Stable Isotope Signatures Illuminate Diverse Nutritional Strategies in Rhizoctonias and Their Orchid Partners.

Understanding the nutritional ecology of orchids, particularly those in symbiosis with rhizoctonias, presents a complex challenge. This complexity arises partly from the absence of macroscopic fruit bodies in rhizoctonias, which impedes the acquisition of their stable isotope data. In this study, we investigated the fungal associations and isotopic signatures in the pelotons of Stigmatodactylus sikokianus (associated with non-ectomycorrhizal [non-ECM] rhizoctonias) and Chamaegastrodia shikokiana (associated with ECM rhizoctonias). Our research reveals elevated levels of 13C enrichment in S. sikokianus plants and their pelotons, similar to those found in fully mycoheterotrophic orchids and their mycobionts. Interestingly, C. shikokiana plants and their pelotons exhibited even higher levels of 13C and 15N enrichment than many other fully mycoheterotrophic species. Our findings imply that both ECM and saprotrophic mycobionts, including certain rhizoctonias, can fulfill the carbon needs of highly mycoheterotrophic orchids. This finding also indicates that 13C enrichment can be an indicator of mycoheterotrophy in at least some rhizoctonia-associated orchids, despite the typically low 13C enrichment in non-ECM rhizoctonias. Our demonstration of partial mycoheterotrophy in S. sikokianus suggests a broader prevalence of this nutritional strategy among orchids, given that almost all orchids are associated with non-ECM rhizoctonias.

Variable, life stage-dependent mycorrhizal specificity and its developmental consequences in the fully myco-heterotrophic orchid Cyrtosia septentrionalis.

The degree of specificity between fully myco-heterotrophic (MH) orchids and mycorrhizal fungi is regarded as high, but some species undergo a mycobiont shift as they transition from juvenile to adult plant. We investigated morphological and physiological aspects of the interaction between the fully MH Cyrtosia septentrionalis and its four known mycobionts to elucidate developmental consequences of variable, life-stage-dependent specificity. Of five randomly sampled germinated seeds co-cultured with each mycobiont, Physisporinus sp. 'TK-10' colonized all, Armillaria gallica and Desarmillaria tabescens colonized one, and A. mellea subsp. nipponica colonized none. Whereas 16.2% of aseptically germinated seeds exhibited onward growth when co-cultured with TK-10, just 1.5-2.6% did so with the other species. Even so, A. gallica colonized and formed internal rhizomorphs within rhizomes established with TK-10, suggesting that this mycobiont can replace and potentially can oust the latter. We infer that the orchid can associate with Armillaria and Desarmillaria mycobionts throughout its life, but that TK-10 enhances early growth. However, because TK-10 has a higher wood-rotting capacity than A. gallica, rapid resource exhaustion may cause nutrient supply shortages. We hypothesize that secondary colonization by Armillaria or Desarmillaria species triggers TK-10 displacement and that this mycobiont shift sustains orchid growth for longer.

Do exchangeable hydrogens affect the evaluation of partial mycoheterotrophy in orchids? Insights from δ2H analysis in bulk, α-cellulose, and cellulose nitrate samples.

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δ2H), carbon (δ13C), and nitrogen (δ15N) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δ13C, δ15N, and δ2H analyses on bulk samples. Additionally, we conducted δ2H analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δ2H of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δ13C, its δ2H was higher than those of the autotrophs. Notably, among all response variables, δ15N and δ2H exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δ15N and δ2H benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

Relictithismia kimotsukiensis, a new genus and species of Thismiaceae from southern Japan with discussions on its phylogenetic relationship.

The family Thismiaceae, known as "fairy lanterns" for their urn- or bell-shaped flowers with basally fused tepals, consists of non-photosynthetic flowering monocots mainly in tropical regions, extending into subtropical and temperate areas. Here, we propose a new mycoheterotrophic genus, Relictithismia Suetsugu & Tagane (Thismiaceae), with its monotypic species Relictithismia kimotsukiensis Suetsugu, Yas.Nakam. & Tagane from Kimotsuki Mountains in the Osumi Peninsula, Kagoshima Prefecture, Kyushu Island, southern Japan. Relictithismia resembles Haplothismia Airy Shaw in having a cluster of tuberous roots, a feature previously observed only in this genus within the family Thismiaceae. However, it differs in having solitary flowers (vs. 2-6-flowered pseudo-raceme in Haplothismia), anther thecae largely separated (vs. connate), and the presence of an annulus (vs. absent). Additionally, Relictithismia differs from the geographically overlapping genus Thismia Griff. in its stamen structure and the position of the annulus. In Relictithismia, the stamens lack connectives, and its free filaments arise from the annulus located inside the perianth mouth, while in Thismia, the stamens typically have connate connectives, forming a staminal tube pendulous from the annulus located at the mouth of the floral tube. Our morphological and phylogenetic data indicated that R. kimotsukiensis holds an early-diverging position within the family, situated outside the Old World Thismia clade. This paper offers an extensive description and color photographs of R. kimotsukiensis, complemented by notes on its phylogenetic relationship and evolutionary history.

