Forensic Science International 306 (2020) 110061

Using museum pelt collections to generate pollen prints from high-riskregions: A new palynological forensic strategy for geolocation

Sophie Warnya,b,*, Shannon Fergusona,b, Mark S. Hafnerb, Gilles Escarguelc

aDepartment of Geology and Geophysics, E235 Howe-Russell, Louisiana State University, Baton Rouge, LA, 70803, USAbMuseum of Natural Science, 109 Foster Hall, Louisiana State University, Baton Rouge, LA, 70803, USAc Laboratoire d’écologie des hydrosystèmes naturels et anthropisés, UMR CNRS 5023, Université Claude Bernard Lyon 1, Boulevard du 11 novembre 1918,F69622, Villeurbanne Cedex, France

A R T I C L E I N F O

Article history:Received 12 March 2019Received in revised form 24 October 2019Accepted 11 November 2019Available online 27 November 2019

Keywords:GeolocationForensicPalynologyMuseum collectionsHomeland securityAnti-terrorism

A B S T R A C T

The use of pollen as a forensic tool for geolocation is a well-established practice worldwide in casesranging from the provenance of drugs and other illicit materials to tracking the travel of individuals incriminal investigations. Here we propose a novel approach to generation of pollen databases that usespollen vacuumed from mammal pelts collected historically from international areas that are nowdeemed too high risk to visit. We present the results of a study we conducted using mammal peltscollected from Mexico. This new investigative technique is important because, although it would seemthat the ubiquitous and geo-specific nature of pollen would make pollen analysis among the mostpromising forensic tools for law enforcement and intelligence agencies, it is not the case. The process isnotoriously slow because pollen identification is a tedious task requiring trained specialists(palynologists) who are few in number worldwide, and the reference materials necessary for geolocationusually are rare or absent, especially from regions of the world that are no longer safe to visit because ofwar or threat of terrorism. Current forensic palynological work is carried out by a few highly trainedpalynologists who require accurate databases of pollen distribution, especially from sensitive areas, to dotheir jobs accurately and efficiently. Our project shows the suitability of using the untapped museum peltresources to support homeland security programs. This first palynological study using museum peltsyielded 133 different pollen and spore types, including 8 moss or fern families, 12 gymnosperm generaand 112 angiosperm species. We show that the palynological print from each region is statisticallydifferent with some important clustering, demonstrating the potential to use this technique forgeolocation.

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1. Introduction

There has been ample discussion recently on the use of pollen asa geolocation tool for trade compliance, criminal investigations,and counter terrorism endeavors [1–5]. Geolocation based onpollen is possible because each plant has a pollen grain that isspecific to it, and each pollen grain has a unique morphology.Pollen is also composed of chemically resistant organic compoundsthat preserve remarkably well and resist microbial attack, hightemperature, and pressure from burial and even acid digestion.Pollen has been continuously produced in vast quantities for about300 million years (e.g., [6]) over much of the Earth, even in some

* Corresponding author at: Department of Geology and Geophysics, E235 Howe-Russell, Louisiana State University, Baton Rouge, LA, 70803, USA.

E-mail address: swarny@lsu.edu (S. Warny).

http://dx.doi.org/10.1016/j.forsciint.2019.1100610379-0738/© 2019 Elsevier B.V. All rights reserved.

parts of Antarctica (e.g. [7–9]). The diversity and relativeabundance of pollen species from a specific region constitutewhat is called “a pollen print” (analogous to a finger print) for thatlocation. The pollen print from a region can be unique, which thenallows the pollen print from a trace evidence to be linked to thatspecific region. Indeed, all objects and organisms on Earth,including humans, are constantly receiving pollen “rain” on theirsurfaces, allowing palynologists to tie that object, organism, orperson to a place where the plants that produce that pollen grew.This technique is commonly used in Europe and New Zealand inforensic investigations (e.g., [10,11]), but the U.S. has not used thistechnique until recently [12], when various U.S. governmentalagencies became increasingly interested in seeing forensicpalynology developed as a forensic tool. The U.S. Department ofHomeland Security (DHS) recently hired two full time forensicpalynologists and their work has allowed the identification of aJane Doe and a Baby Doe in recent high-profile murder cases and

