Geographic distribution of Dalbergia miscolobium (dots) across the Brazilian Cerrado biome (grey) in Eastern Tropical South America. Each dot represents a record of D. miscolobium and each red asterisk a sampled population. The geographic distribution of D. miscolobium was estimated through field trips, herbarium records and databases of floristic checklists. The six phytogeographical provinces proposed by Ratter et al. [11] have been delimited: ‘S’, Southern, ‘C & SE’, Central and southeastern, ‘N & NE’, North and northeastern, ‘CW’, Central-western, ‘FWM’, Far western mesotrophic sites and ‘DA’, Disjunct Amazonian. The codes of the provinces are the same of the original publication. doi:10.1371/journal.pone.0082198.g001 

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... 3. Results from population structure analyses of Dalbegia miscolobium populations (A to E) and the phytogeographical provinces proposed by Ratter et al. [11] that are superimposed to our sampling area (F). In each panel is represented a result of a different analysis: A) Bayesian Analysis of Population Structure (BAPS) with cpDNA only; B) BAPS with nrDNA only; C) BAPS with cpDNA and nrDNA concatenated; D) BAPS with cpDNA and nrDNA concatenated with a prior of k = 5; E) Analysis with the Monmonier’s algorithm implemented in Barrier 2.2. In panels A to E, the polygons are the result of Voronoi tessellation and each one is correspondent to a population whose code is the same as in Table 1. Polygons with the same color belong to the same BAPS cluster. In panel E, the red lines correspond to the consensus barriers among the cpDNA and nrDNA distance matrices. The barriers are numbered in order of their appearance which is related to their probability of existence. The larger abbreviations in panels C and D are for the phylogeographic groups as detailed in Results and Table 2. In panel F, the black traces are for the phytogeographical provinces of Ratter et al. [11] and the blue trace is correspondent to Group 3 from their Twinspan analysis. The codes of the provinces are the same of the original publication. For names of provinces see Figure 1 and for further details about them, refer to the text. doi:10.1371/journal.pone.0082198.g003  ...

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... for the Cerrado to date. Remarkably, the observed phylogeographic structure shows a clear similarity to the Cerrado phytogeographical provinces proposed by Ratter et al. [11] which have been delimited based on a comprehensive sampling effort in the biome. Based on Twinspan and consensus analyses, Ratter et al . proposed five and four provinces respectively [11], when considering only those that are superimposed to our sampling area ( Figure 3) and our data strongly support them. The main difference between Ratter’s four consensus provinces and five Twinspan provinces is the status of province ‘C & SE’ (Figure 1), which is a single province in the consensus analysis but is subdivided into two in the Twinspan analysis, named 2 and 3 (Figure 3D, F) groups. In this region our genetic data also allowed the identification of two phylogeographical groups, CC and CE1 + 2, which correspond to groups 3 and 2, respectively in the Twinspan analysis. Therefore, the observed structure of D. miscolobium is in accordance to the Cerrado phytogeographical patterns observed by Ratter et al. [11]. Some other longstanding phytogeographical hypotheses also are corroborated by our data. Several different authors have proposed Southern Cerrados and Northeastern Cerrados as completely different units from the remaining Cerrado [10,11,63,65,66], and this pattern was also observed in our data, although a wider sampling of Northeastern Cerrados would be desirable to confirm its characteristics found by our study. In some degree, these patterns were also observed by two other phylogeographical studies [21,22]. With regard to the separation between Eastern and Western Central Cerrados, not only the phytogeographical provinces [9–11] but also phylogeographic studies have showed it [20,22]. Finally, another striking phylogeographic pattern we observed is the consistent separation into two groups of the populations in the southern limits of the Cerrado (below 20 u S). They can consistently be separated into an eastern group (our SC group) and a western group (part of the CW2 group; Figure 2C, D), a pattern that have been reported by Durigan et al. [67] based on floristic data. Such ‘genealogical concordances’ [17,68] between phylogeographic and phytogeographical patterns and provinces have been found in other regions of the globe (e.g. [18,69,70]), but this is the first time it is explicitly shown in the Cerrado biome. Given the relatively recent diversification of D. miscolobium , estimated at the Pliocene/Pleistocene (see results), these ‘genealogical concordances’ suggest that a shared and persistent pattern of species diversification might have been present on the Cerrado over time. Ecological as well as historical biogeographic factors have acted collectively in shaping species diversification and distribution in the biome and the relative importance of each of these factors has been the object of considerable debate [3,4,71–73]. Castro and Martins [10] and Ratter et al. [11] proposed that climate (mainly duration of the dry season and mean temperature), soil fertility and drainage are the main factors responsible for the subdivisions of the Cerrado in phytogeographical provinces observed, with variation in altitude and past climatic changes also playing a part in the observed diversification. In a recent study, Simon et al . [73], by analyzing time-calibrated phylogenies and plant adaptations, highlighted the importance of fire and the emergence of C4 flammable grasses to the diversification of the Cerrado flora, as has also been pointed out by others [6]. For Southern Cerrados, the duration of the dry season and the contact with other vegetation types, as the Atlantic Forest, were hypothesized as probable determinant factor in differentiating eastern and