Journal of Biogeography (J. Biogeogr.) (2013) 40, 37–49 ORIGINAL ARTICLE Phylogeny and comparative phylogeography of Sclerurus (Aves: Furnariidae) reveal constant and cryptic diversification in an old radiation of rain forest understorey specialists Fernando M. d’Horta1*, Andrés M. Cuervo2, Camila C. Ribas3, Robb T. Brumfield2 and Cristina Y. Miyaki1 1 Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Brazil, 2 Department of Biological Sciences and Museum of Natural Science, Louisiana State University, Baton Rouge, USA, 3PCAC Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil ABSTRACT Aim To evaluate the role of historical processes in the evolution of Sclerurus leaftossers by integrating phylogenetic and phylogeographical approaches. Location Humid forests of the Neotropical region. Methods We reconstructed the evolutionary history of Sclerurus based on DNA sequences representing all species and 20 of the 26 recognized subspecies using one autosomal nuclear locus and three protein-coding mitochondrial gene sequences. Phylogenetic relationships were inferred using Bayesian and maximum-likelihood methods. We used Bayesian coalescent-based approaches to evaluate demographic changes through time, and to estimate the timing of diversification events. Based on these results, we examined the temporal accumulation of divergence events using lineage-through-time plots. Results The monophyly of all Sclerurus species was strongly supported except for Sclerurus mexicanus, which was paraphyletic in relation to Sclerurus rufigularis, and for the sister pair Sclerurus scansor–Sclerurus albigularis, which were not reciprocally monophyletic in the nuclear tree. We found remarkably deep phylogeographical structure within all Sclerurus species, and overall this structure was congruent with currently recognized subspecies and Neotropical areas of endemism. Diversification within Sclerurus has occurred at a relatively constant rate since the Middle Miocene. Main conclusions Our results strongly support the relevance of physiographical (e.g. Nicaragua Depression, Isthmus of Panama, Andean Cordillera, great rivers of Amazonia) and ecological barriers (open vegetation corridor) and ecological gradients (elevational zonation) to the diversification of Neotropical forest-dwelling organisms. Despite the high congruence among the spatial patterns identified, the variance in divergence times suggests multiple speciation events occurring independently across the same barrier, and a role for dispersal. The phylogenetic patterns and cryptic diversity uncovered in this study demonstrate that the current taxonomy of Sclerurus underestimates the number of species. *Correspondence: Fernando Mendonça d’Horta, Rua do Matão 277, 05508-090, São Paulo, SP, Brazil. E-mail: fmhorta@usp.br ª 2012 Blackwell Publishing Ltd Keywords Amazonia, Andes, areas of endemism, biogeographical barriers, cryptic diversity, elevational zonation, Great American Interchange, molecular clock, Neotropics, Sclerurinae. http://wileyonlinelibrary.com/journal/jbi doi:10.1111/j.1365-2699.2012.02760.x 37 F. M. d’Horta et al. INTRODUCTION The origin of the high biodiversity that characterizes the Neotropics has long been of interest to biogeographers (Wallace, 1853). Many historical and ecological mechanisms have been hypothesized to explain the origin of the biogeographical patterns within this region (e.g. Chapman, 1917; Haffer, 1969; Ayres & Clutton-Brock, 1992; Bush, 1994; Fjeldså et al., 1999; Brumfield, 2012). In the Neotropical region different taxa often show similar distributions, which are used to define areas of endemism (Cracraft, 1985). Based on the conceptual framework of vicariance biogeography (Platnick & Nelson, 1978) some studies on the diversification of Neotropical birds used congruence among area relationships to infer the biogeographical processes (e.g. Cracraft & Prum, 1988; Marks et al., 2002; Aleixo & Rossetti, 2007). However, ignoring temporal information obscures the connection between biogeographical patterns and their underlying causes (Donoghue & Moore, 2003) because a single pattern of area relationships can be achieved through different processes. In the last decade temporal inferences have often been incorporated in studies of diversification of Neotropical bird lineages (e.g. Aleixo, 2004; Brumfield et al., 2007; Derryberry et al., 2011). Although many of these taxa have experienced the same history of landscape evolution, the results of these studies revealed differences among spatio-temporal patterns of diversification. The explanation of this may involve stochastic processes (e.g. dispersal and extinction) and ecological differences among taxa, such as dispersal ability and ecophysiological constraints (Burney & Brumfield, 2009). Here we present a comprehensive study of the historical diversification of the Sclerurus leaftossers (Aves: Furnariidae). Sclerurus is an old, monophyletic group (Derryberry et al., 2011) that comprises six species of strictly understorey, leaflitter specialists, highly sensitive to habitat disturbance (Stotz et al., 1996). The geographical ranges of Sclerurus species are dissected by the major biogeographical barriers of the Neotropics and encompass the main Neotropical