Journal of Heredity Advance Access published online on October 30, 2008
Journal of Heredity, doi:10.1093/jhered/esn092
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Phylogeographical Structure of the Neotropical Forest Tree Hymenaea courbaril (Leguminosae: Caesalpinioideae) and Its Relationship with the Vicariant Hymenaea stigonocarpa from Cerrado
From the Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP: 486, Belo Horizonte, MG, 31270-901, Brazil (Simões Ramos and Lovato); and the Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP: 486, Belo Horizonte, MG, 31270-901, Brazil (Pires de Lemos-Filho)
Address correspondence to Maria B. Lovato at the address above, or e-mail: lovatomb{at}icb.ufmg.br.
The phylogeography of Hymenaea courbaril var. stilbocarpa from Atlantic Forest and riverine forests of the Cerrado biome in central and southeastern Brazil was investigated. The data were compared with those of its congeneric Hymenaea stigonocarpa, a typical tree from savanna. In the Cerrado, H. courbaril var. stilbocarpa is found in sites contiguous with those of H. stigonocarpa, and they share common life-history attributes. The psbC/trnS3 region of the chloroplast DNA was sequenced in 149 individuals of H. courbaril var. stilbocarpa. High genetic variation was found in this species, with the identification of 18 haplotypes, similarly to what was found in H. stigonocarpa with 23 haplotypes in the same geographic region. Populations of H. courbaril var. stilbocarpa could be structured in 3 phylogeographic groups. Spatial analysis of molecular variation indicated that 46.4% of the genetic variation was due to differences among these groups. Three haplotypes were shared by H. courbaril var. stilbocarpa and H. stigonocarpa, and only 10.5% of the total genetic variation could be attributed to between-species difference. We surmise that during the glacial times, H. courbaril var. stilbocarpa populations must have gone extinct in most parts of the southern of its present-day occurrence area. After climate amelioration, these areas were probably recolonized from northern and eastern. The relatively similar phylogeographic structure of vicariant Hymenaea species suggests that they were subjected to the same impacts during the Quaternary climatic fluctuations. The sharing of haplotypes and the genetic similarity between the 2 Hymenaea species suggest the existence of ancestral polymorphism and/or hybridization.
Key Words: Hymenaea courbaril • Hymenaea stigonocarpa • phylogeography • cpDNA • Cerrado • Atlantic forest
The Amazon and Atlantic forests are the major rain forests of South American and encompass the most diverse tropical forests in the world. Between these 2 forests lies a corridor of seasonal and open vegetation that includes the Cerrado in central Brazil, the Caatinga in northeastern Brazil, and the Chaco in Argentina and Paraguay (Prado and Gibbs 1993). The Cerrado is the second largest biome in Brazil extending more than 2 million km2. It is composed of a mosaic of subunits that vary from grasslands to dry forests and is mostly dominated by semideciduous arboreal savanna. Along the rivers that dissect this mosaic, there are strips of mesic riverine forests. This forest provides an important connection between the flora of Amazonia and the Atlantic Forest (Oliveira-Filho and Ratter 1995).
The Quaternary biogeographical history of southeast and central Brazil is complex and poorly understood, due to existence of few palynological records for these regions. In general, pollen data suggest that the last glacial period was cooler and drier than present-day conditions, resulting in an extension of savanna vegetation and reduction in rain forest size (Behling 2002). At the last glacial maximum (LGM), around 27 500 to c. 14 500 14C years ago, cold temperatures and hard frosts made the climate too severe to support Cerrado vegetation or semideciduous forests in this region. At this time, large areas of Atlantic semideciduous forest were replaced by subtropical grasslands, and Cerrado vegetation was displaced further north (Behling and Lichte 1997; Behling 1998). During the early Holocene, with climate amelioration, grasslands began to be replaced by different forms of Cerrado vegetation and by semideciduous forests in regions with a short annual dry season and by rain forests in regions without significant dry periods. Forest expansion from existing gallery forests was recorded between 8800 and 7500 year before present (B.P.) in Lago do Pires (17°57'S, 42°13'W) and at approximately 5500 year B.P. the climate became more humid and development toward modern forests and a more diverse Cerrado began (Behling 1995).
