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Tobacco pollen: archaeological and forensicapplicationsVaughn M. Bryant a , Sarah M. Kampbell b & Jerome Lynn Hall ca Palynology Laboratory, Department of Anthropology, Texas A&M University, CollegeStation, Texas, 77843-4352, USAb Princeton University, 129 Dickinson Hall, Princeton, New Jersey, 08544, USAc University of San Diego, 5998 Alcalá Park, San Diego, California, 92110-2492, USA

Available online: 17 Nov 2011

To cite this article: Vaughn M. Bryant, Sarah M. Kampbell & Jerome Lynn Hall (2011): Tobacco pollen: archaeological andforensic applications, Palynology, DOI:10.1080/01916122.2011.638099

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Tobacco pollen: archaeological and forensic applications

Vaughn M. Bryanta*, Sarah M. Kampbellb and Jerome Lynn Hallc

aPalynology Laboratory, Department of Anthropology, Texas A&M University, College Station, Texas 77843-4352, USA;bPrinceton University, 129 Dickinson Hall, Princeton, New Jersey 08544, USA; cUniversity of San Diego, 5998 Alcala Park,

San Diego, California 92110-2492, USA

The fossil pollen contents found in the charred dottle residue from 20 clay pipe bowls recovered from a cesspit at ahouse in Amsterdam, the Netherlands (which was owned and used by the artist Rembrandt Harmenszoon van Rijn),were collected and studied. The clay pipes date from approximately the same period when Rembrandt lived in thehouse and used the cesspit. We believe that some of the pollen in the dottle residue may have come from the tobacco,some possibly from airborne sources at Rembrandt’s house and/or from debris in the cesspit in which the pipes werediscarded. The results are compared with a similar study of pipe dottle from Native American clay pipes conductedmore than 25 years ago. We also briefly review the origin and spread of domestic tobacco in an effort to determinepotential sources of the tobacco imported and used in the Netherlands during the lifetime of Rembrandt and theperiod when he occupied the house. The pollen content of modern brands of commercial pipe tobacco is studied andthe potential for using pollen found in tobacco products as evidence in forensic circumstances is reviewed.

Keywords: pollen; pipe dottle; tobacco; Rembrandt; forensics

1. Introduction

1.1. Origin

Tobacco is one of the most widely cultivated plantstoday. As in the past, various domesticated species andwild forms of tobacco grow in regions as far north asSouthern Canada and as far south as Patagonia at thesouthern tip of South America (Goodspeed 1954).When Columbus landed on the island of Hispaniola in1492, he became the first known European to beintroduced to tobacco. In 1514, priest Bartolome deLas Casas ‘‘reconstructed’’ lost notes about Columbus’travels. He noted that, on November 6th, Columbusmentioned that two members of his group (Luis deTorres and Rodrigo de Jerez) had seen and alsosmoked certain ‘‘herbs’’ that natives had put into a dryleaf, rolling it up and then lighting it. Columbus alsoreported that the natives chewed or sucked the otherend while taking in the smoke, which he said ‘‘dullstheir flesh’’. He concluded the discussion in his journalentry by saying it is an intoxicating agent that preventsthem from feeling weary. The Spaniards called it‘‘tobaco’’, which was the word the natives used for thepipe in which the tobacco was smoked (Brooks 1952).

Tobacco is a member of the genus Nicotiana in theSolanaceae, known as the nightshade plant family. Thegenus was named in honor of Jean Nicot, the Frenchambassador to the Portuguese court who introduced

tobacco and smoking to the French court during theearly 1600s (Gilman and Xun 2004). The primarygenus in this family is Solanum, which has more thanhalf of all Solanaceae species; Nicotiana and a fewother genera make up the bulk of the other species. Ofthe more than 95 species of Nicotiana most areindigenous to South America, 15 species are native toAustralia and/or areas of the South Pacific, oneoriginated in Africa and the other few species are allnative to Mexico, Central America or the AmericanSouthwest (Goodspeed 1954). Of the more than 95species, humans have selected only a few (mainlyNicotiana rustica and N. tabacum) for cultivation andwide use.

When Columbus returned to Spain he broughtexamples of many New World plants includingtobacco. At that time in Europe, however, most plantsin the Solanaceae were considered poisonous or wereassociated with superstition (Heizer 1969) such as themandrake (Mandragora officinarum). Most of thenewly introduced plants in the nightshade family,such as tobacco, potatoes and tomatoes, were thereforenot considered useful initially. This changed duringColumbus’ second voyage to the New World when oneof the passengers, Fray Ramon Pane, saw Indians onthe island of Hispaniola grinding tobacco leaves andthen inhaling them in their noses as snuff. He alsonoted that they chewed the leaves mixed with lime

*Corresponding author. Email: vbryant@neo.tamu.edu

Palynology

2012, 1–16, iFirst article

ISSN 0191-6122 print/ISSN 1558-9188 online

� 2012 AASP – The Palynological Society

http://dx.doi.org/10.1080/01916122.2011.638099

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powder made from ground mussel shells (Loewe 1988).In 1518, Fray Pane returned to Spain with tobaccoseeds and the following year Hernan Cortes reportedthe widespread use and importance of tobacco tradeamong the natives in the region of Yucatan, Mexico.Tobacco was introduced in Germany as early as 1520and, by 1535, the Spanish were growing tobacco atplantations throughout the Caribbean and also in thePhilippines (Winter 2000).

In the mid-1500s, tobacco seeds were imported andplants were being grown in Portuguese East Africa andin India. Reports also note that by 1554 tobacco wasavailable and used in Holland (TABAMEX 1989). Inthe late 1500s, the English began manufacturing andexporting inexpensive white ball clay pipes for smokingtobacco. By the mid-1600s, tobacco had become aproduct that was traded, grown and consumedglobally. The original English colonies in NorthAmerica were established in hopes of reaping profitsfrom the sale of tobacco. In 1612, English farmers inVirginia shipped 9000 kg of tobacco grown from theseeds of a native tobacco species (N. rustica), whichthey obtained from local Native Americans. TheDutch began growing tobacco (N. rustica) in Utrechtby 1615 and, in 1631, the Dutch were raising tobaccoin New Amsterdam and shipping it back to Holland(Loewe 1988).

The Rustica variety of tobacco is believed to haveoriginated on the eastern slopes of the AndesMountains in South America, where it may havebeen used by natives, and was eventually cultivatedlong before the other primary domesticated type calledN. tabacum var. orinoco. Although the Rustica varietywas the first type grown and shipped back to Englandby the original English colonialists in Virginia, therewere complaints that it was too weak and had a‘‘byting taste’’. For these reasons, John Rolfe obtainedseeds of a new type of South American tobacco (N.tabacum var. orinoco) from the Spanish in Trinidad,which had a pleasing taste and was stronger than theRustica variety (McCusker 1988). He began growingthe new tobacco species in Jamestown in 1612 andshipped his first harvest of the ‘‘new’’ tobacco toEngland in 1613. It was an immediate success and soonbecame the favored type and was known as ‘‘Virginiatobacco’’. Today tobacco is grown commercially allover the world and most of it is some variety of theoriginal tobacco species, N. tabacum. This singlespecies is favored for a variety of reasons, includingit being the most robust species of tobacco with thelargest leaves and producing a favorable tobacco forchewing or smoking. By the late 1990s, the USTobacco Germplasm Catalog listed more than 2358known hybrid forms and varieties of N. tabacum thatare grown throughout the world. Of those many

varieties, 617 of them originated in the USA andare most widely grown in the USA today (Winter2000).

