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Barcode Wales: DNA barcoding the nation's native flowering plants and conifers

Volume 9 Number 1 - January 2012

Natasha de Vere

The ability to identify plant species is fundamental to our understanding of the world around us. To conserve plants, their habitats and ecosystems we need to be able to identify and monitor species.

“Objective 1 of the Global Strategy for Plant Conservation is that: "plant diversity is well understood, documented and recognized".

Correct identification is also vital in order for us to use plants for food, medicine or materials. Identification of plants often relies on morphological examination, but around the world there is a shortage of taxonomic experts able to identify species. Beyond this, it is often desirable to be able to identify species from material where morphological approaches are difficult or impossible to use; for example, from pollen, roots, seeds, or fragments of tissue. In these situations DNA-based identification systems can be used and in 2003 Paul Hebert coined the term 'DNA barcoding' (Hebert et al., 2003).

DNA barcoding uses short sections of DNA to act as a unique identifier for species. The aim of DNA barcoding is to have global agreement on the regions of DNA and protocols used for different groups of living things in order to create an international resource for species identification. To begin with, reference DNA databases are developed using species identified by a taxonomic expert; unknown DNA sequences can then be compared to these to make an identification. Open Science is key, DNA barcodes, their associated information and protocols for their development should be available to everyone, from researchers, regulatory authorities to the general public.

There are now initiatives throughout the world for DNA barcoding species from all of the kingdoms of life, such as the International Barcode of Life initiative (IBOL), which works across 25 countries and aims to DNA barcode 5 million specimens from 500,000 species within five years. The Consortium of the Barcode of Life (CBOL) is devoted to promoting DNA barcoding as a global standard for DNA-based species identification and the Barcode of Life Data System (BOLD) provides a key repository for DNA barcodes and their associated data (Ratnasingham and Hebert, 2007). In 2009, the Plant Working Group of CBOL proposed two sections of genes within the chloroplast genome, rbcL and matK, for plant DNA barcoding, with the suggestion that more markers may be required (CBOL Plant Working Group, 2009). In December 2011 the nuclear gene ITS was announced as the official barcode for fungi during the 4th International Barcode of Life Conference in Adelaide, Australia.

Projects are now underway to DNA barcode the world's plant species. For example, the New York Botanic Garden is DNA barcoding the world's trees, whilst the Universities of Adelaide and British Columbia are working on grasses. Some projects are concentrating on floristic regions: Korea University is working on the flora of Korea, the University of Johannesburg on the flora of the Kruger National Park, the Smithsonian Institute on tropical forestry plots and institutions throughout China are working together to DNA barcode their flora. The Royal Botanic Garden Edinburgh is DNA barcoding British bryophytes and at the National Botanic Garden of Wales, we are DNA barcoding the native flowering plants and conifers of Wales, with our partners at the National Museum Wales (Hollingsworth et al., 2011).

Botanic gardens are increasingly using DNA barcoding as an identification tool for plants in their collections”

Barcode Wales
Wales has 1,143 native and archaeophyte (species introduced before 1500 AD) flowering plants and conifers, found within 455 genera, 95 families and 34 orders. In order to create a reference database of DNA barcodes for these, it is vital to start with correctly identified source material. Taxonomist  Dr Tim Rich, from the National Museum Wales is responsible for checking every species that we DNA barcode. We have concentrated on using herbarium specimens for DNA barcoding. The herbaria of the world provide an incredibly valuable resource of plant species that have been collected and identified by expert taxonomists. We have developed special protocols for extracting DNA from herbarium specimens to make full use of this resource.

For the resulting DNA barcodes to be of most use, it is very important that along with the DNA sequence there is a full record of when, where and by whom the plant was collected. All users of the DNA barcodes need to have access to this information, along with a scan of the herbarium voucher. Where fresh material is collected for DNA barcoding an associated herbarium voucher must always be made. The only exception to this is for endangered species where creating a voucher is not possible on conservation grounds; in this case a photograph is used instead. We also need to DNA barcode more than one specimen for each species to allow for errors and any variation between individuals within the species. The level of variation within the species should be low however, as the idea behind DNA barcoding is to use regions of DNA that differ between species but which are the same within a species. For our Barcode Wales project we aim to DNA barcode at least three samples for each species using the DNA barcode markers rbcL and matK.

Over the last three years we have sampled 4,272 plant specimens, 3,637 from the National Museum Wales herbarium (NMW) and 635 freshly collected from throughout Wales. We have 5,723 DNA barcodes, 3,304 for rbcL and 2,419 for matK. Of the 1,143 species of Wales we have DNA barcoded 98% using rbcL and 90% with rbcL and matK. Our first scientific publication describing our results and protocols will be available soon (de Vere et al., in press) and all of our DNA barcodes, along with their associated collection information and scans of their herbarium vouchers will be accessible shortly on the Barcode of Life Data System in the Plants of Wales project.

The Barcode Wales project provides a valuable resource for researchers wanting to identify species using DNA-based approaches. Our DNA barcodes for Wales also provide a stock of barcodes that can be incorporated into other projects. The 1,143 species of Wales represents 77% of the native flowering plants of the UK. We have just begun DNA barcoding the rest of the UK flora, working with the Royal Botanic Garden Edinburgh.

Once the native flora of the UK is complete we will begin on the non-native species.”

The range of applications using plant DNA barcoding is as broad as our imaginations, with some great applications already being developed. Ecological applications include understanding below-ground biodiversity by DNA-barcoding roots (Kesanakurti et al., 2011), reconstructing past landscapes using plant remains (Sonstebo et al., 2010) and identifying invasive species (Bleeker et al., 2008). DNA barcoding has been used to verify the contents of plant products including medicinal plants (Chen et al., 2010), berries (Jaakola et al., 2010), olive oil (Kumar et al., 2011), tea (Stoeckle et al., 2011) and kitchen spices (De Mattia et al., 2011). DNA sequences obtained for DNA barcoding have also been used to create phylogenetic trees for phylogenetic community ecology research (Kress et al., 2009).

