Demographical expansion of Handroanthus ochraceus in the Cerrado during the Quaternary: implications for the genetic diversity of Neotropical trees

Demographical expansion of Handroanthus ochraceus in the Cerrado during the Quaternary:... Abstract Phylogeographical studies provide insights into the dispersal dynamics of species needed to understand the effects of Quaternary climate changes on the spatial patterns of genetic diversity. We used a multi-model inference approach coupling ecological niche modelling (ENM) with a relaxed random walk model to reconstruct the spatio-temporal history of lineage dispersal of the Neotropical tree species Handroanthus ochraceus. We sampled 24 populations throughout the Cerrado Biome and analysed polymorphisms at three intergenic chloroplast regions and ITS. Coalescent analyses revealed demographical expansion since c. 380 ka. Although ENM predicted no range expansion, coalescent simulations reinforce the pattern of range expansion because demographical expansion was the most likely scenario able to produce the observed spatial pattern of genetic diversity of H. ochraceus. Its most recent common ancestor dated from ~1.9 Ma, and lineages cyclically dispersed from the Southeast and West towards Central–West Brazil. Most dispersal events occurred from populations at the edges of the Cerrado towards Central Brazil. Populations at the edge of the historical refugium show higher genetic diversity and were the source of migrants to central populations. A wide historical climatic refugium through time for H. ochraceus may have allowed dispersal of lineages among populations of Central Brazil, maintaining their historical connectivity and genetic diversity. INTRODUCTION Neotropical vegetation was affected by Palaeogene–Neogene events that might have driven demographical dynamics of species from savannas and adjacent rainforests (Pennington et al., 2004; Hoorn et al., 2010; Rull, 2011). For instance, the Andean orogeny in the Palaeogene increased the latitudinal temperature gradient in South America, and uplift of the Central Brazilian Plateau in the Miocene–Pliocene transition caused the geographical reorganization of the Central Brazil ecosystems that may have favoured species diversification by vicariance (e.g. Luebert & Weigend, 2014; Luebert & Muller, 2015). Quaternary climate oscillations also affected species distributions and diversity through space and time in the Neotropics (Rull et al., 2013; Rull, Vegas-Vilarrúbia & Montoya, 2015). However, due to differences in climatic tolerances and niche characteristics, different species responded individualistically to the common drier and colder conditions of glacial periods (Cheng et al., 2013; Summerhayes & Charman, 2014). The pollen fossil record suggests that south-eastern Brazil was cooler and drier during the glacial periods than it is at present, and savannas shifted in distribution northward due to drier and colder conditions (Behling, 2002, 2003). The grasslands and subtropical Araucaria forest spread northward, replacing the Atlantic Forest and savannas to latitudes up to 20°S and retreating to the south only after the glaciation (Salgado-Labouriau, 1997; Behling, 2003). Modern wet climatic conditions in central Brazil, with short dry periods, and in all main Neotropical biomes became established during the late Holocene (Behling, 2003). Phylogeographical analyses of Neotropical species show the effect of Quaternary climatic change, with demographical expansion of some seasonally dry tropical forest species (e.g. Caetano et al., 2008; Collevatti et al., 2012a, 2016; Vieira et al., 2015), but retraction of others (e.g. Melo et al., 2016). Savanna species also show contrasting phylogeographical patterns in the Neotropics, with demographical retraction during the Last Glacial Maximum (LGM) with multiple refugia (e.g. Collevatti et al., 2012b, 2015a; Lima et al., 2014), or stability over time (e.g. Souza et al., 2017; Lima et al., 2017). Nevertheless, the response of Neotropical plant species to Quaternary climate changes remains poorly understood due to the low number of studies explicitly testing it (e.g. Collevatti et al., 2012b, 2015a; Lima et al., 2014, 2017; Souza et al., 2017). Moreover, the wide variation in response among species hinders a synthesis of the biogeography of the Neotropics and the effects of Quaternary climate changes in species distribution dynamics. Thus, study of the demographical history of plant species is important for understanding the role of Quaternary climate changes to unravel biogeographical patterns in Neotropical species, as we address here for a Neotropical savanna species. Range expansion may lead to spatial genome sorting as the species tracks suitable environments, resulting in lower genetic diversity in new colonizing areas (Hewitt, 1996). In addition, the spread of a low-frequency allele that migrates on the wave of advance of a population in expansion (allele surfing: Excoffier & Ray, 2008; Arenas et al., 2012), and density-dependent processes due to fast colonization and founder events may cause low genetic diversity in new colonizing areas (see Excoffier, Foll & Petit, 2009; Waters, Fraser & Hewitt, 2013). Reconstructing historical demography is essential to elucidate the evolution and divergence of tropical plant lineages (Knowles & Maddison, 2002). For the Neotropics, this is often compromised because of the lack of fossil records for most species (but see an example in Lima et al., 2014 for a swamp palm species). Thus, integrating direct spatio-temporal reconstruction of lineage diffusion (Lemey et al., 2009, 2010) in a multi-model framework (see Collevatti et al., 2015a, b) may give clues to the pathways of lineage dispersal across the Neotropics through the Quaternary. Here, we reconstruct the demographical history and dispersal dynamics of Handroanthus ochraceus (Cham.) Standl. (Bignoniaceae) through the Quaternary using a multi-model inference approach following Collevatti et al. (2015a, b) to understand how the Quaternary climate changes affected the spatial pattern in genetic diversity. Because Neotropical savanna species showed range retraction during the LGM followed by further expansion with multiple refugia we expect that H. ochraceus will show a pattern of range and demographical retraction during glacial phases and range expansion from the LGM to the present day. Because of population isolation in refugia we expect low historical genetic connectivity among populations leading to high population differentiation. We also hypothesize that the south-eastern and western Cerrado were important refugia for savanna species, as shown for a related species, Tabebuia aurea (Collevatti et al., 2015a), and suggested by palaeovegetation reconstruction (Ab’Saber, 2000). Thus, H. ochraceus will have the dispersal origin of extant lineages in the south-eastern and western Cerrado. MATERIAL AND METHODS Population sampling and DNA extraction Handroanthus ochraceus (syn. Tabebuia ochracea) is a widespread savanna tree species, occurring in seasonal savannas and seasonally dry forests in Brazil, Paraguay, northern Argentina, Bolivia, Peru, Colombia, Venezuela and Mesoamerica. Locally it is distributed in well-delimited patches at high density. The species is hermaphroditic and pollinated mainly by large bees such as bumblebees (Bombus spp.), carpenter bees (Xylocopa spp.) and Centris spp., and its winged seeds are wind-dispersed (Gibbs & Bianchi, 1993). We extensively sampled H. ochraceus throughout the Brazilian savanna (Fig. 1; see also Supporting Information Table S1 in Appendix S1 and Fig. S1 in Appendix S2) and collected 468 individuals from 24 populations. We focused our sampling efforts on the Cerrado biome, especially in savannas of Central–West Brazil, due to the higher abundance of the species there and our focus on savanna biogeography. In addition, although occurrence maps (Fig. S1) show records of H. ochraceus in north-eastern Brazil in the Caatinga biome and in the south-east in the Atlantic Forest biome, populations in these regions are rare. North-east Brazil is dominated by xeromorphic steppe vegetation with seasonally dry forests and some islands of savanna where H. ochraceus may occur, but the high level of anthropic disturbance has removed most natural vegetation. In the Atlantic Forest of south-east Brazil most savanna enclaves disappeared due to agriculture and urban expansion. We sampled only adult individuals to avoid the effects of high kinship on estimation of genetic parameters. Vouchers collected were compared and deposited in herbarium UFG (Universidade Federal de Goiás, exsiccate J.A Rizzo 4471, UFG 4491). The distance between population pairs ranged from 20 kmto 2393 km. In populations with fewer than 30 individuals we sampled and sequenced all of them (Table 1). We also sampled and sequenced individuals of Handroanthus impetiginosus (exsiccate N.R. Cunha 221, UFG 27029), Handroanthus serratifolius (exsiccate J.M. Rezende 535, UFG 24877) and Cybistax antisyphillitica (exsiccate L.F. Souza 3227, UFG 1020) to include as outgroups in order to estimate the divergence time to the most recent common ancestor (Table S1). Genomic DNA was extracted from leaves or cambium tissues following the standard 2% CTAB protocol (Doyle & Doyle, 1990). Table 1. Genetic diversity based on Arlequin v3.11 software and demographical parameters based on coalescent analysis performed with Lamarc 2.1.9 software for 24 populations of Handroanthus ochraceus Population  N  k  cpDNA  SD  k  ITS  π  SD  Combined cpDNA and ITS data  h  π  h  θ  95%CI  Ne  95% CI  AGE  27  8  0.781  0.0014  0.0009  9  0.618  0.0023  0.0016  0.0017  0.0004–1.0497  6.9E04  1.8E04–4.3E07  ARA  13  4  0.423  0.0012  0.0008  8  0.808  0.0068  0.0066  0.0077  0.0042–2.8443  3.1E05  1.7E05–1.2E08  BAG  05  1  0.000  0.0000  0.0000  1  0.000  0.0000  0.0000  0.0000  0.0000–0.0004  1.8E03  4.1E02–1.6E04  BAR  16  3  0.242  0.0001  0.0002  11  0.961  0.0019  0.0032  0.0090  0.0000–1.2457  3.7E04  7.2E02–50515031.09  BOD  24  5  0.249  0.0002  0.0002  10  0.652  0.0749  0.0396  0.0006  0.0001–5.3013  2.5E04  5.0E03–2.2E08  CAR  16  4  0.350  0.0010  0.0008  4  0.295  0.0022  0.0016  0.0006  0.0002–0.0013  2.5E04  8.8E03–5.5E04  CHG  20  12  0.916  0.0004  0.0004  4  0.616  0.0017  0.0013  0.0011  0.0006–0.0038  4.3E04  2.5E04–1.5E05  FAZ  31  7  0.723  0.0008  0.0005  2  0.064  0.0001  0.0003  0.0007  0.0002–0.0013  2.7E04  6.9E03–5.4E04  GSV  9  5  0.806  0.0009  0.0007  2  0.000  0.0000  0.0000  0.0004  0.0001–0.0015  1.7E04  3.9E03–6.2E04  MIM  13  5  0.808  0.0015  0.0010  2  0.000  0.0000  0.0000  0.0005  0.0001–0.0011  1.9E04  5.6E03–4.6E04  PAN  24  4  0.370  0.0002  0.0002  6  0.380  0.0013  0.0011  0.0005  0.0004–1.0401  2.1E04  1. 5E04–4.2E07  PLC  32  13  0.846  0.0035  0.0020  5  0.603  0.0014  0.0011  0.0013  0.0008–0.0018  5.1E04  3.4E04–7.5E04  PMS  30  15  0.784  0.0023  0.0013  3  0.381  0.0007  0.0007  0.0022  0.0003–0.0043  8.9E04  1.2E04–1.8E05  PNE  17  9  0.904  0.0022  0.0014  6  0.721  0.0024  0.0018  0.0036  0.0013–0.0099  1.5E05  5.3E04–4.0E05  POT  14  3  0.385  0.0001  0.0002  3  0.385  0.0007  0.0008  0.0002  0.0001–0.0007  1.0E04  2.5E03–2.7E04  PTU  18  8  0.860  0.0034  0.0020  6  0.576  0.0023  0.0017  0.0021  0.0007–0.0054  8.5E04  3.0E04–2.2E05  SAF  12  2  0.167  0.0003  0.0003  6  0.618  0.0022  0.0035  0.0007  0.0004–2.9817  2.8E04  1.6E04–1.2E08  SCA  25  14  0.813  0.0042  0.0024  8  0.446  0.0018  0.0014  0.0032  0.0019–0.0077  1.3E05  7.7E05–3.1E06  SDO  23  5  0.324  0.0002  0.0002  5  0.260  0.0037  0.0024  0.0010  0.0004–0.0019  4.2E04  1.8E04–7.9E04  SEB  21  5  0.338  0.0003  0.0003  8  0.692  0.0071  0.0042  0.0012  0.0007–0.7750  4.7E04  2.8E04–3.1E07  SEC  16  10  0.897  0.0012  0.0013  3  0.143  0.0018  0.0014  0.0008  0.0003–0.0020  3.1E04  1.2E04–7.9E04  SEL  31  7  0.574  0.0012  0.0008  4  0.295  0.0006  0.0007  0.0006  0.0003–0.0018  2.6E04  1.1E04–7.4E04  STZ  12  5  0.576  0.0011  0.0008  4  0.473  0.0009  0.0009  0.0004  0.0001–0.0010  1.7E04  5.8E03–4.2E04  SUM  19  5  0.672  0.0001  0.0002  2  0.000  0.0000  0.0000  0.0001  0.0000–0.0009  4.3E03  1.6E03–3.8E04  Mean  –  6.6  0.575  –  –  5.1  0.416  –  –  –  –  –  –  SD  –  3.8  0.275  –  –  2.8  0.280  –  –  –  –  –  –  Overall  468  106  0.927  0.0044  0.0024  78  0.637  0.0063  0.0036  0.0401  0.0337–0.0430  1.6E06  1.4E06–1.7E06  Population  N  k  cpDNA  SD  k  ITS  π  SD  Combined cpDNA and ITS data  h  π  h  θ  95%CI  Ne  95% CI  AGE  27  8  0.781  0.0014  0.0009  9  0.618  0.0023  0.0016  0.0017  0.0004–1.0497  6.9E04  1.8E04–4.3E07  ARA  13  4  0.423  0.0012  0.0008  8  0.808  0.0068  0.0066  0.0077  0.0042–2.8443  3.1E05  1.7E05–1.2E08  BAG  05  1  0.000  0.0000  0.0000  1  0.000  0.0000  0.0000  0.0000  0.0000–0.0004  1.8E03  4.1E02–1.6E04  BAR  16  3  0.242  0.0001  0.0002  11  0.961  0.0019  0.0032  0.0090  0.0000–1.2457  3.7E04  7.2E02–50515031.09  BOD  24  5  0.249  0.0002  0.0002  10  0.652  0.0749  0.0396  0.0006  0.0001–5.3013  2.5E04  5.0E03–2.2E08  CAR  16  4  0.350  0.0010  0.0008  4  0.295  0.0022  0.0016  0.0006  0.0002–0.0013  2.5E04  8.8E03–5.5E04  CHG  20  12  0.916  0.0004  0.0004  4  0.616  0.0017  0.0013  0.0011  0.0006–0.0038  4.3E04  2.5E04–1.5E05  FAZ  31  7  0.723  0.0008  0.0005  2  0.064  0.0001  0.0003  0.0007  0.0002–0.0013  2.7E04  6.9E03–5.4E04  GSV  9  5  0.806  0.0009  0.0007  2  0.000  0.0000  0.0000  0.0004  0.0001–0.0015  1.7E04  3.9E03–6.2E04  MIM  13  5  0.808  0.0015  0.0010  2  0.000  0.0000  0.0000  0.0005  0.0001–0.0011  1.9E04  5.6E03–4.6E04  PAN  24  4  0.370  0.0002  0.0002  6  0.380  0.0013  0.0011  0.0005  0.0004–1.0401  2.1E04  1. 