The present study shows indications of the action of balancing selection on drought response-related genes in Neotropical tree species, although demographic changes in the populations, causing the same kind of departure from the standard neutral model, cannot be excluded, at least for some species (i.e. P. quinata, E. grandiflora and at least one population of E. falcata).
Extensive nucleotide diversity was found for six PIP gene fragments in five tropical tree species; the sequences obtained are useful for the detection of SNP polymorphisms, for genetic diversity analyses and for testing departures from expectations derived from the neutral theory of molecular evolution . In one case (PIP2.1) the same primer pair was used to amplify two congeneric species (Table 2.b), providing a direct comparison between orthologs. For these two genes (EfaPIP2.1 and EgrPIP2.1, table 1) the level of polymorphism in E. falcata was lower than in E. grandiflora, but departures from mutation-drift equilibrium were detected in both species (Table 3), although not for the same statistics.
Standard neutral model tests on approximately half of the gene amplicons gave significant results. In genes and populations with significant departures from mutation-drift equilibrium the general trend is towards population size bottlenecks and/or balancing selection. However, sliding window analyses show that this trend does not apply uniformly to the entire sequence and that different regions of the genes may be subject to different processes. In one case (EfaPIP1.1; Figure 3), sites responsible for the positive and significant test statistics seem to be mostly restricted at the exon-intron boundary, possibly indicating selection on intron splicing sites or other regulatory functions by intronic sequences. Interestingly, when matched against small RNA databases, the intron fragment contained in EfPIP1.1 sequences is similar to immature Arabidopsis thaliana miRNA ath-MIR863 and to Oryza sativa miRNA osa-MIR420 (E-values of 0.048 and 0.064, respectively), indicating a putative functional role for the intron.
Two pieces of evidence favour balancing selection, rather than demography, as an explanation for the diversity patterns observed in this study, at least for a subset of the gene amplicons.
First, simulations of bottlenecks of variable sizes and ages, resulting in the same amounts of genetic diversity as observed in current populations, cannot produce statistics (Tajima's D, Fu and Li's F*) as high as those observed on real data for C. guianensis (Figure 4 left pane), unless moderate to strong and recent bottlenecks are assumed. D* values are however compatible with all demographic scenarios. For E. falcata (Figure 4 right pane), short bottlenecks are generally compatible with the observed Tajima's D, but not long bottlenecks (F* values are compatible with most demographic scenario in E. falcata, indicated by the fact that this statistic is not significant; table 3 and figure 2). As the species studied here were sampled in largely undisturbed portions of Neotropical forests, and are not known to have historically been exploited for timber, strong, short and very recent bottlenecks seem unlikely. However, bottlenecks hundreds of generations ago would be compatible with processes dating to the Late Pleistocene-Early Holocene, which would correspond to large-scale floristic changes in Neotropical forests . The contrasting behaviour of D*, and F* and D, can be explained by what the statistics actually test and by the structure of our data. Both F* and D rely on estimation of the difference between an estimate of θ based on average pairwise sequence divergence and another estimate based on the number of segregating sites (singletons for F*, global estimate for D), whereas D* compares two estimates of numbers of segregating sites (common versus singleton). The former account for the depth of sequence divergence, whereas the latter does not. It appears that, at least in some cases, purely demographic scenarios cannot account for the excess sequence divergence observed in our data, while they can account for the observed number of segregating sites. This is an indication of exceedingly long genealogical branches in our data, which again favours balancing selection as opposed to bottlenecks. Moreover, all the tests applied have been shown to have similar power in the detection of recent bottlenecks , so that they are expected to give similar results under a purely demographic scenario.
