The oldest indigo-dyed fabric ever found has been discovered in Peru, pushing back the use of this blue coloring to at least 6,200 years ago. 
 
 Previously, the oldest sample of blue-dyed fabric dated to around 4,400 years ago in Egypt, with the oldest written references to blue dye going back to around 5,000 years ago in the Middle East. The discovery in Peru, however, shines a spotlight on the Americas, which are less-discussed in terms of firsts, said study researcher Jeffrey Splitstoser, an archaeologist and textile expert at The George Washington University. 
 
 "The people of the Americas were making scientific and technological contributions as early and in this case even earlier than people were in other parts of the world," Splitstoser told Live Science. "We always leave them out. I think this finding just shows that that's a mistake." [Gallery: See Images of the Oldest Indigo] 
 
 The dyed fabric pieces are small scraps made of woven cotton. They were excavated by archaeologists Tom Dillehay and Duccio Bonavia between 2007 and 2008 from a prehistoric site called Huaca Prieta, which is north of the city of Trujillo in coastal Peru. Huaca Prieta was a prehistoric dwelling that was covered by a mound and turned into a temple, Splitstoser said. The temple was made of a sort of concrete mixed from ash, shells and sand; over the years, many layers of this material had been applied to the structure as local people renovated and rebuilt the temple. The fabric scraps were found in bundles lining the ramp that led up to the top of the temple, embedded in the concrete-like layers. They all date to between 4,000 and 6,200 years ago. 
 
 "They were literally sealed under these new layers of building, but because the building material had so much ash in it, it leached into the textiles, making them a very dirty, sooty color," Splitstoser said. 
 
 The blue color didn't appear until conservationists washed the textiles. Almost all blue dye in nature comes from the compound indigoid, Splitstoser said, which can be made by many plants. But the first tests on the fabric yielded no sign of indigoid. Splitstoser was stumped. 
 
 He persevered, finding another chemist — Jan Wouters of University College London — with more sensitive equipment. Wouters, using a sensitive technique called high-performance liquid chromatography, was able to tease out the chemical makeup of the dye to discover that it was, in fact, indigo. He tested eight tiny samples of the blue cotton and confirmed indigo in five of them. 
 
 "That's when we realized then that we had the world's oldest indigo, by far," Splitstoser said. 
 
 The fabric pieces were all cut or torn before they were deposited on the temple ramp, which probably represented a ritual "killing," by peoples who viewed objects as living, Splitstoser said. 
 
 "We see that all over the Andes. They not only ritually killed textiles, but they ritually killed ceramics. Anything that was buried was broken," he said. [Photos: Journey Into the Tropial Andes] 
 
 Some of the fabrics showed signs of being wetted and then squeezed out, possibly as part of the ritual, Splitstoser said. The fabrics weren't just blue — they were woven in patterns made of blue-dyed yarn, natural off-white cotton and bright-white thread made from milkweed, a very rare textile in South America, Splitstoser said. The yarn had also been dipped in red and yellow ochre, an iron pigment often used in rock art. Unlike the indigo, the ochre would have run when wetted. 
 
 "If you were to pour water on those and then squeeze it, you'd get colored water pouring out of the textiles, which might have been part of the show," Splitstoser said. No one knows what these rituals might have represented to the people who invented them; the era in which the textiles were made was one of a drying climate, Splitstoser said, so perhaps the rituals had to do with rain or water. 
 
 The discovery of indigo dye more than 6,000 years ago couldn't have been mere happenstance. Indigo dye is quite complicated to make, Splistoser said. Many dyes are made from flowers and require simply boiling the blossoms in water to extract the color, he said. 
 
 "Indigo does not work that way," he said. "If you put the leaves — and it's leaves, not flowers — in water, nothing will happen." 
 
 Instead, the leaves have to be fermented. Then, the fermented mixture must be aerated so that a solid compound falls out of the mixture to the bottom of the tub. This mixture can be taken, dried and stored. To reconstitute it requires an alkaline substance, often urine, which makes white indigo, a water-soluble compound. Yarn dipped in white indigo will turn yellow, green and finally blue, "like magic," Splitstoser said. 
 
 "This was probably a technology that was invented by women," he said, as women were typically in charge of weaving and dying in Andean cultures. A few of the later fabric scraps were of fine quality and more complex decoration, he said, but most of the scraps were likely simple squares or rectangles made by locals, who also wove fishing nets and fabric bags. 
 
 "They weren't primitive," Splitstoser said. 
 
 The researchers reported their findings today (Sept. 14) in the journal Science Advances. 
 
 Original article on Live Science. ||||| A George Washington University researcher has identified a 6,200-year-old indigo-blue fabric from Huaca, Peru, making it one of the oldest-known cotton textiles in the world and the oldest known textile decorated with indigo blue. 
 