Phylogeographic studies have suggested that past fragmentation of the Atlantic forest (Cardoso et al. 2000; Lira et al. 2003; Salgueiro et al. 2004; Ledru et al. 2007) and reduction of the southern Cerrado (Collevatti et al. 2003; Ramos et al. 2007) have influenced the current genetic structure of tree species that occur in this part of the Neotropics. All these studies analyzed populations of species occurring in just one of these biomes. Here, we investigate the phylogeographic structure of the forest tree Hymenaea courbaril in a large part of its occurrence in central and southeast Brazil. The phylogeographic data of this species were compared with to those obtained for Hymenaea stigonocarpa, a congeneric species from savanna (Cerrado). Studies of the comparative phylogeography of phylogenetically closely related species showing particular ecological attributes and sharing common life-history traits can be useful to establish the effects of past climatic changes across of these biomes.
The genus Hymenaea pertains to tribe Detarieae (family Leguminosae–Caesalpinioideae), constituting a genus of resin-producing trees reputed to be the main source of fossil amber in the Neotropics (Langenheim et al. 1973). The genus includes 13 species that are distributed in Mesoamerica, the West Indies, and most of South America and one species in East Africa-Madagascar. Hymenaea courbaril, divided into 6 varieties, is distributed over a vast geographical area of tropical America and the Antilles. Hymenaea courbaril var. stilbocarpa occurs in the Atlantic forest and riverine forest of the Cerrado biome. In contrast, H. stigonocarpa is restricted to the Brazilian Cerrado (Lee and Langenheim 1975). Hymenaea stigonocarpa and H. courbaril var. stilbocarpa are considered vicariant species (Heringer et al. 1976).
Hymenaea trees are exploited for their good-quality timber, used for shipbuilding, furniture, etc (Lee and Langenheim 1975). Hymenaea courbaril is also on the official list of Brazilian endangered medicinal species (IBAMA 1992). Hymenaea courbaril and H. stigonocarpa exhibit similar life-history traits, such as pollination biology, mating system, and seed dispersion. They are predominantly outcrossers, mainly pollinated by bat species (Lee and Langenheim 1975; Crestana et al. 1985; Gibbs et al. 1999, Dumphy et al. 2004). Seeds of H. courbaril are mainly dispersed by agoutis (Asquith et al. 1999), and this is probably also true for those of H. stigonocarpa.
Analyses of chloroplast DNA (cpDNA) variation are a useful tool to reconstruct historical events such as population expansions and contractions, migration, and colonization (McCauley 1995; Ennos et al. 1999) and can provide insight into ancient and contemporary hybridization (Rieseberg and Soltis 1991; Rieseberg et al. 1996). In the present study, we used cpDNA sequences from the psbC/trnS3 to analyze the phylogeographic structure of H. courbaril var. stilbocarpa. The same markers were previously analyzed in H. stigonocarpa (Ramos et al. 2007), and here, they were used for comparative phylogeography. In particular, we test the following hypotheses: 1) the genetic structure of H. courbaril var. stilbocarpa should reflect the shift in vegetation patterns across central and southeast Brazil brought about by climatic change during the Quaternary; 2) because in the Cerrado biome, H. courbaril var. stilbocarpa is found in sites contiguous with those of H. stigonocarpa, although at distinct habitats, forest and savanna, and that they have similar life-history attributes, we hypothesized that both species could have been subjected to the same climatic changes in the past, and thus, they could share some similar phylogeographic patterns. This prediction is based on palynological evidence suggesting that both forest and savanna of the studied region were constricted and displaced during the Quaternary. Alternatively, if the distinct habitats suffered differential impacts of the climatic changes, H. courbaril var. stilbocarpa and H. stigonocarpa could show significant differences in their phylogeographic structure.