western [67]. Studies based on molecular phylogenies have pointed that the diversification on the neotropical region resulted from many evolutionary forces acting in different spatial and temporal scales, which include the climatic changes of Quaternary, Neogene tectonic events and paleogeographical reorganizations [3,12,74– 76]. None of these hypotheses can be discarded, and, indeed, the complex interaction among these factors across the Cerrado appears to have determined the occurrence of the biome itself [72], as well as the phytogeographical and phylogeographical patterns that occur within it. But our results suggest that some of those driving forces have persisted over time, and lead to the recent intra-specific diversification of D. miscolobium in a similar way to the events of speciation that are behind the current phytogeographic patterns. Future study on genetic, morphological, and physiological diversity of widespread Cerrado species might provide the basis for better understanding the contribution of each of these factors to the observed patterns of diversification. The pattern of cpDNA diversity of D. miscolobium in the southern Cerrado (SC group) is also very similar to that reported for three other tree species studied in the Cerrado, Caryocar brasiliense [19], Hymenaea stigonocarpa [22] and Plathymenia reticulata [21]. In this region, D. miscolobium presents populations with low genetic diversity, probably descent from northernmost sources, a star-like network and mismatch distributions not significantly different from the demographic and expansion model distribution. Together, these patterns point to a recent range expansion [77] of the species to southern Cerrados, from northernmost sources. The retraction of the Cerrado vegetation in the present-day southern portion of Cerrado during the colder and drier periods of the LGM has been proposed by paleoenvironmental studies [14,78] and it was one of the least probable regions of the Cerrado to persist during the LGM, according to a modeling study [79]. The subtropical SDTF [2,80] and/or grassland species [14,81] from the southernmost regions may have expanded northwards during the LGM, replacing the Cerrado species. Subsequent climate amelioration [15] may have allowed the range expansion of the Cerrado species southwards again. Thus, even though, among the Cerrado species, D. miscolobium seems to better tolerate lower temperatures, it seems to have been similarly affected by the extremely arid and cold conditions of the LGM. It is suggestive that maybe, the entirely Cerrado biota, including its more cold- tolerant species, were unable to resist to the coldest conditions of LGM times in the southern portion of the Cerrado. This same pattern was not observed for nrDNA data, whose variation, in SC group, has contrasting patterns, that is, high genetic diversity and genetic uniqueness. Rapid expansion of those populations coupled with the high rate of mutation of nrDNA, nrDNA paralogue evolution, seed/pollen bias due to cpDNA gene flow only by seeds and even hybridization/introgression are among the factors that could have been responsible for those results [29,82]. Analyses with more markers with different evolutionary rates could help to reveal the causes of these differences between cpDNA and nrDNA diversity levels in southern Cerrado. In this regard, next- generation sequencing to produce maps of model species of Leguminosae could help to find useful markers in D. miscolobium . The heterogeneous distribution of genetic diversity of D. miscolobium is in accordance to the commonly reported heterogeneity of Cerrado biome in terms of species distribution [3,8,11,23]. Also, the high level of differentiation among populations observed in D. miscolobium has been reported to three other species [19,21,22]. Together with the widespread occurrence of exclusive haplotypes, these results reinforce the importance to treat the different Cerrado provinces as different biodiversity assemblages. Among these, two centres of genetic diversity deserve some attention: the CW2 and the CE1 + CE2 regions. The high diversity of CE1 + CE2 regions have been reported in the plant phylogeographic studies of Hymenaea stigonocarpa [22] and Plathymenia reticulata [21] as well as in animal [3,83,84] and plant [85] endemism studies. CE1 + CE2 regions are also relatively close to the recently proposed principal Cerrado refugium [79]. The spatial heterogeneity and/or climate stability of this region, especially in its northern portion, could have helped to give rise to and maintain this diversity. Together with the CW1 + CW2 groups, the CE1 + CE2 were the only groupings of populations that did not show mismatch distributions of sequences in accordance with a recent population expansion for both cpDNA and nrDNA, in a sign that they might have been more stable than the other groups (Table 3). Based on that, we reinforce the important role that the central region of the Cerrado could have in maintaining the genetic diversity of widespread Cerrado species. With regard to CW2 group, this is the first time a phylogeographic study has performed a wide sampling in the south-western portion of the Cerrado and we have found unexpected high levels of genetic diversity and also signs of population stability on it. This region is currently under considerable economic growth and therefore, research should focus on it in order to aid conservation efforts and its sustainable exploitation. These many patterns found rise novel questions for future consideration. Do these biogeographic patterns persist in other widespread Cerrado species? What would have occurred to species endemic to these provinces, especially those found commonly in the southern Cerrado? How did the different ecological and historical factors lead to the diversification of the Cerrado biota? Phylogeography of the Cerrado is in its infancy, and many studies are still needed to improve our understanding of the historical biogeography of this rich and threatened biome to help in its conservation and sustainable ...

