Heterochromatic patterns of Citrus revisited: a new look at species origins and karyotype evolution

Most Citrus L. ‘species’ have arisen through reticulated evolution, resulting in many hybrid species with constant chromosome number (2 n = 18) and confused phylogenetic relationship. Before the molecular era, those hybrids were classified in systems with high numbers of species, when in fact the number of true species is quite small. Cytogenetic and molecular markers have been important tools for characterizing and understanding the hybrids origin, especially with the high variation in the heterochromatin patterns reveled by CMA/DAPI double staining technique. We aimed to determine the heterochromatin pattern distribution of 12 Citrus accessions using CMA/DAPI flourochromes and correlate the results with the currently available cytogenetic, molecular, and phylogenomics data to investigate their possible origins and karyotype evolution. We also performed FISH with 35S rDNA probes in some accessions to improve our discussion. The distinct heteromorphic distribution patterns of CMA + /DAPI - bands among these accessions’ karyotypes confirm their hybrid origins. The hybrid species were then classified into two groups (I and II) based on heterochromatin patterns and phylogenetic relationship. Most hybrids in group I, with class A chromosomes, had C. maxima as an ancestor, while those in group II were descendants of species belonging to the subgenus papeda. We were able to correlate our finds with the available cytogenetic and molecular data, contributing to our understanding of the origins of Citrus hybrid species and their karyotype evolution.