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Sampling Populations and DNA Extraction
Young leaves were collected from 149 adult individuals from 15 populations of H. courbaril, ranging in distribution between 12–23°S and 40–54°W (Table 1 and Figure 1). Following proposition of Lee and Langenheim (1975) for H. courbaril geographic distribution and morphology, the analyzed samples are representatives of the var. stilbocarpa. Populations were sampled in the States of Minas Gerais (MG)—PTM, CPM, FBM, FBMII, FUM, RPM and MOM, Espirito Santo (ES)—RLM and SEM, São Paulo (SP)—SPM, Goiás (GO)—ARM and NIM, Mato Grosso do Sul (MS)—MSM, Bahia (BA)—PAM and in the Federal District (DF)—PNM. Leaves were collected and stored in labeled plastic bags at –20 °C until DNA extraction. Populations of H. stigonocarpa used for comparative analyses were from the same region as H. courbaril, ranging in distribution between 10°–23°S and 41°–50°W (Ramos et al. 2007). In 4 of the sampled sites, populations of H. courbaril var. stilbocarpa co-occur with those of H. stigonocarpa.
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Total DNA was extracted following the protocol of Doyle JJ and Doyle JL (1987) using the modifications suggested by Ferreira and Grattapaglia (1995). The protocol uses 2% of cationic detergent cationic hexadecyl trimetyl ammonium bromide, 100 mM Tris–HCl pH 8.0, 1.4 M NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% polyvinylpyrrolidone, and 2% β-mercaptoetanol. Quantity and quality of DNA were assessed by visualization on a 0.8% agarose gel.
cpDNA Sequencing
Screening for variation in the cpDNA of H. courbaril var. stilbocarpa used polymerase chain reaction (PCR) amplification with the same 9 universal primers, and results were similar to those found for H. stigonocarpa (Ramos et al. 2007). Thus, the same sequence used in H. stigonocarpa, psbC/trnS3 (CS) (Demesure et al. 1995), was amplified for all individuals of H. courbaril var. stilbocarpa.
PCRs were carried out on a 25-µl final volume, containing 10 ng template DNA, 1x PCR buffer (IC—Phoneutria, Belo Horizonte, Brazil), 200 µM deoxynucleoside triphosphates, 0.5 µM each primer, 5 µg of bovine serum albumin, and 1 U Taq polymerase (Phoneutria). After amplification, PCR products were visualized on 1% agarose gels stained with ethidium bromide and purified using polyethylene glycol 20%/2.5 M NaCl precipitation. To sequence the CS region, psbC (Demesure et al. 1995) and RCS 5'-AAGATATGCCAGATTCCACC-3' (Ramos et al. 2007) primers were used.
The sequencing reaction was conducted in a 10-µl reaction containing 3 µl of purified PCR product, 2 µl of milliQ water, 1 µl of primer (5 µM), and 4 µl of ET-DYE Terminator Kit (Amersham Biosciences, Piscataway, NJ). The thermocycling program was as follows: 35 cycles of 25 s at 95 °C, 15 s at 54 °C, and 3 min at 60 °C. Sequencing products were precipitated and cleaned with ammonium acetate and ethanol and then dried at room temperature, dissolved in loading buffer (formamide 70% and 1 mM EDTA), and run on MegaBACE sequencer (80-s injection time, 240-min run length).
Data Analysis
Consensus sequences were assembled for each individual using at least 2 forward and 2 reverse sequences made from independent PCR products, using the softwares Phred v. 0.20425 (Ewing and Green 1998; Ewing et al. 1998), Phrap v. 0.990319 (http://www.phrap.org/), and Consed 12.0 (Gordon et al. 1998). Multiple sequence alignments were made using Clustal X (Thompson et al. 1997) implemented in MEGA 3.0 (Kumar et al. 2004). Clustal alignments were also checked and edited by hand to minimize software artifacts.