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... advances have been made in disentangling biogeographical patterns across the globe, but the diversification of many biodiversity rich areas of the world remain poorly studied [1]. One such region is the Cerrado biome, a savanna vegetation that, together with the Seasonally Dry Tropical Forests (SDTF), covers a huge diagonal seasonally dry area in the interior of Eastern Tropical South America (ETSA) [1–3]. The Cerrado is typically found in dystrophic soils, often latosols, under a climate characterized by a marked dry season, whereas the SDTFs typically occur in areas with more fertile soils and longer dry seasons [2–4]. The Cerrado is the most diverse savanna in the world, particularly in terms of the number of plant species present [5], and it has high levels of endemisms and species adapted to stressed environments [3,5,6]. Its almost 1.8 million km 2 of original area has been reduced by more than 50% in the last years [7], making the Cerrado one of the main global hotspots for biodiversity conservation [5]. Despite this relevance, many details about the historical biogeography of the Cerrado biome are unresolved [4,8]. The Cerrado has a highly heterogeneous biota and many studies have tried to subdivide it into biogeographic provinces (see [4,8] for reviews). Three broad phytogeographical studies have found similar patterns [9–11]. In studies analyzing 145 Cerrado areas, Castro [9] and Castro and Martins [10] recognized three main phytogeographic groups, a Southern Cerrados group (named ‘S ̃o Paulo’), a Central Cerrados group (named ‘Planalto Central’) and the Northeastern Cerrados. Largely concordant with it, Ratter et al. [11], based on the floristic similarity of a broader sampling effort involving 376 Cerrado areas, identified six phytogeographic provinces (see names and delimitation of them in Figure 1). Their results were largely concordant and they suggested that climate and soil, as well as past climatic events, might be among the main factors responsible for the patterns found. With regard to the impact of past climates on the Cerrado biota, many controversies exist, and expansion [12], stability [13], and reduction [14,15] of its extension during the Last Glacial Maximum (LGM) have been proposed. Phylogeographical approaches would help to evaluate the persistence of these patterns at the intra-specific level and to help unravel the causes for them. Phylogeographical studies have proved to be valuable indepen- dent sources of evidence in comprehending biogeographical patterns and in reconstructing past vegetation dynamics [16]. Concordances between phylogeographical and biogeographical patterns and paleodata have been found in numerous instances (e. g. [17,18]), thus helping understand the evolution and shared history of many species. Nevertheless, only a few studies have addressed the phylogeography of species from the Cerrado [1,8] and phylogeographical studies that have focused on Cerrado trees are scarce [19–22]. Essentially, these studies found phylogeographic structuring and high levels of differentiation among populations in the respective species analyzed. Three of them observed major groups of populations subdivided longitudinally [20–22], and three [19,21,22] observed also patterns of recent range expansion of northern resources towards the southern Cerrado. These expansions have been hypothesized to have occurred after the return of wetter and warmer climatic conditions after the last glacial period. A better understanding of the evolutionary patterns of the Cerrado could be achieved with more phylogeographical studies of species with distinct ecological requirements together with wider samplings across the biome, including the peripheral regions. In the present study, we focused on a widespread tree species adapted to the coldest regions of the biome, and encompassing a large portion of the biome, including the southwest portion. The species chosen for this study, Dalbergia miscolobium Benth. (Papilionoideae, Fabaceae), is endemic and widespread in the Cerrado (Figure 1). It is among the 121 most representative tree species of the biome and is the 9 th and 12 th most common tree species in the southeastern and the southern Cerrado, respectively [23]. Unlike other Cerrado trees subjects of phylogeographical studies so far, D. miscolobium seems to be more tolerant to the cold. It is common in the coldest (subject to eventual frosts during winter) and southernmost portions of the Cerrado, and is widely found at higher altitudes ( . 1,000 m) in the biome [24]. In ...

