Journal of Archaeological Science (2001) 28, 55–60doi:10.1006/jasc.2000.0553, available online at http://www.idealibrary.com on

Light Microscopy and Determination of Eryngium yuccifoliumMichaux Leaf Material in Twined Slippers from Salts Cave,Kentucky

Angela Gordon

Department of Anthropology, Campus Box 1114, Washington University, St Louis, MO 63130, U.S.A.

Richard C. Keating

Missouri Botanical Garden, P.O. Box 299, St Louis, MO 63166, U.S.A.

(Received 20 October 1999, revised manuscript accepted 28 January 2000)

Fourteen samples of slippers composed of twined botanical fibres were recovered from Salts Cave, Mammoth CaveNational Park, Kentucky. They were investigated by light microscopy methods for species identification. Specimenswere hydrated, sometimes cleared, dissected, and subjected to dyes which allowed identification of histological features.Comparisons were made with several botanical species considered likely candidates for identification. In archaeologicalsamples with sufficient preservation, all were determined to be Eryngium yuccifolium Michx. (rattlesnake master)Apiaceae, a common component of the tallgrass prairie. Identification was made on the basis of shared anatomicalcharacteristics, including paracytic stomata, marginal bristle-like teeth, and parallel venation. � 2001 Academic Press

Keywords: LIGHT MICROSCOPY, ERYNGIUM YUCCIFOLIUM, ARCHAEOLOGY, FIBRE, SALTS CAVE,FOOTWEAR.

Introduction

W hile many researchers are now turning tothe use of scanning electron microscopesand other technology-intensive methods, in

many cases it remains feasible to use light microscopyfor fibre identification. The benefits of this approachare a decrease in the costs of identification, a wideraccessibility of the techniques and equipment, and thepotential for colour differentiation using diagnosticdyes.

Several authors focus mostly or solely on fibreidentification including methods using SEM (e.g.,Catling & Grayson, 1982; Florian, 1990; Jakes et al.,1994), while others include short methodology sectionswithin larger frameworks of textile analysis (e.g.,Andrews & Adovasio, 1996; Korber-Grohne, 1978;Kuttruff et al., 1995). One of the most completediscussions comes from outside the field of archaeol-ogy, from the London police forensics laboratory(Catling & Grayson, 1982). Unfortunately, the authorsfocus solely on fibre plants currently being growncommercially. Florian (1990) focuses specifically onarchaeological fibre identification. She discusses everyaspect of laboratory work, from preparation of thesample, cutting sections, wet-mount preparation,

550305–4403/01/010055+06 $35.00/0

methods of observation, staining, and preparation ofpermanent slides (Florian, 1990: 33–37).

Few studies report conclusive fibre identificationsfrom the wide range of woven slippers found in theMidwest and Midsouth regions of the United States, anotable exception being Kuttruff et al. (1998), a study inwhich ‘‘the majority of fibers identified are fromErynigium yuccifolium’’ (Kuttruff et al., 1998: 72).Reports from the 1930s also identify Eryngium yuccifo-lium Michx. (rattlesnake master) in woven and twinedslippers, but little is known about the basis for identifi-cation (see Dellinger, 1936; Jones, 1936). The botanicalsource of fibre in many other slippers remains unknown.

This paper describes the light microscopy study offibre samples from a collection of twined, vegetal fibreslippers from Salts Cave, Mammoth Cave NationalPark, Kentucky. Through comparisons of anatomicalfeatures using simple microtechnical methods, theauthors demonstrate methods of identification ofthe botanical fibre source and provide identificationof the botanical source of the Salts Cave material.

Salts CaveSalts Cave, part of the Mammoth Cave system,Mammoth Cave National Park, Kentucky

� 2001 Academic Press

56 A. Gordon and R. C. Keating

(37�11�30�N, 86�03�00�W), is a dry cave, with little orno fluctuation in temperature or humidity. This con-stant environment, as well as the exsiccating effect ofsome of the cave minerals, preserves normally perish-able prehistoric materials, such as plant remains, fibreartefacts, and human palaeofaeces (Watson, 1969).The heaviest period of prehistoric use is solidly datedto the Early Woodland period, with more than 57radiocarbon dates from Salts and Mammoth Cavesclustered around 2500 (Kennedy, 1996).

Archaeological SamplesThe slippers analysed during this research were col-lected as part of the 1963 field season of the IllinoisState Museum—Cave Research Foundation SaltsCave archaeological project. In all, 10 slippers werecollected during the 1963 season (Watson, 1969), andhave been dated to approximately 2500 by associ-ation with other material from Salts and MammothCaves (Kennedy, 1996). Other textile fragments werealso recovered (Watson, 1969).

