CALIBRATED UV REFLECTANCE PHOTOGRAPHY OF GLAUCIPPE SULPHUREA

EVELYN AYRE1 AND GEORGE BEVAN2

1Conservator, Ottawa, Ontario, Canada [email protected] 2Department of Classics, 49 Bader Lane, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021 [email protected]

Abstract.—Ultraviolet (UV) reflective and absorbent markings on wings of male sulphurea are important visual markers used in mating to differentiate them from other . The objective of our study was to determine whether these markings deteriorate in museum collections over time. We first characterized quantitatively the UV reflective and UV absorbent wing markings from fresh and naturally aged male H. glaucippe sulphurea using UV reflectance microphotography, which was calibrated with handmade reflectance standards. The results of calibrated UV reflectance photography were then compared qualitatively with the same markings using visible light photography, transmitted and reflected visible light microscopy, and scanning electron microscopy (SEM). A UV-converted Nikon D200 with a Baader Ultraviolet Venus lens filter was used to record UV reflective and UV absorbent wing markings of the specimens. The handmade reflectance standards were prepared using magnesium oxide, plaster, and carbon, photographed alongside the specimens, and used to calibrate the photographs. The easily and affordably produced handmade reflectance standards were effective in calibrating the UV reflectance digital photographs, which allowed for each pixel of the digital photographs to be used for optical densitometry measurements. Quantitative data from calibrated UV reflectance photography demonstrated little evidence of deterioration in the UV reflective markings, although there was clear deterioration in the UV absorbent markings. This quantitative data, along with the calibrated UV photographs themselves, offered complementary documentation to visible light microscopy and SEM images. Results show that both visible-spectrum and UV markings fade in naturally aging museum specimens. We conclude that by using calibrated UV reflectance photography, a relatively inexpensive technique, a baseline and eventual degradation of wing markings may be quantified and may provide valuable data to clarify the mechanisms behind this degradation. With the rate of change quantified, and the mechanisms of fading understood, it is hoped that preventative measures can be taken in the future to remedy this loss of valuable data in collections.

Key words.—Lepidoptera, markings, photography, standards, ultraviolet Associate Editor.—Christine Johnson

INTRODUCTION Photo documentation allows for the preservation of unique visual information contained in delicate Lepidoptera specimens, which may otherwise change or disappear as a specimen stored or displayed in a natural history museum ages. Photographs can capture and preserve wing patterns, anatomical details, and a sense of surface texture that then serve as a complementary record of the specimen, an archive of the specimen, or a surrogate should the specimen be lost or degrade to the point at which the original appearance has been irretrievably altered. Many researchers, conservators, and collections managers recognize the need to photograph individual specimens in collections for posterity through conventional photography. With the advent of high-resolution UV-sensitive digital cameras, additional information can be obtained from the photographic record, such as UV markings on wings, some that are visible only in the UV range of the electromagnetic spectrum. These important characteristics can now be captured relatively inexpensively and can, consequently, be used to record the spatial distribution of UV wing markings across a much greater number of samples than was possible using UV-sensitive film photography.

Collection Forum 2016; 30(1):34–50 E 2016 Society for the Preservation of Natural History Collections 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 35

