Exposing the Illegal Trade in Cycad Species (Cycadophyta: Encephalartos) at Two Traditional Medicine Markets in South Africa Using DNA Barcoding1 J

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771 ARTICLE Exposing the illegal trade in cycad species (Cycadophyta: Encephalartos) at two traditional medicine markets in South Africa using DNA barcoding1 J. Williamson, O. Maurin, S.N.S. Shiba, H. van der Bank, M. Pfab, M. Pilusa, R.M. Kabongo, and M. van der Bank

Abstract: Species in the cycad genus Encephalartos are listed in CITES Appendix I and as Threatened or Protected Species in terms of South Africa’s National Environmental Management: Biodiversity Act (NEM:BA) of 2004. Despite regulations, illegal plant harvesting for medicinal trade has continued in South Africa and resulted in declines in cycad populations and even complete loss of sub-populations. Encephalartos is traded at traditional medicine markets in South Africa in the form of bark strips and stem sections; thus, determining the species traded presents a major challenge due to a lack of characteristic plant parts. Here, a case study is presented on the use of DNA barcoding to identify cycads sold at the Faraday and Warwick traditional medicine markets in Johannesburg and Durban, respectively. Market samples were sequenced for the core DNA barcodes (rbcLa and matK) as well as two additional regions: nrITS and trnH-psbA. The barcoding database for cycads at the University of Johannesburg was utilized to assign query samples to known species. Three approaches were followed: tree-based, similarity-based, and character-based (BRONX) methods. Market samples identified were Encephalartos ferox (Near Threatened), Encephalartos lebomboensis (Endangered), Encephalartos natalensis (Near Threatened), Encephalartos senticosus (Vulnerable), and Encephalartos villosus (Least Concern). Results from this study are crucial for making appropriate assessments and decisions on how to manage these markets. Key words: cycads, core barcoding regions, muthi, nrITS, trnH-psbA. Résumé : Les espèces appartenant au genre Encephalartos des cycadales figurent a` l’Annexe I de la convention For personal use only. CITES en tant qu’espèces menacées ou protégées sur la base d’une loi de 2004 en Afrique du Sud (« National Environmental Management : Biodiversity Act », ou NEM : BA). En dépit de cette règlementation, des activités illégales de récolte pour des fins médicinales se sont poursuivies en Afrique du Sud et ont entraîné un déclin dans les populations de cycadales et même une perte complète de certaines sous-populations. L’Encephalartos est vendu dans les marchés de remèdes traditionnels en Afrique du Sud sous forme de bandes d’écorce et de sections de tiges; il est ainsi très difficile d’identifier les espèces vendues en raison de l’absence de parties végétales pouvant servir a` l’identification. Dans ce travail, les auteurs présentent une étude de cas sur l’emploi des codes a` barres de l’ADN pour l’identification des cycadales vendues dans les marchés de remèdes traditionnels Faraday et Warwick, respectivement situés a` Johannesbourg et Durban. Les échantillons de ces marchés ont été séquencés pour les séquences usuelles (rbcLa et matK) ainsi que pour deux régions addition- nelles (nrITS et trnH-psbA). La base de données pour les codes a` barres des cycadales a` l’Université de Johan- nesbourg a été employée pour assigner les séquences des échantillons a` des espèces connues. Trois approches ont été explorées : les méthodes fondées sur les arbres, la similarité et les caractères (BRONX). Les échantillons

Received 2 February 2016. Accepted 13 May 2016. Corresponding Editor: Sarah Adamowicz.

Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 J. Williamson,* O. Maurin,* S.N.S. Shiba, M. Pilusa, R.M. Kabongo, and M. van der Bank. The African Centre for DNA Barcoding, Department of Botany & Plant Biotechnology, University of Johannesburg, APK Campus, P.O. Box 524, Auckland Park, 2006, South Africa. H. van der Bank. The African Centre for DNA Barcoding, Department of Zoology, University of Johannesburg, APK Campus, P.O. Box 524, Auckland Park, 2006, South Africa. M. Pfab. South African National Biodiversity Institute, Pretoria National Botanical Garden, P/Bag X101, Silverton, 0184, South Africa. Corresponding author: M. van der Bank (email: [email protected]). *These authors contributed equally to this work. 1This paper is part of a special issue entitled Barcodes to Biomes. Copyright remains with the author(s) or their institution(s). This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Genome 59: 771–781 (2016) dx.doi.org/10.1139/gen-2016-0032 Published at www.nrcresearchpress.com/gen on 13 July 2016. 772 Genome Vol. 59, 2016

ont été identiﬁés comme appartenant aux espèces suivantes : Encephalartos ferox (presque menacée), Encephalartos lebomboensis (en danger), Encephalartos natalensis (presque menacée), Encephalartos senticosus (vulnérable) et Encephalartos villosus (préoccupation mineure). Les résultats obtenus seront essentiels pour l’évaluation et la prise de décisions en ce qui a trait a` la gestion de ce commerce. [Traduit par la Rédaction] Mots-clés : cycadales, régions typiques de codage a` barres, muthi, nrITS, trnH-psbA.