The slippers are fairly simple and unadorned. Theyare constructed of a closed, chevron twine pattern,with an open heel, and are described in detail by King(1974), Miller (1988), and Watson (1969).

The samples for this study were originally chosen byVolney Jones at the University of Michigan, AnnArbor, in 1964 (see Table 1), but Jones was not able tocomplete the identification (King, 1974: 35). The smallsamples removed by Jones from the slippers and othertextile fragments represent both warp and weft fibres.

Methods

Reference materials

For this research, we assembled comparative fibrespecimens from several likely candidate species. The

following sources were consulted to determine thelikely candidates: Henning (1966), King (1974), Miller(1988), Orchard (1920), Scholtz (1975), Watson (1969),Whitford (1941, 1943), and Young (1910). Earlyinvestigations indicated that the source was probablynot an inner bast fibre, because there are stomatavisible in some of the archaeological material. There-fore, although we did compare some of the bast fibrecandidates, we focused more heavily on the plants thatyield either leaf fibres or outer-stem fibres.

The following comparative specimens were providedby Marie Standifer of Louisiana State University inBaton Rouge, Louisiana: Tilia americana L. (Americanbasswood), Salix niger L. (black willow), Sabal minor(Jacquin) Persoon (palmetto), and Andropogon gerardiiVitman (big blue stem grass). Bill Davitt, of theMissouri Botanical Garden’s Litzsinger Road EcologyCenter, St Louis, Missouri, helped to identify andcollect Asimina triloba (L.) Dunal (pawpaw), Apo-cynum cannabinum L. (Indian hemp), Asclepias syriacaL. (common milkweed), and Laportea canadensis (L.)Wedd. (wood nettle). The authors collected Eryngiumyuccifolium Michaux (rattlesnake master) from plant-ing at the Cahokia Mounds Interpretive Center,Illinois, and later from Litzsinger Road EcologyCenter, Missouri, and Hamilton Valley, Kentucky.

Table 1. Results of fibre identification

Sample identification Object Stomata Leaf teeth Identification

SCU Survey Lot 4 Slipper IndeterminateSCU Survey Lot 5 Warp Slipper Present E. yuccifolium

Weft Present Present E. yuccifoliumSCU Survey Lot 12 Warp Slipper Present E. yuccifolium

Weft Present E. yuccifoliumSCU Survey Lot 24 Slipper Present Present E. yuccifoliumSCU Test A Lot 100 Slipper Present E. yuccifoliumSCU Survey Lot 111 Weft Slipper IndeterminateSCU Survey Lot 117 Slipper IndeterminateSCU Test A Lot 137 Warp Slipper Indeterminate

Weft IndeterminateSCU Test A Lot 70 Three ties Present E. yuccifoliumSCU Test A Lot 70 Braid Present E. yuccifoliumSCU Test A Lot 79 Assorted Present E. yuccifolium

Sample identification numbers correspond to those assigned during fieldwork. All samples are from Upper SaltsCave (SCU).

Laboratory techniquesThe majority of this research was done in the PlantAnatomy Laboratory at Missouri Botanical Garden,St Louis, Missouri. Instead of relying on scanningelectron microscopy for fibre identification, we usedthe less expensive and more widely available lightmicroscope.

Dry plant material, such as these archaeologicalsamples, must be restored, or softened, before section-ing (Keating, 1996a: 28). Many solutions are used forsoftening, ranging from gently heating the fibres in

Light Microscopy of Eryngium yuccifolium Michaux, Salts Cave, Kentucky 57

water with a small amount of detergent added as asurfactant, to various chemical preparations (Keating,1996a: 28). For this research, unknown archaeo-logical material and reference materials were treatedidentically and subjected to all treatments.

Specimen hydration was achieved overnight, atroom temperature, by immersion in a proprietarysolution of aqueous p-tertiary octylphenoxy poly-ethyl alcohol and polypropylene glycol (KodakPhotoflo 200�), used in a 1:3 aqueous dilution(Valdes-Reyna & Hatch, 1995). Since the method doesnot require heating or agitation, there is less chance offibre damage occurring. The samples were rinsed bypipetting off the Photoflo solution and adding andpipetting off clean water 3–4 times.

