BIOMECHANICS of PERIDIOLE EJECTION and FUNCTION of the FUNICULAR CORD in BIRD's NEST FUNGI by Maribe

Home , Crucibulum, Crucibulum (gastropod), Cyathus, Cyathus olla, Cyathus stercoreus, Cyathus striatus

ABSTRACT

SPLASH AND GRAB: BIOMECHANICS OF PERIDIOLE EJECTION AND FUNCTION OF THE FUNICULAR CORD IN BIRD’S NEST FUNGI

by Maribeth Hassett

The bird’s nest fungi (Agaricales, Nidulariaceae) package a millions spores into sporangia (referred to as peridioles) that are splashed from their basidiomata by the impact of raindrops. Peridioles are splashed from flute-shaped basidiomata at speeds of 1 to 5 meters per second (11 mph). This study examines the mechanism of peridiole ejection and funicular cord function in Cyathus and Crucibulum using high-speed video. The funicular cord is a highly-extensible bundle of hyphae whose tensile strength is maximized by the modification of clamp connections. The funicular cord remains in a condensed form during flight with an adhesive pad exposed on the projectile surface. The cord unravels when the pad sticks to surrounding vegetation and acts as a brake that quickly reduces the velocity of the projectile. This elaborate mechanism tethers peridioles in a perfect location for browsing by an herbivore and is viewed as a beautiful adaptation for a coprophilous fungus.

SPLASH AND GRAB: BIOMECHANICS OF PERIDIOLE EJECTION AND FUNCTION OF THE FUNICULAR CORD IN BIRD’S NEST FUNGI

A Thesis

Submitted to the

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Master of Science

Department of Botany

Maribeth Hassett

Miami University

Oxford, Ohio

2012

Advisor ______(Dr. Nicholas Money)

Reader______(Dr. Daniel Gladish)

Reader______(Dr. Chun Liang)

Table of Contents

List of Tables………………………………………………..……………………………………iii

List of Figures…………………………………………………………………………………….iv

Acknowledgments…………………………………………………………………….…………..v

Introduction…………………………………………………………….…………..……………...1

Materials and Methods……………………………………………………………...……………..3

Results and Discussion…………………………………….………………………………….…..6

Figures…………………………………….…………………………………………...…………14

References……………………….………………………………………………………..……...30

List of Tables

Table 1. Measured biomechanical data of peridiole ejection based on high-speed video sequences.

Table 2. Predicted biomechanical data based on modeled peridiole trajectories.

Table 3. Measured mechanics of model peridiole discharge using model splash cups.

iii List of Figures

Figure 1: Cyathus fruiting body structure from Brodie (1975).

Figure 2: Species within the Nidulariacae produce fruit bodies of varying shapes.

Figure 3: Figure illustrating the four species used in this study: Cyathus olla, Cyathus stercoreus, Crucibulum laeve, and Cyathus striatus.

Figure 4: Experimental design of splash experiments conducted on bird’s nest fungi.

Figure 5: Splash experiments were performed on model splash for comparison with actual fruit bodies.

Figure 6: Individual frames from video sequence of splash discharge in Cyathus olla at 3,000 fps.

Figure 7: Still frame of Cyathus olla peridiole ejection taken from a high-speed video sequence.

Figure 8: Predicted trajectories of peridioles of four species of bird’s nest fungi based on launch data obtained by high-speed video microscopy.

Figure 9: Splash scenarios resulting from raindrops hitting a fruit body of Cyathus olla at two positions.

Figure 10: Scanning electron micrographs of cross-sectioned peridiole of Cyathus olla.

Figure 11: Scanning electron micrographs of the funicular cord.

Figure 12: Scanning electron micrograph of strand of hyphae from the funicular cord showing a modified clamp connection.