 The discovery marks the earliest use of indigo as a dye, a technically challenging color to produce. According to Jeffrey Splitstoser, lead author of a paper on the discovery and assistant research professor of anthropology at the George Washington University, the finding speaks to the sophisticated textile technology ancient Andean people developed 6,200 years ago. 
 
 "Some of the world's most significant technological achievements were developed first in the New World," said Dr. Splitstoser. "Many people, however, remain mostly unaware of the important technological contributions made by Native Americans, perhaps because so many of these technologies were replaced by European systems during the conquest. However, the fine fibers and sophisticated dyeing, spinning and weaving practices developed by ancient South Americans were quickly co-opted by Europeans." 
 
 The textile was discovered during a 2009 excavation at Huaca Prieta, a desert area that offers nearly pristine archaeological preservation on the north coast of Peru. Experts believe the site was likely a temple where a variety of textiles and other offerings were placed, possibly as part of a ritual. The well-preserved artifacts give a glimpse into ancient civilization and lifestyle and offer an unexpected connection to the 21st century. 
 
 The development of indigo dye was critical for future trends in fashion, fabrics and textile arts, Splitstoser said. 
 
 "The cotton used in Huaca Prieta fabrics, Gossypium barbadense, is the same species grown today known as Egyptian cotton," Dr. Splitstoser said. "And that's not the only cotton connection we made in this excavation -- we may well not have had blue jeans if it weren't for the ancient South Americans." 
 
 The textile is now in the Cao Museum collection in Peru. The paper, "Early Pre-Hispanic Use of Indigo Blue in Peru," published in Science Advances on Sept. 14. ||||| Abstract Archaeological research has identified the use of cultivated cotton (Gossypium barbadense) in the ancient Andes dating back to at least 7800 years ago. Because of unusual circumstances of preservation, 6000-year-old cotton fabrics from the Preceramic site of Huaca Prieta on the north coast of Peru retained traces of a blue pigment that was analyzed and positively identified as an indigoid dye (indigotin), making it the earliest known use of indigo in the world, derived most likely from Indigofera spp. native to South America. This predates by ~1500 years the earliest reported use of indigo in the Old World, from Fifth Dynasty Egypt [ca. 4400 BP (before present)]. Indigo is one of the most valued and most globally widespread dyes of antiquity and of the present era (it being the blue of blue jeans). 
 
 Keywords 
 
 Cotton 
 
 indigo 
 
 textile 
 
 twining 
 
 dye 
 
 preceramic 
 
 Peru 
 
 South America 
 
 blue 
 
 HPLC-PDA 
 
 INTRODUCTION After the arrival of the Spanish in the Americas, few things other than precious gems and metals impressed them more than Inca cumbi (fine tapestry cloth), primarily because of its silk-like texture and use of brilliant colors (1). The superiority of Andean dyes at the time of the conquest should not be surprising, because they were the result of observation and experimentation carried out by native South Americans over several millennia, representing the apogee of one of the world’s oldest and most sophisticated weaving and dyeing traditions. Evidence of the early age and complexity of Andean weaving and dyeing practices comes from cotton textiles decorated with indigo from the Preceramic site of Huaca Prieta on the north coast of Peru, which was occupied between 14,500 and 4000 cal BP (calibrated before present). Huaca Prieta is a large stone and earthen ceremonial mound that was first excavated in the 1940s by J. Bird (2), who established the site’s early age [~5302–1933 cal BP; see the study by Dillehay et al. (3)] and made it famous for producing the oldest known cotton fabrics decorated with recognizable art in the Americas. Although Bird’s dates for the first use of cotton were subsequently surpassed (4, 5), recent work has shown that textiles were produced at Huaca Prieta even earlier than previously documented. The original site for the domestication of cotton, Gossypium barbadense, has not been determined; however, it is thought to be somewhere on the north coast of Peru (6), where it adapted to the region’s arid conditions and grows wild or as an escaped plant. To date, the earliest reported G. barbadense is from the Nanchoc Valley in north Peru, dated to around 7800 cal BP (7). The continued use of G. barbadense for textiles, fish nets, bags, and other materials is documented at numerous other sites dating after 6000–5000 cal BP (8–19). The earliest textiles decorated with blue at Huaca Prieta were excavated from stratum 44 in J. Bird’s HP-3 trench. Although not decorated with indigo, the earliest cotton yarn is from stratum 52, which is radiocarbon-dated at 6882–6657 cal BP [sample AA82121; see the study by Dillehay et al. (3)]. The next radiocarbon-dated level is stratum 35 at 5848–5585 cal BP (sample AA86948 on charcoal from an intact hearth), suggesting that the two textiles from stratum 44 are about 6200 to 6000 years old. They are both weft twining [the fabric structure is spaced, regular, two-strand, Z-twist weft twining with Z(2s) warp and weft yarns; wefts usually enclose two warp yarns, although groupings of one and three yarns are present] decorated with warp stripes where natural colored cotton, which is a light tan, contrasts with cotton dyed blue and cotton plied with a bright-white fiber from the stem of a vine locally known as chivo (Asclepiadaceae sp., a member of the milkweed subfamily). To date, the source of the blue color at Huaca Prieta is unknown. A sample of blue yarn from one of these early blue-striped fabrics, specimen 2009.052.01.B (Fig. 1), and samples of blue yarns from seven other fabrics from Huaca Prieta whose ages range from 6200 to 1500 cal BP, were investigated for the presence of indigoid dyes (see Table 1). All textiles analyzed in Table 1 were associated with thin, continuous, stratigraphically intact floors that were deeply buried in the mound at Huaca Prieta (~10 to 18 m in depth) and without intrusive architecture and other features. Fabrics were dated on the basis of their associations with these floors. For a detailed discussion of the phases at Huaca Prieta and their ages, see the study by Dillehay et al. (3). Fig. 1 Diagram and photo of specimen 2009.052.01.B. Table 1 Huaca Prieta fabrics with blue color that were analyzed, including fabric structure, provenance (level), phase, carbon date, and indigo composition. mAU, milliabsorption units. View this table: Indigoid dyes may be prepared from different plants, such as those belonging to the Indigofera, Strobilanthes, Isatis, and Polygonum genera. All indigoid dyes contain indigotin, the main blue dye component, and indirubin, a structural isomer of indigotin. To date, there are no known features to distinguish between the genera; however, research has shown that, statistically, the proportion of indirubin to indigotin is higher in blue-dyed textiles from China, India, and Latin America as compared to those of European origin, for instance (20, 21). This may be due not only to the indigo-producing plants available or selected but also to the vatting procedure in the dyeing process, which involves creating an alkaline-reducing medium needed to solubilize the indigoid dye. 
 