Fibres were then treated several ways for viewingunder the microscope. Some were simply teased with aprobe to spread them out on the slide, and thenmounted in a 20% aqueous solution of calciumchloride (CaCl2). Calcium chloride has a high refrac-tive index, and also induces differential stainingreactions for plant cell walls using many differentnatural and synthetic dyes. It is also hygroscopic.Specimens mounted in it remain permanently wet(Herr, 1992; Keating, 1996b).

These specimens were stained with either cresylviolet acetate (CVA) or iodine-potassium iodide(I2KI). CVA results in distinctive colours for lignin,cellulose, and collenchyma, while I2KI stains starch toa blue–black, lignin orange, and cellulose magenta(Keating, 1996b). Archaeological specimens differen-tiated these stains less well, suggesting that possiblealterations in cell-wall chemistry have occurred overtime. Staining was very useful for the comparativematerials.

Aniline dyes such as CVA are prepared as 0·1–0·5%dye in a solution of 15% ethanol, an essentially aque-ous solution with enough ethanol to enhance its sol-vent properties and act as a preservative. Specimensare rinsed in 15% ethanol then transferred to mount-ant. Iodine-potassium iodide is prepared as 0·2 gmiodine and 2·0 gm potassium iodide in 100 ml of 15%ethanol. Specimens in I2KI are not rinsed prior totransferring to mountant. Hand sections of dissectionsare stained by inspection and should look overstainedto the unaided eye. The microscope field diaphragm isadjusted for Kohler illumination and the substagediaphragm kept wide open. Focus should be on thesurface of the tissue.

An attempt was made to clear some of the archaeo-logical specimens by placing them in a 5% aqueoussolution of sodium hydroxide (NaOH) and heating ina microwave and a conventional paraffin oven (60�C).This was not very successful in the archaeologicalsamples, but, as with the staining, produced goodclearings of the reference materials.

Both longitudinal sections and transverse sectionsare useful in trying to identify an archaeological fibre.The transverse (cross) sections can sometimes show the

overall arrangement of fibre bundles, soft tissue, andother plant organs better than longitudinal sections.The cross-section of an Eryngium yuccifolium leafshows a distinctive arrangement of structural fibresand vascular bundle caps immersed within islands ofmesophyll tissue.

Unfortunately, the archaeological samples were toofragile to cross-section by hand. Several methods wereused, including encasing a sample in a cube of potatoand using a hand microtome, but none produced across-section without completely crushing any surviv-ing structural elements. Samples were also embeddedupright in Sobo glue, a clear-drying fabric glue, toenable viewing of the cross-sectional structures. Thiswas unsuccessful.

While paraffin embedding is often used to preparepermanent 10–15 �m sections of fragile plant materials(Keating, 1996a: 15), it was not an option for the SaltsCave samples because of the unacceptable quantityof embedded soil granules which would haveseverely damaged the microtome blade. Hence, allidentifications were based on longitudinal sections.

Archaeological samples were compared with theprepared reference materials using an Olympus BX40compound light microscope, with magnification rang-ing from �40 to �400. The arrangements of fibresand soft tissues were compared, and cell lengths andwidths were measured at a magnification of �400.

PhotographyPhotomicrographs were made using bright-field lightmicroscopy. Negatives were made using 35 mm KodakTechnical Pan� film exposed and developed forISO 50.

ResultsThe archaeological material is generally degraded, withmuch of the soft tissue lost, but some epidermal tissueis still present, including the distinctive stomata. Thishelps to narrow the focus to leaf fibres or fibres fromouter stems, because inner-bast fibres lack stomata.Drying and twisting of the archaeological materialbrings the parallel veins into closer proximity andcauses the soft tissue of the leaf areolae to collapsedifferentially. Therefore when one wets dried leaves tore-expand them for microscopy, it is diagnostic to findlittle intact mesophyll and stomate-bearing epidermiscompared to unstressed, modern reference materials.

The archaeological materials share many features,including marginal venation and vascularized marginalbristle-like teeth, parallel venation with oblique crossveins, and paracytic stomata with trapezoidal sub-sidiary cells. The specimens resemble Eryngiumyuccifolium more than any of the other referencematerials. None of the other selected referencematerials have a similar pattern of fibres and softtissue, or similar cell shape and size. Because Eryngium

