Plastid DNA Analysis Reveals Cryptic Hybridization in Invasive Dalmatian Toadflax (Linaria dalmatica) Populations

Gene flow between Dalmatian toadflax (DT) and yellow toadflax (YT), both aggressive invaders throughout the Intermountain West, is creating hybrid populations potentially more invasive than either parent species. To determine the direction of gene flow in these hybrid populations, species-diagnostic cytoplasmic markers were developed. Markers were based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) polymorphisms in the trnT-D chloroplast DNA (cpDNA) region digested with Alu1, and single-nucleotide polymorphisms (SNPs) in the matK and trnL-F chloroplast-barcoding regions. Four hybrid toadflax populations sampled from Colorado, Montana, and Washington contained both DT and YT cytoplasm, with YT predominating; 25 individuals from a fifth hybrid population from Idaho all had identical YT cpDNA haplotypes. Thirteen plants from two Colorado populations, assumed to be DT based on morphology and geographic isolation from any known YT population, were found to have YT cpDNA haplotypes. These results indicate that gene flow between invasive YT and DT populations is more widespread that previously realized and confirms that cryptic introgression of YT alleles has occurred in multiple western U.S. DT populations. The presence of YT genetic material in presumed DT populations may negatively affect host recognition and establishment by biocontrol agents used for toadflax management. Nomenclature: Dalmatian toadflax, Linaria dalmatica (L.) P. Mill. ; yellow toadflax, Linaria vulgaris P. Mill. Management Implications: Yellow toadflax (YT) and Dalmatian toadflax (DT) are not reported to hybridize in their native European ranges, but cross-pollination between these two invasive species has been confirmed in several Rocky Mountain states. This produces novel hybrid-toadflax populations that are more vigorous and robust than either parent species, presenting even greater management challenges. Experimental hand-pollinations under controlled greenhouse conditions to produce hybrid toadflax plants previously showed that hybrids were more likely to result from pollination of YT by DT than vice versa, and results of DNA analysis in this study indicate this is also true when cross-pollination occurs in the field. Hybrid toadflax presents particular problems for biocontrol. Two stem-boring weevils, Mecinus janthinus and Mecinus janthiniformis, are released as approved biocontrol agents on YT and DT, respectively; however, each of these weevils exhibits a strong preference for its natural host toadflax species. Whether weevils will establish on, and control, hybrid toadflax infestations is unknown. An unexpected finding of this study was that some Colorado toadflax populations, presumed to be pure DT based on their morphology and habitat, in fact, contained YT DNA. This is most likely the result of previous hybridization between YT and DT, followed by repeated crossing back to DT, and suggests that hybridization between these two invasive toadflaxes is more widespread and has been occurring for longer than previously realized. The presence of DNA from the nonpreferred toadflax species in a host-plant population may explain why weevil releases fail to establish at apparently favorable sites, and undetected transfer of DNA between YT and DT could undermine the efficacy of biocontrol in managing toadflax invasion.