Identification of Cupressaceae species from airborne pollen grains using chloroplastic markers: implications for reproductive interference evaluation in a remnant natural population of Chamaecyparis pisifera (Sieb. et Zucc.) Endl

Abstract

ABSTRACTMany wild populations of Chamaecyparis pisifera (Cupressaceae) are recently fragmented and surrounded by coniferous plantations. To distinguish the species composition of pollens that are dispersed to female flowers of C. pisifera, genetic markers (Chaps-trnL01 and Chaps-trnL02) were developed based on the differences in trnL (UAA) intron region of chloroplast DNA. This region in C. pisifera contains the site of the Cla I restriction enzyme and allows the distinction of C. pisifera from other Cupressaceae species after digestion of the products with the enzyme resulting in the development of cleaved amplified polymorphic sequence (CAPS) markers. The availability of markers was determined by single-pollen genotyping. Amplification success rates of 75.0, 58.3, and 100% were obtained for the pollens of C. pisifera, Chamaecyparis obtusa, and Cryptomeria japonica, respectively. Then, the markers and genotyping methods were applied to airborne pollen grains sampled from a remnant natural stand of C. pisifera. Over 90% of the pollen grains were identified as belonging to C. obtusa. Thus, we concluded that abundant heterospecific pollen dispersal was present during the flowering periods of C. pisifera. These results suggest that the remnant C. pisifera population might suffer reproductive interference due to the lower chances to be pollinated with conspecific pollens.