The absence of bumblebees on an oceanic island blurs the species boundary of two closely related orchids.

Oceanic islands offer valuable natural laboratories for studying evolution. The Izu Islands, with their recent geological origin, provide an exceptional opportunity to explore the initial evolution on oceanic islands. Another noteworthy aspect is the absence of bumblebee species on most Izu Islands. We used ecological, morphological, and molecular data to investigate the impact of bumblebee absence on the evolution of two closely related orchid species, Goodyera henryi and Goodyera similis, focusing on Kozu Island, the Izu Islands. Our investigation revealed that while G. henryi exclusively relies on a bumblebee species for pollination on the mainland, G. similis is pollinated by scoliid wasps on both the mainland and the island. Intriguingly, all specimens initially categorized as G. henryi on Kozu Island are hybrids of G. henryi and G. similis, leading to the absence of pure G. henryi distribution on the island. These hybrids are pollinated by the scoliid wasp species that also pollinates G. similis on the island. The absence of bumblebees might result in sporadic and inefficient pollination of G. henryi by scoliid wasps, consequently promoting hybrid proliferation on the island. Our findings suggest that the absence of bumblebees can blur plant species boundaries.

Phylogeographical evidence for historical long-distance dispersal in the flightless stick insect Ramulus mikado.

Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.

Autoactivation of mycorrhizal symbiosis signaling through gibberellin deactivation in orchid seed germination.

Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis, in which the plant hormone gibberellin (GA) negatively affects fungal colonization and development, to establish parasitic symbiosis. Although GA generally promotes seed germination in photosynthetic plants, previous studies have reported low sensitivity of GA in seed germination of mycoheterotrophic orchids where mycorrhizal symbiosis occurs concurrently. To elucidate the connecting mechanisms of orchid seed germination and mycorrhizal symbiosis at the molecular level, we investigated the effect of GA on a hyacinth orchid (Bletilla striata) seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead autoactivate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Impact of mycoheterotrophy on the growth of Gentiana zollingeri (Gentianaceae), as suggested by size variation, morphology, and 13C abundance of flowering shoots.

Gentiana zollingeri is an annual photosynthetic plant that employs a mycoheterotrophic growth strategy during its underground seedling stage (initial mycoheterotrophy). Notably, the morphological characteristics of its flowering shoots, such as shoot size, leaf size, and leaf color, are highly variable, and it was hypothesized that these variations may be linked to nutritional mode. The morphological characteristics of G. zollingeri individuals were thus investigated alongside environmental factors, 13C abundance, and diversity of colonizing arbuscular mycorrhizal (AM) fungi. The majority of G. zollingeri flowering individuals were found to exhibit a high affinity for the specific AM fungi that exclusively colonize roots of the mycoheterotrophic seedlings, while other phylogenetically diverse AM fungi could also be detected. The leaves to shoot dry weight ratio (leaf ratio) was negatively correlated with the canopy openness in the habitat, suggesting that leaf development is impeded in sunny conditions. Furthermore, the shoot weight of G. zollingeri was positively correlated with leaf 13C abundance. Given that 13C enrichment can provide indirect evidence of mycoheterotrophy in AM plants, the results suggest that the utilization of carbon obtained through mycoheterotrophy, at least during the underground seedling stage, is crucial for G. zollingeri.

Spiranthes hachijoensis (Orchidaceae), a new species within the S. sinensis species complex in Japan, based on morphological, phylogenetic, and ecological evidence.