Introduction 2007), which in some instances include cycads, puts cy- Cycads, with a fossil record dating back to the early cad populations now further at risk. Approximately two- Permian period (Norstog and Nicholls 1997; Brenner thirds (25 species) of SA’s Encephalartos spp. (known as et al. 2003), are the world’s oldest extant group of seed “isiGqiki-somkovu” in Zulu) are collected for traditional plants and have survived three mass extinction events in medicinal purposes and illegally traded at traditional earth’s history. Furthermore, cycads are classified as the medicine (TM) markets, mainly for the protection they world’s most threatened plant group (Donaldson 2011). offer from evil spirits (Cousins et al. 2011, 2012). Large arbo- Yet, conservation of cycads presents a challenge as rescent species appear to be harvested by removing bark threats such as habitat transformation (e.g., replacement strips from adult individuals, whereas smaller arborescent by commercial crops), illegal trade, and harvesting activ- and subterranean species are harvested by removing the ities subject them to human-induced extinction (Donaldson entire plant, stripping off all its characteristic features and Bösenberg 1999; Donaldson 2008). (Donaldson 2008; Cousins et al. 2012). This makes identifi- African cycads are represented by three genera: Cycas cation very challenging or impossible in many cases. (only Cycas thouarsii R.Br. distributed in eastern Africa), Previous studies described the trade of cycads at urban Encephalartos Lehm., and the monotypic genus Stangeria TM markets, but only morphological-based identifica- T. Moore. Encephalartos, comprised of 68 species and sub- tions were used, and in several cases plants could not be species, is endemic to Africa, with South Africa (SA) being identified to species level (Cousins et al. 2012, 2013; a diversity hotspot, with 37 species. Of these, approxi- Williams et al. 2014). Here, we used DNA barcoding to mately 70% are threatened with extinction (Retief et al. determine the diversity of cycads sold at SA’s two largest 2014). Due to risk associated with international trade in TM markets (Faraday in Johannesburg and Warwick in wild-collected specimens, all SA Encephalartos spp. are Durban). The value of applying DNA barcoding to cycad listed in CITES (Convention on International Trade in identification and the conservation implications of the Endangered Species of Wild Fauna and Flora) Appendix I. results are discussed. This number includes three Extinct in the Wild (EW), For personal use only. Material and methods 12 Critically Endangered (CR), four Endangered (EN), seven Near Threatened (NT), eight Vulnerable (VU), and three Sampling Our sample strategy involved sampling all the stalls Least Concern (LS) species (http://redlist.sanbi.org). Notably, where cycads were sold to ensure all possible Encephalartos two of the three species (Encephalartos brevifoliolatus Vorster spp. present at the TM markets were collected. Only openly and Encephalartos nubimontanus P.J.H. Hurter) that are classi- displayed products were purchased from the traders. The fied as EW acquired this Red List Status between 2003 and price per bark/stem fragment (160 cm × 10 cm) varied 2010 (Cousins et al. 2012). Furthermore, Encephalartos cerinus between 30 and 40 ZAR (2.60–3.47 CAD). In total, 45 un- Lavranos & D.L. Goode, Encephalartos inopinus R.A. Dyer, known cycad bark specimens were obtained between Encephalartos latifrons Lehm., and Encephalartos msinganus January and May 2015 from 12 stores at Faraday TM mar- Vorster, listed as CR, have population sizes in the wild of ket in Johannesburg, Gauteng Province, and 20 stores at less than 100 individuals (Donaldson 2008). Consequently, the Warwick TM market, located on the periphery of SA is now at risk of losing 50% of its cycad species within the Durban Central Business District, KwaZulu-Natal Prov- next 2–10 years. The South African National Biodiversity ince (SA; Fig. 1). Warwick is the largest TM market in the Institute (SANBI) identified this as the “South African cycad country, with nearly 500 traders (Mander 1998), whereas extinction crisis” (Cousins et al. 2012). Faraday is the second largest, with over 400 traders and Continuous trading of South African Encephalartos spp.,

Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 the wholesale epicenter for the trade in traditional med- predominantly for the horticultural trade, has led to a icine in Gauteng (Williams et al. 2007a, 2007b). All spec- rapid decline of wild populations (Donaldson and Bösenberg imens collected were processed and analyzed at the 1999). Wild cycad populations are particularly vulnera- African Centre for DNA Barcoding (ACDB) at the Univer- ble to harvesting due to the slow growth rate of individ- sity of Johannesburg. uals. Habitat loss plays a minor role in depletion of populations, whereas invasive species have a direct im- DNA extraction, ampliﬁcation, and sequencing pact on approximately 10% of cycad habitats (Donaldson DNA was extracted from 0.2–0.8 g of bark material 2008). Furthermore, with an estimated 27 million users of using the 2× CTAB method described by Doyle and Doyle traditional plant-based treatments in SA (Mander et al. (1987). Polyvinyl pyrolidone (2%) was added to reduce the

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Fig. 1. (A) A stand with various plant products at the Faraday traditional medicine (TM) market, Johannesburg, South Africa (Credit: Zandisile Shongwe). (B) A fresh fragment of Encephalartos trunk available at the TM market (Credit: Olivier Maurin). (C) Conﬁscated material following illegal harvesting for the horticultural trade of mature Encephalartos specimens (Credit: Eastern Cape Department of Economic Development, Environmental Affairs and Tourism). (D) Results of illegal harvesting on mature Encephalartos specimen in the wild (Credit: Mpumalanga Tourism and Parks Agency).

For personal use only. effect of high polysaccharide concentrations in the sam- annealing (Savolainen et al. 1995). PCR amplifications ples. To avoid problems of PCR inhibition, all DNA sam- were done using the following programs: for matK, rbcLa, ples were purified using QIAquick purification columns and trnH-psbA, pre-melt at 95 °C for 2:30 min, denatur- (Qiagen Inc., Hilden, Germany) according to the manu- ation at 95 °C for 30 s, annealing at 55 °C for 30 s, exten- facturer’s protocol. sion at 72 °C for 2 min (for 35 cycles), followed by a final Polymerase chain reaction (PCR) amplifications were extension at 72 °C for 10 min; for nrITS the protocol done using the core DNA barcodes (matK and rbcLa) and consisted of pre-melt at 94 °C for 2 min, denaturation at the additional regions trnH-psbA and nrITS recommended to 94 °C for 1 min, annealing at 52 °C for 1 min, extension at enhance the performance of the core barcodes (CBOL 72 °C for 3 min (for 28 cycles), followed by a final exten- Plant Working Group 2009; Hollingsworth et al. 2009). sion at 72 °C for 7 min. Amplified products were purified Primer pairs used for the PCR amplification of matK, using QIAquick columns (Qiagen Inc., Germany) follow- rbcLa, and trnH-psbA regions were GYM F1A:GYM R1A ing the manufacturer’s protocol. (Hollingsworth et al. 2011), rbcLa-F (Levin et al. 2003): Bidirectional cycle sequencing reactions were performed rbcLa-R (Kress and Erickson 2007), and NY1493:NY1494 using the BigDye Terminator V3.1 kit (Applied Biosys- (Sass et al. 2007), respectively. The nrITS region (internal tems, Inc. Warrington,® Cheshire, UK). Cycle sequencing transcribed spacer 1, 5.8S ribosomal RNA, and internal transcribed spacer 2) was amplified in two overlapping products were precipitated in ethanol and sodium ace- Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 pieces using two internal primers with a pair of external tate to remove excess dye terminators before sequencing primers: NY90F:NY729R and 728F:514R (White et al. 1990; on an ABI 3130xl genetic analyser. Complementary strands Sun et al. 1994). All reactions were done using Ready-Mix were assembled and edited using Sequencher v.5.1 (Gene Mastermix (Advanced Biotechnologies, Epsom, UK), with Codes Crop., Ann Arbor, Mich., USA). All protein-coding the addition of 4.5% of dimethylsulphoxide to minimize regions were translated to verify that no stop codons problems with secondary structures and improve an- were present. Alignment was performed for trnH-psbA nealing (Palumbi 1996). We also added 3.2% bovine se- and nrITS using Multiple Sequence Comparison by Log- rum albumin to all PCR reactions to stabilize enzymes, Expectation (MUSCLE v.3.8.31; Edgar 2004) and adjusted reduce problems with secondary structure, and improve manually in PAUP* (version 4.0b10; Swofford 2002). The