2 S. Warny et al. / Forensic Science International 306 (2020) 110061

cold cases [13–15]. In all these cases, the key was to be able tocompare the pollen assemblage found on a perpetrator, victim, orobject to the pollen print from various locations to find the bestpossible match. And this is where one of the major impediments towidespread use of forensic palynology lies. Data needed togenerate pollen prints from the U.S. and many other locations(e.g. Europe, Australia) are readily available, but they areessentially unavailable and/or inaccessible from many areasoutside the U.S. where drug activity and terrorism may occur. InAugust 2012, DHS tasked The MITRE Corporation to conduct anadvanced study to determine the feasibility of applying palyno-logical analysis of pollen to forensics geolocation. While conduct-ing the study, MITRE surveyed the palynology and forensiccommunities and discovered that several disparate pollen data-bases covering the neotropics are under development by theresearch community, including Illinois State Museum’s NeotomaPaleoecology Database, Louisiana State University’s Pollen Data-base maintained by their Center for Excellence in Palynology(CENEX), and the Neotropical Pollen Database maintained byFlorida Institute of Technology. However, these databases are beingdeveloped for different reasons (e.g., paleoecology, paleontology,paleoclimatology, palynology). As a result, they are unfortunatelydevoid of key meta-data important for forensics geolocation(Hwang and Masters, 2012). In some cases, certain areas of theglobe might be difficult to access due to war or other conflicts. Inthose instances, it may not be safe to travel to these areas to collectpollen and generate pollen prints. Here is where our idea of usingcarefully preserved historical museum pelt collections becomes apotential major intelligence asset for the forensic palynologicalfield and an unparalleled way of generating pollen prints from

Fig. 1. Overview of the scientific methods propos

specific geographic locations while also showcasing a novel use ofhistorical museum collections.

2. Investigative approach and material studied

Our approach is based on our hypothesis that a pollen print fora specific location can be created using the pollen trapped in thefur of a mammal collected from that same location. If true, thenthe thousands of mammal pelts housed in historical museumcollections today represent a vast resource for generation ofpollen prints from locations worldwide. We tested the hypothesison specimens of mammals collected in Mexico because themammal collection in our LSU Museum of Natural Science(augmented by specimens in the mammal collection at the NewMexico Museum of Natural History) together hold 4598 skins ofmammals from 564 unique localities spread across all 31 states ofMexico and the Federal District. These priceless and irreplaceablespecimens were collected over the past �70 years by severalgenerations of curators and students who were able to worksafely in all those areas of Mexico. Many of these areas are nolonger accessible today because of drug activities and other illicitoperations. Our selection of Mexico as the test-bed for our projectwas also influenced by the fact that pollen prints for locations inMexico might assist law enforcement agencies, such as the DHS,in determining the geographic attribution of items, includingdrugs, brought into the U.S. illegally. To build this first pelt-basedpollen-print database, we proceeded in 6 global steps (Fig. 1)focusing on 19 Mexican localities distributed widely across thecountry. Details of our analyses are described in the methodologysection below.

ed. Step 7 is out of the scope of this project.

