www.sciencemag.org/cgi/content/full/333/6042/631/DC1

Supporting Online Material for

Floral Acoustics: Conspicuous Echoes of a Dish-Shaped Leaf Attract Bat Pollinators

Ralph Simon,* Marc W. Holderied, Corinna U. Koch, Otto von Helversen

*To whom correspondence should be addressed. E-mail: ralph.simon@uni-ulm.de

Published 29 July 2011, Science 333, 631 (2011) DOI: 10.1126/science.1204210

This PDF file includes:

Materials and Methods Fig. S1 References

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/333/6042/631/DC1)

Movie S1

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Supporting online Material

Material and methods Preparation of leaf replicas

We collected foliage leaves (FL; N = 10) and dish-shaped leaves (DL; N = 12) of Marcgravia evenia either in Cuba (provinces Holguin and Guantanamo) or from the Botanical Garden Frankfurt. Morphological measurements of leaves were taken with a sliding rule. To produce replicas two-piece silicone casts of foliage and dish-shaped leaves (N = 6 each) were taken (Silagum-Putty®, DMG, Hamburg, Germany). To obtain the positive form we filled the casts with a bis-acryl composite material (Protemp™ 3 Garant™, 3M ESPE AG, Seefeld, Germany). Excess material was removed with a fine grinding drill (Proxxon FBS 240/E, Niersbach, Germany). Both Silagum-Putty® and Protemp™ 3 Garant™ are self-cured two-component materials used in dental laboratories that harden within a few minutes. Once hardened Protemp™ 3 Garant™ is a hard synthetic material similar to tooth enamel. Echo-acoustic measurements

We measured the echoes of original foliage leaves (N = 5) and original concave leaves (N = 3) as well as the echoes of all replicas (N = 6 each) in a distance of 20 cm. We measured the leaves’ impulse responses with a method called MLS (Maximum Length Sequence) described in detail in previous work (5, 13). Power spectral density of the echoes was calculated after correcting for the frequency response of the speaker and microphone. We found no significant difference in target strength between natural leaves and leaf replicas indicating that the material had no major influence on the reflected echo (t-test with mean target strength averaged for measurements from different directions, original leaves vs. leaf replicas: DL: -19.7±1.7 dB vs. -19.1±1.1 dB; t7 = 0.718; p = 0.496; FL: -21.6±0.5 dB vs. -22.1±1.5 dB; t10 = 0.768, p = 0.460; see also black curve in Fig. 2E, F). We calculated detection distances using the sonar equation (26): DT = SL + TLA + TLS + TS (dB), where DT is the detection threshold, SL is the source level of the bat's call, TLA is the transmission loss owing to absorption, TLS is the transmission loss owing to spherical spreading and TS is the target strength of the leaf. TLA and TLS are functions of distance. We calculated detection distances for a source level of 90 dB SPL (which is a conservative estimate for the echolocation call intensity of a nectar-feeding bat; Holderied MW, unpublished results) and assumed a detection threshold of 0 dB (27). TLA and TLS were calculated for a frequency of 80 kHz, a temperature of 20°C and 70% humidity. To deduce the catchment volume for every leaf we calculated the detection distance for every direction (n = 81, 2° steps from -80° to 80°), then rotated this detection directionality around 0 degree and calculated the volume of the resulting shape.

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Behavioural experiment To test whether dish-shaped leaves reduce search time for a nectar source, we conducted

behavioural experiments with three adult males of the flower-visiting bat Glossophaga soricina (Phyllostomidae). Experiments were carried out in a fully air-conditioned room at the University of Erlangen-Nuremberg (temperature: 27°C; humidity 75%) during the dark phase of a 12h/12h light/dark regime. The experimental room was divided into two compartments connected through a small computer-controlled door (diameter 20 cm). The smaller compartment (size 5 m x 2 m x 3 m length x width x height; henceforth called roosting compartment) was the roosting quarter of the bat in training and it offered pollen and water ad libitum. Behavioural experiments were conducted in the larger compartment (henceforth called test compartment; 6 m x 5 m x 3 m; see Fig. S1A).

Pre-training phase: In a pre-training period (10 to 15 days) bats learned to commute between roosting compartment and the test compartment every time the door opened. To motivate bats we installed computer-controlled feeders in both compartments (one in the roosting compartment and two in the test compartment; see pre-training feeders in Fig. S1A) that alternately offered small amounts of sugar water. Feeders were PVC cylinders with a 2cm deep cylindrical hole, into which we could release small sugar water rewards (10 - 20 µl) from a reservoir using computer-controlled valves (magnetic pinch valve type 2 / 2 - NC: Gr.1, ASCO Joucomatic, Ölbronn-Dürrn, Germany). Computer-controlled infrared light barrier at the front opening of each feeder registered bat visits. To establish a preference for the test compartment its two feeders gave more concentrated (17%; mass/mass) sugar water than the feeder in the roosting compartment (12%; mass/mass). The sugar water was a mixture of glucose, fructose and sucrose (37:37:26%) dissolved in water, which resembles the sugar composition of floral nectar.

Only one of the two pre-training feeders in the test compartment offered a reward at any given time, and to accustom the bats to the different leaf replicas this feeder was accompanied by a replica of a dish shaped or a foliage leaf. This rewarded feeder changed between trials in a pseudorandom sequence and the replica was shown or hidden using computer-controlled servomotors at each feeder accordingly. Dish-shaped and foliage leaf replicas were used alternately. The bats interacted with this computer-controlled environment by repeatedly visiting the feeder in the roosting compartment, which would release a reward every 20 seconds. Every 90 seconds the door between the two compartments opened after a visit to the feeder in the roosting compartment. Then the bat flew into the test compartment picked up a reward at the pre-training feeder showing the leaf replica and flew back into the roosting compartment. This triggered the closing of the door and concluded the trial. The connecting door, all light barriers and valves were connected to a PC via a USB data acquisition device (NI USB-6501, National Instruments, Austin, Texas) and controlled by the program SEARCHlab written by Ralph Simon in LabVIEW (National Instruments, Austin, Texas).

