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.

Mode of carbon gain and fungal associations of Neuwiedia malipoensis within the evolutionarily early-diverging orchid subfamily Apostasioideae.

BACKGROUND AND AIMS: The earliest-diverging orchid lineage, Apostasioideae, consists only of two genera: Apostasia and Neuwiedia. Previous reports of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages. METHODS: We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundances (δ13C, δ15N, δ2H, δ18O) of different developmental stages with those of autotrophic reference plants. KEY RESULTS: Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and the 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults were predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition. Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon. CONCLUSIONS: The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows various habitat options in the earliest-diverging orchid lineage, Apostasioideae. While A. nipponica relies on the heterotrophic carbon gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic carbon gain as an adult, allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur.

Organellar phylogenomics at the epidendroid orchid base, with a focus on the mycoheterotrophic Wullschlaegelia.

BACKGROUND AND AIMS: Heterotrophic plants have long been a challenge for systematists, exemplified by the base of the orchid subfamily Epidendroideae, which contains numerous mycoheterotrophic species. METHODS: Here we address the utility of organellar genomes in resolving relationships at the epidendroid base, specifically employing models of heterotachy, or lineage-specific rate variation over time. We further conduct comparative analyses of plastid genome evolution in heterotrophs and structural variation in matK. KEY RESULTS: We present the first complete plastid genomes (plastomes) of Wullschlaegelia, the sole genus of the tribe Wullschlaegelieae, revealing a highly reduced genome of 37 kilobases, which retains a fraction of the genes present in related autotrophs. Plastid phylogenomic analyses recovered a strongly supported clade composed exclusively of mycoheterotrophic species with long branches. We further analyzed mitochondrial gene sets, which recovered similar relationships to those in other studies using nuclear data, but the placement of Wullschlaegelia remains uncertain. We conducted comparative plastome analyses among Wullschlaegelia and other heterotrophic orchids, revealing a suite of correlated substitutional and structural changes relative to autotrophic species. Lastly, we investigated evolutionary and structural variation in matK, which is retained in Wullschlaegelia and a few other 'late stage' heterotrophs and found evidence for structural conservation despite rapid substitution rates in both Wullschlaegelia and the leafless Gastrodia. CONCLUSIONS: Our analyses reveal the limits of what the plastid genome can tell us on orchid relationships in this part of the tree, even when applying parameter-rich heterotachy models. Our study underscores the need for increased taxon sampling across all three genomes at the epidendroid base, and illustrates the need for further research on addressing heterotachy in phylogenomic analyses.

Plastid genome evolution in leafless members of the orchid subfamily Orchidoideae, with a focus on Degranvillea dermaptera.

PREMISE: Leafless, heterotrophic plants are prime examples of organismal modification, the genomic consequences of which have received considerable interest. In particular, plastid genomes (plastomes) are being sequenced at a high rate, allowing continual refinement of conceptual models of reductive evolution in heterotrophs. However, numerous sampling gaps exist, hindering the ability to conduct comprehensive phylogenomic analyses in these plants. METHODS: Using floral tissue from an herbarium specimen, we sequenced and analyzed the plastome of Degranvillea dermaptera, a rarely collected, leafless orchid species from South America about which little is known, including its phylogenetic affinities. RESULTS: The plastome is the most reduced of those sequenced among the orchid subfamily Orchidoideae. In Degranvillea, it has lost the majority of genes found in leafy autotrophic species, is structurally rearranged, and has similar gene content to the most reduced plastomes among the orchids. We found strong evidence for the placement of Degranvillea within the subtribe Spiranthinae using models that explicitly account for heterotachy, or lineage-specific evolutionary rate variation over time. We further found evidence of relaxed selection on several genes and of correlations among substitution rates and several other "traits" of the plastome among leafless members of orchid subfamily Orchidoideae. CONCLUSIONS: Our findings advance knowledge on the phylogenetic relationships and paths of plastid genome evolution among the orchids, which have experienced more independent transitions to heterotrophy than any other plant family. This study demonstrates the importance of herbarium collections in comparative genomics of poorly known species of conservation concern.

Exploring mycorrhizal diversity in sympatric mycoheterotrophic plants: a comparative study of Monotropastrum humile var. humile and M. humile var. glaberrimum.