Ultraviolet light is electromagnetic radiation with wavelengths between 100 and 400 nm (ISO 2007). Ultraviolet reflectance photography (UVR) is a photographic technique that captures ultraviolet wavelengths reflected from objects and produces a monochrome representation of the image that we can interpret visually. The human eye is unable to detect near-UV light waves due to ocular filters that remove wavelengths shorter than 400 nm (Dyer et al. 2004). Similarly, digital cameras have internal cutoff filters (ICFs) in front of the camera sensor that act in the same way as ocular filters in the eye. The ICFs remove light wavelengths shorter than 400 nm or longer than 700 nm to minimize Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021 improper light metering in the visible range. For the silicon sensor to record UV, or infrared (IR) wavelengths, the camera’s ICF must be removed, usually by a commercial service (Life Pixel 2012). Removal of the ICF still leaves the Bayer filter in place, and thus an array of red, green, and blue filters allows the sensor to record color information from the monochrome photosites. With the ICF removed and the camera sensitivity extended into the UV range, a lens made of quartz fluorite is used, which allows the transmission of UV wavelengths. This lens is fitted with an additional filter that removes visible and IR light to isolate UV wavelengths. Past UVR imaging was done with photographic film sensitive to both near-UV (320–400 nm) and visible light (Dyer et al. 2004). Filters placed in front of the lens removed infrared and visible light. Humans are unable to directly perceive UV light and may misinterpret photographs that capture wavelengths in the near-UV spectrum because the appearance of an object in the UV range does not necessarily correlate to the appearance of the same object in visible light. Markings observed under “normal” light may be perceived incorrectly because of poorly controlled contrast or artifacts of poor lighting. For this reason, calibrated UV reflectance gray-scale standards should be included in the photographs to ensure that the images are not falsely “enhanced” in post-processing (Dyer et al. 2004). UV reflectance (UVR) photography is distinct from, and must not be confused with, UV fluorescence (UVF) photography, also called ultraviolet-induced visible fluorescence. UVF photography records the fluorescence produced when electrons in an object are excited by a light source that emits UV wavelengths, which results in a visible light photon (fluorescence) being released. UV fluorescence is often used in conservation to provide qualitative visual information that can aid in identification of a variety of materials and was recently found to be effective in early detection of feathers fading due to light exposure (Pearlstein et al. 2015). By contrast, UV reflectance photography captures UV light reflected by an object through a filtering system that removes visible (including UV fluorescence photons) and infrared wavelengths and functionally allows the transmission of UV wavelengths only (Elen 2012; see Fig. 1) and the imaging of what is “visible” in the UV range. UVA wavelengths, 315–400 nm (ISO 2007), are important because many , as well as some birds and lizards, have ultraviolet visual receptors (Silberglied 1979) and use these perceived UVA wavelengths to detect markings invisible to humans (Kevan et al. 1973, Knuttel and Fieldler 2000). Many flowers have evolved “bulls-eye”-like markings in the UVA range to attract pollinator insects (Silberglied 1979). Lepidoptera can see in the UVA range and also have markings that reflect UVA light on their wings. These structures that reflect UVA are created by the laminar structures and pigments of wing scales. Many families of Lepidoptera, Papilionidae, , and Nymphalidae (Bybee et al. 2011) and some moths and larvae (Silberglied 1979) produce wing markings or have other structures that are visible only in UV wavelengths. UVR photographs reveal the spatial distribution of these markings on Lepidoptera and therefore provide an important 36 COLLECTION FORUM Vol. 30(1) Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 1. UV reflectance digital photography (adapted from Elen). complement to visible-light photography (Kevan et al. 2001). UVR documentation of butterflies has proven useful to researchers for several reasons. UVR photographs of butterfly wings, if assessed properly, can (1) serve as regular morphological characters in systematics if assessed properly (Knuttel and Fieldler 2000), (2) reveal differences in UV reflectance wing patterns in butterflies that are morphologically similar, (3) demonstrate intraspecific variability (Silberglied 1979), and (4) show variations in UVR wing patterns that have been correlated to diet differences in butterflies (Silberglied 1979). UVR markings play a role in interspecies and intraspecies communication, such as mate recognition and sexual selection (Robertson and Monteiro 2005). UVR in wing patterns has also been useful in butterfly , and in detecting butterfly gynandromorphs, specimens that exhibit both male and female characteristics (Allyn and Downey 1977). UVR markings are also useful in determining Lepidoptera phylogeny. Given the significance of these UV markings in the study of Lepidoptera, it is important to understand if and how they degrade over time in natural history collections. Given the number of specimens, a relatively low-cost method is needed. UVR photography using a converted DSLR camera along with handmade UV reflectance standards offers conservators and scientists alike an opportunity to document these marking in such collections, to document their degradation, and potentially to isolate environmental factors that could be responsible for this degradation.

