence of three mini-interluvia within the M-T: Madeira-Jiparaná (MJ), Aripuanã-Jiparaná (AJ) and Aripuanã-Tapajós (AT) (Fig. 1). A maximum of 5 individuals per species were collected at each sampling point, with the total sample as follows: Willisornis poecilinotus (n = 45), Schifornis turdina (n = 23), and Glyphorynchus spirurus (n = 25) (see Appendix). Specimens were deposited in the bird collection of the National Institute for Amazonian Research (INPA), Manaus, Brazil, where tissue samples (muscle, heart and liver) were stored in liquid nitrogen for molecular analyses. Material and MetHodS We chose to study these three target species primarily because they are common, easy to collect, and widely distributed across the entire Amazon basin. Although all three are suboscine passerines, they represent three distinct families and, as such, the results obtained in this study can be assumed to be instances of independent evolution, and, thus, support the generality of our conclusions. All three species can be found in the same habitat (terra irme forest) but they difer in a variety of ecological attributes: Glyphorynchus spirurus – his is a polytypic species widely distributed in Neotropical lowland forests, occurring in Amazonia, Central America and along the Atlantic coast of Brazil (Ridgely & Tudor 1994). Marantz et al. (2003) recognized thirteen subspecies, six of which occur in the Brazilian Amazon. hree of these occur Species studied and sampling design We studied three species of passerine birds belonging to three diferent families: Glyphorynchus spirurus (Furnariidae), Willisornis poecilinotus (hamnophilidae), and Schifornis turdina (Tityridae). We sampled birds at 12 sites between the Tapajós and Madeira rivers and ive sites on the left bank of the Madeira River (LM; Figure 1), with the inal number of localities sampled per species difering among the three species (see Results). Individuals were collected along the Madeira, Aripuanã, Jiparaná and Roosevelt (the latter representing the largest tributary of the Aripuanã) rivers; the widths of these FiGure 1. Collection points and the interluvia sampled. MadeiraJiparaná (MJ), Aripuanã-Jiparaná (AJ), Aripuanã-Tapajós (AT) and Left bank of the Madeira River (LM). Revista Brasileira de Ornitologia, 22(4), 2014 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias 363 within or adjacent to the Madeira basin: G. s. castelnaudii (west of the Madeira River to the Andes), G. s. albigularis (south-eastern Bolivia and Peru), and G. s. inornatus, which occurs throughout the Brazilian portion of the Madeira-Tapajós interluvium (Peters 1951; Marantz et al. 2003). With a body mass ranging from 10.5 to 21g (typically 12.6-14.8 g in central Amazonia; Bierregaard, 1988), this is the smallest woodcreeper (Marantz et al. 2003), and it is the smallest of the three species we studied. It occurs in both terra irme and seasonally looded forests (várzea and igapó) (Marantz et al. 2003) and it is moderately sensitive to environmental perturbation (Ferraz et al. 2007). Recently, Fernandes et al. (2013) found that populations of G. s. inornatus are delimited by the Aripuanã and Jiparaná rivers. Willisornis poecilinotus – A species endemic to the Amazon basin, with seven subspecies recognized (Peters 1951; Zimmer & Isler 2003). Only one subspecies (W. p. griseiventris) is recognized from the middle and upper Madeira River basin; and there is no evidence of vocal or morphological diferentiation across the Madeira, Aripuanã and Jiparaná rivers (Isler & Whitney 2011), although Bates (2000) found genetic diferentiation (based on analyses of isozymes) across the Madeira River. Occurs in the understory of terra irme forest, where it is a regular follower of army ant swarms (Zimmer & Isler 2003). It is larger on average than Glyphorynchus, with a body mass ranging from 15 to 19 g (Zimmer & Isler 2003). Schifornis turdina – Nyári (2007), with no samples from the middle or lower Madeira River basin described close geographic proximity in the upper Madeira of two genetically distinct forms, showing no obvious vocal or plumage diferences; the author proposed recognizing them as distinct species (S. amazona and S. turdina), as adopted by the Brazilian Ornithological Records Committee (2014). Schifornis turdina (in the polytypic sense used here) occurs in the