5E04–4.2E07  PLC  32  13  0.846  0.0035  0.0020  5  0.603  0.0014  0.0011  0.0013  0.0008–0.0018  5.1E04  3.4E04–7.5E04  PMS  30  15  0.784  0.0023  0.0013  3  0.381  0.0007  0.0007  0.0022  0.0003–0.0043  8.9E04  1.2E04–1.8E05  PNE  17  9  0.904  0.0022  0.0014  6  0.721  0.0024  0.0018  0.0036  0.0013–0.0099  1.5E05  5.3E04–4.0E05  POT  14  3  0.385  0.0001  0.0002  3  0.385  0.0007  0.0008  0.0002  0.0001–0.0007  1.0E04  2.5E03–2.7E04  PTU  18  8  0.860  0.0034  0.0020  6  0.576  0.0023  0.0017  0.0021  0.0007–0.0054  8.5E04  3.0E04–2.2E05  SAF  12  2  0.167  0.0003  0.0003  6  0.618  0.0022  0.0035  0.0007  0.0004–2.9817  2.8E04  1.6E04–1.2E08  SCA  25  14  0.813  0.0042  0.0024  8  0.446  0.0018  0.0014  0.0032  0.0019–0.0077  1.3E05  7.7E05–3.1E06  SDO  23  5  0.324  0.0002  0.0002  5  0.260  0.0037  0.0024  0.0010  0.0004–0.0019  4.2E04  1.8E04–7.9E04  SEB  21  5  0.338  0.0003  0.0003  8  0.692  0.0071  0.0042  0.0012  0.0007–0.7750  4.7E04  2.8E04–3.1E07  SEC  16  10  0.897  0.0012  0.0013  3  0.143  0.0018  0.0014  0.0008  0.0003–0.0020  3.1E04  1.2E04–7.9E04  SEL  31  7  0.574  0.0012  0.0008  4  0.295  0.0006  0.0007  0.0006  0.0003–0.0018  2.6E04  1.1E04–7.4E04  STZ  12  5  0.576  0.0011  0.0008  4  0.473  0.0009  0.0009  0.0004  0.0001–0.0010  1.7E04  5.8E03–4.2E04  SUM  19  5  0.672  0.0001  0.0002  2  0.000  0.0000  0.0000  0.0001  0.0000–0.0009  4.3E03  1.6E03–3.8E04  Mean  –  6.6  0.575  –  –  5.1  0.416  –  –  –  –  –  –  SD  –  3.8  0.275  –  –  2.8  0.280  –  –  –  –  –  –  Overall  468  106  0.927  0.0044  0.0024  78  0.637  0.0063  0.0036  0.0401  0.0337–0.0430  1.6E06  1.4E06–1.7E06  N, sample size; k,–number of haplotypes; h,–haplotype diversity; π,–nucleotide diversity; SD,–standard deviation; θ,–coalescent or mutation parameter; Ne,–effective population size; 95% CI, 95% credibility interval. View Large Figure 1. View large Download slide Geographical distribution of haplotypes of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Different colours were assigned for each haplotype according to the figure legend. The circle size represents the sample size in each population and the circle sections represent the haplotype frequency in each sampled population. For details on population codes and localities see Table S1 in Appendix S1. Figure 1. View large Download slide Geographical distribution of haplotypes of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Different colours were assigned for each haplotype according to the figure legend. The circle size represents the sample size in each population and the circle sections represent the haplotype frequency in each sampled population. For details on population codes and localities see Table S1 in Appendix S1. Genetic data To generate genetic data, we sequenced three intergenic spacers of chloroplast DNA (cpDNA): psbA-trnH, trnC-ycf6 and trnS-trnG (Shaw et al., 2005) and the region ITS1 + 5.8S + ITS2 (hereafter ITS) from nuclear ribosomal DNA (nrDNA) (Desfeux & Lejeune, 1996). PCR conditions and amplifications followed Collevatti et al. (2012a). PCR fragments were sequenced using the BigDye Terminator v3.1 kit (Applied Biosystems), and sequenced in the forward and reverse directions using a GS 3500 Genetic Analyzer (Applied Biosystems), according to the manufacter’s instructions. Sequences were analysed, and consensus sequences were obtained using the software SeqScape v2.7 (Applied Biosystems) and aligned with the software ClustalΩ (Sievers et al., 2011). Polymorphisms at mononucleotide microsatellites were excluded due to ambiguous alignment and higher mutation rates. Long indels (usually > 5 bp) were coded as one evolutionary event (one character). Genetic diversity and population structure We estimated nucleotide (π) and haplotype (h) diversity for each population and the populations overall following Nei (1987) using the software ArlequinVer 3.11 (Excoffier, Laval & Schneider, 2005a). To test for population differentiation, we performed an analysis of molecular variance (AMOVA, Excoffier, Smouse & Quattro, 1992) and estimated FST values. Analyses were performed using the software Arlequin v3.11 and statistical significance was tested via a non-parametric permutation test (10000 permutations). Population structure was also assessed using Bayesian clustering implemented in the software BAPS v6.0 (Corander & Marttinen, 2006; Corander et al., 2008). The cpDNA and ITS partitions were analysed separately. We performed population admixture analysis based on mixture clustering with ‘not fixed number of clusters (K)’ with an upper limit of K = 24. Phylogeographical reconstruction Population demography All coalescent analyses were performed with cpDNA and nrDNA ITS partitions concatenated, but giving separate priors. No evidence of heterozygous individuals was found when ITS sequences were analysed using SeqScape v2.7 (Applied Biosystems). Thus, recombination was neglected in all coalescent analyses. To set the priors, evolutionary model selection for both chloroplast and ITS regions was performed based on the Akaike Information Criterion implemented in the software jModelTest2 (Darriba et al., 2012). For chloroplast regions, the model HKY+G+I (−lnL = 3625.8751, wBIC = 0.5466) was selected, with gamma shape equal to 0.067, kappa equal to 1.95 and pinv = 0.404. For ITS, the evolutionary model TPM3uf+G (−lnL = 1755.8913, wBIC = 0.3426) was selected with gamma shape equal to 0.558. To study the dynamics in effective population size and genetic connectivity among populations, we estimated the mutation or coalescent parameter theta (θ = 2μNe for haploid genomes, θ = 4μNe for diploid genomes), and the immigration rate among all population pairs (M = 2Nem/θ for haploid genomes, M = 4Nem/θ for diploid genomes). Estimations were based on a Bayesian model using the Markov chain Monte Carlo (MCMC) approach implemented in Lamarc 2.1.10 software (Kuhner, 2006). Each analysis was run with 20 initial chains of 4000 steps and three final chains of 50000 steps. The chains were sampled every 100 steps. We used default settings for the initial estimate of theta. The program was run four times for each parameter to certify convergence among runs and validate the analyses using Tracer v1.6 (Rambaut & Drummond, 2007), and combined results were then generated using LogCombiner 1.8.2 (Drummond et al., 2012). Results were considered when the effective sample size (ESS) was ≥ 200 (and when marginal posterior probability densities were unimodal and converged among runs. Effective population size was obtained from θ (Kingman, 1982) using a generation time of 15 years based on flowering time measured in permanent plots established for demography dynamics studies (R. G. Collevatti, unpublished data). To understand changes in population size through time we performed a Coalescent Extended Bayesian Skyline Plot (EBSP) analysis (Heled & Drummond, 2008) implemented in BEAST 1.8.3 (Drummond & Rambaut, 2007), combining data from different partitions. We used the substitution models reported above and the relaxed molecular clock model (uncorrelated lognormal) for both chloroplast and ITS regions. The ucld.stdev parameter (standard deviation of the uncorrelated lognormal relaxed clock) and the coefficient of variation were inspected for among-branch rate heterogeneity within the data. In all runs ucld.stdev was greater than 1.25 and the coefficient of variation frequency histogram viewed in Tracer spanned zero (~1.6–2.5) showing heterogeneity among branches. Mutation rates for both chloroplast and ITS regions were the same as used for two taxonomically related species, Handroanthus impetiginosus (Collevatti et al., 2012a) and Tabebuia aurea (Collevatti et al., 2015a). Four independent analyses were run for 30 million generations. Convergence and stationarity were checked, and the independent runs were combined. Results were considered when ESS ≥ 200. We performed Fu’s FS tests of neutrality implemented in Arlequin 3.11. Negative FS values indicate an excess of rare alleles or new mutations in the genealogy resulting from either population expansion or selective sweeps. Lineage dispersal To reconstruct the spatio-temporal history of H. ochraceus lineage dispersal, we used the relaxed random walk model (RRW; Lemey et al., 2009, 2010) implemented in the software BEAST 1.8.3, which analyses molecular sequence evolution, the demographical model and lineage dispersal in space and time simultaneously (Lemey et al., 2010). We used the two sequence partitions with unlinked priors but sharing the same location rate matrix (Lemey et al., 2009, 2010). We used Bayesian Stochastic Search Variable Selection (BSSVS), which considers a limited number of rates (at least k − 1, k = 24 localities) to explain the phylogenetic diffusion process and a Symmetric Substitution Model that uses a standard continuous-time Markov chain (CTMC) in which the transition rates between locations are reversible. Priors for sequence evolution were the same as described above for EBSP. For tree priors, we used the Coalescent GMRF Bayesian Skyride model (Minin, Bloomquist & Suchard, 2008) and for location state rate the prior CTMC Rate Reference (Ferreira & Suchard, 2008). Four runs were performed with 30 million generations, stability was analysed using Tracer 1.6 and results were considered when ESS ≥ 200. The annotated tree (maximum clade credibility tree) was generated with 50% burnin and the spatio-temporal reconstruction was performed using SPREAD 1.0.6 (Bielejec et al., 2011). We also analysed the well-supported transition rates using the Bayes factor (BF) test implemented in SPREAD. Transition rates between localities were considered only for BF > 8.0. We ran the analysis for each sequence region, cpDNA and ITS, to detect different contributions by seed and pollen dispersal. Coalescent tree and time to most recent common ancestor We obtained a coalescent tree based on Bayesian coalescent analysis implemented in the software BEAST 1.8.3 to estimate time to the most recent common ancestor (TMRCA). For this analysis, we included the outgroup sequences of H. impetiginosus, H. serratifolius and C. antisyphillitica. We used the same priors of EBSP, except that for tree prior we used Coalescent Expansion Growth based on the results of EBSP (see Results below). To date the tree we used the same mutation rates used for EBSP, i.e. the same mutation used for the related species Handroanthus impetiginosus (Collevatti et al., 2012a) and Tabebuia aurea (Collevatti et al., 2015a). MCMC conditions and number of runs also remained unchanged. The independent runs were analysed using Tracer 1.6 and the results were considered when ESS ≥ 200. We also ran an empty alignment (sampling only from priors) to verify the sensitivity of the results to the given priors. The analysis showed that our data were informative because posterior values (e.g. posterior probability) were different from those obtained from empty alignment. Setting demographical scenarios Palaeodistribution modelling We obtained 888 occurrence records of H. ochraceus across the Neotropics from the online databases GBIF (Global Biodiversity Information Facility, http://www.gbif.org/) and Species Link (http://splink.cria.org.br/), which were mapped in a grid of cells of 0.5° × 0.5° (longitude × latitude) (Table S2, Fig. S1). The resolution of 0.5° × 0.5° was the same as for the climatic variables used in the ecological niche modelling (ENM) approach. The climatic layers were represented by five bioclimatic variables (annual mean temperature, mean diurnal temperature range, isothermality − mean diurnal range/temperature annual range, precipitation of wettest month and precipitation of driest month), selected by factorial analysis with Varimax rotation from 19 bioclimatic variables obtained from the ecoClimate database (www.ecoclimate.org;Lima-Ribeiro et al., 2015). We also obtained subsoil (30–100 cm) data related to soil fertility from the Harmonized World Soil Database (version 1.2, FAO/IIASA/ISRIC/ISS-CAS/JRC 2009, available at http://www.fao.org/docrep/018/aq361e/aq361e.pdf). Using factorial analysis we selected subsoil pH (30–100 cm). We assume subsoil pH to be constant between the LGM and pre-industrial age, which was used in ENMs as a ‘constraint variable’ to better model the environmental preferences of H. ochraceus. The bioclimatic variables were obtained for pre-industrial (representing current climate conditions), mid-Holocene (6 ka) and LGM (21 ka) from five coupled Atmosphere-Ocean General Circulation Models (AOGCMs) (Table S3). Estimates of the current and past potential distribution of H. ochraceus were obtained by modelling its ecological niche using 12 algorithms (Table S4). When needed, we randomly selected pseudo-absences across the Neotropical grid cells keeping prevalence equal to 0.5 (see Stokland, Halvorsen & Stoa, 2011). The ENMs were then built on the current climatic scenario and projected onto the climatic conditions during both the mid-Holocene (6 ka) and LGM (21 ka). All ENM procedures were run in the integrated computational platform BIOENSEMBLES, which provides predictions based on the ensemble approach (see Araújo & New, 2007; Diniz-Filho et al., 2009). For each algorithm and AOGCM, models were built using 75% of occurrences and tested against the remaining records (25%). To avoid modelling bias, this procedure was repeated 50 times for each combination of ENM and AOGCM by randomly splitting training (75%) and test data (25%). After eliminating the models showing poor performance [True Skill Statistics (TSS) < 0.5, Allouche Tsoar & Kadmon, 2006, see Table S5], we computed the binary maps using thresholds established by the area under the receiver operating characteristic (ROC) curve (maximizing specificity + sensitivity). The frequency of a predicted presence was used as a measure of consensus suitability from 50 initial models. Next, we applied a hierarchical ANOVA using the predicted suitability from all models (12 ENMs × 5 AOGCMs × 3 Times) as the response variable to disentangle the effects of climate change on species distribution through the time from predictive uncertainties in the potential distribution due to modelling components (i.e. ENMs, AOGCMs). For this, the ENM and AOGCM components were nested into the time component, but crossed by a two-way factorial design within each time period (see Terribile et al., 2012 for details about hierarchical design). Because we do not have repetitions, the residual term from ANOVA represents the interaction between AOGCM and ENM (AOGCM × ENM; Sokal & Rohlf, 1995). Finally, the full ensemble was obtained for each time period using the predictive performances (TSS) to compute a weighted mean of suitabilities (Table S5), from which historical refugia were mapped (i.e. all grid cells with suitability values ≥ 0.5 during the three time periods). The threshold of 0.5 corresponds to the suitability value after excluding the lowest 10th percentile of suitability values related to the species occurrence records. We performed sensitivity analyses using thresholds of 0.4, 0.6 and 0.7, corresponding to the 5th, 12th and 17th percentiles, respectively, and the results were virtually indistinguishable. The result differed only for a threshold > 0.7, resulting in a small refugium in Central–West Brazil. Demographical scenarios The ENMs resulted in 60 predictive maps (12 ENMs × 5 AOGCMs) for each time period, which were used to support alternative demographical scenarios. By keeping the ENM and AOGCM constant across time periods, we computed the range shift across the last glacial cycle (i.e. the difference in predicted