GENOMIC RESOURCES ARTICLE Complete plastome sequences from Bertholletia excelsa and 23 related species yield informative markers for Lecythidaceae Ashley M. Thomson1,2*, Oscar M. Vargas1* , and Christopher W. Dick1,3 Manuscript received 1 October 2017; revision accepted PREMISE OF THE STUDY: The tropical tree family Lecythidaceae has enormous ecological and 11 January 2018. economic importance in the Amazon basin. Lecythidaceae species can be difficult to identify 1 Department of Ecology and Evolutionary Biology, University of without molecular data, however, and phylogenetic relationships within and among the most Michigan, Ann Arbor, Michigan 48109, USA diverse genera are poorly resolved. 2 Faculty of Natural Resources Management, Lakehead University, Thunder Bay, Ontario P7B 5E1, Canada METHODS: To develop informative genetic markers for Lecythidaceae, we used genome 3 Smithsonian Tropical Research Institute, Panama City skimming to de novo assemble the full plastome of the Brazil nut tree (Bertholletia excelsa) 0843-03092, Republic of Panama and 23 other Lecythidaceae species. Indices of nucleotide diversity and phylogenetic signal 4 Author for correspondence: [email protected] were used to identify regions suitable for genetic marker development. *These authors contributed equally to this work. RESULTS: The B. excelsa plastome contained 160,472 bp and was arranged in a quadripartite Citation: Thomson, A. M., O. M. Vargas, and C. W. Dick. 2018. structure. Using the 24 plastome alignments, we developed primers for 10 coding and non- Complete plastome sequences from Bertholletia excelsa and 23 related species yield informative markers for Lecythidaceae. coding DNA regions containing exceptional nucleotide diversity and phylogenetic signal. We Applications in Plant Sciences 6(5): e1151. also developed 19 chloroplast simple sequence repeats for population- level studies. doi:10.1002/aps3.1151 DISCUSSION: The coding region ycf1 and the spacer rpl16-rps3 outperformed plastid DNA markers previously used for barcoding and phylogenetics. Used in a phylogenetic analysis, the matrix of 24 plastomes showed with 100% bootstrap support that Lecythis and Eschweilera are polyphyletic. The plastomes and primers presented in this study will facilitate a broad array of ecological and evolutionary studies in Lecythidaceae. KEY WORDS Amazonian trees; Bertholletia excelsa; DNA barcoding; genetic markers; Lecythidaceae; plastome. Lecythidaceae (sensu lato) is a pantropical family of trees with et al., 2013). The most species-rich genus, Eschweilera Mart. ex DC., three subfamilies: Foetidioideae, which is restricted to Madagascar; with approximately 99 species (Mori, 2017), is also the most abun- Barringtonioideae, found in the tropical forests of Asia and Africa; dant tree genus in the Amazon basin (ter Steege et al., 2013), and E. and the Neotropical clade Lecythidoideae, which contains ap- coriacea (DC.) S. A. Mori is the most common tree species in much proximately 234 of the approximately 278 known species in the of Amazonia (ter Steege et al., 2013). Lecythidaceae provide impor- broader family (Mori et al., 2007, 2017; Huang et al., 2015; Mori, tant ecological services such as carbon sequestration and are food 2017). Neotropical Lecythidaceae are understory, canopy, or emer- resources for pollinators (bats and large bees) and seed dispersers gent trees with distinctive floral morphology and woody fruit cap- (monkeys and agouties) (Prance and Mori, 1979; Mori and Prance, sules. Among Lecythidaceae species are the iconic Brazil nut tree, 1990). Bertholletia excelsa Bonpl.; the oldest documented angiosperm tree, Tools for species- level identification and phylogenetic analyses of Cariniana micrantha Ducke (dated at >1400 years old in Manaus, Lecythidaceae could significantly advance research on Amazon tree Brazil; Chambers et al., 1998); the cauliflorous cannonball tree diversity. However, despite their ease of identification at the family commonly grown in botanical gardens, Couroupita guianensis level, species- level identification of many Lecythidaceae (especially Aubl.; and important timber species (e.g., Cariniana legalis (Mart.) Eschweilera) is notoriously difficult when based on sterile (i.e., with- Kuntze). Lecythidaceae is the third most abundant family of trees in out fruit or floral material) herbarium specimens, and flowering of the Amazon forest, following Fabaceae and Sapotaceae (ter Steege individual trees often occurs only at multi-year intervals (Mori and Applications in Plant Sciences 2018 6(5): e1151; http://www.wileyonlinelibrary.com/journal/AppsPlantSci © Thomson et al. Applications in Plant Sciences is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 1 of 11 Applications in Plant Sciences 2018 6(5): e1151 Thomson et al.