Figure 13: Scanning electron micrographs of clamp connections from the funicular cord showing extracellular bands around two septa

iv Acknowledgments

I would like to thank my advisor, Dr. Nicholas Money, for the opportunity to work on this project. I am thankful for his dedication, encouragement, and sense of humor that made this project rewarding and enabled me to grow professionally. I am also grateful for the insight and guidance given to me by Dr. Chun Liang and Dr. Daniel Gladish during the duration of this project. In addition, I am thankful that I had the opportunity to grow as educator under the tutelage of Dr. Susan Barnum. Thanks to Matt Duley for helping me with my scanning electron micrographs. A special thanks to Zachary Sugawara and Jessica Stolze-Rybczynski whose preliminary data made this project possible. Furthermore, I had the privilege of collaborating with Dr. Mark W. F. Fischer whom helped me develop my quantitative research skills. I am also especially grateful for the love and support from my family.

v Introduction and historical context

The bird’s nest fungi (Agaricales, Basidiomycota) produce basidiomata adapted for splash dispersal, utilizing the force of a raindrop to launch their spore containing peridioles to remarkable heights. Species within this family package hundreds of thousands of basidiospores inside spore dispersing packets known as peridioles. Ejected peridioles become tethered to vegetation, placing them in a perfect position to be eaten by grazing herbivores, a perfect location for grazing herbivores. Peridioles are tethered by a specialized structural organ known as the funicular cord, an extendible bundle of hyphae that is twisted together to form a strengthened cord-like structure. Brodie illustrates the structural anatomy of the fruit body of Cyathus in detail (1) (Figure 1). Structural adaptations for splash dispersal and attachment of peridiole vary among the five genera of bird’s nest fungi. Species of Cyathus and Nidula produce a flute-shaped fruit body. Species of Crucibulum produce a shallow, saucer-shaped fruit body. Species of Nidularia and Mycocalia produce sac-like fruit bodies that split open upon maturity to reveal peridioles embedded in a gelatinous pad. Peridioles of Nidularia, Mycocalia, and Nidula lack a funiculus but are covered in an adhesive coat, while Cyathus and Crucibulum produce peridioles with funiculus (Figure 2). Species that produce a flute-shaped fruit body with a funiculus are believed to be most effective at launching peridioles long distances via splash dispersal (1). The first mention of the bird’s nest fungi in literature was by Clusius in 1601 (8), followed by several classic works including Tulasne (18), Sachs (17), Eidam (11), and Brefeld (2). These works inspired several descriptive monographs including White (20) and C.J. Lloyd (13). These monographs detailed the morphology of the fruit bodies but lacked information on the splash mechanism. Researchers at this time were puzzled by the function of the unusual anatomical structures found in this family and what role these structures might play in spore dispersal. The mechanics of splash dispersal of the bird’s nest fungi was misunderstood by early investigators. The Tulasne brothers wrote detailed descriptions of the morphology of Cyathus species including a description of the funiculus. The brothers were unable however, to determine the function of the funiculus and the relationship between the structure of the fruit body and its function (3). In 1927 Martin (14) was first to suggest that raindrops might splash peridioles from 1 the fruit bodies into the air and onto vegetation. He conducted simple splash experiments by holding a pipette containing water a meter above the fruit bodies and observed that the force of a raindrop was able to eject the peridioles. Interest in the bird’s nest fungi was renewed again in the 1940s when several researchers learned that bird’s nest fruit bodies’ function as spectacular splash cups. In 1941, Zenker (21) published a paper describing a parasitic fungus that he named Leptostroma camelliae adhering on Camellia leaves. William Diehl (9) corrected this mistake and discovered that this purported parasitic fungus was Cyathus. Zenker had mistaken Cyathus peridioles for the hyphae of a parasitic fungus. Diehl marveled at how high he found the peridioles on foliage and wondered how they were launched vertically to such great heights. Also in 1941, Dodge (10) received several inquiries describing bird’s nest peridioles found four meters above ground. Dodge (10) speculated that the funiculus might function as an attachment organ for peridioles but his work made no mention of splash dispersal. He estimated what force would be strong enough to propel the peridioles three to five meters in the air. It was not until 1941 that Buller (7) revealed the relationship between the vase- shape fruit body and peridiole ejection. Buller combined previous knowledge of morphology of fruit bodies, and work done by Diehl (9), to solve the puzzle of splash dispersal. He was the first to describe splash dispersal of peridioles and the correct function of the funiculus. Buller’s student, Harold Brodie (3,4,5,6) continued his work and dedicated his career and many publications to the study of the bird’s nest fungi. Brodie (5) conducted simple splash experiments on several species of bird’s nest fungi, however was limited to ambiguous measurements of peridiole ejection because he had was unable to visualize peridiole launch. He hypothesized about the events that occurred during peridiole ejection and was left with many unanswered questions about the biomechanics of splash dispersal in the bird’s nest fungi. He was puzzled by the mechanics of the funicular cord, and without high-speed video, was unable to describe in detail the events that occurred during splash dispersal (1). Since peridiole ejection occurs at great speeds, tools such as high-speed video can enable visualization and analysis of splash dispersal in the bird’s nest fungi. Previous researchers have only hypothesized about the events that occur during peridiole ejection and their impact on vegetative substrates. There has been little research on this fungal

