Our study on the genetic diversity and population structure C. legalis, an endemic and endangered tree from the Brazilian Atlantic Forest, shows that, despite intense illegal logging and deforestation of this ecosystem, the species still exhibits a degree of genetic resilience. In particular, the Cabruca agroforestry system has proven to be extremely important both for retaining genetic diversity and for connecting populations by gene flow. However, some populations evaluated already show signs of genetic erosion, most likely in response to the anthropogenic disturbances experienced by the species over the years [65].
In this context, we believe that, because this is a species with a long generation time and high life expectancy, there may be a delayed genetic response (genetic extinction debt), masking the real magnitude of the harmful effects of anthropogenic disturbances [20]. Therefore, we emphasize caution when interpreting the results, due to a possible genetic time lag, and highlight that, in addition to the forest remnants of the Atlantic Forest, conservation efforts must consider the Cabruca agroforestry system as a key piece for the genetic conservation of C. legalis.
Patterns of genetic diversity and inbreeding coefficient among populations and ontogenetic stages
The low average proportion of loci in Hardy–Weinberg equilibrium at both ontogenetic stages may indicate the action of microevolutionary forces, such as genetic drift, migration, selection, or non-random reproduction in the populations [68].
Moreover, the patterns of genetic diversity observed in C. legalis show marked variation among the evaluated populations. This may reflect both human activities, such as different intensities of selective logging between areas, and historical processes (e.g., pollen and seed dispersal) related to the establishment and natural dynamics of populations across different locations [34, 50, 60, 65]. For instance, the ST, JS, and FR populations with adult individuals exhibited the highest values for the genetic diversity estimators evaluated here (A, AP, Ar, HO, and HE) and the lowest inbreeding rates, suggesting a relatively efficient maintenance of genetic variability in these areas. The values observed for these genetic variability estimators are within the upper range observed in neotropical tree species that maintain high levels of genetic diversity, even in fragmented landscapes [36, 53]. Thus, although a myriad of factors of natural or anthropogenic origin may influence this pattern, we believe that the greater genetic connectivity between these populations (see FST and Nm) is favoring the maintenance of genetic diversity in these populations [49].
In contrast, BC and VCL populations showed reduced genetic diversity and high levels of inbreeding, which may be indicative of isolation effects, genetic drift, and crosses between related individuals [30, 49]. This pattern is consistent with studies in other plant species subjected to fragmentation, in which reduced diversity and increased inbreeding are associated with habitat loss and decreased effective population size [30, 49]. Although the general pattern observed in adult populations is maintained in the juveniles of the VCL population, it is worth noting that the pattern is only partially retained in BC. In the latter case, the values of A, AP, Ar, and HE in BC are statistically equal to those reported in genetically more diverse populations. These findings reveal that within the same population, there may be genetic differences between individuals of different ontogenetic stages depending on the sensitivity of the genetic variability estimator used in responding to environmental changes [36, 53, 66].
For example, the adult population of BC is made up of only 13 individuals, which would partially explain the low HO value and high inbreeding found in the following generation of young individuals [61]. In this context, even if new alleles potentially arrived from neighboring populations and increased the number of individuals, which would explain the increase in estimators calculated from the number and/or allele frequency, BC is still at risk of genetic erosion via genetic drift and crosses between related individuals [61]. In this sense, the inbreeding coefficients observed in adult and juvenile populations, particularly in BC and VCL (f > 0.43) are considered high and suggest potential risks of inbreeding depression, reduced reproductive vigor and impairment of natural regeneration in the long term [61].
Furthermore, the absence of significant differences in the average of genetic diversity parameters between adults and juveniles, except for the inbreeding coefficient, may indicate either genetic resilience to anthropogenic disturbances or a time lag in the genetic response (genetic time lag), as already reported for C. legalis and other tropical plant species [4, 53, 54, 61]. On the other hand, the lower inbreeding found in juveniles may suggest a slight recovery in outcrossing patterns. However, considering the still high levels of overall inbreeding (adults f = 0.34 and juveniles f = 0.28), there is clear evidence of crosses between close relatives or limitations in the activity of dispersers is affecting the populations [1, 4]. We hypothesize that the low number of reproductive individuals in populations favors an increase in genetic relatedness among individuals, generating the observed pattern of inbreeding, as already described for other populations of C. legalis [34, 62].
Paradoxically, although fragmentation reduces C. legalis populations over time, it can also increase gene flow distances, which would help explain the slight reduction in inbreeding in juveniles [4, 36]. This hypothesis is because C. legalis flowers are pollinated by bees of the genera Melipona and Trigona [45], while its seeds are dispersed by gravity and wind [10]. Thus, it would be expected that bees would expand their foraging range in environments with low density of C. legalis, and that the wind would favor the dispersal of seeds over greater distances in more fragmented environments, promoting greater gene flow and, consequently, reducing inbreeding [26, 48, 62]. Despite this apparent attenuation of inbreeding in juveniles, values still range from moderate to high, which suggests source limitation (reproductive individuals), reflecting a restricted parental base [1, 4].
