Integrative evidence on the hybridization between <i>Cenostigma microphyllum</i> and <i>C. pyramidale</i> (Leguminosae) in the Caatinga dry forest

Interspecific hybridization plays an important role in plant evolution, contributing to taxonomic uncertainty through intermediate phenotypes or the emergence of novel traits. The characterization of hybridization is important to elucidate systematic relationships and its role in the diversification of lineages. The genus Cenostigma comprises neotropical legume trees with phylogenetic inconsistencies, and individuals showing intermediate morphology between sympatric species, suggesting natural hybridization. We tested this hypothesis by investigating two endemic species from the Caatinga dry forest in northeast Brazil (C. microphyllum and C. pyramidale) using molecular markers (nuclear and plastid SSRs), geometric morphometrics, non-targeted metabolomics, and ecological analyses. We detected a high plastidial genetic structure among populations, not related to species boundaries but to their geographic distribution. The geometric morphometric analysis showed a clustering of pure individuals of both species with hybrids in an intermediate position, demonstrating the hybridization of these species in Caatinga. Nuclear DNA and metabolite diversity supported the separation of the two species into three clusters, with a subdivision of C. pyramidale in populations from the north (Pernambuco) and south (Bahia). Metabolomics revealed a fourth group formed mostly by hybrids. Later generation hybrids were detected as intermediate morphological forms, and gene flow was assumed as asymmetric among species and populations, being higher from C. pyramidale to C. microphyllum in populations from Bahia State. Ecological data indicated niche overlap. Hence, interspecific gene flow occurs among Cenostigma tree species, contributing to the evolution of the dry forest. Given the karyotypic and genomic similarity among species, as well as molecular and ecological evidence, we infer that the hybrids are fertile, allowing introgression and contributing to systematic complexity in Cenostigma. Hybridization did not significantly increase chemodiversity in terms of novel compounds but differentiated hybrids from parental species. In summary, we highlight the importance of multiple evidence, particularly genetic, morphological, and metabolomic traits, in the identification of hybrids and its evolutionary impact in natural environments.