Evidence of introduced honeybees (Apis mellifera) as pollen wasters in orchid pollination.

Biological invasions threaten global biodiversity, altering landscapes, ecosystems, and mutualistic relationships like pollination. Orchids are one of the most threatened plant families, yet the impact of invasive bees on their reproduction remains poorly understood. We conduct a global literature survey on the incidence of invasive honeybees (Apis mellifera) on orchid pollination, followed by a study case on Australian orchids. Our literature survey shows that Apis mellifera is the primary alien bee visiting orchids worldwide. However, in most cases, introduced honeybees do not deposit orchid pollen. We also test the extent to which introduced honeybees affect orchid pollination using Diuris brumalis and D. magnifica. Diuris brumalis shows higher fruit set and pollination in habitats with both native and invasive bees compared to habitats with only introduced bees. Male and female reproductive success in D. magnifica increases with native bee abundance, while conversely pollinator efficiency decreases with honeybee abundance and rises with habitat size. Our results suggest that introduced honeybees are likely involved in pollen removal but do not effectively deposit orchid pollen, acting as pollen wasters. However, Apis mellifera may still contribute to pollination of Diuris where native bees no longer exist. Given the global occurrence of introduced honeybees, we warn that certain orchids may suffer from pollen depletion by these invaders, especially in altered habitats with compromised pollination communities.

Mimicking orchids lure bees from afar with exaggerated ultraviolet signals.

Flowers have many traits to appeal to pollinators, including ultraviolet (UV) absorbing markings, which are well-known for attracting bees at close proximity (e.g., <1 m). While striking UV signals have been thought to attract pollinators also from far away, if these signals impact the plant pollinia removal over distance remains unknown. Here, we report the case of the Australian orchid Diuris brumalis, a nonrewarding species, pollinated by bees via mimicry of the rewarding pea plant Daviesia decurrens. When distant from the pea plant, Diuris was hypothesized to enhance pollinator attraction by exaggeratedly mimicking the floral ultraviolet (UV) reflecting patterns of its model. By experimentally modulating floral UV reflectance with a UV screening solution, we quantified the orchid pollinia removal at a variable distance from the model pea plants. We demonstrate that the deceptive orchid Diuris attracts bee pollinators by emphasizing the visual stimuli, which mimic the floral UV signaling of the rewarding model Daviesia. Moreover, the exaggerated UV reflectance of Diuris flowers impacted pollinators' visitation at an optimal distance from Da. decurrens, and the effect decreased when orchids were too close or too far away from the model. Our findings support the hypothesis that salient UV flower signaling plays a functional role in visual floral mimicry, likely exploiting perceptual gaps in bee neural coding, and mediates the plant pollinia removal at much greater spatial scales than previously expected. The ruse works most effectively at an optimal distance of several meters revealing the importance of salient visual stimuli when mimicry is imperfect.

Auditing data resolves systemic errors in databases and confirms mycorrhizal trait consistency for most genera and families of flowering plants.

Nearly 150 years of research has accumulated large amounts of data on mycorrhizal association types in plants. However, this important resource includes unreliable allocated traits for some species. An audit of six commonly used data sources revealed a high degree of consistency in the mycorrhizal status of most species, genera and families of vascular plants, but there were some records that contradict the majority of other data (~ 10% of data overall). Careful analysis of contradictory records using rigorous definitions of association types revealed that the majority were diagnosis errors, which often stem from references predating modern knowledge of mycorrhiza types. Other errors are linked to inadequate microscopic examinations of roots or plants with complex root anatomy, such as phi thickenings or beaded roots. Errors consistently occurred at much lower frequencies than correct records but have accumulated in uncorrected databases. This results in less accurate knowledge about dominant plants in some ecosystems because they were sampled more often. Errors have also propagated from one database to another over decades when data were amalgamated without checking their suitability. Due to these errors, it is often incorrect to designate plants reported to have inconsistent mycorrhizas as "facultatively mycorrhizal". Updated protocols for resolving conflicting mycorrhizal data are provided here. These are based on standard morphological definitions of association types, which are the foundations of mycorrhizal science. This analysis also identifies the need for adequate training and mentoring of researchers to maintain the quality of mycorrhizal research.

