Ploidy has been well studied and used extensively in the genus Opuntia to determine species boundaries, detect evidence of hybridization, and infer evolutionary patterns. We carried out chromosome counts for all members of the Humifusa clade to ascertain whether geographic patterns are associated with differences in ploidy. We then related chromosomal data to observed morphological variability, polyploid formation, and consequently the evolutionary history of the clade. We counted chromosomes of 277 individuals from throughout the ranges of taxa included within the Humifusa clade, with emphasis placed on the widely distributed species, Opuntia humifusa (Raf.) Raf., 1820 s.l. and Opuntia macrorhiza Engelm., 1850 s.l. We also compiled previous counts made for species in the clade along with our new counts to plot geographic distributions of the polyploid and diploid taxa. A phylogeny using nuclear ribosomal ITS sequence data was reconstructed to determine whether ploidal variation is consistent with cladogenesis. We discovered that diploids of the Humifusa clade are restricted to the southeastern United States (U.S.), eastern Texas, and southeastern New Mexico. Polyploid members of the clade, however, are much more widely distributed, occurring as far north as the upper midwestern U.S. (e.g., Michigan, Minnesota, Wisconsin). Morphological differentiation, although sometimes cryptic, is commonly observed among diploid and polyploid cytotypes, and such morphological distinctions may be useful in diagnosing possible cryptic species. Certain polyploid populations of Opuntia humifusa s.l. and Opuntia macrorhiza s.l., however, exhibit introgressive morphological characters, complicating species delineations. Phylogenetically, the Humifusa clade forms two subclades that are distributed, respectively, in the southeastern U.S. (including all southeastern U.S. diploids, polyploid Opuntia abjecta Small, 1923, and polyploid Opuntia pusilla (Haw.) Haw., 1812) and the southwestern U.S. (including all southwestern U.S. diploids and polyploids). In addition, tetraploid Opuntia humifusa s.l., which occurs primarily in the eastern U.S., is resolved in the southwestern diploid clade instead of with the southeastern diploid clade that includes diploid Opuntia humifusa s.l. Our results not only provide evidence for the polyphyletic nature of Opuntia humifusa and Opuntia macrorhiza, suggesting that each of these represents more than one species, but also demonstrate the high frequency of polyploidy in the Humifusa clade and the major role that genome duplication has played in the diversification of this lineage of Opuntia s.s. Our data also suggest that the southeastern and southwestern U.S. may represent glacial refugia for diploid members of this clade and that the clade as a whole should be considered a mature polyploid species complex. Widespread polyploids are likely derivatives of secondary contact among southeastern and southwestern diploid taxa as a result of the expansion and contraction of suitable habitat during the Pleistocene following glacial and interglacial events.
Keywords: Cactaceae; Opuntia humifusa; Opuntia macrorhiza; Pleistocene refugia; chromosome numbers; polyploid complex; polyploidy.