Electronic Supplementary Material (ESI) for Analytical Methods. This journal is © The Royal Society of Chemistry 2014

ELECTRONIC SUPPLEMENTARY INFORMATION FOR

NONLETHAL SKIN SWABBING OF CUTANEOUS NATURAL

PRODUCTS FOR HPLC FINGERPRINTING

Thomas P. Umile,a Patrick J. McLaughlin,b Kendall R. Johnson,a Shaya Honarvar,b

Alison L. Blackman,c Elizabeth A. Burzynski,a Robert W. Davis,a Thais L. Teotonio,c

Gail W. Hearn,b Christine A. Hughey,c Anthony F. Lagalante,a and Kevin P.C.

Minbiolea*

a Department of Chemistry, Villanova University, Villanova, PA 19085 USA

b Department of Biology, Drexel University, Philadelphia, PA 19104 USA

c Department of Chemistry, James Madison University, Harrisonburg, VA 22807 USA

* corresponding author contact information:

email: [email protected]

tel: 610-519-4873

fax: 610-519-7167

1

SWABBING OF ON BIOKO ISLAND

Swabbing of Bioko Frogs. specimens were collected at night by hand from four different locations on Bioko Island. These sites ranged in elevation from 0-1400 m asl.

Arena Blanca (0-100 m asl) consists of dense lowland tropical forest and brackish swamps, and represented 4% of total sampling effort. Risule and Moeri were comparable sites (400-700 m asl) consisting of a patchwork of disturbed and pristine submontane forest, and represented 85% of total sampling effort. Moka (1300-1400 m asl) is a mixture of montane tropical forest with large areas converted for agriculture, and represented 11% of total sampling effort. A total of 47 specimens were collected for swabbing across all sites. Frog cataloging information is listed below (Table S1).

2

Fig. S1 Map of sampling sites on Bioko Island, Equatorial . The figure details the number and species of specimens from each site. Inset shows Bioko Island with focal area containing sample sites.

3 Family Genus Species Map/PCO4ID Swabs Sample4Site Body4Size Voucher4Specimens Healthy4Release Arthroleptidae Arthroleptis variabilis Avar 2 Moeri Medium 2 0 Arthroleptis poecilonotus Apoe 3 Risule Small 0 3 Bufonidae Didynamipus sjoestedti Dsjo 5 Moeri Tiny 0 5 paradorsalis Apar 5 Risule Medium 0 5 Hyperolius kuligae Hkul 9 5JKRisule;K4JKMoka Small 5 4 Hyperolius ocellatus Hoce 4 2JKArenaKBlanca;K2JKRisule Small 4 0 Petropedetidae Phrynobatrachus auritus Paur 2 Risule Medium 0 2 Pipidae Xenopus fraseri Xfra 3 Risule Large 3 0 Ranidae Hylarana albolabris Halb 4 Risule Large 4 0 Rhacophoridae Chiromantis rufescens Cruf 6 Risule Large 0 6

Table S1. Summary of species sampled for cutaneous bacteria and small molecules on

Bioko Island.

Fig. S2 Overlays of UV-Vis spectra of fingerprint compounds from a) A. paradorsalis and b) D. sjostedti (inset legends refer to the corresponding retention time in min)

4

Fig. S3 Overlay of representative total wavelength chromatograms from swab extract and skin extract from a single frog specimen

5 Table S2 (follows) A comprehensive table of compounds found on each frog specimen, along with other specimen-specific notes. For each specimen, sample site and collection data are provided, along with notes regarding the location and/or microhabitat they were discovered in before capture. Where possible, sex was determined for each specimen. For each specimen, an "X" indicates a small molecule (“metabolite”), bacteria, or tissue sample was taken, while an "O" indicates no sample was taken. In the cases were two swab samples were taken, a starred X (“X*”) indicates the sample that was collected first.

For the “Specimen” column, an "X" indicates a museum specimen was preserved,

"Healthy Release" indicates the specimen was released healthy to the site of capture within 24 hours, and "Skin Sample" indicates a skin tissue sample was taken and it was prepared as a museum specimen