forest biomes from southern Brazil to central Mexico. Herein we analyse the evolutionary history, divergence time and diversification patterns of Sclerurus leaftossers across the Neotropical region. We maximized the geographical sampling of all taxa to minimize problems with potential cryptic diversity. Thus, we provide a comprehensive temporal and spatial analysis of the history of this group. MATERIALS AND METHODS Sampling We reconstructed the phylogeny of Sclerurus based on 119 ingroup samples representing all six species in the genus and 20 of the 26 subspecies (Remsen, 2003) (see Appendix S1 in Supporting Information). The outgroup taxa were defined based on previous phylogenies (Irestedt et al., 2009; Derryberry et al., 2011). We included two species of Geositta miners 38 (Geositta poeciloptera and Geositta tenuirostris), that is the sister-group of Sclerurus, and representatives of the closely related subfamilies, e.g. Xenops minutus (Furnariinae) and Lepidocolaptes angustirostris (Dendrocolaptinae). For most samples, we sequenced a total of 2962 bp from fragments of four loci: one autosomal nuclear locus, b-fibrinogen intron 7 (Fib7), and three protein-coding mitochondrial DNA (mtDNA) genes: cytochrome b (cyt b) and NADH dehydrogenase subunits 2 (ND2) and 3 (ND3). DNA extraction, amplification, sequencing and alignment procedures are described in Appendix S2. Phylogenetic analyses The phylogenetic analyses were performed separately for mtDNA (2408 bp) and for Fib7 (914 bp). To estimate gene trees we used maximum-likelihood (ML) and Bayesian inference (BI) approaches. To select the simplest model of molecular evolution with the highest likelihood for the data, we applied a likelihood-ratio test (LRT) performed using Modeltest 3.7 (Posada & Crandall, 1998). The ML searches were performed using RAxML 7.0.4 (Stamatakis, 2006), assuming a general time-reversible (GTR) model of evolution with distributed rate heterogeneity, four rate categories, and estimation of the proportion of invariable sites. The ML analyses of mtDNA were carried out considering three partitions (one partition per gene: cyt b, ND2 and ND3). To determine if analyses had become trapped in local optima, we conducted 10 independent ML searches. The robustness of the nodes was determined by 1000 bootstrap replicates, using the ‘fast bootstrap’ algorithm of RAxML. The BI with Markov chain Monte Carlo (MCMC) sampling was implemented in MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). As in the ML analyses, a partitioned analysis was conducted with three locus-specific models, estimating parameters independently for each partition (nst = 6; rates = invgamma). We conducted two independent, parallel analyses with four simultaneous chains each for 10 million generations, sampling parameters and trees every 1000 generations. We evaluated the convergence between analyses by comparing the posterior probabilities of clades using awty (Nylander et al., 2008). The first 2.5 million generations were discarded as burn-in and the posterior probabilities were estimated from the remaining trees. Temporal patterns of lineage diversification The timing of diversification events within Sclerurus was estimated using a subset of the mtDNA sequence matrix containing all species and one exemplar of each intra-specific clade that represented divergent populations (Appendix S1). Divergence times were estimated using the Bayesian approach implemented in beast 1.5.1 (Drummond & Rambaut, 2007) with the uncorrelated relaxed clock model (uncorrelated lognormal). The calibration used was based on the results of dating of the Furnariidae radiation obtained by Derryberry et al. (2011) in a very comprehensive study that included 97% Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd Sclerurus phylogeny and phylogeography of furnariid species and used similar genetic markers (ND2, ND3, CO2 and Fib7). We used the confidence intervals obtained by Derryberry et al. (2011) to set lower and upper limits of uniform prior distributions of ages for the most recent common ancestor for five clades within the Sclerurus phylogeny. Based on the resulting time-calibrated tree from beast, the temporal accumulation of divergence events was examined using lineage-through-time (LTT) plots to test for changes in lineage-splitting events along the evolutionary history of Sclerurus. Given the deep phylogeographical structure within Sclerurus species (see Results), we used the least inclusive terminal clades for this analysis. We used phylogenetic and likelihood-based statistical methods implemented in R 2.13.0 (R Development Core Team, 2011): packages ape 2.7 (Paradis et al., 2004), laser 2.2 (Rabosky, 2006) and geiger (Harmon et al., 2008). We compared diversification events of Sclerurus among 10 major biogeographical areas to examine the spatial and temporal congruence of divergence in co-occurring lineages. The 10 biogeographical areas considered are: Central America – north (CAN; Mexico to Nicaragua), Central America – south (CAS; Costa Rica to Darién, north-western Colombia), Chocó (CHO; from Darién to western Ecuador), base of the Andes (BAN; eastern Andean foothill forests from southern Colombia to Bolivia), western Amazonia – south (WAS; lowland forests from the right bank of the Amazon/Ucayali rivers to the left bank of the Madeira River), western Amazonia – north (WAN; lowland forests from the left bank of the Amazon/Ucayali rivers to the right bank of the Negro River), Brazilian shields – west (BSW; the right bank of the Madeira River to the left bank of the Xingu River), Brazilian shields – eastern (BSE; the right bank of the Xingu River to the eastern limits of Amazonia), Guiana Shield (GUY; northern Amazonia from the left bank of the Negro River to the eastern limit of Amazonia) and Atlantic forest (ATF; southern Brazil, eastern Paraguay and north-eastern Argentina to north-eastern Brazil). These areas were designated based on their geological, geographical and palaeoecological similarities and distinctions (Bates, 2001; Campbell et al., 2006; Aleixo & Rossetti, 2007) and based on patterns of distributions for terrestrial vertebrates (Haffer, 1974; Cracraft, 1985). Comparative demography We examined the molecular signature of past demographic events in Amazonian populations of each species using the mtDNA and Fib7 data. We used a Bayesian approach implemented in phase 2.0 (Stephens et al., 2001; Stephens & Donnelly, 2003) to identify heterozygous haplotypes of Fib7. Heterozygous indel positions were not found. To evaluate the existence of significant evidence of recombination in Fib7, the pairwise homoplasy index (PHI) test (Bruen et al., 2006) was performed for each lineage using SplitsTree 4.10 (Huson & Bryant, 2006). We calculated population genetic summary statistics and estimated parameters such as genetic diversity (Q) and population growth rate (g) using the lamarc package (Kuhner, 2006). lamarc implements a MCMC method for Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd sampling genealogies and calculates a likelihood curve used to determine the values of Q and g that maximize the probability of originating the empirical data. lamarc analyses were performed with five replicates of 10 short chains each (500 genealogies sampled each 50 interactions and a burn-in of 1000 genealogies), and two long chains (20,000 genealogies sampled each 50 interactions and a burn-in of 1000 genealogies). For these analyses we did not consider the migration effect among populations. Because the PHI test indicated that the null hypothesis of no recombination could not be rejected, the parameter r (recombination) was not considered in the lamarc analyses. In addition, we used Tajima’s D (Tajima, 1989), Fu’s FS test (Fu, 1997) and R2 (Ramos-Onsins & Rozas, 2002) to test the scenarios of population expansion. The significance of the tests was determined based on 10,000 coalescent simulations, which assumed neutrality and equilibrium conditions. These analyses were performed using DnaSP 4.10.9 (Rozas et al., 2003). RESULTS Data characteristics An alignment of 2048 bp of mtDNA (119 individuals) contained cyt b (1022 bp; 117 individuals), ND2 (1041 bp; 118 individuals) and ND3 (345 bp; 119 individuals). We obtained 914 bp of the nuclear intron Fib7 from 107 individuals. No indels were present in the mitochondrial alignment, but some were identified in the Fib7 dataset. All sequences were deposited in GenBank under the accession numbers JQ903619–JQ904023. The mtDNA dataset was characterized by 826 (40.3%) variables and 785 (38.3%) parsimony-informative sites, and the Fib7 dataset by 93 (10.2%) variables and 73 (8.0%) parsimony-informative sites. The GTR + I + C evolutionary model (pinv = 0.5353 and a = 1.3229) was selected for all partitions of mtDNA, while the HKY + C model (C = 0.2708) was selected for Fib7. The uncorrected mtDNA distance between Sclerurus and its sister genus Geositta (Irestedt et al., 2009; Derryberry et al., 2011) was 18.4%. Within Sclerurus, mitochondrial genetic distances ranged from 16.1% (South American Sclerurus mexicanus versus Sclerurus scansor) to 2.7% (S. scansor versus Sclerurus albigularis). Although pronounced genetic structure was found within all species, the mean genetic distances among populations within species were quite variable, ranging from 12.1% between the two major clades of the broadly defined S. mexicanus (Central versus South American populations) to 0.16% among S. scansor populations (Appendix S3). Phylogeny of Sclerurus species The phylogenetic ML and BI analyses of the mitochondrial and nuclear data sets strongly support the monophyly of the genus Sclerurus. Two main clades were identified within Sclerurus (Fig. 1). The first includes four light-throated species segre39 F. M. d’Horta et al. (a) (b) S. scansor/ albigularis 94/1 92/0.99 S. mexicanus (South America) 100/1 87/1 73/0.96 58 59/0.63 95/1 98/1 S. mexicanus (Central America) S. guatemalensis 97/1 0.01 S. caudacutus 80/0.90 Geositta poeciloptera Xenops minutus Figure 1 Phylogenetic relationships of Sclerurus leaftossers. Maximum likelihood tree inferred from (a) 2408 bp of mitochondrial DNA (mtDNA; cyt b, ND2 and ND3) and (b) from 914 bp of Fib7. Maximum likelihood bootstrap (1000 replicates) and Bayesian inference posterior probability values are shown at the nodes. Illustrations are from nominate subspecies, except for Sclerurus mexicanus from South America, which is represented by subspecies S. m. obscurior (courtesy of Lynx Edicions; Handbook of the birds of the world, Vol. 8, 2003). 