range size between the present and LGM – 21 ka), and classified the 60 predictive maps according to three general demographical scenarios using a threshold difference of ± 200 cells: (1) ‘Range Stability’, no difference in range size through time; (2) ‘Range Expansion’, range size was smaller at the LGM than at present; and (3) ‘Range Retraction’, range size was larger at the LGM than at present. Along with the three hypotheses supported by ENMs (see above), a fourth biogeographical hypothesis of ‘Multiple Refugia’ was derived from palaeoclimatic (e.g. Ab’Saber, 2000) and palaeovegetation reconstructions (e.g. Salgado-Labouriau, 1997). Simulation of demographical scenarios The demographical scenarios were simulated based on coalescent analysis (Kingman, 1982) implemented in DIYABC v2.1.0 (Cornuet et al., 2008; Cornuet, Ravigne & Estoup, 2010), following the framework described by Collevatti et al. (2012a, 2013a, 2015b). For model calibration, we used the demographical parameters estimated with Lamarc software and the same priors for sequence evolution used in Bayesian analyses (see above). The number of generations until the LGM (21 ka) was calculated using the generation time of H. ochraceus (15 years, R. G. Collevatti, unpublished data). Population dynamics were simulated backwards, with 24 demes, from t0 (present) to t1400 generations ago (at 21 ka). At t0, all demes had the same population size N0 = 100000 and reached N1, the effective population size at t1400 (21 ka), according to our theoretical expectation for each demographical scenario (see Figure 2 for details). Given the high variation in H. ochraceus effective population sizes (Table 1), we performed simulations with different initial deme sizes, N0 = 100, 1000, 10, 000 and 100000 for all scenarios. However, we detected differences among scenarios only when N0 = 100000. The demographical scenario predicted by ‘Range Expansion’ was then simulated by applying an exponentially negative population growth from the present to 21 ka, reaching N1400 = 10000, if N0 = 100000. By contrast, the ‘Range Retraction’ scenario was simulated applying exponentially positive population growth during the same period, reaching N1400 = 1000000, if N0 = 100000. The scenario of ‘Range Stability’ predicted no change in effective population size through time, i.e. N1400 = 100000 if N0 = 100000. The ‘Multiple Refugia’ scenario considers that savannas shifted in range and retracted during the LGM, forming fragments of different sizes (Ab’Saber, 2000). Thus, to model ‘Multiple Refugia’, we considered that current populations (at t0) are descendant from lineages in different demes at t1400 generations ago (LGM) with different effective population sizes: N1400 = 1000000, 100000, 10000, 1000, 100 or 10. Migration was simulated considering all current deme descendants from lineages originally in deme 1 at t generations ago, meaning that while the coalescent tree builds back through time, there is a 0.01 per generation chance that each lineage in deme x will migrate to deme 1. We also simulated different migration rates. Values < 0.01 were not sufficient to show any demographical variation at the time scale we are working and values > 0.1 retrieved equal likelihoods for all models. Figure 2. View largeDownload slide Demographical scenarios simulated for Handroanthus ochraceus using the software DIYABC 2.1.0 and their geographical representation. The simulations were performed for 24 demes. LIG, last interglacial; LGM, last glacial maximum; Pres, present day; N0, effective population size at time t0 (present); N1, effective population size at time t1400 (1400 generations ago). td is the time to most recent common ancestor of all lineages. Figure 2. View largeDownload slide Demographical scenarios simulated for Handroanthus ochraceus using the software DIYABC 2.1.0 and their geographical representation. The simulations were performed for 24 demes. LIG, last interglacial; LGM, last glacial maximum; Pres, present day; N0, effective population size at time t0 (present); N1, effective population size at time t1400 (1400 generations ago). td is the time to most recent common ancestor of all lineages. The relative fits of the models were calculated using approximate Bayesian computation (ABC; Excoffier, Estoup & Cornuet, 2005b; Beaumont, 2008) implemented in the software DIYABC v2.1.0. The number of distinct haplotypes, number of segregating sites, mean and variance of the pairwise differences, and mean and variance of the number of the rarest nucleotide of segregating sites, retrieved across 4 million simulations, were compared with those of the observed data sets, for cpDNA and ITS separately. In the software DYABC, the posterior probability of each scenario was retrieved to compare different scenarios that can explain the observed data set. We used the relative proportion of each scenario in the simulated data set closest to the observed data set (hereafter ‘direct approach’) and the logistic regression (hereafter ‘logistic approach’) of each scenario probability on the deviation between simulated and observed summary statistics (Cornuet et al., 2008; see also the DIYABC website, http://www1.montpellier.inra.fr/CBGP/diyabc/). Spatial patterns in genetic diversity We used spatially explicit analyses to detect spatial patterns in observed genetic diversity in response to late Quaternary climate oscillations, for both cpDNA and ITS. We first tested whether genetic differentiation across H. ochraceus populations (linearized FST) correlates with their geographical distance (on a log scale) using the Mantel test. The analysis was performed using the software Arlequin v3.11 and statistical significance was tested across 10000 random permutations. Next, we used quantile regressions to analyse the relationships of climatic suitability and stability through time with genetic diversity (Cade & Noon, 2003). For this, we calculated the difference of ensemble suitability between the LGM and the present day as a measure of climate stability through time. We then analysed whether historical changes in the geographical range of a species generated a cline spatial pattern in haplotype (h) and nucleotide (π) diversities, as well as in effective population size (Ne), due to expansion of climatically suitable conditions. We obtained the distance between each sampled population and the centroid of historical refugium and of the predicted distribution at 21 ka and the present day, and then performed quantile regressions of genetic parameters against these spatial distances and against climatic stability. RESULTS Genetic diversity and population structure The combined data of chloroplast intergenic spacers generated 2133 bp and the nrDNA ITS generated 610 bp, resulting in 2299 bp when microsatellites and ambiguous alignments were eliminated (1772 bp for chloroplast and 527 for ITS). We found 106 different chloroplast and 78 ITS haplotypes for the 468 individuals of H. ochraceus, respectively (Fig. 1). Some populations showed low haplotype and nucleotide diversities (Table 1), despite the high number of individuals sampled. Most haplotypes were exclusive of one population (Fig. 1, see also Table S6) and two chloroplast and ITS haplotypes were very widespread, H1 and H5 (Fig. 1). The AMOVA showed significant genetic differentiation among populations for both cpDNA (FST = 0.742; P < 0.001) and nuclear ITS (FST = 0.544; P < 0.001). Bayesian clustering showed an optimal partition of four clusters for both ITS and cpDNA (Fig. 3), but with no clear geographical pattern. For ITS (Fig. 3A), almost all populations were clustered together in one group (cluster I, in green), but individuals from populations ARA and SEB from Northwest, SCA from Southeast, and PAN and BAR from Central-West were also grouped in cluster II (red). The chloroplast genome (Fig. 3B) showed a higher admixture, with most populations grouped in cluster III (light green), but with admixture with cluster I (blue) and cluster IV (orange). Populations PLC and PMS formed a different cluster for both ITS (cluster IV, blue) and chloroplast data (cluster II, red). Overall, we found high genetic differentiation among populations but with high admixture indicating historical connectivity. Figure 3. View large Download slide Geographical distribution of Bayesian clustering of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Each colour represents an inferred cluster (four clusters for ITS and cpDNA), also represented in the bar graphs below the maps. In the bar graphs, each individual bar represents an individual plant and colour indicates the cluster. The dark grey area represents the limits of the Brazilian Cerrado Biome. For details on population codes and localities see Table S1 in Appendix S1. Figure 3. View large Download slide Geographical distribution of Bayesian clustering of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Each colour represents an inferred cluster (four clusters for ITS and cpDNA), also represented in the bar graphs below the maps. In the bar graphs, each individual bar represents an individual plant and colour indicates the cluster. The dark grey area represents the limits of the Brazilian Cerrado Biome. For details on population codes and localities see Table S1 in Appendix S1. Phylogeographical reconstruction Population demography We found low values of θ for each population and for the populations as a whole (Table 1), but high effective population sizes. However, migration was negligible (less than one migrant per generation) between all pair of populations (Tables S7 and S8). The EBSP showed population demographical expansion (Fig. S2a) at c. 380 ka, with lower growth rates after c. 150 ka. Fu’s FS test also showed significant signal of population expansion following a retraction for both ITS (FS = −25.520, P = 0.0001) and cpDNA (FS = −24.848, P = 0.0002). However, the negative FS values potentially indicate selective sweeps. Overall, coalescent analyses showed demographical expansion through time and high effective population sizes. Lineage dispersal We recovered one most probable ancestral location for H. ochraceus haplotypes (Fig. S2b) in population SCA in Southeast Brazil. Lineages started to disperse at c. 520 ka (Fig. S2b) to populations at the eastern (PLC and PAN), central (MIM and AGE) and western Cerrado (POT and FAZ). Most dispersal events (28 of 41) occurred in the Middle Pleistocene, after c. 400 ka, matching the demographical expansion revealed by EBSP (Figure 4). In addition, most dispersal events occurred during interglacial periods (47 of 71 dispersal events). The Bayes factor analysis showed that all links among localities are well supported (all BF > 8.0). The mean dispersal rate was 1.01 km/year [SD = 0.939, 95% highest posterior density interval 0.019–2.946 km/year]. The RRW analyses revealed historical connectivity among populations that coincides with the timing of population demographical expansion. Figure 4. View large Download slide Spatio-temporal dynamics of Handroanthus ochraceus lineage diffusion among the 24 populations sampled in the Brazilian savanna, for 1.350 Ma, 900 ka, 650 ka and 300 ka until the present day. Arrows between locations represent branches in the tree along which the relevant location transition occurs. The map was adapted from the .kml file provided by SPREAD software generated using Google Earth (http://earth.google.com). The δ18O curve corresponds to the composite benthic stable oxygen isotope ratios (Lisiecki and Raymo, 2005). For details on population codes and localities see Table S1 in Appendix S1 and Figure 1. Figure 4. View large Download slide Spatio-temporal dynamics of Handroanthus ochraceus lineage diffusion among the 24 populations sampled in the Brazilian savanna, for 1.350 Ma, 900 ka, 650 ka and 300 ka until the present day. Arrows between locations represent branches in the tree along which the relevant location transition occurs. The map was adapted from the .kml file provided by SPREAD software generated using Google Earth (http://earth.google.com). The δ18O curve corresponds to the composite benthic stable oxygen isotope ratios (Lisiecki and Raymo, 2005). For details on population codes and localities see Table S1 in Appendix S1 and Figure 1. Time to most recent common ancestor Haplotypes of H. ochraceus showed a recent TMRCA (Fig. 5), c. 1.9 Ma [95% credibility interval (CI) = 0.1–2.3 Ma], which coincides with the coalescence of haplotypes from populations PLC and PMS with all other haplotypes. Most divergences occurred after c. 1.1 Ma (Fig. 5) and geographically distant populations (e.g. SEC and BOD) shared haplotypes and common ancestors. Figure 5. View largeDownload slide Relationships and TMRCA of Handroanthus ochraceus lineages. The light pink bar corresponds to the 95% highest posterior probability of the median time to the common ancestor; numbers above branches are support values for the nodes (posterior probability) and numbers below are the TMRCA. The time scale is in millions of years ago (Ma). Figure 5. View largeDownload slide Relationships and TMRCA of Handroanthus ochraceus lineages. The light pink bar corresponds to the 95% highest posterior probability of the median time to the common ancestor; numbers above branches are support values for the nodes (posterior probability) and numbers below are the TMRCA. The time scale is in millions of years ago (Ma). Setting demographical scenarios Palaeodistribution modelling Handroanthus ochraceus shows a clear preference for hot and dry climates (Fig. S3), matching the general current conditions of Neotropical savannas. Such climate space was equally available at the LGM, mid-Holocene and the present day (Fig. 6A). ENMs predicted only a slightly difference in the potential distribution of H. ochraceus through time (Fig. 6, see also Fig. S4), with a slightly higher range size at the LGM (Fig. 6A) compared to both the mid-Holocene (Fig. 6B) and the present day (Fig. 6C). In addition, a wide region across Central–West and Northeast Brazil probably acted as an historical refugium maintaining populations of H. ochraceus during the climate changes of the last glaciation (Fig. 6D). Overall, the ENMs showed quasi-stability in the geographical range of H. ochraceus through time. Figure 6. View largeDownload slide Consensus maps of the 60 models expressing the potential distribution for Handroanthus ochraceus, based on ecological niche modelling during the (A) LGM (21 ka), (B) mid-Holocene (6 ka) and (C) present day, and (D) for historical refugium through time (from the LGM to the present day). Black dots indicate the 24 populations sampled. Figure 