6 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10

9 8 7 6 5 4 3 2 1 Specimen No. Xenopus fraseri Xenopus fraseri Xenopus fraseri Phrynobatrachus auritus Phrynobatrachus auritus boulengeri Leptopelis sp. Leptopelis modestus Hyperolius ocellatus Hyperolius ocellatus Hyperolius ocellatus Hyperolius ocellatus Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hyperolius kuligae Hylarana albolabris Hylarana albolabris Hylarana albolabris Hylarana albolabris Didynamipus sjostedti Didynamipus sjostedti Didynamipus sjostedti Didynamipus sjostedti Didynamipus sjostedti Chiromentis rufescens Chiromantis rufescens Chiromantis rufescens Chiromantis rufescens Chiromantis rufescens Chiromantis rufescens Arthroleptis variabilis Arthroleptis variabilis Arthroleptis sylvaticus Arthroleptis poecilonotus Arthroleptis poecilonotus Arthroleptis poecilonotus Afrixalus paradorsalis Afrixalus paradorsalis Afrixalus paradorsalis Afrixalus paradorsalis Afrixalus paradorsalis Species Sampling Sampling 9/19/12 9/19/12 9/19/12 9/16/12 9/16/12 9/16/12 9/16/12 9/16/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/16/12 9/16/12 9/16/12 9/21/12 9/21/12 9/21/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/21/12 9/21/12 9/19/12 9/17/12 9/19/12 9/21/12 9/21/12 9/21/12 9/21/12 9/17/12 9/17/12 9/19/12 9/19/12 9/19/12 9/19/12 9/19/12 9/17/12 9/17/12 9/19/12 Date Mud puddle near cement water tank Mud puddle near cement water tank Mud puddle near cement water tank Fern on edge of trail Fern on edge of trail Orange and black ventral mottling, black vocal sack Behind latrine at MWC Banana leaf above cement tank In amplexus PJM438with Di-chromatic coloration In amplexus PJM436with Di-chromatic coloration Outside metal drum Metal drum, guarding nests Cement water tank Cement water tank Cement water tank Non-dichromatic coloration; yellow ventral side Cement water tank Metal drum, guarding nests Metal drum, guarding nests Cement water tank Cement water tank Cement water tank Cement water tank Leaf litter alongside trail Leaf litter alongside trail Leaf litter alongside trail Leaf litter alongside trail Leaf litter alongside trail Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank White blackw/ pattern Green Orange & Yellow Ventral Side Fern on edge of trail Fern on edge of trail Fern on edge of trail Cement water tank Cement water tank Cement water tank Cement water tank Cement water tank Notes Sex M M M M M M M M M M M M M M M M M M M M M M M M F F F F F F F F F ------Risule Risule Risule Risule Risule Risule Moka Risule Arena Blanca Arena Blanca Risule Risule Moka Moka Risule Risule Risule Risule Risule Moka Moka Risule Risule Risule Risule Moeri Moeri Moeri Moeri Moeri Risule Risule Risule Risule Risule Risule Moeri Moeri Moeri Risule Risule Risule Risule Risule Risule Risule Risule Location X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X X X X X X X X X X X X X X X X X X X X X X X X X Metabolite)Swab X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X* X X X X X X X X X X X X X X X X X X X X X X Skin)Bacteria)Swab O O O O O O O O O O O O O O O O O O O O O X X X X X X X X X X X X X X X X X X X X X X X X X X Tissue)Sample Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample

X X X X X X X X X X X X X X X X X X X Specimen

Retention Chromaphore Time (min) (nm ) + 7.58 271 + 7.74 268 + 7.78 none + 7.80 none + 7.82 none + 7.83 274, 351 + 7.86 268 + 7.98 280 + + + 8.00 272, 357 + 8.02 261 + 8.08 none + + 8.09 274, 354 + + + 8.13 none + + + + + + 8.17 none + 8.18 271 + + + 8.20 none + 8.21 269 + + + 8.25 none + 8.29 271 + 8.33 270 + 8.38 280 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10

9 8 7 6 5 4 3 2 1 Specimen No. + + + + + 8.51 274, 354 + 8.54 none + 8.59 none + + + + 8.65 none + 8.67 271 + + 8.71 none + 8.75 none + + 8.80 none + 8.89 none + + + + 9.01 279 + + + + + + + 9.04 none + 9.06 280 + 9.26 288 + + 9.27 282 + 9.28 260 + 9.39 none + + 9.43 288 + + + 9.44 none + 9.45 273 + 9.59 264 + 9.64 none + 9.66 280 + + + + 9.68 none + + 9.85 271 + 9.86 none + + 9.94 none + 10.05 280 + + + + + 10.25 298 + + 10.26 252 + + + 10.37 267 + + 10.37 272 + 10.38 none + 10.43 none + 10.47 279 + + + + + + 10.47 none + + 10.54 324 + + + + + 10.69 300 + 10.70 none + + 10.77 none + + + + 10.80 none + + + + 10.82 none + + + + + 10.82 298 + + + 10.84 none + + + + + 10.86 280 + + + 10.88 none + + 10.93 279 + + 10.97 280 + + + 10.99 274 + + + + + + + + + + + + + + + + + + + 11.00 none + 11.02 279 + + + + + 11.04 none + + + + 11.08 280 + + + 11.09 none + + + + 11.12 none + + 11.13 280 + + + + + + 11.16 298 + + + 11.17 none + + + 11.20 none + 11.28 279 + + + + + 11.32 298 + + + + 11.34 none 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10