Molecular diversity indices (
, nucleotide diversity; h, haplotype diversity; and k, mean number of nucleotide substitutions) were calculated using MEGA 3.0 and ARLEQUIN software ver. 3.01 (Excoffier et al. 2005). The haplotype network was constructed using the program DNAsp 3.99 (Rozas et al. 2003). The haplotypic richness was estimated by RAREFAC that uses the technique of rarefaction to correct for sample size (Petit et al. 1998). Typically, rarefaction is used to standardize allelic richness to the smallest N in a comparison (Petit et al. 1998). However, the MSM population was not included in this analysis due to its small sample size (N = 2), and a rarefaction size of N = 4 was used.
To perform the comparative analysis with H. stigonocarpa, we used sequences of 175 individuals from different populations of this species and 2 outgroup taxa, Hymenaea aurea and Hymenaea reticulata, as described in a previous study (Ramos et al. 2007). Intraspecific and interspecific relationships were inferred by the construction of haplotype networks using the median-joining (MJ) algorithm (Bandelt et al. 1999) implemented in the NETWORK 4.1 software (http://www.fluxus-engineering.com).
Estimates of differentiation and FST statistics were calculated taking into account the pairwise distance between cpDNA haplotypes. The program spatial analysis of molecular variation (SAMOVA, Dupanloup et al. 2002) was used to analyze the population structure. This method defines groups of populations that are geographically homogenous and maximally differentiated from each other, through a priori definition of the number of groups (K) of populations, and generates F statistics (FSC, FST, and FCT) using an analysis of molecular variation (AMOVA; Excoffier et al. 1992). By exploring the behavior of the indices FCT and FSC for different values of K, it is possible to identify the optimum number of population groups (Dupanloup et al. 2002). For each value of K, 100 simulated annealing processes were used, ranging from K = 2 to K = 8.
Pairwise comparisons of FST between populations, the genetic differentiation among species, populations, and groups were analyzed using an AMOVA implemented in the ARLEQUIN software ver. 3.01 (Excoffier et al. 2005). Tests of neutrality were performed using Tajima's D (Tajima 1989) and Fu's Fs (Fu 1997) tests with 10 000 simulation steps using ARLEQUIN software ver. 3.01 (Excoffier et al. 2005). The demographic history of the H. courbaril var. stilbocarpa was investigated by plotting a mismatch distribution analysis. This distribution is usually multimodal in samples drawn from populations of relatively stable size over time and unimodal in populations that experienced a recent demographic expansion (Rogers and Harpending 1992). Although models of mismatch distribution were developed for animal mitochondrial DNA, they have been used with cpDNA data in several species of plants for test demographic expansions in Quaternary (Hwang et al. 2003; Cheng et al. 2005; Lorenz-Lemke et al. 2006; Taylor and Keller 2007).
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Genetic Diversity in H. courbaril var. stilbocarpa
Amplification of the noncoding cpDNA region CS produced a fragment of
1600 bp, of which 535 bp were sequenced for all individuals. The aligned CS region presented 9 indels in the positions 11, 12, 13, 158, 415, 416, 434, 438, and 504 (Table 2). There were 516 conserved positions and (excluding 9 indels) 10 variable sites (9 parsimony informative sites) in the positions 80, 261, 297, 322, 359, 433, 465, 470, 492, and 525 (Table 2). This region exhibited a high AT content (58.9%), with the presence of several mononucleotide repeats, as is generally found in noncoding cpDNA regions.
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Eighteen haplotypes were found (Figure 1B and Table 2) defined by the 10 sites and 9 indels. Diversity was high with total haplotype diversity (Hd), nucleotide diversity (
), and the mean number of nucleotide differences (k) equal to 0.80, 0.003, and 1.48, respectively. Haplotype diversity for each population (h) ranged from 0 to 0.83, haplotypic richness (A) from 0 to 2.183, nucleotide diversity from 0 to 0.00279, and the mean number of nucleotide differences from 0 to 1.47 (Table 1). The 2 most diverse populations were the CPM and RPM for all diversity indices. Conversely, populations SPM, MSM, and RLM exhibited only one haplotype each (diversity indices = 0) (Table 1).