FungalRoot: global online database of plant mycorrhizal associations.

Testing of ecological, biogeographical and phylogenetic hypotheses of mycorrhizal traits requires a comprehensive reference dataset about plant mycorrhizal associations. Here we present a database, FungalRoot, which summarizes publicly available data about vascular plant mycorrhizal type and intensity of root colonization by mycorrhizal fungi, accompanied with rich metadata. We compiled and digitized data about plant mycorrhizal colonization in nine widespread languages. The present version of the FungalRoot database contains 36 303 species-by-site observations for 14 870 plant species, tripling the previously available compiled information about plant mycorrhizal associations. Based on these data, we provide a recommended list of genus-level plant mycorrhizal associations, based on the majority of data for species and careful analysis of conflicting data. The majority of ectomycorrhizal and ericoid mycorrhizal plants are trees (92%) and shrubs (85%), respectively. The majority of arbuscular and nonmycorrhizal plant species are herbaceous (50% and 70%, respectively). Our publicly available database is a powerful resource for mycorrhizal scientists and ecologists. It features possibilities for dynamic updating and addition of data about plant mycorrhizal associations. The new database will promote research on plant and fungal biogeography and evolution, and on links between above- and belowground biodiversity and ecosystem functioning.

Global mycorrhizal plant distribution linked to terrestrial carbon stocks.

Vegetation impacts on ecosystem functioning are mediated by mycorrhizas, plant-fungal associations formed by most plant species. Ecosystems dominated by distinct mycorrhizal types differ strongly in their biogeochemistry. Quantitative analyses of mycorrhizal impacts on ecosystem functioning are hindered by the scarcity of information on mycorrhizal distributions. Here we present global, high-resolution maps of vegetation biomass distribution by dominant mycorrhizal associations. Arbuscular, ectomycorrhizal, and ericoid mycorrhizal vegetation store, respectively, 241 ± 15, 100 ± 17, and 7 ± 1.8 GT carbon in aboveground biomass, whereas non-mycorrhizal vegetation stores 29 ± 5.5 GT carbon. Soil carbon stocks in both topsoil and subsoil are positively related to the community-level biomass fraction of ectomycorrhizal plants, though the strength of this relationship varies across biomes. We show that human-induced transformations of Earth's ecosystems have reduced ectomycorrhizal vegetation, with potential ramifications to terrestrial carbon stocks. Our work provides a benchmark for spatially explicit and globally quantitative assessments of mycorrhizal impacts on ecosystem functioning and biogeochemical cycling.

Misdiagnosis of mycorrhizas and inappropriate recycling of data can lead to false conclusions.

We draw attention to a worrying trend for the uncritical use of 'recycled' mycorrhizal data to compile host species lists that include obvious errors or undertake risky analyses that correlate mycorrhizal colonisation levels with environmental or physiological factors despite inherent limitations in datasets. We are not suggesting that all meta-studies are wrong, only that more care should be taken to resolve what can safely be done with recycled mycorrhizal data in the future. We also recommend that mycorrhizal species lists should be checked against standard references since the majority of EM hosts and NM plant belong to families that are well resolved. However, additional research is required in cases where plant families have multiple root types within genera or occur in habitats where mycorrhizal associations are often suppressed (see Brundrett & Tedersoo, 2018). We hope that the mycorrhizal science community will work together more closely in the future to develop and enforce standards for mycorrhizal diagnosis and to share carefully corrected datasets for realistic meta-studies.

Evolutionary history of mycorrhizal symbioses and global host plant diversity.