Second, the tests on chloroplast SSRs generally refuted past bottlenecked populations, even if not all loci detect signatures of population expansion. Variability in the results from different loci is due to the fact that, if all loci describe the same evolutionary process, their statistics are multiple stochastic outcomes from the same probability function. As demography should influence all portions of the nuclear genome, as well as organellar genomes, in the same direction although with different sensitivity for the intensity and timing of demographic events , it is unlikely that events in opposing directions would be detected at different loci (recombination tends to smooth diversity estimates across genes, by reducing the variance of θ estimators ). Thus we can tentatively conclude that the departures from equilibrium at PIP loci are due to balancing selection. A caveat should be added that, as SSRs generally have different mutation rates than coding sequences, comparison of results from these two types of data may be misleading. In particular, chloroplast SSRs tend to mutate more quickly than nuclear, non-repeated sequences, although more slowly than nuclear SSRs , and the demographic events suggested by these markers may be more recent than the results of forces acting on sequence diversity. A further concern is that effective population size and demographic dynamics are not the same for chloroplast and nuclear loci, making the former, being haploid, more sensitive to demographic change. However, a recent demographic event detected in SSRs should also be detectable in sequences. Significant values for tests of departures from standard neutral equilibrium of opposite sign seem to support balancing selection on the expressed loci, while cases where demographic signatures are detected at chloroplast markers but not at PIP loci can be conservatively attributed to differences in marker sensitivity. Note, however, that this argument does not hold if the populations have undergone a bottleneck relatively far in the past (more than approximately 50 generations, the time beyond which the SSR-based methods used here cannot detect a bottleneck). In this case, fast-evolving SSRs may display the effects of the expansion following the bottleneck, while slow-evolving sequences may still display the effect of the bottleneck itself.
The observed departures from hypothetical selective neutrality in spite of a relative paucity of non-synonymous mutations is not surprising, given that regulatory sequences, potentially undergoing selection, may lie anywhere along gene sequences and that introns can have a function besides gene regulation. Among the gene fragments shown by mutation-drift equilibrium tests to putatively experience selection, VsePIP2.1 and EfaPIP1.1 do not contain non-synonymous mutations, and so, for these loci, selection may not be acting directly on the portion of protein-coding sequence analysed here. Selection may however affect other properties of the transcribed sequence, such as codon composition or intron functions. As PIP sequences are conserved among and within species  selection could rather act on regulatory regions. Alternatively, these polymorphism patterns may reflect selection on neighbouring sites, although the estimated population parameters suggest that recombination would quickly break down associations between selected sites and associated neutral sites, such that selection signatures may not extend beyond a few hundred base pairs from the site under selection. For genes that did not display any departure from expectations of the standard neutral model, full-length sequencing, including the promoter region, is advisable. In general, evolutionary patterns may diverge among different parts of a gene  and there is evidence of balancing selection in the promoter region of the TFL1 gene in Arabidopsis thaliana . On the other hand, demographic events could be responsible for significant departures from the standard neutral model in the whole genome, including gene regions. Moreover, loci outside the sequenced region, but in linkage disequilibrium (LD) with it, may also affect the results of mutation-drift equilibrium tests. This possibility cannot be ruled out for the current data set, as the fragments are too short for testing the decay of LD with distance, although, in some species, population recombination rates are relatively high (Table 3), indicating that LD should rapidly fall to zero. This is generally the case for forest trees, where LD falls below 0.2 within 200-300 base pairs ). Therefore the main issue when observing significant mutation-drift equilibrium tests in genes is to discover whether selection or past demography underlie the observed levels of diversity.
For C. guianensis population SE, all tests for departure from mutation-drift equilibrium on locus PIP1.1 gave strongly significant results, while no chloroplast SSR marker did. This pattern is consistent with the maintenance of polymorphism by long-term balancing selection. It is interesting to note that the samples of population SE were collected from a hybridisation zone between C. guianensis and C. procera. Bayesian assignation methods, applied to independent loci (SSRs), show however that the sampled trees belong with a probability of 1 to C. guianensis populations , perhaps pointing to a stable and selectively advantageous introgression of C. procera genes into a C. guianensis genetic background. Actually, sequences of CguPIP1.1 obtained in pure C. procera stands show the same haplotype found in population SE of C. guianensis (Casalis et al. in preparation). If this hypothesis holds true, it may also explain the pattern of polymorphism observed for the two populations of this species: the NW population would display the "typical" status of the species, while the SE population, which would have undergone historical introgression, would carry an extra allele derived from the sister species C. procera (n.b. the taxonomic status of the latter species is under revision; P.M. Forget, personal communication). Further analyses of the diversity of PIP genes in the two species will test this hypothesis (M. Casalis et al. in prep.).
