Genetic variation of dipterocarps: from molecular phylogenies to the identification of the origin of timber Reiner Finkeldey, Oliver Gailing, Iskandar Siregar, Ulfah Siregar, Randy Villarin, Cuiping Cao, Nga Phi Nguyen, Hani Nuroniah, Yanti Rachmayanti Büsgen Institute Forest Genetics and Forest Tree Breeding 1 Structure of presentation l Background – Dipterocarpaceae l Interspecific variation: molecular phylogeny of Dipterocarpaceae l Intraspecific variation of selected Shorea spp. l Application: Molecular methods to trace the origin of wood l Conclusions and outlook l Literature 2 Background: Dipterocarpaceae The pantropical distribution of Dipterocarpaceae Monotoideae Dipterocarpoideae Pakaraimoideae 3 Genera; 40 Species 13 Genera; 470 Species 1 Genus; 1 Species 3 Background: Dipterocarpaceae Dipterocarpoideae in insular Southeast-Asia Phylogenie der Dipterocarpaceae Number of endemic species Number of non-endemic species 4 Background: Dipterocarpaceae Dipterocarpoideae in Southeast-Asia l Widely distributed – Mainland and insular SE-Asia l Dominating lowland rainforests and monsoon forests in SE-Asia – 50-80% of wood volume – Keystone group l Important timber species – Trade name: Meranti, Balau, Keruing, Kapur l Many endangered taxa Dipterocarp forest on Borneo 5 Background: Dipterocarpaceae Dipterocarp: growth habit l Medium-sized – emergent trees – Mainly climax species – Often long, straight boles – Slow to fast growth Dipterocarpus Shorea selanica (<60 years) 6 condoriensis Background: Dipterocarpaceae Dipterocarpaceae – Reproductive biology l Hermaphrodites l Zoogamous (mainly insect- pollinated) – Bees, butterflies – Thrips l Self compatible, mixed mating system – t typical 60-90% 7 Dipterocarpus condoriensis Background: Dipterocarpaceae Dipterocarp fruits l Typical 2 (0-5) winged diaspores – Medium-sized to very large l recalcitrant l No efficient means of seed dispersal 8 Background: Dipterocarpaceae Exploitation of dipterocarps Local International 9 Hintergrund: Dipterocarpaceae Use of dipterocarps By-products Wood window frames charcoal furniture plywood 10 resin Phylogeny of Dipterocarpaceae Order: Malvales; Family Dipterocarpaceae l Blume (1825): related to Tiliaceae l Cronquist (1968) & Takhtajan (1969): Theales l Dahlgren (1975): Malvales l Maguire et al. (1977), Dayanandan et al. (1999): Malvales (Sarcolaenaceae) l Chase et al. (1993); Alverson et al. (1998); Bayer et al., (1999): Malvales (Bombacaceae, Tiliaceae, Sterculiaceae and Malvaceae) 11 Phylogeny of dipterocarps Malvales; family Dipterocarpaceae rbcL sequences 12 (nach CHASE et al., 1993) Material Material for studies on inter- and intraspecific variation l Over 3000 samples from dipterocarps in SE-Asia – Dried leaves l Silica gel l Long-term storage at -60°C – Extracted DNA – Herbarium material maintained l Mainly with partners l Total of 116 species – All genera of Dipterocarpoideae – From six countries l More than 40 locations l Focus on Indonesia (>2500 samples) – Mainly natural forests (>80%), few plantations, arboreta, botanical gardens 13 Phylogeny of Dipterocarpaceae Methods: cpDNA polymorphisms (Indrioko et al., 2006) – PCR-RFLP ofcpDNA regions : trnLF, rbcL, petB, psbA, psaA (Tsumura et al., 1996) – cpSSRs (Microsatellites): ccmp2, ccmp6, ccmp10 (Weising et al., 1999) – Sequences: trnL; trnLF trnL region 14 PCR RFLPs cpSSRs Sequences Outgroup: Upuna Outgroup: Monotes Upuna borneensis Cotylelobium lanceolatum Monotes kerstingii Vatica bantamensis Cotylelobium lanceolatum Vatica bella Upuna borneensis Vatica granulata Anisoptera costata 99 Vatica pauciflora Anisoptera marginata 99 Anisoptera reticulata Vatica rassak 68 Vatica bantamensis 95 Vatica venulosa Vatica bella Anisoptera costata Vatica granulata Anisoptera marginata 99 Vatica pauciflora 99 Anisoptera reticulata 83 Vatica rassak 100 94 Dipterocarpus grandiflorus Vatica venulosa Dipterocarpus oblongifolius Dipterocarpus grandiflorus Dipterocarpus retusus 100 Dipterocarpus oblongifolius Dipterocarpus rigidus Dipterocarpus retusus 71 100 Dipterocarpus tempehes Dipterocarpus rigidus Dryobalanops aromatica Dipterocarpus tempehes Dryobalanops lanceolata Dryobalanops aromatica 99 Hopea bancana Dryobalanops lanceolata 99 Hopea celebica Hopea bancana Hopea odorata Hopea celebica 80 92 Hopea sangal Hopea odorata Hopea dryobalanoides 85 Hopea sangal <50 Hopea nigra Hopea dryobalanoides <50 <50 Hopea griffithii Hopea nigra Hopea mengarawan <50 Hopea griffithii 67 Parashorea lucida Hopea mengarawan 100 66 Parashorea globosa Parashorea lucida Parashorea globosa 94 Shorea blumutensis Shorea guiso Shorea blumutensis Shorea montigena Shorea guiso Shorea scaberrima Shorea montigena Shorea seminis Shorea scaberrima Shorea acuminata Shorea seminis Shorea andulensis Shorea acuminata Shorea javanica Shorea andulensis 58 Shorea johorensis Shorea javanica Shorea leprosula 67 Shorea johorensis Shorea macroptera Shorea leprosula Shorea macroptera Shorea mecistopteryx 98 95 69 Shorea mecistopteryx Shorea ovalis 69 63 Shorea ovalis Shorea palembanica 63 Shorea palembanica Shorea parvifolia Shorea parvifolia Shorea platyclados Shorea platyclados Shorea xanthophylla 67 <50 Shorea xanthophylla Shorea balangeran Shorea balangeran Shorea selanica 50 Shorea selanica 59 Shorea splendida Shorea splendida Shorea macrophylla Shorea macrophylla 58 Shorea pinanga 55 Shorea pinanga 50 Shorea stenoptera <50 Shorea stenoptera Shorea acuminatissima Shorea acuminatissima Shorea dasyphylla Shorea dasyphylla Shorea faguetiana Shorea faguetiana 77 82 Shorea multiflora Shorea multiflora 15 Shorea fallax Shorea fallax Shorea materialis Shorea materialis 98 98 Shorea virescens Shorea virescens 60 61 Phylogeny of Dipterocarpaceae Diagnostic characters of endemic species Monotes kerstingii Cotylelobium lanceolatum Upuna borneensis Anisoptera costata Upuna borneensis Anisoptera marginata 68 99 Anisoptera reticulata Vatica bantamensis Vatica bella 99 Vatica granulata Vatica pauciflora 83 94 Vatica rassak Vatica venulosa Dipterocarpus grandiflorus Dipterocarpus oblongifolius 71 Dipterocarpus retusus 100 Dipterocarpus rigidus Dipterocarpus tempehes Dryobalanops aromatica 99 Dryobalanops lanceolata Hopea bancana Hopea celebica Hopea odorata 85 Hopea sangal <50 Hopea dryobalanoides Hopea nigra <50 Hopea griffithii 66 Hopea mengarawan Anisoptera reticulata 100 Parashorea lucida Parashorea globosa