6. View largeDownload slide Consensus maps of the 60 models expressing the potential distribution for Handroanthus ochraceus, based on ecological niche modelling during the (A) LGM (21 ka), (B) mid-Holocene (6 ka) and (C) present day, and (D) for historical refugium through time (from the LGM to the present day). Black dots indicate the 24 populations sampled. The models were well evaluated from TSS (Table S5). Analysis of uncertainty using a hierarchical ANOVA showed high proportional variance from the modelling method (ENMs) and AOGCM (Table S9, Fig. S5), but variation was spatially structure and higher outside the occurrence range of H. ochraceus, indicating that the ENMs were able to detect the effects of climate changes on the distribution dynamics of H. ochraceus through the last glaciation, despite the AOGCM variation. Demographical scenarios and simulations The scenario of ‘Range Stability’ was the most frequent hypothesis from ENM predictions (45%, see Table S10) followed by ‘Range Retraction’ (40%) and ‘Range Expansion’ (15.0%). Simulations of demographical scenarios showed only a slight difference using a direct approach (Fig. S6) for both cpDNA (Fig. S6a) and ITS (Fig. S6c). However, using a logistic approach, scenario 4 (Multiple Refugia) was the most likely one to predict the observed parameters for cpDNA (posterior probability = 1.00, 95% CI = 1.00–1.0, Fig. S6b) and scenario 1 (Range Expansion) was the most likely one for ITS (posterior probability = 0.91, 95% CI = 0.84–0.97, Fig. S6d, see also Table S11 for posterior probabilities). The simulations thus showed that H. ochraceus had a smaller range size at the LGM than at the present day for both molecular markers. Spatial patterns in genetic diversity Pairwise differentiation was high for almost all population pairs (Table S12), but was not correlated with geographical distance for either chloroplast (r2 = 0.078, P = 0.180) or ITS (r2 = 0.054, P = 0.670). Areas with higher climatic instability through time (higher delta suitability 0–21 ka) showed lower nucleotide and haplotype diversity (Fig. S7). In addition, populations at greater distances from the centroid of the climatic refugium showed higher nucleotide and haplotype diversities (Fig. S8). Effective population size was not significantly related to the distance from the centroid or to climatic suitability. DISCUSSION Our results show that H. ochraceus lineages dispersed cyclically from Southeast and West towards Central–West Brazil. Most dispersal events occurred from populations at the edges of the Cerrado biome towards Central Brazil. Populations at the edge of the historical refugium showed higher genetic diversity and were the source of migrants to populations at the centre of the refugium. The palaeodistribution dynamics showed a large historical refugium throughout Central Brazil and range stability through time. The slight difference in range size through the last glacial cycle is due mainly to range expansion towards the Amazon Basin at the LGM. All sampled populations remained in environmentally suitable areas through the last glacial cycle, inside the large climatic refugium, which may have favoured high historical connectivity among populations. In fact, the climatic niche space of H. ochraceus indicates its preference for hot and dry conditions that prevailed during the last glacial cycle in its geographical range. The lack of dispersal of new lineages after c. 130 ka, the low number of migrants per generation (Nm < 1.0) and the high number of haplotypes with restricted geographical distribution show that, despite the high historical connectivity, recent connectivity is low. The lack of recent connectivity may be due to widespread fragmentation due to anthropogenic disturbance. Although RRW predicts spatial displacements occurring earlier than ENMs, they both converge to consistent patterns, showing that our approach is reliable in reconstructing the phylogeography of H. ochraceus. The dispersal results also indicated that populations currently found in Northern and Northeastern Brazil received migrants mainly from the Southeast (e.g. see arrow direction in Fig. 4). Such dispersal routes have probably occurred because suitable climatic conditions were available across a wider region during warming phases, allowing the connectivity of populations. Diffusion models suggested that inverse dynamics occurred during cooling phases (e.g. 200 and 260 ka), when lineages dispersed in the opposite direction, from North-Northeast toward Central and Central-West Brazil. This region was hotter and drier, thus retaining more suitable niches for H. ochraceus during the cooler phases. A potential continuous historical refugium across Neotropical savannas may be the key factor allowing uninterrupted lineage dispersal among populations of Central Brazil, as predicted by the RRW model. Note that a similar result was reported for a related species, Tabebuia aurea (Collevatti et al., 2015a), reinforcing the idea that dispersal from Southeast to Northeast might have been an important route for savanna plant species in the Quaternary. In addition, population SCA was also one of the ancestral localities of T. aurea lineages (together with CAC, see Collevatti et al., 2015a). However, the dispersal of lineages present in a population would not be detected as a new gene flow event. Thus, population connectivity could be maintained after 130 ka but our results show no dispersal of new lineages. Despite the difference in demographical scenarios between cpDNA (Multiple Refugia) and ITS (Range Expansion), both molecular markers predicted population expansion through time (i.e. from the LGM to present day) as the most likely general demographical scenario for H. ochraceus, matching our theoretical prediction. The difference between the two molecular markers is probably due to evolutionary rates. Besides substitution rates, which are higher in ITS than in chloroplast intergenic regions, and concerted evolution in nrDNA that may decrease genetic variation, ITS has four times the effective size of the chloroplast genome and may be differentially affected by drift. However, ENMs revealed quasi-stability in geographical range, with a slightly larger range at the LGM compared to the present day. Note that H. ochraceus showed different geographical range dynamics through time compared to other taxonomically related species such as T. aurea (Collevatti et al. 2015a) and T. roseoalba (Melo et al., 2016) that showed range retraction during the LGM. Moreover, H. ochraceus has a more recent common ancestor (c. 1.9 Ma) than other related species, such as T. aurea (4.0 ± 2.5 Ma, Collevatti et al. 2015a), T. roseoalba (4.9 ± 1.9 Ma, Melo et al., 2016) and H. impetiginosus (4.7 ± 1.1 Ma, Collevatti et al., 2012a), probably due to differences in demographical history. Because favourable climatic conditions for H. ochraceus (i.e. hotter and drier) were more widely available during glacial than interglacial periods in the Neotropics, effective population size has increased in the last c. 380 ka, despite glacial cycles. Besides, the increasing number of lineages, especially in the last c. 500 ka, may have resulted from the demographical expansions across cyclical glacial periods, during which wider areas of suitability were available for colonization. Lineage dispersal was mainly from populations SCA and PAN (see Fig. 4). This diffusion pattern explains the widespread geographical distribution of some haplotypes (e.g. H1 and H5 for ITS, and H1 and H5 for cpDNA, see Fig. 1) and the restricted geographical distribution of other haplotypes that diverged more recently (e.g. H76, H77 and H78 for ITS, and H104, H105 and H106 for cpDNA, see Fig. 1), leading to high population differentiation. The analyses also indicated in situ diversification of haplotypes in many populations, such as PLC, PMS, PNE, SEB, SEC, SEL, STZ, SUM (see Fig. S2b), and a higher diversification after c. 200 ka, which coincides with the demographical population growth. The lack of dispersal events after c. 130 ka, also indicated by coalescent analyses in Lamarc software, may have led to the restricted geographical distribution of many haplotypes. Although our findings show possible long-distance dispersal between distant populations such as SCA and SEC and SEC and CAR, it is important to note that these events are probably due to stepping-stone dispersal and would be detected only with continuous spatial sampling. However, continuous large-scale sampling is hindered by the fragmentation of savanna in Brazil. Besides, mean rate of dispersal estimates of 1.01 km/year using RRW is congruent with the findings for the related species T. aurea (1.17 km/yr, Collevatti et al., 2015a). Handroanthus ochraceus and T. aurea share the same pollinator species and seed dispersal syndrome, and they co-occur in many localities. Thus, we expect that long-distance dispersal be due mainly to pollen dispersal (see Braga & Collevatti, 2011), which may also explain the widespread distribution of ITS haplotypes. Despite the demographical expansions through time, ENMs suggested that all populations are within the historical refugium, i.e. areas of high climatic suitability throughout the study period (see Fig. 6D), favouring population persistence and genetic connectivity. If this stable area was maintained during the recurrent glaciations of the Quaternary, we can predict long-term genetic connectivity among H. ochraceus populations, which is also suggested by a high density of well-supported links among populations inside this historical refugium. Thus, the low haplotype diversity within some populations may be an effect of selective sweeps, as indicated by the negative FS values (e.g. Kapralov & Filatov, 2007). In addition, despite differences in effective population size and inheritance, the chloroplast genome showed higher diversity than ITS nrDNA, although the latter has four times the effective size. This may be due to concerted evolution in nrDNA that may decrease genetic variation (Ohta, 1984). Moreover, recent anthropogenic disturbance may also have caused bottlenecks, decreasing genetic diversity in H. ochraceus populations because some populations, such as BAG, POT, CAR, which are isolated remnants in pasture or crop plantations, have low genetic diversity. We believe that our results are not an artefact of sampling effort because populations with larger sample sizes had the same number of haplotypes as populations with smaller sample sizes. The pattern of lineage spread and extinction may explain the low genetic diversity in many populations, even in the centre of the species’ geographical range. In fact, populations in more unstable areas had lower genetic diversity and populations further from the centroid, at the edge of the climatic refugium, showed higher genetic diversity. This reinforces the multiple refugia demographical scenario. We hypothesize that these populations, at the edge of historical refugia, may represent ‘stable rear edges’, i.e. relict populations that have persisted in suitable local habitats across the Quaternary glaciation cycles (see Petit et al., 2003; Hampe & Petit, 2005). Note that due to the coarse resolution of climatic layers (0.5° × 0.5°) the ENM may not be able to predict species presence on the local scale (small refugia). Finally, the scenario of demographical expansion through time for H. ochraceus was also supported for other Brazilian savanna species such as Tabebuia aurea (Collevatti et al., 2015a), Caryocar brasiliense (Collevatti et al., 2012b) and Dipteryx alata (Collevatti et al., 2013b) and for a Brazilian savanna swamp palm species, Mauritia flexuosa (Lima et al., 2014). Phylogeographical studies of savanna species (e.g. Ramos et al., 2007; Novaes et al., 2010, 2013; Collevatti et al., 2012b) have shown high levels of genetic differentiation among populations and evidence of colonization of Southeastern Brazilian savannas during warmer interglacial periods of the Quaternary. However, our results for H. ochraceus and T. aurea (Collevatti et al. 2015a) do not support that populations from Southeast Brazil were recently colonized because population SCA, in Southeast Brazil, was the ancestral locality for extant lineages of the two species and had old lineages. Our results also match the spatial pattern in genetic diversity for other savanna tree species, such as C. brasiliense (Collevatti et al., 2012b) and T. aurea (Collevatti et al., 2015a), and reinforces the idea that relict populations may have persisted in suitable local habitats across the Quaternary glaciation cycles. Nevertheless, there is high variation in spatial patterns of genetic diversity in savanna trees, and some species show a central–peripheral pattern, such as Eugenia dysenterica (Lima et al., 2017) and Dimorphandra mollis (Souza et al., 2017). In conclusion, our findings suggest that the pattern of genetic diversity in H. ochraceus may be the result of range stability and population demographical expansion through the Quaternary. The general dynamics of lineage dispersal followed suitable climatic conditions, towards the Northern and Central–West regions during warming periods in the early Quaternary, and an opposite direction during glacial phases. The same pattern was also found for a taxonomically related species, T. aurea (Collevatti et al. 2015a). We also provide evidence of a general pattern of distribution dynamics and lineage dispersal in savanna tree species and emphasize that the south-eastern and western Cerrado were important refugia for savanna species. Our study also shows that our multi-model inference approach allows us to infer the different processes affecting phylogeographical patterns through time. ACKNOWLEDGMENTS This work was supported by grants to the research network GENPAC (Geographical Genetics and Regional Planning for natural resources in Brazilian Cerrado) supported by CNPq/MCT/CAPES/FAPEG (project nos. 564717/2010-0, 563727/2010-1 and 563624/2010-8), and Rede Cerrado CNPq/PPBio (project no. 457406/2012-7). We thank Thiago F. Rangel for providing access to the computational platform BIOENSEMBLES and the World Climate Research Programmer’s Working Group on Coupled Modelling (Table S3) for making available their model outputs. We also thank three anonymous reviewers for their helpful comments. SUPPORTING INFORMATION Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Appendix S1. 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Demographical expansion of Handroanthus ochraceus in the Cerrado during the Quaternary: implications for the genetic diversity of Neotropical trees