2 family has not been updated since the 1970’s, and many questions about its splash dispersal mechanism are still unanswered. This thesis reports both the structural and mechanical aspects of splash dispersal in the species Cyathus striatus, Cyathus stercoreus, Cyathus olla, and Crucibulum laeve using modern techniques. A thorough analysis of the dispersal mechanisms of Nidulariacae was conducted, using high-speed video and scanning electron microscopy. The objective was to answer the following key questions using a combination of observational and measured mechanical data based on splash experiments: - What are the mechanics of peridiole discharge in different species? - How does fruit body shape affect peridiole ejection? - How does drop entry affect peridiole ejection? - How does the funicular cord function?

Materials and Methods Specimen preparation for splash experiments Mature fruit bodies of the bird’s nest fungi species; Cyathus olla, Cyathus striatus, Cyathus stercoreus, and Crucibulum laeve (Basidiomycota, Agaricomycotina, Agaricomycetes, Agaricomycetidae, Agaricales, Nidulariaceae) were collected from mulch applied to landscaping at Miami University in Oxford, OH USA (Figure 3). Specimens were pinned to pieces of corkboard for stability during splash experiments. Four sheets of glass were placed around the fruit bodies to protect the high-speed camera from splashing. The camera was placed outside the glass enclosure and focused on the fruit body in order to capture the splash event. Water drops were released from a burette positioned 1.2 m above the fruit bodies to simulate raindrops. Water drops , approximately 6 mm in diameter, hit the fruit bodies at a mean velocity of 4.4 +/- 0.05 m/s (Figure 4).

Model Fruit bodies Species of bird’s nest fungi produce fruit bodies of various shapes. In addition to experiments with fresh specimens, models of fruit bodies were fabricated to enhance our analyses in various ways. Models resembling species of Cyathus, Crucibulum, and Nidula were created by cutting Eppendorf tubes with a heated scalpel to create splash cups with heights varying from 8 to 15 mm and openings of 5 to 8 mm. The plastic on the bottom of the cups was 3 melted and fixed to the bases of inverted Petri dishes. Clear plastic balls (3/32” nylon white; Small Parts Manufacturing, Portland, OR) were used as model peridioles (Figure 5). Fresh specimens of the more open types of fruit body produced by Nidularia and Mycocalia were not collected for use in this study due to geographical limitations. Model fruit bodies resembling species of Nidularia or Mycocalia were created using Stick Tack (ShurTech Brands, Avon, OH) modeling clay. Mucilage was modeled using 0.8% agar since Nidularia and Mycocalia produce peridioles that are embedded in mucilage. Approximately 10-15 plastic balls, previously mentioned, were embedded in the mucilage inside the model fruit body to model peridioles (Figure 5). Previous researchers have been unable to visualize the attachment of peridioles to vegetation due to the high velocity of peridiole ejection. In order to observe the mechanics involved with the attachment of peridioles to vegetation, fruit bodies were surrounded by model vegetation. Fruit bodies were pinned to corkboard and surrounded by 2’’ pieces of metal floral wire to visualize attachment of peridioles with high-speed video.

High-Speed Videography and Data analysis High-speed videos of peridiole discharge in Cyathus and Crucibulum were captured with a FASTCAM-ultima APX-RS camera (Photron, San Diego, CA). The camera enabled the visualization of the initial ejection of the peridiole and also the impact of the peridiole on model vegetation. Videos were analyzed using Photron Fast Cam Viewer and Image-Pro Plus 6.2 (Leeds Precision Instruments, Minneapolis, MN). Velocity measurements of the peridiole and drop, along with the ejection angle of launched peridioles, were calculated using VideoPoint 2.0. The amount of kinetic energy was found using the data measured from VideoPoint 2.0. The kinetic energy utilized to achieve peridiole launch using determined using the following equation

2 1 Kinetic Energy = 2 × mass ×(velocity) In this equation, mass denotes the mass of the peridiole measured with an accuracy of +/- 0.1 mg. Velocity refers to the velocity of the peridiole after ejection from the fruit body. To determine the efficiency of the splash discharge mechanism, the kinetic energy of the discharged peridiole was divided by the kinetic energy of the water droplet before it hit the fruit body.