MATERIALS AND METHODS Butterfly Preparation Eleven dry-preserved males of the pierid butterfly, Hebomoia glaucippe sulphurea, were prepared for UV reflectance photography to examine UV reflective and UV absorbent wing markings. Male H. glaucippe sulphurea have predominantly white wings with UV reflective orange markings and UV absorbent black markings at the apex of the forewings. Nine fresh, dried specimens from Bachan Island, Indonesia, were purchased from Thorne’s Shoppe Ltd.; two older specimens that had been on continuous display in unfiltered daylight for more than three years, perhaps as long as 12 years (A. Brewster, Cambridge Butterfly Conservatory, pers. comm.), were provided by Cambridge Butterfly Conservatory. All 11 specimens were rehydrated, spread, and pinned to display the dorsal wing surface and each assigned an identifying letter (Fig. 2). 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 37 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 2. Fresh butterfly preparation, pinning and flattening.

Handmade Reflectance Standards Each photograph received a reflectance standard in the frame because the UV response for each color channel on a converted DSLR camera is not perfectly linear. This set of standards with a range of nominal reflectance values established by spectrophotometry allowed us to estimate accurately the camera response and calculate an appropriate linearized calibration (Stevens et al. 2007). Commercially produced reflectance calibration standards, such as those made by Spectralon, are costly. Therefore, we developed a method for preparing handmade reflectance standards following Dyer et al. (2004), which was adapted from Kevan et al. (1973). Five handmade reflectance calibration standards were prepared by mixing magnesium oxide (Anachemia Chemicals, Montreal, 38 COLLECTION FORUM Vol. 30(1)

Table 1. Proportions of magnesium (MgO), plaster, and carbon used in preparing handmade reflectance standards.

Patch MgO (%) Weight MgO (g) Plaster (%) Weight plaster (g) Carbon (%) Weight carbon (g) 1 70 12.8 26 3.55 4 0.45 2 60 10.74 33 4.56 7 0.7 3 60 10.74 31 4.28 9 1.02 4 40 7.16 30 4.14 30 3.39 5 20 3.59 30 4.14 50 5.07 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Canada), plaster (Burma Casting Plaster of Paris), and carbon (lamp black amorphous carbon) in several proportions to create five different standard patches that ranged from most UV-reflective (Patch 1) to most UV-absorbent (Patch 5). Magnesium oxide was used for its highly reflective properties, and carbon black was used for its absorptive properties, across the wavelengths perceived by insects and humans (Kevan et al. 1973). To create the five standards, we mixed together the magnesium oxide, plaster, and carbon powders by hand. An electronic balance was used to weigh each powder, instead of measuring by volume amount as described by Dyer et al., as this is a more suitable method for measuring powder. We factored in the density of each component into the conversion: magnesium oxide 3.58 g/cm3 (Anachemia Chemicals 2011), plaster 2.76 g/cm3 (ICPS 2004), and carbon 2.26 g/cm3 (Reade 2012) (Table 1). When mixing by hand, magnesium oxide tended to form hard clumps, which may have caused some variation in our final mixtures. We recommend using a mechanical mixer, such as a coffee grinder, to blend the dry powders beforehand. After dry powders were mixed, H2O was added until the mixture had a wet, sour cream–like consistency. The mixture was then poured into small 5 ml plastic cups and left to dry overnight. Once dry, the calibration standards were popped out of the plastic cups.

Spectrophotometry The spectral reflectance of the five prepared reflectance standards was measured with a spectrophotometer to determine if they exhibited relatively flat spectral curves across 300–700 nm wavelengths. Season Tse, Senior Conservation Scientist at the Canadian Conservation Institute in Ottawa, Ontario, took spectrophotometric measurements using a Cary 3 UV-Vis spectrophotometer, which has 2 nm spectral bandwidth with double beam reverse with R928 PMT detector, on 22 March 2013. Wavelengths were measured in the range 200–700 nm, every 1 nm, with 0.1 save time. A glass slide was placed on the sample port to prevent carbon particles from the calibration standards contaminating the integrating sphere. A baseline correction was done to account for this (S. Tse, Canadian Conservation Institute, unpubl. data). These values were later used to calibrate the pixel intensities to a linear-response model in the ImageJ software to retrieve accurate quantitative values for the reflectance of the UV reflective markings. Spectrophotometry showed a uniform change in spectral reflectance with the variation in carbon, magnesium oxide, and calcium sulfate (plaster) contents. Between 300 and 400 nm, all five calibration standards dropped from a noisy 100% reflectance at 300 nm, to staggered reflectance that corresponds to the different quantities of the components of the mixtures at around 340 nm. The calibration standards showed relatively flat reflectance across the 350–520 nm range. The spectra showed that calibration standard no. 2 and calibration standard no. 3 exhibited very similar reflectance across the 300–700 nm range. See Figure 3 and Table 2. 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 39 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 3. Handmade calibration standards, percent reflectance, 300–400 nm as measured Cary UV-Vis Spectrophotometer.

