Phylogenetic, population structure, and population demographic analyses reveal that Vicia sepium in Japan is native and not introduced.

Vicia sepium (bush vetch) is a perennial legume widely distributed throughout the Eurasian continent. However, its distribution in Japan is limited to Mt. Ibuki and small parts of central and southern Hokkaido. Therefore, each Japanese V. sepium lineage has been considered to have been introduced separately from Europe. Here, we examined whether the species was introduced or not on the basis of cpDNA sequences and genome-wide SNPs from Japanese and overseas samples. Both the cpDNA haplotype network and the nuclear DNA phylogenetic tree showed that Japanese V. sepium is monophyletic. Furthermore, although the nuclear DNA phylogenetic tree also showed that each lineage is clearly monophyletic, genetic admixture of the genetic cluster dominated in the Hokkaido lineage was also detected in the Mt. Ibuki lineage. Population divergence analysis showed that the two lineages diverged during the last glacial period. The Mt. Ibuki lineage showed a sudden population decline 300-400 years ago, indicating that some anthropogenic activity might be involved, while the Hokkaido lineage showed a gradual population decline from 5000 years ago. Consequently, these two lineages show low current genetic diversity compared with overseas lineages. These results show that the Japanese V. sepium is not introduced but is native.

Mycorrhizal communities of two closely related species, Pyrola subaphylla and P. japonica, with contrasting degrees of mycoheterotrophy in a sympatric habitat.

Mycoheterotrophic plants typically form associations with a narrow range of mycorrhizal fungi. Consequently, mycorrhizal specialization is often considered to be an important step in mycoheterotrophic evolution. However, it remains unclear whether such specialization is likely to occur in plants of the genus Pyrola, which are generally associated with fungi in multiple ectomycorrhizal families. Here, we investigated the mycorrhizal communities of a nearly fully mycoheterotrophic Pyrola species (Pyrola subaphylla), a closely related partially mycoheterotrophic Pyrola species (Pyrola japonica), and a co-occurring autotrophic ectomycorrhizal tree, Quercus crispula, which is their potential carbon source, in a cool-temperate Japanese forest. High-throughput DNA sequencing revealed that numerous common ectomycorrhizal OTUs interact with the two Pyrola species and Q. crispula, thereby providing an opportunity to exploit a certain amount of carbon from common mycorrhizal networks. In addition, not only P. japonica but also P. subaphylla exhibited exceptionally high alpha mycobiont diversity, with 52 ectomycorrhizal OTUs belonging to 12 families being identified as P. subaphylla mycobionts and 69 ectomycorrhizal OTUs in 18 families being detected as P. japonica mycobionts. Nonetheless, the beta mycobiont diversity of P. subaphylla and P. japonica individuals was significantly lower than that of Q. crispula. Moreover, the beta mycobiont diversity of P. subaphylla was found to be significantly lower than that of P. japonica. Therefore, despite their seemingly broad mycorrhizal interactions, the two Pyrola species (particularly P. subaphylla) showed consistent fungal associations, suggesting that mycorrhizal specialization may have developed during the course of mycoheterotrophic evolution within the genus Pyrola.

Evidence for newly discovered albino mutants in a pyroloid: implication for the nutritional mode in the genus Pyrola.

PREMISE: Difficulties in comparing extremely divergent features in fully mycoheterotrophic plants with those in closely related chlorophyllous plants have complicated attempts to reveal the evolutionary patterns and processes of fully mycoheterotrophic plants. Albino mutants of partially mycoheterotrophic plants, generally observed in Orchidaceae, have provided an ideal model for investigating the evolution of mycoheterotrophy within similar genetic backgrounds. In 2018, we found a putative albino population of Pyrola (Ericaceae). Here we aimed to reveal the identity of the albino pyroloid and confirm its fully mycoheterotrophic status. METHODS: To reveal the putative albino pyroloid's identity, we examined its morphology and sequenced its chloroplast DNA. In addition, we assessed the trophic status of the putative albino pyroloid by analyzing chlorophyll fluorescence, chlorophyll concentration, and natural 13 C and 15 N abundances. RESULTS: We identified albino individuals as P. japonica-otherwise a partially mycoheterotrophic species. We confirmed their albino status by their considerably lower chlorophyll fluorescence and concentrations than those of sympatrically occurring chlorophyllous plants. 13 C abundance in the albino individuals was significantly higher than in the green individuals of P. japonica. CONCLUSIONS: This first report of albino mutants from partially mycoheterotrophic species in angiosperms other than orchids will play a valuable role in further studies focused on mycoheterotrophy. For instance, their δ13 C and δ15 N values represent a reference for fully mycoheterotrophic plants in Pyrola. Our findings also indicate the strong dependence of some leafy Pyrola species on fungal C during their entire life cycle.

Corrigendum: New microsatellite markers recognize differences in tandem repeats among four related Gastrodia species (Orchidaceae) [Genes Genet. Syst. (2019) 94, p. 225-229].

Tables 3 and 4 on p. 228 should be replaced with the corrected Tables 3 and 4 shown bellow.

New microsatellite markers recognize differences in tandem repeats among four related Gastrodia species (Orchidaceae).