4 Mathematical modeling Mathematical models of peridiole trajectories were created using MATHEMATICA 6 (Wolfram Research, Inc., Champaign, IL). To generate equations for the x- and y- positions of the spore mass as functions of time, Mathematica was used to symbolically (no numbers)   integrate Newton' s second Law, ΣF = ma , where the forces were taken to be gravity (mg) in the   minus y-direction and Stokes Law drag opposing the motion: Fdrag = −6πrηv . In this equation, r is the effective radius of the peridiole (found by calculating the radius of a sphere with the same volume as the peridiole (and any attached water), η is the viscosity of air = 18.07 x10-6 Pa s or (N/m2) s = 180.7 x10-6 poise, and  v is the velocity of the peridiole. Since velocity is the time-derivative of position and acceleration is the time-derivative of velocity, the equations € −6πrηvx = max and

−6πrηvy − mg = may € can be solved for the x- and y-positions as functions of time. These equations were used to model the flight of a peridiole given the size, mass, and launch speed and angle of the projectile. The € resulting flights were compared to the observed flights. Modeling of peridiole trajectories also revealed the predicted maximum height and maximum range for each species.

Scanning electron microscopy Fresh fruit bodies of four species of bird’s nest fungi were collected in Butler County, Ohio. Mature peridioles were extracted from each individual fruit body with forceps. Each peridiole was cross-sectioned using a razor blade leaving the funicular cord intact. The funicular cord was kept intact so that basidiospores and the funicular cord of each peridiole could be fixed at the same time. Peridioles were immersion fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer for one hour at room temperature. Samples were then rinsed 4 x 15 minutes at room temperature with 0.05M sodium cacodylate buffer. Samples were ethanol dehydrated (25% ethanol solution for 20 mins; 50% 20 mins; 75% 20 mins; 95% 30 mins;

5 100% 60 mins; 100% 60 mins) and further prepared by critical point drying. Dried samples were mounted on an adhesive tab on the stub and silver painted. Finished samples were sputter coated 3 times at 20 nm each, once not tilted, and then tilted on opposing sides to ensure all sides of peridiole were coated with gold. Samples were viewed using Zeiss Supra 35 VP FEG SEM.