 RESULTS The presence of an indigoid dye has been firmly indicated in five of eight examined samples (Table 1) representing two plain weaves and three twined textiles. An example of chromatographic analysis that is of particular interest is given in Fig. 2, and the spectral analysis of the chromatographic peaks is shown in Fig. 3; both refer to the sample from specimen 2009.065.01.A. The HPLC (high-performance liquid chromatography) chromatogram of sample 2009.052.01.B&C, the oldest one of the collection analyzed, as well as the full set of chromatograms and peak integration tables, is included in the Supplementary Materials (section S1). Of particular interest in the analysis of sample 2009.065.01.A is the presence of four dye components. Besides the generally expected indigotin and indirubin, two components that show ultraviolet-visible (UV-vis) spectra closely related to indirubin (Fig. 3, indirubin-like) were observed at trace amounts, but with significantly different retention times. These components have never been reported in other analyses of indigoid dyes worldwide. In three samples, the high relative proportion of indirubin reflects earlier findings on pre-Columbian fabrics (21). In one plain weave textile, only indirubin was found. In one sample, 2009.018.01, indigotin and indirubin were observed at trace amounts. Fig. 2 Chromatographic analysis of sample 2009.065.01.A, recorded at 600 nm. Fig. 3 UV-vis spectral identification of the four peaks from Fig. 2 The source of the indigoid dye at Huaca Prieta was most likely Indigofera spp.; however, other indigo-producing plants native to the tropics of South America are grown as dye plants today, including Justicia colorifera (cuaja tinta or tinta montes), Koanophyllon tinctorium Arruda, and Cybistax antisyphilitica Martius (yangua). These plants produce blues similar to those of Indigofera, and the dye produced by yangua, for instance, contains both indigotin and indurbin (22). Although these plants are not grown on the north coast of Peru today, their distribution 6000 to 7000 years ago is unknown. 
 
 DISCUSSION Early examples of the use of blue yarns that were most likely colored with indigo are known, but dye analysis had heretofore been unavailable. The composition of the indigoid dyes identified in the fabrics presented here reflects that of earlier findings in Latin American and Asian contexts, in that proportions of indirubin relative to indigotin are significantly higher as compared to European productions. To date, there is no firm evidence to explain these differences, but plant species, harvesting, dye preparation, and actual dyeing, as well as differential conservation processes of essential dye components, may have, alone or in combination, contributed to this observation. One interesting hypothesis, requiring further confirmation, is that ancient vat dyeing technologies favored the formation and uptake by the yarn of indirubin. This would have resulted in a more purplish hue produced by a reddish indirubin and a bluish indigotin. Why no indigoid dye components were found in three of the samples remains an unresolved issue. Limits of peak detectability, too uncertain UV-vis spectral confirmation, sample size, and material losses are all associated with attempts to detect compounds at the limit of detectability. However, the main driver for the present analytical campaign was to see whether indigoid dyes could be detected in textile fragments of the given archaeological context. The conclusion is clear: yes. To date, the earliest identified blue dyes in the Americas were from Ocucaje and the Paracas Necrópolis dating to around 2500–2250 cal BP (23). In the Necrópolis textiles, studies have found combinations of indigo with either Relbunium ciliatum (South American madder) or a variety of yellow dyes, to make violet and green, respectively (21). In Old World fabrics, there are Egyptian textiles with indigo-dyed bands from the Fifth Dynasty (ca. 4400 BP) (24). Early examples of indigo also are known from Xinjiang, China, where indigo has been positively identified as early as 3000 BP (25, 26). It is believed that indigo might have been used earlier in the Near East; however, actual examples with positive identifications of indigo are not available, making the indigo found at Huaca Prieta the earliest known use of indigo. 
 