Gastrodia is the most species-rich genus among mycoheterotrophic plants, and is thus an essential taxon to understand the mechanism of species diversification in mycoheterotrophs. In this study, we developed microsatellite markers with high transferability for four Gastrodia species to examine genetic differentiation and similarity among species, populations and individuals. The 12 microsatellite markers developed from a G. fontinalis library showed high transferability for the ramets that identified G. nipponica, G. kuroshimensis and G. takeshimensis. In addition to the high transferability of these markers, we observed low allele variation within a sampled population of each species and allele differences among the four species. The 12 markers described here will be useful for investigating the genetic differences among and within the Gastrodia species, which evolved by a limitation of gene flow.

First molecular phylogenetic insights into the evolution of Eriocaulon (Eriocaulaceae, Poales).

Eriocaulon is a genus of c. 470 aquatic and wetland species of the monocot plant family Eriocaulaceae. It is widely distributed in Africa, Asia and America, with centres of species richness in the tropics. Most species of Eriocaulon grow in wetlands although some inhabit shallow rivers and streams with an apparent adaptive morphology of elongated submerged stems. In a previous molecular phylogenetic hypothesis, Eriocaulon was recovered as sister of the African endemic genus Mesanthemum. Several regional infrageneric classifications have been proposed for Eriocaulon. This study aims to critically assess the existing infrageneric classifications through phylogenetic reconstruction of infrageneric relationships, based on DNA sequence data of four chloroplast markers and one nuclear marker. There is little congruence between our molecular results and previous morphology-based infrageneric classifications. However, some similarities can be found, including Fyson's sect. Leucantherae and Zhang's sect. Apoda. Further phylogenetic studies, particularly focusing on less well sampled regions such as the Neotropics, will help provide a more global overview of the relationships in Eriocaulon and may enable suggesting the first global infrageneric classification.

Development of microsatellite markers for the completely cleistogamous species Gastrodia takeshimensis (Orchidaceae) that are transferable to its chasmogamous sister G. nipponica.

We developed microsatellite markers to compare the genetic variation between the putatively cleistogamous Gastrodia takeshimensis (Orchidaceae) and its chasmogamous sister species G. nipponica. We expected low genetic variation in G. takeshimensis in view of its hypothesized cleistogamy. Eighteen primer pairs were developed from a G. takeshimensis genomic DNA library, and their characteristics were tested for G. takeshimensis and G. nipponica. Seven loci were polymorphic in G. nipponica, whereas all loci showed no polymorphism in G. takeshimensis. Genetic diversity was thus not detected in G. takeshimensis, and it seems to have been lost by repeated selfing in the completely closed flower. The 18 markers described here will be useful for investigating the genetic variation between a cleistogamous species and its chasmogamous sister species.

Comparative morphological analysis of two parallel mycoheterotrophic transitions reveals divergent and convergent traits in the genus Pyrola (Pyroleae, Ericaceae).

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.

Genetic analysis of Japanese and American specimens of Scirpus hattorianus suggests its introduction from North America.

Scirpus hattorianus is a possible alien species in Japan, and a clarification of its unclear taxonomy is required to reveal its origin. It is not known whether the plants initially described from Japan represent the same species distributed in North America. To clarify the origin of the species, we attempted to sequence old specimens collected about 80 years ago using newly designed primer pairs specific for short sequences, including the variable sites. Chloroplast sequences of ndhF were compared among Japanese and North American S. hattorianus, and the closely related species, S. atrovirens, S. flaccidifolius, and S. georgianus. We succeeded in sequencing all samples, and two haplotypes were detected in S. hattorianus: one was unique to the species and the other, detected from specimens potentially collected from the same population as the types, was shared by both North American S. hattorianus and two closely related species, S. atrovirens and S. flaccidifolius. Our results suggest that Japanese S. hattorianus is an alien species that was introduced from North America at least twice.

Development of microsatellite markers for partially and putative fully mycoheterotrophic varieties of Pyrola japonica sensu lato (Ericaceae).

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.

Variation in vegetative morphology tracks the complex genetic diversification of the mycoheterotrophic species Pyrola japonica sensu lato.

PREMISE OF THE STUDY: Although the evolution of full mycoheterotrophy has attracted many plant researchers, molecular phylogenetic studies that focus on the transition from partial to full mycoheterotrophy are limited to a few taxa. Pyrola japonica sensu lato is an ideal model for examining the evolution of mycoheterotrophy, owing to its variable leaf size, which suggests that the species comprises several transitional stages. METHODS: To elucidate the molecular and morphological changes that occur during the evolutionary transition between partial and full mycoheterotrophy in P. japonica s.l. from 18 populations in Japan, we estimated a parsimony network of plastid haplotypes based on three noncoding regions, measured the leaf size and scape color of the shoots, and compared morphology among haplotypes. KEY RESULTS: The seven haplotypes exhibited star-like relationships, and at least three divergent haplotypes were associated with differences in morphology. The first was mainly observed in large-leaved and green-scaped populations, whereas the second was observed in extremely small-leaved and reddish-scaped populations, which indicated a high degree of mycoheterotrophy, and the last was detected among mixed populations with both green- and reddish-scaped shoots with intermediate leaf sizes. In addition, the inconsistent association between the haplotypes and morphology suggests a complex relationship. CONCLUSIONS: 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.