Genetic diversity and population structure of Nuphar submersa (Nymphaeaceae), a critically endangered aquatic plant endemic to Japan, and implications for its conservation.

Nuphar submersa (Nymphaeaceae) is a critically endangered freshwater macrophyte indigenous to central Japan, with only four small extant populations represented across its entire range. We investigated the genotypic and genetic diversity as well as the genetic structure of all extant individuals of N. submersa based on analysis of 15 microsatellite loci. Among 278 individual ramets, 52 multilocus genotypes were detected: 30 genotypes in Nikko City (NIK), 18 in Nasukarasuyama City (NAS), 3 in Mooka City (MOK), and 1 in Sakura City (SAK). The average number of alleles per locus ranged from 1.20 to 1.93, whereas the observed and expected heterozygosities ranged from 0.11 to 0.33 and from 0.10 to 0.24, respectively. With the exception of SAK, all populations contained multiple clones, but our results indicated low levels of within-population genetic diversity. The populations NIK and NAS comprised few large or middle-sized genets and many small genets. The populations NIK and NAS were suggested to comprise large old, old fragmented, and/or young small genets resulting from seedling establishment. All four populations were differentiated, and gene flow between the populations was restricted (average level of gene flow (Nm) = 0.122, G'  = 0.639). Of the total genetic diversity, 67.20 and 9.13% were attributable to inter- and intra-population diversity, respectively. STRUCTURE analysis revealed two or three well-differentiated groups of populations. Cluster I comprised one population (NIK) and cluster II comprised the remaining populations at K = 2. The populations NIK, NAS, and the remaining populations were assigned to clusters I, II, and III, respectively, at K = 3. For conservation practices, we recommend that each cluster be regarded as a different management unit. We further suggest that artificial gene flow among MOK and SAK populations is an appropriate option, whereas NIK should not be reinforced with genotypes from the remaining populations.