Contents Summary 1108 I. Introduction 1108 II. Mycorrhizal plant diversity at global and local scales 1108 III. Mycorrhizal evolution in plants: a brief update 1111 IV. Conclusions and perspectives 1114 References 1114 SUMMARY: The majority of vascular plants are mycorrhizal: 72% are arbuscular mycorrhizal (AM), 2.0% are ectomycorrhizal (EcM), 1.5% are ericoid mycorrhizal and 10% are orchid mycorrhizal. Just 8% are completely nonmycorrhizal (NM), whereas 7% have inconsistent NM-AM associations. Most NM and NM-AM plants are nutritional specialists (e.g. carnivores and parasites) or habitat specialists (e.g. hydrophytes and epiphytes). Mycorrhizal associations are consistent in most families, but there are exceptions with complex roots (e.g. both EcM and AM). We recognize three waves of mycorrhizal evolution, starting with AM in early land plants, continuing in the Cretaceous with multiple new NM or EcM linages, ericoid and orchid mycorrhizas. The third wave, which is recent and ongoing, has resulted in root complexity linked to rapid plant diversification in biodiversity hotspots.

High-resolution secondary ion mass spectrometry analysis of carbon dynamics in mycorrhizas formed by an obligately myco-heterotrophic orchid.

Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre-lysed pelotons. We used high-resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched (13) CO2 to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post (13) CO2 labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.

Limited carbon and mineral nutrient gain from mycorrhizal fungi by adult Australian orchids.

PREMISE OF THE STUDY: In addition to autotrophic and fully mycoheterotrophic representatives, the orchid family comprises species that at maturity obtain C and N partially from fungal sources. These partial mycoheterotrophs are often associated with fungi that simultaneously form ectomycorrhizas with trees. This study investigates mycorrhizal nutrition for orchids from the southwestern Australian biodiversity hotspot. METHODS: The mycorrhizal fungi of 35 green and one achlorophyllous orchid species were analyzed using molecular methods. Nutritional mode was identified for 27 species by C and N isotope abundance analysis in comparison to non-orchids from the same habitat. As a complementary approach, (13)CO(2) pulse labeling was applied to a subset of six orchid species to measure photosynthetic capacity. KEY RESULTS: Almost all orchids associated with rhizoctonia-forming fungi. Due to much higher than expected variation within the co-occurring nonorchid reference plants, the stable isotope approach proved challenging for assigning most orchids to a specialized nutritional mode; therefore, these orchids were classified as autotrophic at maturity. The (13)CO(2) pulse labeling confirmed full autotrophy for six selected species. Nonetheless, at least three orchid species (Gastrodia lacista, Prasophyllum elatum, Corybas recurvus) were identified as nutritionally distinctive from autotrophic orchids and reference plants. CONCLUSIONS: Despite the orchid-rich flora in southwestern Australia, partial mycoheterotrophy among these orchids is less common than in other parts of the world, most likely because most associate with saprotrophic fungi rather than ectomycorrhizal fungi.

Rampant gene loss in the underground orchid Rhizanthella gardneri highlights evolutionary constraints on plastid genomes.

Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ∼110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.

Carbon and nitrogen supply to the underground orchid, Rhizanthella gardneri.

*Rhizanthella gardneri is a rare and fully subterranean orchid that is presumably obligately mycoheterotrophic. R. gardneri is thought to be linked via a common mycorrhizal fungus to co-occurring autotrophic shrubs, but there is no experimental evidence to support this supposition. *We used compartmentalized microcosms to investigate the R. gardneri tripartite relationship. (13)CO(2) was applied to foliage of Melaleuca scalena plants and [(13)C-(15)N]glycine was fed to the common mycorrhizal fungus, and both sources traced to R. gardneri plants. *In our microcosm trial, up to 5% of carbon (C) fed as (13)CO(2) to the autotrophic shrub was transferred to R. gardneri. R. gardneri also readily acquired soil C and nitrogen (N), where up to 6.2% of C and 22.5% of N fed as labelled glycine to soil was transferred via the fungus to R. gardneri after 240 h. *Our study confirms that R. gardneri is mycoheterotrophic and acquires nutrients via mycorrhizal fungus connections from an ectomycorrhizal autotrophic shrub and directly from the soil via the same fungus. This connection with a specific fungus is key to explaining why R. gardneri occurs exclusively under certain Melaleuca species at a very limited number of sites in Western Australia.