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alignments used for further analysis are available as sup- tion, a sequence of each complete sample was queried plementary data, Data file S12. against this reference database. We employed the scoring function of one for each matching pretext/text/postext Encephalartos DNA barcode library combination. Thus, the score for each query could vary An Encephalartos DNA barcode library was prepared between 0 and 1, where the reference terminal with the and used to identify the unknown TM market samples. highest score is considered the correct identification This library was assembled from 260 taxa (excluding (Little 2011). market samples) representing 48 southern African species of known provenance that were identified by an Data analysis of query market samples Encephalartos taxonomist, Piet Vorster. We have also con- Three approaches were followed to assign query mar- sulted with various authorities from the Environmental ket samples to known species, namely tree-based method, Management Inspectorate (EMI) and the Green Scorpions similarity-based method, and character-based method. In the (Environmental law enforcement) based in the different tree-based method, query samples were assigned to spe- provinces to assist with and verify species identifica- cies if they form a monophyletic cluster with identified tions. Between two and six individuals per species were specimens in the Encephalartos DNA barcode tree. For this sampled to incorporate species-level variation into the approach we used maximum parsimony (MP; Farris 1972) sampling. Specimens were prepared into herbarium and Bayesian inference (BI; Huelsenbeck and Ronquist vouchers and archived at the South African National Bio- 2001; Ronquist and Huelsenbeck 2003). Lepidozamia Lehm. diversity Institute (SANBI), Pretoria (PRE), and the her- was used as the outgroup in the analysis as its phylo- barium at the University of Johannesburg (JRAU), with genetic placement has consistently been resolved as sister the collection and sequence data deposited in the Bar- to Encephalartos (Treutlein and Wink 2002; Hill et al. 2003; code of Life Data Systems (BOLD; www.boldsystems.org; Bogler and Francisco-Ortega 2004; Chaw et al. 2005; Sangin Ratnasingham and Hebert 2007). Although the barcode et al. 2008; Zgurski et al. 2008). Maximum parsimony library for this study contained 48 of the estimated 68 using PAUP* v. 4.0b1 (Swofford 2002) was implemented to Encephalartos spp. and subsp. occurring in Africa, all of analyze the combined dataset (matK, rbcLa, trnH-psbA, and the 37 species occurring in SA are included, and all other nrITS). Tree searches were conducted using 1000 repli- Encephalartos spp. occurring in the region have been bar- cates of random taxon addition, retaining 10 trees at coded and are available on BOLD (Rousseau 2012). Voucher each step, with tree-bisection-reconnection (TBR) branch specimen information and GenBank accession numbers swapping and MulTrees in effect. Support for clades was are listed in the supplementary data, Table S12. estimated using bootstrap analysis (Felsenstein 1985) with To test the efficiency of the Encephalartos DNA barcode 1000 replicates, simple sequence addition, TBR swapping,

For personal use only. library, we calculated species discrimination using BRONX with MulTrees in effect, but saving a maximum of version 2.0 (Barcode Recognition Obtained with Nucleo- 10 trees per replicate. The following categories were tide eXposé’s; Little and Stevenson 2007; Little 2011), used to classify bootstrap support (BS): low (50%–74%), which is an alignment-free algorithm that relies on the moderate (75%–84%), and high/strong (85%–100%). De- presence/absence of particular characters in the barcode layed transformation character optimization (DELTRAN) sequences for identification, instead of using them all was used to calculate branch lengths, due to reported (Van Velzen et al. 2012), thereby accounting for within- errors with accelerated transformation optimization taxon variation (Little 2011). For this, a unified reference (ACCTRAN) in PAUP* v.4.0b1 (http://paup.csit.fsu.edu/ database was constructed for southern African Encephalartos problems.html). spp. using all four plant DNA markers. The generated Bayesian inference (Huelsenbeck and Ronquist 2001; markers in the dataset were combined using 18 “N” codes Ronquist and Huelsenbeck 2003) was performed using insulation between the marker sequences. No context MrBayes v.3.1.3, as implemented on the Cyber infrastruc- combinations that included IUPAC ambiguity codes were ture for Phylogenetic Research (CIPRES) Science Gateway used. The combined Encephalartos dataset was reduced to v.3.1 (Miller et al. 2009; www.phylo.org) on the combined reference sequences of a series of characters defined by data set. Prior to BI, a hierarchical likelihood ratio test flanking pretext and postext of the default six base implemented in MODELTEST v.3.06 (Posada and Crandall

Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 lengths using the fasta2bdb.pl function. This sidelines 1998) was used to determine the appropriate substitu- the alignment difﬁculties assigned to variable markers (i.e., tion model for each of the four gene regions sequenced trnH-psbA and nrITS). The resulting BRONX character data- based on the Akaike information criterion (AIC; Sugiura base comprised of a list of terminals consisting of all 1978). The following models were selected: rbcLa =HKY+Y, observed sequence fragments and their ﬂanking context matK =TVM+G,trnH-psbA =HKY+I+G,nrITS = K81uF + (pretext and postext). To calculate species discrimina- I + G. A partitioned analysis was run since different models

2Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/gen- 2016-0032.