1.2. Tobacco and pollen

A recent report by the Food and Agriculture Organi-zation of the United Nations (UN report 2010) notedthat by the end of 2010 the projected annual worldproduction of commercial tobacco will exceed 7.1million tons. The largest tobacco growers todayinclude China (which grows 40%), India (9%), Brazil(8%), USA (7.5%), Turkey (4%) and all areas inAfrica combined (3.5%). The remaining tobacco isgrown in minor amounts in 95 other countries. Becausetobacco is grown, cured and converted into productsfor consumption in over 100 different countries, thepotential pollen composition of various tobaccoproducts might differ widely depending on theircountry of origin (van Liemt 2002). We also knowthat, based on tobacco leaf morphology, potentialpollen recovery is significant since tobacco leavesexcrete sticky exudates from special glands and theleaves are covered with tiny trichomes that shouldeffectively trap pollen grains (Goodspeed 1954).

An initial study of tobacco products by Donaldsonand Stephens (2010) has suggested that tobaccoproduced in different geographical regions shouldreveal distinguishable ‘‘pollen prints’’ useful for linkingthose products to specific locations. More importantly,they suggest that tobacco products that might beassociated with, or recovered from, a crime scene couldbe examined for their pollen contents with the resultinginformation providing critical clues linking suspects,victims or objects to a primary or secondary crimescene.

To test their ideas, Donaldson and Stephens (2010)examined pollen recovered from two tobacco sources.One sample was called 1R5F and consisted of a blendof USA tobacco leaves which is used as a referencestandard for cigarette emission testing. It was obtainedfrom research facilities at the University of Kentucky.The second sample was from a Chinese brand ofcigarette called ‘‘Hongtashan1’’, which is manufac-tured in the Yunnan province from what is assumed tobe locally produced tobacco. Donaldson and Stephens(2010) reported that they found an average of 24,341pollen grains per gram of the 1R5F sample and anaverage of only 2841 pollen grains per gram in theHongtashan tobacco. They also noted that pollencould be recovered from individual cigarettes; theirresults confirm that differences in the distribution andpercentages of key pollen types exist as well as thepresence of different pollen taxa. They then suggestedthat these data should be sufficiently encouraging to

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warrant the use of this type of analysis as a forensictool.

In another study which began during the 1970s,Patricia Hall investigated the potential of searching forfossil pollen in pipe dottle (charred material) that couldbe examined from the inside of smoking pipesrecovered from North American archaeological sites,or from smoking pipes collected during the 1700s and1800s that are on display in museums. Some of herpollen data remain unpublished, but other parts of herstudy were published (Hall 1984).

Before beginning her study, Patricia Hall wanted toknow if tobacco leaves retain pollen from their ownflowers as well as pollen dispersed by other nearbyplants. She selected the fresh leaves from four nativespecies of mature tobacco plants (N. acuminata var.multifloral, N. trigonophylla, N. glauca and N. bigelovii)during the mid-to-late growing season in variousnatural habitats of California. Each tobacco specieswas collected in a different ecological zone in fourdifferent counties of California. Selected leaves fromeach natural tobacco plant were cut into small portionsuntil 0.2 g were obtained from each sample. Leaf areafor each 0.2 g sample was extrapolated to establish apollen concentration value per cm2 of tobacco leaf.

After adding one tablet of Lycopodium spores(12,500 spores) to each sample and processing thesamples using potassium hydroxide solution (KOH),acetolysis and hydrofluoric acid (HF), Hall (1984)determined that the leaves of all four species of nativetobacco plants provided excellent pollen traps not onlyfor Nicotiana, but also for the pollen dispersed by 13other known taxa as well as those from a number ofunidentified plant sources. She calculated that herrecovery rate for tobacco leaves ranged from 4,207pollen grains per gram in N. glauca to 114,999 pollengrains per gram of leaf in N. acuminata. Recoveredpollen taxa on the leaves included mostly wind-pollinated types, but at least one insect-pollinatedgenus from the rose family was found.

Hall (1984) also searched for pollen in threebrands of commercial pipe tobacco. Volunteerssmoked these in three wooden pipes over severalweeks to develop sufficient dottle that could beremoved and examined. She then processed 0.4 g ofdottle from each of the three pipes and the sameamount of fresh commercial tobacco from each ofthe three brands to compare pollen recovery results.After adding one tablet of Lycopodium (12,500spores), she used the same processing techniqueminus the HF. Analysis revealed about 86,662 pollengrains per gram in the unsmoked EdgeworthTobacco1, about 59,756 pollen grains per gram inFive Brothers Tobacco1 and 78,977 pollen grainsper gram in Revelation Tobacco1.

Following this experiment, Hall (1984) removed,processed and examined pollen grains trapped incharred pipe dottle from five Native American smok-ing pipes. She noted that searching for pollen in theprocessed pipe dottle was a daunting task, because evenafter processing there were charred clumps of debris(degraded and distorted pollen) that could not beidentified. There were also so many Lycopodium sporesthat she often had to count for an hour or more beforefinding a single pollen grain. The study revealed between39 and 93 trapped pollen grains in each of the dottlesamples. From this she extrapolated that the potentialnumber of pollen grains in Native American pipecharred dottle would probably range from about 1,552to 4,474 pollen grains per gram of dottle. Some of theinsect-pollinated pollen types in the charred pipematerial included Nicotiana in three samples andAcacia pollen in one. The other pollen types foundin the five samples were from fairly ubiquitous wind-pollinated types such as grass, pine, composites, alderand Cheno-Ams.

Hall’s (1984) study was unique for two reasons;first, it appears to be the first attempt to recover andexamine pollen trapped in pipe dottle and second, itwas also the first known attempt to document pollenfrom Native American smoking pipes recovered fromarchaeological sites.

1.3. Rembrandt’s smoking pipes

Rembrandt Harmenszoon van Rijn was born in 1606in Leiden, the Netherlands. He grew up in a middle-class environment and, after successfully completinghis early schooling, enrolled at the University ofLeiden. Although he never graduated (as he wasapparently more interested in learning to paint thanin other studies), Rembrandt apprenticed underPieter Lastman in Amsterdam. He eventually re-turned to Leiden where, at the age of 21, he openeda studio. Within a few years, Rembrandt’s reputationwas such that he received several portrait commis-sions and moved back to Amsterdam where he livedin the house of Hendrick van Uylenburgh, a notedart dealer who recognized Rembrandt’s talent andexpressed interest in selling his paintings. In 1634,Rembrandt married van Uylenburgh’s niece, Saskiavan Uylenburgh, and a few years later they bought ahouse on Jodenbreestraat in 1639 (Figure 1). Here,Rembrandt enjoyed the good life until forced todeclare bankruptcy, an unfortunate move due inlarge part to his lavish spending habits andaccumulated debt. This forced him to sell his homeand first move into rooms at an inn and eventuallyto a smaller house on the Rozengracht, where hespent the rest of his life. Rembrandt died at the age

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of 63, leaving a rich legacy of more than 600paintings as well as many etchings and drawings.

