Genus Asarum (Aristolochiaceae) shows diverse floral morphology and is hypothesized to have diversified as a result of pollinator-mediated selection. Yet most aspects of their reproductive ecology, including pollinators, remain unclear. This study focuses on A. costatum and A. minamitanianum in Japan, a sister species pair having remarkable differences in calyx lobe length (10-20 mm and 70-180 mm, respectively). The objectives of this study are to elucidate multiple aspects of reproductive ecology of these two species and obtain evolutionary insights into floral organ elongation. We adopted combined approaches, including field observations, molecular analyses and cultivation experiments, such as pollinator observation for 3 years, fine-scale spatial genetic analysis of 769 individuals, paternity analysis based on 566 seeds over 4 years, and control pollination experiments. Both Asarum species had strong spatial genetic structures, indicating limited seed dispersal. Pollinator observation revealed that flies and ground-dwelling insects visited flowers of both species, but that the pollinator fauna differed between the species. The visitation rate of flies was extremely low but was more than twice as high in the species with an elongated floral appendage. Paternity analysis revealed A. minamitanianum was predominantly outcrossing, while A. costatum showed a wide range of selfing rates among fruits. These two Asarum species are likely adapted to fly pollination in the shady forest understorey, where available pollinator fauna is limited. In addition, although its function remains unclear, the elongated calyx lobe of A. minamitanianum could have evolved for effective pollen dispersal by attracting fly visitors.

Clinal variation is a major pattern of observed phenotypic diversity and identifying underlying demographic processes is a necessary step to understand the establishment of clinal variation. The wild ginger series Sakawanum (genus Asarum) comprises four taxa, which exhibit intertaxonomic clinal variation in calyx lobe length across two continental islands isolated by a sea strait. To test alternative hypotheses of the evolutionary history and to determine the implications for the formation of clinal variation, we conducted approximate Bayesian computation (ABC) analysis and ecological niche modeling (ENM). ABC analysis indicated that the scenario assuming multiple admixture events was strongly supported. This scenario assumed two admixture events occurred between morphologically distinct taxa, likely leading to the generation of intermediate taxa. One of the admixture events was estimated to have occurred during the last glacial maximum (LGM), during which the taxa were estimated to have formed a common refugia in southern areas by ENM analysis. Although four taxa are currently distributed allopatrically on different islands and trans-oceanic dispersal appears unlikely, the formation of a land bridge and the geographic range shift to refugia would have allowed secondary contact between previously isolated taxa. This study suggests that clinal variation can be shaped by demographic history including multiple admixtures due to climatic oscillations.

To investigate the therapeutic effect of high-dosage gamma-aminobutyric acid (GABA) on acute tetramine (TET) poisoning, 50 Kunming mice were divided into 5 groups at random and the antidotal effects of GABA or sodium dimercaptopropane sulfonate (Na-DMPS) on poisoned mice in different groups were observed in order to compare the therapeutic effects of high-dosage GABA with those of Na-DMPS. Slices of brain tissue of the poisoned mice were made to examine pathological changes of cells. The survival analysis was employed. Our results showed that both high-dosage GABA and Na-DMPS could obviously prolong the survival time, delay onset of convulsion and muscular twitch, and ameliorate the symptoms after acute tetramine poisoning in the mice. Better effects could be achieved with earlier use of high dosage GABA or Na-DMPS. There was no significant difference in prolonging the survival time between high-dose GABA and Na-DMPS used immediately after poisioning. It is concluded that high-dosage GABA can effectively antagonize acute toxicity of teramine in mice. And it is suggested that high-dosage GABA may be used as an excellent antidote for acute TET poisoning in clinical practice. The indications and correct dosage for clinical use awaits to be further studied.