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© 2018 The Linnean Society of London, Biological Journal of the Linnean Society
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Abstract

Abstract Phylogeographical studies provide insights into the dispersal dynamics of species needed to understand the effects of Quaternary climate changes on the spatial patterns of genetic diversity. We used a multi-model inference approach coupling ecological niche modelling (ENM) with a relaxed random walk model to reconstruct the spatio-temporal history of lineage dispersal of the Neotropical tree species Handroanthus ochraceus. We sampled 24 populations throughout the Cerrado Biome and analysed polymorphisms at three intergenic chloroplast regions and ITS. Coalescent analyses revealed demographical expansion since c. 380 ka. Although ENM predicted no range expansion, coalescent simulations reinforce the pattern of range expansion because demographical expansion was the most likely scenario able to produce the observed spatial pattern of genetic diversity of H. ochraceus. Its most recent common ancestor dated from ~1.9 Ma, and lineages cyclically dispersed from the Southeast and West towards Central–West Brazil. Most dispersal events occurred from populations at the edges of the Cerrado towards Central Brazil. Populations at the edge of the historical refugium show higher genetic diversity and were the source of migrants to central populations. A wide historical climatic refugium through time for H. ochraceus may have allowed dispersal of lineages among populations of Central Brazil, maintaining their historical connectivity and genetic diversity. INTRODUCTION Neotropical vegetation was affected by Palaeogene–Neogene events that might have driven demographical dynamics of species from savannas and adjacent rainforests (Pennington et al., 2004; Hoorn et al., 2010; Rull, 2011). For instance, the Andean orogeny in the Palaeogene increased the latitudinal temperature gradient in South America, and uplift of the Central Brazilian Plateau in the Miocene–Pliocene transition caused the geographical reorganization of the Central Brazil ecosystems that may have favoured species diversification by vicariance (e.g. Luebert & Weigend, 2014; Luebert & Muller, 2015). Quaternary climate oscillations also affected species distributions and diversity through space and time in the Neotropics (Rull et al., 2013; Rull, Vegas-Vilarrúbia & Montoya, 2015). However, due to differences in climatic tolerances and niche characteristics, different species responded individualistically to the common drier and colder conditions of glacial periods (Cheng et al., 2013; Summerhayes & Charman, 2014). The pollen fossil record suggests that south-eastern Brazil was cooler and drier during the glacial periods than it is at present, and savannas shifted in distribution northward due to drier and colder conditions (Behling, 2002, 2003). The grasslands and subtropical Araucaria forest spread northward, replacing the Atlantic Forest and savannas to latitudes up to 20°S and retreating to the south only after the glaciation (Salgado-Labouriau, 1997; Behling, 2003). Modern wet climatic conditions in central Brazil, with short dry periods, and in all main Neotropical biomes became established during the late Holocene (Behling, 2003). Phylogeographical analyses of Neotropical species show the effect of Quaternary climatic change, with demographical expansion of some seasonally dry tropical forest species (e.g. Caetano et al., 2008; Collevatti et al., 2012a, 2016; Vieira et al., 2015), but retraction of others (e.g. Melo et al., 2016). Savanna species also show contrasting phylogeographical patterns in the Neotropics, with demographical retraction during the Last Glacial Maximum (LGM) with multiple refugia (e.g. Collevatti et al., 2012b, 2015a; Lima et al., 2014), or stability over time (e.g. Souza et al., 2017; Lima et al., 2017). Nevertheless, the response of Neotropical plant species to Quaternary climate changes remains poorly understood due to the low number of studies explicitly testing it (e.g. Collevatti et al., 2012b, 2015a; Lima et al., 2014, 2017; Souza et al., 2017). Moreover, the wide variation in response among species hinders a synthesis of the biogeography of the Neotropics and the effects of Quaternary climate changes in species distribution dynamics. Thus, study of the demographical history of plant species is important for understanding the role of Quaternary climate changes to unravel biogeographical patterns in Neotropical species, as we address here for a Neotropical savanna species. Range expansion may lead to spatial genome sorting as the species tracks suitable environments, resulting in lower genetic diversity in new colonizing areas (Hewitt, 1996). In addition, the spread of a low-frequency allele that migrates on the wave of advance of a population in expansion (allele surfing: Excoffier & Ray, 2008; Arenas et al., 2012), and density-dependent processes due to fast colonization and founder events may cause low genetic diversity in new colonizing areas (see Excoffier, Foll & Petit, 2009; Waters, Fraser & Hewitt, 2013). Reconstructing historical demography is essential to elucidate the evolution and divergence of tropical plant lineages (Knowles & Maddison, 2002). For the Neotropics, this is often compromised because of the lack of fossil records for most species (but see an example in Lima et al., 2014 for a swamp palm species). Thus, integrating direct spatio-temporal reconstruction of lineage diffusion (Lemey et al., 2009, 2010) in a multi-model framework (see Collevatti et al., 2015a, b) may give clues to the pathways of lineage dispersal across the Neotropics through the Quaternary. Here, we reconstruct the demographical history and dispersal dynamics of Handroanthus ochraceus (Cham.) Standl. (Bignoniaceae) through the Quaternary using a multi-model inference approach following Collevatti et al. (2015a, b) to understand how the Quaternary climate changes affected the spatial pattern in genetic diversity. Because Neotropical savanna species showed range retraction during the LGM followed by further expansion with multiple refugia we expect that H. ochraceus will show a pattern of range and demographical retraction during glacial phases and range expansion from the LGM to the present day. Because of population isolation in refugia we expect low historical genetic connectivity among populations leading to high population differentiation. We also hypothesize that the south-eastern and western Cerrado were important refugia for savanna species, as shown for a related species, Tabebuia aurea (Collevatti et al., 2015a), and suggested by palaeovegetation reconstruction (Ab’Saber, 2000). Thus, H. ochraceus will have the dispersal origin of extant lineages in the south-eastern and western Cerrado. MATERIAL AND METHODS Population sampling and DNA extraction Handroanthus ochraceus (syn. Tabebuia ochracea) is a widespread savanna tree species, occurring in seasonal savannas and seasonally dry forests in Brazil, Paraguay, northern Argentina, Bolivia, Peru, Colombia, Venezuela and Mesoamerica. Locally it is distributed in well-delimited patches at high density. The species is hermaphroditic and pollinated mainly by large bees such as bumblebees (Bombus spp.), carpenter bees (Xylocopa spp.) and Centris spp., and its winged seeds are wind-dispersed (Gibbs & Bianchi, 1993). We extensively sampled H. ochraceus throughout the Brazilian savanna (Fig. 1; see also Supporting Information Table S1 in Appendix S1 and Fig. S1 in Appendix S2) and collected 468 individuals from 24 populations. We focused our sampling efforts on the Cerrado biome, especially in savannas of Central–West Brazil, due to the higher abundance of the species there and our focus on savanna biogeography. In addition, although occurrence maps (Fig. S1) show records of H. ochraceus in north-eastern Brazil in the Caatinga biome and in the south-east in the Atlantic Forest biome, populations in these regions are rare. North-east Brazil is dominated by xeromorphic steppe vegetation with seasonally dry forests and some islands of savanna where H. ochraceus may occur, but the high level of anthropic disturbance has removed most natural vegetation. In the Atlantic Forest of south-east Brazil most savanna enclaves disappeared due to agriculture and urban expansion. We sampled only adult individuals to avoid the effects of high kinship on estimation of genetic parameters. Vouchers collected were compared and deposited in herbarium UFG (Universidade Federal de Goiás, exsiccate J.A Rizzo 4471, UFG 4491). The distance between population pairs ranged from 20 kmto 2393 km. In populations with fewer than 30 individuals we sampled and sequenced all of them (Table 1). We also sampled and sequenced individuals of Handroanthus impetiginosus (exsiccate N.R. Cunha 221, UFG 27029), Handroanthus serratifolius (exsiccate J.M. Rezende 535, UFG 24877) and Cybistax antisyphillitica (exsiccate L.F. Souza 3227, UFG 1020) to include as outgroups in order to estimate the divergence time to the most recent common ancestor (Table S1). Genomic DNA was extracted from leaves or cambium tissues following the standard 2% CTAB protocol (Doyle & Doyle, 1990). Table 1. Genetic diversity based on Arlequin v3.11 software and demographical parameters based on coalescent analysis performed with Lamarc 2.1.9 software for 24 populations of Handroanthus ochraceus Population  N  k  cpDNA  SD  k  ITS  π  SD  Combined cpDNA and ITS data  h  π  h  θ  95%CI  Ne  95% CI  AGE  27  8  0.781  0.0014  0.0009  9  0.618  0.0023  0.0016  0.0017  0.0004–1.0497  6.9E04  1.8E04–4.3E07  ARA  13  4  0.423  0.0012  0.0008  8  0.808  0.0068  0.0066  0.0077  0.0042–2.8443  3.1E05  1.7E05–1.2E08  BAG  05  1  0.000  0.0000  0.0000  1  0.000  0.0000  0.0000  0.0000  0.0000–0.0004  1.8E03  4.1E02–1.6E04  BAR  16  3  0.242  0.0001  0.0002  11  0.961  0.0019  0.0032  0.0090  0.0000–1.2457  3.7E04  7.2E02–50515031.09  BOD  24  5  0.249  0.0002  0.0002  10  0.652  0.0749  0.0396  0.0006  0.0001–5.3013  2.5E04  5.0E03–2.2E08  CAR  16  4  0.350  0.0010  0.0008  4  0.295  0.0022  0.0016  0.0006  0.0002–0.0013  2.5E04  8.8E03–5.5E04  CHG  20  12  0.916  0.0004  0.0004  4  0.616  0.0017  0.0013  0.0011  0.0006–0.0038  4.3E04  2.5E04–1.5E05  FAZ  31  7  0.723  0.0008  0.0005  2  0.064  0.0001  0.0003  0.0007  0.0002–0.0013  2.7E04  6.9E03–5.4E04  GSV  9  5  0.806  0.0009  0.0007  2  0.000  0.0000  0.0000  0.0004  0.0001–0.0015  1.7E04  3.9E03–6.2E04  MIM  13  5  0.808  0.0015  0.0010  2  0.000  0.0000  0.0000  0.0005  0.0001–0.0011  1.9E04  5.6E03–4.6E04  PAN  24  4  0.370  0.0002  0.0002  6  0.380  0.0013  0.0011  0.0005  0.0004–1.0401  2.1E04  1. 5E04–4.2E07  PLC  32  13  0.846  0.0035  0.0020  5  0.603  0.0014  0.0011  0.0013  0.0008–0.0018  5.1E04  3.4E04–7.5E04  PMS  30  15  0.784  0.0023  0.0013  3  0.381  0.0007  0.0007  0.0022  0.0003–0.0043  8.9E04  1.2E04–1.8E05  PNE  17  9  0.904  0.0022  0.0014  6  0.721  0.0024  0.0018  0.0036  0.0013–0.0099  1.5E05  5.3E04–4.0E05  POT  14  3  0.385  0.0001  0.0002  3  0.385  0.0007  0.0008  0.0002  0.0001–0.0007  1.0E04  2.5E03–2.7E04  PTU  18  8  0.860  0.0034  0.0020  6  0.576  0.0023  0.0017  0.0021  0.0007–0.0054  8.5E04  3.0E04–2.2E05  SAF  12  2  0.167  0.0003  0.0003  6  0.618  0.0022  0.0035  0.0007  0.0004–2.9817  2.8E04  1.6E04–1.2E08  SCA  25  14  0.813  0.0042  0.0024  8  0.446  0.0018  0.0014  0.0032  0.0019–0.0077  1.3E05  7.7E05–3.1E06  SDO  23  5  0.324  0.0002  0.0002  5  0.260  0.0037  0.0024  0.0010  0.0004–0.0019  4.2E04  1.8E04–7.9E04  SEB  21  5  0.338  0.0003  0.0003  8  0.692  0.0071  0.0042  0.0012  0.0007–0.7750  4.7E04  2.8E04–3.1E07  SEC  16  10  0.897  0.0012  0.0013  3  0.143  0.0018  0.0014  0.0008  0.0003–0.0020  3.1E04  1.2E04–7.9E04  SEL  31  7  0.574  0.0012  0.0008  4  0.295  0.0006  0.0007  0.0006  0.0003–0.0018  2.6E04  1.1E04–7.4E04  STZ  12  5  0.576  0.0011  0.0008  4  0.473  0.0009  0.0009  0.0004  0.0001–0.0010  1.7E04  5.8E03–4.2E04  SUM  19  5  0.672  0.0001  0.0002  2  0.000  0.0000  0.0000  0.0001  0.0000–0.0009  4.3E03  1.6E03–3.8E04  Mean  –  6.6  0.575  –  –  5.1  0.416  –  –  –  –  –  –  SD  –  3.8  0.275  –  –  2.8  0.280  –  –  –  –  –  –  Overall  468  106  0.927  0.0044  0.0024  78  0.637  0.0063  0.0036  0.0401  0.0337–0.0430  1.6E06  1.4E06–1.7E06  Population  N  k  cpDNA  SD  k  ITS  π  SD  Combined cpDNA and ITS data  h  π  h  θ  95%CI  Ne  95% CI  AGE  27  8  0.781  0.0014  0.0009  9  0.618  0.0023  0.0016  0.0017  0.0004–1.0497  6.9E04  1.8E04–4.3E07  ARA  13  4  0.423  0.0012  0.0008  8  0.808  0.0068  0.0066  0.0077  0.0042–2.8443  3.1E05  1.7E05–1.2E08  BAG  05  1  0.000  0.0000  0.0000  1  0.000  0.0000  0.0000  0.0000  0.0000–0.0004  1.8E03  4.1E02–1.6E04  BAR  16  3  0.242  0.0001  0.0002  11  0.961  0.0019  0.0032  0.0090  0.0000–1.2457  3.7E04  7.2E02–50515031.09  BOD  24  5  0.249  0.0002  0.0002  10  0.652  0.0749  0.0396  0.0006  0.0001–5.3013  2.5E04  5.0E03–2.2E08  CAR  16  4  0.350  0.0010  0.0008  4  0.295  0.0022  0.0016  0.0006  0.0002–0.0013  2.5E04  8.8E03–5.5E04  CHG  20  12  0.916  0.0004  0.0004  4  0.616  0.0017  0.0013  0.0011  0.0006–0.0038  4.3E04  2.5E04–1.5E05  FAZ  31  7  0.723  0.0008  0.0005  2  0.064  0.0001  0.0003  0.0007  0.0002–0.0013  2.7E04  6.9E03–5.4E04  GSV  9  5  0.806  0.0009  0.0007  2  0.000  0.0000  0.0000  0.0004  0.0001–0.0015  1.7E04  3.9E03–6.2E04  MIM  13  5  0.808  0.0015  0.0010  2  0.000  0.0000  0.0000  0.0005  0.0001–0.0011  1.9E04  5.6E03–4.6E04  PAN  24  4  0.370  0.0002  0.0002  6  0.380  0.0013  0.0011  0.0005  0.0004–1.0401  2.1E04  1. 