Although numerous scholars have contributed tothe wealth of literature on Rembrandt, adding datesand points of interest to his biography, few haveexplored beyond the mundane details of his family life,births, and deaths, changing artistic approaches or

problems of personal fortunes. This study chronicles asmall aspect of his personal life by examining thepollen contents of a number of smoking pipesrecovered during archaeological excavations asso-ciated with his original home in Amsterdam.

Rembrandt lived in the house on Jodenbreestraat,now known as the Museum Het Rembrandthuis, during

Figure 1. A view of Rembrandt’s house in Amsterdam today (photograph courtesy of J. Howe).

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the period 1639–1658 (Tissink 2003). While living here,he completed many of his most famous paintings andpersonal commissions. Unfortunately, this house alsowitnessed some of his personal and financial tragediesincluding the deaths of his wife and three of hischildren and, eventually, his declaration of bankruptcy(Tissink 2003). In June 2004, one of us (JH) visited HetRembrandthuis and noticed a small display of claysmoking pipes and a Westerwald jug excavated fromthe cesspit in 1997 under the direction of thearchaeological department of Dienst Amsterdam Beh-eer. These pipes had not been studied and JH returnedin January 2005 to study both pipes stored at HetRembrandthuis and a larger collection of pipes housedat the Noorderkerk (Northern Church) Museum.

The cesspit or latrine from which the pipes wererecovered is located in the courtyard immediatelyoutside the kitchen (Figure 2) and is actually theoverflow of a sewer from the house, which would have

been ‘‘a novelty in wealthier 17th century houses’’(Baart 1997). Archaeological excavation of this featureyielded 10 smoking pipes, ceramic wares and a smallassortment of wooden and metal artifacts. Findingpipes and other debris in the remains of a cesspitshould not be considered unusual, since pit toilets werereceptacles for broken and discarded household itemsas well as for human waste.

Smoking pipes are common elements found inEuropean latrines and garbage middens dating to thepost-Medieval period, and especially after shipmentsof tobacco began arriving in Europe from theAmerican colonies after the early 1600s (Andersenand Moltsen 2007). The Dutch were beginning tobreak the English monopoly in Virginia by the late1630s, gaining a sizable share of the lucrative tobaccomarket in the following years as demonstrated by asingle Dutch company that imported at least 27,000 kgof Virginian tobacco in 1640 (Pagan 1982). By the

Figure 2. The area excavated outside the kitchen area above the cesspit at Rembrandt’s house (photograph courtesy of J. Hall).

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1650s, Dutch and English relations became strained;this made it difficult for the Dutch to gain access to theNew World Virginian tobacco markets, although theywere not entirely excluded (Pagan 1982).

During the seventeenth century, Amsterdam be-came a focal point for the tobacco trade in Europe.Imports came not only from Virginia, but also fromtobacco-growing areas of Spanish America and thePortuguese colony of Brazil, as well as from Germanyand Dutch tobacco producers (Price 1961; Roessingh1978). Tobacco was not only marketed in Amsterdambut it was also processed there, which greatly increasedits availability and value (Goodman 1993). Smokingwas such an integral component of seventeenth centuryDutch culture and society that it captured the creativeattention of many painters, including, inter alia, FransHals (1580–1666), Pieter Claesz (1597–1660), AdriaenBrower (1605–1638), Adriaen van Ostade (1610–1685),David Teniers (the younger) (1610–1690), Willem Kalf(1619–1693), Jan Steen (1626–1679) and Pieter deHooch (1629–1684).

Holland’s major cities (especially Amsterdam) werereplete with pipe manufacturers; in southwestern Hol-land, half the labor force in Gouda was employed in pipeproduction (Schama 1997). This was largely due toEuropean cultural perspectives on recreation and health,both of which incorporated tobacco. Not long after itsintroduction to European society, smoking became thesocial pastime of men, women and children and wasconsidered, albeit erroneously, a prophylactic and cura-tive for just about every known disease including theplague. Most of these pipes are white, made of kaolinclay, molded as a single piece and had stems of varyinglengths. Clay pipes, entire or partial, have long beenregarded as valuable tools for dating archaeological sites.These types of pipes were frequently manufactured,smoked and then thrown away often within a matter ofa year or two or less if they became broken (Noel-Hume2001). These types of pipes were mass produced,inexpensive and extremely fragile, hence were commonin latrines during this period in Europe (Higgins 1997).

2. Material and methods

2.1. Rembrandt pipe samples

When JH first examined the pipes they had little, if any,post-depositional debris trapped in their bowls. Each hadbeen smoked however, evidenced by dottle adhered to thebowl interior and the blackening of the outer bowl lip. Toremove this charred dottle, the interior wall of each pipebowl was carefully scraped with a wooden satay stick.This produced a total of 20 individual samples, none ofwhich weighed more than 0.1 g. The intention of thesampling was first to remove any post-depositional dirtthat entered the pipe bowls after they were discarded into

the latrine. This was done during cleaning shortly afterthe excavation was completed in 1997, thereby exposingthe remains of the charred tobacco dottle and someunburned tobacco that adhered to the inner walls of eachpipe bowl. It was hoped that, by sampling the thin,charred layers next to the inside bowl of each pipe,archaeologists might learn something about the origin ofthe tobacco used or perhaps gather information on localpollen rain during the time the pipes were in use.

We were encouraged to undertake this studybecause pollen grains are extremely durable andmany taxa often remain preserved in sediments forthousands and even millions of years. Additionally,experiments have shown that pollen heated in ceramiccontainers can survive temperatures in the range of170–3008C, but will become oxidized in open flamesand in most oxygen-rich fires (Sengupta 1975; Ghoshet al. 2006). Fossil pollen collected from seventeenthand early eighteenth century houses, privies andgardens (Kelso 1998; Kelso et al. 2006) and fromitems recovered from cargo containers found in ancientshipwreck sites (Jones et al. 1998) have providedimportant insights about past lifestyles, dietary prac-tices and other cultural activities. Furthermore, Hall(1984) demonstrated that pipe dottle contains residualpollen grains.

2.2. Sources of uncertainty

Throughout the examination and analysis phases of thisproject we were aware of inherent problems, the first ofwhich was proper sampling and possible contamination.Because we were not able to learn the precise laboratorytreatment used to clean the bowls of each pipe prior toour examination and sampling in 2005, we do not knowwhat precautions were taken to avoid atmospheric pollencontamination. Additionally, although the pipes werestored in covered containers since their initial recoveryand cleaning, we cannot be certain about the differentlocations or procedures used to store the pipes before wewere granted permission to sample them. Therefore, theymay have been exposed to pollen contamination duringthe post excavation phases. Finally, we had problemsfinding a sterile place to sample the pipes; pipe dottle fromeach bowl was therefore removed at one of two locations(depending on whether they were stored at Het Re-mbrandthuis or at the Bureau Monumenten & Archeologielaboratory at the Noorderkerk or North Church).Because we were not allowed to remove the pipes fromtheir storage locations and because there were noavailable sterile environments (such as a fume hood ora clean bench area), sampling was carried out as carefullyas possible on open tables (Figure 3). The samplingtherefore does not preclude the potential of a limitedamount of pollen contamination from modern sources.

