*2.4. Methods: Microscopy and Fourier-Transform Infrared Spectroscopy*

Each of the ancient samples was visually inspected and photographed under magnification (10×–200×) using a Dino-Lite AM4815ZT digital microscope. The samples from the Lambayeque textile were further examined using scanning electron microscopy with energy dispersive spectroscopy thanks to the short-term loan of a JEOL (JEOL USA, Peabody, MA, USA) JCM-7000 NeoScope benchtop instrument, used in our curriculumbased undergraduate research experience (CURE) in September 2019. SEM images were collected under high-vacuum conditions with a landing voltage of 10.0 kV at a working distance of approximately 12 mm. Animal hair—presumably camelid wool—was identified on the basis of visible scales and the presence of S and N, while cellulose—presumably cotton—was identified based on visible morphological features including convolutions similar to other cotton species and a marked lack of nodal structures, along with a lack of significant S and N signals.

When SEM-EDS was not available and fibers were not readily identifiable visually by optical microscopy, fibers were characterized using attenuated total reflectance Fouriertransform infrared spectroscopy. Measurements were obtained using a Pike Technologies (Madison, WI, USA) MIRacle ATR attachment with a ZnSe crystal in a Shimadzu IRTracer spectrometer (Shimadzu Corp, Kyoto, Japan). Spectra were collected by averaging 32 scans at a resolution of 4 cm−<sup>1</sup> over the range of 4000–600 cm−1. The presence of the amide I and II bands around 1600 and 1500 cm−1, respectively, were considered characteristic of animal hair protein. Absorbance bands at 1020 cm−<sup>1</sup> combined with the presence of an –OH stretching band around 3300 cm−<sup>1</sup> were indicative of cellulose.

#### **3. Results**

The purpose of this study was twofold. First, we sought to identify the colorants present in the yarn samples from the ancient Peruvian textiles to add to the existing body of knowledge about the dyes used over the time periods described above, ideally to determine the relationships between the primary and secondary colors. Second, we applied ambient ionization mass spectrometry methods for these identifications, specifically to compare this approach to HPLC with diode array detection, which is the most commonly reported means of identifying dyes in archaeological and historical textiles. The two approaches differ significantly in how much material is needed for analysis, how many colorants can be detected and identified, and the length of time needed to complete the analysis. The results from the analyses of the ancient Peruvian textiles are summarized in Table 3 for the samples from the collections of the Michael C. Carlos Museum and in Table 4 for the Paracas samples from the MNAAHP. Results of the analyses of the Peruvian dyes reference collection are presented in Table 5. Complete results, including those for the fiber identifications, are found in Supplementary Tables S1 and S2.


**Table 3.** Results for MCCM textile samples. Details in Supplementary Table S1.

**Table 4.** Results of the analysis of the dyes in the Paracas Necropolis samples from Museo Nacional de Arqueología, Antropología, e Historia del Perú (MNAAHP), Lima, Peru. Details in Supplementary Table S2.



**Table 5.** Results of analysis of reference samples from the Antúnez de Mayolo Peruvian dye plant collection.