The spectrophotometry measurements in the range 345–355 nm for each calibration standard were averaged in Excel to determine the mean percentage reflectance values for each calibration standard. This range, 345–355 nm, was selected because this is the range surrounding the Baader U-Filter’s peak transmission, the filter that was used in photography capture. This range was also chosen because trial spectrophotometry measurements below 345 nm showed that handmade standards no. 2 and no. 3 had nearly

Table 2. Percentage reflectance of calibration standards as determined by spectrophotometer, values used to determine percentage reflectance value used in optical densitometry.

Wavelength Calibration Calibration Calibration Calibration Calibration (nm) standard no. 1 standard no. 2 standard no. 3 standard no. 4 standard no. 5 355 39.79 29.87 28.89 15.63 13.11 354 41.45 30.82 29.53 15.81 13.36 353 40.43 30.42 29.07 16.07 13.75 352 40.31 30.90 29.32 15.79 13.92 351 41.06 30.85 29.97 16.38 13.84 350 41.34 31.11 30.37 16.60 13.61 349 41.45 30.74 31.38 16.82 13.97 348 41.10 31.83 30.49 17.70 14.86 347 41.97 32.65 31.22 17.65 14.77 346 42.42 31.76 31.54 18.15 14.75 345 41.98 32.05 31.10 18.09 15.55 Mean (Value used in 41.21 31.18 30.26 16.79 14.14 optical densitometry) Standard deviation 0.79 0.81 0.97 0.96 0.75 40 COLLECTION FORUM Vol. 30(1) identical measurements and, hence, could not be used for calibration for UVR photography with shorter wavelengths.

UV Reflectance Photography Prior to photographing the specimens, each specimen was placed on an insect pinning block with a hole cut in the bottom groove of the pinning block, so that the wings were held only by the pinning block’s angled sides. A small specimen identifier label was placed in the frame next to the butterfly. The five prepared reflectance standards were placed to Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021 the right of the butterfly specimen, on a single layer of CoroplastH, to be at a slightly higher plane than the butterfly to eliminate some of the surface texture that would affect the calibration. Thus, the standards appear slightly out of focus in the photograph. A small square of black velvet was also included as the darkest patch in calibration, representing a highly absorbent surface. A ruler was included in the frame as a reference for calculating the absolute areas measured in histogram measurements in Image J. Two Vivitar 285 HV lights, attached to a copy stand at approximately 45u angles to the main shaft, were fitted with xenon bulbs with UV blocking removed to maximize the bulbs’ UV output. A UV-Converted Nikon D200 10 megapixel camera body, converted by Life Pixel to better capture UV wavelengths and produce images with higher resolution, mounted on the copy stand was used. The lens was a Coastal optics 60 mm quartz fluorite lens, fitted with a Baader Ultraviolet Venus filter to selectively allow wavelengths to pass in the 320–400 nm range and with peak transmission at 360 nm. This filter is also extremely efficient at removing all light in the IR spectrum (Baader 2012). The specimens were photographed in a single session, one by one, using Camera Control Pro 2 Nikon software version 2.8.0 to fire the camera at F4, 1/60, ISO 1600. All images were captured as raw files, with the highest camera resolution possible. All raw files were archived, and TIFF files (an uncompressed format) were generated and archived, following digital image archiving protocol at the Canadian Conservation Institute (M. Choquette, Canadian Conservation Institute, pers. comm.). Nine fresh butterflies of the same species (“Pierid A, B, C, D, E, F, G, H, and I”) were photographed seven times with the same lights, camera, lens, and reflectance standards. We followed the same procedure for two older H. glaucippe sulphurea male specimens (“Pierid O” and “Pierid P”).