Phylogeographic Structure of cpDNA Haplotypes and Geographical Differentiation in H. courbaril var. stilbocarpa
The phylogenetic relationships among the 18 haplotypes can be observed in the network in Figure 2, analyzed by the MJ method. The most frequent haplotypes were H2, H34, H26, H32, and H1, occurring in 38%, 20%, 13%, 8%, and 6% of all sampled individuals, respectively. H34, H26, H32, and H1 were linked to H2 by a single nucleotide substitution in positions 525, 434, 492, and 80, respectively (Figure 2). Most haplotypes (13) were exclusive of only one population (Table 2). This is the case of haplotypes H29, H30, and H31 found only in the CPM population and haplotypes H35, H36, and H37 found exclusively in the PAM population (Table 2).
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The SAMOVA analyses of H. courbaril var. stilbocarpa data indicated that there were distinct genetically defined groups of sampling areas. The analysis using K = 2 resulted in FCT = 0.464 and FSC = 0.347; for K = 3, FCT value remained 0.464 and FSC decreased to 0.262. Between K = 4 and K = 8, the FCT maintained similar values, ranging from 0.437 to 0.516, but resulted in groups containing only one population. Thus, our SAMOVA analysis indicated the same FCT values for both configurations, with 2 or 3 groups. The configuration with K = 3 gave a lower value of FSC than with that with K = 2, indicating more-similar populations within groups. So, we selected this configuration with 3 groups to explain the genetic structure in H. courbaril. Thus, the 3 geographical groups are western, composed of ARM, NIM, PNM, PTM, SPM, MSM, FBM, FBMII, and FUM; central group composed of CPM and RPM populations; and northeastern composed of PAM, RLM, SEM, and MOM populations (Figure 1B). The haplotype H2 is found in the western and central groups, and the haplotype H1 is restricted to the central group. Haplotype H34 is found not only in northeastern group but also in FUM population (western group) and RPM (central group). An FST value of 0.604 indicated that 60.4% of the variation was due to differences among all populations (Table 3). The fixation indices (FST) calculated for each pair of populations ranged from 0.11 to 1.00, and most values were significant (P < 0.05).
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Sequence variation demonstrated significant deviation from expectations of neutrality by Fu's test (Fs = –9.40, P < 0.001) but was nonsignificant in Tajima's test (D = –0.95, P > 0.17) for the total sample. A unimodal histogram of the genetic differences between pairs of individuals in a mismatch distribution considering all the analyzed individuals (with a peak at about one difference) (Figure 4), suggested a recent population expansion.
Combined Analysis of H. courbaril var. stilbocarpa and H. stigonocarpa
Forty haplotypes (19 variable sites) were identified considering 175 individuals of H. stigonocarpa, 149 of H. courbaril var. stilbocarpa, 1 of H. aurea, and 1 of H. reticulata. The phylogenetic relationship among the 40 observed haplotypes is shown by the network in Figure 3, analyzed by the MJ method. The comparison of geographic distribution of haplotypes between H. courbaril var. stilbocarpa and H. stigonocarpa can be seen in Figure 1. The results revealed that H. courbaril var. stilbocarpa and H. stigonocarpa, besides showing similar phylogeographic structure, shared 3 haplotypes, H1, H2, and H8, the most frequent of which in both species being H2 (Figures 1 and 3). Both haplotypes of outgroups, H24 (H. aurea) and H25 (H. reticulata), were associated directly with haplotype H2. The H2 haplotype is present in about 38% of all H. courbaril var. stilbocarpa individuals sampled and is most frequent in western group (59.8% of the individuals). In H. stigonocarpa, this haplotype occurs in 76.1% of western group and in 33% of all individuals sampled of this species. In H. courbaril northeastern group, the most frequent haplotype is H34 (76.3%), which is directly related to the other haplotypes found in this group (H35, H36, H37, H39, and H40). The H8 haplotype, the most frequent in the eastern group of H. stigonocarpa, is found in a single individual of H. courbaril var. stilbocarpa from the RPM population (central group) (Table 2). H1 is the most frequent haplotype in the central group of H. stigonocarpa and is present in the CPM and RPM populations of central group of H. courbaril var. stilbocarpa. Only the H34 haplotype is shared between the 3 groups of H. courbaril var. stilbocarpa, being found in one individual from the population FUM (western group) and one from the population RPM (central group).