understory of terra irme and sandy-belt campinarana forests. Body mass averages 31 g (Snow 2004). his species is sensitive to forest fragmentation, disappearing from small forest fragments (Ferraz et al. 2007). volumes using a hermo Hybaid PCR Express thermal cycler under the following conditions: (1) an initial denaturing step at 94°C for 5 min; (2) 35 cycles of the following: 1 min at 92°C, 1 min at 48°C, and 1 min at 72°C; (3) a 10-min extension step at 72°C. Following PCR, correct fragment size and the presence of a single ampliication product was conirmed via electrophoresis on 1% agarose gel. After ampliication, the PCR products were puriied using a salt protocol (Sambrook et al. 1989). Sequencing was performed by the chain termination method (Sanger et al. 1977), using a Big Dye Termination Kit (Applied Biosystems) following the manufacturer’s speciications. he products of the sequencing reaction were precipitated with Tris-HCl and alcohol, and resuspended in formamide and resolved by capillary electrophoresis in an ABI 3130xl automatic sequencer (Applied Biosystems). All sequences have been deposited in GenBank (accession numbers: HM164938 – HM165034). extraction, ampliication and sequencing of dna Phylogenetic analysis of DNA sequence data was performed using maximum parsimony (MP) and maximum likelihood (ML) via PAUP* 4.0b10 (Swoford 2002) and Bayesian inference (BI) implemented in MRBAYES 3.0b4 (Hulsenbeck & Ronquist 2001). Maximum parsimony analysis was performed using a heuristic search with the following options: TBR branchswapping with 10 trees held at each step. Support for nodes was assessed using 1000 bootstrap replicates. Maximum likelihood was performed using the model parameters determined in the program Modeltest DNA was extracted from breast muscle (approximately 0.2 g) using a standard phenol chloroform protocol (Sambrook et al. 1989). The mitochondrial cytochrome b was ampliied via the polymerase chain reaction (PCR) using the primers: forward H16064 5′-ATCTCARCCTGATGAAAYTTYGG-3′, revers e L14993 5′-AAGTGGTAAGTCTTCAGTCTTTGGTT-3′, both of which were designed exclusively for this project. All ampliication reactions were performed in 25 μl alignment Sequences of DNA were visualized and edited using the Bioedit program (Hall, 1999). Alignments were performed in Clustal X within Bioedit (Hall, 1999). We used recommended precautions and are conident that all sequences represent mitochondrial DNA for the following reasons: (1) DNA was extracted only from tissue samples, which have high ratios of mitochondria to nuclei relative to blood or skin samples; (2) no stop codons occurred within the cytochrome b of any of the sequences; (3) sequences contain no insertions or deletions relative to one another or to other known avian cytochrome b sequences; (4) sequences in both DNA fragments from each individual were identical and unambiguous in their region of overlap; (5) in phylogenetic analyses, no samples appeared in unexpectedly basal portions of the tree or had exceptionally short or long branch lengths, both of which, if present, would indicate a fast evolving gene or an early diverged gene (a pseudogene, for example). Phylogenetic analyses Revista Brasileira de Ornitologia, 22(4), 2014 364 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias (Posada & Crandall 1998). he support for nodes in the likelihood tree was assessed using 500 bootstrap iterations. For BI analyses, two independent runs of 8,000,000 generations each were performed; for each run four Markov chains were simulated. Trees were sampled every 500 generations and the irst 4,000 samples were discarded as burn-in. Because there is strong evidence that geographic distributions of Amazonian birds are bounded by large rivers that form areas of Neotropical endemism, we used individuals from populations from other interluvia as outgroups for the three species studied. For the analysis of Glyphorynchus spirurus we used two individuals collected in the headwaters of the Negro River (Appendix) as the outgroup. For Willisornis poecilinotus, we used one individual collected in the Solimões-Negro River interluvium and for Schifornis turdina, we used as outgroups one individual collected north of Manaus and also one sequence of Schifornis virescens from GenBank (accession number AF453816; Appendix). Phylogenetic