Reflectography Calibration and Optical Densitometry The images were calibrated and analyzed using Image J 1.46 r software. Each photograph was converted from its raw format into JPEG format temporarily and then opened in ImageJ. The image’s color channels were split, the blue and red channels closed, and the individual channels saved as TIFF for use in analysis. The relative signal strengths of each channel were compared. In this case, the green channel offered the best contrast and the most linear response when compared with the blue and red channels, which was not unexpected because 50% of the individual photosites on the Bayer filter make up the green channel. It should be noted that this is not always true, and that the red and blue channels can contain more information depending on exact setup used (Richards 2010). What is more, it has been observed that the blue and red channels also have a bias to the short-wave and long-wave sides of the UVA spectrum with the Baader filter (Richards 2010). A Gaussian blur filter with 10.00-sigma radius was applied to a rectangular selection containing the calibration standards. This was done to achieve a homogenous surface texture on the calibration standards, which is better for the 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 41 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 4. Measured square selections, in turquoise, at the apex of male Hebomoia glaucippe sulphurea, dorsal right forewing. calibration. Next, the scale was set by drawinga1cmline on a known 1 cm length (on the ruler in the frame) and then setting this measurement for future measurements. Then a circular selection measuring 1 cm in diameter was drawn. This circular selection was placed in turn on each calibration standard (no. 1–5 plus black velvet), and these were measured, giving uncalibrated mean pixel values for the circular selection. Using the calibrate function, these values were paired with percentage reflectance values, previously determined through spectrophotometry, for each calibration standard using a linear function. A graph representing this calibration function was produced for each image with y-axis percentage reflectance and x-axis pixel value. The black velvet was not measured in spectrophotometry, but black velvet is a Lambertian reflector (matte) and can be assigned 0% reflectance. This calibrated the image so that any future measurements of any area would produce a calibrated value for percentage reflectance. A 1 cm diameter circular area of the five handmade calibration standards no. 1–5 plus the square of black velvet were then measured with the calibration in place. This gave a high root mean square value for all the photographs, . 0.95. A smaller square selection, 2 mm 3 2 mm, was used to measure the reflectance of a UV reflective marking and a UV absorbent marking. These markings were measured on the apex (top corner) of the right forewing (top wing) on each butterfly (see Fig. 4). This square measured area was saved as a TIFF image to record the image contents of the measured area. It was also saved as an image text file, recording the pixel-by-pixel percentage reflectance values in the square selection area. The reflectance measurements taken of the calibrated image for the UV reflective area and for the UV absorbent area were compared across all 11 butterflies. The mean reflectance for all butterflies, all fresh butterflies, and the two old butterflies was calculated for both UV reflected and UV absorbent markings. Then reflectance values for each pixel in the measured areas, for both a UV absorbent marking and a UV reflective marking, of each specimen (Pierid A–I, O, and P) were retrieved from the photograph in Image J. This produced a text file listing these values. A total of 1,600 values (from 1,600 pixels in the measured area) were retrieved. These values were then imported into Excel for statistical analysis. The pixel-by-pixel values for the 42 COLLECTION FORUM Vol. 30(1) measured square selections for the fresh butterflies versus the older butterflies were compared statistically to address the questions “Do UV reflectance markings degrade over time?” and “Do UV absorbent markings degrade over time?”

Visible Light Microscopy Reflected visible light microscopy and transmitted visible light microscopy were used to examine the structure of the H. glaucippe sulphurea wings. Pierid A and Pierid O were microimaged with an Olympus DP72 camera mounted to an Olympus BX51 microscope Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021 with transmitted light at 1003 and 2003. The left forewing dorsal surface was examined and photographed. The same wing area on Pierid A and Pierid O was examined through reflected light microscopy using an Olympus BH optical microscope. Digital photographs were taken through this microscope using a Moticam camera, with Motic Images Plus 2.0 ML software.