Mycoheterotrophic plants (MHPs) rely on their mycorrhizal fungus for carbon and nutrient supply, thus a shift in mycobionts may play a crucial role in speciation. This study aims to explore the mycorrhizal diversity of two closely related and sympatric fully MHPs, Monotropastrum humile var. humile (Mhh) and M. humile var. glaberrimum (Mhg), and determine their mycorrhizal associations. A total of 1,108,710 and 1,119,071 ectomycorrhizal fungal reads were obtained from 31 Mhh and 31 Mhg, and these were finally assigned to 227 and 202 operational taxonomic units, respectively. Results show that sympatric Mhh and Mhg are predominantly associated with different fungal genera in Russulaceae. Mhh is consistently associated with members of Russula, whereas Mhg is associated with members of Lactarius. Associating with different mycobionts and limited sharing of fungal partners might reduce the competition and contribute to their coexistence. The ectomycorrhizal fungal communities are significantly different among the five forests in both Mhh and Mhg. The distinct mycorrhizal specificity between Mhh and Mhg suggests the possibility of different mycobionts triggered ecological speciation between sympatric species.

Effects of fungicide treatments on mycorrhizal communities and carbon acquisition in the mixotrophic Pyrola japonica (Ericaceae).

Pyrola japonica, a member of the family Ericaceae, is a mixotroph that grows on forest floors and obtains carbon (C) from both its photosynthesis and its mycorrhizal fungi. Its mycorrhizal community is dominated by Russulaceae. However, the mechanism of its C acquisition and its flexibility are not well understood. Our aim was to assess the impact of disturbance of the mycorrhizal fungal communities on C acquisition by P. japonica. We repeatedly applied a fungicide (Benomyl) to soils around P. japonica plants in a broad-leaved forest of central Japan, in order to disturb fungal associates near roots. After fungicide treatment, P. japonica roots were collected and subjected to barcoding by next-generation sequencing, focusing on the ITS2 region. The rate of mycorrhizal formation and α-diversity did not significantly change upon fungicide treatments. Irrespective of the treatments, Russulaceae represented more than 80% of the taxa. Leaves and seeds of the plants were analysed for 13C stable isotope ratios that reflect fungal C gain. Leaf and seed δ13C values with the fungicide treatment were significantly lower than those with the other treatments. Thus the fungicide did not affect mycorrhizal communities in the roots, but disturbed mycorrhizal fungal pathways via extraradical hyphae, and resulted in a more photosynthetic behaviour of P. japonica for leaves and seeds.

Stoichiometry of carbon, nitrogen and phosphorus is closely linked to trophic modes in orchids.

BACKGROUND: Mycorrhiza is a ubiquitous form of symbiosis based on the mutual, beneficial exchange of resources between roots of autotrophic (AT) plants and heterotrophic soil fungi throughout a complex network of fungal mycelium. Mycoheterotrophic (MH) and mixotrophic (MX) plants can parasitise this system, gaining all or some (respectively) required nutrients without known reciprocity to the fungus. We applied, for the first time, an ecological stoichiometry framework to test whether trophic mode of plants influences their elemental carbon (C), nitrogen (N), and phosphorus (P) composition and may provide clues about their biology and evolution within the framework of mycorrhizal network functioning. RESULTS: We analysed C:N:P stoichiometry of 24 temperate orchid species and P concentration of 135 species from 45 plant families sampled throughout temperate and intertropical zones representing the three trophic modes (AT, MX and MH). Welch's one-way ANOVA and PERMANOVA were used to compare mean nutrient values and their proportions among trophic modes, phylogeny, and climate zones. Nutrient concentration and stoichiometry significantly differentiate trophic modes in orchids. Mean foliar C:N:P stoichiometry showed a gradual increase of N and P concentration and a decrease of C: nutrients ratio along the trophic gradient AT < MX < MH, with surprisingly high P requirements of MH orchids. Although P concentration in orchids showed the trophy-dependent pattern regardless of climatic zone, P concentration was not a universal indicator of trophic modes, as shown by ericaceous MH and MX plants. CONCLUSION: The results imply that there are different evolutionary pathways of adaptation to mycoheterotrophic nutrient acquisition, and that the high nutrient requirements of MH orchids compared to MH plants from other families may represent a higher cost to the fungal partner and consequently lead to the high fungal specificity observed in MH orchids.

Habitat-related plastome evolution in the mycoheterotrophic Neottia listeroides complex (Orchidaceae, Neottieae).