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... advances have been made in disentangling biogeographical patterns across the globe, but the diversification of many biodiversity rich areas of the world remain poorly studied [1]. One such region is the Cerrado biome, a savanna vegetation that, together with the Seasonally Dry Tropical Forests (SDTF), covers a huge diagonal seasonally dry area in the interior of Eastern Tropical South America (ETSA) [1–3]. The Cerrado is typically found in dystrophic soils, often latosols, under a climate characterized by a marked dry season, whereas the SDTFs typically occur in areas with more fertile soils and longer dry seasons [2–4]. The Cerrado is the most diverse savanna in the world, particularly in terms of the number of plant species present [5], and it has high levels of endemisms and species adapted to stressed environments [3,5,6]. Its almost 1.8 million km 2 of original area has been reduced by more than 50% in the last years [7], making the Cerrado one of the main global hotspots for biodiversity conservation [5]. Despite this relevance, many details about the historical biogeography of the Cerrado biome are unresolved [4,8]. The Cerrado has a highly heterogeneous biota and many studies have tried to subdivide it into biogeographic provinces (see [4,8] for reviews). Three broad phytogeographical studies have found similar patterns [9–11]. In studies analyzing 145 Cerrado areas, Castro [9] and Castro and Martins [10] recognized three main phytogeographic groups, a Southern Cerrados group (named ‘S ̃o Paulo’), a Central Cerrados group (named ‘Planalto Central’) and the Northeastern Cerrados. Largely concordant with it, Ratter et al. [11], based on the floristic similarity of a broader sampling effort involving 376 Cerrado areas, identified six phytogeographic provinces (see names and delimitation of them in Figure 1). Their results were largely concordant and they suggested that climate and soil, as well as past climatic events, might be among the main factors responsible for the patterns found. With regard to the impact of past climates on the Cerrado biota, many controversies exist, and expansion [12], stability [13], and reduction [14,15] of its extension during the Last Glacial Maximum (LGM) have been proposed. Phylogeographical approaches would help to evaluate the persistence of these patterns at the intra-specific level and to help unravel the causes for them. Phylogeographical studies have proved to be valuable indepen- dent sources of evidence in comprehending biogeographical patterns and in reconstructing past vegetation dynamics [16]. Concordances between phylogeographical and biogeographical patterns and paleodata have been found in numerous instances (e. g. [17,18]), thus helping understand the evolution and shared history of many species. Nevertheless, only a few studies have addressed the phylogeography of species from the Cerrado [1,8] and phylogeographical studies that have focused on Cerrado trees are scarce [19–22]. Essentially, these studies found phylogeographic structuring and high levels of differentiation among populations in the respective species analyzed. Three of them observed major groups of populations subdivided longitudinally [20–22], and three [19,21,22] observed also patterns of recent range expansion of northern resources towards the southern Cerrado. These expansions have been hypothesized to have occurred after the return of wetter and warmer climatic conditions after the last glacial period. A better understanding of the evolutionary patterns of the Cerrado could be achieved with more phylogeographical studies of species with distinct ecological requirements together with wider samplings across the biome, including the peripheral regions. In the present study, we focused on a widespread tree species adapted to the coldest regions of the biome, and encompassing a large portion of the biome, including the southwest portion. The species chosen for this study, Dalbergia miscolobium Benth. (Papilionoideae, Fabaceae), is endemic and widespread in the Cerrado (Figure 1). It is among the 121 most representative tree species of the biome and is the 9 th and 12 th most common tree species in the southeastern and the southern Cerrado, respectively [23]. Unlike other Cerrado trees subjects of phylogeographical studies so far, D. miscolobium seems to be more tolerant to the cold. It is common in the coldest (subject to eventual frosts during winter) and southernmost portions of the Cerrado, and is widely found at higher altitudes ( . 1,000 m) in the biome [24]. In ...

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... of Bridgewater et al. [23], D. miscolobium was found in 82% of the 22 floristic surveys conducted in the southern Cerrado and in 75% of the 73 sites studied in the south-eastern Cerrado. On the other hand, the species was encountered in only 37% and 28% of the north-eastern and central-western Cerrado provinces, respectively. It is a tree pollinated by bees and its seeds are wind- dispersed [25]. It is an important timber species, and is a rapid colonizer of suitable environments. Here, we undertook an extensive sampling of D. miscolobium populations encompassing a large portion of the Cerrado biome in order to investigate the species’ phylogeographical structure and to verify its congruence with phylogeographical and biogeographical patterns reported to the Cerrado biome so far. More specifically, we aimed to answer the following questions: 1) is the phylogeographical structure of this wide range species congruent with the Cerrado biogeographical provinces proposed by Ratter et al. [11]? Considering that the historical casual factors that contributed to determine the phytogeographical provinces of Cerrado may also have contributed to the phylogeographical structure of D miscolobium we predicted some congruency between them; 2) in what extension is the phylogeographical structure of D. miscolobium similar to other Cerrado tree species studied so far? Based on previous studies [20–22] we predicted that the southern populations present lower genetic diversity than populations from central Cerrado, which in turn would present high diversity levels, consistent with a recent colonization of southernmost populations