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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. SPECIES 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 Genus 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 Plant Taxonomy, Academic Press, 1963 WILLIAM E. HILLIS 1921 - 2008 Heartwood and Tree 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 Dalbergia madagascariensis showing vessels (X64) PERICOPSIS SPP Hans Beekman Victor Declerk Tervuren Xylarium Bioscience Engineering Royal Museum for Central Ghent University Africa Ghent Tervuren Belgium Belgium Michael C. Wiemann USDA Center for Wood Anatomy Research, Forest 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 Dalbergia melanoxylon 23 II East Africa Milicia excelsa 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: ARAUCARIACEAE 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 Argentina 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/Brazil Araucaria heterophylla (Salisb.) Franco 10 Florida Agathis australis (D. Don) Lindl. 20 New Zealand Wollemia nobilis W.G.Jones, K.D.Hill & 2 Australia 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 Cedrela 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 Cedrela odorata. 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 Trees, Volume 2: South America: Nomenclature, Taxonomy and Ecology. Academic Press. Synonymous species? LOOCV - 70.2% ENTANDROPHRAGMA 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 Swietenia spp Cedrela spp Pterocarpus spp Pericopsis spp Dalbergia spp Caesalpinia spp Guibourtia 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 Swietenia mahagoni 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** Agathis australis Kauri wood, New Zealand > 50,000* * Radiocarbon dating ** Historical context Acacia koe Aniba 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 / Guitar kits Dalbergia nigra Violin bows Dalbergia retusa Guitars Dalbergia stevensonii Knife handles Dalbergia spruceana Beads Diospyros sp. Incense Quercus sp. Oils Pericopsis sp. Wood from Shipwrecks Platymiscium sp. Antique furniture 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 Europe 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 Pericopsis elata 1 Pinaceae Pinus koraiensis 1 Leguminosae Platymiscium pleiostachyum 1 Rosaceae Prunus africana 1 Leguminosae Pterocarpus erinaceus 1 Santalaceae Osyris lanceolata 1 Leguminosae Pterocarpus santalinus 1 Meliaceae 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 Guibourtia Tessmannii 1 Meliaceae Swietenia macrophylla 1 Juglandaceae Oreomunnea pterocarpa 1 Meliaceae Swietenia mahagoni 1 Oleaceae Fraxinus mandshurica 1 Zygophyllaceae Bulnesia sarmientoi 1 Lauraceae Aniba rosaeodora 1 Zygophyllaceae Guaiacum spp 5 Thymelaeaceae Aquilaria spp 26 Thymelaeaceae Gonystylus 40 Thymelaeaceae Gyrinops 8 -251 CITES listed timber species -*Four App I species baroni * Madagascar rosewood Dalbergia caerensis cochinensis * Siamese rosewood congestiglora decipularis * Tuplipwood granadillo latifolia * Bombay blackwood madagascariensis maritima melanoxylon * African blackwood nigra * Brazilian rosewood retusa * Cocobolo 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 illegal logging 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-fir 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? [email protected].
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  • Silvicultural Contributions Towards Sustainable Management And

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    1 Silviculture in Southern Ecuador (Unpublished) Silvicultural contributions towards sustainable management and conservation of forest genetic resources in Southern Ecuador Condiciones silviculturales para manejo sostenible y conservacion de recursos geneticos forestales en el Sur de Ecuador Sven Guenter, Bernd Stimm & Michael Weber* Chair of Silviculture and Forest Planning, Technische Universität München, D - 85354 Freising - Weihenstephan, GERMANY, Tel. 0049 8161 71 4689 , Fax 0049 8161 71 4616, Email: [email protected] *Corresponding author Jun 28 2004 Lyonia, Volume #, Pages [ # - # ], 2004 Sven Guenter, Bernd Stimm & Michael Weber* 2 Resumen Bosques con una diversidad alta de arboles por area normalmente son caracterizados por una abundancia baja de muchas especies. Este hecho tiene implicaciones fuertes hacia un manejo sustentable de recursos genéticos forestales como tambien hacia la conservación. Depues de la selección de especies prioritarias utilizando criterios ecológicos y económicos, se inició una evaluación de la variación genética dentro de cada especie prioritaria. Por ello se estudió la fenología de Cedrela montana, Prumnopitys montana, Myrica pubescens y Clethra revoluta durante porun plazo de dos años en dos sitios diferentes, ECSF en el Valle de la Estacón Cientifíca San Francisco y El Bosque, cerca de San Pedro de Vilcabamba. Adicionalmente se realizaro inventarios forestales en la Reserva Estación Científica San Francisco y en la Reserva La Ceiba en el bosque seco. Los resultados demuestran una variación alta de datos de floración y fructificación, no solo entre las dos areas geograficas, sino también dentro de ellas. Como consecuencia, esa variación se debe considerar en planes de manejo y conservación. Los inventarios demostraron que hay varias especies con subpopulaciones sin contacto o flujo de genes entre ellas o que son demasiado pequeñas para conservación estatica de recursos genéticos.