 MATERIALS AND METHODS All dye analyses were performed with HPLC and photodiode-array detection (HPLC-PDA) (27) on yarn extracts prepared with the oxalic acid hydrolysis method (28). This method allows for the simultaneous detection of components present in yellow, red, green, blue, and black dyes while avoiding degradation of acid-vulnerable components, such as those bearing glucosidic moities. Analytical results are expressed as a percentage of composition of the components, resulting from the integration of chromatographic peaks at the wavelength indicated. For indigoid dyes, the selected wavelengths were 288 and 600 nm. A full description of sample preparation and HPLC-PDA analysis methods is given in the Supplementary Materials (section S2). Preceding the indication of the wavelength of detection in Table 1 is the response of the smallest chromatographic peak in milliabsorption units. This helps to evaluate the level of reliability of the analytical result. For acceptance, the peak height should be at least twice as high as the average background noise. This method, which leads to a lower-limit acceptance of peaks than the usual standard, can be used in dye analysis practice because besides a peak’s retention time, its UV-vis spectrum must confirm the identification of the component suggested by that retention time. The average background noise is typically 0.025 mAU; thus, a positive detection, pending spectral confirmation, always involves a peak height of at least 0.050 mAU. The identification of individual dye components results from a combination of retention time in the chromatographic system and online UV-vis spectrometry, recorded between 200 and 800 nm, and from comparison of these data with standards. Conclusions are based on chromatographic data obtained at 600 nm, because this wavelength of detection excludes any influence of UV-absorbing noncoloring contaminants in the quantitative estimations of indigoid dye components; however, each analysis was screened for absorptions using the whole detector’s wavelength range. 
 
 SUPPLEMENTARY MATERIALS Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1501623/DC1 section S1. Chromatographic analysis of all samples, recorded at 600 nm. section S2. Description of sample preparation and HPLC-PDA analysis methods. 
 
 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited. 
 
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 Acknowledgments: We thank the Ministerio de Cultura, Lima, Peru, for granting us permission to work at Huaca Prieta. We are grateful to C. Galvéz and J. Briceño (Dirección Regional del Ministerio de la Cultura, Trujillo) for their support. Funding: This project was financed by the NSF, the National Geographic Society, Vanderbilt University, and the Lapinski and O’Leary families. Competing interests: The authors declare that they have no competing interests. Author contributions: J.C.S. analyzed the textiles, recognized their significance, conceived the study, and coordinated the project. T.D.D. managed excavations at Huaca Prieta and supervised the radiocarbon dating of all strata used to date the textiles. J.W. (60%) and A.C. (40%) planned and performed the experiments and collected and analyzed all data associated with HPLC-PDA analysis. All authors contributed to the writing of the manuscript. The approximate individual author contribution breakdown is as follows: J.C.S. (31%), T.D.D. (30%), J.W. (30%), and A.C. (9%). Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
– The dye that makes blue jeans blue has been around a lot longer than anyone thought. Archaeologists digging in Peru found a piece of cotton fabric dyed with indigo that is 6,200 years old, according to a press release. That's 1,500 years older than the previous oldest discovered use of indigo from Egypt. And Live Science reports the Peru textile predates even the oldest known reference to indigo dye in the Middle East by 1,200 years. "Some of the world's most significant technological achievements were developed first in the New World," professor Jeffrey Splitstoser says in the press release. "We may well not have had blue jeans if it weren't for the ancient South Americans." The indigo-dyed textile is made of what is now known as Egyptian cotton. It was found during a 2009 dig at a site in Peru called Huaca Prieta. According to a paper published Wednesday in Science Advances, it was intact thanks to "unusual circumstances of preservation" at the site. The fabric was embedded in a concrete-like substance used to build what researchers believe was a temple. Indigo is a notoriously difficult dye to make, as it requires the leaves of various plants to be fermented, then aerated, and then doused with an alkaline substance—usually urine in ancient times. (A newly discovered Nazca Line in Peru depicts a weird creature.)