Supporting Online Material for

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1 Supporting Online Material for Heat Exchange from the Toucan Bill Reveals a Controllable Vascular Thermal Radiator This PDF file includes: Materials and Methods Figs. S1 to S3 Table S1 References Glenn J. Tattersall, Denis V. Andrade, Augusto S. Abe Published 24 July 2009, Science 325, 468 (2009) DOI: /science Other Supporting Online Material for this manuscript includes the following: (available at Movies S1 and S2

2 1 Supporting Online Material Materials and Methods Animals. Toucans (N=6, 4 adults, 2 juveniles aged 2 months) were obtained from a local breeder in Rio Claro, Brazil. All animals were housed in outdoor enclosures with access to fresh fruit and toucan pellets (Alcon Club, Camboriú, SC, Brazil), and maintained at their natural ambient light:dark cycles. Experiments conformed with local authority and institutional (UNESP) animal care and use procedures. Collection of Thermographic Data. Thermographic images were collected using a thermal imaging camera (Mikron Instruments, Model 7515) with a video acquisition program (MikrospecRT, Mikron Instruments) as described previously (S1). Throughout all experiments, images were collected every 10s. The thermal imaging camera was mounted above the toucan such that the entire bill could be observed independent of the angle of the bird. The camera was calibrated against an internal thermocouple (NIST standard). Emissivity was assumed to be 0.97 (S2). A total of 110 hours (12-24 hours per toucan, >30000 frames in total) of video data was collected and analysed, with approximately 6 hours obtained per day per toucan. Time lapsed video files were generated during exposure to a range of T a (10-35ºC). Experimental Protocol. On the day of experimentation, a bird was removed by hand from the aviary and transferred to a perch inside an environmental chamber (FANEM Ltd., Sao Paulo). Birds were placed in the chamber in the early morning, and allowed 1-2 hours to acclimate. Generally, the birds exhibited a very warm bill immediately after handling, evidence of an increased heat load associated with activity, which subsided within minutes and with further habituation. On the first day, the T a was either raised or lowered (randomly) in 2 degree increments down to 8-10ºC or up to 35-36ºC. On the second day of experimentation, the temperature was altered as above, in order to expose the bird to the remaining range of ambient temperatures. Birds were given 1-2 days of rest between experiments. Bill Allometry. Actual bill lengths were only available from the 6 animals studied, however, digital photographs were made available from a toucan breeder, with accompanying body mass and age. To assess bill size from photographic data, we determined the ratio of bill length:eye diameter from photographs of toucans of known ages and body masses, and used predictive equations to calculated eye diameter from body mass (S3), obtaining bill length from the ratio above. This allowed for estimates of bill length allometric exponents (b) within Ramphastos toco using non-linear regression analysis (i.e., BL=aM b ). Mass scaling exponents for bill length were compared against that expected for isometric relationships (b=0.33) (S4). Analysis of Thermographic Images. For each toucan, hours of data (recorded at 10s intervals) were obtained, representing exposures to T a from 10 to 35ºC at 1ºC increments. Using MikroSpec RT software (Mikron Instruments), regions of interest on the toucan s body were drawn for each frame to obtain the average surface temperature of: the eye (including the surrounding naked peripalpebral region), the dorsal feathers (as an average assessment surface temperature where minimal heat would exchange), the proximal region of the bill (the third of the upper bill closest to the cranium), and the

3 2 distal region of the bill (the distal two-thirds of the upper bill). Since multiple frames were obtained from each toucan for each T a recorded, minimum, average, and maximum observed surface temperatures were made possible obtained. Values for surface temperature were converted into estimates of total body and bill heat loss as described previous (S5,S6). Breathing Frequency, Expired Air Temperatures, and Gaping Assessment. Breathing frequency (fr) and exhaled air temperatures (T exp ) were obtained using the thermal imaging camera since the nostrils face caudally and warm up adjacent feathers in phase with each breath. T exp was estimated as the maximum value for feather temperature caudal to the nostrils. One minute intervals (sampled at 22 Hz) of the nostril feather temperature were obtained at approximately 1ºC increments between 10 and 35ºC. Gaping was assessed from the thermographic video files as the threshold T a when the bill exhibited a noticeable and sustained gape. Estimation of Heat Loss. Radiative heat exchange (Q r ) and convective heat exchange (Q c ) were determined as described previously (S5, S6) using average values (for adult and juvenile toucans separately) from the major exchange surfaces (eye/peripalpebral region, external surface of feathers, proximal and distal portions of the bill) and summed to determine whole body heat loss (Q t ) based on the following equations: Q t i = Q + ri 1 i 1 Q ci [1] Q Q r c 4 4 = εσ A( T T ) [2] s a = h A( T Ta ) [3] c s where 1 through i refer to the different body regions, T s is the surface temperature of the respective body region (ºK), T a is the ambient temperature (ºK), A is the surface area of (m 2 ), ε is the combined emissivity of the object and the environment (assumed to be 0.97), σ is the Stephan-Boltzman constant, and h c is the convective heat transfer coefficient for that particular body region (W m -2 ºK -1 ), determined using: h c Nu k = [4] D where D is the critical dimension of the body region (i.e., height of bill in metres), k is the thermal conductivity of air (W m -1 ºK -1 ), determined for each T a : k = e [5] 6 T a and Nu is the Nusselt number determined for each region surface: n N u = cr e [6]