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were selected. Analyses were run with 2 000 000 generations 94); samples St004_1, St004_2, St004_3, St014_1, St014_2, with a sampling frequency of 200. The log-likelihood scores and St014_3 clustered with Encephalartos natalensis R.A. Dyer were plotted against their likelihoods to determine the & I. Verd. (BI = 0.85); the remaining samples (St008_1, point where likelihoods converged on a maximum value, St032_1, St032_2, St032_3, St039a_1, St039b_1, St101_1, and the trees before the convergence were discarded as St101_1, St102_1, St102_3, St103_1, St103_2, St117a_1, and burn in (1000 trees). The remaining trees were imported St117b_1) are allied with Encephalartos aemulans Vorster, into PAUP*v.4.0b10 to generate a majority-rule consensus Encephalartos lebomboensis I. Verd., and Encephalartos senticosus tree showing posterior probabilities (PP) of all the ob- Vorster (BI = 0.73). served bi-partitions. The following scale was used to eval- uate the posterior probability values (PPs): well supported Blast results (0.95–1.0) and weakly supported (<0.95). The identification success was dependent on the marker 2 The NCBI’s MegaBlast algorithm (www.blast.ncbi.nlm. (Table S3 ). The highest-scoring hit from each query on the nih.gov/Blast.cgi), using the default setting, was used to basis of E-value and maximum identity was taken as the assign query barcodes to species. Identification was made barcoding identification. All query samples could be taking both the E-value and maximum identity into con- identified to genus level, whereas species identification sideration. was ambiguous in 54% of the cases as two or more equally Lastly, we used the character-based method BRONX high-scoring hits were assigned per query. The trnH-psbA (version 2.0) of Little (2011) to assign query market sam- region did not show promise as a barcoding locus for ples to known species. The function Bronx.pl was used to Encephalartos because of its inability to provide specific iden- match the context of the query sequence with the corre- tification for taxa; in all cases when using trnH-psbA, a dif- sponding invariant flanking context in the reference ferent species was identified that did not match what was database. On the basis of these flanking regions, the vari- found when using rbcLa, matK, and (or) nrITS. Furthermore, able regions were scored in the query sequence integrat- trnH-psbA also occurs in more than one copy in cycads (Sass ing the variability observed within the taxon in the et al. 2007). scoring procedure. The technique in effect imitates the BRONX results procedure used in conventional morphological system- The combined Encephalartos dataset displays an aver- atics, where each composite exposé is equivalent to a age sequence length of 3474 base pairs (bp) with the taxonomic morphological description and where some shortest fragment at 1785 bp and the longest at 3784 bp, characters provide context for others (Little 2011). representing 48 species of the genus Encephalartos. The Results 45 combined (rbcLa, matK, trnH-psbA, and nrITS) query

For personal use only. market samples displayed a mean sequence length of DNA barcode success and efficacy of Encephalartos DNA barcode library 3098 bp with the minimum at 1269 bp and the maximum Recovery of DNA barcodes from TM market products was at 3631 bp. Overall, no individual query sequences matched effective, with sequencing success for rbcLa, matK, trnH- the length of the longest sequences in the reference data- psbA, and nrITS being 100%, 100%, 89%, and 89%, respec- base. Plant material poorly conserved is routinely ex- tively. Of the 46 Encephalartos spp. represented by two or pected at TM markets and greatly influence the fragment more individuals in the complete sample (Reference library, size generated. However, for this specific reason the with 2 rbcLa and 10 trnH-psbA sequences lacking in the data- BRONX analysis is explicitly designed to use only unam- set), BRONX could distinguish 40 species (87%) from all biguous context/text in the scoring, which caters for the other species using a combination of the four above- imperfect overlaps between the query and reference data- mentioned markers (Table S22). base (Little 2011). This is adequately reflected in the results (Fig. 3; Table S42), as genus-level tests of identifi- Tree-based results cation were 100% successful, with a species-level identifi- The parsimony analysis of the combined dataset (rbcLa, cation success rate of over 90%. Query sequences JW4, JW5, matK, trnH-psbA, and nrITS) resulted in four equally par- JW8, and JW24 yielded ambiguous species-level identifi- simonious trees (tree length, 675 steps; CI = 0.75; RI = cations. This can be attributed to the lack of nrITS se- 0.95). Of the 4068 included characters, 3604 (88.6%) were

Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 quences for these samples. constant, and 464 (11.4%) were variable. Of the variable sites, 305 (7.5% of the total) were potentially parsimony Encephalartos spp. identified at Faraday and the Warwick informative. The combined MP analysis is congruent TM markets with the BI analysis, and therefore results are displayed Utilizing the barcoding database for cycads at the Uni- on the BI tree (Fig. 2). Market samples are retrieved in versity of Johannesburg and combining the tree-based, four major clades: all samples from Warwick are in- blast, and BRONX results, we positively identified five cluded in a clade containing Encephalartos aplanatus Vor- Encephalartos spp. currently traded at Faraday and the ster and Encephalartos villosus Lem. (BI = 1.0); sample St104_1 Warwick TM markets, viz., E. lebomboensis (EN; Lebombo grouped with Encephalartos ferox G. Bertol (BI = 1.0; BP = cycad), E. senticosus (VU; Jozini cycad), E. natalensis (NT;

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Fig. 2. The 50% majority-rule consensus tree obtained from the Bayesian inference (BI) analysis of the combined barcode dataset for reference library of southern African Encephalartos spp. plus market samples. We mapped the bootstrap values from the maximum parsimony (MP) tree onto the BI majority-rule consensus tree because the MP and BI analyses produced similar topologies. Numbers above nodes are BI posterior probabilities above 50%, and those below nodes are MP bootstrap support values. The phylogenetic position of market samples is indicated using red branches. For personal use only. Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16

Natal giant cycad), E. ferox (NT; Tongaland or Kozi cycad), the plant kingdom (Potgieter and Cresswell 1989). Very and E. villosus (LC; poor man’s cycad). similar to the rhino poaching crises that SA is currently facing is the devastating loss of cycads—a tragedy largely Discussion unnoticed. Encephalartos spp. face several threats, with As stated by Douglas Goode, considered to be the the largest being the illegal removal of adult plants from world’s ﬁnest cycad artist and a renowned expert on the the wild for private cycad collections, horticultural pur- group, cycads could be referred to as the “rhino horn” of poses, and as parental stock for seedling propagation for

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Fig. 3. Summary of BRONX results (supplementary data, Table S42) for Encephalartos spp. encountered at Faraday and Warwick traditional medicinal markets. Number in parenthesis indicates the number of samples identified. For personal use only. both domestic and international trade (Donaldson 2003, identifier for species and provides identification of a speci- 2008). Cycads have also been poached for use in TM mar- men to species even if only a small fragment of plant ma- kets where these plants are used for traditional purposes terial is available. Furthermore, the power and strength of (Cousins et al. 2012, 2013; Williams et al. 2014). Harvest- DNA barcoding has been demonstrated in many botanical ing of plant species from the wild for traditional medi- studies ranging from the discovery of unknown species, cine is having an adverse effect on biodiversity (Dold and resolving taxonomic problems, species adulteration in herbal Cocks 2002; Botha et al. 2004) and is mostly ignored by products (Srirama et al. 2010; Baker et al. 2012; Newmaster provincial and national conservation agencies, even though et al. 2013), and TM market surveys (Kool et al. 2012; Arun Dev results from several studies have shown that there is cur- et al. 2014); DNA barcoding has also proven to be of great rently a lucrative trade in cycad species at TM markets in utility in scrutinizing the illegal trade of endangered SA. For example, Cunningham and Davis (1997) found that plant species (Pryer et al. 2010; Subedi et al. 2013). How- 30% of the 54 TM shops investigated in KwaZulu-Natal ever, the taxonomic value of DNA barcoding has rarely Province sold Encephalartos, averaging a 50 kg size bag been tested on phylogenetically closely related species, annually; 6% of the 50 TM shops surveyed in the Johan- for example Encephalartos. Sass et al. (2007) assessed the nesburg area (Williams 2003) sold Encephalartos speci- efficacy of the approach across cycad genera and recog- mens. Cousins et al. (2011) reported that Encephalartos nized that none of the proposed markers (rbcLa and matK) Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 spp. were sold by 26.4% and 13.2% of traders at Faraday provided unique identifiers for all species tested and con- and Warwick TM markets, respectively, with an esti- cluded that nrITS showed the most promise in terms of mated nine metric tons traded at Warwick in 2009. variability. Nicolalde-Morejón et al. (2011) evaluated suc- Correctly identifying species at TM markets is a major cessful species-level molecular identification in the three challenge since plants are normally traded as plant parts extant cycad genera occurring in Mexico (Ceratozamia, Dioon, and plant-derived products, either separately or in mix- and Zamia) and found that at least three chloroplast re- tures. The emergence of DNA barcoding now provides a gions and one nuclear region were needed to achieve >70% tool to tackle this challenge. DNA barcoding involves using unique species identification. Also, the number of spe- a short, agreed-upon region of a genome as a unique cies within genera with horticultural appeal is often