Scanning Electron Microscopy Scanning electron microscopy (SEM) was used to image a UV reflective marking and a UV absorbent marking, on the dorsal left forewing, on a fresh specimen (Pierid A) and on an aged specimen (Pierid O). This was done to investigate the microstructures responsible for the coloration and UV reflectance or absorbance of the wing markings. Pierid O was imaged to compare with Pierid A to see if any deterioration occurs to these micro-wing structures. The top left-hand corner of the top left forewing was cut off Pierid A and Pierid O and mounted on metal discs with Nisshim Em Co. Ltd. double-sided conductive carbon tape. These were then sputter-coated with gold. A JEOL 840 Scanning Electron Microscope at 10 kV energy was used to image the samples. Images were captured at 5003, 1,5003, 10,0003, and 30,0003 magnification.

RESULTS UV Reflectance Photography UVR photographs suggested that the UV reflective orange markings were unaffected by aging. Older specimens (Pierid O and Pierid P) and all of the fresh specimens (Pierids A–I) exhibited consistently brilliant UV reflective markings as captured in UVR photography. However, a notable difference between aged and fresh specimens could be observed in the appearance of the UV absorbent black markings. Pierid O’s and P’s black markings were washed out in comparison to the fresh specimen’s markings (see Fig. 5).

UV Reflectography Calibration and UV Optical Densitometry The measured calibrated percentage reflectance values echoed what could be seen visually in the photographs: the UV reflective markings were consistently bright across all fresh specimens and older specimens; the UV absorbent markings were consistently dark across all fresh specimens, but slightly lighter on the older specimens (see Table 3). We used an ANOVA in XLSTAT on averaged pixel reflectance values across fixed sampling areas in the images to compare the reflectance values for the UV absorbent markings and UV reflective markings across specimens. A Tukey multiple post-hoc comparison was made to compare between all samples both within the fresh Pierid group (Pierids A–I) and the aged group (Pierids O and P) and between members of the fresh and aged groups. There were significant differences of in reflectance values between all individual specimens (P , 0.05), but no detectable difference between aged and fresh groups with 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 43 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 5. UV reflectance photographs, green channel: (A) Pierid F and (B) Pierid O. Note: corner of left forewing was snipped off for SEM imaging. a95% confidence interval. This statistical analysis showed that all nine specimens were significantly different in the reflectance values of both their UV absorbent and UV reflective markings, and that there was no detectable difference in reflectance values between aged and fresh specimens (see Fig. 6). These parametric techniques were inadequate to deal with the high level of variance in the data derived from averaging large numbers of pixel values.

Table 3. Percentage UV reflectance of UV-absorbent and UV-reflective markings on male Hebomoia glaucippe sulphurea specimens Pierid A–I, O, and P.

% Reflectance % Reflectance UV-reflective UV-absorbent Pierids (orange) marking (black) marking A 47.28 3.14 B 44.17 2.79 C 44.22 3.19 D 43.93 3.73 E 44.33 3.49 F 42.49 3.60 G 43.95 3.30 H 43.55 2.63 I 43.30 2.96 O 43.93 4.38 P 43.52 4.97 Mean Pierids A–I 44.14 3.21 Mean: O and P 43.73 4.68 Mean: all specimens 44.06 3.47 44 COLLECTION FORUM Vol. 30(1) Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 6. (A) Percentage reflectance of UV reflective markings from pixel-by-pixel analysis, box plot chart. (B) Percentage reflectance of UV absorbent markings from pixel-by-pixel analysis, box plot chart. + mean, ¤ maximum/minimum. Because standard parametric techniques could not confirm the difference between the average calibrated pixel intensities of the markings of the aged and fresh specimens, but qualitative inspection of the calibrated photographs showed that the UVA markings of the older Pierids (O and P) usually had a higher mean reflectance (i.e., they were a lighter shade), Agglomerative Hierarchical Clustering analysis, a nonparametric method, was 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 45 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 7. (A) UV Reflective markings dendrogram, from pixel-by-pixel analysis and (B) UV Absorbent markings dendrogram, from pixel-by-pixel analysis. selected in the XLSTAT package with a Euclidian distance metric (spatial difference) and agglomeration using Ward’s method (complete linkage to others). Clustering analysis, while semiquantitative, can often identify differences in populations with high variance when parametric methods fail (Johnson and Wichern 2007). Analysis of the calibrated pixel intensity UV reflective markings produced four classes in cluster analysis, but no distinction was made between aged and fresh specimens. This represents the high variability between all specimens and supports the hypothesis that there is no discernible degradation that occurs to these UVR markings. Pierid A was placed in its own class with a high amount of dissimilarity. There is no obvious explanation for why this was the case. It is possible that this butterfly was younger when it was collected, or that its diet was different, or that there was a slight difference in the photography capture setup (i.e., the butterfly wing may have been curled) that caused it to have a higher reflectance value. By contrast, the cluster analysis of the UV absorbent markings placed the aged Pierids in their own class that was distinct from the others (see Fig. 7). This analysis tends to supports the hypothesis that the UV absorbent markings do indeed deteriorate. 46 COLLECTION FORUM Vol. 30(1) Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 8. Reflected visible light microscopy of orange and black scales on dorsal left forewings: (A) Pierid A, 5003 and (B) Pierid O, 2003.

