Furthermore many intermediate plants did not show additive
Furthermore, many intermediate plants did not show additive combinations of parental allozymes, which would be expected in F1 hybrids. The absence of some characteristic parental bands suggests that sexual reproduction has occurred among the hybrid plants. Genetic variations within and among the intermediate populations are likely to be brought about by the sexual reproduction among the hybrids beyond the F1. We suppose that extensive segregation has occurred from intercrosses between parent species or between independently produced hybrids as well as backcrossing of hybrids with one of the parent species. During our field study, we observed that some populations of the J-O (GI-3, OK-6, KA-2) and J-S groups (NI-5, HI-2a, TO-2, TO-3b, MY-1bc) had lower fruit set and pollen stainability than their putative parent species (T. Shiga and Y. Kadono, unpublished data). This observation agrees well with the fact that interspecific hybrids are often less fertile than their parents (cf. Arnold, 1997), and suggests that the J-O and J-S plant groups were of hybrid origin. All of the combinations of crosses among N. japonica, N. oguraensis, N. subintegerrima and intermediate plants produced F1 progeny in our crossability test (T. Shiga and Y. Kadono, unpublished data). The chromosome number of Nuphar is 2n=34, and it has been interpreted as EZLink Sulfo-NHS-SS-Biotin Kit with x=17 (Langlet and Søderberg, 1927, Heslop-Harrison, 1953, Okada and Tamura, 1981). Pollinators of Nuphar have been known to be flies, bees and beetles and they do not identify floral morphological differences among species (Lippok et al., 2000). It is probable that interspecific crosses frequently occur, if the populations of different species stand in neighbor. Reproductive isolation mechanisms might be weak in Nuphar species and promote introgressive hybridization among these plants. Genotypic diversities of populations of the putative parental species were lower than that of intermediate populations. Some populations of J-O and J-S groups included plants that were genetically same as the plants of putative parental species. Introgressive hybridization may occur and include plants of parental species in some populations. Shiga and Kadono (2004) identified the two intermediate morphological groups by averages of morphological characters of each population. Further morphology and molecular analyses based on individual data are needed to discuss the hybridizaion and introgression events in each population. The three Japanese Nuphar species were well distinguished by patterns of allele frequencies and PCO analysis (Table 2, Fig. 3). Although N. japonica had species-specific alleles with low frequencies, N. oguraensis and N. subintegerrima had diagnostic loci and were distinguishable. Although a recent taxonomic study of Nuphar worldwide treated N. subintegerrima as a synonym of N. japonica (Padgett et al., 1999, Padgett et al., 2002), the present results indicate that these two species differed in genetical features. Several isozymes in our study (MDH, PGI and TPI) revealed duplicated loci (Fig. 2). Isozyme duplication has also been reported in isozyme studies of N. pumila var. ozeensis (Miki) H.Hara and intermediate plants, identified as “N. subintegerrima” (, Murayama et al., 1998). discussed that intermediate populations of Nuphar in Japan are of hybrid origin based on allozyme data of in which enzyme loci of an intermediate population are duplicated. However, our allozyme analysis revealed that isozyme duplication was not restricted to intermediate plants, but also occurred in MDH, PGI and TPI in N. japonica, N. oguraensis and N. subintegerrima. Furthermore, these isozymes were also duplicated in N. pumila (Shiga and Kadono, 2007a). Although the Nuphar species have been considered diploid (Langlet and Søderberg, 1927, Okada and Tamura, 1981), these isozyme duplications suggest that sect. Nuphar is genetically polyploid. It is probable that these duplications occurred early in the evolution of the genus Nuphar.