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The 2 species are very similar according to the AMOVA considering the pairwise distance between haplotypes. This indicated that only 10.5% of the genetic variation found is due to differences between the species, 51.7% of it was due to differences among populations belonging to the same species, and 37.8% within populations (Table 3).
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Hymenaea courbaril var. stilbocarpa exhibited high diversity in the 535-bp sequence of the region psbC/trnS3, with diversity indices very similar to those of the congeneric H. stigonocarpa (h = 0.804,
= 0.003, Ramos et al. 2007). The genetic divergence among H. courbaril var. stilbocarpa populations (FST = 0.650) was similar to values that are typically observed for angiosperm tree species (mean GST = 0.646, Petit et al. 2005). Similar among-population differentiation was reported for H. stigonocarpa (FST = 0.692, Ramos et al. 2007). The phylogeographic analysis of H. courbaril var. stilbocarpa suggested the existence of a distinct group located in the northeast of the sampled area, comprising populations of Bahia, north of Minas Gerais, and Espirito Santo states. The genetic discontinuity found in this region of the Atlantic forest is in agreement with previously described patterns for several animal and plant species (Costa 2003; Lira et al. 2003; Moraes-Barros et al. 2006; Ledru et al. 2007; Tchaicka et al. 2007). The location of the split coincides with those described by Costa (2003) for small mammals and by Tchaicka et al. (2007) for a canid species. In a study with Podocarpus species, Ledru et al. (2007) also delimited a northeast group with a split around 15°S. All these data suggest large-scale historical vicariance events promoting genetic differentiation. The most common haplotype in the eastern group of the congeneric H. stigonocarpa (H8, Ramos et al. 2007) was not found in the northeastern group of H. courbaril var. stilbocarpa. However, the haplotypes found in these 2 groups of both species are closely related (Figure 3), suggesting that the fragmentation effects clearly detected for H. courbaril var. stilbocarpa in this latitude can also had affected populations of H. stigonocarpa. The western group of H. courbaril var. stilbocarpa has a close phylogeographical relationship with the western group of H. stigonocarpa (Figure 3), being both characterized by high frequency of the H2 haplotype and their derived haplotypes. The central position and high occurrence of H2 in the network would suggest that it is the most ancestral haplotype according to coalescence theory (Posada and Crandall 2001). This premise is supported by the relationships of H2 with outgroups (H. reticulata and H. aurea). Further evidence that H2 may be the oldest haplotype is given by the geographic proximity of the Amazon rain forest to western groups of both species, H. courbaril var. stilbocarpa and H. stigonocarpa. According to Langenheim et al. (1973), the center of Neotropical distribution of Hymenaea genus is the Amazonian hylaea.