5E04–4.2E07  PLC  32  13  0.846  0.0035  0.0020  5  0.603  0.0014  0.0011  0.0013  0.0008–0.0018  5.1E04  3.4E04–7.5E04  PMS  30  15  0.784  0.0023  0.0013  3  0.381  0.0007  0.0007  0.0022  0.0003–0.0043  8.9E04  1.2E04–1.8E05  PNE  17  9  0.904  0.0022  0.0014  6  0.721  0.0024  0.0018  0.0036  0.0013–0.0099  1.5E05  5.3E04–4.0E05  POT  14  3  0.385  0.0001  0.0002  3  0.385  0.0007  0.0008  0.0002  0.0001–0.0007  1.0E04  2.5E03–2.7E04  PTU  18  8  0.860  0.0034  0.0020  6  0.576  0.0023  0.0017  0.0021  0.0007–0.0054  8.5E04  3.0E04–2.2E05  SAF  12  2  0.167  0.0003  0.0003  6  0.618  0.0022  0.0035  0.0007  0.0004–2.9817  2.8E04  1.6E04–1.2E08  SCA  25  14  0.813  0.0042  0.0024  8  0.446  0.0018  0.0014  0.0032  0.0019–0.0077  1.3E05  7.7E05–3.1E06  SDO  23  5  0.324  0.0002  0.0002  5  0.260  0.0037  0.0024  0.0010  0.0004–0.0019  4.2E04  1.8E04–7.9E04  SEB  21  5  0.338  0.0003  0.0003  8  0.692  0.0071  0.0042  0.0012  0.0007–0.7750  4.7E04  2.8E04–3.1E07  SEC  16  10  0.897  0.0012  0.0013  3  0.143  0.0018  0.0014  0.0008  0.0003–0.0020  3.1E04  1.2E04–7.9E04  SEL  31  7  0.574  0.0012  0.0008  4  0.295  0.0006  0.0007  0.0006  0.0003–0.0018  2.6E04  1.1E04–7.4E04  STZ  12  5  0.576  0.0011  0.0008  4  0.473  0.0009  0.0009  0.0004  0.0001–0.0010  1.7E04  5.8E03–4.2E04  SUM  19  5  0.672  0.0001  0.0002  2  0.000  0.0000  0.0000  0.0001  0.0000–0.0009  4.3E03  1.6E03–3.8E04  Mean  –  6.6  0.575  –  –  5.1  0.416  –  –  –  –  –  –  SD  –  3.8  0.275  –  –  2.8  0.280  –  –  –  –  –  –  Overall  468  106  0.927  0.0044  0.0024  78  0.637  0.0063  0.0036  0.0401  0.0337–0.0430  1.6E06  1.4E06–1.7E06  N, sample size; k,–number of haplotypes; h,–haplotype diversity; π,–nucleotide diversity; SD,–standard deviation; θ,–coalescent or mutation parameter; Ne,–effective population size; 95% CI, 95% credibility interval. View Large Figure 1. View large Download slide Geographical distribution of haplotypes of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Different colours were assigned for each haplotype according to the figure legend. The circle size represents the sample size in each population and the circle sections represent the haplotype frequency in each sampled population. For details on population codes and localities see Table S1 in Appendix S1. Figure 1. View large Download slide Geographical distribution of haplotypes of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Different colours were assigned for each haplotype according to the figure legend. The circle size represents the sample size in each population and the circle sections represent the haplotype frequency in each sampled population. For details on population codes and localities see Table S1 in Appendix S1. Genetic data To generate genetic data, we sequenced three intergenic spacers of chloroplast DNA (cpDNA): psbA-trnH, trnC-ycf6 and trnS-trnG (Shaw et al., 2005) and the region ITS1 + 5.8S + ITS2 (hereafter ITS) from nuclear ribosomal DNA (nrDNA) (Desfeux & Lejeune, 1996). PCR conditions and amplifications followed Collevatti et al. (2012a). PCR fragments were sequenced using the BigDye Terminator v3.1 kit (Applied Biosystems), and sequenced in the forward and reverse directions using a GS 3500 Genetic Analyzer (Applied Biosystems), according to the manufacter’s instructions. Sequences were analysed, and consensus sequences were obtained using the software SeqScape v2.7 (Applied Biosystems) and aligned with the software ClustalΩ (Sievers et al., 2011). Polymorphisms at mononucleotide microsatellites were excluded due to ambiguous alignment and higher mutation rates. Long indels (usually > 5 bp) were coded as one evolutionary event (one character). Genetic diversity and population structure We estimated nucleotide (π) and haplotype (h) diversity for each population and the populations overall following Nei (1987) using the software ArlequinVer 3.11 (Excoffier, Laval & Schneider, 2005a). To test for population differentiation, we performed an analysis of molecular variance (AMOVA, Excoffier, Smouse & Quattro, 1992) and estimated FST values. Analyses were performed using the software Arlequin v3.11 and statistical significance was tested via a non-parametric permutation test (10000 permutations). Population structure was also assessed using Bayesian clustering implemented in the software BAPS v6.0 (Corander & Marttinen, 2006; Corander et al., 2008). The cpDNA and ITS partitions were analysed separately. We performed population admixture analysis based on mixture clustering with ‘not fixed number of clusters (K)’ with an upper limit of K = 24. Phylogeographical reconstruction Population demography All coalescent analyses were performed with cpDNA and nrDNA ITS partitions concatenated, but giving separate priors. No evidence of heterozygous individuals was found when ITS sequences were analysed using SeqScape v2.7 (Applied Biosystems). Thus, recombination was neglected in all coalescent analyses. To set the priors, evolutionary model selection for both chloroplast and ITS regions was performed based on the Akaike Information Criterion implemented in the software jModelTest2 (Darriba et al., 2012). For chloroplast regions, the model HKY+G+I (−lnL = 3625.8751, wBIC = 0.5466) was selected, with gamma shape equal to 0.067, kappa equal to 1.95 and pinv = 0.404. For ITS, the evolutionary model TPM3uf+G (−lnL = 1755.8913, wBIC = 0.3426) was selected with gamma shape equal to 0.558. To study the dynamics in effective population size and genetic connectivity among populations, we estimated the mutation or coalescent parameter theta (θ = 2μNe for haploid genomes, θ = 4μNe for diploid genomes), and the immigration rate among all population pairs (M = 2Nem/θ for haploid genomes, M = 4Nem/θ for diploid genomes). Estimations were based on a Bayesian model using the Markov chain Monte Carlo (MCMC) approach implemented in Lamarc 2.1.10 software (Kuhner, 2006). Each analysis was run with 20 initial chains of 4000 steps and three final chains of 50000 steps. The chains were sampled every 100 steps. We used default settings for the initial estimate of theta. The program was run four times for each parameter to certify convergence among runs and validate the analyses using Tracer v1.6 (Rambaut & Drummond, 2007), and combined results were then generated using LogCombiner 1.8.2 (Drummond et al., 2012). Results were considered when the effective sample size (ESS) was ≥ 200 (and when marginal posterior probability densities were unimodal and converged among runs. Effective population size was obtained from θ (Kingman, 1982) using a generation time of 15 years based on flowering time measured in permanent plots established for demography dynamics studies (R. G. Collevatti, unpublished data). To understand changes in population size through time we performed a Coalescent Extended Bayesian Skyline Plot (EBSP) analysis (Heled & Drummond, 2008) implemented in BEAST 1.8.3 (Drummond & Rambaut, 2007), combining data from different partitions. We used the substitution models reported above and the relaxed molecular clock model (uncorrelated lognormal) for both chloroplast and ITS regions. The ucld.stdev parameter (standard deviation of the uncorrelated lognormal relaxed clock) and the coefficient of variation were inspected for among-branch rate heterogeneity within the data. In all runs ucld.stdev was greater than 1.25 and the coefficient of variation frequency histogram viewed in Tracer spanned zero (~1.6–2.5) showing heterogeneity among branches. Mutation rates for both chloroplast and ITS regions were the same as used for two taxonomically related species, Handroanthus impetiginosus (Collevatti et al., 2012a) and Tabebuia aurea (Collevatti et al., 2015a). Four independent analyses were run for 30 million generations. Convergence and stationarity were checked, and the independent runs were combined. Results were considered when ESS ≥ 200. We performed Fu’s FS tests of neutrality implemented in Arlequin 3.11. Negative FS values indicate an excess of rare alleles or new mutations in the genealogy resulting from either population expansion or selective sweeps. Lineage dispersal To reconstruct the spatio-temporal history of H. ochraceus lineage dispersal, we used the relaxed random walk model (RRW; Lemey et al., 2009, 2010) implemented in the software BEAST 1.8.3, which analyses molecular sequence evolution, the demographical model and lineage dispersal in space and time simultaneously (Lemey et al., 2010). We used the two sequence partitions with unlinked priors but sharing the same location rate matrix (Lemey et al., 2009, 2010). We used Bayesian Stochastic Search Variable Selection (BSSVS), which considers a limited number of rates (at least k − 1, k = 24 localities) to explain the phylogenetic diffusion process and a Symmetric Substitution Model that uses a standard continuous-time Markov chain (CTMC) in which the transition rates between locations are reversible. Priors for sequence evolution were the same as described above for EBSP. For tree priors, we used the Coalescent GMRF Bayesian Skyride model (Minin, Bloomquist & Suchard, 2008) and for location state rate the prior CTMC Rate Reference (Ferreira & Suchard, 2008). Four runs were performed with 30 million generations, stability was analysed using Tracer 1.6 and results were considered when ESS ≥ 200. The annotated tree (maximum clade credibility tree) was generated with 50% burnin and the spatio-temporal reconstruction was performed using SPREAD 1.0.6 (Bielejec et al., 2011). We also analysed the well-supported transition rates using the Bayes factor (BF) test implemented in SPREAD. Transition rates between localities were considered only for BF > 8.0. We ran the analysis for each sequence region, cpDNA and ITS, to detect different contributions by seed and pollen dispersal. Coalescent tree and time to most recent common ancestor We obtained a coalescent tree based on Bayesian coalescent analysis implemented in the software BEAST 1.8.3 to estimate time to the most recent common ancestor (TMRCA). For this analysis, we included the outgroup sequences of H. impetiginosus, H. serratifolius and C. antisyphillitica. We used the same priors of EBSP, except that for tree prior we used Coalescent Expansion Growth based on the results of EBSP (see Results below). To date the tree we used the same mutation rates used for EBSP, i.e. the same mutation used for the related species Handroanthus impetiginosus (Collevatti et al., 2012a) and Tabebuia aurea (Collevatti et al., 2015a). MCMC conditions and number of runs also remained unchanged. The independent runs were analysed using Tracer 1.6 and the results were considered when ESS ≥ 200. We also ran an empty alignment (sampling only from priors) to verify the sensitivity of the results to the given priors. The analysis showed that our data were informative because posterior values (e.g. posterior probability) were different from those obtained from empty alignment. Setting demographical scenarios Palaeodistribution modelling We obtained 888 occurrence records of H. ochraceus across the Neotropics from the online databases GBIF (Global Biodiversity Information Facility, http://www.gbif.org/) and Species Link (http://splink.cria.org.br/), which were mapped in a grid of cells of 0.5° × 0.5° (longitude × latitude) (Table S2, Fig. S1). The resolution of 0.5° × 0.5° was the same as for the climatic variables used in the ecological niche modelling (ENM) approach. The climatic layers were represented by five bioclimatic variables (annual mean temperature, mean diurnal temperature range, isothermality − mean diurnal range/temperature annual range, precipitation of wettest month and precipitation of driest month), selected by factorial analysis with Varimax rotation from 19 bioclimatic variables obtained from the ecoClimate database (www.ecoclimate.org;Lima-Ribeiro et al., 2015). We also obtained subsoil (30–100 cm) data related to soil fertility from the Harmonized World Soil Database (version 1.2, FAO/IIASA/ISRIC/ISS-CAS/JRC 2009, available at http://www.fao.org/docrep/018/aq361e/aq361e.pdf). Using factorial analysis we selected subsoil pH (30–100 cm). We assume subsoil pH to be constant between the LGM and pre-industrial age, which was used in ENMs as a ‘constraint variable’ to better model the environmental preferences of H. ochraceus. The bioclimatic variables were obtained for pre-industrial (representing current climate conditions), mid-Holocene (6 ka) and LGM (21 ka) from five coupled Atmosphere-Ocean General Circulation Models (AOGCMs) (Table S3). Estimates of the current and past potential distribution of H. ochraceus were obtained by modelling its ecological niche using 12 algorithms (Table S4). When needed, we randomly selected pseudo-absences across the Neotropical grid cells keeping prevalence equal to 0.5 (see Stokland, Halvorsen & Stoa, 2011). The ENMs were then built on the current climatic scenario and projected onto the climatic conditions during both the mid-Holocene (6 ka) and LGM (21 ka). All ENM procedures were run in the integrated computational platform BIOENSEMBLES, which provides predictions based on the ensemble approach (see Araújo & New, 2007; Diniz-Filho et al., 2009). For each algorithm and AOGCM, models were built using 75% of occurrences and tested against the remaining records (25%). To avoid modelling bias, this procedure was repeated 50 times for each combination of ENM and AOGCM by randomly splitting training (75%) and test data (25%). After eliminating the models showing poor performance [True Skill Statistics (TSS) < 0.5, Allouche Tsoar & Kadmon, 2006, see Table S5], we computed the binary maps using thresholds established by the area under the receiver operating characteristic (ROC) curve (maximizing specificity + sensitivity). The frequency of a predicted presence was used as a measure of consensus suitability from 50 initial models. Next, we applied a hierarchical ANOVA using the predicted suitability from all models (12 ENMs × 5 AOGCMs × 3 Times) as the response variable to disentangle the effects of climate change on species distribution through the time from predictive uncertainties in the potential distribution due to modelling components (i.e. ENMs, AOGCMs). For this, the ENM and AOGCM components were nested into the time component, but crossed by a two-way factorial design within each time period (see Terribile et al., 2012 for details about hierarchical design). Because we do not have repetitions, the residual term from ANOVA represents the interaction between AOGCM and ENM (AOGCM × ENM; Sokal & Rohlf, 1995). Finally, the full ensemble was obtained for each time period using the predictive performances (TSS) to compute a weighted mean of suitabilities (Table S5), from which historical refugia were mapped (i.e. all grid cells with suitability values ≥ 0.5 during the three time periods). The threshold of 0.5 corresponds to the suitability value after excluding the lowest 10th percentile of suitability values related to the species occurrence records. We performed sensitivity analyses using thresholds of 0.4, 0.6 and 0.7, corresponding to the 5th, 12th and 17th percentiles, respectively, and the results were virtually indistinguishable. The result differed only for a threshold > 0.7, resulting in a small refugium in Central–West Brazil. Demographical scenarios The ENMs resulted in 60 predictive maps (12 ENMs × 5 AOGCMs) for each time period, which were used to support alternative demographical scenarios. By keeping the ENM and AOGCM constant across time periods, we computed the range shift across the last glacial cycle (i.e. the difference in predicted range size between the present and LGM – 21 ka), and