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over-estimated. For instance, 894 species were initially Donaldson (2010d). It qualifies as EN due to the extent described for the genus Viburnum; this number has now of past decline and its narrow distribution range. The been drastically reduced to only 172 (Clement and Donoghue E. senticosus population has declined by >30% at least in 2012). Such over-estimation of species might also be pos- parts of its range (near Sitegi in Swaziland and Mkuze in sible within Encephalartos. SA) during the previous 60 years. There is evidence of In the current study, using DNA barcoding, five continuing decline, which means that it also qualifies as Encephalartos spp. were identified at Faraday and the VU under criterion C1 (Donaldson 2010e). Warwick TM markets, viz., E. ferox, E. lebomboensis, Both Crouch et al. (2003) and Cousins et al. (2012) iden- E. natalensis, E. senticosus, and E. villosus. All samples col- tified E. ghellinckii Lem. at the Warwick market, and this lected at Warwick TM market were identified as E. villosus species was also identified at Faraday along with E. ngoyanus (poor man’s cycad), which coincides with the findings of I. Verd. by Cousins et al. (2013). We did not encounter any Williams et al. (2014), i.e., that it is the most abundant other Encephalartos spp. at these two markets other than species sold as bark fragments at Warwick. Encephalartos the five species mentioned above. Moreover, we identi- villosus, presently categorized as LC (Donaldson 2010a), is fied an additional species sold at the Faraday market, one of the most common cycads in SA, with numerous E. lebomboensis, which was not encountered by Cousins large subpopulations throughout KwaZulu-Natal in easy et al. (2013). This might be due to the fact that TM traders access from/to Warwick medicinal market. It is evident are aware that the trade in cycads is illegal and therefore that many hundreds of plants have been taken from tend to hide their stock of cycad fragments. Also, their habitats; in the Eastern Cape, large habitat areas Encephalartos spp. sold at Faraday and Warwick could have been cleared for pineapple plantings, and in KwaZulu- have been overlooked or missed as our collections at the Natal many E. villosus habitats have been converted to two markets, especially at Warwick, were conducted as banana plantations. It should be noted that relative snap shots and not over a longer period. Finally, Cousins abundance in a cycad species should never compromise et al. (2012, 2013) based their identification on stem- and conservation efforts; thus, care must be taken that the leaf-based morphological characters, together with har- incessant removal of E. villosus from the wild be moni- vesting localities records. The authors interviewed TM tored to ensure that the species does not become threat- traders regarding harvesting locality, and these data ened in the near future. were then use to distinguished species by plotting har- The extensive trade in E. natalensis fragments is con- vesting localities on a map that was overlaid with the cerning. Williams et al. (2014) also identified E. natalensis geographical distribution of the 14 Encephalartos spp. oc- as one of the cycads traded in the largest quantities at curring in KwaZulu-Natal. Although this approach to identify Faraday. Encephalartos natalensis has declined in certain Encephalartos spp. in the TM trade is useful, numerous For personal use only. parts of its range, which includes the Qumbu and Ta- Encephalartos spp. are morphologically very similar, and bankulu areas of the northern part of the Eastern Cape, closely related species are often difficult to distinguish through most of KwaZulu-Natal up to the upper catch- from one another (Golding and Hurter 2003; Grobbelaar ment areas of the Mkuze and Umfolozi rivers near Vry- 2004). Therefore, morphological and harvesting recollec- heid in SA. The overall population decline is estimated to tions alone will unlikely provide conclusive identifica- be <30% over the past 60 years (Donaldson 2010b). If this tions that could be used in law enforcement programs decline continues and the overall population numbers and prosecutions. Also, plants traded at TM markets are drop below 10 000, this species could be listed as Vulner- mainly harvested from wild resources by specialized col- able (Donaldson 2010b). lectors and reach the market via a middle man; thus, One sample at Faraday was identified as E. ferox. It is exact harvesting localities are not always available. a widespread species occurring in northern KwaZulu- To summarize, all Encephalartos spp. (E. ferox, E. lebomboensis, Natal Province of SA and southern Mozambique. How- E. natalensis, and E. senticosus) identified at Faraday and ever, according to Donaldson (2010c), E. ferox needs E. villosus identified at Warwick have distribution ranges monitoring as numerous plants have been removed and extending into KwaZulu-Natal. This coincides with the sold alongside roads in Mozambique. Donaldson (2010c) demographics of traders and customers at these two TM estimated that population reduction could increase markets. Traders from Warwick are mainly Zulu speak- Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 to >30% over three generations in which case it would ing from KwaZulu-Natal and neighbouring countries qualify as VU under criterion A2 (Donaldson 2010c). such as Swaziland and Mozambique, whereas the major- It is of specific concern that E. lebomboensis and E. senticosus ity of the traders at Faraday are migrants from KwaZulu- are also sold at Faraday. Encephalartos lebomboensis occurs Natal, with 90% speaking Zulu. Two reasons proposed by in Swaziland and Mozambique as well as in northeastern Williams (2003) for the movement of traders from War- KwaZulu-Natal and the Lebombo Mountains of southern wick to Faraday are, first, that many traders realized that Mpumalanga Province of SA. The E. lebomboensis popula- the market in Durban was becoming overcrowded with tion has declined by at least 50% in the past 90 years, with traders, and hence concluded that there was compara- only 5000 mature plants recorded in the wild in 2010 by tively more business at Faraday. Second, many traders