S. Warny et al. / Forensic Science International 306 (2020) 110061 3

3. Methods

3.1. Research sampling protocol and method

The uniqueness of this project is based on the medium fromwhich we extracted the pollen. To date, studies designed to createpollen print maps have used soil samples, herbaria or honey to citea few, to gather pollen assemblages from one location [14,16–18].As discussed above, the problem is the lack of available samplesfrom geographic locations of interest that are no longer accessible.The medium we selected to investigate as a potential pollen sourcefrom such location is the pelt of mammals collected at knownlatitude and longitude by Museum curators and students. Ourstudy used museum collections that include 564 Mexican localities(with precise GPS coordinates). We divided Mexico into its 19principal biogeographic provinces based on studies by Arriaga et al.[19] and Morrone et al. [20]. Each province has different types ofvegetation because each is characterized by different factors suchas altitudes or proximity to the coast. In each of these 19biogeographic provinces, we selected 12–15 mammals fromvarious locations (see Table 1 in supplementary documentation).This number is based on a previous evaluation we conducted todetermine how many mammals had to be vacuumed in order tohave a higher probability of gathering 300 pollen grains perlocation. We then vacuumed the dust (that includes the pollen)from the fur using a forensic trace evidence vacuum (Staplex ModelEC-1), chemically extracted the pollen from the dust samples (seebelow for the chemical technique used to maximize the pollenyield). The residues recovered were mounted on microscope slides,and each specimen recovered was identified and tabulated to thegenus and species level when possible. The resulting pollen andspore database was analyzed statistically. It is important to notethat the specimens of mammals that we vacuumed have beenstored in airtight cabinets since their arrival from Mexico, socontamination from local pollen is not an issue. Specimens in ourcollection were collected in approximately equal numbers from all12 months of the year, and because the animals we selected do notbathe, we can assume that the pollen trapped in their furrepresented a multi-month, if not full year, sample of pollen.We also selected various types of mammals to ensure that we haddifferent habitats represented for each location (Table S1 insupplemental documents). The most common species vacuumedwere cottontails (Sylvilagus), mice (Baiomys, Chaetodipus, Liomys,

Table 1Operating Procedure Summary.

Operating Procedure

Removal of filters 1. Carefully remove top plug of p2. Spray EtOH through the small3. Use a coin to separate the bot4. Use tweezer to remove the fil

Palynological processing 1. Place filter in a 15 ml test tube2. Slowly add an acetolysis solut3. Place 10 min on a heating blo4. Wash the residue 3x with EtO5. Add 8 ml of HF to dissolve sili6. Place 30 min on a heating blo7. Wash the residue 3x with EtO8. Add 15 ml of HCl to dissolve c9. Wash the residue 3x with EtO10. Add a drop of Safranin-O col

Slide preparation 1. Transfer the residue of organi2. Add 3 drops of glycerin3. Cover with paper and let evap4. Pipette one to two drops of th5. Spread to the area that will b

Peromyscus), rats (Dipodomys, Oryzomys, Sigmodon), woodrats(Neotoma), squirrels (Otospermoph, Sciurus, Spermophilus, Xero-spermop), chipmunks (Tamias) gophers (Cratogeomys, Pappoge-omys, Thomomys) and opossums (Caluromys, Chironectes, Didelphis,Philander). Each of these animal is known for having a very smallhome range, so the location where they were trapped is essentiallythe area from which their fur trapped the pollen. Following thisreasoning, we excluded species such as bats from our mammalsampling strategy.

3.2. Palynological processing method

The filter used to vacuum each set of mammals from a specificlocation was chemically processed independently following aforensic technique established by V. Bryant (Texas A&M, CollegeStation, TX) to maximize recovery. This procedure is described indetail below and synthesized in Table 1 in the form of an operatingprocedure summary. First, the top plug of the vacuum filter wasremoved leaving a mm-wide opening through which ethanol(EtOH) was sprayed onto the filter to saturate it. This step is crucialas it prevents loss of pollen when the vacuum’s capsule is opened.The top half of the capsule was then opened using a coin toseparate the top portion of the capsule from the bottom part, andtweezers were used to carefully remove the 7-micron thick filter soas to prevent it from tearing. The cotton underlining on which thefilter sits in the capsule should not be grabbed. The filter shouldimmediately be placed into a new, clean 15 ml test tube, and thetest tube should be filled with glacial acetic acid. The tubes are thencentrifuged and decanted. An acetolysis solution is then added tothe tube using a solution made of 9 parts acetic anhydride and 1part sulfuric acid. This step will dissolve the filter (as it is made ofcellulose) and enhance the morphological features of the pollenwalls. Note that the acetolysis solution has to be added very slowlybecause EtOH present in the tube has a tiny amount of water, andthe addition of a few drops of acetolysis will cause a “pop” thatcould surprise the person holding the tubes and cause it to drop,loosing the filtered content. Once the acetolysis solution is addedto the tube, it should sit in a heating block for 10 min, stirringhalfway through. The solution is again centrifuged and decanted,then washed three times with ethanol. The solution is centrifugedand decanted between each wash. Under a HF-grade hood, about7–8 ml of HF should be added to the solution to dissolve any sandy/silicate sediment that is mixed with the pollen residues. That

