PHYTOCHEMICAL ANALYSIS OF TIMBERS USING DIRECT ANALYSES IN REAL TIME, TIME OF FLIGHT MASS SPECTROMETER (DART TOFMS)

February 28, 2017 Seattle, WA

Ed Espinoza and Pam McClure National Fish and Wildlife Forensic Laboratory Ashland, Oregon U.S.A. ID EQUATION : COLONIAL MODEL

Geographic Anatomy / Identification Provenance Morphology

Known Suspected but required herbarium sample for taxonomic classification Identification

VOUCHER SPECIMEN SPECIES ID EQUATION: A NEW MODEL

Geographic Anatomy DNA / Identification Provenance Chemotypes

Known / Determines Determines Identification Inferred with Species Isotopes SPECIES ID EQUATION AT NFWFL: ANATOMY PLUS CHEMOTYPE

Geographic Anatomy Chemotypes Identification Provenance

Unknown Determines Determines Identification Genus Species

A single spectrum is useful for determining presence or absence of specific molecules Afrormosin

Dihydromethoxyflavone

Dalbergin Caviunin

Benzoic Acid Dalbergichromene or isomer

(3S)-Sativanon (-)-Alpinetin or isomer or isomer

KNApSAcK: A Comprehensive Species-Metabolite Relationship Database http://kanaya.naist.jp/knapsack_jsp/top.html TONY SWAIN 1922 - 1987

 Chemical , Academic Press, 1963

WILLIAM E. HILLIS 1921 - 2008  Heartwood and Exudates Spinger-Verlag 1987 Many spectra are useful for characterizing species or populations

Hierarchical Cluster Analysis Heat Map Multivariate Analysis

Supervised and Non- Non-supervised inferences supervised statistics Caviunin m/z 375.108

D. nigra

D. spruceana WHERE ARE THESE PHYTOCHEMICALS?

Transverse section of madagascariensis showing vessels (X64) SPP

Hans Beekman Victor Declerk Tervuren Xylarium Bioscience Engineering Royal Museum for Central Ghent University Ghent Tervuren Belgium Belgium

Michael C. Wiemann USDA Center for Anatomy Research, Products Laboratory, Madison, WI 53726 U.S.A. PERICOPSIS AND LOOK-ALIKES

Species n CITES Provenance P. angolensis ** 22 East Africa P. elata 60 II Central Africa P laxiflora 5 Central Africa P. mooniana 10 S.E. Asia 23 II East Africa 20 Central Africa Haplormosia monophylla 7 Central Africa

“ …P. angolensis is a subspecies species of P. elata…”

Plant resources of tropical Africa. Prota 7(1): Timbers, (Eds: D. Louppe, A.A. Oteng-Amoaki, M. Brink). Backhuys Publishers, CTA, Wageningen, 2008. Subspecies? 2-O-Methylangolensin

286.114 KDA GRAPH OF PERICOPSIS AND LOOK-ALIKES

LOOCV = 84.62% DOES THE CHEMOTYPE SUPPORT THE HYPOTHESIS THE P ANGOLENSIS IS A SUBSPECIES OF P ELATA?

LOOCV = 92.59%

“…taxonomy is a process that synthesizes evidence from all relevant comparative sources…”

Willi Hennig. 1966. Phylogenetic Systematics. University of Illinois Press, Urbana 1913 - 1976 Species Identification: COLLABORATORS

Philip D. Evans Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, Canada

Ignacio A. Mundo Laboratorio de Dendrocronología e Historia Ambiental, IANIGLA, CCT CONICET Mendoza

Michael C. Wiemann USDA Center for Wood Anatomy Research, Forest Products Laboratory, Madison, WI 53726 U.S.A. ARAUCARIACEAE SPECIMENS STUDIED