BACKGROUND: Mycoheterotrophs, acquiring organic carbon and other nutrients from mycorrhizal fungi, have evolved repeatedly with substantial plastid genome (plastome) variations. To date, the fine-scale evolution of mycoheterotrophic plastomes at the intraspecific level is not well-characterized. A few studies have revealed unexpected plastome divergence among species complex members, possibly driven by various biotic/abiotic factors. To illustrate evolutionary mechanisms underlying such divergence, we analyzed plastome features and molecular evolution of 15 plastomes of Neottia listeroides complex from different forest habitats. RESULTS: These 15 samples of Neottia listeroides complex split into three clades according to their habitats approximately 6 million years ago: Pine Clade, including ten samples from pine-broadleaf mixed forests, Fir Clade, including four samples from alpine fir forests and Fir-willow Clade with one sample. Compared with those of Pine Clade members, plastomes of Fir Clade members show smaller size and higher substitution rates. Plastome size, substitution rates, loss and retention of plastid-encoded genes are clade-specific. We propose to recognized six species in N. listeroides complex and slightly modify the path of plastome degradation. CONCLUSIONS: Our results provide insight into the evolutionary dynamics and discrepancy of closely related mycoheterotrophic orchid lineages at a high phylogenetic resolution.

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.

Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons.

Stable isotope signatures of fungal sporocarps have been instrumental in identifying carbon gains of chlorophyllous orchids from a fungal source. Yet, not all mycorrhizal fungi produce macroscopic sporocarps and frequently fungi of different taxa occur in parallel in orchid roots. To overcome this obstacle, we investigated stable isotope signatures of fungal pelotons extracted from orchid roots and compared these data to the respective orchid and reference plant tissues. Anoectochilus sandvicensis and Epipactis palustris represented specialized or unspecialized rhizoctonia-associated orchids. Epipactis atrorubens and Epipactis leptochila are orchids considered ectomycorrhiza-associated with different preferences for Basidio- and Ascomycota. 13 C enrichment of rhizoctonia pelotons was minor compared with plant tissues and significantly lower than enrichments of pelotons from ectomycorrhizal Epipactis species. 15 N values of pelotons from E. leptochila and E. atrorubens showed similar patterns as known for respective sporocarps of ectomycorrhizal Ascomycota and Basidiomycota, however, with an offset towards lower 15 N enrichments and nitrogen concentrations. Our results suggest an explicit fungal nutrition source of orchids associated with ectomycorrhizal fungi, whereas the low 13 C enrichment in rhizoctonia-associated orchids and fungal pelotons hamper the detection of carbon gains from fungal partners. 15 N isotopic pattern of orchids further suggests a selective transfer of 15 N-enriched protein-nitrogen into orchids.

At the core of the endomycorrhizal symbioses: intracellular fungal structures in orchid and arbuscular mycorrhiza.

Arbuscular (AM) and orchid (OrM) mycorrhiza are the most widespread mycorrhizal symbioses among flowering plants, formed by distinct fungal and plant species. They are both endosymbioses because the fungal hyphae can enter inside the plant cell to develop intracellular fungal structures that are surrounded by the plant membrane. The symbiotic plant-fungus interface is considered to be the major site of nutrient transfer to the host plant. We summarize recent data on nutrient transfer in OrM and compare the development and function of the arbuscules formed in AM and the pelotons formed in OrM in order to outline differences and conserved traits. We further describe the unexpected similarities in the form and function of the intracellular mycorrhizal fungal structures observed in orchids and in the roots of mycoheterotrophic plants forming AM. We speculate that these similarities may be the result of convergent evolution of mycorrhizal types in mycoheterotrophic plants and highlight knowledge gaps and new research directions to explore this scenario.

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales).

PREMISE: Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome. METHODS: We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω). RESULTS: Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK. CONCLUSIONS: We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.

Rethinking convergence in plant parasitism through the lens of molecular and population genetic processes.