58 A. Gordon and R. C. Keating

Figure 1. Photomicrographs of (a, c, e) leaf material of extant Eryngium yuccifolium and (b, d, f, g) comparable archaeological leaf material.(a) Cleared whole mount of leaf margin showing marginal venation (arrow) and vascularized marginal bristle-like tooth. (b) Whole mount ofarchaeological material comparable to (a) (Survey Lot 5). (c) Leaf surface whole mount showing parallel venation with smaller, oblique crossveins (arrow C), and areole structure (arrow A). (d) Archaeological material, comparable to (c) (Survey Lot 24). (e) Epidermal surface ofcleared leaf showing paracytic stomata with trapezoidal or ‘‘winged’’ subsidiary cells (arrow). (f) Comparable stomata (Survey Lot 12).(g) Comparable stomata (Survey Lot 5). Note: epidermal cells become less polyhedral and more elongated in vicinity of veins. Scale bars:(a)–(d)=1 mm; (e)–(g)=50 �m.

Light Microscopy of Eryngium yuccifolium Michaux, Salts Cave, Kentucky 59

is also frequently mentioned in association with slip-pers both from Salts Cave and from other locations(e.g. Kuttruff et al., 1998), and because of Jones’tentative identification of these samples as Eryngium(King, 1974: 38), we focused heavily on this speciesfairly early in this process.

The results show that nine of the 14 analysedsamples can be classified as Eryngium yuccifolium, anidentification based on a combination of features.The strongest evidence comes from the vascularizedmarginal bristle-like teeth present in two of the samples(Figure 1(a), (b)). A major characteristic that definesE. yuccifolium within its genus is the presence of single,widely spaced marginal leaf teeth (Coulter & Rose,1900: 42). A second characteristic is the parallelvenation within the leaf, with smaller, oblique crossveins (Figure 1(c), (d)). The paracytic stomata, withtrapezoidal subsidiary cells, are present in both thearchaeological and reference material (Figure 1(e), (f),(g)), and support the identication as E. yuccifolium.

The nine classifiable samples, representing four slip-pers, have either the characteristic stomata/subsidiarycell pattern and size (Figure 1(f), (g)), leaf teeth, orboth (see Table 1). Together with the general patternsof fibres and intervening soft tissue (Figure 1(c), (d)),these nine samples can confidently be identified asE. yuccifolium.

As a further basis of identificaiton, measurements ofepidermal cells overlying fibre bundles in both archaeo-logical and modern E. yuccifolium samples were madeusing an ocular micrometer at �400. As can be seen inFigure 2, there is a significant degree of similaritybetween the archaeological and modern samples. Themean length and width of the archaeological cells(N=45) are 110·64 �m (..=20·61) and 20·39 �m(..=3·37), respectively. The modern mean lengthand width (N=40) are 115·63 �m (..=27·10) and21·25 �m (..=2·88), respectively.

Of the five other samples, three strongly resemble E.yuccifolium (Lot 137, warp and weft, and Lot 111),based on general characteristics and similarity to theidentified archaeological specimens, but do not haveeither of the strongly diagnostic features. It is possible

that these samples could be positively identified usingSEM, although the use of SEM does not guarantee anidentification (see Kuttruff et al., 1995).

ConclusionThis research demonstrates the feasibility of usinglow-cost, widely available light microscopy techniquesfor archaeological fibre identification. In addition, theidentification of Eryngium in the Salts Cave slippersadds to a growing body of work on the use ofEryngium yuccifolium as a prehistoric fibre source,specifically, its use in the manufacture of footwear.Other slippers or sandals in museum collections inMissouri (Kuttruff et al., 1998), Ohio, and Kentucky(Dellinger, 1936) have been shown to contain E.yuccifolium, while extensive collections from Arkansasand elsewhere in the Southeast remain unanalysed. Theaffordability and widespread availability of lightmicroscopes makes them a valuable alternative to SEMidentifications for future research in this area.

AcknowledgementsA. Gordon is grateful to Patty Jo Watson and Gayle J.Fritz for support throughout this project. MarieStandifer and Bill Davitt kindly provided referencematerial, and the Missouri Botanic Garden allowed theuse of their Plant Anatomy Laboratory. The authorsalso wish to thank the anonymous reviewers for theirdetailed and helpful comments.

15210

30

Cell dimensions (µm)

Wid

th (

µm)

50

25

20

1901701501301109070

Figure 2. Dimensions of epidermal cells (�m) overlying fibre bundlesin E. yuccifolium (�, N=40) and archaeological slipper fibres (�,N=45). Measured at �400.

ReferencesAndrews, R. L. & Adovasio, J. M. (1996). The origins of fiber

perishables production east of the Rockies. In (J. B. Petersen, Ed.)A Most Indispensable Art: Native Fiber Industries from EasternNorth America. Knoxville: University of Tennessee, pp. 30–49.

