Exo‐ and endophytic fungi enable rapid transfer of nutrients from ant waste to orchid tissue

Summary The epiphytic orchid Caularthron bilamellatum sacrifices its water storage tissue for nutrients from the waste of ants lodging inside its hollow pseudobulb. Here, we investigate whether fungi are involved in the rapid translocation of nutrients. Uptake was analysed with a 15N labelling experiment, subsequent isotope ratio mass spectrometry (IRMS) and secondary ion mass spectrometry (ToF‐SIMS and NanoSIMS). We encountered two hyphae types: a thick melanized type assigned to ‘black fungi’ (Chaetothyriales, Cladosporiales, and Mycosphaerellales) in ant waste, and a thin endophytic type belonging to Hypocreales. In few cell layers, both hyphae types co‐occurred. 15N accumulation in both hyphae types was conspicuous, while for translocation to the vessels only Hypocreales were involved. There is evidence that the occurrence of the two hyphae types results in a synergism in terms of nutrient uptake. Our study provides the first evidence that a pseudobulb (=stem)‐born endophytic network of Hypocreales is involved in the rapid translocation of nitrogen from insect‐derived waste to the vegetative and reproductive tissue of the host orchid. For C. bilamellatum that has no contact with the soil, ant waste in the hollow pseudobulbs serves as equivalent to soil in terms of nutrient sources.


Introduction
The canopy of tropical forests is a challenging habitat with often harsh environmental conditions.High radiation and limited availability of water and nutrients force organisms to develop a multitude of strategies to grow and reproduce (Nakamura et al., 2017).This is especially true for epiphytes which, unlike terrestrial plants, lack direct soil contact.To be able to meet their nutritional needs, vascular epiphytes have evolved remarkable morphological adaptations: for example, aerial roots with a velamen radicum, leafabsorbing trichomes, leaf arrangements for litter-trapping, adventitious roots or leaves forming tanks with water and nutrientstoring phytotelmata (Benzing, 1970(Benzing, , 2000;;Zotz & Hietz, 2001;Zotz & Richter, 2006;Zotz et al., 2021).Such adaptations enable vascular epiphytes to take up nutrients from clouds, rainfall, throughfall and stemflow water and/or from decomposing organic matter from plants, insects, birds or other organisms (Benzing, 1990;Winkler & Zotz, 2010;Zotz & Winkler, 2013;Gotsch et al., 2015;Leroy et al., 2017;Sun et al., 2020).Epiphytes also frequently engage in mutualistic relationships with other organisms to escape nutrient scarcity, for example with mycorrhizal fungi (Rains et al., 2003;Looby et al., 2020), or with ants.
Specific and obligate mutualisms between ants and plants with distinct morphological plant structures that serve as nesting space for ants (domatia) occur only in a relatively small number of plant species (myrmecophytes), and epiphytes account for a significant proportion (Chomicki & Renner, 2015).An important reason for the latter might be that the association with ants enables access to nutrient resources.Irrespective of a mutualistic relationship with plants, ant nests and nest environments are hotspots of nutrients (C, N, P and K) and cations (Al, Ca, Mg and Na).A key role plays ant waste generated from plant material, faeces, carcasses and metabolic products typical for ants (Beattie, 1985;Blüthgen et al., 2001;Farji-Brener & Werenkraut, 2017).In myrmecophytes, ants accumulate such organic waste in the nesting sites inside the host plant (Treseder et al., 1995;Fischer et al., 2003;Solano & Dejean, 2004;Defossez et al., 2011;Bazile et al., 2012;Lucas et al., 2018).The transfer of nutrients from such ant waste to plants has been demonstrated in ground-rooted myrmecophytes (Fischer et al., 2003;Solano & Dejean, 2004;Defossez et al., 2011;Bazile et al., 2012) as well as in myrmecophytic epiphyte genera like Dischidia sp.(Apocynaceae) with its domatia from folded leaves (Treseder et al., 1995), in Lecanopteris sp.(Polypodiaceae) with its hollow rhizomes (Gay, 1993), or Hydnophytum, Myrmecodia and Squamellaria sp.(Rubiaceae), which exhibit a prominent caudex with natural cavities (Huxley, 1978;Rickson, 1979;Chomicki & Renner, 2019).The importance of nutrient supply from ants to plants may be different depending on ecology and habitat of the host plant, suggesting that epiphytes with missing soil connection and limited access to mineral nutrients benefit to a much higher degree than ground-rooted plants (Janzen, 1974;Rico Gray, 1987;Gay, 1993;Treseder et al., 1995;Gegenbauer et al., 2012).
Remarkably, for some epiphytes, access to nutrients may even be more important than water storage tissue.This is the case in species of two myrmecophytic orchid genera (Myrmecophila and Caularthron) which sacrifice a considerable part of water storage tissue to form hollow pseudobulbs that are used by ants as nesting space even though water is the most limiting resource for growth and survival of epiphytes (Zotz et al., 2021).In case of Caularthron, up to 50% of pseudobulb tissue is lost during pseudobulb maturation (G.Zotz, unpublished;Gegenbauer et al., 2012), which raises the question of a trade-off between water and nutrients.Indeed, carbon uptake from the debris of ants placed in pseudobulbs has been demonstrated for Myrmecophila tibicinis (Orchidaceae; Rico Gray et al., 1989) and nitrogen transfer from ants to plants in Caularthron bilamellatum (Orchidaceae; Gegenbauer et al., 2012).In the latter, a multitude of organic and inorganic nitrogen sources were taken up by hollow pseudobulbs under field conditions and even translocated into seeds.Enzyme kinetics suggested the presence of an active uptake system but when investigating the morphology and anatomy of the pseudobulbs highly specialized surface structures as known from epiphytic Rubiaceae living with ants (Janzen, 1974;Huxley, 1978;Rickson, 1979), were not found.Instead, we frequently observed a conspicuous colonization by fungal hyphae in the ant debris as well as in the pseudobulb tissue close to the cavity (fig.2D in Gegenbauer et al., 2012).This is prompting us to suggest that these fungi may facilitate plant nutrient uptake from ant waste as it is well known from rootfungi associations.Usually, ant-associated fungi are restricted to ant waste and dead cells and do not penetrate living plant cells (Defossez et al., 2009;Voglmayr et al., 2011;Vasse et al., 2017;Mayer et al., 2018).
In the present study, we aimed at investigating whether the endophytic fungi in the tissue of pseudobulbs play a crucial role in the mutualistic association between the epiphytic orchid Caularthron bilamellatum and its inhabiting ants.We hypothesize that the endophytic fungi are the 'active uptake system' mediating the rapid translocation of nutrients from the ant waste in the cavity of the pseudobulb into the vegetative and reproductive plant tissue.
To address this aim, we conducted a labelling experiment on C. bilamellatum pseudobulbs under field conditions using 15 N stable isotope tracing for a period of 1, 2, 4 and 8 d.Mature pseudobulbs from plants inhabited by different ant species were labelled by placing a solid organic 15 N source into the hollow chamber.Ants were barred from entering and spreading the label. 15N uptake into plant tissue was then analysed using isotope ratio mass spectrometry (IRMS), as well as secondary ion mass spectrometry (ToF-and NanoSIMS).Further, the tissue was screened for fungal endophytes using light microscopy (LM), scanning electron microscopy (SEM) and X-ray micro-computed tomography (micro-CT).Fungi were isolated from ant waste and plant tissue, cultured and sequenced for identification.We aimed at correlating fungal morphology in LM with secondary ion mass spectrometry images and to trace 15 N uptake from the pseudobulb cavity to vessels in the plant tissue.Finally, we tested whether ant presence enhances nutrient uptake capabilities by comparing ant-inhabited pseudobulbs rich in detritus and fungi with closed pseudobulbs free of ants.