The systematics of the Old World Spiranthes sinensis (Pers.) Ames species complex (Orchidaceae) has been complicated by its wide distribution and morphological variations. Within the species complex, S. australis Lindl. has been generally accepted as the only Spiranthes Rich. species distributed on the Japanese mainland. The present study provides morphological, phylogenetic, and ecological evidence for the recognition of S. hachijoensis Suetsugu as a new species of the S. sinensis species complex on the Japanese mainland. Spiranthes hachijoensis is morphologically similar to S. hongkongensis S.Y. Hu & Barretto and S. nivea T.P. Lin & W.M. Lin, sharing a degenerated rostellum, pollinia without a viscidium, and distinctly trilobed stigma. However, the taxon can be morphologically distinguished from S. hongkongensis by its glabrous rachis, ovaries, and sepals, and from S. nivea by its papillate labellum disc, larger papillate basal labellum callosities, and glabrous rachis, ovaries, and sepals. The autogamy and flowering phenology (i.e., earlier flowering) of S. hachijoensis are most likely responsible for premating isolation from the sympatric S. australis. A MIG-seq-based high-throughput molecular analysis indicated that the genetic difference between S. hachijoensis and its putative sister species S. sinensis is comparable to, or even greater than, the genetic difference between pairs of other species within the S. sinensis species complex. Our multifaceted approach strongly supports the recognition of S. hachijoensis as a morphologically, phenologically, phylogenetically, and ecologically distinct species.

Mitochondrial genome sequencing and analysis of the invasive Microstegium vimineum: a resource for systematics, invasion history, and management.

Premise of the Research: Plants remain underrepresented among species with sequenced mitochondrial genomes (mitogenomes), due to the difficulty in assembly with short-read technology. Invasive species lag behind crops and other economically important species in this respect, representing a lack of tools for management and land conservation efforts. Methodology: The mitogenome of Microstegium vimineum, one of the most damaging invasive plant species in North America, was sequenced and analyzed using long-read data, providing a resource for biologists and managers. We conducted analyses of genome content, phylogenomic analyses among grasses and relatives based on mitochondrial coding regions, and an analysis of mitochondrial single nucleotide polymorphism in this invasive grass species. Pivotal Results: The assembly is 478,010 bp in length and characterized by two large, inverted repeats, and a large, direct repeat. However, the genome could not be circularized, arguing against a "master circle" structure. Long-read assemblies with data subsets revealed several alternative genomic conformations, predominantly associated with large repeats. Plastid-like sequences comprise 2.4% of the genome, with further evidence of Class I and Class II transposable element-like sequences. Phylogenetic analysis placed M. vimineum with other Microstegium species, excluding M. nudum, but with weak support. Analysis of polymorphic sites across 112 accessions of M. vimineum from the native and invasive ranges revealed a complex invasion history. Conclusions: We present an in-depth analysis of mitogenome structure, content, phylogenetic relationships, and range-wide genomic variation in M. vimineum's invasive US range. The mitogenome of M. vimineum is typical of other andropogonoid grasses, yet mitochondrial sequence variation across the invasive and native ranges is extensive. Our findings suggest multiple introductions to the US over the last century, with subsequent spread, secondary contact, long-distance dispersal, and possibly post-invasion selection on awn phenotypes. Efforts to produce genomic resources for invasive species, including sequenced mitochondrial genomes, will continue to provide tools for their effective management, and to help predict and prevent future invasions.

Transcriptomic heterochrony and completely cleistogamous flower development in the mycoheterotrophic orchid Gastrodia.

Cleistogamy, in which plants can reproduce via self-fertilization within permanently closed flowers, has evolved in > 30 angiosperm lineages; however, consistent with Darwin's doubts about its existence, complete cleistogamy - the production of only cleistogamous flowers - has rarely been recognized. Thus far, the achlorophyllous orchid genus, Gastrodia, is the only known genus with several plausible completely cleistogamous species. Here, we analyzed the floral developmental transcriptomes of two recently evolved, completely cleistogamous Gastrodia species and their chasmogamous sister species to elucidate the possible changes involved in producing common cleistogamous traits. The ABBA-BABA test did not support introgression and protein sequence convergence as evolutionary mechanisms leading to cleistogamy, leaving convergence in gene expression as a plausible mechanism. Regarding transcriptomic differentiation, the two cleistogamous species had common modifications in the expression of developmental regulators, exhibiting a gene family-wide signature of convergent expression changes in MADS-box genes. Our transcriptomic pseudotime analysis revealed a prolonged juvenile state and eventual maturation, a heterochronic pattern consistent with partial neoteny, in cleistogamous flower development. These findings indicate that transcriptomic partial neoteny, arising from changes in the expression of conserved developmental regulators, might have contributed to the rapid and repeated evolution of cleistogamous flowers in Gastrodia.

Monotropastrum kirishimense (Ericaceae), a new mycoheterotrophic plant from Japan based on multifaceted evidence.

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.