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A stand with various plant products at the Faraday traditional medicine market, Johannesburg. Photo by Zandisile Shongwe.

CYCADS TRACKED THROUGH DNA BARCODES

BY JANICE WILLIAMSON, HERMAN VAN DER BANK, RONNY SETTING THE SCENE: The cycad fossil record KABONGO AND MICHELLE VAN DER BANK dates back to the early Permian (280 million years THE AFRICAN CENTRE FOR DNA BARCODING (ACDB), ago) and possibly the Carboniferous (320 million UNIVERSITY OF JOHANNESBURG years ago) making them the world’s oldest extant seed plants.

Cycads flourished during the Mesozoic the next two-10 years, a predicament Encephalartos brevifoliolatus and – the age of the dinosaurs – but, unlike recently described by SANBI’s Michele E. nubimontanus, two of the three the dinosaurs, have survived three mass Pfab as “the South African cycad South African species classified as extinction events in Earth’s history. extinction crisis”. Extinct in the Wild, became extinct between 2003 and 2010. Numerous The devastating loss of cycads, a tragedy South African representatives of the populations of cycads have been largely unnoticed, is very similar to that order Cycadales, include Stangeria virtually obliterated by ‘collectors’. For of the rhino poaching crisis. Cycads are eriopus and 37 Encephalartos species example, populations of Encephalartos classified as the world’s most threatened of which three are Extinct in the Wild, cerinus, first described in 1989, have plant group. Cycad artist Douglas twelve Critically Endangered, four recently been plundered so heavily, Goode stated in 1989: “cycads could Endangered, seven Near Threatened, that in less than 30 years, this species be referred to as the rhino horn of the nine Vulnerable, and only three species is on the verge of becoming Extinct in plant kingdom”. South Africa is currently of Least Concern (see March 2016 issue the Wild with only an estimated 20 to at risk of losing 50% of its cycads within of Veld & Flora). 70 plants remaining! JUNE 2016 | VELD & FLORA 215

LEGISLATION AND CYCADS material) as a unique identifier for each project was launched in 2010 at the species. These short gene sequences – African Centre for DNA Barcoding South African legislation is among some known as DNA barcodes – can be used (ACDB), University of Johannesburg, of the world’s strictest laws controlling to identify a species, even if only a small with the aim of creating a DNA cycad trafficking. All Encephalartos fragment of plant material is available. In barcode reference library for African species are listed in Appendix I of the 2009, the Consortium for the Barcode of Encephalartos species in an attempt to Convention on International Trade in Life (CBOL) Plant Working Group proposed control illegal cycad collection. This Endangered Species of Wild Fauna and the standardised use of the gene sequences project is in collaboration with the Flora (CITES) and as Threatened or rbcLa and matK, found in chloroplasts, as International Barcode of Life and the Protected Species in terms of the National the core DNA plant barcodes. Barcode of Wildlife projects (see Box 1). Environmental Management: Biodiversity Act (NEM:BA; Act 10 of 2004). The library, representing 64 of the 65 HOW CAN DNA BE USED? African Encephalartos species, is now Conservation officials are often left complete. For each species we have helpless, however, when monitoring Accurate identification is essential to barcoded at least five duplicate samples illegal trafficking of species and products enforce the highest penalties for illegal from different geographical regions. derived from cycad species. Helpless, for cycad trafficking. The emergence Thus, we are now able to compare a reasons including: of DNA barcoding is providing an small fragment of cycad material – a invaluable tool in tackling this challenge. fragment of stem, seed, seedlings or • Traders deliberately misidentifying Various botanical studies have already mature specimen without leaves that threatened cycads as more common demonstrated the impact of DNA cannot be identified morphologically – species that are less likely to attract barcoding, which has been used to with the cycad DNA library in order to the attention of law enforcement discover and identify unknown species, provide accurate identification. agencies; resolve taxonomic problems, detect substitution in herbal products in North • Species-level identification is America, aid with traditional medicine nearly impossible when plants market surveys – allowing identification BOX 1: LARGE-SCALE are stripped of their leaves for of commercialised medicinal plants in BARCODING INITIATIVES transport purposes; and, northern Morocco – and scrutinise the illegal trade of endangered plant species, International Barcode of Life Project • It is impossible to identify species such as wild-harvested orchids in Nepal. (www.ibol.org): In 2011, the Barcode of from fragments, such as bark Life Project was launched in Canada. material, sold on the umuthi The largest biodiversity genomics (traditional medicine) markets. THE SOUTH AFRICAN initiative ever undertaken, it is an CONNECTION international effort to build a digital identification system for the planet’s WHAT IS DNA BARCODING? In response to an increase in cycad estimated 10-100 million species. poaching, South Africa’s officials are Barcode of Wildlife Project (www. DNA barcoding involves using a short, turning to DNA barcoding to safeguard barcodeofwildlife.org) – The Barcode agreed-upon region of a genome (genetic our cycads. A cycad DNA barcoding of Wildlife Project, funded by Google and managed by the Consortium for the Barcode of Life, aims to demonstrate the value of DNA- barcoding to investigate and prosecute wildlife crime in five developing countries. Goals of the project include: 1) to build a public database of reference barcode records for traded species and their close relatives and look-alikes, against which the barcode sequences of confiscated material can be compared; 2) to train researchers, technicians, border inspectors, prosecuting attorneys in the participating countries in DNA barcoding and its applications; and, 3) to test DNA barcodes as a real- world tool for enforcement through implementation of operational, cost- Confiscated material following illegal harvesting of mature Encephalartos specimens. effective barcoding programmes. Photo by Eastern Cape Department of Economic Development, Environmental Affairs and Tourism. JUNE 2016 | VELD & FLORA 216