Sample collection and experimental set-up
We collected 15 mature, hollow pseudobulbs of Caularthron bilamellatum (Rchb.f.) R.E.Schult.(Orchidaceae) growing on Annona glabra (Annonaceae) trees (Fig. 1a) at each of six different sampling plots along the southern shore of Barro Colorado Island (BCI), Republic of Panama.A sampling plot was defined as an isolated group of trees inhabited by a single ant species.In total, 90 pseudobulbs were taken for the labelling experiment.In rare cases, the formation of an opening slit during maturation fails and the pseudobulb remains closed and inaccessible to ants and microorganisms.Two such samples could be obtained to investigate nutrient uptake in uninhabited pseudobulbs.The experimental set-up took place at the laboratories of the Smithsonian Tropical Research Station (STRI).
Pseudobulb incubation Upon maturation, pseudobulbs of C. bilamellatum desiccate, forming a hollow chamber (Fig. 1b, Supporting Information Fig. S1) and a slit at the base allowing ants to access the cavity.After the natural opening at the base of the pseudobulbs was enlarged, the remaining ants were manually removed and the agar disc containing the high molecular weight 15 N label was placed on the ant debris in the apical region of the pseudobulb cavity.The opening was then sealed with parafilm leaving a small slit mimicking the original state to maintain the natural micro-climate within the cavity.For each plot, two pseudobulbs per time point (1, 2, 4 and 8 d) were incubated under field conditions but kept free of ants (= 8 per plot), in total 48 pseudobulbs.Table S1 gives the sample size per sampling plot.Upon reaching the intended incubation time, pseudobulbs were cut open, the remains of the agar discs removed and rinsed twice with 10 mM CaCl 2 solution and distilled water to remove label adhering to the surface of the pseudobulb cavity.Several small samples of each pseudobulb's apical region were cut out and either fixed in 70% EtOH for sectioning or dried for 24 h at 60°C for IRMS analysis.Also, leaf tips c. 10-15 cm distant from the labelling site were harvested and dried.Unfortunately, some samples were lost during shipping to Vienna leaving 40 pseudobulb and 37 leaf samples for IRMS.
For a 3D reconstruction of the waste distribution, apical pseudobulb parts were analysed with a microXCT-200 X-ray tomography (micro-CT) system (Zeiss Microscopy, Jena, Germany).Sample preparation and reconstruction were performed according to Staedler et al. (2013).