divergence analyses Phylogenetic divergence was estimated in the program BEAST v1.6.1 (Drummond & Rambaut 2007) using the coalescent constant population size tree prior (Drummond et al. 2002), the uncorrelated lognormal relaxed molecular clock model (Drummond et al. 2006), and the HKY (Hasegawa et al. 1985) models of molecular evolution, including gamma-distributed rate heterogeneity among sites and invariant sites. After preliminary runs, we adjusted priors and MCMC operators to assure optimum performance. To assess the robustness of estimates and investigate the inluence of the tree prior, we also performed analyses under the exponential (Drummond et al. 2002) and the Bayesian skyline (Drummond et al. 2005) tree priors. To convert divergence time estimates into units of millions of years, we used the mean substitution rate of 0.01105 substitutions/site/lineage/million years as proposed by Weir & Schluter (2008). For each set of priors, two independent MCMC analyses were run for 100 million generations, subsampling every 100 thousand generations. After a 10% burn-in, convergence of parameter estimates was assessed using the Gelman-Rubin statistic implemented in the module coda in the statistical package R (R Development Core Team 2011). Independent chains were combined, and marginal posterior parameter means and their associated 90% highest probability density intervals (90% HPD) together with efective sample size (ESS) for each divergence time estimate were calculated in the statistical package R (R Development Core Team 2011). reSultS We found signiicant phylogeographic structure among populations within all three study species. he Madeira River clearly separates genetically distinct populations in all of them. Within the Madeira-Tapajós interluvium, the degree of structure varied among species (see below). In each species, tree topologies were identical for all four tree-building algorithms, thus we only show the tree resulting from the Bayesian inference analyses. he pairwise genetic p-distance between individuals from opposite banks of the three rivers ranged from 3.1 to 5.5% but the variation within interluvia was low (0.0–0.09%). Results for each species were as follows: Glyphorynchus spirurus We sequenced a total of 946 bp for 27 individuals of G. spirurus. Parsimony, maximum likelihood, and Bayesian inference analyses suggested a genetic structure in the form of monophyletic groups on opposite banks of the Madeira, Aripuanã and Jiparaná rivers, each supported by high bootstrap values (MP = 100, ML = 100, BI = 1.00). No barrier efect was found on opposite banks of the Roosevelt River. Parsimony analysis yielded two equally parsimonious trees (length = 170, CI = 0.8235, RI = 0.9504). From 130 variable sites, 110 were parsimony informative. Maximum likelihood (-ln L = 2012.00097) and Bayesian inference resulted in a topology very similar to that of the parsimony analysis. Levels of genetic divergence (uncorrected p-distance) between individuals of diferent clades ranged from 3.2% (populations of AJ versus MJ) to 5.5% (populations of LM versus AT) and levels of divergence between individuals within the same interluvium ranged from 0.0–0.03% (Figure 2). Coalescent analyses in the program BEAST indicate a 6.5 mya (1.9 – 32.7, 90% HDP) divergence between populations on the left and right banks of the Madeira River. Willisornis poecilinotus We sequenced a total of 956 bp for 46 Willisornis poecilinotus individuals. he results of parsimony, maximum likelihood, and Bayesian inference analyses were concordant, thus indicating a strong phylogenetic signal supported by high bootstrap values (MP = 100, ML = 100, BI = 1.00) (Figure 3). Parsimony analysis yielded 100 equally parsimonious trees (length = 94, CI = 0.8723, RI = 0.9634). From 71 variable sites, 41 were parsimony informative. Maximum likelihood (-ln L = 1757.25307) and Bayesian inference resulted in a topology very similar to that of the parsimony analysis. he level of genetic divergence (uncorrected p-distance) Revista Brasileira de Ornitologia, 22(4), 2014 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias between individuals of the two clades separated by the Madeira River, RM (right bank of Madeira River) versus LM (left bank of Madeira River), was 3.4% (Figure 3). Levels of divergence between individuals in