—Lecythidaceae plastome markers • 2 of 11 Prance, 1987). As a complement to other approaches, DNA barcod- as supplemental barcodes for vascular plants (Kress et al., 2005; ing (Dick and Kress, 2009; Dexter et al., 2010) may help to identify Lahaye et al., 2008; Li et al., 2011). However, an evaluation of a sub- species or clades of Lecythidaceae. set of these markers (ITS, psbA-trnH, matK, rbcL, rpoB, rpoC1, and A combination of two protein-coding plastid regions (matK trnL) on Lecythidaceae in French Guiana (Gonzalez et al., 2009) and rbcL) has been proposed as a core plant DNA barcode showed poor performance for species identification. Furthermore, (Hollingsworth et al., 2009), although other coding and non- the use of traditional markers (plastid ndhF, trnL-F, and trnH-psbA, coding plastome regions (psbA-trnH, rpoB, rpoC1, trnL, and ycf5) and nuclear ITS) for phylogenetic analysis has produced weakly and the ITS of nuclear ribosomal genes have been recommended supported trees (Mori et al., 2007; Huang et al., 2015), indicating a C C trnS-GGA trnG-U psbZ trnL trnF-G A -U trnM-CAU AA AA psbC psbD petN trnC-GC trnT-GGU psa yc rbcL rps4 trnT-UGU f aB 3 A accD ndhJ ps t rn atp rnS-UGA psaI 4 V t a U ycf4 tp E -U s1 B A ce ndhK CA rp - m C ndhC C A A pet C oB rp A trnfM psbM rnE-UU t petL trnY-GU pet LSC trnD-GU G rpoC1 2 C psa o rpl33J rp rps18 psbJ p s rps2 psbFbL psbE atpI trnW-C rpl20 trnP atpH -U C rps12 G A atpF UCU G trnR- trnG-GCC clpP atpA psbB psbT psbH psbI psbK petB psbN trnS-GCU trnQ-UUG petD rps16 rpoA Bertholletia excelsa rps11 rpl36 infA trnK-UUU rps8 chloroplast genome matK rpl14 rpl16 psbA rps3 160,472 bp trnH-GUG trnH-GUG rpl22 rp rps19 s3 rpl2 rpl22 3 rpl2 U rps19 trnI-CA rpl2 rpl23 trnI-CAU ycf2 IR A B IR ycf2 yc f15 trnL-CAA ndhB trnL-CAA ycf15 rps7 trnV rps7 - G nd SSC trn t rr AC hB r t rnI-GAU R r rn n n4.5 1 -A A 6 rps rrn - CG U rps1 7 5 G C 2 orf42 rrn23 G h psaC ndhE GAC nd 16 n trnN-GUU ycf rr r trnV- n ps15 nd dh 1 ndhI C ndhF GAU hA - H G dhD U n trnI trnA- rrn23 t photosystem I rnN-GUU photosystem II 1 ycf 5 cytochrome b/f complex . 2 4 n G rrn5 ATP synthase rr C rpl3 ccsA NADH dehydrogenase RuBisCO large subunit trnR-A trnL-UAG RNA polymerase ribosomal proteins (SSU) ribosomal proteins (LSU) clpP, matK other genes hypothetical chloroplast reading frames (ycf) ORFs transfer RNAs ribosomal RNAs FIGURE 1. Plastome map of the Brazil nut tree, Bertholletia excelsa. Genes outside of the circle are transcribed clockwise; genes inside of the circle are transcribed counterclockwise. Gray bars in the inner ring show the GC content percentage. http://www.wileyonlinelibrary.com/journal/AppsPlantSci © 2018 Thomson et al. Applications in Plant Sciences 2018 6(5): e1151 Thomson et al.—Lecythidaceae plastome markers • 3 of 11 need to develop more informative markers and/or increase molec- edulis Seem.) from the Barringtonioideae. The sampling included ular sampling. all 10 Lecythidoideae genera (Appendix 1). Silica-dried leaf tissue The main objectives of this study were to (1) assemble, anno- from herbarium- vouchered collections was collected by Scott Mori tate, and characterize the first complete plastome sequence of and colleagues and loaned by the New York Botanical Garden. Total Lecythidaceae from the iconic Brazil nut tree, B. excelsa; (2) obtain genomic DNA was extracted from 20 mg of dried leaf tissue using a robust backbone phylogeny for the Neotropical clade using newly the NucleoSpin Plant II extraction kit (Machery-Nagel, Bethlehem, assembled draft plastome sequences for an additional 23 species; Pennsylvania, USA) with SDS lysis buffer. Prior to DNA library and (3) develop a novel set of informative molecular markers for preparation, 5 μg of total DNA was fragmented using a Covaris DNA barcoding and broader evolutionary studies. S- series sonicator (Covaris Inc., Woburn, Massachusetts, USA) following the manufacturer’s protocol to obtain approximately 300- bp insert sizes. We prepared the sequencing library using the METHODS NEBNext DNA library Prep Master Mix and Multiplex Oligos for Illumina Sets (New England BioLabs Inc., Ipswich, Massachusetts, Plant material and DNA library preparation USA) according to the manufacturer’s protocol. Size selection was carried out prior to PCR using Pippin Prep (Sage Science, Beverly, We performed genomic skimming on 24 Lecythidaceae species, in- Massachusetts, USA). Molecular mass of the finished paired-end cluding 23 Lecythidoideae and one