4 3 using c and n values of and when R e 4000, and and when R e <4000. R e was determined using from the following: R e V D = [7] υ where V is air velocity (m s -1 ), D is the critical dimension, and υ is the kinematic viscosity of air (m 2 s -1 ), determined from T a : 5 υ = 1.088e e [8] 8 T a The bill was modelled as a cylinder, the eye as a plate, and the feathered body as a sphere. For simplicity, the entire feathered region of the body was modelled as sphere (S5), and feet were ignored in total heat loss calculations since they were not reliably visible to the thermal camera. Statistical Analysis. For surface temperature differentials, one-way ANOVA (repeated measures, with T a as the main effect) was used, with Holm-Sidak post hoc tests employed where appropriate. If normality was violated, log transformation was used and normality confirmed on residuals. An α of 0.05 was used as the critical level for statistical tests.

5 4 Figure S1. Rapid and reversible changes in bill perfusion are readily observed from thermal images. A-D depict dorsal views and E-F represent frontal images. A, a toucan at 20ºC with minimal heat loss from the bill; B, the same toucan two minutes later with profound bill perfusion; C, a toucan at 25ºC with moderate heat loss from the bill; D, the same toucan two minutes later with a nearly fully perfused bill; E, a toucan at 10ºC with near complete vasoconstriction within the bill; F, a toucan at 35ºC demonstrating greater perfusion of the distal bill region relative to the proximal region; G, a toucan at 22ºC with an open gape demonstrating that the interior of the bill can be perfused, while the exterior is vasoconstricted; H, a toucan at 35ºC exhibiting wing spreading and thermal gaping with a full vasodilated bill.

6 5 T exp - Ta fr (% of Rest) 14 A 12 ** *** 10 * * **** C 2 30s B * Ambient Temperature ( C) C D Figure S2. Ventilatory responses in toco toucans exposed to changing ambient temperature. Expired air temperature (mean ± SE), depicted as T exp T a is shown in A, and breathing frequency (fr), expressed as percent of resting value (values at 24ºC) is shown in B. These values were made possible by phasic changes (inset in A) in feather temperature (arrows in D) immediately caudal to the nostrils (compare absence in C to presence in D). Asterisks denote significant (P<0.05) differences from T a = 33ºC values.

7 6 Surface Temperature ( C) Back Eye Proximal Bill Distal Bill Flight Speed Time (min) Flight Speed (km h -1 ) Figure S3. Activity-induced vasodilation of the bill. Surface temperatures (back feathers, eye, proximal bill, and distal bill) from a toco toucan in an outdoor aviary during flight, demonstrating a substantial and rapid diversion of heat toward peripheral tissues (i.e., the bill). During this 10 minute flight, the toucan exhibited an average flight speed of 17 km h -1. Ambient temperature is indicated by the dotted line.

8 7 Table S1. Morphological and thermoregulatory parameters in adult and juvenile toucans Adults Juveniles Body Mass (g) 676 ± Bill Length (cm) 18.7 ± Bill Height (cm) 5.9 ± Bill Width (cm) 4.1 ± Bill Area (cm 2 ) 293 ± Predicted Bill Area (cm 2 ) (S7) 7.6 ± Predicted Body Surface Area (cm 2 ) (S7) 622 ± Proximal Vasodilation Threshold (ºC) 20.8 ± Distal Vasodilation Threshold (ºC) 25.1 ± Gaping Threshold (ºC) 33.1 ± Reported values are mean (N=4) ± sem. Reported values are mean (N=2)

9 References S1. G. R. Scott, V. Cadena, G. J. Tattersall, W. K. Milsom, Journal of Experimental Biology 211, 1326 (2008). S2. J. R. Speakman, S. Ward, Zoology 101, 224 (1998). S3. H. C. Howland, S. Merola, J. R. Basarab, Vision Research 44, 2043 (2004). S4. W. A. Calder, Size, function, and life history (Harvard University Press, Cambridge, Mass., 1984), pp. xii, 431 p. S5. P. K. Phillips, A. F. Sanborn, Journal of Thermal Biology 19, 423 (1994). S6. K. Blaxter, Energy metabolism in animals and man (Cambridge University Press, Cambridge, 1989), pp S7. G. E. Walsberg, J. R. King, Journal of Experimental Biology 76, 185 (1978). 8