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Second, we cannot be certain that pipes recoveredfrom the latrine are contemporary with Rembrandt’soccupation of the house (1639–1658) and can thereforeonly assume their use by members of or visitors to thehousehold. Nevertheless, some pipe morphologies fitwell into typological series, suggesting manufacture oruse during this period. Although unlikely, pipes with amanufacturing design that fits well into the time ofRembrandt’s residence on Jodenbreestraat may notactually have been used or discarded during thatperiod. Likewise, we may not assume that a pipe whosebowl form or heel mark predates Rembrandt’s knowndates of occupancy was not used or discarded duringhis tenure. Additionally, we cannot be certain that theartist smoked these or any other pipes. Unfortunately,no records acknowledge whether Rembrandt partookof tobacco in any way. What we do know is that hisstudio was used by a number of students, and thatclients frequently visited the house. Traffic throughout

the house and to the courtyard cesspit would thereforehave been continuous. The clay smoking pipes couldhave been discarded by anyone; however, even if notused by Rembrandt, their presence suggests tobaccowas a pastime at Het Rembrandthuis.

Third, a variety of factors may have affected latrinedeposits. Human activities around the house and/orchanges in the natural environment could have shiftedor altered the cesspit’s contents. Because this featurewas connected to a local canal by a narrow drainingpassageway, high tidal actions may have pushed canalwaters back into the latrine. This would not only haveresulted in flushing of its contents, but also possiblyintroduced mold, fungi and pollen. An additional(albeit remote) possibility exists that the pipes them-selves may have been intrusive as a result of beingwashed in with tidal activity.

Fourth, pollen control samples of surface soilsfrom areas near the actual archaeological excavations

Figure 3. Jerome Hall removing dottle from the bowl of a clay pipe recovered from Rembrandt’s cesspit (photograph courtesyof J. Hall).

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are typically used to provide some information on localpollen spectra. Those data serve as guides to determinepotential pollen concentration values and possibledifferential pollen loss due to edaphic and/or climaticconditions. No control surface soil samples werecollected during the Rembrandthuis excavations how-ever, but we believe this lack of information has notgreatly affected our analysis of the dottle materialsscraped from the pipe bowl interiors.

Lastly, during the eighteenth century workers duginto and removed some of the original seventeenthcentury latrine deposits. It is possible that they couldhave exposed extant sediments to airborne pollencontamination during that time. Nevertheless, becausethis study focuses only on the thin layer of charred dottlefrom inside each pipe, it is doubtful that exposure to theaforementioned conditions were sufficient to introducemodern pollen contaminants to our samples.

2.3. Sample processing

After thoroughly drying each pipe, cleaning it anddiscarding any remaining soil and deposits within eachbowl, the thin charred layer of dottle from each pipewas carefully removed and placed in a small sterileZiploc plastic bag which was then labeled and sealed(Figure 4). Each of the 20 pipe content samples wasprocessed in the same manner. First, we removed thecontents of each bag and placed it into a sterile 10 mlbeaker under a fume hood in a contamination-freelaboratory. For each sample, the small amount ofdeposit was removed by tapping the bag lightly toconcentrate all material in one corner and then pouringit into the plastic beaker. Each sample bag was rinsedwith ethanol (ETOH) and the contents added to thedry sample in each beaker. Next, the contents of eachbeaker were concentrated by centrifugation and placedin separate 1.5 ml Eppendorf centrifuge tubes.

The extraction procedure for these samples con-sisted of the following steps.

(1) Material from each sample was placed in a1.5 ml Eppendorf centrifuge (ET) tube that waslabeled in ink and etched with an ID numberusing a diamond marking pen to ensure correctsample identification. None of the originalsample was transferred out of the ET untilextraction was completed and residue fromeach vial was removed and placed on micro-scope slides for analysis. Likewise, O-ring screwtops for each ET were in place at all timesexcept when chemicals were added or decantedmaterials were pipetted off after each step. Thisensured that none of the samples were subjectto intrusive pollen. Likewise, all work tookplace in a sealed Palynology Laboratory andwas conducted under a fume hood using sterileequipment and surgical gloves. Glycerine-coated slides left exposed in various locationswithin the lab were checked weekly for signs ofpollen contamination. None were noted duringor after the project was completed. Thus, to ourknowledge, no pollen contamination occurredduring processing.

(2) After being concentrated into the ET, sampleswere rinsed in glacial acetic acid. After cen-trifuging, the glacial acetic acid was carefullypipetted out.

(3) Next, the samples were treated with acetolysiscomprising 1 part sulfuric acid to 9 partsacetic anhydride. After placing samples in aheating block at 808C for 10 minutes,they were centrifuged and the mixture waspipetted out.

(4) The samples were again rinsed in glacial aceticacid which, after centrifuging, was pipetted out.

Figure 4. Samples received by the TAMU Palynology Laboratory for analysis. The material from each pipe bowl was placed ina sealed Zip-loc plastic bag (photograph courtesy of V. Bryant).

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(5) The samples were rinsed twice in distilledwater.

(6) Samples were rinsed with ETOH which, aftercentrifuging, was pipetted out.

(7) Finally, one drop of diluted Saffranin-O stainin ETOH was added. The sample was thencentrifuged and the ETOH and excess stainwere removed. The residue was then mixed withtwo drops of glycerine and samples were placedin the heating block for a few minutes to enablethe remaining ETOH to evaporate. When allprocessing was completed, one or more slidesfrom each sample were prepared for laterexamination.

Due to the quantity of original material in eachsample (50.1 g/sample in most cases) and thesuspected minimal number of fossil pollen and sporespresent, Lycopodium sp. tracer spores were not added.Likewise, neither hydrofluoric acid nor heavy liquidseparation techniques were used, again due to smallsample sizes. However, initial processing of samplesthrough a NITEXWoven Nylon screen (openings witha diagonal length of 100 mm) was undertaken. Becausesome pollen grains are larger than 100 mm, charcoalfragments and other debris trapped on the screensurface were monitored to ensure that no pollen wasdetected. None was found.

2.4. Modern tobacco samples

Modern pipe tobaccos were purchased, processed andexamined to record typical pollen spectra from bothforeign and domestic products. Unfortunately, wediscovered that all pipe tobacco products consist ofvarying ‘‘blends’’ with all the geographical origins ofthe blend either unstated or unknown. These includedinter alia tobacco types listed by common names suchas English, Virginia, Golden Virginia, Light Virginia,Sweet Virginia, Brightleaf Virginia, Burley, Cavendish,Black Cavendish, Brown Cavendish, Perique, NaturalTuscan, Turkish, Oriental, Latakia, Macedonia Bright,Yenidje, Cyprian, Syrian Latakia, Cuban Corojo,Dominican Republic Criollo and Matured Red Virgi-nia. Although we had hoped to sample tobaccos fromknown geographical regions, no brands of certainorigin were found. This search for unique regionalproducts was predicated on the study by Donaldsonand Stephens (2010), where these authors suggest thattobacco products contain unique pollen spectra thatcould be used as markers for both the brand and thegeographical region of manufacture.

We used 2 g of each modern pipe tobacco blend asour sample, which was removed from the productpackage under a fume hood using sterile equipment.