Training phase: When bats had learned to switch between the two compartments the two pre-training feeders in the test compartment were replaced by a background of artificial foliage. Within this background the bats had to search for a feeder, which offered a sugar water reward. The artificial foliage was mounted on a 2 m x 2 m chipboard, which was

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covered with sound absorbing Basotect® mats (BASF, Ludwigshafen, Germany; see Fig 1). To build artificial foliage that was reproducible and generated clutter echoes for many different directions, we installed individual plastic disc reflectors (diameter 38 mm) on wooden sticks at a spacing of 60 mm (distance of the centre of the reflectors). On these sticks plastic discs were inclined by nine different angles from 5-45° in 5° steps (numbers in green circles in Fig. S1B) and in four different inclination directions: up, down, left or right (arrow directions in Fig. S1B). In total there were thus 36 different plastic disc inclinations (9 angles x 4 directions). Each of these was used once to fill a field of 6 x 6 discs (Fig. S1B). The complete artificial foliage background consisted of 16 identical copies of this field (Fig. S1C). We added one row of discs at the bottom and one to the right of the background to achieve the full field of 25 x 25 plastic discs (see Fig. S1B, C).

Within this grid of plastic discs a feeder could be hidden at 64 positions (small black circles in Fig 3B). To avoid that the feeder was recognized as a protruding structure the four plastic plates directly adjacent to these positions were placed closer to the Basotect® mats. They stuck out only 4 cm (light green in Fig 3B), while all other discs were placed 8 cm from the backplane (dark green in Fig 3B), which allowed hiding the feeder completely within the artificial foliage. To reduce the feeder's echo strength it was wrapped in a thin layer of Basotect® (Fig. S1D). For the same reason the feeder had no valves or sugar water reservoir but was manually filled with higher amounts (25 µl - 50 µl) of 17 % (mass/mass) sugar water. During pre-training we individually identified the amount of sugar water that maintained sufficient levels of motivation in all three bats. This reward was then kept constant for each individual throughout the experiments.

As soon as the bats had learned to search for the feeder within this artificial foliage and that feeder position changed from trial to trial, data collection commenced. We measured the time it needed from entering the test compartment until finding the hidden feeder. Infrared light barriers registered both events. The connecting door was closed after the bat entered the test compartment, and opened again once the feeder was found. The bat then returned to the roosting compartment and the door was kept closed while we refilled the feeder with sugar water and changed its position in a predefined, randomized order.

To test if a replica of a dish-shaped leaf reduced search time, the feeder was presented in three arrangements: 1. with a replica of a dish-shaped leaf (Fig. S1F), 2. with a replica of a foliage leaf (Fig. S1E) and 3. without any leaf (control; Fig. S1D). For each of these treatments, we presented the feeder once at each of the 64 positions within the foliage background amounting to a total of 192 trials per animal. We conducted on average 30 trials per day with equal trial numbers for each feeder arrangements in changing order. Feeder positions were randomly drawn without replacing and neighbouring positions were excluded from subsequent trials to avoid possible confounding effects by the bats’ spatial memory. All trials exceeding 3 minutes search time were discarded because this indicated bats had ceased searching for the feeder. This happened in less than 3% of all trials and never for feeders with a replica of a dish-shaped leaf. Discarded trials were repeated at subsequent days.

To exclude scent cues we used sugar water as reward and the feeder was cleaned every day. While we cannot entirely exclude the possible use of odour cues, we are still confident

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that the observed differences in search time were due to differences in echo-acoustic conspicuousness because we used the same feeder throughout all experiments and any potential odour cues would have been equally available for all feeder arrangements. The behavioural experiments complied with the German laws on animal experimentation and with the institutional guidelines of the University Erlangen-Nuremberg.

Supplementary Movie S1 The first sequence of this movie shows field observations of nectar-feeding bats (Monophyllus redmani, Phyllostomidae) visiting an inflorescence of Marcgravia evenia. The second sequence documents one trial of the behavioural experiments. We recorded videos using an infrared sensitive camcorder (Sony Mini DV Camcorder, Tokyo, Japan) and custom built synchronised infrared flashlights for illumination.

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Fig. S1. Experimental setup for the behavioural experiments. (A) The experimental room was divided into a roosting and a test compartment, which were connected by a computer-controlled door. Positions of pre-training feeders and the artificial clutter background are indicated (not drawn to scale). (B) Schematic of one 6 x 6 block of artificial foliage (highlighted) plus some adjacent foliage. Green circles stand for plastic discs. They bear a number indicating the inclination angle and an arrow for the inclination direction. Dark green discs protruded 8 cm and light green discs 4 cm from the backplane. Small circles indicate possible feeder positions. The complete artificial foliage background consisted of 16 identical copies of the 6x6 block plus one feeder row at the bottom and to the right. (C) Photograph of the complete artificial foliage background installed on Basotect® mats. (D-F) Feeder types surrounded by artificial foliage, i.e. four green plastic discs. (D) Feeder without reflector, (E) bearing a replica of a foliage leaf, and (F) bearing a replica of a dish-shaped leaf.

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