Isotope ratio mass spectrometry
The 15 N : 14 N isotopic ratios were determined using an elemental analyser (EA 110; CE Instruments, Milan, Italy) coupled to an isotope ratio mass spectrometer (Delta Plus; Finnigan MAT, Bremen, Germany) as described previously (Gegenbauer et al., 2012).
Nitrogen isotope ratio deviation (δ) values were calculated as: R sample and R standard are the molar ratios of 15 N : 14 N of the sample and the standard (atmospheric air; Peterson & Fry, 1987).
The measured 15 N enrichment per sample was calculated to atom% excess as (APE): Secondary ion mass spectrometry (ToF-SIMS and NanoSIMS) Four 70% EtOH fixed samples of pseudobulbs inhabited by Azteca cf velox, one labelled for 24 h, one for 8 d and two unlabelled controls were embedded in an epoxy resin (Araldite 502; EMS, Hatfield, PA, USA) and sectioned with a Reichert-Jung UltraCut E Ultramicrotome using glass knives into 0.5-1-μm semithin sections.Sections were deposited either onto antimonydoped silicon wafer platelets (7.1 × 7.1 × 0.75 mm; Active Business Co., Brunnthal, Germany) or indium tin oxide (ITO)coated glass slides (7.1 × 7.1 × 1.1 mm; Praezisions Glas & Optik GmbH, Iserlohn, Germany) and kept at 80°C for 1 h.For NanoSIMS analysis, sections were sputter-coated with an AuPd (80/20) layer of 30 nm (nominal thickness) to prevent electric charging through the NanoSIMS measurement process.For ToF-SIMS, the sections remained uncoated.
ToF-SIMS At the Institute of Chemical Technologies and Analytics of the Technical University of Vienna, a TOF-SIMS 5 instrument (Ion-Tof, Münster, Germany), utilizing 25 keV Bi + as primary ions in imaging mode was used to analyse negative secondary ions.The ion beam was chopped into eight short pulses (burst mode), which was necessary to attempt discrimination of several ion species at the nominal masses 26 and 27.Areas of 300 × 300 μm 2 were scanned using a raster of 2048 × 2048 pixels.Before measurement, an area of 900 × 900 μm 2 was presputtered with 1 keV Cs + ions to remove c. 300 nm from the specimen surface that was altered from exposure to air (measurable as a change of secondary ion yield with depth).During measurement, the same parameters were used to remove c. 1 nm after each x-y image (i.e.2048 × 2048 mass spectra).An electron flood gun (20 eV) was used for charge compensation.
To determine the local nitrogen isotope composition, the secondary ion counts of 12 C 14 N − and 12 C 15 N − of all eight bursts were integrated and used for calculation as their signal intensities were considerably stronger than those of 14 N − and 15 N − .It should be noted that the mass resolving power achieved in the burst mode (M/M ≈ 1300, according to the 10% valley definition) was insufficient for complete separation of all mass 26 and 27 ionic species.As such, we consider the results obtained by ToF-SIMS as semiquantitative, in contrast to the values obtained by NanoSIMS.However, ToF-SIMS is superior to NanoSIMS in the achievable size of the field of view that can be scanned without significant lens aberration (up to 500 × 500 μm 2 vs c. 100 × 100 μm 2 , respectively).Accordingly, we utilized ToF-SIMS for obtaining survey images with high throughput and NanoSIMS for high-accuracy isotope analysis within particular measurement areas predefined by ToF-SIMS.
NanoSIMS NanoSIMS measurements were performed on an NS50L instrument (Cameca, Gennevilliers, France) at the Large-Instrument Facility for Environmental and Isotope Mass Spectrometry of the University of Vienna.Before data acquisition, analysis areas were presputtered utilizing a high-intensity, slightly defocused Cs + ion beam (100-200 pA beam current, c. 1.5 μm spot size) to a primary ion fluence of 6.2E16 ions per cm 2 , which enabled the establishment of the steady-state secondary ion signal intensity regime.Data were acquired as multilayer image stacks by sequential scanning of a finely focused Cs + primary ion beam (c.80 nm probe size at 2 pA beam current) over areas between 60 × 60 and 73 × 73 μm 2 at 512 × 512 pixel image resolution and a primary ion beam dwell-time of 7.5 ms (pixel × cycle) −1 .The detectors were positioned to enable parallel detection of 12 C − , 12 C 2 − , 12 C 14 N − , 12 C 15 N − , 31 P − and 32 S − secondary ions.The mass spectrometer was tuned to achieve a mass resolving power of > 9000, according to Cameca's definition (Hoppe et al., 2013), which enabled selective detection of the targeted secondary ion species (i.e. 12 C 14 N − and 12 C 15 N − ) without contributions from any other potentially formed secondary ions species at M26 and M27 (Table S2).
NanoSIMS images were processed using the WINIMAGE software package v.2.0.8 provided by Cameca.Before stack accumulation, the individual images were aligned to compensate for positional variations arising from primary ion beam and/or sample stage drift.Secondary ion signal intensities were dead time corrected on a per-pixel basis.Nitrogen isotope composition images displaying the 15 N/( 14 N + 15 N) isotope fraction, designated as 15 N atom%, were inferred from the 12 CN − secondary ion signal intensity distribution images via per-pixel calculation of

Fungus culture
Fungus cultures were obtained from the tissue as well as from ant waste of six living pseudobulbs inhabited by Azteca cf velox.Fungal isolation from ant waste was done as described in Voglmayr et al. (2011) and Nepel et al. (2014).For isolation of fungi from living host tissue of pseudobulbs, a slightly modified protocol of Bougoure et al. (2005) was used.Thin sections of surface sterilized pseudobulbs were placed on 2% malt extract agar (MEA) plates supplemented with antibiotics (0.1% Streptomycin, 0.1% Penicillin G; Sigma Aldrich).Hyphal tips from outgrowing hyphae were transferred to new MEA plates.Once free from bacterial and yeast contamination, hyphae were transferred onto new plates and grown at room temperature.