The results for PIP genes of E. falcata (the species for which the largest number of different genes was obtained) were divergent for the two populations. In summary (Table 3), population NW displays significant results for D and F* for genes PIP1.1 and PIP2.1, while population SE displays a significant test for gene PIP1.2 and the statistic D*. The results are almost perfectly complementary for the two populations: they show significant results for different genes and different statistics. Moreover, test statistics for population NW largely show incompatibility with demographic scenarios. As stated above, D and F* are more sensitive than D* to the extent of sequence divergence, and thus to balancing selection. Therefore, the simplest explanation for the observed pattern is that balancing selection is detected in population NW for two genes, while demography (i.e. a past population bottleneck) drives diversity patterns in population SE. The fact that chloroplast SSRs tend to support population expansion, rather than bottleneck, for population NW provides a further hint that the positive values of the mutation-drift equilibrium tests are due to selection rather than demography. Selection on the PIP1.1 gene would overcome the baseline demographic signal, which would provide negative statistics which are detected by SSRs (n.b. SSRs can only detect recent bottlenecks, while older ones may still affect sequence diversity). A similar argument can be applied to the only interspecific comparison, for gene PIP2.1 in the two Eperua species. Here, estimates of θ from pairwise sequence differences (θπ) are higher in E. falcata than in E. grandiflora, while estimates from haplotype diversity (θW) are higher in E. grandiflora (Table 3). This apparent contradiction may be explained by the fact that E. falcata has a smaller number of more divergent haplotypes than E. grandiflora (A; table 3). Such results tend to favour different explanations for the diversity patterns in the two species: the historical preservation of highly divergent haplotypes in E. falcata, which is compatible with balancing selection (see above) and the presence of larger numbers of less differentiated haplotypes for E. grandiflora, which may be compatible with recovery from an old bottleneck or population expansion.
Finally, for P. quinata, departure from mutation-drift equilibrium was found for Fu and Li's D*, but the species displayed very little polymorphism at cpSSR loci, and therefore it was difficult to evaluate alternative hypotheses. The observed value of D* was, however, entirely compatible with demographic scenarios. Moreover, sample sizes per sampling site were relatively small for this species (see Methods) and, although tests for population differentiation were all non-significant, statistical power was low. Consequently, the departures from mutation-drift equilibrium may in this case be an artefact caused by hidden population structure. Large year to year variation (up to 100%) in overall rainfall and length of dry season may provide balancing selection, while extensive gene flow (bat pollination, seed dispersal by convection currents) may reduce population differentiation.
Among the cases described in this study, balancing selection may be the most common trend, but it is currently hard to outline why this should be the case. One possibility is that the sampled populations may be further structured along local ecological gradients at a smaller geographic scale, and that different environments favour alternative alleles, thus maintaining genetic diversity within populations. In this case, variation of ecological conditions at a spatial scale smaller than the size of populations may lead to the coexistence of different genetic optima in sub-structured populations, providing patterns that mimic balancing selection. As stated above, hidden population subdivision at loci responding to selective gradients can bias tests such as those applied here . However, since we used PIP genes themselves to group individuals into non differentiated populations, this is likely to be a relatively minor problem in our data set. Alternatively, the wide variation in environmental conditions over time, both between seasons and over longer climatic cycles, may prevent selection from fixing a particular variant, explaining the balancing selection patterns. In general, although selective explanations of observed patterns of diversity should not be taken for granted unless solid evidence is provided, there is no reason either to assume that the most likely explanation should be a demographic one .
To test for the presence of selection, further studies could look for association between haplotype and adaptive traits related to water stress, as well as for selection in other candidate genes. Indeed, a large number of genomic or proteomic studies have identified candidate genes for water stress tolerance. For example dehydrin genes are up-regulated by drought stress (BjDHN2 and BjDHN3 in Brassica juncea ; PgDhn1 in Picea glauca ) and alcohol dehydrogenase (Adh) transcripts are induced by anoxia and hypoxia [40, 41]. Contrasting the results for these genes with analyses of neutral loci to detect demographic events will also be fundamental to disentangle demographic versus adaptive interpretations of tests for departure from the standard neutral equilibrium model. As this is the first genomic study of gene sequences in non-plantation tropical trees, genomic resources that would allow such comparisons among different classes of genomic regions are not yet available.
Another limitation of our study is the length of the fragments analysed for each gene. Statistical tests, such as those we applied, gain more from increases in sequence length than from increases in sample size. Again, the complete lack of genomic resources for non-plantation tropical trees slows down the accumulation of sequence information, although isolation of larger sequences (Vedel et al. in prep.), as well as large-scale genome sequencing (Duret et al. in prep.) are under way. On the other hand, analysing larger sample sizes than generally suggested for this kind of studies was necessary, as the underlying distribution of genetic diversity and delimitation of populations was also unknown for these species. This information was necessary prior to the application of statistical tests. In spite of these limitations, the current data set allowed us to detect informative trends in sequence diversity. The results reported here should actually prove the feasibility of such research programmes, and are expected to prime more extensive investigations into the genomics of non-model tropical trees.