Results and Discussion Splash experiments using high-speed video High-speed video recordings of peridiole ejection were made from four species of bird’s nest fungi: Cyathus striatus, Cyathus olla, Cyathus stercoreus, and Crucibulum laeve. High- speed video provided new information on splash discharge and, for the first time, allowed precise measurement of peridiole ejection speeds (Figure 6). The experiments verified that the fruit bodies of all four species operated as splash cups as A. H. R. Buller discovered in the 1940s (7). Analysis of the video recordings provided data on the velocity and angle of ejection. The average velocity of launched peridioles varied among species (Table 1). Peridioles of Cyathus olla were ejected at the slowest speeds, averaging 1.5 m/s, and Cyathus striatus was fastest a mean velocity of 3.6 m/s. The average ejection angle was similar for all four species. Theoretically, an ejection angle of 45 degrees would launch peridioles to the farthest distances in the absence of surrounding obstacles, but the videos showed that most launches were closer to vertical. The majority of ejection angles ranged between the angles of 70 to 90 degrees, with a mean angle for all species of 70 degrees (Figure 7). Based on these data, the splash dispersal of bird’s nest fungi appears to be adapted for maximum height rather than maximum distance. Peridiole launch is achieved by harnessing the kinetic energy of a falling raindrop. The kinetic energy (KE) of the drop and the ejected peridiole were calculated using the formula , using the measured mass and velocity of the drop and peridiole. The kinetic energy of the discharged peridiole was divided by the kinetic energy of the water droplet before it hit the fruit body to calculate the amount of energy utilized from the raindrop to achieve launch. The kinetic energy utilized in the peridiole launch was only 1% of the energy in the falling drop (Table 1). Models for the trajectory of discharged peridioles were developed using measurements of average peridiole size, mass, ejection velocity, and ejection angle. Trajectory plots indicate predicted maximum height and maximum distance based on these data (Figure 8). The plots 6 show that a high proportion of the peridioles ejected at angles between 70 and 90 degrees are propelled to heights from 12 to 49 cm. The modeling data are summarized in Table 2. In the absence of high-speed recordings of peridiole discharge, Brodie (5) was unable to measure launch speeds and other parameters. He conducted splash experiments and was able to measure the distances that peridioles were ejected from fruit bodies. He concluded that most species launched peridioles between 0.45 and 0.75 meters with the maximum horizontal range of 2.4 meters. Fruit body shapes vary among the five genera of bird’s nest fungi. These differences may affect the mechanics of peridiole discharge. Fresh fruit bodies of Cyathus and Crucibulum were collected for the present study and models of fruit bodies produced by genera that could not be collected were used to extend the range of observations. The shape and size of plastic splash cups corresponded to the morphology of the fruit bodies used in the experiments. Statistical comparisons (single classification ANOVA) between the initial velocity of the ejected beads and the peridioles showed that bead velocity matched the speed of peridiole ejection for Cyathus olla (P = 0.42), but was slower than the ejection speeds for the other fungi in this study (P < 0.005). Mean ejection angles of the beads from the model cups was the same as the mean ejection angles of the peridioles from all four fungal species (P ³ 0.54). Model splash cups resembling the shapes produced by Cyathus and Crucibulum ejected plastic beads (peridioles) at steep angles and velocities similar to Cyathus olla. Model splash cups resembling the mucilaginous pads produced by Nidularia and Mycocalia ejected beads at much shallower angles and slower velocities (Table 3). Splash experiments on fresh specimens of Nidularia and Mycocalia are needed to determine the validity of the models as surrogates for the real fruit bodies. Brodie (5) suggests that certain species of bird’s nest fungi may be better adapted for splashing peridioles over longer distances. Brodie placed the genera of bird’s nest fungi in the following series based on their effectiveness at launching peridioles: Mycocalia, Nidularia, Nidula, Crucibulum, and Cyathus. In the present study it was observed that all four species functioned as effective splash cups; however, several structural adaptations of Cyathus striatus appear to facilitate the upward thrust of the drop that affects peridiole ejection by increasing the launch speed. Cyathus striatus forms a deep vase-shaped fruit body with vertical striations on the inner wall of the cup. These striations may allow the water drop to increase in momentum as it

7 thrusts from the cup. Small hairs, known as setae, line the outer rim of the cup and may also serve as an adaptation that directs water upwards at a faster rate. Although this study did not examine fresh fruit bodies of Mycocalia, Nidularia, or Nidula, observations on models suggest that these species launch peridioles at slower speeds than Cyathus and Crucibulum. Previous studies suggested that particular cup angles alter the distance of peridiole discharge (1, 5). Brodie created artificial splash cups with modeling clay and found that cups resembling Cyathus species, with a wall angle between 60 and 75 degrees discharged water drops over the greatest distances. He noted that projectiles launched at an angle of 45 degrees would travel the longest distances, and suggested that surface tension forces between the water and the inner wall of the cup might reduce the ejection angle to this angle, or even lower (5). Amador et al. (1) found that the majority of plants produce splash cups with an opening angle of 27 to 54 degrees. Their study found that an ejection angle of 40 degrees was most efficient for seed dispersal, disseminating propagules over the greatest distance. Interestingly, the majority of peridioles of species of Cyathus and Crucibulum, are launched at an angle between 70 and 90 degrees, reflecting a steep launch to a great height but limiting horizontal range. The adaptive significance, if any, of these differences in launch characteristics is not known, but we can make the following logical inference from the ecology of the bird’s nest fungi. Vertical launches may increase the probability that peridioles clear vegetation next to the fruit bodies and adhere to surrounding grass culms, and the stems of other plants, positioning them for consumption by grazing herbivores. One feature of splash discharge that was not emphasized in previous studies is the importance of the position of drop impaction on the fruit body. High-speed video recordings show that peridioles are never launched when the first drop from the burette impacts the fruit body. The funiculus may need to be hydrated to facilitate purse rupture, which is necessary to allow the peridioles to be released from the inner surface of the fruit body (5). The displacement of water within the splash cup by a drop entering the cup may also be required to create the upward thrust that expels the peridioles. Interestingly, drops hitting the center of the cup did not eject the peridioles. Peridioles were only ejected when drops fell off-center and hit the rim of the fruit body (Figure 9).