Species CITES No. Origin

Araucaria araucana (Molina) K.Koch App I 21 Argentina

Araucaria angustifolia (Bertol.) Kuntze 23 Argentina/

Araucaria heterophylla (Salisb.) Franco 10 Florida

Agathis australis (D. Don) Lindl. 20

Wollemia nobilis W.G.Jones, K.D.Hill & 2 J.M.Allen HEAT MAP KERNEL DISCRIMINANT ANALYSIS

LOOCV = 87.1 % HIERARCHICAL CLUSTER ANALYSIS W. nobilis (WARD’S METHOD; BOOT N=100)

A. australis

A. angustifolia

A. heterophylla

A. angustifolia

A. araucana C. odorata

SPECIES IDENTIFICATION

CEDRELA

Collaborators Kathelyn Paredes and Pieter Zuidema, Wageningen University The Netherlands SEVEN (7) SPECIES TESTED

Species CITES n Sample Origen angustifolia (C. lilloi) App III 17 Bolivia Cedrela balancea ** 17 Bolivia Cedrela fissilis App III 63 Bolivia Cedrela montana 3 Bolivia Cedrela huberi 6 Bolivia . App III 31 C & S America Cedrela saltensis 10 Bolivia Σ = 147 ** Syn with C. fissilis

Taxonomic source M.M. Grandtner; Julien Chevrette (21 September 2013). Dictionary of , Volume 2: : Nomenclature, Taxonomy and Ecology. Academic Press. Synonymous species? LOOCV - 70.2% SPP

Hans Beekman Victor Declerk Tervuren Xylarium Bioscience Engineering Royal Museum for Ghent University Central Africa Ghent Tervuren Belgium Belgium

ENTANDROPHRAGMA KDA GRAPH

LOOCV = 61.7% FAMILY PICTURE Bulnesia and Guiacum spp spp Cedrela spp

Pterocarpus spp

Pericopsis spp

Dalbergia spp

Caesalpinia spp spp

Diospyros spp PERSISTENCE OF THE CHEMOTYPES

http://forseadiscovery.eu/sites/default/files/attachments/documents/poster_fsd.pdf PERSISTENCE OF HEARTWOOD CHEMOTYPES

Species Source Years BP Sunken torpedo boat, Florida, US ~ 50** Dalbergia spp. Madagascar ~ 220* Swietenia mahagoni High chest, Princeton Art Museum, US ~ 245**

Aquilaria spp. Imperial Vault of Heaven, China ~ 262** Quercus faginea Sunken Spanish galleon, Portugal > 500** australis Kauri wood, New Zealand > 50,000*

* Radiocarbon dating ** Historical context Acacia koe roseadora UMMARY OF CASES S : Araucaria araucana Aquilaria sp. > 60 LAW ENFORCEMENT CASES (~ 390 ITEMS) Caesalpinia echinata > 40 IDENTIFICATIONS FOR PARTNERS Cedrela odorata Timber Dalbergia cochichinensis Dalbergia sp. from Madagascar Veneers / kits Violin bows Dalbergia retusa Dalbergia stevensonii Knife handles Dalbergia spruceana Beads sp. Incense Quercus sp. Oils Pericopsis sp. Wood from Shipwrecks Platymiscium sp. Antique Platymiscium yucatanum Santalum sp. We have provided ID’s to Swietenia mahagoni USDA/APHIS and CBP Tabebuia sp. Etc. CONCLUSIONS OF HEARTWOOD ANALYSIS USING DART TOFMS

 ~152 species (> 3000 specimens) have been characterized using DART TOFMS.

 ~ 129 species (~87 %) have species–specific chemotypes.

 Wood anatomy & chemotype profiles are useful for making species identification of CITES species.

 Environmental or temporal effects does not significantly alter the chemotype (data was not shown) CHALLENGES ~40,000 TROPICAL TREE SPECIES (~100,000 DESCRIBED SPECIES)

 Indo-Pacific Region ~20,000  Amazon ~16,000  Africa ~5,000  Central 124

 Inside Wood has description of 7,159 species

 (~18% of all tropical species).

