Revealing the Diversity and Varietal Relationships of Regional Cacao and Close Relatives in the Northwestern Colombian Amazon: Insights for Conservation and Agroforestry Resilience

Understanding the genetic diversity and structure of regional cacao and its close relatives is essential for strengthening conservation strategies and enhancing the resilience of Amazonian agroforestry systems. This study evaluated the genetic diversity, population structure, and varietal relationships of 48 sexually derived regional accessions of Theobroma cacao, T. grandiflorum, and T. bicolor with desirable morpho-agronomic traits, together with eight universal T. cacao reference clones, all cultivated in farmer-managed agroforests of the northwestern Colombian Amazon, using a panel of 15 SSR markers. The loci exhibited substantial allelic richness (mean Na = 8.53) and consistently high expected heterozygosity (Hexp = 0.74), with numerous private alleles indicating species- and lineage-specific divergence. Bayesian clustering, ΔK inference, and minimum spanning networks identified four genetically coherent subpopulations corresponding to the three species and a distinct lineage within T. cacao, strongly aligned with the discriminant analysis of principal components (DAPC) results. Analysis of Molecular Variance (AMOVA) revealed that most genetic variation occurred among subpopulations (56.68%), while pairwise FST (Wright’s fixation index) values confirmed strong interspecific differentiation and significant divergence within T. cacao. No isolation-by-distance pattern was detected. These findings demonstrate that regional Theobroma germplasm maintained in smallholder agroforests constitutes a valuable reservoir of genetic diversity that complements universal reference clones. By documenting species-level divergence and lineage-specific variation, this study supports the integration of farmer-managed genetic resources into conservation planning and highlights their importance for the long-term resilience of Amazonian cacao-based agroforestry landscapes.