40 Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd Sclerurus phylogeny and phylogeography gated into two clades: (1) a sister pair of cross-Andes lowland species, Sclerurus caudacutus and Sclerurus guatemalensis; and (2) a sister pair of a circum-Amazonian species of foothill montane forests, S. albigularis and an Atlantic forest species, S. scansor (Fig. 1). The second main clade in Sclerurus includes the rufous-throated species: the Amazonian lowland Sclerurus rufigularis that forms a polytomy with two deeply divergent S. mexicanus clades, one from Central America (Mexico to Darién) and the other from South America (Fig. 1). Except for S. mexicanus, all currently recognized species of Sclerurus were found to be monophyletic according to the mtDNA gene tree with high statistical support (Fig. 1a). Although the Fib7 gene tree largely reflects the same phylogenetic structure of mtDNA, the reciprocal monophyly between S. scansor and S. albigularis was not recovered. However, because the geographical sampling for S. albigularis was the least comprehensive of all, our current results do not allow us to draw strong conclusions about the phylogeography of that species (Fig. 1b). Intra-specific differentiation in Sclerurus leaftossers A striking pattern recovered by the wide geographical sampling is the pervasive population genetic structure within all Sclerurus species (Fig. 2). We found congruence between c. 80% of the subspecies sampled and the intra-specific lineages in the mtDNA tree, suggesting an overall good correspondence between phenotypic and phylogeographical breaks in Sclerurus. Exceptions were the subspecies of S. mexicanus from north-western South America and Darién (see below). However, the observed phylogeographical patterns are even more complicated than those suggested by the currently recognized subspecies. These patterns are largely consistent with the known Neotropical areas of endemism (Figs 2 & 3) and are bounded by either known physiographic barriers such as the Nicaragua Depression, the Andes, the Madeira, Negro and Amazon/Solimões rivers, open diagonal (Caatinga, Cerrado and Chaco), or by ecological gradients such as different elevational zones along the Andean foothills (Figs 2 & 3). Temporal patterns of divergence Divergence time estimates were obtained for 27 nodes distributed throughout the Sclerurus phylogeny (Fig. 3). Diversification within the genus was estimated to have occurred over the last 12 million years and at a relatively constant rate. A pure-birth model of diversification provided the best fit to the data [diversification rate = 0.28, Akaike information criterion (AIC) = )11.41, DAIC = 0, log-likelihood = 6.71], although the LTT curve tended to border the lower end of the expected diversification rate under that model (Fig. 4). Likelihood ratio tests did not reject the pure-birth model as the best fit to the data in comparison with birth– death and two-rate models. Intra-specific lineages of different species separated by a common geographical feature were compared to test for spatial and temporal congruence. Few Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd patterns were common in both spatial and temporal aspects within Sclerurus (Fig. 3, Table 1). The divergence between lineages from Andean foothills and Amazonian lowlands (red line) occurred at similar times both in S. albigularis (Fig. 3, node 19) and in South American S. mexicanus (Fig. 3, node 6). Also, the split associated with the Amazon River (orange line) occurred at similar times in both S. rufigularis (Fig. 3, node 10) and S. caudacutus (Fig. 3, node 23). Historical demography in Amazonia Tests based on summary statistics suggest distinct demographic scenarios for different populations of Sclerurus leaftossers (Table 2). The FS results for both mtDNA and Fib7 datasets, as well as the R2 result for the mtDNA dataset, indicate recent population expansion within the S. rufigularis population from WAN. Significant values were also obtained for the S. caudacutus and S. rufigularis populations from BSW. For S. caudacutus the FS test was significant for Fib7, and the R2 test was significant for Fib7 and mtDNA. The FS and R2 tests within S. rufigularis were only significant for the mtDNA dataset. Tajima’s D-test results were not significant for any of the studied populations (Table 2). A similar scenario emerged from lamarc analyses (Fig. 5). Populations of S. rufigularis and S. peruvianus from GUY and BAN, respectively, exhibited 95% confidence intervals (CI) of the population growth rate (g) that overlapped with zero (Fig. 5); hence, the null hypothesis of constant effective population size was not rejected. On the other hand, positive g values were obtained for populations associated with WAN, WAS and BSW, suggesting scenarios of population expansion (Fig. 5). DISCUSSION Origin, diversification and systematics Sclerurus is one of the oldest genus-level lineages in the Furnariidae radiation (mean estimated age of 22 Ma; c. 26 Ma in Derryberry et al., 2011), yet the genus is relatively homogeneous in morphology and coloration, and has comparatively few species. Our