Identity and specificity of the fungi forming mycorrhizas with the rare mycoheterotrophic orchid Rhizanthella gardneri.

Fully subterranean Rhizanthella gardneri (Orchidaceae) is obligately mycoheterotrophic meaning it is nutritionally dependent on the fungus it forms mycorrhizas with. Furthermore, R. gardneri purportedly participates in a nutrient sharing tripartite relationship where its mycorrhizal fungus simultaneously forms ectomycorrhizas with species of Melaleuca uncinata s.l. Although the mycorrhizal fungus of R. gardneri has been morphologically identified as Thanatephorus gardneri (from a single isolate), this identification has been recently questioned. We sought to clarify the identification of the mycorrhizal fungus of R. gardneri, using molecular methods, and to identify how specific its mycorrhizal relationship is. Fungal isolates taken from all sites where R. gardneri is known to occur shared almost identical ribosomal DNA (rDNA) sequences. The fungal isolate rDNA most closely matched that of other Ceratobasidiales species, particularly within the Ceratobasidium genus. However, interpretation of results was difficult as we found two distinct ITS sequences within all mycorrhizal fungal isolates of R. gardneri that we assessed. All mycorrhizal fungal isolates of R. gardneri readily formed ectomycorrhizas with a range of M. uncinata s.l. species. Consequently, it is likely that R. gardneri can form a nutrient sharing tripartite relationship where R. gardneri is connected to autotrophic M. uncinata s.l. by a common mycorrhizal fungus. These findings have implications for better understanding R. gardneri distribution, evolution and the ecological significance of its mycorrhizal fungus, particularly in relation to nutrient acquisition.

Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions.

The diversity of mycorrhizal fungi associated with an introduced weed-like South African orchid (Disa bracteata) and a disturbance-intolerant, widespread, native West Australian orchid (Pyrorchis nigricans) were compared by molecular identification of the fungi isolated from single pelotons. Molecular identification revealed both orchids were associated with fungi from diverse groups in the Rhizoctonia complex with worldwide distribution. Symbiotic germination assays confirmed the majority of fungi isolated from pelotons were mycorrhizal and a factorial experiment uncovered complex webs of compatibility between six terrestrial orchids and 12 fungi from Australia and South Africa. Two weed-like (disturbance-tolerant rapidly spreading) orchids - D. bracteata and the indigenous Australian Microtis media, had the broadest webs of mycorrhizal fungi. In contrast, other native orchids had relatively small webs of fungi (Diuris magnifica and Thelymitra crinita), or germinated exclusively with their own fungus (Caladenia falcata and Pterostylis sanguinea). Orchids, such as D. bracteata and M. media, which form relationships with diverse webs of fungi, had apparent specificity that decreased with time, as some fungi had brief encounters with orchids that supported protocorm formation but not subsequent seedling growth. The interactions between orchid mycorrhizal fungi and their hosts are discussed.

Diversity and classification of mycorrhizal associations.