To each sample we added two Lycopodium sporetablets (batch # 124961; 12,542 spores/tablet). Proces-sing was identical to that used for the dottle from theRembrandt pipes with two exceptions. First, eachsample was placed in a 5% KOH mixture and thenheated at 808C for 10 minutes followed by a rinse inconcentrated HCl and two water washes. Next, eachsample was passed through a 150 mm stainless steelscreen to remove large fragments of plant material.These steps were then followed by the regularprocessing steps mentioned above. Residues werethen stained and mounted in glycerine for analysis.

3. Results

3.1. Rembrandt pipe samples

Initial examination revealed that silicates remained inall processed samples, due to the fact that hydrofluoricacid was not used. Although these amounts differedamong samples, they did not hinder the search forpollen. In retrospect there may have been other stepsthat could have been taken to remove sample debris:sonication to separate clumps; the introduction of amild solution of KOH oxidation; and the use of HF.Heavy liquid separation was used on a few samples,but offered little help in concentrating the pollen andremoving debris; it was therefore abandoned. Theheavy liquid separation technique tended to yieldcharcoal flecks and burned particles along with pollen.In the years since this project began we have gainedextensive experience with many types of forensicsamples, most of which consist of extremely smallamounts of material from a wide variety of matrices.Many of those forensic samples are even smaller thanthe amounts available for this study (Bryant and Jones2006). In future studies of pipe dottle material, wewould therefore use a different set of procedures whichwe believe might yield a better removal of debris andlead to better analysis results.

One unanticipated challenge to which resolutionwas problematic was residue clumping. In some of theprepared samples, tiny charcoal fragments, smallpieces of remaining organic debris, silicate particlesand pollen were all clumped together. It is possible thatthis may have been caused by ionic valances resultingfrom atoms which, during various processing stages,either lost or gained electrons in their outermost ringand thus acquired a positive or negative charge inrelation to other nearby atoms.

In the only other similar study of pipe dottle, Hall(1984) noted that numerous charred clumps of debrisand distorted pollen grains prevented identification ofprobable sample pollen. Hall also noted that it was amistake to add Lycopodium spores in hopes of usingthem to calculate pollen concentration values.

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Unfortunately, she noted that there were so manyLycopodium spores added and so few original pollengrains that she often had to scan through debris andthen count hundreds of Lycopodium spores beforefinding even a single identifiable pollen grain. Based onher report, we decided not to add Lycopodium sporesto our pipe dottle samples, but we did add them to themodern pipe tobacco samples we processed.

3.2. Modern tobacco samples

Finding and counting the pollen, even in modern pipetobacco, was not an easy task. Many of the pollengrains were deformed and broken. In addition, ourattempts to remove the fragments of tobacco leavesfrom each sample met with limited success. Even afterusing 5% KOH and acetolysis on each sample, theamount of remaining organic material was significant.We decided not to try other oxidizing procedures forfear of losing some or all of the pollen. To count thepollen in each sample, we therefore had to thin out theresulting residues and prepare a number of slides fromeach sample to enable adequate separation of the pollenand tracer Lycopodium spores from the remainingdebris.

3.3. Pollen counts

Sample analyses were conducted using a NikonOptiphot binocular microscope at magnifications overthe range 400–1000. Normally, a pollen grain count of200–300 (when possible) per sample generally providesan adequate reflection of pollen contents (Jones andBryant 1998). Larger pollen counts offer slight increasesin accuracy and often reveal additional pollen taxapresent in very low frequencies (Traverse 2007). Insamples containing very small amounts of initialsample, such as these dottle samples, the contents ofamphorae recovered from shipwrecks and manyforensic samples reaching pollen counts of 200–300pollen grains per sample is often impossible (Jones et al.1998; Bryant 2009). Hall (1984) noted between 39 and93 grains in the examination of five pipe dottle samples.In our study of the Rembrandt pipe samples, pollencounts ranged from a low of 0 to a high of 151 in sample65. Frequently, in small samples such as the pipe dottleand many types of forensic samples, the total numbersof pollen grains are minimal (Bryant 2009). In thosesituations, interpretations can be discussed in generalterms but they are often insufficient to provide anaccurate image of the local environmental setting.Nevertheless, the presence of only a few pollen grainsfrom some rare pollen taxon or from some insect-pollinated type might be of significant importance inunderstanding the complexity of a sample.

All pollen types were identified to family or genuslevel whenever possible, although some remain uni-dentified. The pollen spectrum of each sample is shownin Table 1. Pollen counts from modern commercialpipe tobacco samples are shown in Table 2. Compar-isons between samples and pollen reference materialsin the Texas A&M Palynology Laboratory providedthe basis for identifications. Composite pollen wasdivided into five categories: Artemisia; high spine(composite pollen with spines longer than 2.5 mm);low spine (composite pollen with spines shorter than2.5 mm); Centaurea; and fenestrate types such asTaraxacum. Periporate pollen belonging to taxa inthe Chenopodiaceae and genus Amaranthus werecombined into the category called Cheno-Ams (Martin1963) because precise identification to the generic levelis generally not possible at the light microscope (LM)level without an extensive reference collection of localtaxa (McAndrews and Swanson 1967). When pollengrains could not be identified due to degradationfactors such as folding, breaking or loss of exteriorornamentation features, they were listed as ‘‘indeter-minate(s)’’. A variety of fungal spore taxa weredetected in most samples.

4. Discussion

4.1. Rembrandt smoking pipe samples

Most of the pollen taxa found in the modern pipesamples do not aid in determining a specific origin forthe tobacco used; neither do various pollen spectrafrom the pipe dottle samples provide specific cluesabout the origin or cultural aspects associated with theuse of the pipes by Rembrandt or others. It issuspected that some of the pollen recovered from thepipe dottle represents contaminants which eitherentered the tobacco during its use or were derivedfrom intrusive materials in the latrine after the pipeswere discarded. As previously mentioned, care wastaken to clean the pipe bowls prior to sampling thecharred dottle.

Pollen spectra from the Rembrandt house pipesamples reveal a wide variety of types; however, onlytwo pollen types are considered unusual for the regionof Amsterdam. One is the single Myrtaceae pollengrain found in sample 65, which may have beenassociated with the original tobacco that was smokedor might have come from another source that wasdumped into the cesspit and then became trapped inone of the discarded pipe bowls. The other unusualpollen type found in pipe dottle sample 65 is one pollengrain from the hop bush plant (Dodonaea). NeitherDodonaea nor species of Myrtaceae are known to growin Amsterdam today. Precautions taken during proces-sing also suggest neither pollen type originated as

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contaminants. We also note that no Myrtaceae or hopbush pollen was found in any of the three modern pipetobacco samples we examined (Table 2); however,Donaldson and Stephens (2010) found both types inthe modern tobacco samples they examined.