the same 365 interluvium ranged from 0.0–0.09%. Coalescent analyses in the program BEAST indicate a 2.6 mya (0.8 – 13.8, 90% HDP) divergence between populations on the left and right banks of the Madeira River. FiGure 2. Species–area relationships (a) and Bayesian inference phylogeny (b) estimated for Glyphorynchus spirurus. Numbers at the tips of branches refer to localities where individuals were sampled. Bayesian inference (BI) posterior probabilities and genetic distance (uncorrected p-distance) values are indicated in the branches. Note grouping of a sample from the Aripuanã-Tapajós interluvium (location 15) with those of the Madeira-Jiparaná interluvium (see Discussion). FiGure 3. Species–area relationships (a) and Bayesian inference phylogeny (b) estimated for Willisornis poecilinotus. Numbers at the tips of branches refer to localities where individuals were sampled. Bayesian inference (BI) posterior probabilities and genetic distance (uncorrected p-distance) values are indicated in the branches. Revista Brasileira de Ornitologia, 22(4), 2014 366 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias Schiffornis turdina We sequenced a total of 968 bp for 25 Schifornis turdina individuals. Parsimony, maximum likelihood and Bayesian inference analyses suggested genetic structure on opposite banks of the Madeira River supported by high bootstrap values (MP = 100, ML = 98, BI = 1.00) (Figure 4). Parsimony analysis yielded 48 equally parsimonious trees (length = 175, CI = 0.9371, RI = 0.9214). From 159 variable sites, 67 were parsimony informative. Maximum likelihood (-ln L = 2043.26577) and Bayesian inference resulted in a topology very similar to that of the parsimony analysis consensus topology. he maximum divergence (uncorrected p-distance) between individuals of the RM and LM clades was 3.1% (Figure 4). Levels of divergence among individuals of the same interluvium ranged from 0.0–0.3%. Coalescent analyses in the program BEAST indicate a 3.1 mya (1.0 – 15.2, 90% HDP) divergence between populations on the left and right banks of the Madeira River. FiGure 4. Species–area relationships (a) and Bayesian inference phylogeny (b) estimated for Schifornis turdina. Numbers at the tips of branches refer to localities where individuals were sampled. Bayesian inference (BI) posterior probabilities and genetic distance (uncorrected p-distance) values are indicated in the branches. diScuSSion Strong genetic diferentiation in the face of highly conserved phenotype is at the heart of numerous descriptions of “cryptic species” in recent years (Whitney et al. 2013a, b, c, d, e) and appears to be a frequent phenomenon in the Amazon. In S. turdina, diferentiation on opposite banks of the middle and lower reaches of the Madeira River is consistent with that detected earlier in the upper Madeira (Nyári 2007) and associated with species level taxa. In all three studied species, the observed molecular groups are monophyletic and parapatrically distributed, their geographic distributions are delimited by rivers, and the observed phylogenetic divergence between clades on opposite banks of the Madeira River (3.1–5.5%) is consistent with interspeciic divergences in other avian taxa separated by the same geographic barrier (Ribas et al. 2012). Based on coalescent analyses (see Methods), we estimated mean divergences of 6.5 mya, 2.6 mya and 3.1 mya between populations on left and right banks of the Madeira River for G. spirurus, W. poecilinotus and S. turdina, respectively. he separation of the lineages in all three species of passerines are clearly ancient, all lineages are diagnosable by multiple molecular synapomorphies, and all lineages are parapatrically distributed and likely represent phylogenetic species. However, it is also clear that a more detailed analysis evaluating species status and establishing species boundaries is necessary. Irrespective of taxonomy, the pattern of geographic variation delimited by rivers is clear for all three taxa studied. Our results indicate genetically distinct populations on opposite banks of the Madeira River. For all three species analyzed in this study we found sister Revista Brasileira de Ornitologia, 22(4), 2014 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias clades on opposite banks of the Madeira River, and for G. spirurus, as documented