from more stable northernmost areas. Dalbergia miscolobium is not included in the Brazilian Official List of Threatened Plants. The collection of samples of leaves in conservation units was realized with the legal authorization of the ‘Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renov ́veis’ (IBAMA; populations DFE, MUC and PER - licenses 192/2003, 110/2005, 26.627.1) and of the ‘Instituto Estadual de Florestas – MG’ (population RPE – license 018/05). The collection of the samples in localities out of conservation units had authorization for collection and transport of IBAMA (licenses 099/2004 and 016/2006, 14035-2, 293770) and of the owners of the lands. Also we had authorization for the access to sampling of component of genetic patrimony of the Brazilian ‘Conselho de Gest ̃o do Patrim ˆnio Gen ́tico’ (CGEN/MMA) number 03/ 2004. The geographic distribution of D. miscolobium was estimated through field trips, databases of floristic checklists (personal communications by Jos ́ Felipe Ribeiro and Ary T. Oliveira- Filho) and herbarium records (Figure 1). Expeditions were carried out from 2004 to 2010 to sample D. miscolobium across the Brazilian Cerrado. Leaf samples were collected from 287 adult trees in 32 populations covering most of its geographic distribution range, encompassing seven Brazilian states (Table 1 and Figure 1). Our sampling covered all the four Cerrado phytogeographic provinces proposed by Ratter et al. [11] in which the species is found: Southern (S), Central and southeastern (C & SE), North and northeastern (N & NE) and Central-western (CW) (Figure 1). We sampled a lower number of populations in the N & NE province, where the species is scarcely found (see Introduction). Three to 14 individuals were sampled in each population. Populations were sampled in natural reserves and remnant fragments of the Cerrado (Table 1). Genomic DNA was isolated by a modified cetyl trimethylammonium bromide (CTAB) extraction method [26] and quantification was carried out by visual inspection of agarose gels. The following regions were chosen for DNA sequencing: the trnL intron of the chloroplast DNA (cpDNA) and the internal transcribed spacer (ITS) region of the 18S–26S nuclear ribosomal DNA (nrDNA) genes (which includes the ITS1 and ITS2 intergenic spacers and the 5.8S gene). The trnL intron and ITS region were amplified and sequenced using primers as described by Taberlet et al. [27] and Delgado-Salinas et al. [28], respectively. The cpDNA region was chosen, because it presented specific amplification and the highest variability among 15 non-coding cpDNA regions evaluated (information is available on request). Special care was taken with regard to the specificities of the ITS region and the main guidelines proposed by Feliner and Rossell ́ [29] were followed in the amplification and analysis of this region. The amplification of both regions was performed in 25 m l total volume containing 10–20 ng of DNA, 1X PCR buffer with 2.0 mM MgCl 2 , 200 m M of each dNTP, 0.2 ng bovine serum albumin (BSA), 0.5 m M of each primer, 1 U Taq DNA polymerase (Phoneutria, Belo Horizonte, Brazil), and autoclaved deionized water. The nrDNA region reaction mixture also contained 2% dimethyl sulfoxide (DMSO) and 1 M Betaine (N3-trimethyglycine). Thermal conditions of the reaction were as follows: initial denaturation at 94 u C for 2 min, followed by 35 cycles at 94 u C for 1 min, 56 u C (cpDNA) or 60 u C (nrDNA) for 1 min, and 72 u C for 1 min, and a final elongation at 72 u C for 7 min. Each PCR product was then double-strand sequenced using the DYEnamic ET dye terminator sequencing Kit (GE Healthcare, UK). Sequencing reactions were analyzed on a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer’s instructions. Sequences were assembled using the package PHRED/ PHRAP/CONSED to generate consensus sequences. The cpDNA and nrDNA sequences were deposited in GenBank under accession numbers JQ612719 to JQ612730 and JQ582850 to JQ582876, respectively. The consensus sequences were aligned using CLUSTAL-W [30] implemented in the MEGA 5 program [31], followed by careful manual adjustment. The ends of the sequences were pruned to eliminate fragments that could not be obtained for all individuals and to preserve only high confidence bases. For nrDNA sequences, all variable sites were carefully checked in the original electropherograms and, when heterozygous nucleotide positions identified by double peaks were present, samples were re-sequenced independently in order to confirm the genotype. To reconstruct the nrDNA haplotypes, we used PHASE 2.1 software [32], which uses a Bayesian statistical method to infer haplotypes of individuals that have more than one polymorphic site based on genotypic data. It was run under default conditions, allowing for multiallelic loci (-d option) and for 10,000 iterations. In the first run, one multiallelic site was shown to be highly homoplasious in our data and, based on this, we removed this site prior to further analysis. Only haplotypes recovered with . 0.90 posterior probability were used in subsequent analyses, a precaution adopted by other studies that have used PHASE [33]. To perform subsequent genetic analyses, the output of PHASE was transformed through the pipeline described by Machado et al. [34]. Genetic diversity indices were estimated in ARLEQUIN 3.5 [35] and in DNAsp 5.10 [36]. The hypothesis of population expansion was tested by pairwise mismatch distributions [37] and the neutrality tests Tajima’s D [38] and Fu’s F S [39]. These analyses were performed with the ARLEQUIN and DNAsp programs. Allelic richness was computed in Contrib [40] and index of differentiation among populations was estimated by D [41] in SMOGD [42]. Both the indices and the tests were performed for the whole species as well as for the phylogeographic groups of populations delimited in the analyses (see Results and Table 2 for details about their composition). Analyses of molecular variance (AMOVA; [43]) using pairwise differences were conducted in ARLEQUIN to assess population genetic structure in D. miscolobium based on cpDNA and nrDNA data. The phylogenetic relationships among haplotypes were inferred using the Median-Joining (MJ) network method [44] and Statistical Parsimony [45] as implemented in ...