Although most of the pollen we found in the dottlesamples occurs in only one or two of the samples, somecommon taxa, such as those from grasses, composites,pine, and Cheno-Ams occur in a wider number of the20 individual pipe samples. Major types are similar tothose found by Donaldson and Stephens (2010) in theirstudy of tobacco pollen and therefore possibly reflectweedy plants growing in agricultural fields wheretobacco was grown and harvested. Similar to their

study, there was an absence of Nicotiana pollen (or anyother similar pollen type in the Solanaceae family) inthe Rembrandt house samples (Gish 2000; Hollowayand Dean 2000). This is not considered unusual sincetobacco plants are usually ‘‘topped’’ in the agriculturalfields, meaning that the flowering inflorescence isremoved prior to blooming (a practice stimulatingleaf growth). On the other hand, Native Americantobacco grown without removing the flowers shouldcontain large quantities of Nicotiana pollen. That wasrevealed by Hall’s study (1984) where she found thatthe leaves of wild species of tobacco, used by NativeAmericans, produced an average number of Nicotianapollen grains ranging from a low of 417 pollen grains

Table 1. Pollen counts from the Rembrandt house pipe bowl samples.

Pipe samples 44 46 47 48 49 50 51 52 53 56 57 58 59 60 61 62 63 64 65 66

Pollen taxaAesculus (horse chestnut) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Alnus (alder) 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 5 0APIACEAE (umbel family) 0 1 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 7 0ASTERACEAE (dandelion type) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0ASTERACEAE (ragweed type) 1 1 0 0 1 5 1 0 1 0 0 1 0 0 0 4 3 1 8 3ASTERACEAE (sunflower type) 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 3Betula (birch) 1 0 2 0 0 2 4 0 2 0 0 0 0 1 0 5 0 0 2 0BRASSICACEAE (mustards) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 4 0Castanea (chestnut) 0 0 1 0 0 3 1 0 0 0 0 0 0 0 0 1 0 0 0 0Celtis (hackberry) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0Centaurea (knapweed) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Cheno-Am 0 0 0 0 0 5 2 0 0 0 0 0 0 0 0 2 0 0 8 4Corylus (hazelnut or filbert) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0CUPRESSACEAE (cedar) 1 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0CYPERACEAE (sedge) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0ERICACEAE (ericads) 0 0 0 0 0 5 5 0 0 0 0 0 0 1 0 7 0 0 2 3Dodonaea (hop bush) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Juglans (walnut) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 2 0LAMIACEAE (mint family) 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Ligustrum (privet) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0LILIACEAE (lily) 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0Liquidambar (sweetgum) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0Melilotus (clover) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0MYRTACEAE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Pinus (pine) 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Plantago (plantain) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0POACEAE (grass) 3 2 0 0 0 5 2 0 0 0 0 0 0 0 0 8 1 0 7 1Polygonum (knotweed) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Populus (aspen) 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0Quercus (oak) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0Tilia (linden) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0Triticum (wheat) 0 0 0 0 0 12 6 0 0 0 0 0 0 0 0 3 0 0 12 1Typha latifolia (cattail) 0 0 0 0 1 1 1 0 0 0 0 0 0 1 1 1 0 0 0 0Ulmus (elm) 0 0 1 0 0 2 1 0 0 0 0 0 0 0 0 3 0 0 0 0Viola (violet) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0Indeterminate 3 3 4 1 1 5 4 2 0 0 0 2 1 6 5 12 1 2 10 0Unknown A 0 0 0 0 0 10 0 0 0 0 0 0 0 5 2 9 0 0 66 0Unknown 2 1 2 0 0 1 2 0 0 0 0 0 0 2 0 0 0 0 4 0

Total pollen 12 11 10 1 5 66 32 2 3 0 0 4 1 16 8 57 5 3 151 15Total fern spores 0 0 0 0 0 1 6 0 0 0 0 0 0 0 0 2 0 1 7 1Total fungal spores 27 17 23 3 18 382 146 2 1 1 1 27 1 33 13 57 3 52 137 39

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per gram of leaf to a high of 4755 pollen grains pergram of leaf.

Pollen types recovered from the Rembrandt housesamples may reflect local environments, or may simplybe part of the pollen spectrum in tobacco that wassmoked. Key insect-pollinated types in these samplesinclude several types of monocolpate, reticulate grainssimilar to those found in the lily family (Liliaceae). It ispossible that the pollen came from either the tobaccoor might be a contaminant from local flowers (theNetherlands is famous for its tulips and other flowersin the lily family). Another insect-pollinated typefound in five of the pipe samples (50, 51, 60, 62 and65) is from some genus in the ericad family (Ericaceae).

There are a number of different genera of berries,shrubs and trees belonging to the family Ericaceae,many of which grow in northern Europe and theNetherlands. Those pollen grains may have originatedfrom the tobacco or from plants growing in or aroundthe latrine where the pipes were used and discarded.Although the source is uncertain, all ericad pollen ismorphologically similar in size and ornamentation;this suggests that those pollen grains did not comefrom airborne contamination since the Ericaceaeproduce limited amounts of insect-dispersed pollen.Other pollen types that may reflect shrubs andornamentals growing near the Rembrandt houselatrine or pollen from the tobacco products include

Table 2. Pollen counts from modern commercial pipe samples. TT is a brand called Turkish Tobacco, purportedly grown andproduced in Turkey. VR is Virginia Ribbon made from a blend of Virginia tobaccos and burley. JS is Jefferson Street comprisinga blend of burley, Turkish and Cavendish tobaccos. The true geographical origin of these tobaccos is unknown.

Modern pipe tobaccoPollen taxa TT % VR % JSAcer (maple) 1 0.4% 0 0.0% 0 0.0%Alnus (alder) 1 0.4% 0 0.0% 0 0.0%APIACEAE (umbel family) 2 0.8% 0 0.0% 0 0.0%Artemisia (wormwood) 18 7.5% 0 0.0% 0 0.0%ASTERACEAE (dandelion type) 1 0.4% 0 0.0% 2 1.0%ASTERACEAE (ragweed type) 6 2.5% 95 39.4% 29 14.1%ASTERACEAE (sunflower type) 6 2.5% 10 4.1% 11 5.4%Betula (birch) 0 0.0% 0 0.0% 1 0.5%BRASSICACEAE (mustard family) 6 2.5% 1 0.4% 0 0.0%Castanea (chestnut) 0 0.0% 0 0.0% 1 0.5%Casuarina (she oak) 0 0.0% 8 3.3% 0 0.0%Cedrus (cedar) 1 0.4% 0 0.0% 0 0.0%Celtis (hackberry) 0 0.0% 1 0.4% 0 0.0%Centaurea (thistle) 2 0.8% 0 0.0% 6 2.9%Cheno-Ams (goosefoot) 29 12.1% 33 13.7% 21 10.2%Cirsium (thistle) 1 0.4% 0 0.0% 0 0.0%Citrus (orange, lemon, etc.) 0 0.0% 3 1.2% 0 0.0%CYPERACEAE (sedge) 2 0.8% 1 0.4% 0 0.0%ERICACEAE (ericads) 1 0.4% 0 0.0% 1 0.5%FABACEAE (legumes) 1 0.4% 0 0.0% 0 0.0%Ligustrum (privet) 1 0.4% 0 0.0% 0 0.0%Magnolia (magnolia) 0 0.0% 2 0.8% 0 0.0%Olea (olive) 2 0.8% 0 0.0% 1 0.5%Picea (spruce) 1 0.4% 0 0.0% 0 0.0%Pinus (pine) 25 10.4% 2 0.8% 5 2.4%Plantago (plantain) 5 2.1% 0 0.0% 0 0.0%POACEAE (grass) 50 20.8% 47 19.5% 53 25.9%Quercus (oak) 5 2.1% 1 0.4% 7 3.4%ROSACEAE (rose family 0 0.0% 8 3.3% 49 23.9%Triticum (wheat) 4 1.7% 0 0.0% 2 1.0%Typha/Sparganium (monad type) 0 0.0% 3 1.2% 0 0.0%Typha latifolia type 1 0.4% 0 0.0% 0 0.0%Ulmus/Zelkova (elm) 0 0.0% 1 0.4% 0 0.0%Viola (violet) 64 26.7% 2 0.8% 12 5.9%Zea mays (maize) 0 0.0% 2 0.8% 3 1.5%Unknown pollen 2 0.8% 13 5.4% 1 0.5%Degraded beyond recognition 2 0.8% 8 3.3% 0 0.0%Totals 240 100.0% 241 100.0% 205 100.0%Lycopodium spores counted 48 187 171Pollen concentration per gram 62,710 16,163 15,035

