RESSALVA Atendendo a solicitação do autor, o texto completo desta tese será disponibilizado somente a partir de 30/03/2024. UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE ENGENHARIA CAMPUS DE ILHA SOLTEIRA LORENA FRIGINI MORO CAPO TIMBER TRACKING OF Jacaranda copaia (Aubl.) D. Don. (BIGNONACEAE) FROM AMAZON FOREST USING DNA FINGERPRINT Ilha Solteira 2022 Campus de Ilha Solteira POST-GRADUATION PROGRAM IN AGRONOMY LORENA FRIGINI MORO CAPO TIMBER TRACKING OF Jacaranda copaia (Aubl.) D. Don. (BIGNONACEAE) FROM AMAZON FOREST USING DNA FINGERPRINT Doctoral thesis presented to the Engineering Faculty of Ilha Solteira – UNESP as part of the requirements to obtain the title of PhD in Agronomy. Supervisor Prof. Dr. Alexandre Magno Sebbenn Ilha Solteira 2022 DEDICATION I dedicate this thesis to my father, Jorge Luiz Moro Capo (in memory) who supported me and made it all possible. ACKNOWLEDGEMENTS First of all, I would like to thank my supervisor, Alexandre Magno Sebbenn, with all my heart, because without him I would not have been able to finish my PhD. He was much more than a mentor, he was a friend and reached out to me both at work and in life. Alexandre was very professional, but above all, he was a friendly shoulder and knew how to give me space when I needed it, but he also pulled me back to reality when necessary. I am grateful for all the support and support given by the German Federal Ministry of Food and Agriculture within the framework of the “Large-scale project on genetic verification of wood”. To the Genomic and Sequencing Facility of Bordeaux, where the genotyping was carried out, (grants from the Conseil Regional d'Aquitaine n° 20030304002FA and 20040305003FA, European Union, FEDER n° 2003227 and Investissements d'avenir, n° ANR- 10-EQPX-16 - 01 and CEBA: ANR-10-LABX-25-01 to UMR EcoFoG). ICMBIO permits No. 47960, 49369, CGen registration No. A16E4DA (Brazil), MAYA/VMABCCGDF/DGBAP/MEG No. 0280/2016 (Bolivia), R.D. No. 001-2016- SERNANP-DGANP, R.D. No. 001A-2015 -SERFOR-DGGSPFFS-DGSPF and Contract No. 001-2016-SERFOR-DGGSPFFS-DGSPF (Peru) were awarded for the collection, transport and genetic analysis of samples. We are grateful to colleagues at IIAP, INRA French Guiana, Station Cirad Paracou, Office National des Forêts and Institut Thünen for their assistance during field and laboratory work. In Bolivia, the samples were identified by the Museo de Historia Natural Noel Kempff Mercado. Thank you very much. To my family and friends, I am deeply grateful for all the emotional support and strength they have given me. Every moment I had a demonstration of affection and pride for getting where I am. And, last but not least, I thank my partner who literally fought with me daily so that I would continue and not let this dream end without my realizing it. RESUMO A floresta Amazônica e outras florestas tropicais ao redor do mundo estão atualmente sendo intensivamente desmatadas para e abertura de áreas para uso na agropecuaria intensiva e extração de madeira, o que em geral ocorre ilegalmente em áreas protegidas como reservas e áreas indigenas, resultando em problemas ecológicos, ambientais e econômicos. Com o objetivo de parar o desmatamento e a comercialização de madeira de corte ilegal de florestas tropicais, novas leis foram introduzidas em muitos países. Aqui investigamos a utilidade da impressão digital de DNA de marcadores SNPs nucleares e citoplasmáticos para rastrear a origem da madeira extraida e comercializada da árvore neotropical Jacaranda copaia. Amostras de 832 indivíduos de 43 populações da Bolívia, Brasil, Guiana Francesa e Peru foram utilizadas para investigar o poder de marcadores SNPs, sendo 113 nucleares (nSNPs), 11 cloroplastidiais (CpSNPs) e quatro mitocondriais (MtSNPs) para determinar corretamente o país, população e região dentro do Brasil e Peru de origem. A diferenciação genética (𝐺𝑆𝑇 ′ ) entre todas as populações, entre populações de diferentes países e entre regiões dentro dos países foi alta (0,506-0,698), especialmente para locos CpMtSNP (> 0,9), mostrando um forte padrão genético de isolamento por distância entre populações, o que é favoravel à determinação correta do local de origem de amostras de mandeira de indivíduos de J. copaia. Para testes de auto- atribuição foi possivel determinar corretamente, com 100% de precisão, o país, população e região de origem de todas as amostras quando foram utilizados todos os locos SNPs ou apenas os nSNPs. Os resultados mostraram que o uso de todos os marcadores SNPs ou nSNPs é uma ferramenta