8 The rim of the fruit body plays an essential role in splash dispersal, acting as an obstacle that fractures the drop, forcing water into the cup and away from the cup. When a drop hit the rim of a splash cup, some of the water was shed from the outer rim, while the water entering the cup was directed upwards by the fluted form of the fruit body. Presumably, this upward thrust of water was sufficiently forceful to rupture the purse and liberate the peridioles. Drops hitting the direct center of the fruit body resulted in the displacement of water over the rim in all directions. Drops hitting the fruit body at an eccentric position and directly on the rim are most likely to eject peridioles. It is the upward thrust of water that is essential for the ejection of the peridioles. Brodie briefly mentioned the function of the fruit body rim of bird’s nest species and the role that a flattened rim may play in dispersal. Fruit bodies of Cyathus olla produce this kind of rim. He hypothesized that a flattened rim would reduce the ejection angle resulting in a more horizontal discharge. Observations with high-speed videography do not support this hypothesis: the average ejection angle was similar for all of the species in the present study regardless of rim shape. Brodie conducted experiments using model splash cups with varying wall angles but did not consider the effects of the position of drop impaction (5). As previously mentioned, both splash scenarios, a drop hitting center or off center, create very different splash results (Figure 9). Peridioles are ejected only when a raindrop strikes off center, on the rim of the cup, and never when the drop hits dead center. If Brodie’s experiments were limited to the analysis of drops hitting the center of the artificial cups his investigations would, ultimately, be irrelevant to the mechanics of splash dispersal in these fungi. A study by Amador et al. (1) discusses the importance of drop positioning in plants that produce splash cups to disperse their seeds. This study found that off-center drops launched seeds 3 to 5 times faster than the incoming drop velocity. Although velocity enhancements of this magnitude were not observed here, the present study does confirm that off-center drops striking the rim result in peridiole launch. Amador and colleagues also suggested that seeds encapsulated in water would travel further distances by increasing their mass. We observed that launched peridioles were typically surrounded by the stream of water that caused ejection, but it seems unlikely that this small amount of water would increase dispersal distance.

9 Buller and Brodie were the first to accurately describe the function of the funiculus, a morphological structure specalized for the attachment of peridioles to vegetation. Presumably, the fixation of peridioles to vegetation is a key aspect of spore dissemination, placing peridioles in the perfect location for grazing by an herbivore. Consumption by an herbivore carries the spores over much greater distances than the initial dispersal by the splash mechanism, providing an opportunity for the development of new colonies far from the parent mycelium. Furthermore, after traveling through the digestive tract, peridioles are deposited into an ideal nutritive environment. Brodie has suggested that passage through the digestive tract of an herbivore is necessary for spore germination (5). There have been few studies on this process in bird’s nest fungi and it seems that some species may grow on plant debris without interacting with an animal vector. The only species that form cord structures are within the genera Cyathus and Crucibulum. Based on the hypothesis that the cord is a specialized structure that aids peridiole consumption by herbivores, it seems likely that Cyathus and Crucibulum are, primarily, coprophilous fungi, while species of the non-corded Nidula, Nidularia, and Mycocalia may be lignicolous. High-speed video recordings of splash discharge from fruit bodies surrounded by plant wires (to model vegetation) indicated how the funicular cord is deployed. Previous investigators were unable to determine whether the funicular cord extended as the peridiole left the fruit body, or whether it unraveled after the hapteron, the sticky end of the funicular cord, attached to vegetation (3,5,7). Our experiments showed that the funicular cord remains in compacted form when the peridioles are splashed from their fruit bodies and unravels only after the hapteron makes contact with an obstacle. The momentum created by the centripetal motion of the peridiole after the hapteron adheres to vegetation deploys the funicular cord and secures the peridole to the obstacle.