phylogeographical results demonstrated deep genetic divergence within Sclerurus species, suggesting that the low phenotypic diversity in Sclerurus may obscure cryptic species-level diversity. Our results provide an opportunity for a better taxonomic interpretation of Sclerurus diversity, particularly in S. mexicanus. In some cases, intra-specific divergence times, as observed in S. mexicanus (3.09–5.49 Ma) and S. caudacutus (2.43– 3.97 Ma), surpassed the divergence times between distinct species, as in S. scansor and S. albigularis (1.74–2.81 Ma), and between many sister species pairs in the Furnariidae (Derryberry et al., 2011) and other passerine birds (Roy et al., 1997; Garcı́a-Moreno et al., 1999; Chesser, 2004). The high genetic diversity within Sclerurus species, the long time of isolation of differentiated populations and the potential geographical overlap in some of these forms highlight the need for an 41 F. M. d’Horta et al. (a) (c) (b) (d) Figure 2 Geographical distribution of genetic variation in Sclerurus species in the Neotropics. Phylogeographies are depicted as maximum likelihood trees based on a mitochondrial DNA concatenated dataset with bootstrap and posterior probability values at the node. Individuals are labelled as in Appendix S1 and coloured according to geography and lineage. Each map depicts the overall geographical range of each species and the location of sampled localities coloured and labelled as in each tree and Appendix S1: (a) Sclerurus albigularis and S. scansor; (b) S. caudacutus and S. guatemalensis; (c) S. mexicanus of Central America (upper right), and S. obscurior, S. andinus, S. peruvianus and S. macconnelli of South America (lower left); and (d) S. rufigularis. integrative systematic revision of this group to properly reflect this diversity in its taxonomic classification. The non-monophyly of S. mexicanus given the phylogenetic uncertainty of its two major clades in relation to S. rufigularis, as well as the relatively ancient time of isolation among major clades, and the elevational parapatry of divergent lineages along the Andes slopes (see below) support a redefinition of species boundaries in this complex. First, the Central American lineages that include samples referable to the subspecies Sclerurus mexicanus mexicanus and Sclerurus mexicanus pullus (M1 to M8, Fig. 2c), should be regarded minimally as one full species. Second, divergent South American populations of S. mexicanus exhibit elevational parapatry on both sides of the Andes, which is a strong indication of evolutionary isolation and supports the elevation of these populations to species rank. 42 In particular, along the elevational gradient of the western Andes two deeply divergent clades were identified: one including the birds from the lowlands of Esmeraldas, Ecuador, near the type locality of the taxon Sclerurus mexicanus obscurior (M9 and M10, Fig. 2c); and other from the highlands (c. 1100– 2000 m) of Ecuador, Colombia and Venezuela (M11–M14, Fig. 2c) corresponding to the range of Sclerurus andinus. In the eastern Andean slope we also identified two parapatric lineages along the elevational gradient: one referable to the taxon Sclerurus macconnelli is widespread throughout the Amazonian lowlands (M15–M24, Fig. 2c), while Sclerurus peruvianus includes populations from the Andean foothills and outlying ridges (M25–M33, Fig. 2c). Therefore, we suggest the recognition of five species within the current S. mexicanus: (1) Sclerurus mexicanus Sclater, 1856 Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd Sclerurus phylogeny and phylogeography Figure 3 Chronogram of Sclerurus diversification in the Neotropics from beast analysis based on the concatenated mitochondrial DNA dataset. The bars on nodes represent the 95% confidence intervals around the mean divergence times, and are coloured according to the biogeographical break separating sister clades. Individuals analysed are identified as in Appendix S1. Biogeographical areas are: CAN (Central America – north), CAS (Central America – south), CHO (Chocó), NAN (northern Andes), BAN (base of the Andes), WAS (western Amazonia – south), WAN (western Amazonia – north), BSW (Brazilian shields – west), BSE (Brazilian shields – eastern), GUY (Guiana Shield), and ATF (Atlantic forest). (type locality: Ituribisci River, Guyana), comprising the Amazonian lowlands and, probably, the central Atlantic forest, corresponding to the ranges of subspecies Sclerurus mexicanus macconnelli and Sclerurus mexicanus bahiae, respectively; (5) Sclerurus peruvianus Chubb, 1919 (type locality: Yurimaguas, Loreto, Peru), comprising the populations of eastern Andean foothills from Bolivia to eastern Colombia, also reaching the lowlands and outlying ridges in north-western Amazonia. Hereafter we will follow this classification and refer to mexicanus taxa of Central American as S. mexicanus, and South American taxa as S. obscurior, S. andinus, S. macconnelli and S. peruvianus. Biogeography Figure 4 Lineage-through-time plot (logarithmic scale) and 95% confidence intervals (according to the inset legend) under the pure-birth model of Sclerurus lineage diversification in the Neotropics. (type locality: Veracruz, Mexico) of Central America, comprising the