previously (Fernandes et al. 2013), the data further indicated sister clades on opposite banks of the smaller Aripuanã and Jiparaná rivers. Our data therefore reinforce the importance of rivers as geographic barriers, and suggest a hierarchical efect in which larger rivers divide older clades whereas smaller rivers are associated with more recent divergences. For G. spirurus, an individual collected on the right bank of the Aripuanã River that grouped in the clade Madeira/ Jiparaná (MJ) provides evidence of upstream gene low across both of the same rivers (Jiparaná and Aripuanã) that delimit diferentiated populations in their lower reaches. Since rivers naturally tend to be narrower in the upper reaches, this result suggests that river width is important in determining a river’s likelihood of delimiting distributions and further strengthens the hypothesis of a hierarchical efect of river width in structuring populations (Hafer 1974, 1997). Assuming roughly equal rates of substitution, then Willisornis poecilinotus and Schifornis turdina populations may have diferentiated across the Madeira River at about the same time; however, Glyphorynchus spirurus would appear to have diferentiated much earlier. his implies that not all sympatric bird taxa necessarily share the same evolutionary scenario. Although the Madeira River currently delimits the distributions of the left- and right-bank clades of all three species, the Madeira River might not necessarily be the primary agent that has driven the observed divergence. It may simply represent current limits of distribution for clades that have diverged due to other abiotic or biotic forces, independent of the formation of the Madeira River itself. Another non-exclusive possibility is that rates of molecular substitutions are 2–3 times faster in G. spirurus than in W. poecilinotus and S. turdina; however, such an elevated substitution rate appears to be a rare phenomenon in passerine birds, and has been suggested only for one case of an Old World species (Nectarinia humbloti; Warren et al. 2003). Finally, a third explanation suggested previously (Willis 1969, Fernandes et al. 2012, 2014) is that changes in the courses of rivers might confuse the phylogenetic pattern. here is evidence that the course of rivers in the Madeira basin changed throughout history, but remained stable for long periods of time (Latrubesse 2002). he period of stability could be enough to cause diferentiation until their course was modiied again and became stable for another long period of time thus causing both spatial and temporal incongruences among phylogenies of codistributed species (Fernandes 2013). A comparative analysis including additional species and sampling nuclear markers is likely to shed more light on this issue, but at least three other studies have found populations 367 separated by the Madeira River not to be reciprocally monophyletic (Aleixo 2004, Patané et al. 2009, SousaNeves et al. 2013), as recovered herein for G. spirurus, W. poecilinotus, and S. turdina, hence supporting a more complex scenario of diferentiation and a broad range of phylogeographic patterns for the same region. Despite the importance of rivers for avian diferentiation, even the largest Amazonian rivers are not barriers for all species and smaller rivers are less likely to be barriers than larger rivers. here are several potential explanations for this phenomenon. Molecular studies suggest that populations of canopy species are less structured than those of understory birds (Capparella 1988; Burney & Brumield 2009). he latter authors showed that genetic divergence is signiicantly smaller across the Andes and two Amazonian rivers (Amazon and Madeira rivers) in canopy birds than in understory species. Burney & Brumield (2009) further suggested that there is a negative relationship between dispersal propensity and genetic structure. Species that occupy the understory are supposed to be less efective dispersers, which may be one reason why there are more species of understory birds, and that they are more locally distributed. However, we found diferences in genetic structure among understory species, suggesting that other factors may also inluence the diversiication of birds. One might also expect the degree of sensitivity to disturbance or habitat specialization on primary terra irme