DNA extraction and sequencing
Mycelium growth and DNA extraction was performed according to Voglmayr & Jaklitsch (2011).The nuclear ITS-partial LSU rDNA was amplified and sequenced as a single stretch as described in Voglmayr et al. (2011) from 21 pure fungus cultures.Taxonomic identification of the sequences was done using a BLAST search in GenBank.All sequences are deposited in Gen-Bank (http://www.ncbi.nlm.nih.gov/genbank/).Strains and Gen-Bank accession numbers are listed in Table 1.

Statistics
Statistical analysis was performed with RSTUDIO TEAM (2020) v.2022.07.2.The overall effect of treatment and time points was tested with a generalized linear model (GLM).Pairwise comparisons were made with a Kruskal-Wallis rank-sum test and a subsequent Dunn's post hoc analysis.

Pseudobulb morphology
The most prominent anatomical feature of the cavity surface is the large thick-walled and suberized dead cells of the desiccated former water storage tissue (Gegenbauer et al., 2012fig. 2C).They sometimes exceed 1 mm in diameter and are visible to the naked eye as a rough surface, especially in the apical region.These cells form deep depressions in which ant waste accumulates (Fig. 1c).Closed pseudobulbs that had not formed a slit miss those structures on the cavity surface.Instead, it was partially covered with the residues of the tissue that had desiccated (Fig. S2a).

Ant inhabitants and appearance of the pseudobulb interior
Of the six sampling plots, two were inhabited by Azteca cf velox (Dolichoderinae), which was the most common ant species encountered.Other ant species found in one plot each were another larger Azteca sp., Camponotus atriceps (Formicinae) and Pheidole sp.(Myrmicinae).One plot turned out to be ant free but with signs of former ant presence in pseudobulbs, suggesting recent abandonment.
In each plot, 10-15 pseudobulbs were examined.The appearance of the interior of the pseudobulbs varied depending on the inhabiting ant species (Table S1).The abandoned ant-free pseudobulbs (Fig. S2b) had the same characteristics as the Azteca-inhabited ones.The ones inhabited by Camponotus exhibited a thick, dark brown and mud-like layer of ant waste comprised of dead ants, insect remains and fungal hyphae and were populated by a variety of organisms like coccids, small snails, nematodes and protists (Fig. S2c).The inside of all pseudobulbs inhabited by Pheidole was of light colour, exhibiting a rough surface but with little to no detritus (Fig. S2d).In the pseudobulbs of the three plots inhabited by different Azteca species, the waste area in the apical region was blackish-brown and heavily pervaded by melanized fungal hyphae (Fig. S2e-g).Cardboard-like structures (=carton) which are typical for nests of Azteca ants were present.

Identity of fungi and distribution
At least two strikingly distinct hyphal types were commonly encountered in ant-inhabited pseudobulbs.
Type 1 comprises thick and distinctly septate melanized hyphae with a diameter of 4-8 μm.These hyphae pervaded the ant waste on the surface of the pseudobulb cavity and the layers of dead cells.They also grew through numerous pits 1-3 cell layers into the adjacent living tissue (Fig. 1e,h).We identified the isolates as members of the Ascomycota from Chaetothyriales, Cladosporiales and Mycosphaerellales orders (Table 1; Fig. S3d).Obviously, these melanized hyphae were responsible for the characteristic dark brown colour of pseudobulb cavities inhabited by ants of the genus Azteca (Figs 1b, S2e-g), and Camponotus atriceps (Fig. S2c).Very few to no such hyphae were observed in pseudobulbs inhabited by Pheidole sp., which explains the light colour of the chamber surface (Table S1; Fig. S2d).
Type 2 are thin, hyaline and indistinctly septate hyphae with a diameter of 1-2 μm.Most isolates from this type could be assigned to the ascomycete order Hypocreales (Fig. 1d,f,g; Table 1).Hyphae of this type were observed to grow along the cell walls (Figs 1d, S3a-c), several layers deep into the living cells and into cells of the vascular bundles (Fig. 1g).Few hyphae of this type were also found in the dead cells together with thick melanized hyphae of type 1 and from time to time in the detritus covering the pseudobulb cavity (Fig. 1h).All visible endophytes were, without exception, limited to the cell layers of the innermost third of the pseudobulb tissue and never found in the part close to the epidermis.They were especially prominent in Azteca and Pheidole sp.inhabited ones, while being less frequent in abandoned pseudobulbs and those colonized by Camponotus atriceps (Table S1).
Both hyphae types co-occurred in the layers of dead cells and 1-3 layers in the living tissue.This zone is called the 'interface zone' (Fig. 2).
Closed pseudobulbs, which had failed to form a natural opening, were completely free of fungal hyphae on the chamber surface and in living tissue.
The translocation of 15 N into leaves was quick and 15 N enrichment significant compared with unlabelled plant tissue at all time points (atom% 15 N leaves day 1, 2, 4, 8: GLM estimate 0.009, t-value 3.61, df = 43, P < 0.001).However, labelling duration had no significant impact on 15 N enrichment (Fig. 3c).Interestingly, 15 N enrichment of leaves of the abandoned pseudobulbs (AB) did not differ significantly from Camponotus atriceps (CAM) inhabited ones.Both had a significantly lower 15 N APE than pseudobulbs inhabited by Pheidole sp.(PHE) and all Azteca species (AZ1, AZ2, AZA; P < 0.01, df = 6; Kruskal-Wallis rank-sum test, Dunn post hoc test; Fig. 3d; Table 2).Unfortunately, no data on leaf samples of the two closed pseudobulbs could be obtained.