In this context, our findings emphasize the importance of sustainable management initiatives aimed at promoting gene flow between genetically distinct populations and implementing strategic reforestation efforts in the region. In this way, it would be possible to carry out a genetic rescue of compromised populations, favoring intraspecific genetic variability and maximizing the adaptive resilience of C. legalis in the face of environmental changes and anthropogenic pressures in the Atlantic Forest [18, 63].
Genetic structure and historical gene flow (Nm) between populations
The results obtained for genetic differentiation and historical gene flow from the FST and Nm estimators indicate significant variations in the degree of genetic structure of the populations analyzed, both for adults and juveniles [5]. The high FST values and low Nm values between VCL and the other populations suggest a clear barrier to historical gene flow [5, 37]. Certainly, a myriad of factors potentially influences this observed pattern. For example, isolation by geographic distance and forest fragmentation can create obstacles to gene flow, resulting in a significant genetic differentiation between populations [8, 37, 58]. On the other hand, different colonization histories or anthropogenic disturbances such as selective logging can also lead to genetic differentiation between populations [27, 41].
In contrast, the FST and Nm values between the JS and FR, ST and FR, and ST and JS population pairs indicate a moderate genetic structure, suggesting a more efficient historical gene flow between these populations at both ontogenetic stages [5, 37]. Taken together, the results of our study suggests that the pattern of genetic differentiation observed among C. legalis populations in fragmented landscapes of the Atlantic Forest may be more complex than the simple spatial separation of populations [12, 58].
In this context, although the Mantel test did not detect a statistically significant correlation between geographic distance and genetic differentiation for adults and juveniles, the correlation coefficients suggest a moderate to strong relationship, which may have biological relevance. The lack of statistical significance may be partly related to the low number of population pairs (n = 10), which reduces the statistical power of the test and limits the detection of significant patterns, especially in contexts with high spatial variability. Therefore, although it is not possible to state that isolation by distance is the main factor of genetic structuring in C. legalis, the observed coefficients suggest that spatial structure may still contribute to the detected genetic patterns, together with other ecological and historical factors [12, 58].
Finally, although the Nm results are convergent with other analyses (e.g. DAPC and genetic network analysis), it is important to highlight that the use of Nm values derived from the classical equation Nm = (1 – FST)/(4FST) is based on highly simplified assumptions, such as equal and constant population sizes, symmetrical migration, and equilibrium between drift and gene flow [71]. The violating these assumptions can lead to substantial biases, making such estimates unreliable indicators of recent gene flow. Therefore, in this study, Nm values are presented as indirect indicators of historical gene exchange but should not be interpreted as direct measures of contemporary gene flow. To overcome these limitations, we also employed complementary approaches, such as DAPC and genetic network analysis (EDENetworks), that allowed a more nuanced interpretation of connectivity among populations beyond what FST alone can reveal.
Genetic connectivity and population clusters
Genetic network analysis also revealed an interesting pattern of connectivity between populations. The JS and FR populations from both ontogenetic stages appeared as the most central in the network, maintaining stronger connections with the other populations, which may be indicative of these populations acting as genetic”hubs,”facilitating the exchange of genetic material between different locations [51, 55]. This pattern of centrality of these populations inserted into Cabruca agroforestry highlights the crucial role of this production system in maintaining the genetic diversity of C. legalis in fragmented landscapes [34]. Thus, our study highlights the relevance of Cabruca for maintaining biodiversity at the genetic level, corroborating other studies reporting the importance of this production system in conserving biodiversity at the species level [11, 16, 52].
In contrast, the VCL population, which is an environmentally protected area, demonstrated the lowest genetic connection for both ontogenetic stages, reflecting its genetic isolation in relation to the other populations, as already evidenced in the FST and Nm analyses.
Furthermore, Discriminant Principal Component Analysis (DAPC) revealed a clear differentiation between adult populations, with the formation of five distinct genetic clusters, corroborating the results of FST, Nm, and networks. The JS and FR populations clustered closely, suggesting a significant degree of genetic sharing between them. In contrast, VCL appeared as the most isolated population for adults, which reinforces the idea that this area experiences a greater genetic barrier, potentially reflecting the genetic isolation and low genetic diversity observed [4, 19].
Regarding juveniles, the formation of six genetic clusters, with the subdivision of JS into two groups, indicates the complexity of genetic interactions between populations. The overlap of the JS and FR clusters may suggest that, although there is some differentiation, there is also considerable genetic flow between them. The BC and VCL populations are in the same quadrant, while ST remained more isolated. These patterns indicate that gene flow between juvenile populations was not homogeneous, which may reveal different rates of seed dispersal and pollination between populations, reflecting the observed genetic structure [4]. In summary, our results point to a complex genetic structure in C. legalis, with a large variation in connectivity among populations. The centrality of some populations, such as JS and FR, and the isolation of VCL suggest that maintaining genetic connectivity and implementing conservation strategies that promote gene flow are essential for the long-term conservation of this endangered species [61, 62].