Imaging Wing-Marking Substructures Visible light microscopy.—Transmitted visible light microscopy revealed UV reflective orange scales had a round oval shape, whereas UV absorbent black scales had jagged edges with three or four rounded points. Transmitted light microscopy also showed that the wing scales were translucent. Comparison between the fresh Pierid A and the older Pierid O revealed clues about the degradation of scales. Pierid A’s black scales were a deep, nearly opaque black, and its orange scales were brilliant orange-red. The black scales of Pierid O showed a dramatic loss of coloration compared to Pierid A, in some areas appearing completely transparent. Also visible was an apparent loss of scales, as areas missing scales allowed more light to pass through the wing. Many black scales were curled inward. Compared to Pierid A, Pierid O’s orange scales appeared less vibrant, duller, but were not curling as seen in the black scales and had no other changes to their morphology. When viewed through reflected visible light microscopy at 1003 magnification, the contrast between the very matte quality of the black scales and the shimmery orange scales was apparent. The black scales appeared like black velvet, profoundly black. At 5003 could be seen a hint of the scale’s substructure: striations were visible in the scales. The orange scales, by contrast, appeared luminously orange, radiant and flame-like, with a slight shimmer (see Fig. 8). Differences could also be seen in reflected visible light microscopy when comparing aged specimen markings to new specimen markings (see Fig. 8). Pierid O’s black scales appeared grayish and particularly faded at the edges. The orange scales appeared less orange than in the fresh specimen (Pierid A), but they retained a shimmery quality. Scanning electron microscopy.—SEM imaging revealed details about the morphology of the UV reflective and UV absorbent scales, as well as the differences between the fresh specimen (Pierid A) and the older specimen (Pierid O). Black UV absorbent markings were composed of ground scales with four rounded points. Visible were straight lamellar ridges with perpendicular struts containing an interstitial substance that was not discernible due to the instrument’s resolution limits. By visual inspection, Pierid A’s black marking appeared to have more of this interstitial substance than Pierid O. Pierid O’s scales were curled inward, unlike Pierid A’s, which lay flat. Striations on lamellar ridges were more visible in Pierid O (see Fig. 9A–D). 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 47 Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

Figure 9. Scanning Electron Microscopy of dorsal left forewings. (A–D) Black markings: (A) Pierid A, 1,5003, (B) Pierid O, 5003, (C) Pierid A, 10,0003, (D) Pierid O, 10,0003. (E and F) Orange markings: (E) Pierid A, 10,0003, (F) Pierid O, 1,5003.

The orange UV reflective marking’s scales were formed of undulating lamellar ridges (see Fig. 9E, F). Some areas of these markings seemed to have more undulations in the lamellar ridges. There was little difference between Pierid A’s and Pierid O’s orange UV reflective markings, though Pierid O’s lamellar ridges seemed to have fewer undulations. This may have been a local variation in the wing marking (i.e., this may not have been the 48 COLLECTION FORUM Vol. 30(1) exact same spot imaged as for Pierid A), or it could be that this was due to this being an older specimen. Pigment granules were visible below the lamellar ridges.

