Pyrola japonica s.l. has at least three separate genetic lineages that have different leaf morphologies. The genetic lineages and their coexistence could have led to the variable leaf size and suggest the possibility that gene flow from partial to full mycoheterotrophs could reverse the evolutionary transition to full mycoheterotrophy.

The genus Pyrola includes species with different degree of mycoheterotrophy; some species possess individuals that rely on all carbon through their associations with fungi (full mycoheterotrophy, FM), whereas some species obtain carbon through both fungi and photosynthesis by itself (partial mycoheterotrophy, PM). To investigate how plant functional traits of photosynthesis and reproduction are related to the degree of mycoheterotrophy in the initial stage of the transition from PM to FM, we determined morphological traits in FM (or nearly FM) and PM species in two independent lineages, P. picta and P. japonica complexes. We used herbarium specimens and examined leaf number, leaf area, flower number, and scape length in FM or nearly FM species (P. aphylla and P. subaphylla) and PM species (P. picta s.l. and P. japonica). We found a leaf area reduction in FM (or nearly FM) species in both lineages, suggesting that this is a convergent trait. The number of flowers was not significantly different between FM (or nearly FM) and PM species in both lineages. On the other hand, differences in the variation between FM (or nearly FM) and PM species were found in some traits between the two lineages. The FM (or nearly FM) species in one lineage only possessed rudimentary leaves, whereas that in the other linage possessed a few small, ordinary leaves in addition to those with only rudimentary leaves. The scape length of the FM (or nearly FM) species was significantly longer than that of PM species in one lineage, whereas it was shorter in the other lineage. The different and common variations are divergent and convergent traits, respectively, that could be associated with the transition to FM in Pylora. In addition, shoots of both PM species occasionally lacked ordinary leaves, possibly indicating possession of these shoots is preadaptation for the transition to FM in Pyrola.

We conclude that the relative difference in light availability between summer and winter leaves is a critical factor for regulation of leaf shedding, consistent with the theory of maximization of the whole-plant photosynthesis.

Premise of the study:Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum (Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan.Methods and Results:Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in at least one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged from 0.00 to 0.92 and 0.00 to 0.71, respectively.Conclusions:The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.

We developed microsatellite markers to compare the genetic variation and reproductive biology between the partially mycoheterotrophic Pyrola japonica var. japonica and the putative fully mycoheterotrophic P. japonica var. subaphylla. Fifteen primer pairs were developed for P. japonica sensu lato and they were tested on 77 ramets from three populations of the two varieties. Thirteen loci were polymorphic in at least one of the two var. japonica populations, whereas only four loci were polymorphic in the var. subaphylla population. The considerably lower genetic variation of the var. subaphylla population may be attributed to frequent selfing and/or inbreeding. The markers developed in this study will be useful for comparing the genetic diversity of P. japonica s. l. populations and measuring gene flow within and between populations and varieties.