Most mycorrhizas are 'balanced' mutualistic associations in which the fungus and plant exchange commodities required for their growth and survival. Myco-heterotrophic plants have 'exploitative' mycorrhizas where transfer processes apparently benefit only plants. Exploitative associations are symbiotic (in the broad sense), but are not mutualistic. A new definition of mycorrhizas that encompasses all types of these associations while excluding other plant-fungus interactions is provided. This definition recognises the importance of nutrient transfer at an interface resulting from synchronised plant-fungus development. The diversity of interactions between mycorrhizal fungi and plants is considered. Mycorrhizal fungi also function as endophytes, necrotrophs and antagonists of host or non-host plants, with roles that vary during the lifespan of their associations. It is recommended that mycorrhizal associations are defined and classified primarily by anatomical criteria regulated by the host plant. A revised classification scheme for types and categories of mycorrhizal associations defined by these criteria is proposed. The main categories of vesicular-arbuscular mycorrhizal associations (VAM) are 'linear' or 'coiling', and of ectomycorrhizal associations (ECM) are 'epidermal' or 'cortical'. Subcategories of coiling VAM and epidermal ECM occur in certain host plants. Fungus-controlled features result in 'morphotypes' within categories of VAM and ECM. Arbutoid and monotropoid associations should be considered subcategories of epidermal ECM and ectendomycorrhizas should be relegated to an ECM morphotype. Both arbuscules and vesicles define mycorrhizas formed by glomeromycotan fungi. A new classification scheme for categories, subcategories and morphotypes of mycorrhizal associations is provided.

Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats.

An innovative ex situ fungal baiting method using soil collected from field sites which allows the simultaneous detection of mycorrhizal fungi for multiple terrestrial orchids is presented. This method demonstrated that coarse organic matter (> 2 mm) in the litter and topsoil was the most important reservoir of inoculum of these fungi. A new in situ seed baiting method using multi-chambered packets to simultaneously assess germination for different orchid species within soil is also introduced. These in situ and ex situ methods are compared using seed of orchids in the genera Monadenia, Microtis, Caladenia, Pterostylis and Diuris, using urban Banksia woodland sites with high or low weed cover. Both these seed baiting methods detected compatible fungi for these orchids, but common orchids germinated more frequently than those which were uncommon at the field sites. Germination rates were not significantly affected by weed cover even though adult orchids were rare in areas with high weed cover. The two new seed baiting methods vary in efficiency and applicability depending on the situation where they are used. However, the ex situ method allowed the time-course of germination to be observed, resulting in the production of more protocorms and facilitation of the isolation of mycorrhizal fungi. These techniques provide valuable new tools for detection of compatible mycorrhizal fungi to assist orchid research and conservation.

Coevolution of roots and mycorrhizas of land plants.

Here, the coevolution of mycorrhizal fungi and roots is assessed in the light of evidence now available, from palaeobotanical and morphological studies and the analysis of DNA-based phylogenies. The first bryophyte-like land plants, in the early Devonian (400 million years ago), had endophytic associations resembling vesicular-arbuscular mycorrhizas (VAM) even before roots evolved. Mycorrhizal evolution would have progressed from endophytic hyphae towards balanced associations where partners were interdependent due to the exchange of limiting energy and nutrient resources. Most mycorrhizas are mutualistic, but in some cases the trend for increasing plant control of fungi culminates in the exploitative mycorrhizas of achlorophyllous, mycoheterotrophic plants. Ectomycorrhizal, ericoid and orchid mycorrhizas, as well as nonmycorrhizal roots, evolved during the period of rapid angiosperm radiation in the Cretaceous. It is hypothesised that roots gradually evolved from rhizomes to provide more suitable habitats for mycorrhizal fungi and provide plants with complex branching and leaves with water and nutrients. Selection pressures have caused the morphological divergence of roots with different types of mycorrizas. Root cortex thickness and exodermis suberization are greatest in obllgately mycorrhizal plants, while nonmycorrhizal plants tend to have fine roots, with more roots hairs and relatively advanced chemical defences. Major coevolutionary trends and the relative success of plants with different root types are discussed. Contents Summary 275 I. Introduction 276 II. Mycorrhizal Fungi 276 III. The Dawn of Mycorrhizas 279 IV. Mycorrhizal Associations of Living and Extinct Plants 282 V. Evolution of Roots 288 VI. The Root as a Habitat for Fungi 290 VII. Mycorrhizal Evolution Trends 295 Acknowledgements 298 References 298.