forest to predict the importance of rivers in driving or maintaining allopatric diferentiation. Ferraz et al. (2007) analyzed thirteen years of capture/recapture data for birds in the reserves managed by the Biological Dynamics of Forest Fragmentation Project (BDFFP), located in the Brazilian state of Amazonas north of Manaus. hese authors derived measures of the vulnerability of a species to isolation and sensitivity due to fragment size. hese two measures relect sensitivity to environmental change. Among the 54 species examined by Ferraz et al. (2007), G. spirurus was the least sensitive to the size of the fragment and one of the ten species least vulnerable to isolation. By contrast, S. turdina was among the most vulnerable and most sensitive species. Willisornis poecilinotus was not included in the analysis. One would therefore expect G. spirurus, the species least afected by isolation and fragmentation, to have lower genetic divergence across the rivers than the other two species; however, our results contradict the expected pattern. Glyphorynchus spirurus, although occurring in diferent types of forests and not being especially sensitive to disturbance, has populations that are much more strongly structured than are those of the other two species. In this case, sensitivity to disturbance and degree of specialization on primary terra irme forest were not good predictors of the degree of population genetic structure. Revista Brasileira de Ornitologia, 22(4), 2014 368 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias he Jiparaná and Aripuanã rivers separate populations of G. spirurus, the smallest species we examined (average body mass 13.7 g), but not the populations of two other species, W. poecilinotus (15–19 g) and S. turdina (30–35.5 g). he Jiparaná and Aripuanã rivers also appear to limit the distributions of populations in other very small birds, including Hemitriccus minor (Sardelli 2005), Hypocnemis rondoni (Isler et al. 2007; Tobias et al. 2008; Whitney et al. 2013a), Herpsilochmus stotzi (Whitney et al. 2013b), Hylophylax naevius (Fernandes et al. 2014) and Picumnus aurifrons (Cohn-Haft et al. 2007), all of which weigh on average less than 13 g each. In other parts of the world, tiny birds make spectacular long-distance migrations and even in Amazonia, where most species tend to be sedentary (Stotz et al. 1996), certain species, such as those adapted to river islands (Remsen & Parker, 1983; Rosenberg, 1990), are likely to be excellent dispersers, independent of size. However, there is evidence that at least two other heavier bodied species have structured populations delimited by the Aripuanã and Jiparaná rivers as well: hamnophilus aethiops (23-30g; hom & Aleixo 2015) and Malacoptila rufa (36-44g; Ferreira 2013). As discussed by Smith et al. (2014) diferences in life history attributes, efective population sizes, lineage ages, and dispersal rates can together account for highly disparate responses of avian lineages across important physical barriers in the Neotropics such as the Andes and some large Amazonian rivers, including the Madeira River. Further tests, controlling for phylogeny, habitat, wing shape and loading, and behavioral responses to open spaces, will be necessary to determine the extent to which body mass is a useful predictor of genetic population structure in suboscine passerines. acKnoWledGMentS AMF was supported during his masters studies by a fellowship from Conselho Nacional de Desenvolvimento Cientiico e Tecnologico (CNPq). Financial support for both laboratory and ield work was granted by IEB (Instituto de Educação do Brasil, programa BECA). he research was conducted in the Laboratório de Evolução e Genética Animal (LEGAL) with portion of the analyses being done in the High Performance Computational Facility of the University of Puerto Rico. We want to thank F. A. Carvalho for essential help and aiding in preparing the igures, L. N. Naka, T. C. H. Cole, C. A. Marantz and an anonymous reviewer for useful and inspiring comments on earlier versions of this manuscript. 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Population/locality taxon: Voucher/Genbank accession numbers 0/ AM: right bank of lower Juruá, RESEX Baixo Juruá, comunidade Socó. 3°36’S; 66°4’W 1/ AM:”Campo do Lago Preto”, left bank of Madeira river, 39 km W Novo Aripuanã. 