The autotrophic lifestyle of photosynthetic plants has profoundly shaped their body plan, physiology, and gene repertoire. Shifts to parasitism and heterotrophy have evolved at least 12 times in more than 4000 species, and this transition has consequently left major evolutionary footprints among these parasitic lineages. Features that are otherwise rare at the molecular level and beyond have evolved repetitively, including reduced vegetative bodies, carrion-mimicking during reproduction, and the incorporation of alien genetic material. Here, I propose an integrated conceptual model, referred to as the funnel model, to define the general evolutionary trajectory of parasitic plants and provide a mechanistic explanation for their convergent evolution. This model connects our empirical understanding of gene regulatory networks in flowering plants with classical theories of molecular and population genetics. It emphasizes that the cascading effects brought about by the loss of photosynthesis may be a major force constraining the physiological capacity of parasitic plants and shaping their genomic landscapes. Here I review recent studies on the anatomy, physiology, and genetics of parasitic plants that lend support to this photosynthesis-centered funnel model. Focusing on nonphotosynthetic holoparasites, I elucidate how they may inevitably reach an evolutionary terminal status (i.e., extinction) and highlight the utility of a general, explicitly described and falsifiable model for future studies of parasitic plants.

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.

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.

Association of mycoheterotrophic Gentianaceae with specific Glomus lineages.

Some plant species took an alternative evolutionary pathway in which they lost their photosynthetic capacity to depend exclusively on carbon supplied by arbuscular mycorrhizal fungi (AMF) in an association called mycoheterotrophy. Among them is Voyriella parviflora, a species of the family Gentianaceae, which is found in tropical regions such as the Amazon basin. Here, we assessed the identity of AMF symbionts associated with this species. DNA was isolated from eight Gentianaceae specimens and from litter and surrounding roots of photosynthetic plants. The atp1 gene was amplified by Sanger sequencing to determine the taxonomic affiliation of the mycoheterotrophic plants. A 280 bp region of the 18S rRNA gene of AMF was amplified with primers NS31/AML2 by high-throughput sequencing. The mycoheterotrophic specimens were assigned to V. parviflora with a bootstrap support of 72%. Glomus was the most abundant AMF genus, both in the mycoheterotrophic plants and in the litter and roots of photosynthetic plants. In addition, a few Glomus genotypes were abundantly enriched in the mycoheterotrophic plants, with only a few specimens colonized by Gigaspora, Acaulospora, and Scutellospora in a low proportion. These genotypes formed a cluster within a larger clade, suggesting that V. parviflora shows a preferential association with a narrow Glomus lineage which is not phylogenetically close to a previously identified V. parviflora's associated lineage. Furthermore, detecting fungi from other families suggests that V. parviflora is colonized by other genera, although with low frequency. These findings provide new insights into the association between AMF and mycoheterotrophic species and highlight the importance of considering trap culture-independent approaches in understanding this symbiosis.

The Gastrodia menghaiensis (Orchidaceae) genome provides new insights of orchid mycorrhizal interactions.

BACKGROUND: To illustrate the molecular mechanism of mycoheterotrophic interactions between orchids and fungi, we assembled chromosome-level reference genome of Gastrodia menghaiensis (Orchidaceae) and analyzed the genomes of two species of Gastrodia. RESULTS: Our analyses indicated that the genomes of Gastrodia are globally diminished in comparison to autotrophic orchids, even compared to Cuscuta (a plant parasite). Genes involved in arbuscular mycorrhizae colonization were found in genomes of Gastrodia, and many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids. The highly expressed genes for fatty acid and ammonium root transporters suggest that fungi receive material from orchids, although most raw materials flow from the fungi. Many nuclear genes (e.g. biosynthesis of aromatic amino acid L-tryptophan) supporting plastid functions are expanded compared to photosynthetic orchids, an indication of the importance of plastids even in totally mycoheterotrophic species. CONCLUSION: Gastrodia menghaiensis has the smallest proteome thus far among angiosperms. Many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids.

Phylogenomics, plastome degradation and mycoheterotrophy evolution of Neottieae (Orchidaceae), with emphasis on the systematic position and Loess Plateau-Changbai Mountains disjunction of Diplandrorchis.