The process takes less than 24 hours which wild population these plants from the reception of the material originate. The ACDB laboratory is now FURTHER READING to identification. We have assisted in moving towards building a genetic CBOL Plant Working Group. 2009. several police cases related to cycads, profile of cycad species using AFLPs. A DNA barcode for land plants. as well as tracked the illegal trade of This is a long-term project since we Proceedings of the National Academy cycads at traditional medicine markets would need to create a comprehensive of Sciences USA 106(31): 12794–12797. (see Box 2). AFLP genotype reference library of all wild populations. The project is feasible Cousins, S.R., Williams, V.L. & Witkowski, Unfortunately, population-level and would significantly improve cycad E.T.F. 2012. Uncovering the cycad taxa markers (e.g. amplified fragment length law enforcement in South Africa. (Encephalartos species) traded for polymorphisms - AFLPs) are not yet However, cost and accessibility to the traditional medicine in Johannesburg available for Encephalartos species, various cycad populations around the and Durban, South Africa. South African making it impossible to infer from country are presently limiting factors. Journal of Botany 78: 129–138.

Kool, A., De Boer, H.J., Krüger, Å., Rydberg, A., Abbad, A., Björk, L., & Martin, G. 2012. Molecular identification of commercialized medicinal plants in Southern Morocco. PLoS One 7: e39459.

Newmaster, S.G., Grguric, M., Shanmughanandhan, D., Ramalingam, S. & Ragupathy, S. 2013. DNA barcoding detects contamination and substitution in North American herbal products. BMC Medicine 11: 222–235.

Retief, K., West, A. & Pfab, M. 2015. Are Dinosaur sculpture at Kirstenbosch. Photo by André Wessels. you involved in the illegal cycad trade? Veld & Flora 101(1): 13–15.

Rayner, T. & Pires, A. 2016. Protecting BOX 2: EXPOSING THE ILLEGAL TRADE IN highly threatened South African cycads from extinction: a new approach. Veld ENCEPHALARTOS SPECIES AT THE FARADAY AND & Flora 102(1): 14–16. WARWICK TRADITIONAL MEDICINE MARKETS USING DNA BARCODING Subedi, A., Kunwar, B., Choi, Y., Dai, Y., van Andel, T., Chaudhary, R.P., de Boer Background: Illegal plant harvesting for the medicinal trade has increased in South H.J. & Gravendeel, B. 2013. Collection Africa and resulted in the decline of cycad populations, including complete loss of sub- and trade of wild-harvested orchids populations. Encephalartos is traded at traditional medicine markets in the form of bark in Nepal. Journal of Ethnobiology and strips and stem sections, thus determining the actual species traded presents a major Ethnomedicine 9(1): 64. challenge due to lack of characteristic plant identification markers. Methodology: We used DNA barcoding to identify cycads sold at Faraday, and Warwick CONTACT DETAILS traditional medicine markets. Market samples were sequenced for the core DNA Michelle van der Bank, African Centre barcodes (rbcLa and matK), as well as the two additional regions trnH-psbA and nrITS. for DNA Barcoding (ACDB), University of The barcoding database for cycads at the University of Johannesburg was utilised to Johannesburg, APK Campus, assign query samples to known species. Tel: 011 559 2495/ 3477, Results: Our study indicated that there is currently a lucrative trade in cycad species at Email: [email protected], traditional medicine markets. Market samples identified were: E. ferox (Near Threatened), Website: www.acdb.co.za E. lebomboensis (Endangered), E. natalensis (Near Threatened), E. senticosus (Vulnerable), and E. villosus (Least Concern). ACKNOWLEDGEMENTS Significance: The study validates the use of DNA barcoding in identifying Encephalartos We would like to thank the Government species rapidly, with incredible accuracy, particularly when morphological evidence is of Canada through Genome Canada lacking. The study highlights the undercover trade in Encephalartos species at traditional and the Ontario Genomics Institute medicine markets, which are not adequately researched and recognised in official (2008-OGI-ICI-03), the Consortium government documents or databases. Now solutions are being provided to address for the Barcode of Life (CBOL) through conservation of our most endangered plant species. the Google’s Global Impact Award Source: Williamson, J. et al. Exposing the illegal trade in Encephalartos species at the Programme and the University of Faraday and Warwick traditional medicine markets in South Africa using DNA barcoding Johannesburg (Analytical Facility) for (Submitted to Genome). financial support.