lastic capsule opening to saturate the filter and assure that pollen will not disperse in step 3tom from the top part of the capsule and provide access to the filterter (do not grab the cotton underliner)

filled with glacial acetic acid, centrifuge, decantion (9 parts acetic anhydride and 1 part sulfuric acid)ck, stirring every 5 min, centrifuge, decantH, centrifuge and decant between each washcatesck, stirring every 10 min, centrifuge, decantH, centrifuge and decant between each washarbonatesH, centrifuge and decant between each washorant if you wish to stain the organic matter

c matter (mostly pollen) to a 2 ml vial (use EtOH to help with the transfer)

orate any residual EtOH (�12 h)e pollen/glycerin mixture and place on a microscope slidee covered by a cover-slip, apply the cover-slip and seal

4 S. Warny et al. / Forensic Science International 306 (2020) 110061

solution should sit on a heating block for 30 min, then the solutionshould be centrifuged, decanted, and followed by three sets ofEtOH washes, each separated by centrifuging and decanting.Fifteen ml of HCl should then be added to the tube to dissolve anyCaCO3 dust vacuumed from the fur, and further concentrate thepollen and spore residue. The solution is centrifuged, decanted andwashed with EtOH three times. At this time, the majority of theorganic residue left in the tube is composed of the pollen andspores vacuumed from the pelt. That residue is stained withSafranin-O (one drop) and rinsed with EtOH wash, centrifuged anddecanted one last time. The Safranin-O allows to enhance thepollen’s morphological features. The residue is then transferred to2 ml vials. About three drops of glycerin are added to the vial. If thevial still smells like EtOH, the vial should be covered with a paper toallow any remaining ethanol to evaporate for up to 12 h. Theremaining residue is mixed, and 1–2 drops of that residue/glycerinmixture is mounted on a microscope slide. It is important to havethe pollen spread out on the slides (and not on top of one another),so the residue should be diluted as needed with glycerin (i.e., byadding an additional drop of glycerin if further dilution is needed).

3.3. Statistical analysis method

The slides were analyzed using an Olympus BX41 microscopeunder 600� and 1000� magnification using oil immersionobjectives. The specimens recovered were identified and tabulatedin each sample. The raw counts were analyzed statistically usingPAST v. 2.17c. The analysis was done using the gymnosperms plusangiosperms dataset. The spores were excluded because none ofthe 8 spore taxa being observed were found in more than onesample, thus, no sample association could be derived from thespore dataset. Spores were only recovered in significant amountsfrom sample named “PACs”. The analyses were based on presence/absence (not abundance) of taxa within samples. The existence ofsample groups were identified by combination of an OrdinationAnalysis (Principal Coordinate Analysis) and Hierarchical (UPGMA)and non-hierarchical (Neighbor-Joining) Cluster Analyses, in allcases based on a Dice (=Sørensen) similarity matrix. The Dicesimilarity between two samples is d ¼ 2M

2MþNwhere M is the numberof matches between the two samples (i.e., number of taxasimultaneously present – but not absent – in both samples) andN is the number of taxa present in only one of the two samples.Dice similarity ranges from 0 (i.e., when the two compared samplesare totally distinct in their taxonomical composition) to 1 (i.e.,when the two compared samples are perfectly similar in theirtaxonomical composition). This similarity matrix has been usedextensively by ecologists and biogeographers in recent decadesdue to its interesting geometric and sampling properties. Thereason it was selected over other classical indices such as theEuclidean, Mahalanobis or Chi-square distances, is that theseclassic methods are not suitable for ecological binary (presence/absence) data analysis. Indeed, by giving a double weight to co-occurrences (i.e., M) as an indication of similarity between samples(i.e., N) as an indication of difference between them (contrary tothe closely related and equally popular Jaccard similarity indexwhich gives a simple weight to co-occurrences), the Dice similarityis relatively immune to false absences as (wrong) evidence ofdifference between samples, an appealing property in the case ofthe present study [21]. In addition, preliminary computationsshow that the Dice similarities computed for this dataset are notinfluenced (i.e., biased) by sample size differences (i.e., differencein the number of pollen counted in each sample), a mandatorycondition in order to interpret the Dice coefficient as a coefficientof compositional similarity (i.e., all sampling effort being equal).The sample groups (=clusters) identified through Ordination +Cluster analyses are compared through one-way ANOSIM, a non-