ToF-SIMS
A large-area composite ToF-SIMS image (900 × 300 μm 2 ) of a C. bilamellatum pseudobulb inhabited by Azteca cf velox and labelled with 15 N for 24 h gives an overview of the label distribution.It shows a highly heterogeneous 15 N uptake with pronounced hotspots visible in the detritus layer and in dead cells.Cross sections of thick hyphae (type 1) and smaller spots probably depicting the small hyaline Hypocreales (type 2) could be resolved (Fig. 4a).However, many structures identified as hyphae remained unlabelled.Apart from hyphae, 15 N was translocated deep into living tissue along cell walls and the active nuclei close to a vessel about eight layers of living cells inwards from the pseudobulb chamber (Fig. 4a).IRMS measurement of the corresponding bulk samples yielded 10.3 APE 15 N for pseudobulb tissue and 0.009 APE 15 N for leaves.From a second sample inhabited by Azteca cf velox which had been labelled for 8 d, an area of 500 × 500 μm 2 was imaged. 15N was almost exclusively found in hyphae, which could clearly be attributed to either thick type 1 or thin type 2 (Hypocreales).The corresponding morphological structures were validated in LM images (Figs 4b-e, S5) The 15 N label content in thin hyphae reached values of up to nominally 50 atom% but varied largely.The IRMS determined bulk values for this sample were 8.4 APE 15 N for pseudobulb tissue and 0.05 APE 15 N for leaves.

NanoSIMS
The same two samples were used for close-up views on the cellular level in NanoSIMS.Of particular interest were the vessel deep inside the pseudobulb tissue, the innermost living cell layer bordering the pseudobulb cavity and the interface zone where both hyphae types co-occurred.After a labelling time of 24 h, vessel protoplast exhibited a considerable enrichment (0.15-3.5 APE 15 N) while cell walls only showed moderate-to-low values (0.02-0.5 APE 15 N).A ring-like structure representing a crosssectioned thin fungal hyphae (type 2) was also highly enriched (2.9 APE 15 N).The highest 15 N enrichment was observed in the two active cell nuclei near the vessel (5.0 APE 15 N and 4.8 APE 15 N; Fig. 5a; Table 3a).
Within the innermost living cell layer close to the pseudobulb cavity, hyphae of type 2 (Hypocreales) were strongly labelled (3.0, 1.4 APE 15 N, respectively).Cell walls had an enrichment of 0.4 APE 15 N.An area free of plasmatic structures and interpreted as the vacuole, exhibited the lowest values (0.06 APE 15 N; Fig. 5b; Table 3b).