ranges of S. m. mexicanus Sclater, 1856 and S. m. pullus Bangs, 1902; (2) Sclerurus obscurior Hartert, 1901 (type locality: Lita, Esmeraldas, Ecuador) of the Chocó lowlands of Ecuador and Colombia; (3) Sclerurus andinus Chapman, 1914 (type locality: Buenavista, above Villavicencio, Colombia) of the humid Andean slopes of western Ecuador, Colombia and western Venezuela; (4) Sclerurus macconnelli Chubb, 1919 Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd Central–South America disjunction Despite the uncertainty of evolutionary relationships in the rufous-throated group, our estimates of divergence times indicate that the split between the Central (i.e. S. mexicanus) and all the South American lineages (i.e. S. obscurior, S. andinus, S. macconnelli, S. peruvianus and S. rufigularis) occurred during the Middle Miocene, between 6.5 and 10.5 Ma (nodes 2 and 3; Fig. 3, Table 1), and divergence between S. guatemalensis clades occurred during Late Pliocene–Early Pleistocene (from 1.0 to 1.9 Ma; node 22; Fig. 3, Table 1). Two hypotheses invoke specific timings of the origin of the Panamanian land bridge between Central and South America. It is generally held that the final closure of the 43 F. M. d’Horta et al. Isthmus of Panama occurred around 3.5 Ma (Coates et al., 1992). An emerging alternative hypothesis based on recent fossil dating suggests that the final closure occurred between 12 Table 1 Ages and 95% confidence intervals (CI) for nodes of the time-calibrated phylogeny of Sclerurus leaftossers (Fig. 3). Minimum and maximum ages were determined for five nodes following Derryberry et al. (2011); see text for details. Node 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Age (Ma) 12.89 8.74 7.98 4.73 2.55 1.42 1.19 0.92 0.66 2.98 1.78 1.41 2.17 4.23 6.76 2.27 0.90 0.59 1.74 1.34 5.73 1.47 3.20 2.02 0.92 1.08 0.65 CI (Ma) Calibration 11.26–15.11 6.99–10.51 6.47–9.77 3.61–5.95 1.92–3.31 1.06–1.83 0.85–1.54 0.62–1.24 0.40–0.94 2.28–3.80 1.24–2.33 0.89–1.94 1.53–2.84 3.09–5.49 5.52–8.01 1.74–2.81 0.61–1.23 0.36–0.83 1.30–2.24 0.95–1.77 4.80–6.68 1.00–1.98 2.43–3.97 1.52–2.56 0.61–1.23 0.75–1.43 0.42–0.92 11.26–16.55 and 20 Ma (Farris et al., 2011; Montes et al., 2012). Considering the ecological characteristics of Sclerurus (e.g. presumed low dispersal ability), this new temporal hypothesis is attractive because the dispersion between Central and South America, followed by the differentiation (node 2; Fig. 3, Table 1), would have occurred after the final closure of the Isthmus of Panama, and not before, as under the traditional geological dating. Differences in divergence times across the isthmus have also been observed in other terrestrial organisms (Koepfi et al., 2007; Cody et al., 2010; Smith & Klicka, 2010; Johnson & Weckstein, 2011). 6.56–11.87 Central America disjunction 5.40–8.60 1.72–3.43 The main phylogeographical break identified within S. mexicanus sensu stricto is associated with the Nicaragua Depression, the biogeographical barrier that defines the limits between the CAN and CAS. This barrier is important for a number of unrelated taxa (e.g. Duellman, 1999; Marshall & Liebherr, 2000; Pérez-Emán, 2005). The estimated divergence time between CAN and CAS lineages of S. mexicanus was substantial (from 3.09 to 5.49 Ma; node 14; Fig. 3, Table 1), placing the differentiation of S. m. mexicanus (CAN) and S. m. pullus (CAS) in the Late Miocene–Early Pliocene. Narrow landmasses (Kirby & MacFadden, 2005) and marine gaps occurred in this region during most of the Miocene (Coates & Obando, 1996). The estimated divergence time between the two Central American S. mexicanus lineages show a great overlap with that estimated for Cerropidion snakes (from 3.1 to 6.0 Ma; Castoe et al., 2009), but is more recent than estimates for other snakes, Bothriechis and Atropoides (5.7–9.9 and 6.8–10.6, respectively; Castoe et al., 2009), suggesting more than one vicariance event across the Nicaraguan Depression. 3.69–6.68 Table 2 Results of summary statistical tests applied to populations of Sclerurus leaftossers associated with distinct regions of Neotropics, for mitochondrial DNA (mtDNA; cyt b, ND2 and ND3), and to Fib7. Tests Region Species Marker n D Base of the Andes (BAN) S. peruvianus Guiana shields (GUY) S. rufigularis Western Amazonia – north (WAN) S. rufigularis Western Amazonia – south (WAS) S. caudacutus Brazilian shields – west (BSW) S. caudacutus mtDNA Fib7 mtDNA Fib7 mtDNA Fib7 mtDNA Fib7 mtDNA Fib7 mtDNA Fib7 5 10 6 10 8 8 11 16 5 10 7 14 )1.16 1.32 )0.53 0.05 )1.60 )0.43 )0.45 )0.41 )0.68 )0.85 )1.18 0.36 S. rufigularis ns ns ns ns ns ns ns ns ns ns ns ns FS R2 1.21 ns )0.63 ns 1.10 ns )0.91 ns )8.19** )11.67** )0.48 ns )1.75 ns )0.75 ns )5.99** )2.94* )1.12 ns 0.33 ns 0.22 ns 0.20 ns 0.16 ns 0.06** 0.11 ns 0.19 ns 0.16 ns 0.14* 0.11* 0.12* 0.17 ns n, number of sequences; D, Tajima’s (1989) D-test; FS, Fu’s (1997) FS-test; R2, Ramos-Onsins & Rozas’ (2002) R2-test. ns, not significant; *P < 0.05; **P < 0.01. 