parametric test of significant difference between two or moregroups, also based on the Dice similarities between samples. TheANOSIM statistics, called R, ranges from 0 to 1; the closer to 1, thehigher the similarities between groups of samples. Significance ofthe R value is estimated through random permutation of theavailable dataset (here, performing 99,999 permutations). Thelower the resulting p-value, the more likely the differencesobserved between the groups cannot be explained by chance (i.e.,the more likely the taxonomical composition of each group is notrandomly drawn from a single parent assemblage). Finally, the taxamost contributing to the taxonomic composition differencesbetween the sample groups are identified through a Dicesimilarity-based SIMPER analysis. An arbitrary cut-off at 66 % ofthe inter-group dissimilarity is fixed, corresponding to 25–50 % ofthe analyzed taxa (i.e., in all group comparisons, 25–50% of thestudied taxa explain 2/3 of the taxonomic composition differencesbetween these groups, making these taxa the main drivers of theamong-group differences).

4. Results

4.1. Palynological results

Of the 19 localities selected for analysis (=19 samplesprocessed), 7 did not provide sufficient pollen yields for the study,but the remaining 12 samples provided excellent recovery ofpollen and spores. The material recovered gives us a detailedprofile of the type of vegetation found in each of the localitiesstudied. From the 12 productive samples, we recovered a total of133 different pollen and spore types. These include spores of 8different ferns of the family Polypodiaceae, pollen representing 12genera or families of gymnosperms (Ephedra, Taxodium, Cupressa-ceae, and species of Abietaceae including various species fromgenera such as Pinus, Abies, and Picea). Not surprisingly,angiosperms were the most common species recovered (112species), the most common of which were specimens from generaand families such as Acacia, Alchornea, Alnus, Alternanthera,Amaranthaceae, Anacardiaceae, Arecaceae, Artemisia, Asteraceae,Betula, Brassicaceae, Banksia (Proteaceae), Bursera, Cactaceae,Carya, Caryophyllaceae, Casuarina, Ceanothus, Celtis, Combretum,Croton, Cyperaceae, Dodonaea viscosa, Eucalyptus, Euphorbiaceae,Fabaceae, Ficus, Fraxinus, Graminae (other than Zea mays), Helio-carpus, Ligustrum, Liliaceae, Liquidambar, Lythrum, Mimosa, Morus,Myrica, Myrtaceae, Onagraceae, Phacelia, Pistacia, Polygonaceae,Prosopis, Quercus, Ranunculaceae, Rhamnaceae, Rosaceae, Salix,Spermacoce, Ulmus, Zanthoxylum and Zea mays.

4.2. Statistical analysis

The SIMPER analysis conducted on the palynological dataidentified 59 pollen species that together were responsible for themajor compositional differences among the 12 productive samplesand separated them into three groups (red, green and blue). Onecluster (red) includes the SMO and PAC-n samples (Fig. 1, Table 2),and are located in the central portion of Mexico on a North-Southtransect. A second cluster (green) includes samples PACs, APS, CLFand APNsw. The larger cluster (blue) includes VOLc on one side andGMn, SONn, CAB and BC on the other. Eighteen taxa characterizethe red cluster, the key species are Ceanothus, Combretum,Dodonaea viscosa, Lilaceae, Liquidambar, Rosaceae, Zanthoxylumand a variety of Pinus species. The green cluster is characterized by12 taxa, primarily by Cupressaceae, Ulmus, Quercus, Prosopis,Pistacia, Morus, Ligustrum, Ficus, Fabaceae, Cyperaceae, andAnacardiaceae. Finally, 21 taxa characterize the difference betweenthe green and blue clusters, meaning that the blue cluster can beaccurately isolated from the green one. The principal taxa that

Fig. 2. Statistical analysis (cluster).