precisa e útil para alfândegas e policias nacionais e internacionais averiguarem se o local de extração declarado na documentação de exportação de madeira de J. copaia da Floresta Amazônica tem origem legal ou ilegal. Palavras-chave: comércio de madeira; desenvolvimento de marcadores moleculares; impressão digital de DNA; Jacaranda copaia; polimorfismo de nucleotídeo único; rastreamento de madeira. ABSTRACT Amazon and other tropical forest are actually subject to strong deforestation, generally originated from illegal logging, resulting in ecological, environmental and economic problems. Aiming stop deforestation and timber commercialization of illegal logging of tropical forest, new laws has been introduced in many countries. Here we investigated the utility of DNA fingerprinting of nuclear and cytoplasmatic SNPs markers to timber tracking the intensive logged and commercialized of the Amazonian Neotropical tree Jacaranda copaia. Samples of 832 individuals from 43 populations from French Guiana, Brazil, Peru, and Bolivia were used to investigate the power of 113 nuclear SNPs, 11 CpSNPs and four MtSNPs loci to determine the country, population and region within Brazil and Peru origin. The genetic differentiation (𝐺𝑆𝑇 ′ ) among all populations, contries, and regions within coutries was generaly high (0.506- 0.698), specialy for CpMtSNP (> 0.9) loci, and there is a strong isolation by distance pathern among populations, favoring the group or individual samples tracking to correct site. For self- assignment tests, we were able to 100% correct determine country, population and region site origin of all samples using all SNPs and nSNPs. Our results show that the use of all SNP or nSNP markers are suitable to correct determination of country and population site of J. copaia timber origin and very useful tool for customs and local and international policies. Keywords: DNA fingerprinting, Jacaranda copaia; marker development; single nucleotide polymorphism; timber tracking; timber trade. SUMMARY 1 INTRODUCTION ...................................................................................................... 9 2 REVIEW OF LITERATURE ..................................................................................... 10 2.1 Jacaranda genus ....................................................................................................... 10 2.2 Treaties and regulations concerning timber trade ................................................ 11 2.3 Illegal Timber Control ............................................................................................. 12 2.3.1 Genetic Assignment .................................................................................................. 14 2.3.2 DNA Fingerprinting ................................................................................................. 14 2.4 REFERENCES ........................................................................................................... 15 3 DNA FINGERPRINTING USING SNPS MARKERS ......................................... 20 3.1 INTRODUCTION ...................................................................................................... 22 3.2 MATERIALS AND METHODS ............................................................................... 23 3.2.1 Sampling .................................................................................................................... 23 3.2.2 DNA extraction and SNPs analysis ......................................................................... 28 3.2.3 Genetic diversity and population differentiation .................................................. 28 3.2.4 Spatial genetic structure .......................................................................................... 29 3.2.5 Genetic assignment ................................................................................................... 29 3.3 RESULTS ................................................................................................................... 29 3.3.1 Genetic diversity and genetic differentiation ......................................................... 29 3.3.2 Genetic assignment ................................................................................................... 38 3.3.3 Genotype self-assignment tests ................................................................................ 41 3.3.4 Timber assignment test ............................................................................................ 