classified the 60 predictive maps according to three general demographical scenarios using a threshold difference of ± 200 cells: (1) ‘Range Stability’, no difference in range size through time; (2) ‘Range Expansion’, range size was smaller at the LGM than at present; and (3) ‘Range Retraction’, range size was larger at the LGM than at present. Along with the three hypotheses supported by ENMs (see above), a fourth biogeographical hypothesis of ‘Multiple Refugia’ was derived from palaeoclimatic (e.g. Ab’Saber, 2000) and palaeovegetation reconstructions (e.g. Salgado-Labouriau, 1997). Simulation of demographical scenarios The demographical scenarios were simulated based on coalescent analysis (Kingman, 1982) implemented in DIYABC v2.1.0 (Cornuet et al., 2008; Cornuet, Ravigne & Estoup, 2010), following the framework described by Collevatti et al. (2012a, 2013a, 2015b). For model calibration, we used the demographical parameters estimated with Lamarc software and the same priors for sequence evolution used in Bayesian analyses (see above). The number of generations until the LGM (21 ka) was calculated using the generation time of H. ochraceus (15 years, R. G. Collevatti, unpublished data). Population dynamics were simulated backwards, with 24 demes, from t0 (present) to t1400 generations ago (at 21 ka). At t0, all demes had the same population size N0 = 100000 and reached N1, the effective population size at t1400 (21 ka), according to our theoretical expectation for each demographical scenario (see Figure 2 for details). Given the high variation in H. ochraceus effective population sizes (Table 1), we performed simulations with different initial deme sizes, N0 = 100, 1000, 10, 000 and 100000 for all scenarios. However, we detected differences among scenarios only when N0 = 100000. The demographical scenario predicted by ‘Range Expansion’ was then simulated by applying an exponentially negative population growth from the present to 21 ka, reaching N1400 = 10000, if N0 = 100000. By contrast, the ‘Range Retraction’ scenario was simulated applying exponentially positive population growth during the same period, reaching N1400 = 1000000, if N0 = 100000. The scenario of ‘Range Stability’ predicted no change in effective population size through time, i.e. N1400 = 100000 if N0 = 100000. The ‘Multiple Refugia’ scenario considers that savannas shifted in range and retracted during the LGM, forming fragments of different sizes (Ab’Saber, 2000). Thus, to model ‘Multiple Refugia’, we considered that current populations (at t0) are descendant from lineages in different demes at t1400 generations ago (LGM) with different effective population sizes: N1400 = 1000000, 100000, 10000, 1000, 100 or 10. Migration was simulated considering all current deme descendants from lineages originally in deme 1 at t generations ago, meaning that while the coalescent tree builds back through time, there is a 0.01 per generation chance that each lineage in deme x will migrate to deme 1. We also simulated different migration rates. Values < 0.01 were not sufficient to show any demographical variation at the time scale we are working and values > 0.1 retrieved equal likelihoods for all models. Figure 2. View largeDownload slide Demographical scenarios simulated for Handroanthus ochraceus using the software DIYABC 2.1.0 and their geographical representation. The simulations were performed for 24 demes. LIG, last interglacial; LGM, last glacial maximum; Pres, present day; N0, effective population size at time t0 (present); N1, effective population size at time t1400 (1400 generations ago). td is the time to most recent common ancestor of all lineages. Figure 2. View largeDownload slide Demographical scenarios simulated for Handroanthus ochraceus using the software DIYABC 2.1.0 and their geographical representation. The simulations were performed for 24 demes. LIG, last interglacial; LGM, last glacial maximum; Pres, present day; N0, effective population size at time t0 (present); N1, effective population size at time t1400 (1400 generations ago). td is the time to most recent common ancestor of all lineages. The relative fits of the models were calculated using approximate Bayesian computation (ABC; Excoffier, Estoup & Cornuet, 2005b; Beaumont, 2008) implemented in the software DIYABC v2.1.0. The number of distinct haplotypes, number of segregating sites, mean and variance of the pairwise differences, and mean and variance of the number of the rarest nucleotide of segregating sites, retrieved across 4 million simulations, were compared with those of the observed data sets, for cpDNA and ITS separately. In the software DYABC, the posterior probability of each scenario was retrieved to compare different scenarios that can explain the observed data set. We used the relative proportion of each scenario in the simulated data set closest to the observed data set (hereafter ‘direct approach’) and the logistic regression (hereafter ‘logistic approach’) of each scenario probability on the deviation between simulated and observed summary statistics (Cornuet et al., 2008; see also the DIYABC website, http://www1.montpellier.inra.fr/CBGP/diyabc/). Spatial patterns in genetic diversity We used spatially explicit analyses to detect spatial patterns in observed genetic diversity in response to late Quaternary climate oscillations, for both cpDNA and ITS. We first tested whether genetic differentiation across H. ochraceus populations (linearized FST) correlates with their geographical distance (on a log scale) using the Mantel test. The analysis was performed using the software Arlequin v3.11 and statistical significance was tested across 10000 random permutations. Next, we used quantile regressions to analyse the relationships of climatic suitability and stability through time with genetic diversity (Cade & Noon, 2003). For this, we calculated the difference of ensemble suitability between the LGM and the present day as a measure of climate stability through time. We then analysed whether historical changes in the geographical range of a species generated a cline spatial pattern in haplotype (h) and nucleotide (π) diversities, as well as in effective population size (Ne), due to expansion of climatically suitable conditions. We obtained the distance between each sampled population and the centroid of historical refugium and of the predicted distribution at 21 ka and the present day, and then performed quantile regressions of genetic parameters against these spatial distances and against climatic stability. RESULTS Genetic diversity and population structure The combined data of chloroplast intergenic spacers generated 2133 bp and the nrDNA ITS generated 610 bp, resulting in 2299 bp when microsatellites and ambiguous alignments were eliminated (1772 bp for chloroplast and 527 for ITS). We found 106 different chloroplast and 78 ITS haplotypes for the 468 individuals of H. ochraceus, respectively (Fig. 1). Some populations showed low haplotype and nucleotide diversities (Table 1), despite the high number of individuals sampled. Most haplotypes were exclusive of one population (Fig. 1, see also Table S6) and two chloroplast and ITS haplotypes were very widespread, H1 and H5 (Fig. 1). The AMOVA showed significant genetic differentiation among populations for both cpDNA (FST = 0.742; P < 0.001) and nuclear ITS (FST = 0.544; P < 0.001). Bayesian clustering showed an optimal partition of four clusters for both ITS and cpDNA (Fig. 3), but with no clear geographical pattern. For ITS (Fig. 3A), almost all populations were clustered together in one group (cluster I, in green), but individuals from populations ARA and SEB from Northwest, SCA from Southeast, and PAN and BAR from Central-West were also grouped in cluster II (red). The chloroplast genome (Fig. 3B) showed a higher admixture, with most populations grouped in cluster III (light green), but with admixture with cluster I (blue) and cluster IV (orange). Populations PLC and PMS formed a different cluster for both ITS (cluster IV, blue) and chloroplast data (cluster II, red). Overall, we found high genetic differentiation among populations but with high admixture indicating historical connectivity. Figure 3. View large Download slide Geographical distribution of Bayesian clustering of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Each colour represents an inferred cluster (four clusters for ITS and cpDNA), also represented in the bar graphs below the maps. In the bar graphs, each individual bar represents an individual plant and colour indicates the cluster. The dark grey area represents the limits of the Brazilian Cerrado Biome. For details on population codes and localities see Table S1 in Appendix S1. Figure 3. View large Download slide Geographical distribution of Bayesian clustering of Handroanthus ochraceus for (A) ITS and (B) cpDNA, based on the sequencing of 468 individuals from 24 populations from the Brazilian savanna. Each colour represents an inferred cluster (four clusters for ITS and cpDNA), also represented in the bar graphs below the maps. In the bar graphs, each individual bar represents an individual plant and colour indicates the cluster. The dark grey area represents the limits of the Brazilian Cerrado Biome. For details on population codes and localities see Table S1 in Appendix S1. Phylogeographical reconstruction Population demography We found low values of θ for each population and for the populations as a whole (Table 1), but high effective population sizes. However, migration was negligible (less than one migrant per generation) between all pair of populations (Tables S7 and S8). The EBSP showed population demographical expansion (Fig. S2a) at c. 380 ka, with lower growth rates after c. 150 ka. Fu’s FS test also showed significant signal of population expansion following a retraction for both ITS (FS = −25.520, P = 0.0001) and cpDNA (FS = −24.848, P = 0.0002). However, the negative FS values potentially indicate selective sweeps. Overall, coalescent analyses showed demographical expansion through time and high effective population sizes. Lineage dispersal We recovered one most probable ancestral location for H. ochraceus haplotypes (Fig. S2b) in population SCA in Southeast Brazil. Lineages started to disperse at c. 520 ka (Fig. S2b) to populations at the eastern (PLC and PAN), central (MIM and AGE) and western Cerrado (POT and FAZ). Most dispersal events (28 of 41) occurred in the Middle Pleistocene, after c. 400 ka, matching the demographical expansion revealed by EBSP (Figure 4). In addition, most dispersal events occurred during interglacial periods (47 of 71 dispersal events). The Bayes factor analysis showed that all links among localities are well supported (all BF > 8.0). The mean dispersal rate was 1.01 km/year [SD = 0.939, 95% highest posterior density interval 0.019–2.946 km/year]. The RRW analyses revealed historical connectivity among populations that coincides with the timing of population demographical expansion. Figure 4. View large Download slide Spatio-temporal dynamics of Handroanthus ochraceus lineage diffusion among the 24 populations sampled in the Brazilian savanna, for 1.350 Ma, 900 ka, 650 ka and 300 ka until the present day. Arrows between locations represent branches in the tree along which the relevant location transition occurs. The map was adapted from the .kml file provided by SPREAD software generated using Google Earth (http://earth.google.com). The δ18O curve corresponds to the composite benthic stable oxygen isotope ratios (Lisiecki and Raymo, 2005). For details on population codes and localities see Table S1 in Appendix S1 and Figure 1. Figure 4. View large Download slide Spatio-temporal dynamics of Handroanthus ochraceus lineage diffusion among the 24 populations sampled in the Brazilian savanna, for 1.350 Ma, 900 ka, 650 ka and 300 ka until the present day. Arrows between locations represent branches in the tree along which the relevant location transition occurs. The map was adapted from the .kml file provided by SPREAD software generated using Google Earth (http://earth.google.com). The δ18O curve corresponds to the composite benthic stable oxygen isotope ratios (Lisiecki and Raymo, 2005). For details on population codes and localities see Table S1 in Appendix S1 and Figure 1. Time to most recent common ancestor Haplotypes of H. ochraceus showed a recent TMRCA (Fig. 5), c. 1.9 Ma [95% credibility interval (CI) = 0.1–2.3 Ma], which coincides with the coalescence of haplotypes from populations PLC and PMS with all other haplotypes. Most divergences occurred after c. 1.1 Ma (Fig. 5) and geographically distant populations (e.g. SEC and BOD) shared haplotypes and common ancestors. Figure 5. View largeDownload slide Relationships and TMRCA of Handroanthus ochraceus lineages. The light pink bar corresponds to the 95% highest posterior probability of the median time to the common ancestor; numbers above branches are support values for the nodes (posterior probability) and numbers below are the TMRCA. The time scale is in millions of years ago (Ma). Figure 5. View largeDownload slide Relationships and TMRCA of Handroanthus ochraceus lineages. The light pink bar corresponds to the 95% highest posterior probability of the median time to the common ancestor; numbers above branches are support values for the nodes (posterior probability) and numbers below are the TMRCA. The time scale is in millions of years ago (Ma). Setting demographical scenarios Palaeodistribution modelling Handroanthus ochraceus shows a clear preference for hot and dry climates (Fig. S3), matching the general current conditions of Neotropical savannas. Such climate space was equally available at the LGM, mid-Holocene and the present day (Fig. 6A). ENMs predicted only a slightly difference in the potential distribution of H. ochraceus through time (Fig. 6, see also Fig. S4), with a slightly higher range size at the LGM (Fig. 6A) compared to both the mid-Holocene (Fig. 6B) and the present day (Fig. 6C). In addition, a wide region across Central–West and Northeast Brazil probably acted as an historical refugium maintaining populations of H. ochraceus during the climate changes of the last glaciation (Fig. 6D). Overall, the ENMs showed quasi-stability in the geographical range of H. ochraceus through time. Figure 6. View largeDownload slide Consensus maps of the 60 models expressing the potential distribution for Handroanthus ochraceus, based on ecological niche modelling during the (A) LGM (21 ka), (B) mid-Holocene (6 ka) and (C) present day, and (D) for historical refugium through time (from the LGM to the present day). Black dots indicate