Table 2Pollen and spores raw counts per location.

S. Warny et al. / Forensic Science International 306 (2020) 110061 5

allow differentiation of blue from green are Alnus, Brassicaceae,Carya, Celtis, Mimosa, Picea, and Abies.

Although three groups emerged (Fig. 2), the statistical analysisperformed (see methodology) plots the 12 pollen print samples in12 independent locations (see one-way ANOSIM graph, Fig. 3). Inother words, other than the three clusters, the biogeographicalstructuring shows 12 very different assemblages with fewsimilarities between sites. Hence, geolocation of a sample shouldbe possible by comparing taxa found in trace evidences to taxafound in one of the twelve assemblages recovered from these 12biogeographical locations.

5. Discussion

Most common techniques to gather pollen print data fromcurrently at-risk locations include directly sampling from herbari-um to build pollen reference material. But with almost half amillion species extant today, this task is very time-consuming andunpractical. The recovery of 133 different types of pollen andspores from the pelts and their clustering per region demonstratesthe high potential of this technique to gather geolocationintelligence data. But, if adopted, it is important to keep somepotential limits in mind. The most important one is considering theyear the historical material was collected in the context ofvegetative cover change. For the present study, pelts were collectedbetween 1943 and 2008 during 22 annual field seasons to variousregions of Mexico (Fig. 4). Of these, 38 % were collected recentlybetween 2003 and 2008, 28 % between 1990 and 1999, and the

remaining of the pelts were collected between 1943 and 1998.Climate during the past 70 years has not changed enough to affectthe vegetative cover. Holocene research along the coast of the Gulfof Mexico has clearly shown that the last change in vegetationlinked to climate occurred several hundreds to thousands of yearsago. For instance, vegetation changes in Texas were seen at �8400,5400, 4100, 3800, 2200, 1800, 1200, and 800 yr [22,23]. Changes inLouisiana were mostly seen at 3700 yr [24]. Records from centralMexico [25] showed that El Niño–Southern Oscillation decoupledfrom the Intertropical Convergence Zone (ITCZ) in late Holocenebetween 900 and 1550 CE, causing a dry Medieval ClimateAnomaly and a wet early Little Ice Age. Data from the ChihyahuanDeseart in Northern Mexico [26] showed the latest climaticchanges occurred at 221 yr B.P. (Little Ice Age equivalent), 3815–4251 yr B.P. (early Neoglacial), 6110–6721 yr B.P. (mid-Holocene),and 8269 yr B.P. Another study discusses Holocene climatevariability from southwestern Mexico [27]. Their record suggestedthat Pacific moisture became increasingly more influential throughENSO since about 4300 yr and noted the presence of two otherlarge climatic anomalies 10.3 and 8.2 kyr ago. These and otherreviews of the literature show that if vegetation changes occurredduring the past 70 years, they are not likely to have been linked tothe climate. However, that does not preclude anthropogenicimpact on vegetative cover that could affect patterns of species

Fig. 3. Statistical analysis (PCA).

6 S. Warny et al. / Forensic Science International 306 (2020) 110061

diversity. Although this cannot be fully excluded from our area ofstudy, all field research and collecting were performed in mostlyrural areas where human impact on vegetation is expected to beminimal.

Despite these limitations, the study hightlights the invaluableand irreplaceable value of Natural History Museums collections,one that is often not fully perceived, possibly because theresearch importance of these collection has not yet beenappreciated to its full potential. Warny [3] discussed thedramatic consequences that can result from such a perceptionwith the example of the Chicago’s Field Museum that saw itsresearch and collections suffer $3 million in cuts, endangeringthe preservation of its rare collections. Although the main goal ofthe present project was to provide a new way to create pollenmaps, it also shows the importance of preserving natural sciencecollections as new research venues and applications for historical

museum collections can arise. In the case presented herein, onesuch new venue is addressing the paucity of pollen referencedata when dealing with war zones, terrorism and criminalactivities. Extensive studies like the one discussed here can beused to generate accessible database for field agents and theintelligence community that would assist in the geographicattribution of trace evidence from locations worldwide as thistechnique can be transferred to any regions of interest, buildingon years of fieldwork by museum curators and graduate students.Database created using the technique proposed here would beusable by non-palynologists because the comparison betweenpollen prints from the control sample (mammal collections) tothe trace evidence would be done based on pollen morphology.Museum of Natural History collections include specimens thathave been collected over decades, if not centuries, from allaround the world.