44 Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd Sclerurus phylogeny and phylogeography GUY WAN BAN WAS BSW(1) BSW(2) g -2000 -1000 0 the coalescent processes, selection, demographic processes and ecological traits. Also, differential rates of molecular evolution between lowland and montane lineages (Gillman et al., 2009) could potentially affect our interpretation of the influence of the Andean uplift on the diversification of Sclerurus. Such a pattern is suggested in our data, especially in the South American clade formed by S. obscurior (lowlands), S. andinus (montane), S. macconnelli (lowlands) and S. peruvianus (montane), where there appears to be a gradient of branch lengths that decrease with increasing elevation (see Fig. 2c). 1000 2000 3000 4000 5000 6000 12000 Elevational zonation in the trans-Andean foothills Figure 5 Values of exponential population growth rate (g) of Sclerurus lineages from distinct Amazonian biogeographical regions. Light grey bars represent populations for which 95% confidence intervals of g include zero, hence demographic stability is inferred; dark grey bars represent populations that have experienced demographic expansion. GUY: Guiana Shield (S. rufigularis); WAN: western Amazonia – north (S. rufigularis); WAS: western Amazonia – south (S. caudacutus); BAN: base of the Andes (S. peruvianus); BSW: Brazilian shields – west (1, S. rufigularis; 2, S. caudacutus). Disjunctions across the Andes The importance of the Andes in the differentiation of the Neotropical biotas is unquestionable, but their function as a primary (Chapman, 1917) or secondary (Haffer, 1967) barrier has been the subject of debate (e.g. Brumfield & Capparella, 1996; Ribas et al., 2007). The sister species pair S. caudacutus–S. guatemalensis, as well as S. obscurior–S. macconnelli/peruvianus, are isolated by the Andes (Fig. 2b,c, respectively). Divergence time estimates between these two cross-Andes pairs are largely similar, overlapping between 4.8 and 6.0 Ma (Fig. 3; nodes 21 and 4). The estimates of divergence time between cis- and transAndean lineages of lowland species in these Sclerurus taxa (Fig. 3, Table 1) and Pyrilia (6.8–8.8 Ma; Ribas et al., 2005), suggest that those lineages diverged between the Late Miocene and Early Pliocene. On the other hand, pairs of montane lineages, i.e. S. andinus and S. peruvianus (up to 2200 m) diverged during the Late Pliocene (1.9–3.3 Ma; node 5; Fig. 3, Table 1) or, in the case of the Pionus corallinus–Pionus mindoensis group (up to 3000 m; Ribas et al., 2007) during the Pleistocene. These results are congruent with the expected differential effect that the Andean uplift may have had in species with distinct elevational ranges. Additionally, the divergence times are compatible with the temporal evolution of the Andes (Gregory-Wodzicki, 2000). In contrast, Ribas et al. (2007) estimated the divergence time between cis- and trans-Andean lineages of lowland lineages of Pionus as being between 0.34 and 1.41 Ma, indicating dispersal of populations after the Andes had already reached high elevations. Large variation among divergence times across barriers could arise for a number of reasons, such as the stochasticity of Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd Along the continuum of humid forests from the Chocó lowlands to the slopes of the western Andes, two lineages seem to be segregated elevationally: S. obscurior restricted to the Chocó lowlands (M09 and M10, Fig. 2c), and S. andinus found locally from about 1000 m (often up to 2000 m) along the western slopes of the Andes from Ecuador to Venezuela (M11– M14, Fig. 2c). The two lineages are potentially syntopic at an intermediate point of the elevational and ecological gradient, where no obvious physical barrier is in place. Furthermore, our results indicate that the lowland Chocó (i.e. S. obscurior) and the Andean foothill species (i.e. S. andinus) have been evolutionarily isolated for a substantial length of time; they last shared a common ancestor in the Early Pliocene, between 3.6 and 6.0 Ma (node 4; Fig. 3, Table 1). The Andean foothill lineage originated during the Late Pliocene–Early Pleistocene, between 1.9 and 3.3 Ma (node 5; Fig. 3, Table 1). In addition, another case of elevational parapatry is observed between two divergent, non-sister lineages of the South American mexicanus group: S. macconnelli and S. peruvianus. These two species are in close geographical proximity in southern Peru and Bolivia but seem to occupy different elevations along the cis-Andean foothills (M25–M33 versus M15–M24; Fig. 2c). Although elevational zonation is a prominent characteristic of the Andean avifauna (Terborgh, 1977; Graves, 1985), so far only a few studies have documented ecological replacement of closely related, ecologically equivalent species along elevational gradients (Dingle et al., 2006; Cadena, 2007). Cis-Andean South America disjunctions Several birds, including Sclerurus, exhibit high congruence in the geographical range boundaries of divergent populations across cis-Andean forests. However, our results and the available data strongly suggest that these biogeographical patterns emerged more than once as a result of distinct sequences of cladogenetic events (e.g. Marks et al., 2002; Aleixo, 2004; Ribas et al., 2011). Recent studies have shed new light on the processes of landscape change in Amazonia since the Miocene (Hoorn et al., 1995, 2010; Rossetti et al., 2005; Campbell et al., 2006). The available data support