5°09’S; 60°44’W G. spirurus: INPA A 808/HM164938 2/ AM: right bank Purús river, Ussuã stream, tributary of Mucuim river (right bank). 7°13’S; 64°10’W 3/ RO: left bank of the Madeira river, near Jacy Paraná, ca. 45 km southwest Porto Velho. 9°10’S; 64°23’W W. poecilinotus: INPA A 420/HM164985,421/HM164980, 422/HM164994, 424/HM164975. S. turdina: INPA A 395/ HM165029, 413/HM165028 W. poecilinotus: INPA A 101/HM164978 G. spirurus: INPA A 349/HM164942, 359/HM164941. W. poecilinotus: INPA A 345/HM164983, 347/HM164999, S. turdina INPA A 348/HM165032 4/ RO: left bank of the Madeira river, ca. 20 km N Abunã. G. spirurus: INPA A 173/HM164943, 191/HM164960. W. 9°31’S; 65°21’W poecilinotus: 182/HM164984 5/ AM: left bank of Aripuanã river, Arauazinho stream, 130 km G. spirurus: INPA A 461/HM164951, 466/HM164950, S Novo Aripuanã. 6°18’S; 60°24’W 510/HM164952, 553/HM164963, 562/HM164962. W. poecilinotus: INPA A 465/HM164974, 472/HM164967, 475/HM165009, 504/HM164971. S. turdina: INPA A 533/ HM165014 6/ AM: left bank of lower Roosevelt river, conluence with G. spirurus: INPA A 906/HM164945. W. poecilinotus: INPA A Aripuanã river. 7°35’S; 60°43’W 902/HM164976, 904/HM164992. S. turdina: INPA A 903/ HM165017. Revista Brasileira de Ornitologia, 22(4), 2014 Rivers acting as barriers for bird dispersal in the Amazon Alexandre M. Fernandes, Mario Cohn-Haft, Tomas Hrbek and Izeni Pires Farias 7/ AM: right bank of lower Roosevelt river, conluence with Aripuanã river. 7°38’S; 60°40’W 8/ RO: right bank lower Jiparaná river, comunidade Demarcação, ca. 20 km southeast Calama. 8°09’S; 62°47’W 9/ RO: left bank of lower Jiparaná river, opp. Comunidade Demarcação, ca. 20 km southeast Calama. 8°14’S; 62°46’W 10/ RO: right bank of Madeira river, 9.5 km southeast Porto Velho. 8°52’S; 64°0’W 11/ RO: right bank of Madeira river, near Jacy Paraná, ca. 45 km southwest Porto Velho 12/ RO: right bank of Madeira river, ca. 20 km N Abunã. 9°35’S; 65°21’W 13/ AM: right bank of Aripuanã, Extremo stream, 135 km S Novo Aripuanã. 6°18’S; 60°20’W 14/ AM: right bank of middle Aripuanã, conluence with Roosevelt river. 7°37’S; 60°40’W 15/ AM: Floresta Estadual do Sucunduri, right bank of upper Sucunduri river. 8°34.5’S; 59°08.5’W 16/ AM: Parque Estadual do Sucunduri; right bank of Bararati river. 8°21’S; 58°37’W Outgroup/ AM: right bank of upper Negro river, 3 km SW São Gabriel da Cachoeira. 0°8’S; 67°5’W Outgroup/ AM: left bank upper Negro river, 10 km east São Gabriel da Cachoeira. 0°10’S; 66°59’W Outgroup/ AM: left bank middle Solimões river; RDS Amanã, Comunidade Nova Canaã, Centro Grande stream. 2°36’S; 64°52’W Outgroup/ AM: ca. 60 km N Manaus; highway BR-174, km 43; Campina reserve/INPA G. spirurus: INPA A 895/HM164948. W. poecilinotus: INPA A 894/HM164997, 896/HM165007, 900/HM165003, 898/ HM165001. S. turdina: INPA A 893/HM165015 G. spirurus: INPA A 875/HM164946, 877/HM164958. W. poecilinotus: INPA A 876/HM164989, 884/HM164990, 907/ HM165008. S. turdina: INPA A 885/HM165016 G. spirurus: INPA A 870/HM164964, 886/HM164940. W. poecilinotus: INPA A 871/HM164982, 872/HM164996, 873/HM165000, 878/HM164988, 881/HM165010, 882/ HM164977. S. turdina: INPA A 874/HM165034, 880/ HM165021, 883/HM165018 G. spirurus: INPA A 329/HM164959. W. poecilinotus: INPA A 307/HM164966, 308/HM164970, 326/HM164968, 327/ HM164973, 334/HM164972 W. poecilinotus: INPA A 367/HM165002, 368/HM164969. S. turdina: INPA A 371/HM165030, 372/HM165025, 374/ HM165026 G. spirurus: INPA A 208/HM164949. W. poecilinotus: INPA A 248/HM165005, 264/HM165006, 265/HM164993. S. turdina: INPA A 249/HM165031, 266/HM165012 G. spirurus: INPA A 536/HM164944, 559/HM164947, 561/ HM164955. W. poecilinotus: INPA A 478/HM164987, 479/ HM164979. S. turdina: INPA A 525/HM165027, 527/ HM165023, 538/HM165024 G. spirurus: INPA A 890/HM164954. W. poecilinotus: INPA A 887/HM164995, 888/HM164981, 891/HM165004. S. turdina: INPA A 892/HM165033, 889/HM165019 G. spirurus: INPA A 845/HM164957. W. poecilinotus: INPA A 849/HM164998. S. turdina: INPA A 846/HM165013, 848/ HM165022, 850/HM165020 G. spirurus: INPA A 852/HM164953, 855/HM164956. W. poecilinotus: INPA A 856/HM164986, 857/HM164991 G. spirurus: INPA A 1153/HM164939 G. spirurus: INPA A 1118/HM164961 W. poecilinotus: INPA A 398/HM164965 S. turdina: INPA A 777/HM165011 Revista Brasileira de Ornitologia, 22(4), 2014 View publication stats 371