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privet (Ligustrum) and several other unidentified pollentypes which come from insect-pollinated sources.

Included in the pollen spectra from the pipesamples are traces from tree species. Some are fromwind-pollinated trees such as pines (Pinus), oaks(Quercus), chestnut (Castanea), hackberry (Celtis),elm (Ulmus), walnut (Juglans), aspens (Populus) andbirch (Betula). None of those individual pollen typesoccur in any of the 20 samples in significant amounts.Whether these pollen grains were from smokedtobacco or from airborne sources could not bedetermined. However, most of those same pollen typeshave been recovered from modern commercial pipetobacco samples which we examined and which wereexamined by Hall (1984) or by Donaldson andStephens (2010). Other tree pollen types found in thesepipe samples come from trees that rely on insectpollination such as linden (Tilia), horse chestnut(Aesculus) and sweetgum (Liquidambar). Those pollengrains may have come from the tobacco products orfrom some of the trees that may have grown inRembrandt’s yard close to the latrine. Unfortunately,descriptions of Rembrandt’s property during the timehe lived there do not mention which trees were plantedin his yard.

Cereal pollen is uncommon to commercial tobaccoas evidenced by the study by Donaldson and Stephens(2010) and the samples of commercial pipe tobacco weexamined. It is likely that large cereal pollen grainsfound in the Rembrandt pipe samples are from wheat(Triticum) as they are somewhat larger than those fromwild grasses found in the Netherlands, or couldrepresent pollen from other cultivated cereals innorthern Europe such as barley (Hordeum) and rye(Secale).

Wheat is primarily self-pollinating but producesample pollen that may become airborne. During theharvesting and threshing process, large amounts ofpollen from closed inflorescences are normally dis-lodged and incorporated with the kernels (Greig 1982).Wheat pollen can also be dislodged and deposited onvarious surfaces during transport from the field tostorage areas, or when converted into flour at gristmills. Our own lab experiments and analyses revealthat milled flour and baked breads contain ampleamounts of wheat pollen. It is therefore possible thatwheat pollen in the Rembrandt house pipe samplesmay have come from tobacco products, from pollen inwheat flour used in the Rembrandt household or fromlatrine fill.

In summary, pollen recovered from the 20 Re-mbrandt pipe dottle samples do not provide substan-tive insight into either the geographical origin of thetobacco that was smoked or information about thepotential flora surrounding the house during the period

that Rembrandt occupied it. As noted from taxa listedin Tables 1 and 2, many pollen types recovered frompipe dottle samples are also found in modern examplesof commercial pipe and cigarette tobacco.

4.2. Commercial pipe tobacco samples

There have been few if any actual forensic orarchaeological studies that focused primarily on pollencontents in tobacco. To our knowledge, no one has yettried to use pollen data from tobacco to link anyone toa crime scene or as a guide to the geographical originof the tobacco. We know of only one unpublishedreport (Hall 1984) focusing on the recovery of pollencontents from archaeological remains consisting ofcharred dottle in Native American smoking pipes. In adifferent study, Donaldson and Stephens (2010)examined differences between pollen types found in atobacco sample grown in the United States versusthose found in samples packaged, and possibly grown,in China. Their recent study was an exploratory effortto determine whether pollen could be found in moderncommercial tobacco products and, if so, whetherdifferent brands might reflect their countries of origin.Results indicated that: (1) pollen can be recoveredfrom tobacco in cigarettes; (2) different tobacco brandscontain varying percentages of common pollen types;and (3) pollen types can be unique to each brand.

In the two tobacco brands examined by Donaldsonand Stephens (2010), high amounts of composite(Asteraceae), grass (Poaceae) and Cheno-Ams pollenwere found. They also noted that both tobacco brandscontained large grass pollen grains, which theybelieved were from species of domestic cereals suchas wheat or rice. Other taxa in their two tobaccosamples included pollen from various weedy plantsthat one would expect to find in the normal pollen rainof open agricultural fields located in temperate regions.Some weedy pollen types in their samples includedthose from the mustard family (Brassicaceae), plan-tains (Plantaginaceae), umbels (Apiaceae), sedges(Cyperaceae) and pinks (Caryophyllaceae). Theirsamples also contained airborne pollen types fromtrees but their total arboreal pollen counts were low,suggesting those grains may have arrived by longdistance transport or from wooded areas located somedistance from agricultural fields. Arboreal pollen typesincluded in the counts of their two samples werelimited to species of pine (Pinus), oaks (Quercus) andpollen grains from unidentified genera in the Myrta-ceae. Conspicuously absent in either sample wasNicotiana (tobacco).

One goal of the Donaldson and Stephens (2010)study was to calculate pollen concentration values fortwo tobacco samples. Using Lycopodium tracer spores,

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they noted values ranging from 2841 grains per gram inthe Chinese cigarette sample to a high of 24,341 grainsper gram in the US sample. These figures are lowerthan concentration values in three commercial tobaccobrands sampled by Hall (1984), whose analysisproduced 86,662 pollen grains per gram in Edge-worth1 tobacco, 78,977 pollen grains per gram inRevelation1 tobacco and 59,756 pollen grains pergram in Five Brothers1 tobacco. In our study ofpollen in modern brands of pipe tobacco, we foundpollen concentration values ranging from about 15,000per gram in one sample to over 60,000 per gram inanother. This, combined with the results from previousstudies, suggests a wide variation in the amount ofpollen trapped in various brands of commercialtobacco. It is therefore doubtful that pollen concentra-tion values are a useful tool for identifying geographi-cal origins of specific tobacco brands.

The second goal of the Donaldson and Stephens(2010) study was to determine if pollen in commercialbrands could be used as a unique indicator for thegeographical origin of the tobacco. Their data suggestthat the differences in percentages of various pollentaxa are sufficiently pronounced and geographicallyunique to warrant the supposition that pollen intobacco could be used to identify major tobacco-growing regions of the world. Our study of moderncommercial pipe tobacco calls that assumption intoquestion.