Analysis of funicular cord structure and peridiole anatomy using scanning electron microscopy Peridioles of Crucibulum laeve, Cyathus striatus, Cyathus strecoreus, and Cyathus olla, were studied with scanning electron microscopy. Peridioles were cross-sectioned to examine internal anatomy and the funicular cord was studied in the same samples. Basidiospores of bird’s

10 nest fungi were enclosed entirely within the peridioles, presumably to protect the spores and ensure germination. Also, it is likely that there are ecological advantages of splashing millions of spores contained within a single peridiole in one place. Spores are produced in a central mucilaginous layer of hyphae and enclosed within three subsequent layers of hyphae. Hyphal layering may be an adaptation to allow spores to pass through the digestive tract of an herbivore and germinate in fecal matter. Spore morphology of the bird’s nest fungi has been studied previously (5, 12, 14, 19), but detailed scanning electron micrographs of internal peridiole anatomy do not exist for the species Cyathus olla. Cyathus olla produces the largest peridioles of the species we examined, and therefore was the easiest to prepare for viewing with the scanning electron microscope. It was observed that this species produces three morphologically distinct hyphal layers, with numerous spores embedded in a central mucilage-filled region. The scar on the spore surface marking its connection to the sterigma was visible in spores of Cyathus olla (Figure 10). The key structural element of the funciulus is the funicular cord, which has been described as a strengthened cord-like attachment organ (3). The funicular cord is composed of thousands of individual strands of hyphae twisted together, rather than a bundle of individual strands aligned parallel to each other. Engineers have known for centuries that cords are strengthened by twisting fibers together. Twisted structures exhibit unique properties including tensile strength, flexibility, and elastic behavior (16). The funicular cord must retain a certain level of tensile strength in order to withstand the centripetal force of the flying peridiole as it winds around vegetation (Figure 11). Clamp connections are characteristic of dikaryotic hyphae formed by basidiomycetes and are produced by hyphae that form the funicular cord. This process generates two internal cross- walls, or septa, within an individual hypha (15). The septa created during clamp formation allow migration of dividing nuclei and create separate dikaryotic compartments (Figure 12). Brodie (5) first noted the interesting structure of clamp connections within the funicular cord using light microscopy. Due to the limited resolution of light microscopy, Brodie incorrectly described the clamp connections as double or paired clamps. Fleger and Hooper (12) later observed the clamp connections of Cyathus stercoreus using scanning electron microscopy and noted that the clamp connections were not doubled, but modified with a reinforcing extracellular band. The study by

11 Fleger and Hooper was limited to a single species and there have been no descriptions of clamp connections in other bird’s nest fungi. It was observed that the clamp connections on the funicular cord appear to be modified in Cyathus striatus, Cyathus stercoreus, Cyathus olla, and Crucibulum laeve. An extracellular band was observed around the septal wall between the hyphal compartments and around the septum in the clamp connection. It is possible that both septa create planes of potential weakness on the funicular cord. Based on this assumption, the extracellular bands may act to strengthen the cord, giving it the elasticity required to withstand the force exerted by the peridiole as it swings around vegetation. The presence of reinforcing extracellular bands is unique to the funiculus of the bird’s nest fungi and has not been reported in other species of basidiomycetes that produce clamp connections (Figure 13).

12 Summary of major findings • Bird’s nest fungi launch spore-filled peridioles at speeds of up to 3.6 m/s (13 km/h) to a height of 0.5 m utilizing only a fraction of the kinetic energy of a falling raindrop. • Drop placement is key to splash dispersal: drops that hit the rim of the fruit body are most likely to eject peridioles. • The funicular cord is strengthened by its twisted structure and the secretion of reinforcing bands around hyphal clamp connections. • High-speed video shows that that the funicular cord remains in compacted form when the peridioles are splashed from their fruit bodies and unravels only after the sticky hapteron makes contact with an obstacle.

13 Figure 1.

Figure 1. Cyathus fruiting body structure from Brodie (1975). The image shows a cross section of the vase -shaped fruiting body revealing the peridioles inside. Details of the peridiole structure show external morphology (left) and internal structure (right), including the funicular cord coiled inside the enclosing structure called the purse.

14 Figure 2.

Figure 2. Species within the Nidulariacae produce fruit bodies of varying shapes. The figure includes a digital image of a representative species of the five genera of bird’s nest fungi followed by description of fruit body morphology.

Figure 3.