Discussion
By combining bulk IRMS data and visualization of 15 N uptake by means of ToF-SIMS and NanoSIMS, we could demonstrate that the agar-bound label placed on ant detritus in hollow pseudobulbs of C. bilamellatum was mainly incorporated into specific structures and did not diffuse uniformly between cells.This rules out passive diffusion as the main path of nitrogen transfer.Instead, apoplastic transport along cell walls and enrichment of fungal hyphae was observed, which indicates that fungal hyphae are particularly absorptive structures to take up 15 N from the ant waste site. 15N uptake was considerable and a part of it was even transferred into the leaves within only 24 h.Single hyphae sampled from the inner surface of hollow pseudobulbs where the ant waste was deposited and from the pseudobulb tissue could be cultivated and were assigned to respective fungal orders.The thin hyaline endophytes (type 2) were identified as members of Hypocreales, an order with different lifestyles ranging from plant and insect pathogens to symbiotic endophytes (Saikkonen et al., 1998;Wu & Cox, 2021).The cultures grown from septate and melanized single hyphae (type 1) were assigned to Capnodiales, Chaetothyriales and Mycosphaerellales, orders which belong to the so-called 'black fungi'.Members of this group of phylogenetically unrelated fungi have in common the presence of melanin in the cell wall, the formation of yeast-like daughter cells under certain environmental conditions, and their ability to withstand hostile environments (de Hoog & Hermanides-Nijhof, 1977;Gostinčar et al., 2012).This ability makes black fungi frequent associates of ants with a more or less mutualistic relationship (Schlick-Steiner et al., 2008;Voglmayr et al., 2011;Mayer et al., 2018;Moreno et al., 2019;Blatrix et al., 2021).
First evidence for an involvement of an ant-associated fungus in nitrogen transfer from ant made carton outside the stem to the plant tissue provided a study on the ground-rooting ant-plant Hirtella physophora (Chrysobalanaceae; Leroy et al., 2017).The authors classified the fungus as belonging to Chaetothyriales and suggested that the hyphae outside and the ones inside the plant tissue belong to the same species.The occurrence of > 1 fungal strain involved in nutrient uptake distinguishes our study considerably from the findings in myrmecophytic Hirtella physophora.In that ant-plant, inhabiting ants form galleries stabilized by a fungus classified as member of Chaetothyriales, which mediated transfer of nitrogen from carton to the plant (Leroy et al., 2011(Leroy et al., , 2017)).The authors found endophytic hyphae inside the plant cells and propose that the hyphae on the gallery outside the plant tissue and the ones in the plant tissue belong to the same fungal species (Leroy et al., 2017).
In C. bilamellatum, however, a high abundance of black fungi in the ant waste and the adjacent layer of dead cells originating from the now dried-up original water storage tissue was not correlated with either nutrient uptake or translocation into leaves.Instead, the best predictor for the average nutrient uptake was the abundance of hyphae assigned to Hypocreales forming a net of hyphae in the living tissue but grew alsothough less abundantin the dead cell layer and rarely also directly into the ant waste.High abundance of Hypocreales and low abundance of black fungi resulted in a higher uptake rate than in the reverse  3. (a) Region around a vessel about six cell layers distant from the hollow chamber. 15N is strongly incorporated into active nuclei reaching 5 APE (a1,2), into hyphae with 2.9 APE (a3) and in the protoplast of a vessel with up to 3.5 APE (a4).Cell walls displayed much lower uptake of 0.5 APE (a5) or remained almost unlabelled at 0.02 APE (a6), as did the protoplast of a cell outside the vessel with 0.15 APE (a7).case with high abundance of black fungi and low abundance of Hypocreales.In the interface zone in which both hyphal types co-occur, Hypocreales exhibited an 15 N enrichment more than twice as high as hyphae of black fungi.However, as the incorporation of 15 N is only expected in newly grown hyphal cells, the difference in enrichment probably reflects the very slow growth rate in black fungi compared with Hypocreales (Abdollahzadeh et al., 2020;Quan et al., 2020).
From that pattern, we deduce that in C. bilamellatum Hypocreales are especially important for rapid nutrient transport into living tissue and vascular bundles, and from there into leaves and reproductive parts due to their faster growth and endophytic lifestyle.Remarkably, though variation among replicates was substantial, we found the highest label translocation rates to leaves when both, black fungi and Hypocreales, had a high abundance in the interface zone.This area seems to be an important interface at a fungus-fungus interaction level with a synergistic effect for nutrient transfer.To explain the peaks, a scenario with two simultaneous uptake pathways is conceivable: One pathway involves black fungi (type 1) taking up the high molecular weight 15 N label from the site of the ant waste and processing it in their intermediary metabolism.Amino acids are then released back into the apoplast of cells of interface zone where both hyphae types co-occur.From there, endophytic Hypocreales could absorb the 15 N labelled amino acids and transfer them to the vascular bundles.The second pathway is direct via endophytic Hypocreales hyphae that have been growing into the ant waste.As Hypocreales are less abundant in the layer of dead cells, and even less in the waste than in living cells, direct nutrient uptake will result in a lower amount of 15 N transfer than a 2-way transfer involving both hyphal species at the same time.Due to their rapid growth and hyphal network in the living pseudobulb tissue, Hypocreales are most likely responsible for the amazing speed of nutrient uptake found in our previous study (Gegenbauer et al., 2012).
In general, hypocrealean endophytes are not strictly bound to root symbiosis but can also use other niches, sometimes even simultaneously (Leger & Wang, 2020); therefore, the mechanism underlying endophyte-mediated nitrogen transfer in the pseudobulb tissue of C. bilamellatum is expected to be comparable to that in roots.Ant waste in the hollow pseudobulbs acts as an equivalent to soil in terms of nutrient source for this epiphytic orchid with no contact with the ground, and the stem (=pseudobulb)born endophytic network of Hypocreales mediating nutrient transfer from ant-derived waste to the host plant tissue may be functionally equivalent to mycorrhizal mycelia in roots.
Unfortunately, to date, we do not know whether carbon from plant photosynthates is reciprocally transferred into the hyphae.Nor do we know which of the six Hypocreales strains cultivated from the pseudobulb mediates nutrient transfer.Candidates are Purpureocillium lilacinum and P. lavendulum (Hypocreales, Ophiocordycipitaceae) isolated from the tissue of several C. bilamellatum pseudobulbs, which are known to enhance plant growth and reproductive tissue development in cotton plants as root endophytes (Castillo Lopez & Sword, 2015).Fungal endophytes may improve host resilience to abiotic stress like drought and heat (Rodriguez et al., 2008;González-Teuber et al., 2018;Moghaddam Hosseyni et al., 2021), both of which aretogether with nutrient deficiencyextreme in the natural environment of C. bilamellatum.Information on the variation and direction of nutrient flux within the plant throughout the annual growth cycle is lacking so far but the mature pseudobulbs examined may not exclusively be a nitrogen sink as we demonstrated in this study.They may also act as a nutrient source for adjacent immature pseudobulbs, depending on nutrient distribution and demand within the entire plant (Zotz, 1999).In this scenario, nitrogen would rather be translocated to stronger nutrient sinks within the plant like the emerging new growth than stored in mature pseudobulbs and their leaves.This might be a reason for the strong variation in 15 N transfer to leaves as well as the lack of 15 N accumulation over time above a certain baseline.