the idea that different areas of Amazonia experienced distinct rates of landscape change, with WA (see Fig. 3) being the most dynamic (Hoorn et al., 1995; 45 F. M. d’Horta et al. Rossetti et al., 2005; Campbell et al., 2006). Marine incursions during the Miocene (Räsänen et al., 1995), as well as the development of a mega-wetland system in western Amazonia during the Early to Middle Miocene, caused by the Andean uplift (Hoorn et al., 2010), would have isolated three regions of Amazonia (Bates, 2001; Aleixo & Rossetti, 2007): GUY, BS and BAN (Fig. 3). With the increase in the rate of Andean uplift the sedimentary wedge progressed to the east, promoting the disappearance of the mega-wetland system in western Amazonia and allowing the expansion of terra firme environments through this region. The evolutionary relationships among Sclerurus lineages support the geographical predictions that emerge from this scenario. Three species have lineages associated with WA (S. macconnelli, S. caudacutus, S. rufigularis), but no WA lineage occupies basal positions regarding BA, BS or GUY lineages, whereas in S. macconnelli and S. caudacutus, GUY and BA lineages are basal in relation to WA lineages. In addition, demographic expansion was inferred for populations associated with WA (WAS, S. caudacutus; WAN, S. rufigularis), whereas the hypothesis of demographic stability was not rejected for populations from BA (S. peruvianus) and GUY (S. rufigularis) (Fig. 5). The two populations from BSW also exhibit clear signs of population expansion (Fig. 5), unlike that observed for Xiphorhynchus (Aleixo, 2004) and Psophia (Ribas et al., 2011). These contrasting patterns in BSW are expected considering that this region was partially submerged by the wetland system (Bates, 2001). Some authors support the hypothesis that the end of the WA mega-wetlands took place during the Miocene (Hoorn et al., 1995; Figueiredo et al., 2009), whereas recent studies indicate that the wetland system may have persisted until the Pliocene or Pleistocene (Rossetti et al., 2005; Campbell et al., 2006; Latrubesse et al., 2010). The estimates of divergence time among WA lineages and the historical demographic results are congruent with the scenario of recent evolution of western Amazonian drainage: the divergence between WA and GUY/BS lineages was estimated at 2.2 Ma (95% CI, 1.5–2.8; S. rufigularis, Fig. 3, node 13, Table 1) and 2.02 Ma (1.5–2.6; S. caudacutus, Fig. 3, node 24, Table 1); and the divergence time between WAN and WAS lineages of S. caudacutus was estimated at 0.9 Ma (0.6–1.2; Fig. 3, node 25, Table 1). The demographic results obtained for cis-Andean Sclerurus could be interpreted as temporally congruent with the mechanism proposed by Haffer (1969), but are highly contrasting with the palaeoenvironmental scenarios inferred for this region (Anhuf et al., 2006), which suggest that forests of GUY and parts of the BS region would have been strongly reduced during glacial periods, while those of WA would have remained stable. ACKNOWLEDGEMENTS We thank J. V. Remsen, L. N. Gillman and the anonymous referees who reviewed the manuscript and help us to improve it. We are indebted to the numerous collectors, and the 46 following curators, staff and institutions for providing tissue samples: J. Bates and D. Willard (FMNH), A. Aleixo and F. Lima (MPEG), J. Cracraft and P. Sweet (AMNH), N. Rice (ANSP), G. Graves and J. Dean (USNM), D. López and the GEMA group (IAvH-BT), J. Pérez, M. Lentino and J. Miranda (COP), and J. Klicka (MBM) (see Appendix S1 for institutions in full). Lynx Edicions kindly provided permission to use images from Handbook of birds of the world. This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior, Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico to F.M.H.; by National Science Foundation grants DBI-0400797, DEB-0543562, DEB-0910285 to R.T.B.; and by the Lewis and Clark Exploration Fund, Wilson Ornithological Society, American Ornithologists’ Union, Society of Systematic Biologists, Chapman Fund of the AMNH, Society of Integrative and Comparative Biology, and Idea Wild to A.M.C. We thank the support of local authorities for granting research permits. We finally thank P. E. Vanzolini for his constant support. REFERENCES Aleixo, A. (2004) Historical diversification of a terra-firme forest bird superspecies: a phylogeographic perspective on the role of different hypotheses of Amazonian diversification. Evolution, 58, 1303–1317. 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Author contributions: F.M.H. and A.M.C. conceived the ideas and conducted the laboratory analysis; F.M.H., A.M.C. and C.C.R. analysed the data; F.M.H. and A.M.C. led the writing; and C.C.R., R.T.B. and C.Y.M. provided samples and participated in the discussions and writing. Editor: Len N. Gillman SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Appendix S1 Tissue samples analysed, taxa, collection number and localities. Appendix S2 Procedures of DNA extraction, amplification, sequencing and alignment. Appendix S3 Mean pairwise (p) distances between and within species of Sclerurus leaftossers. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Journal of Biogeography 40, 37–49 ª 2012 Blackwell Publishing Ltd 49