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... of Bridgewater et al. [23], D. miscolobium was found in 82% of the 22 floristic surveys conducted in the southern Cerrado and in 75% of the 73 sites studied in the south-eastern Cerrado. On the other hand, the species was encountered in only 37% and 28% of the north-eastern and central-western Cerrado provinces, respectively. It is a tree pollinated by bees and its seeds are wind- dispersed [25]. It is an important timber species, and is a rapid colonizer of suitable environments. Here, we undertook an extensive sampling of D. miscolobium populations encompassing a large portion of the Cerrado biome in order to investigate the species’ phylogeographical structure and to verify its congruence with phylogeographical and biogeographical patterns reported to the Cerrado biome so far. More specifically, we aimed to answer the following questions: 1) is the phylogeographical structure of this wide range species congruent with the Cerrado biogeographical provinces proposed by Ratter et al. [11]? Considering that the historical casual factors that contributed to determine the phytogeographical provinces of Cerrado may also have contributed to the phylogeographical structure of D miscolobium we predicted some congruency between them; 2) in what extension is the phylogeographical structure of D. miscolobium similar to other Cerrado tree species studied so far? Based on previous studies [20–22] we predicted that the southern populations present lower genetic diversity than populations from central Cerrado, which in turn would present high diversity levels, consistent with a recent colonization of southernmost populations from more stable northernmost areas. Dalbergia miscolobium is not included in the Brazilian Official List of Threatened Plants. The collection of samples of leaves in conservation units was realized with the legal authorization of the ‘Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renov ́veis’ (IBAMA; populations DFE, MUC and PER - licenses 192/2003, 110/2005, 26.627.1) and of the ‘Instituto Estadual de Florestas – MG’ (population RPE – license 018/05). The collection of the samples in localities out of conservation units had authorization for collection and transport of IBAMA (licenses 099/2004 and 016/2006, 14035-2, 293770) and of the owners of the lands. Also we had authorization for the access to sampling of component of genetic patrimony of the Brazilian ‘Conselho de Gest ̃o do Patrim ˆnio Gen ́tico’ (CGEN/MMA) number 03/ 2004. The geographic distribution of D. miscolobium was estimated through field trips, databases of floristic checklists (personal communications by Jos ́ Felipe Ribeiro and Ary T. Oliveira- Filho) and herbarium records (Figure 1). Expeditions were carried out from 2004 to 2010 to sample D. miscolobium across the Brazilian Cerrado. Leaf samples were collected from 287 adult trees in 32 populations covering most of its geographic distribution range, encompassing seven Brazilian states (Table 1 and Figure 1). Our sampling covered all the four Cerrado phytogeographic provinces proposed by Ratter et al. [11] in which the species is found: Southern (S), Central and southeastern (C & SE), North and northeastern (N & NE) and Central-western (CW) (Figure 1). We sampled a lower number of populations in the N & NE province, where the species is scarcely found (see Introduction). Three to 14 individuals were sampled in each population. Populations were sampled in natural reserves and remnant fragments of the Cerrado (Table 1). Genomic DNA was isolated by a modified cetyl trimethylammonium bromide (CTAB) extraction method [26] and quantification was carried out by visual inspection of agarose gels. The following regions were chosen for DNA sequencing: the trnL intron of the chloroplast DNA (cpDNA) and the internal transcribed spacer (ITS) region of the 18S–26S nuclear ribosomal DNA (nrDNA) genes (which includes the ITS1 and ITS2 intergenic spacers and the 5.8S gene). The trnL intron and ITS region were amplified and sequenced using primers as described by Taberlet et al. [27] and Delgado-Salinas et al. [28], respectively. The cpDNA region was chosen, because it presented specific amplification and the highest variability among 15 non-coding cpDNA regions evaluated (information is available on request). Special care was taken with regard to the specificities of the ITS region and the main guidelines proposed by Feliner and Rossell ́ [29] were followed in the amplification and analysis of this region. The amplification of both regions was performed in 25 m l total volume containing 10–20 ng of DNA, 1X PCR buffer with 2.0 mM MgCl 2 , 200 m M of each dNTP, 0.2 ng bovine serum albumin (BSA), 0.5 m M of each primer, 1 U Taq DNA polymerase (Phoneutria, Belo Horizonte, Brazil), and autoclaved deionized water. The nrDNA region reaction mixture also contained 2% dimethyl sulfoxide (DMSO) and 1 M Betaine (N3-trimethyglycine). Thermal conditions of the reaction were as follows: initial denaturation at 94 u C for 2 min, followed by 35 cycles at 94 u C for 1 min, 56 u C (cpDNA) or 60 u C (nrDNA) for 1 min, and 72 u C for 1 min, and a final elongation at 72 u C for 7 min. Each PCR product was then double-strand sequenced using the DYEnamic ET dye terminator sequencing