44 3.4 DISCUSSION ............................................................................................................ 47 3.4.1 Genetic diversity ....................................................................................................... 47 3.4.2 Population genetic differentiation ........................................................................... 47 3.4.3 Genetic assignment and practical application ....................................................... 48 3.4.4 Genotype self-assignment tests ................................................................................ 49 3.4.5 Timber assignment tests .......................................................................................... 50 3.5 CONCLUSION .......................................................................................................... 50 3.6 REFERENCES ............................................................ Erro! Indicador não definido. 3.7 SUPPLEMENTARY MATERIAL ............................................................................ 56 9 1 INTRODUCTION Much of the world's commercial logging from natural forests is illegally sourced, and even the legal source has come from unsustainable logging. This is especially true for tropical forests, where most of the world's plant and animal biodiversity lives. Both illegal and legal harvesting contribute to the loss of biodiversity, diminishing the potential for human resources to find medicinal and future sources of wood. Illegal logging is also an economic problem for the legal market, as extremely low-priced timber competes with legal logging, where costs are higher (GRAY, 2002; DEGEN et al., 2013). Because of this, many international casualties were established in countries around the world to prevent illegal timber imports. To trace the origin of wood, many methods have been tested, such as chemical differences between wood species (PAREDES-VILLANUEVA et al., 2018), transformation cyclotron resonance mass spectrometry (DEKLERCK et al., 2017), wood anatomy and DNA fingerprinting (JOLIVET; DEGEN 2012; DEGEN et al., 2013; LOWE et al., 2016; CHAVES et al., 2018; SEBBENN et al., 2019). Results from different methods showed strong potential for determining species, country, and site origin, in particular using DNA fingerprinting (LOWE et al., 2016; CHAVES et al., 2018) The current work investigated the use of the DNA fingerprint method to track the intensive and high value wood of the Neotropical pioneer tree Jacaranda copaia (Aubl.) D. Don. (Bignonaceae). The wood is light and used for furniture (LOUREIRO et al., 1979). This is a fast-growing species, with an average annual increase in diameter at breast height (DBH) of 2.05 cm and height of 1.98 m, with a great capacity for regeneration in gabs (SAMPAIO et al., 1989). The trees have a straight stem, reaching 106 cm in DBH and 45 m in height (VINSON et al., 2015a). The species occurs from northern to western South America, from Belize to Bolivia and Brazil, French Guiana, Peru (GENTRY, 1992). In Brazil, the species is found in the states of Acre, Amapá, Amazonas, Maranhão, Rondônia, Roraima, Mato Grosso and Pará and populations generally have more than one tree per hectare (VINSON et al., 2015a). The species is hermaphroditic, self-incompatible and about 40 species of bees, butterflies and hummingbird wasps were detected as potential pollinators, although Euglossa spp. and Centris spp. bees were detected as the main pollinators in the Tapajós National Forest, Brazil (MAUES et al., 2008). The fruits can have up to 250 seeds and the winged seeds are dispersed by the wind (MAUES et al., 2008). 50 3.5 CONCLUSION The genetic differentiation (𝐺′ 𝑆𝑇) among all populations, contries, and regions within coutries is generaly high, specialy for CpMtSNP loci, and there is a strong isolation by distance pathern among populations, favoring the group or individual samples tracking to correct site. For self-assignment tests, we were able to 100% correct determine country, population and region site origin of all samples using all SNPs and nSNPs. Our results show that the use of all SNP or nSNP markers are suitable to correct determination of country and population site of J. copaia timber origin and very useful tool for customs and local and international policies. The J. copaia reference database of our study represents a robust assignment tool available to timber companies or governmental agencies to test and validate origin declarations. 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