Figure 3. Figure illustrating the four species used in this study: Cyathus olla, Cyathus stercoreus, Crucibulum laeve, and Cyathus striatus. The digital images are of fruiting bodies and the peridioles that are splashed from them utilizing the energy of raindrops (scale 2mm).

Figure 4.

Figure 4. Experimental design of splash experiments conducted on bird’s nest fungi. Burette was suspended approximately 1.2 m above fruit bodies in order to simulate raindrops. Fruit bodies were surrounded by four sheets of glass to protect the high-speed camera.

17 Figure 5.

Figure 5. Splash experiments were performed on model splash for comparison with actual fruit bodies. (A) Cups resembling Cyathus, Crucibulum, and Nidula were created by cutting eppendorf tubes. (B) Cups resembling Mycocalia and Nidularia were created using a modeling clay filled with a gelatinous agar solution. Clear plastic balls (3/32’’ Nylon White) were used as model peridioles (scale 5mm).

Figure 6.

Figure 6. Individual frames from video sequence of splash discharge in Cyathus olla at 3,000 fps. Peridioles are ejected by the upward displacement of water when a drop hits the fruit body off center and on the rim. (A) Time = 0, (B) time = 4.3 ms, (C) time = 6.7 ms, (D) time =8.0 ms (E) time =11.3 ms, (F) time =15.3 ms.

19 Figure 7.

Figure 7. Still frame of Cyathus olla peridiole ejection taken from a high-speed video sequence. Peridioles of bird’s nest fungi are ejected on average between 70-90 degrees. indicating that peridioles are shot to extreme heights and short distances.

20 Figure 8.

Figure 8. Predicted trajectories of peridioles of four species of bird’s nest fungi based on launch data obtained by high-speed video microscopy. Trajectories of launched peridioles of Crucibulum laeve (A), Cyathus stercoreus (B), Cyathus olla (C) , and Cyathus striatus (D). Each trajectory represents predicted maximum height and distance traveled by peridioles based on the average measured peridiole size, mass, launch angle, and velocity. Figures in parenthesis indicate the actual fraction of peridioles launched at the given range of angles.

21 Figure 9.

Figure 9. Splash scenarios resulting from raindrops hitting a fruit body of Cyathus olla at two positions. (A) Drops hitting the center of the fruit body results in displacement of water in all directions causing no peridiole ejection (B) A drop hitting the fruit body off center and on the rim results in displacement in two directions, over the rim and in an upward thrust, causing peridiole ejection.

Figure 10.

Figure 10. Scanning electron micrographs of cross sectioned peridiole of Cyathus olla. (A) Internal peridiole anatomy showing three distinct hyphal layers. (B) Magnified view of internal peridiole anatomy. (C) Second hyphal layer enclosing central layer embedded with thousands of basidiospores. (D) Central layer of mucilagenous hyphae with basidiospores. Note the apiculi are present on several spores. Scale (A) 0.1 mm, (B) 0.1 mm (C) 0.05 mm, (D) 10 µm.

23 Figure 11.

Figure 11. Scanning electron micrographs of the funicular cord. (A) Hyphal strands of funicular cord of Cyathus olla with numerous clamp connections. (B) Peridiole (p) of Cyathus stercoreus with funicular cord (f) and hapteron (h). (C) Peridiole (p) of Crucibulum laeve showing funicular cord (f) tethered to Oxalis sp. stem (o). (D) Peridiole (p) of Cyathus olla showing the twisted nature of the funicular cord (f). Scale (A) 10 µm, (B) 0.5 mm, (C) 0.5 mm, (D) 0.1 mm

24 Figure 12.

Figure 12. Scanning electron micrograph of strand of hyphae from the funicular cord showing a modified clamp connection. (A) Arrows indicate reinforcing bands around septa. (B) Diagram of basidiomycete clamp connection. Arrows point to the septa, sites of potential weakness. Scale 2mm.

Figure 13.

Figure 13. Scanning electron micrographs of clamp connections from the funicular cord showing extracellular bands around two septa. (A) Crucibulum laeve, (B) Cyathus stercoreus (C) & (D) Cyathus olla. Scale 2 µm.

Table 1. Measured biomechanical data of peridiole ejection based on high-speed video sequences.

27 Table 2. Predicted biomechanical data based on modeled peridiole trajectories.

28 Table 3. Measured mechanics of model peridiole discharge using model splash cups.

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