Kit (GE Healthcare, UK). Sequencing reactions were analyzed on a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer’s instructions. Sequences were assembled using the package PHRED/ PHRAP/CONSED to generate consensus sequences. The cpDNA and nrDNA sequences were deposited in GenBank under accession numbers JQ612719 to JQ612730 and JQ582850 to JQ582876, respectively. The consensus sequences were aligned using CLUSTAL-W [30] implemented in the MEGA 5 program [31], followed by careful manual adjustment. The ends of the sequences were pruned to eliminate fragments that could not be obtained for all individuals and to preserve only high confidence bases. For nrDNA sequences, all variable sites were carefully checked in the original electropherograms and, when heterozygous nucleotide positions identified by double peaks were present, samples were re-sequenced independently in order to confirm the genotype. To reconstruct the nrDNA haplotypes, we used PHASE 2.1 software [32], which uses a Bayesian statistical method to infer haplotypes of individuals that have more than one polymorphic site based on genotypic data. It was run under default conditions, allowing for multiallelic loci (-d option) and for 10,000 iterations. In the first run, one multiallelic site was shown to be highly homoplasious in our data and, based on this, we removed this site prior to further analysis. Only haplotypes recovered with . 0.90 posterior probability were used in subsequent analyses, a precaution adopted by other studies that have used PHASE [33]. To perform subsequent genetic analyses, the output of PHASE was transformed through the pipeline described by Machado et al. [34]. Genetic diversity indices were estimated in ARLEQUIN 3.5 [35] and in DNAsp 5.10 [36]. The hypothesis of population expansion was tested by pairwise mismatch distributions [37] and the neutrality tests Tajima’s D [38] and Fu’s F S [39]. These analyses were performed with the ARLEQUIN and DNAsp programs. Allelic richness was computed in Contrib [40] and index of differentiation among populations was estimated by D [41] in SMOGD [42]. Both the indices and the tests were performed for the whole species as well as for the phylogeographic groups of populations delimited in the analyses (see Results and Table 2 for details about their composition). Analyses of molecular variance (AMOVA; [43]) using pairwise differences were conducted in ARLEQUIN to assess population genetic structure in D. miscolobium based on cpDNA and nrDNA data. The phylogenetic relationships among haplotypes were inferred using the Median-Joining (MJ) network method [44] and Statistical Parsimony [45] as implemented in NETWORK 4.6 (fluxus-engineering.com) and TCS 1.21 [46], respectively. Inser- tion/deletions (indels) were considered as fifth character states and were coded as single mutations, regardless of their size. The phylogeographic structure of populations ...

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... of Bridgewater et al. [23], D. miscolobium was found in 82% of the 22 floristic surveys conducted in the southern Cerrado and in 75% of the 73 sites studied in the south-eastern Cerrado. On the other hand, the species was encountered in only 37% and 28% of the north-eastern and central-western Cerrado provinces, respectively. It is a tree pollinated by bees and its seeds are wind- dispersed [25]. It is an important timber species, and is a rapid colonizer of suitable environments. Here, we undertook an extensive sampling of D. miscolobium populations encompassing a large portion of the Cerrado biome in order to investigate the species’ phylogeographical structure and to verify its congruence with phylogeographical and biogeographical patterns reported to the Cerrado biome so far. More specifically, we aimed to answer the following questions: 1) is the phylogeographical structure of this wide range species congruent with the Cerrado biogeographical provinces proposed by Ratter et al. [11]? Considering that the historical casual factors that contributed to determine the phytogeographical provinces of Cerrado may also have contributed to the phylogeographical structure of D miscolobium we predicted some congruency between them; 2) in what extension is the phylogeographical structure of D. miscolobium similar to other Cerrado tree species studied so far? Based on previous studies [20–22] we predicted that the southern populations present lower genetic diversity than populations from central Cerrado, which in turn would present high diversity levels, consistent with a recent colonization of southernmost populations from more stable northernmost areas. Dalbergia miscolobium is not included in the Brazilian Official List of Threatened Plants. The collection of samples of leaves in conservation units was realized with the legal authorization of the ‘Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renov ́veis’ (IBAMA; populations DFE, MUC and PER - licenses 192/2003, 110/2005, 26.627.1) and of the ‘Instituto Estadual de Florestas – MG’ (population RPE – license 018/05). The collection of the samples in localities out of conservation units had authorization for collection and transport of IBAMA (licenses 099/2004 and 016/2006, 14035-2, 293770) and of the owners of the lands. Also we had authorization