The RPM and CPM populations of H. courbaril formed a distinct group (central) exhibiting the highest diversity. In both populations, the H1 haplotype was present at high frequencies, as it was in the central group of H. stigonocarpa. In addition, both populations contain individuals with the H2 haplotype that was characteristic of the western groups of the 2 Hymenaea species. Also, the RPM population exhibited the H34 haplotype that was the most common haplotype in the northeastern group of H. courbaril var. stilbocarpa. Together, these results suggest that, in these regions, large populations of H. courbaril var. stilbocarpa survived maintaining refugia for the species during the glacial periods. The geographic characteristics of the Rio Doce and Jequitinhonha valleys, regions where the CPM and RPM populations occur, allow typical lowland rain forest species to expand their distribution toward the interior (Oliveira-Filho and Fontes 2000). In these regions, the most favorable climatic conditions during glaciation could have allowed the persistence of Atlantic forest refugia harboring populations of H. courbaril var. stilbocarpa. An alternative, but not mutually exclusive, hypothesis is that this region could have received migrants of populations from western and northeastern groups.
The H. courbaril var. stilbocarpa populations sampled in our study are mainly from semideciduous Atlantic forest (also referred to as seasonally dry forest by Pennington et al. [2006]) and riverine forest in the Cerrado biome. Paleopalinological studies suggest that large areas of the southern and southeastern Brazilian highlands were covered with subtropical grasslands during the last glacial, reflecting a cold dry climate (Behling and Hooghiemstra 2001). This region was 5–7 °C cooler during the LGM than today, with hard frosts which precluded the survival of Cerrado vegetation and semideciduous forests (Behling 1998). More frequent frosts have been suggested as an important factor limiting the development of Cerrado vegetation (Eiten 1972; Silberbauer-Gottsberger et al. 1977). During the glacial times, tropical gallery forests and semideciduous forests may only have existed where frosts were not frequent, probably in the north part of southeastern Brazil. With an increase in temperature at the beginning of Holocene, grasslands were replaced by Cerrado vegetation in regions with long annual dry periods (5–6 months), by semideciduous forests in regions with a short annual dry season (3–5 months), and by rain forests in regions without significant dry periods. Initial expansion probably originated from gallery forests and forest remnants in regions free of hard frosts and strong drought stress (Behling 1998). Our cpDNA data suggest that during LGM H. courbaril populations must have persisted in the most northerly regions and in sites at low elevation. This may explain the higher genetic diversity that was found in the most northeastern populations, PAM and MOM, and the maintenance of populations in regions that correspond to the central group, RPM and CPM. In a recent study, using paleoclimatic models and a few published mitochondrial DNA phylogeographical studies in animals, Carnaval and Moritz (2008) proposed a great area of forest stability (refuge) in the central corridor of Atlantic forest (Bahia) during Pleistocene, extending from the Doce river northward to the São Francisco river. This putative refuge coincides with most populations from central and northeastern groups of H. courbaril var. stilbocarpa.
The expansion of H. courbaril populations southward into the Cerrado biome after reestablishment of climate conditions from the northernmost areas may have initially occurred through riverine forests. Many rain forest species, both Amazonian and Atlantic, are known to expand their distribution into areas with strongly seasonal climates via riverine forests (Oliveira-Filho and Ratter 1995). The population FUM, located at Furnas, south of sampled area, contained the most common haplotype of the western group (H2) and the haplotype H32 that occurs in high frequency in FBM and FBMII populations. In addition, in FUM population, there is a haplotype in low frequency typical of the northeastern group (H34). One population of the savanna species, H. stigonocarpa, was also present in Furnas and contained haplotypes from the 3 groups of this species, haplotypes H1, H2, and H8 (Ramos et al. 2007). This pattern, common to both species, reinforces the hypothesis suggesting that the region was recolonized by different lineages from the more northern populations (Ramos et al. 2007). This fact and the same overall diversity of the 2 species suggest that although they occupy different habitats (savanna and forest) both must have experienced the same impacts of the Quaternary climatic changes. This reinforces evidence of large vegetation changes suggested by paleopalinological studies. Patterns of late Quaternary distribution of Atlantic forest predicted by Carnaval and Moritz (2008) would be useful to construct an alternative hypothesis to southward expansion of H. courbaril var. stilbocarpa. In their maps, they showed historically stable areas of broader sense Atlantic forest located western inland around 20°S. If this model represents a real situation, it is probable that populations of H. courbaril var. stilbocarpa persisted in the southwestern of our sampling area instead of being colonized by northern populations.
