Cytogenetic insights into the diversification of Calanthe: polyploidy, karyotype variation, and genome size evolution.

Changes in chromosome number and genome size arising from hybridization and polyploidization are major drivers of plant diversification. The orchid genus Calanthe, broadly distributed across tropical and subtropical Asia, is known to exhibit complex ploidy variation. Here we document, in South Korean natural populations, both diploid (2n = 40) and triploid (2n = 60) cytotypes among the surveyed species. We further report triploid cytotypes for the first time in South Korean natural populations of C. aristulifera, C. discolor, and C. sieboldii, and document diploid-triploid coexistence in natural populations of C. discolor and C. sieboldii. Nuclear DNA content (1 C values) varied 1.85-fold, spanning 7.85 to 14.50 pg, placing these taxa in the upper distribution range of the subfamily Epidendroideae. In the two mixed-ploidy taxa (C. discolor and C. sieboldii), 1Cx values (monoploid genome size) were broadly comparable between ploidy levels, providing no clear evidence of extensive genome downsizing in triploids. Triploid individuals showed no marked reduction in pollen stainability, with abundant pollen tetrads and aniline-blue-positive staining observed. Overall, our integrative cytogenetic data provide a baseline for subsequent molecular cytogenetic and phylogenomic analyses, facilitating rigorous tests of hypotheses on hybridization, polyploidization, and genome evolution underlying diversification in Calanthe.