CONCLUSIONS UV reflectance photography offered complementary documentation to visible light photography for Lepidoptera with UV reflective markings. This demonstrates what

H. glaucippe sulphurea butterflies are likely to see on the wings of others of their species. Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021 These results show that UV markings in these species exist, and researchers may investigate further the significance of the variations in such UV markings. Though this technique requires special equipment, it is relatively inexpensive and useful. The use of handmade reflectance standards in UV reflectance photography allowed for quantitative measurements of reflectance to be derived from the UV reflectance photographs. These handmade reflectance standards were very inexpensive compared to commercially produced reflectance standards. They were also very easy to make and required only the materials themselves and an electronic balance if the proportions of materials are measured out by weight. Any container with a relatively flat bottom can be used as a mold for the mixed materials. Although the spectra of the handmade standards were not perfectly flat across the entire UVA and visible range, the most relevant range was flat and could be used for optical densitometry for images captured with wavelengths in these ranges. The use of these calibration standards, with their percentage reflectance values across relevant wavelengths as determined through spectrophotometry, allowed calibrated measurements of reflectance to be taken using ImageJ software. When this method was applied to the UV reflectance photographs of the H. glaucippe sulphurea specimens, a noticeable, measurable difference in the reflectance values of the UV absorbent markings on the fresh specimens compared with the older specimens of the same species was perceived. The UV absorbent markings of older specimens were deteriorated. No measurable difference was noted in the reflectance values of the UV reflective markings on the fresh and older specimens. The results of the statistical analysis support what can be seen, qualitatively, in the photographs. SEM and lower magnification microscopy (both transmitted and reflected light) provided complementary qualitative information. SEM images show nanostructural deterioration in the UV absorbent markings on the older specimen, but little difference in the UV reflective markings. Visible light microscopy at lower scale magnification (both transmitted and reflected light) supports this. SEM and lower magnification microscopy also revealed structural differences in the morphology of the UV reflective and UV absorbent scales. These structural differences contribute to the different reflectance of these scales in the UV range and the visible light range. Black markings on butterfly wings are “super-black” with low reflectance as a result of light-absorbing pigmentation and complex nanostructures (Vukusik et al. 2004). The nanostructures of the black markings contain the ovoid melanin-containing (pigment) bodies, but also act as Lambertian reflectors, therefore creating a super-matte surface with low reflectance in the UV range and visible range. It seems that the UV absorbent structures and UV reflective structures serve to maximize the contrast between these two types of wing scales. We examined the deterioration of UV absorbent and UV reflective markings on male H. glaucippe sulphurea specimens. Overall, the UV absorbent, black, melanin pigmentary color scales show clear deterioration, whereas UV reflective, orange, lamellar structural 2016 AYRE AND BEVAN—CALIBRATED UV REFLECTANCE PHOTOGRAPHY 49 color scales do not show any clear deterioration, except in visible light where the orange markings appear to have faded. Calibrated UV reflectance photography, a relatively inexpensive technique, can not only document UV markings, but also accurately detect and quantify the degradation of UV markings and could, in the future, provide valuable data to clarify the mechanisms of this degradation.

ACKNOWLEDGMENTS Downloaded from http://meridian.allenpress.com/collection-forum/article-pdf/30/1-2/34/1505769/0831-4985-30_1_34.pdf by guest on 27 September 2021

We wish to thank the following people for their assistance in this research: Adrienne Brewster, Cambridge Butterfly Conservatory; Dr. Alison Murray, Krysia Spirydowicz, Charles Cooney, Bernie Ziomkiewicz, Ian Longo, and 2013 Master of Art Conservation graduates, Queen’s University; Myle`ne Choquette and Season Tse, Canadian Conservation Institute; Luci Cipera, Canadian Museum of Nature; Alex Gabov, Conservation of Monuments Sculptures and Objects; and Lucas Sprague-Coyle, Rowan and Linden Ayre, John Ayre, Mary Ann Evans and family; financial support from the Ontario Graduate Scholarship program; the Society for the Preservation of Natural History Collections (SPNHC) Fitzgerald Travel Grant, and reviewers for providing helpful feedback on this paper.

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