Conclusion
We were able to demonstrate that two types of fungiexophytic and endophytic onesmediate the remarkably rapid transfer of nutrients from ant waste to the plant tissue in the epiphytic orchid C. bilamellatum.This is the first evidence that a network of two different fungi results in a synergism in terms of nutrient uptake.While studies on nutrient transfer via mycorrhiza in roots are numerous, much less is known about the role of endophytic fungi in the stem of plants for nutrient provision.In nutrientdeficient habitats like a tropical rainforest canopy, the capacity to use nutrients from insect waste may be crucial for survival and reproduction for plants with an arboreal lifestyle.

Fig. 1
Fig. 1 Morphology of Caularthron bilamellatum (Orchidaceae), anatomy of pseudobulbs inhabited by Azteca cf velox and endophytic fungi.(a) Epiphytic growth of C. bilamellatum with ants inhabiting the hollow pseudobulbs (pbs).(b) Longitudinal section through pbs displaying the hollow chamber, which forms upon maturation.Ants can enter through a basal slit (bottom).(c) Micro-CT image of transversal section of pb.Big cells of the former water storage tissue are torn open and filled with ant waste (white).(d) Scanning electron microscopy image of living plant tissue several cell layers outward from the pb cavity.Thin fungal hyphae are growing along and across cell walls.(e) Thick, distinctly septate melanized fungal hyphae (black fungi) growing in and through dead cells adjacent to the pseudobulb cavity.Note abundant pits.(f) Thin hyaline string-like hyphae (Hypocreales) growing in living cells several layers outward from the pb cavity.The cell nucleus can be seen in the upper right corner.(g) Thin hyphae (visible as blue strings) growing deeper into living tissue and towards the vessels (bottom left).(h) Interface c. 2-4 cell layers outward from the pb cavity with both thick (black fungi, middle left) and thin (Hypocreales, middle right) hyphae within living cells, often crossing cell walls.

Fig. 2
Fig. 2 Schema of a pseudobulb of Caularthron bilamellatum colonized by ants.Hyphae of type 1 (Capnodiales, Chaetothyriales and Mycosphaerellales) were found in ant waste and dead cells originating from the former water storage tissue, only rarely in living tissue.Hyphae of type 2 (Hypocreales) mainly colonized the living tissue but were also found in the dead cell layers and rarely in ant waste.The area where both types of hyphae occur together is called 'interface'.

Fig. 3
Fig. 3 15 N Isotope ratio mass spectrometry (IRMS) data for Caularthron bilamellatum pseudobulbs (pbs) and leaves.Box plots show the median as horizontal line, interquartile ranges as coloured box and whiskers as minimum and maximum values.Rings denote outliers and asterisks extreme outliers.Different letters indicate significant differences between groups (Kruskal-Wallis rank-sum test with a Dunn post hoc analysis).(a) Time series for labelled pbs from 1 to 8 d.Pbs from all plots pooled.The incubation time had a significant effect on 15 N APE in pseudobulbs (pbs: GLM estimate 1.53, t-value 2.1, df = 40, P = 0.043, whereas day 1 and 8 differed significantly P = 0.023, Kruskal-Wallis rank-sum test with a Dunn post hoc analysis).(b) Comparison of pbs inhabited by different ant species, each pooled across time series 1-8 d.CL, closed sterile (n = 2); CAM, Camponotus atriceps (n = 4); AB, uninhabited but with signs of former ant presence (n = 7); AZA, Azteca sp.(n = 8); PHE, Pheidole sp.; AZ1 (n = 7) and AZ2 (n = 7), Azteca cf velox.Significant differences were found between CL and CAM vs PHE, AZ1, AZ2, AZA (higher 15 N APE, P < 0.01, df = 6; Kruskal-Wallis rank-sum test with a Dunn post hoc analysis).(c) Time series for leaves (all n = 6 except for AZA with n = 8) carried by pseudobulbs shown in (a).(d) Comparison of leaves carried by pbs shown in (b).No leaf samples for closed pbs could be obtained.AB had a significantly lower 15 N APE than PHE and AZ1, AZ2, AZA (P < 0.01, df = 6; Kruskal-Wallis rank-sum test with a Dunn post hoc analysis).

Fig. 4
Fig. 4 Secondary ion mass spectrometry images of 15 N uptake in Caularthron bilamellatum inhabited by Azteca cf velox.The false-colour scale displays at % 15 N values ranging from 0 (black) to 100 (white).(a) Qualitative ToF-SIMS image of pseudobulb (pb) tissue, cross section.Giant cells bordering the pb chamber can be seen on the left side, and the epidermis would be towards the right (not visible).Red, 12 C 14 N − (natural abundance); green, 12 C 15 N − (label).Pbs were labelled by placing agar discs containing a high molecular weight 15 N source on the surface of the hollow chamber for 24 h.Thick hyphae (black fungi) in the detritus layer and in dead giant cells and thinner hyphae (Hypocreales) in the innermost living cell layer exhibit strong 15 N uptake. 15N is transported towards active cell nuclei and into a vessel.Note that signal intensities are biased by mass interferences.(b, d) Semiquantitative ToF-SIMS image of a longitudinal section through the innermost living cells rich in endophytes displaying 12 C 15 N − normalized to the sum of 12 C 14 N − + 12 C 15 N − after a labelling period of 8 d.Unlabelled background (natural abundance) is shown in black, moderate enrichment up to 20 atom% 15 N in blue.Fungal hyphae are displayed as turquoise to yellow dots and exhibit massive enrichment of 50-60 atom% 15 N. (c, e) Light microscopy image of the corresponding regions.Thick hyphae (black fungi) and thin hyphae (Hypocreales) can be seen within the same cell.All strong 15 N signals can be correlated to hyphae-like structures but not all hyphae incorporated 15 N. BF, black fungi; Hyp, Hypocreales.