Catling, D. M. & Grayson, J. E. (1982). Identification of VegetableFibres. London: Chapman and Hall.

Coulter, J. M. & Rose, J. N. (1900). Monograph of the NorthAmerican Umbelliferae. Systematic and geographic botany, andaboriginal uses of plants. Contributions from the U.S. NationalHerbarium 8(1).

Dellinger, S. C. (1936). Baby cradles of Ozark Bluff dwellers.American Antiquity 1, 197–214.

Florian, M. E. (1990). Identification of plant and animal materials inartifacts. In (M. E. Florian, D. P. Kronkright & R. E. Norton,Eds) The Conservation of Artifacts Made from Plant Materials.Los Angeles: The Paul Getty Trust, pp. 1–27.

Henning, A. E. (1966). Fabrics and related materials fromArnold-Research Cave. The Missouri Archaeologist 28, 41–107.

Herr, J. M. Jr (1992). New uses for calcium chloride solution as amounting medium. Biotechnology Histochemistry 67, 9–13.

Jakes, K. A., Sibley, L. R. & Yerkes, R. (1994). A comparativecollection for the study of fibres used in prehistoric textiles fromeastern North America. Journal of Archaeological Science 21,641–650.

Jones, V. (1936). The vegetal remains of Newt Kash Hollow shelter.In (W. S. Webb & W. D. Funkhouser, Eds) Rockshelters inMenifee County, Kentucky. University of Kentucky Reports inArchaeology and Anthropology, pp. 147–167.

60 A. Gordon and R. C. Keating

Keating, R. C. (1996a). Anther investigations: a review of methods.In (W. G. D’Arcy & R. C. Keating, Eds) The Anther: Form andPhylogeny. Cambridge and New York: Cambridge UniversityPress, pp. 255–271.

Keating, R. C. (1996b). Techniques and Resources for ComparativePlant Anatomy. Privately published, 103 pp.

Kennedy, M. C. (1996). Radiocarbon dates from Salts andMammoth Caves. In (K. C. Carstens & P. J. Watson, Eds) OfCaves and Shell Mounds. Alabama: University of Alabama Press,pp. 48–81.

King, M. E. (1974). The Salts Cave textiles: a preliminary account.In (P. J. Watson, Ed.) Archaeology of the Mammoth Cave Area.New York: Academic Press, pp. 31–40.

Korber-Grohne, U. (1978). Microscopic methods for identificaitonof plant fibres and animal hairs from the Prince’s Tomb ofHockdorf, southwest Germany. Journal of Archaeological Science15, 73–82.

Kuttruff, J. T., DeHart, S. G. & O’Brien, M. J. (1998). 7500 yearsof prehistoric footwear from Arnold Research Cave, Missouri.Science 281(5373), 72–75.

Kuttruff, J. T., Standifer, M. S., Kuttruff, C. & Tucker, S. C. (1995).Investigations of early cordage from Bayou Jasmine, Louisiana.Southeastern Archaeology 14, 69–83.

Miller, J. (1988). Experimental replication of Early Woodlandvegetal fiber slippers. Southeastern Archaeology 7(2), 132–137.

Orchard, W. C. (1920). Sandals and other fabrics from Kentuckycaves. Miscellaneous No. 4, Indian Notes and Monographs.New York: Museum of the American Indian, Heye Foundation.

Scholtz, S. C. (1975). Prehistoric Plies: A Structural and Compara-tive Analysis of Cordage, Netting, Basketry, and Fabric fromOzark Bluff Shelters. Fayetteville: Arkansas Survey ResearchSeries.

Valdes-Reyna, J. & Hatch, S. L. (1995). Anatomical studyof Erioneuron and Dasychloa (Poaceae: Chloridoideae:Eragrostideae) in North America. Sida 16, 413–426.

Watson, P. J. (Ed.) (1969). The Prehistory of Salts Cave, Kentucky.Springfield: Illinois State Museum.

Whitford, A. C. (1941). Textile fibers used in eastern aboriginalNorth America. Anthropological Papers of the American Museumof Natural History 38(1).

Whitford, A. C. (1943). Fiber plants of the North Americanaborigines: the story of what the prehistoric Indian employed fortextiles, ropes, fish nets and lines, bags, burden straps, and sandals.Journal of the New York Botanical Garden 44(518), 25–34.

Young, B. H. (1910). The Prehistoric Men of Kentucky. Louisville:John P. Morton.