for the access to sampling of component of genetic patrimony of the Brazilian ‘Conselho de Gest ̃o do Patrim ˆnio Gen ́tico’ (CGEN/MMA) number 03/ 2004. The geographic distribution of D. miscolobium was estimated through field trips, databases of floristic checklists (personal communications by Jos ́ Felipe Ribeiro and Ary T. Oliveira- Filho) and herbarium records (Figure 1). Expeditions were carried out from 2004 to 2010 to sample D. miscolobium across the Brazilian Cerrado. Leaf samples were collected from 287 adult trees in 32 populations covering most of its geographic distribution range, encompassing seven Brazilian states (Table 1 and Figure 1). Our sampling covered all the four Cerrado phytogeographic provinces proposed by Ratter et al. [11] in which the species is found: Southern (S), Central and southeastern (C & SE), North and northeastern (N & NE) and Central-western (CW) (Figure 1). We sampled a lower number of populations in the N & NE province, where the species is scarcely found (see Introduction). Three to 14 individuals were sampled in each population. Populations were sampled in natural reserves and remnant fragments of the Cerrado (Table 1). Genomic DNA was isolated by a modified cetyl trimethylammonium bromide (CTAB) extraction method [26] and quantification was carried out by visual inspection of agarose gels. The following regions were chosen for DNA sequencing: the trnL intron of the chloroplast DNA (cpDNA) and the internal transcribed spacer (ITS) region of the 18S–26S nuclear ribosomal DNA (nrDNA) genes (which includes the ITS1 and ITS2 intergenic spacers and the 5.8S gene). The trnL intron and ITS region were amplified and sequenced using primers as described by Taberlet et al. [27] and Delgado-Salinas et al. [28], respectively. The cpDNA region was chosen, because it presented specific amplification and the highest variability among 15 non-coding cpDNA regions evaluated (information is available on request). Special care was taken with regard to the specificities of the ITS region and the main guidelines proposed by Feliner and Rossell ́ [29] were followed in the amplification and analysis of this region. The amplification of both regions was performed in 25 m l total volume containing 10–20 ng of DNA, 1X PCR buffer with 2.0 mM MgCl 2 , 200 m M of each dNTP, 0.2 ng bovine serum albumin (BSA), 0.5 m M of each primer, 1 U Taq DNA polymerase (Phoneutria, Belo Horizonte, Brazil), and autoclaved deionized water. The nrDNA region reaction mixture also contained 2% dimethyl sulfoxide (DMSO) and 1 M Betaine (N3-trimethyglycine). Thermal conditions of the reaction were as follows: initial denaturation at 94 u C for 2 min, followed by 35 cycles at 94 u C for 1 min, 56 u C (cpDNA) or 60 u C (nrDNA) for 1 min, and 72 u C for 1 min, and a final elongation at 72 u C for 7 min. Each PCR product was then double-strand sequenced using the DYEnamic ET dye terminator sequencing Kit (GE Healthcare, UK). Sequencing reactions were analyzed on a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer’s instructions. Sequences were assembled using the package PHRED/ PHRAP/CONSED to generate consensus sequences. The cpDNA and nrDNA sequences were deposited in GenBank under accession numbers JQ612719 to JQ612730 and JQ582850 to JQ582876, respectively. The consensus sequences were aligned using CLUSTAL-W [30] implemented in the MEGA 5 program [31], followed by careful manual adjustment. The ends of the sequences were pruned to eliminate fragments that could not be obtained for all individuals and to preserve only high confidence bases. For nrDNA sequences, all variable sites were carefully checked in the original electropherograms and, when heterozygous nucleotide positions identified by double peaks were present, samples were re-sequenced independently in order to confirm the genotype. To reconstruct the nrDNA haplotypes, we used PHASE 2.1 software [32], which uses a Bayesian statistical method to infer haplotypes of individuals that have more than one polymorphic site based on genotypic data. It was run under default conditions, allowing for multiallelic loci (-d option) and for 10,000 iterations. In the first run, one multiallelic site was shown to be highly homoplasious in our data and, based on this, we removed this site prior to further analysis. Only haplotypes recovered with . 0.90 posterior probability were used in subsequent analyses, a precaution adopted by other studies that have used PHASE [33]. To perform subsequent genetic analyses, the output of PHASE was transformed through the pipeline described by Machado et al. [34]. Genetic diversity indices were estimated in ARLEQUIN 3.5 [35] and in DNAsp 5.10 [36]. The hypothesis of population expansion was tested by pairwise mismatch distributions [37] and the neutrality tests Tajima’s D [38] and Fu’s F S [39]. These analyses were performed with the ARLEQUIN and DNAsp programs. Allelic richness was computed in Contrib [40] and index of differentiation among populations was estimated by D [41] in SMOGD [42]. Both the indices and the tests were performed for the whole species as well as for the phylogeographic groups of populations delimited in the analyses (see Results and Table 2 for details about their composition). Analyses of molecular variance (AMOVA; [43]) using pairwise ...