One aspect revealed by our study of pollen inmodern tobacco is that a number of pollen types arefound in almost every commercial brand. Eventhough percentages of some ubiquitous types suchas Asteraceae, Poaceae and Cheno-Ams differ be-tween brands, we remain unconvinced that suchdifferences remain constant for tobacco productsgrown and packaged in specific geographical regions.Of concern is that some analyst might attempt to usea single pollen grain or a group of minor pollen typesin one tobacco product as confirmation of a uniquegeographical region. For example, in our analysis ofVirginia Ribbon brand tobacco we found maize (Zeamays) and magnolia (Magnolia) pollen, but could weconfirm that these types are unique only to tobaccoproducts produced in the tobacco-growing region ofthe eastern USA? Not necessarily, because variousspecies of magnolia trees are endemic to other regionssuch as China and tropical regions in CentralAmerica where tobacco is grown. Likewise, in theTurkish brand tobacco we found wormwood (Arte-misia), olive (Olea), wheat (Triticum) and cedar(Cedrus) pollen, but is that sufficient to proclaimthese as being unique pollen types for Turkishtobacco products? For example, wheat and olivepollen are also found in Jefferson Street brand

tobacco, which is reported as a blend of Virginiatobaccos but which must also include a blend offoreign tobaccos. One interesting discovery is that thepollen of violets (Viola) occurs in almost every sampleof commercial pipe tobacco we examined, and wasfound in one of the Rembrandt house pipe dottlesamples (sample 50). Although the species remainsuncertain, all of the violet pollen we found in bothgroups of samples are similar in size and have a faintstriate pattern. It is suspected that all of these violetpollen grains might come from one region where theygrow in tobacco fields. Perhaps tobacco from thosefields is used as one of the common blending agentsin each of the commercial samples we examined.

Because most pipe tobacco brands are blends ofmore than one type, often grown in different geogra-phical regions, it is doubtful that a pollen signaturefrom any one brand would be reliable as forensicevidence of a specific geographical location or parti-cular place of manufacture. It is further supposed thatannual variations in climatic conditions or differencesin cultivation patterns could alter seasonal pollen rainon any given tobacco crop anywhere in the world.Studies of airborne pollen traps (Hall 1992) indicatethat pollen rain differs geographically and annually. Itis therefore suspected that the percentages of trappedpollen (and even some minor pollen types) found inone year’s tobacco crop could vary significantly frompollen spectra trapped in the tobacco from that sameregion in previous or subsequent years.

Another potential source of pollen in tobacco couldcome from the pollen in honey that is often added totobacco. Reports on contents of tobacco produced byat least five leading manufacturing firms (BritishAmerican Tobacco, Philip Morris, R.J. ReynoldsTobacco Company, Gallaher and Japan TobaccoInternational) note that often sweeteners in the formof maple syrup, fructose or honey are added to tobaccoto produce acids that will neutralize the harsh andbitter taste of natural tobacco and make smoke morepleasing to the delicate tissues of the throat (Dobson2006). These sweeteners are especially prevalent inchewing tobacco, snuff, cigarettes and various pipetobaccos. As reported from laboratory experiments(Talhout et al. 2006), the additional advantage ofadding sugars such as fructose and honey to tobacco isthat the burning sugars create acetaldehyde, whichcreate addictive properties and also aid in the deliveryof nicotine.

Because of potential tobacco blending and thepossible addition of honey containing pollen fromvarious sources, the assumption by Donaldson andStephens (2010) that there is a forensic validity forconsidering pollen in tobacco products as usefulgeographical indicators may be questionable.

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5. Summary

Results of our study of pollen from 20 charred claypipe dottle residues recovered from a cesspit atRembrandt’s house has limited cultural significance.Some or all of the pollen from the residue may havecome from the tobacco, from airborne contaminants atRembrandt’s house or from the cesspit debris in whichthe pipes were discarded. Although Rembrandt’ssmoking pipes provided inconclusive evidence as tothe origin or types of tobacco that may have beensmoked, this study has demonstrated that smallsamples of charred dottle may be examined and mightprovide important archaeological evidence. Additionalstudies of this type should be conducted on otherrecovered smoking pipes from archaeological contexts.In pollen studies of charred dottle recovered fromNorth American aboriginal smoking pipes, Hall (1984)made the same recommendation.

A second part of this study was to examine pollenrecovered from modern brands of commercial pipetobacco. Our original goal was to search for simila-rities between pollen types found in modern tobaccobrands from various geographical locations and thepollen spectra recovered from the Rembrandt pipedottle studies. Unfortunately, even though the pollentypes in the various modern brands of pipe tobaccoand the pollen types recovered from the Rembrandtpipe studies exhibit similarities, it is impossible to linkthe Rembrandt pipe pollen evidence to any specificgeographical region where tobacco has been grown orpackaged for sale.

A byproduct of this study was the realization thatcommercial pipe tobacco brands that we examinedcontain pollen spectra that do not appear to be uniqueto a single geographical region. Instead, we believe thatbecause most pipe tobaccos are blends of tobaccogrown in different geographical regions during differ-ent years, the notion that it is possible to identify aspecific location of production or shipment based onpollen analysis is questionable at best.

We believe that tobacco products could be used asforensic evidence, but only in specific circumstanceswhere a tobacco product (e.g. cigarette, cigar butt, pipetobacco or the remains of chewing tobacco) isdeposited at a crime scene. Perhaps a pollen spectrumfrom one of those specific discarded tobacco productscould be linked to the pollen found in tobacco productsin the possession of a suspect or found in the ashtray ofa suspect’s home or vehicle. In those types of forensicsituations, pollen in a tobacco product might assist inlinking a suspect to a crime scene.

Acknowledgements

We are especially indebted to Patricia Hall, anthro-pologist and botanist, who very kindly gave us access

to some of her unpublished work related to the studyof native tobacco pollen in California and also the datapertaining to some of her experiments extracting andanalyzing pollen from the pipe dottle of ancient pipesonce used by Native Americans. The authors alsogratefully acknowledge the help and assistance of Bobvan Der Boogaert, Jaap van der Veen, MechtildBeckers, Ed de Heer and Eva Vonk of Museum HetRembrandthuis and Wiard Krook and Jerzey Ga-wronski at Bureau Monumenten & Archeologie, head-quartered at the Noorderkerk, Amsterdam.

Author biographies

VAUGHN M. BRYANT achieved degreesfrom the University of Texas at Austin (BAin geography, MA in anthropology, Ph.D. inBotany) and is currently a Professor ofAnthropology and the Director of the TexasA&MUniversity Palynology Laboratory. Heis a former editor and president of AASP andis currently a Trustee and the Secretary of theAASP Foundation.

SARAH M. KAMPBELL has earned aBA in Anthropology, History and Com-puter Applications from the University ofNotre Dame, an MA in Anthropology(Underwater Archaeology) from TexasA&M University, an MA in Historyfrom Princeton University and is

currently pursuing her Ph.D. in History from PrincetonUniversity.

JEROME LYNN HALL is an AssociateProfessor of Anthropology at the Uni-versity of San Diego. He received his BSfrom Abilene Christian University, hisMS from Nova-Southeastern Universityand his Ph.D. from Texas A&M Uni-versity. Dr Hall, a nautical archaeologist,is presently studying the construction of a

first century wooden boat extracted from the Sea ofGalilee, as well as continuing research on a seventeenthcentury northern European merchant shipwreck in theDominican Republic.

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