As described above, the 2 congeneric species, H. courbaril var. stilbocarpa and H. stigonocarpa, share 3 haplotypes H1, H2, and H8. In both species, these haplotypes are found in approximately the same geographic areas. The AMOVA comparing the 2 Hymenaea species indicated that they are very similar. Only 10.5% of genetic variation was due to differences between these species and 51.7% was due to differences among populations belonging to the same species. This demonstrates that there is more divergence among populations of the same species than divergence between species. Maternally inherited markers are frequently shared among holoarctic tree species (Rajora and Dancik 1992; Petit et al. 2002; Palmé et al. 2004; Lexer et al. 2005; Heuertz et al. 2006). The sharing of haplotypes among species can be due to recent origin associated with the presence of ancestral polymorphisms or hybridization and introgression. It has been suggested that differentiation of these Hymenaea species is recent (Langenheim et al. 1973; Lee and Langenheim 1975). Hymenaea stigonocarpa and H. courbaril var. stilbocarpa are considered to be vicariant species by botanists, that is, closely related species that occur in adjacent areas but are ecologically distinct (Heringer et al. 1976). The 2 species co-occur in some regions, have similar flower morphology (Lee and Langenheim 1975), and probably share the same pollinator species (Gibbs et al. 1999). Phenological data show that their flowering times overlap: H. courbaril flowers between November and January and H. stigonocarpa from December to March in the regions we sampled (Lee and Langenheim 1975 and analysis of herbarium collection of Departamento de Botânica, da Universidade Federal de Minas Gerais). Although Lee and Langenheim (1975) considered occurrence of hybridization and introgression in the genus Hymenaea to be possible, we are not aware of hybridization between these species. Thus, the available evidences do not allow us to rule out none of the 2 proposed hypotheses. Both ancestral polymorphism (incomplete lineage sorting) and hybridization would explain the existence of shared cpDNA haplotypes and the genetic similarity we found between H. stigonocarpa and H. courbaril var. stilbocarpa. Given that cpDNA markers exhibit a slow evolution rate, evidence from other markers with a faster rate of evolution such as single sequence repeat would help to elucidate the evolutionary history of these related species. In addition, another important question that emerges from the present data is related to the taxonomic status of these species. Hymenaea courbaril var. stilbocarpa was considered a separate species, H. stilbocarpa, until the last revision of the genus made by Lee and Langenheim (1975). According to these authors, H. stigonocarpa is closely related to H. courbaril, but the 2 are separable by morphological characteristics and distinctive growth form. However, Rizzini (1997) considered that the herbarium material of H. stigonocarpa and H. courbaril var. stilbocarpa show only small differences, although in nature they are better distinguishable by tree size and bark differences. The sharing of haplotypes and the similar phylogeographic patterns between H. stigonocarpa and H. courbaril var. stilbocarpa suggest that their current taxonomic status needs revision. That would necessarily include extensive taxonomic and phylogenetic studies with all the recognized varieties of both species as well the remaining species of Hymenaea.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil grant no. 470674/2004-0); and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).
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Acknowledgments |
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We also thank the Instituto Brasileiro de Meio Ambiente for providing facilities, Reinaldo M. Silva, Renan Milagres, Luciana C. Resende, and Juliano Leal for technical assistance in this study, Fabrício dos Santos Rodrigues for suggestions and sequencing facilities, Ana Y. Ciampi, Rosangela L. Brandão, and Maíra F. Goulart for their help in sample collection. A.C.S. Ramos received a PhD fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil). M.B. Lovato and J.P. Lemos-Filho received research fellowship from CNPq/Brazil.
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Footnotes |
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Corresponding Editor: James Hamrick
Received June 19, 2008
Revised September 10, 2008
Accepted September 25, 2008
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