Fig. 4. Distribution of selected pelt specimens per year of field seasons.

S. Warny et al. / Forensic Science International 306 (2020) 110061 7

6. Conclusion

The paper provides an innovative way to develop database ofpalynological species from currently inaccessible regions ofinterest where there is a significant modern palynological gap.Additionally, the research presented here shows the importance ofpreserving museum collections. Our particular study of mammalpelts collected across Mexico provides new pollen prints (pollenassemblage data) for 12 regions of Mexico, many of which are nolonger safely accessible. The statistical analysis demonstrates thateach region is different from one another with some importantclustering, as to be expected based on the various environmentssampled. The pollen print gathered from the pelt study shouldprovide the type of results that forensic palynologists will needduring their investigations.

Author contributions

S.W. designed the concept of the study, completed palynologi-cal analyses and wrote the first draft of the manuscript. All co-authors provided text and edits to the manuscript. S.F. assistedwith sampling while she conducted her PhD research at LSU. M.H.selected the pelts from his collections. G.E. conducted thestatistical analysis.

Acknowledgements

We are grateful to the U.S. Department of Homeland Security forproviding some of the equipment used in this research, andspecifically to Dr. Vaughn Bryant for introducing us to the field offorensic palynology, teaching us how to process forensic samplesand always being there for us to help identify rare species. Theproject would not have been possible without the mammal peltspecimens collected over 30+ years by Dr. Mark Hafner, anemeritus professor and mammal curator of the LSU Museum ofNatural Science, and for his selection of mammal specimens loanedto us for this project. Thanks are extended to the two reviewers fortheir excellent feedback that greatly improved this manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at https://doi.org/10.1016/j.forsciint.2019.110061.

References

[1] V.M. Bryant Jr, J.G. Jones, D.C. Mildenhall, Forensic palynology in the UnitedStates of America, Palynology 14 (1) (1990) 193–208.

[2] G.M. Hwang, D. Masters, Forensic geolocation challenge: is pollen analysis theanswer, AASP—Palynol. Soc. (2) (2013).

[3] S. Warny, Museums’ role: pollen and forensic science, Science 339 (8) (2013)1149.

[4] F.J. Goodman, J.W. Doughty, C. Gary, C.T. Christou, B.B. Hu, E.A. Hultman, D.G.Deanto, D. Masters, PIGLT: a pollen identification and geolocation system forforensic applications April, 2015 IEEE International Symposium on Technolo-gies for Homeland Security (HST), IEEE, 2015, pp. 1–7.

[5] K.C. Riley, J.P. Woodard, G.M. Hwang, S.W. Punyasena, Progress towardsestablishing collection standards for semi-automated pollen classification inforensic geo-historical location applications, Rev. Palaeobot. Palynol. 221(2015) 117–127.

[6] J.M. Anderson, The biostratigraphy of the Permian and Triassic. Part 3. A reviewof Gondwana Permian palynology with particular reference to the northernKaroo Basin of South Africa, Mem. Bot. Surv. S. Afr. 41 (1977) 1–133.

[7] S. Warny, R. Askin, M. Hannah, B. Mohr, I. Raine, D.M. Harwood, F. Florindo, theSMS Science Team, Palynomorphs recovered from sediment core reveal aremarkably warm Antarctica during the Mid Miocene, Geology 37 (10) (2009)955–958, doi:http://dx.doi.org/10.1130/G30139A.1.

[8] M.J. Amesbury, T.P. Roland, J. Royles, D.A. Hodgson, P. Convey, H. Griffiths, D.J.Charman, Widespread biological response to rapid warming on the AntarcticPeninsula, Curr. Biol. 27 (11) (2017) 1616–1622.