 Includes descriptions of fossil material

 DART TOFMS chemotypes for 152 species

 (~0.5% of all tropical species).

-J. W. Ferry Slik et al. An estimate of the number of tropical tree species. Proceedings of the National Academy of Sciences, 1. Juni 2015; doi: 10.1073/pnas.1423147112 -http://insidewood.lib.ncsu.edu/ IDENTIFICATION FOR ~51% OF CITES SPECIES

Family Genus species n: Family Genus species n: Ebenaceae Diospyros spp 85 Araucariaceae Araucaria araucana 1* Leguminosae Caesalpinia echinata 1 Cupressaceae Fitzroya cupressoides 1* Leguminosae Dalbergia spp 60* Cupressaceae Pilgerodendron uviferum 1* Leguminosae Dipteryx panamensis 1 Pinaceae Abies guatemalensis 1 Leguminosae 1 Pinaceae 1 Leguminosae Platymiscium pleiostachyum 1 Rosaceae africana 1 Leguminosae erinaceus 1 Santalaceae Osyris lanceolata 1 Leguminosae Pterocarpus santalinus 1 Cedrela fissilis 1 Fagaceae Quercus mongolica 1 Meliaceae Cedrela lilloi 1 Fabacea Guibourtia demise 1 Meliaceae Cedrela odorata 1 Fabacea Guibourtia pellegriniana 1 Meliaceae Swietenia humilis 1 Fabacea 1 Meliaceae 1 pterocarpa 1 Meliaceae Swietenia mahagoni 1 Oleaceae Fraxinus mandshurica 1 Zygophyllaceae Bulnesia sarmientoi 1 Aniba rosaeodora 1 Zygophyllaceae spp 5 Aquilaria spp 26 Thymelaeaceae 40 Thymelaeaceae Gyrinops 8

-251 CITES listed timber species -*Four App I species baroni * Madagascar Dalbergia caerensis cochinensis * Siamese rosewood congestiglora decipularis * Tuplipwood granadillo latifolia * Bombay blackwood madagascariensis maritima melanoxylon * African blackwood nigra * Brazilian rosewood retusa * sisso * Indian rosewood spruceana * Amazon rosewood stevensonii * Honduras rosewood tucurensis * Guatemalan rosewood about 170 more species

Includes tree, shrubs and lianas It appears that of the 187 species, about 10 – 15 % are trees Caesalpinia cacalaco echinata App II platyloba sclerocarpa yucatensis missing about 65 species CITATIONS

 2012  1. Analysis of Select Dalbergia and Trade Timber Using Direct Analysis in Real Time and Time-of-Flight Mass Spectrometry for CITES Enforcement  2. Evaluating Agarwood products for 2-(2-phenylethyl)-chromones using Direct Analysis in Real Time Time-of-Flight Mass Spectrometry  2014  3. Distinguishing Wild from Cultivated Agarwood (Aquilaria spp.) Using Direct Analysis in Real Time (DART™) and Time-of-Flight Mass Spectrometry  2015  4. Metabolome chemotypes of CITES protected Dalbergia timbers from Africa, Madagascar and Asia  5. A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures  6. Forensic Analysis of CITES Protected Dalbergia Timber from the Americas  7. Forensic timber identification: it's time to integrate disciplines to combat  2016  8. The Best Practice Guide for Forensic Timber Identification  2017  9. Anatomy and Chemistry Analysis of Selected CITES-Protected Auracariaceae. International Association of Wood Anatomists Journal. 38(2). DOI 10.1163/22941932- 20170171

 In Review  10. Navigating a random forest with wood chemistry: source identification of Western Oregon Douglas- wood cores using mass spectrometry  11. Species verification of the Dalbergia nigra and Dalbergia spruceana samples in the Wood Collection of the Forest Products Laboratory in Madison, Wisconsin Vessels containing phytochemicals Empty vessels

The End?

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