3.1.2. Reds

The source of red dyes in ancient South American textiles has long been of interest [2, 24,27,35,36] and, as such, has been widely studied. Plant reds consistent with those derived from the roots of *Relbunium* or *Galium* species were used throughout the Paracas and Nazca periods, as indicated by the presence of purpurin and other related anthraquinones. Carminic acid from cochineal insects (*Dactylopius coccus*) appears later, specifically in the Wari and Lambayeque textile fragments. Samples from the discontinuous warp and weft textiles attributed to the Wari period showed no evidence of cochineal, only anthraquinone compounds characteristic of the plant reds. This may be due to the difference in preparation of these textiles, where tie- and over-dyeing were used to create the unique patterns of "white" undyed fabric with primary and secondary color designs. The other red yarns from the Wari tapestry weave textiles were prepared with cochineal, as indicated by the presence of carminic acid in those samples. While the Chancay would have had access to cochineal, the reddish-brown yarn from the weaver's basket was clearly dyed with a plant red consistent with a *Relbunium* or *Galium*. Example chromatograms, UV-vis and mass spectra for a *Relbunium* red sample are shown in Supplementary Figure S16. Chromatograms and mass spectra for red yarns from the Antúnez de Mayolo Peruvian dye plant collection reference samples are part of another manuscript in preparation with Degano et al. at the University of Pisa. Detailed results are shown in Supplementary Tables S1 and S2.

Of the anthraquinone aglycones present in the plant reds, purpurin was the major component by HPLC. A peak at *m/z* 239.04 in the negative ion DART mass spectra may be indicative of either xanthopurpurin or alizarin, both dihydroxyanthraquinones differing only in the position of the –OH groups: xanthopurpurin is 1,3-dihydroxyanthraqui-none, while alizarin is 1,2-dihydroxyanthraquinone. Both give nearly identical UV-vis spectra, and under the conditions used for the majority of the HPLC analyses, both also give identical retention times as determined from multiple analyses of standards; the synthesized xanthopurpurin used as a standard may have contained trace impurities. Under collisioninduced dissociation conditions (with the AccuTOF Orifice 1 set to 90 V), reference samples of alizarin and xanthopurpurin yielded different mass spectra consistent with what has been reported [24,37]. However, under these conditions, dyed yarn samples yield complex spectra that do not clearly differentiate between these compounds. Dutra Moresi [38] carried out significant work to characterize the anthraquinones found in various species of *Relbunium*, finding that they contain no alizarin, as determined by HPLC. Negative ion DART mass spectra of reference dyeings prepared from three *Relbunium* species and *Galium antuneziae* [39,40] from Peru showed no consistent differences in the distribution of the characteristic anthraquinone compounds, as indicated in Table 5. Differences that have been attributed to different species may actually be due to differences in dye preparation conditions such as temperature, age of the roots, pH, and mordants. Further complicating the situation is the fluidity of the classification of these plants at the genus level [39,41]. Thus, we have not attempted to differentiate the plant reds and consider all to be "*Relbunium*" for simplicity, though further studies are warranted.

Carminic acid and other glycosides are not well identified with DART-MS [9,25], likely because they cannot be desorbed from the fibers solely by heat. For red samples that do not show the characteristic peaks for the plant red anthraquinones (purpurin, rubiadin, etc.) by DART-MS, we followed up with paper spray MS on the extracted solution used for HPLC analysis. Carminic acid readily ionizes by this method, as do the other glycosides characteristic of cochineal. HPLC of the same extracts showed primarily carminic acid; the other glycosides were either not detected or not identified, as no standards were available for comparison. Example chromatograms, UV-vis spectra, and mass spectra for a cochineal red sample are shown in Supplementary Figure S17.