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Fig. 4. A selection of Encephalartos spp. identiﬁed at the traditional medicinal (TM) markets: (A) E. lebomboensis (Credit: Philip Rousseau), (B) E. ferox (Credit: Olivier Maurin), (C) E. senticosus (Credit: Philip Rousseau), (D) E. villosus (Credit: David Sengani).

are of the opinion that most plants are easily accessible ical identiﬁcation difﬁcult. The combined power of rbcLa,

For personal use only. in the mountains of KwaZulu-Natal, and consequently matK, trnH-psbA, and nrITS to discriminate among south- local people can harvest what they need and do not need ern African Encephalartos spp. has proven to be relatively to buy it, therefore resulting in fewer customers. The high (86%), thus validating the use of DNA barcoding to opposite is true for Faraday; the urban population in identify Encephalartos spp. rapidly when morphological Gauteng does not have free access to medicinal plants evidence is lacking. Furthermore, the technique can be and need to buy it from TM markets. Finally, both TM of great assistance in monitoring the trade in Encephalartos markets sell stock harvested primarily in KwaZulu-Natal, spp., specifically in relation to conservation and law en- with traders at Faraday obtaining most of their stock forcement interventions that may be implemented in from Warwick TM market. The explanation why we iden- the future. tified different Encephalartos spp. at Faraday than at War- wick could be attributed to the fact that at Warwick Acknowledgements traders are more aware that trading in cycads is illegal We would like to thank the Government of Canada and thus hide them under more common species. The through Genome Canada and the Ontario Genomics In- opposite is true for Faraday, where we observed several stitute (2008-OGI-ICI-03), the Consortium for the Barcode heaps of Encephalartos fragments. of Life (CBOL) through the Google’s Global Impact Award Programme, and the University of Johannesburg (Analyt- Conclusion ical Facility) for financial support and various local au- Genome Downloaded from www.nrcresearchpress.com by EAST CHINA UNIV OF SCI & TECHNOLOGY on 11/15/16 This study presents yet another example of the ap- thorities who granted plant collection permits (Cape plicability of DNA barcoding in the identification of Nature 0028-AAA008-00171; Department of Environmental medicinal plants and highlights the undercover trade in Affairs, ToPs Certificate Number 07138 and ToPs permit Encephalartos spp. at TM markets, which is not adequately number 03009; Eastern Cape ANO 3/5/4 – M. van der Bank; recognized in official government documents or data- Gauteng 0039; Limpopo 0090-MKT001-00009; Mpumalanga bases. Five Encephalartos spp. (Fig. 4) were identified as MPB.1305). Zandisile Eugene Shongwe is thanked for tech- currently being traded illegally at Faraday and Warwick. nical assistance in the laboratory and the late Philip Rousseau All of these species were harvested in the form of bark for providing sequence data for the Encephalartos DNA strips and whole stem sections, thus making morpholog- reference library.

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