Fig. 5
Fig. 5 Quantitative NanoSIMS isotope imaging of Caularthron bilamellatum pseudobulb inhabited by Azteca cf velox and labelled with 15 N either for 24 h (a, b) or 8 d (c).The false-colour scale displays atom% 15 N values, regions of interest (ROIs) are marked in purple, and the corresponding δ 15 N and atom% excess (APE) values are given in Table3.(a) Region around a vessel about six cell layers distant from the hollow chamber. 15N is strongly incorporated into active nuclei reaching 5 APE (a1,2), into hyphae with 2.9 APE (a3) and in the protoplast of a vessel with up to 3.5 APE (a4).Cell walls displayed much lower uptake of 0.5 APE (a5) or remained almost unlabelled at 0.02 APE (a6), as did the protoplast of a cell outside the vessel with 0.15 APE (a7).(b) Innermost living cell bordering the pseudobulb chamber in close vicinity to where the labelling substance was placed. 15N is incorporated in hyphae (oval and bar-like structures) to 2-3 APE (b1,2).Thick cell walls show a slight enrichment of 0.45 APE (b3) while the protoplast remained nearly unlabelled at 0.06 APE (b4).(c) Interface harbouring thick and thin hyphae, 2-3 cell layers distant from the pseudobulb cavity.Thin Hypocreales hyphae (c1-3) incorporated up to 50 APE, thick hyphae of black fungi (c4,5) up to 20 APE.An inactive cell nucleus, 0.71 APE (c6) and the cell wall, 2.3 APE (c7) incorporated far lower amounts of 15 N. (d) ( 12 C 14 N − + 12 C 15 N − )/ 12 C 2 − secondary ion intensity ratio image corresponding to the area displayed in (c), revealing intracellular Fig. 5 Quantitative NanoSIMS isotope imaging of Caularthron bilamellatum pseudobulb inhabited by Azteca cf velox and labelled with 15 N either for 24 h (a, b) or 8 d (c).The false-colour scale displays atom% 15 N values, regions of interest (ROIs) are marked in purple, and the corresponding δ 15 N and atom% excess (APE) values are given in Table3.(a) Region around a vessel about six cell layers distant from the hollow chamber. 15N is strongly incorporated into active nuclei reaching 5 APE (a1,2), into hyphae with 2.9 APE (a3) and in the protoplast of a vessel with up to 3.5 APE (a4).Cell walls displayed much lower uptake of 0.5 APE (a5) or remained almost unlabelled at 0.02 APE (a6), as did the protoplast of a cell outside the vessel with 0.15 APE (a7).(b) Innermost living cell bordering the pseudobulb chamber in close vicinity to where the labelling substance was placed. 15N is incorporated in hyphae (oval and bar-like structures) to 2-3 APE (b1,2).Thick cell walls show a slight enrichment of 0.45 APE (b3) while the protoplast remained nearly unlabelled at 0.06 APE (b4).(c) Interface harbouring thick and thin hyphae, 2-3 cell layers distant from the pseudobulb cavity.Thin Hypocreales hyphae (c1-3) incorporated up to 50 APE, thick hyphae of black fungi (c4,5) up to 20 APE.An inactive cell nucleus, 0.71 APE (c6) and the cell wall, 2.3 APE (c7) incorporated far lower amounts of 15 N. (d) ( 12 C 14 N − + 12 C 15 N − )/ 12 C 2 − secondary ion intensity ratio image corresponding to the area displayed in (c), revealing intracellular structures and hyphae with high nitrogen content.Grey areas in (a, b) refer to pixels where an unbiased atom% 15 N visualization is not feasible due to inferior counting statistics, emerging from weak 12 CN-secondary ion signal intensities (mainly within resin areas).

Table 1
Fungal pure cultures of six Caularthron bilamellatum pseudobulbs inhabited by Azteca cf velox.
Samples were taken from living tissue (T) and the inner surface of hollow pseudobulbs (S) and cultured on 2% MEA agar plates.Taxonomic affiliation was determined by sequencing the complete ITS1-5.8S-ITS2nuclear ribosomal DNA and subsequent NCBI BLAST search.New Phytologist (2023) 238: 2210-2223 www.newphytologist.comÓ 2023 The Authors New Phytologist Ó 2023 New Phytologist Foundation

Table 2
Mean 15 N atom% excess (APE) AESE in labelled pseudobulbs and corresponding leaves of Caularthron bilamellatum for each sampling plot separately.

Table 3
NanoSIMS determined atom% excess (APE) and δ 15 N (‰) values of Caularthron bilamellatum pseudobulb (pb) inhabited with Azteca cf velox within the particular regions of interest (ROI) shown in Fig. 5. Pbs were labelled by placing an agar disc with an organic 15 N source into the apex of hollow pbs for 24 h (a, b) or 8 d (c).(a) Vessel about six cell layers inwards from the pb cavity.(b) Innermost living cell layer bordering the pbs cavity.(c) Living cells rich in endophytic fungi 2-3 layers from the pb cavity.