Shorea blumutensis Shorea guiso Shorea montigena Shorea scaberrima Shorea seminis Shorea acuminata Shorea andulensis Shorea javanica 67 Shorea johorensis Shorea leprosula Shorea macroptera 95 69 Shorea mecistopteryx 63 Shorea ovalis Shorea palembanica Shorea parvifolia Shorea platyclados <50 Shorea xanthophylla Shorea balangeran 59 Shorea selanica Shorea splendida Shorea macrophylla 55 Shorea pinanga <50 Shorea stenoptera Shorea acuminatissima Shorea dasyphylla 77 Shorea faguetiana Shorea multiflora 16 Shorea fallax Shorea fallax Shorea materialis 98 60 Shorea virescens Phylogeny of Dipterocarpaceae Phylogeny of tribe Dipterocarpae l Based on sequence variation of two cpDNA regions – trnL Intron – trnL-F intergenic spacer 17 (Nga, 2009) Phylogeny of Dipterocarpaceae Molecular phylogeny based on AFLPs Dipterocarpus retusus Dipterocarpus oblongifolius Dipterocarpus rigidus Dipterocarpus tempehes Dipterocarpus glandiflorus 1 Dipterocarpus glandiflorus 2 Anisoptera reticulata Anisoptera marginata Anisoptera costata 1 Anisoptera costata 2 Cotylelobium lanceolatum Upuna borneensis Vatica bantamensis 1 Vatica bantamensis 2 Vatica venulosa Vatica bella Vatica granulata Vatica rassak Vatica pauciflora 1 Vatica pauciflora 2 Hopea celebica Hopea bancana 1 Hopea bancana 2 Hopea sangal 1 The phylogeny based on morphology Hopea sangal 2 Hopea griffithii Hopea nigra Hopea dryobalanoides 1 and cpDNA variation is also reflected at Hopea dryobalanoides 2 Hopea mengarawan 1 Hopea mengarawan 2 Parashorea lucida AFLPs, indicating genome-wide Parashorea globosa Hopea odorata 1 Hopea odorata 2 Shorea seminis differentiation pattern congruent with Shorea macroptera Shorea montigena Shorea andulensis Shorea balangeran variation at cpDNA Shorea selanica 1 Shorea selanica 2 Shorea xanthophylla 1 Shorea xanthophylla 2 Shorea materialis Shorea macrophylla 1 Shorea macrophylla 2 Shorea splendida 1 Shorea splendida 2 Shorea mecistopteryx 1 Shorea mecistopteryx 2 Shorea mecistopteryx 3 Shorea scaberrima Shorea palembanica 1 Shorea palembanica 2 Shorea leprosula 1 Shorea leprosula 2 Shorea parvifolia 1 Shorea parvifolia 2 Shorea guiso 1 Shorea guiso 2 Shorea platyclados 1 Shorea platyclados 2 Shorea ovalis 1 Shorea ovalis 2 Shorea faguetiana Shorea acuminatissima Shorea javanica Shorea johorensis 1 Shorea johorensis 2 Shorea blumutensis 1 Shorea blumutensis 2 Shorea acuminata 3 Shorea acuminata 1 Shorea acuminata 2 Dryobalanops aromatica Dryobalanops lanceolata 1 Dryobalanops lanceolata 2 Shorea virescens 1 Shorea virescens 2 18 1 (from CAO et al., 2006a) Intraspecific diversity in Indonesian Shorea spp. Sampling locations Indonesia Sumatra SLB (7, 8, 9, 10, 11) Borneo NS (1, 2, 3, 4, 5, 6) Java 19 Diversity within Shorea species Genetic diversity within populations of nine Shorea species in Indonesia at AFLP loci Pop. ID Distribution Sample size Polymorphic loci PPL na ne He I 1. Spar_NS common 26 38 44.71% 1.447 1.176 0.110 0.174 2. Sacu_NS common 32 42 49.41% 1.494 1.159 0.100 0.162 3. Sdas_NS scattered 20 47 55.29% 1.553 1.273 0.164 0.251 4. Sblu_NS rare 21 53 62.35% 1.624 1.266 0.165 0.257 5. Slep_NS common 16 36 42.35% 1.424 1.224 0.134 0.204 6. Smac_NS common 26 45