BACKGROUND: Mycoheterotrophy is a unique survival strategy adapted to dense forests and has attracted biologists' attention for centuries. However, its evolutionary origin and related plastome degradation are poorly understood. The tribe Neottieae contains various nutrition types, i.e., autotrophy, mixotrophy, and mycoheterotrophy. Here, we present a comprehensive phylogenetic analysis of the tribe based on plastome and nuclear ITS data. We inferred the evolutionary shift of nutrition types, constructed the patterns of plastome degradation, and estimated divergence times and ancestral ranges. We also used an integration of molecular dating and ecological niche modeling methods to investigate the disjunction between the Loess Plateau and Changbai Mountains in Diplandrorchis, a mycoheterotrophic genus endemic to China that was included in a molecular phylogenetic study for the first time. RESULTS: Diplandrorchis was imbedded within Neottia and formed a clade with four mycoheterotrophic species. Autotrophy is the ancestral state in Neottieae, mixotrophy independently originated at least five times, and three shifts from mixotrophy to mycoheterotrophy independently occurred. The five mixotrophic lineages possess all plastid genes or lost partial/all ndh genes, whereas each of the three mycoheterotroph lineages has a highly reduced plastome: one lost part of its ndh genes and a few photosynthesis-related genes, and the other two lost almost all ndh, photosynthesis-related, rpo, and atp genes. These three mycoheterotrophic lineages originated at about 26.40 Ma, 25.84 Ma, and 9.22 Ma, respectively. Diplandrorchis had presumably a wide range in the Pliocene and migrated southward in the Pleistocene. CONCLUSIONS: The Pleistocene climatic fluctuations and the resultant migration resulted in the Loess Plateau-Changbai Mountains disjunction of Diplandrorchis. In the evolution of mycoheterotrophic lineages, the loss of plastid-encoded genes and plastome degradation are staged and irreversible, constraining mycoheterotrophs to inhabit understories with low light levels. Accordingly, the rise of local forests might have promoted the origin of conditions in which mycoheterotrophy is advantageous.

Mycoheterotrophic plants preferentially target arbuscular mycorrhizal fungi that are highly connected to autotrophic plants.

How mycoheterotrophic plants that obtain carbon and soil nutrients from fungi are integrated in the usually mutualistic arbuscular mycorrhizal networks is unknown. Here, we compare autotrophic and mycoheterotrophic plant associations with arbuscular mycorrhizal fungi and use network analysis to investigate interaction preferences in the tripartite network. We sequenced root tips from autotrophic and mycoheterotrophic plants to assemble the combined tripartite network between autotrophic plants, mycorrhizal fungi and mycoheterotrophic plants. We compared plant-fungi interactions between mutualistic and antagonist networks, and searched for a diamond-like module defined by a mycoheterotrophic and an autotrophic plant interacting with the same pair of fungi to investigate whether pairs of fungi simultaneously linked to plant species from each interaction type were overrepresented throughout the network. Mycoheterotrophic plants as a group interacted with a subset of the fungi detected in autotrophs but are indirectly linked to all autotrophic plants, and fungi with a high overlap in autotrophic partners tended to interact with a similar set of mycoheterotrophs. Moreover, pairs of fungi sharing the same mycoheterotrophic and autotrophic plant species are overrepresented in the network. We hypothesise that the maintenance of antagonistic interactions is maximised by targeting well linked mutualistic fungi, thereby minimising the risk of carbon supply shortages.

Novel mycorrhizal cheating in a green orchid: Cremastra appendiculata depends on carbon from deadwood through fungal associations.

To date, there has been no robust evidence for the exploitation of saprotrophic non-rhizoctonia fungi by green plants, although some fully mycoheterotrophic orchids are known to exploit them, and mycoheterotrophic evolution has probably occurred through intermediate mixotrophic stages. We investigated the physiological ecology of a fully mycoheterotrophic species Cremastra aphylla and its photosynthetic sister species Cremastra appendiculata, which putatively exploit saprotrophic fungi. Their mycorrhizal partners and ultimate nutritional sources were determined using molecular, stable isotopic, and radiocarbon analysis. Both Cremastra aphylla and Cremastra appendiculata were consistently associated with wood-decaying Psathyrellaceae. In addition, both species were highly enriched in carbon-13 (13 C) and, to a less degree, in nitrogen-15 (15 N). The δ13 C and δ15 N values of Cremastra appendiculata were intermediate between those of Cremastra aphylla and those of autotrophic plants. All Cremastra appendiculata samples and two Cremastra aphylla samples exhibited elevated Δ14 C values due to the acquisition of carbon fixed in wood during the past decades (14 C-enriched bomb carbon). Our multifaceted evidence indicated that both species obtained carbon from deadwood via saprotrophic fungi. Our findings strongly suggest that mixotrophic relationships associated with wood-decaying fungi represent a novel evolutionary pathway for full mycoheterotrophy in orchids.