[9] S.P. Gulick, A.E. Shevenell, A. Montelli, R. Fernandez, C. Smith, S. Warny, S.M.Bohaty, C. Sjunneskog, A. Leventer, B. Frederick, D.D. Blankenship, Initiationand long-term instability of the East Antarctic Ice Sheet, Nature 552 (7684)(2017) 225–229.

[10] D.C. Mildenhall, Forensic palynology in New Zealand, Rev. Palaeobot. Palynol.64 (1–4) (1990) 227–234.

[11] M. Horrocks, K.A. Walsh, Forensic palynology: assessing the value of theevidence, Rev. Palaeobot. Palynol. 103 (1–2) (1998) 69–74.

[12] N. Miroff, Pollen ‘nerds’: U.S. Government Enlists Scientists to Track DrugLoads, Crack Cold Cases, The Washington Post, 2019 August 30, 2019.

[13] S. Augenstein, Forensic palynologist tracks the near-invisible, from nationalsecurity to cold cases, Forensic Mag. (2016). Online https://www.forensicmag.com/article/2016/05/forensic-palynologist-tracks-near-invisible-national-se-curity-cold-cases.

[14] A.R. Laurence, V.M. Bryant, Forensic palynology, Encyclopedia Criminol. Crim.Just. (2014) 1741–1754.

[15] A.R. Laurence, Forensic palynology in the United States: the search forgeolocation, Microsc. Microanal. 24 (S1) (2018) 1186–1187.

[16] V.M. Bryant, D.C. Mildenhall, Forensic palynology: a new way to catch crooks,Contrib. Ser.-Am. Assoc. Stratigr. Palynol. 33 (1998) 145–155.

[17] V.M. Bryant, G.D. Jones, Forensic palynology: current status of a rarely usedtechnique in the United States of America, Forensic Sci. Int. 163 (3) (2006) 183–197.

[18] V.M. Bryant, M.K. Bryant, The role of palynology in forensic, Forensic Archaeol.:Multidiscip. Perspect. (2019) 177.

[19] L. Arriaga, C. Aguilar, D. Espinosa, R. Jimenez, Regionalizacion ecologica ybiogeographica de Mexico, Workshop at the Comision Nacional para elConocimiento y Uso de la Biodiversidad (Conabio) (1997) 99.

[20] J.J. Morrone, D.E. Organista, J.L. Bousquets, Mexican biogeographic provinces:preliminary scheme, general characterizations, and synonymies, Acta Zool.Mex. (2002) 83–108.

8 S. Warny et al. / Forensic Science International 306 (2020) 110061

[21] P. Legendre, L. Legendre, Numerical ecology, Developments in EnvironmentalModelling 24, 3rd edition, Elsevier, Amsterdam, 2012, pp. 1006.

[22] S. Ferguson, S. Warny, J.B. Anderson, A.R. Simms, G. Escarguel, Holocenevegetation and climate evolution of Corpus christi and Trinity bays: implicationson coastal Texas source-to-sink deposition, Geobios 51 (2) (2018) 123–135.

[23] S. Ferguson, S. Warny, J.B. Anderson, A.R. Simms, C. White, Breaching ofMustang Island in response to the 8.2 ka sea-level event and impact on CorpusChristi Bay, Gulf of Mexico: implications for future coastal change, Holocene 28(1) (2018) 166–172.

[24] R.A. Tedford, A Multi-Proxy Approach to Investigating the Latest Holocene(�4,500 yrs. BP) Vegetational History of Catahoula Lake, Louisiana, (2009) .

[25] J. Park, R. Byrne, H. Böhnel, Late holocene climate change in Central Mexico andthe decline of Teotihuacan, Ann. Am. Assoc. Geogr. 109 (1) (2019) 104–120.

[26] P.J. Castiglia, P.J. Fawcett, Large Holocene lakes and climate change in theChihuahuan Desert, Geology 34 (2) (2006) 113–116.

[27] J.P. Bernal, M. Lachniet, M. McCulloch, G. Mortimer, P. Morales, E. Cienfuegos, Aspeleothem record of Holocene climate variability from southwestern Mexico,Quat. Res. 75 (1) (2011) 104–113.