the 24 populations sampled. Figure 6. View largeDownload slide Consensus maps of the 60 models expressing the potential distribution for Handroanthus ochraceus, based on ecological niche modelling during the (A) LGM (21 ka), (B) mid-Holocene (6 ka) and (C) present day, and (D) for historical refugium through time (from the LGM to the present day). Black dots indicate the 24 populations sampled. The models were well evaluated from TSS (Table S5). Analysis of uncertainty using a hierarchical ANOVA showed high proportional variance from the modelling method (ENMs) and AOGCM (Table S9, Fig. S5), but variation was spatially structure and higher outside the occurrence range of H. ochraceus, indicating that the ENMs were able to detect the effects of climate changes on the distribution dynamics of H. ochraceus through the last glaciation, despite the AOGCM variation. Demographical scenarios and simulations The scenario of ‘Range Stability’ was the most frequent hypothesis from ENM predictions (45%, see Table S10) followed by ‘Range Retraction’ (40%) and ‘Range Expansion’ (15.0%). Simulations of demographical scenarios showed only a slight difference using a direct approach (Fig. S6) for both cpDNA (Fig. S6a) and ITS (Fig. S6c). However, using a logistic approach, scenario 4 (Multiple Refugia) was the most likely one to predict the observed parameters for cpDNA (posterior probability = 1.00, 95% CI = 1.00–1.0, Fig. S6b) and scenario 1 (Range Expansion) was the most likely one for ITS (posterior probability = 0.91, 95% CI = 0.84–0.97, Fig. S6d, see also Table S11 for posterior probabilities). The simulations thus showed that H. ochraceus had a smaller range size at the LGM than at the present day for both molecular markers. Spatial patterns in genetic diversity Pairwise differentiation was high for almost all population pairs (Table S12), but was not correlated with geographical distance for either chloroplast (r2 = 0.078, P = 0.180) or ITS (r2 = 0.054, P = 0.670). Areas with higher climatic instability through time (higher delta suitability 0–21 ka) showed lower nucleotide and haplotype diversity (Fig. S7). In addition, populations at greater distances from the centroid of the climatic refugium showed higher nucleotide and haplotype diversities (Fig. S8). Effective population size was not significantly related to the distance from the centroid or to climatic suitability. DISCUSSION Our results show that H. ochraceus lineages dispersed cyclically from Southeast and West towards Central–West Brazil. Most dispersal events occurred from populations at the edges of the Cerrado biome towards Central Brazil. Populations at the edge of the historical refugium showed higher genetic diversity and were the source of migrants to populations at the centre of the refugium. The palaeodistribution dynamics showed a large historical refugium throughout Central Brazil and range stability through time. The slight difference in range size through the last glacial cycle is due mainly to range expansion towards the Amazon Basin at the LGM. All sampled populations remained in environmentally suitable areas through the last glacial cycle, inside the large climatic refugium, which may have favoured high historical connectivity among populations. In fact, the climatic niche space of H. ochraceus indicates its preference for hot and dry conditions that prevailed during the last glacial cycle in its geographical range. The lack of dispersal of new lineages after c. 130 ka, the low number of migrants per generation (Nm < 1.0) and the high number of haplotypes with restricted geographical distribution show that, despite the high historical connectivity, recent connectivity is low. The lack of recent connectivity may be due to widespread fragmentation due to anthropogenic disturbance. Although RRW predicts spatial displacements occurring earlier than ENMs, they both converge to consistent patterns, showing that our approach is reliable in reconstructing the phylogeography of H. ochraceus. The dispersal results also indicated that populations currently found in Northern and Northeastern Brazil received migrants mainly from the Southeast (e.g. see arrow direction in Fig. 4). Such dispersal routes have probably occurred because suitable climatic conditions were available across a wider region during warming phases, allowing the connectivity of populations. Diffusion models suggested that inverse dynamics occurred during cooling phases (e.g. 200 and 260 ka), when lineages dispersed in the opposite direction, from North-Northeast toward Central and Central-West Brazil. This region was hotter and drier, thus retaining more suitable niches for H. ochraceus during the cooler phases. A potential continuous historical refugium across Neotropical savannas may be the key factor allowing uninterrupted lineage dispersal among populations of Central Brazil, as predicted by the RRW model. Note that a similar result was reported for a related species, Tabebuia aurea (Collevatti et al., 2015a), reinforcing the idea that dispersal from Southeast to Northeast might have been an important route for savanna plant species in the Quaternary. In addition, population SCA was also one of the ancestral localities of T. aurea lineages (together with CAC, see Collevatti et al., 2015a). However, the dispersal of lineages present in a population would not be detected as a new gene flow event. Thus, population connectivity could be maintained after 130 ka but our results show no dispersal of new lineages. Despite the difference in demographical scenarios between cpDNA (Multiple Refugia) and ITS (Range Expansion), both molecular markers predicted population expansion through time (i.e. from the LGM to present day) as the most likely general demographical scenario for H. ochraceus, matching our theoretical prediction. The difference between the two molecular markers is probably due to evolutionary rates. Besides substitution rates, which are higher in ITS than in chloroplast intergenic regions, and concerted evolution in nrDNA that may decrease genetic variation, ITS has four times the effective size of the chloroplast genome and may be differentially affected by drift. However, ENMs revealed quasi-stability in geographical range, with a slightly larger range at the LGM compared to the present day. Note that H. ochraceus showed different geographical range dynamics through time compared to other taxonomically related species such as T. aurea (Collevatti et al. 2015a) and T. roseoalba (Melo et al., 2016) that showed range retraction during the LGM. Moreover, H. ochraceus has a more recent common ancestor (c. 1.9 Ma) than other related species, such as T. aurea (4.0 ± 2.5 Ma, Collevatti et al. 2015a), T. roseoalba (4.9 ± 1.9 Ma, Melo et al., 2016) and H. impetiginosus (4.7 ± 1.1 Ma, Collevatti et al., 2012a), probably due to differences in demographical history. Because favourable climatic conditions for H. ochraceus (i.e. hotter and drier) were more widely available during glacial than interglacial periods in the Neotropics, effective population size has increased in the last c. 380 ka, despite glacial cycles. Besides, the increasing number of lineages, especially in the last c. 500 ka, may have resulted from the demographical expansions across cyclical glacial periods, during which wider areas of suitability were available for colonization. Lineage dispersal was mainly from populations SCA and PAN (see Fig. 4). This diffusion pattern explains the widespread geographical distribution of some haplotypes (e.g. H1 and H5 for ITS, and H1 and H5 for cpDNA, see Fig. 1) and the restricted geographical distribution of other haplotypes that diverged more recently (e.g. H76, H77 and H78 for ITS, and H104, H105 and H106 for cpDNA, see Fig. 1), leading to high population differentiation. The analyses also indicated in situ diversification of haplotypes in many populations, such as PLC, PMS, PNE, SEB, SEC, SEL, STZ, SUM (see Fig. S2b), and a higher diversification after c. 200 ka, which coincides with the demographical population growth. The lack of dispersal events after c. 130 ka, also indicated by coalescent analyses in Lamarc software, may have led to the restricted geographical distribution of many haplotypes. Although our findings show possible long-distance dispersal between distant populations such as SCA and SEC and SEC and CAR, it is important to note that these events are probably due to stepping-stone dispersal and would be detected only with continuous spatial sampling. However, continuous large-scale sampling is hindered by the fragmentation of savanna in Brazil. Besides, mean rate of dispersal estimates of 1.01 km/year using RRW is congruent with the findings for the related species T. aurea (1.17 km/yr, Collevatti et al., 2015a). Handroanthus ochraceus and T. aurea share the same pollinator species and seed dispersal syndrome, and they co-occur in many localities. Thus, we expect that long-distance dispersal be due mainly to pollen dispersal (see Braga & Collevatti, 2011), which may also explain the widespread distribution of ITS haplotypes. Despite the demographical expansions through time, ENMs suggested that all populations are within the historical refugium, i.e. areas of high climatic suitability throughout the study period (see Fig. 6D), favouring population persistence and genetic connectivity. If this stable area was maintained during the recurrent glaciations of the Quaternary, we can predict long-term genetic connectivity among H. ochraceus populations, which is also suggested by a high density of well-supported links among populations inside this historical refugium. Thus, the low haplotype diversity within some populations may be an effect of selective sweeps, as indicated by the negative FS values (e.g. Kapralov & Filatov, 2007). In addition, despite differences in effective population size and inheritance, the chloroplast genome showed higher diversity than ITS nrDNA, although the latter has four times the effective size. This may be due to concerted evolution in nrDNA that may decrease genetic variation (Ohta, 1984). Moreover, recent anthropogenic disturbance may also have caused bottlenecks, decreasing genetic diversity in H. ochraceus populations because some populations, such as BAG, POT, CAR, which are isolated remnants in pasture or crop plantations, have low genetic diversity. We believe that our results are not an artefact of sampling effort because populations with larger sample sizes had the same number of haplotypes as populations with smaller sample sizes. The pattern of lineage spread and extinction may explain the low genetic diversity in many populations, even in the centre of the species’ geographical range. In fact, populations in more unstable areas had lower genetic diversity and populations further from the centroid, at the edge of the climatic refugium, showed higher genetic diversity. This reinforces the multiple refugia demographical scenario. We hypothesize that these populations, at the edge of historical refugia, may represent ‘stable rear edges’, i.e. relict populations that have persisted in suitable local habitats across the Quaternary glaciation cycles (see Petit et al., 2003; Hampe & Petit, 2005). Note that due to the coarse resolution of climatic layers (0.5° × 0.5°) the ENM may not be able to predict species presence on the local scale (small refugia). Finally, the scenario of demographical expansion through time for H. ochraceus was also supported for other Brazilian savanna species such as Tabebuia aurea (Collevatti et al., 2015a), Caryocar brasiliense (Collevatti et al., 2012b) and Dipteryx alata (Collevatti et al., 2013b) and for a Brazilian savanna swamp palm species, Mauritia flexuosa (Lima et al., 2014). Phylogeographical studies of savanna species (e.g. Ramos et al., 2007; Novaes et al., 2010, 2013; Collevatti et al., 2012b) have shown high levels of genetic differentiation among populations and evidence of colonization of Southeastern Brazilian savannas during warmer interglacial periods of the Quaternary. However, our results for H. ochraceus and T. aurea (Collevatti et al. 2015a) do not support that populations from Southeast Brazil were recently colonized because population SCA, in Southeast Brazil, was the ancestral locality for extant lineages of the two species and had old lineages. Our results also match the spatial pattern in genetic diversity for other savanna tree species, such as C. brasiliense (Collevatti et al., 2012b) and T. aurea (Collevatti et al., 2015a), and reinforces the idea that relict populations may have persisted in suitable local habitats across the Quaternary glaciation cycles. Nevertheless, there is high variation in spatial patterns of genetic diversity in savanna trees, and some species show a central–peripheral pattern, such as Eugenia dysenterica (Lima et al., 2017) and Dimorphandra mollis (Souza et al., 2017). In conclusion, our findings suggest that the pattern of genetic diversity in H. ochraceus may be the result of range stability and population demographical expansion through the Quaternary. The general dynamics of lineage dispersal followed suitable climatic conditions, towards the Northern and Central–West regions during warming periods in the early Quaternary, and an opposite direction during glacial phases. The same pattern was also found for a taxonomically related species, T. aurea (Collevatti et al. 2015a). We also provide evidence of a general pattern of distribution dynamics and lineage dispersal in savanna tree species and emphasize that the south-eastern and western Cerrado were important refugia for savanna species. Our study also shows that our multi-model inference approach allows us to infer the different processes affecting phylogeographical patterns through time. ACKNOWLEDGMENTS This work was supported by grants to the research network GENPAC (Geographical Genetics and Regional Planning for natural resources in Brazilian Cerrado) supported by CNPq/MCT/CAPES/FAPEG (project nos. 564717/2010-0, 563727/2010-1 and 563624/2010-8), and Rede Cerrado CNPq/PPBio (project no. 457406/2012-7). We thank Thiago F. Rangel for providing access to the computational platform BIOENSEMBLES and the World Climate Research Programmer’s Working Group on Coupled Modelling (Table S3) for making available their model outputs. We also thank three anonymous reviewers for their helpful comments. SUPPORTING INFORMATION Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Appendix S1. 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Biological Journal of the Linnean SocietyOxford University Press

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