9 8 7 6 5 4 3 2 1 Specimen No. + + + 11.38 none + + + + + 11.39 297 + + 11.41 278 + + + + + + 11.42 none + 11.46 none + + + + 11.51 275 + 11.54 270 + + 11.58 none + + + + + 11.60 297 + + + + 11.72 280 + + + + + + + + + + 11.74 none + + 11.77 283 + + 11.80 none + + 11.84 279 + + 11.87 288 + + 11.90 none + + 11.94 279 + + + 11.99 281 + + + + + 11.99 295 + 12.01 267 + + + 12.05 279 + + + + + 12.18 279 + + + 12.22 272 + + + + + + + + + + + + + + + + 12.25 279 + + + 12.31 none + + 12.37 none + + + + 12.46 none + 12.54 272 + + + + + + + 12.60 none + 12.63 none + + 12.69 none + 12.73 279 + + + + + 12.99 none + + + 13.25 281 + + 13.44 280 + + + + 13.49 none + + + + + 13.51 none + + 13.51 280 + + + + 13.54 none + + 13.63 280 + + + + 13.64 none + 13.84 none + 13.88 none + + + + + + + + + + + + 13.94 280 + + + + 13.95 none + + + + + 14.11 280 + + + + + + + + + + + + 14.23 none + + 14.26 none + + + + + + + + + 14.34 none + 14.37 none + 14.40 none + + + + + 14.50 280 + + + + + + 14.63 280 + 15.01 none + + + 15.04 none + 15.08 none + 15.13 none + + + + + 15.13 280 + + 15.17 288 + 15.21 288 + 15.49 none + + + + 15.81 none 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10

9 8 7 6 5 4 3 2 1 Specimen No. + + + + + + + + + + + + + + + + 15.85 none + 15.85 275 + + 15.88 280 + 16.03 none + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 16.48 none + 16.86 none 9/19/16 9/19/16 9/19/16 9/21/16 9/21/16 9/19/16 9/17/16 9/19/16 9/21/16 9/21/16 9/21/16 9/21/16 9/17/16 9/17/16 9/19/16 9/19/16 9/19/16 9/19/16 9/19/16 9/17/16 9/17/16 9/19/16 9/19/16 9/19/16 9/19/16 9/16/16 9/16/16 9/16/16 9/16/16 9/16/16 9/19/16 9/19/16 9/19/16 9/19/16 9/19/16 9/19/16 9/16/16 9/16/16 9/16/16 9/21/16 9/21/16 9/21/16 9/19/16 9/19/16 9/19/16 9/19/16 9/19/16 Sampling Sampling Date Cement water tank Cement water tank Cement water tank - - - - - Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank Banana leaf above cement tank White blackw/ pattern Green Orange & Yellow Ventral Side Fern on edge of trail Fern on edge of trail Fern on edge of trail Cement water tank Cement water tank Cement water tank Cement water tank Cement water tank Mud puddle near cement water tank Mud puddle near cement water tank Mud puddle near cement water tank Fern on edge of trail Fern on edge of trail Unknown species; orange and black mottling, black vocal sack Behind latrine at MWC Banana leaf above cement tank In amplexus Specimenwith 38 Di-chromatic coloration In amplexus Specimenwith 36 Di-chromatic coloration Outside metal drum Metal drum, guarding nests Cement water tank Cement water tank Cement water tank Non-dichromatic coloration; yellow ventral side Cement water tank Metal drum, guarding nests Metal drum, guarding nests Cement water tank Notes Sex M? M? M? M? F? M M M M M M M M M M M M M M M M M M M M F F F F F F F F ------Risule Risule Risule Risule Risule Risule Moka Risule Arena Blanca Arena Blanca Risule Risule Moka Moka Risule Risule Risule Risule Risule Moka Moka Risule Risule Risule Risule Moeri Moeri Moeri Moeri Moeri Risule Risule Risule Risule Risule Risule Moeri Moeri Moeri Risule Risule Risule Risule Risule Risule Risule Risule Location X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X GPS X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Metabolism)Swab O O O O O O O O O O O O O O O O O O O O O X X X X X X X X X X X X X X X X X X X X X X X X X X Skin)Bacteria)Swab Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample

X X X X X X X X X X X X X X Tissue)Sample Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Healthy Release Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample Skin Sample

X X X X X X X X X X X X X X Specimen