At the National Botanic Garden of Wales we have started to develop applications that use our DNA barcodes for Wales in collaboration with partners around the world. We have worked with Dr Sandra Ronca (Aberystwyth University) and Prof. Mike Wilkinson (University of Adelaide) to track the movements of pollinators in threatened habitats by DNA barcoding pollen found on their bodies. We are working with Dr Joel Allainguillaume (University of the West of England) to use DNA barcoding to carry out ecological surveys and Dr Neil Loader (Swansea University) on reconstructing landscapes from plant remains in peat cores. We are also using our DNA barcodes for human health in a project with Jenny Hawkins and Prof. Les Baillie of the Welsh School of Pharmacy (Cardiff University). For her PhD research, Jenny is collecting honey samples from throughout the UK and testing their ability to fight the hospital infections MRSA and Clostridium difficile. She will then DNA barcode the honey to find out what plant species the bees visited to make it. We hope to use this to pinpoint active phytochemicals donated by the plant species that contribute to the honey’s anti-microbial properties.

Many of our applications use 'next generation' DNA sequencing as this allows us to analyse samples containing mixtures of plant species. Our PhD student, Hannah Garbett, co-supervised by Dr Tatiana Tatarinova (Glamorgan University) is developing bioinformatic tools that will help to analyse these large and complicated datasets. As well as using our DNA barcodes for species identification we are working with Prof. Pete Hollingsworth (Royal Botanic Garden Edinburgh) to create the first complete national phylogeny of the flowering plants of the UK.

Public engagement
DNA barcoding is not only a resource for scientists; it is also an excellent way to engage people with their native flora and plant sciences research. At the National Botanic Garden of Wales we are particularly interested in projects that link arts and science. We have worked with botanical artist, Emma Tuck, on a contemporary art exhibition and community artist, Pod Clare, on a Barcode Wales mosaic created by community groups from throughout Wales. We are currently working with Prof. Andrea Liggins (Swansea University) on a photographic exhibition to be shown in botanic gardens and universities throughout China on native Welsh plants and their DNA barcodes.

Barcode Wales is also a mechanism for training the next generation of plant scientists. The workforce assembling the DNA barcodes for the Barcode Wales and Barcode UK projects are undergraduate students who spend a year at the Garden as part of their degree. Their work is supplemented with work experience students from A-level to postgraduate who spend from two weeks to a few months at the Garden.
The Barcode Wales project provides the most complete coverage of DNA barcodes of any national flora, offering a platform for a wide range of applications where accurate species identification is required. It also provides a hub for multi-disciplinary research across the arts, sciences and social sciences and a vehicle for training plant science students and volunteers. The DNA barcodes and their associated information are Open Access to make a resource available to everyone.

Bleeker, W., Klausmeyer, S., Peintinger, M., and Dienst, M. 2008. DNA sequences identify invasive alien Cardamine at Lake Constance. Biological Conservation 141: 692-698.

CBOL Plant Working Group. 2009. A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America 106: 12794-12797.

Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., Luo, K., Li, Y., Li, X., Jia, X., Lin, Y., and Leon, C. 2010. Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. Plos One 5: e8613.

De Mattia, F., Bruni, I., Galimberti, A., Cattaneo, F., Casiraghi, M., and Labra, M. 2011. A comparative study of different DNA barcoding markers for the identification of some members of Lamiacaea. Food Research International 44: 693-702.

de Vere, N., Rich, T.C.G., Ford, C.R., Trinder, S.A., Long, C., Moore, C.W., Satterthwaite, D., Davies, H., Allainguillaume, J., Ronca, S., Tatarinova, T., Garbett, H., Walker, K. and Wilkinson, M.J. (in press) DNA barcoding the native flowering plants and conifers of Wales. Plos One.

Hebert, P.D.N., Cywinska, A., Ball, S.L., and DeWaard, J.R. 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B-Biological Sciences 270: 313-321.

Hollingsworth, P.M., Graham, S.W., and Little, D.P. 2011. Choosing and Using a Plant DNA Barcode. Plos One 6: e19254.

Jaakola, L., Suokas, M., and Haggman, H. 2010. Novel approaches based on DNA barcoding and high-resolution melting of amplicons for authenticity analyses of berry species. Food Chemistry 123: 494-500.

Kesanakurti, P.R., Fazekas, A.J., Burgess, K.S., Percy, D.M., Newmaster, S.G., Graham, S.W., Barrett, S.C.H., Hajibabaei, M., and Husband, B.C. 2011. Spatial patterns of plant diversity below-ground as revealed by DNA barcoding. Molecular Ecology 20: 1289-1302.

Kress, W.J., Erickson, D.L., Andrew Jones, F., Swenson, N.G., Perez, R., Sanjur, O., and Bermingham, E. 2009. Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proceedings of the National Academy of Sciences of the United States of America 106: 18621-18626.
Kumar, S., Kahlon, T., and Chaudhary, S. 2011. A rapid screening for adulterants in olive oil using DNA barcodes. Food Chemistry 127: 1335-1341.

Ratnasingham, S., and Hebert, P.D.N. 2007. BOLD: The Barcode of Life Data System ( Molecular Ecology Notes 7: 355-364.

Sonstebo, J.H., Gielly, L., Brysting, A.K., Elven, R., Edwards, M., Haile, J., Willerslev, E., Coissac, E., Rioux, D., Sannier, J., Taberlet, P., and Brochmann, C. 2010. Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate. Molecular Ecology Resources 10: 1009-1018.

Stoeckle, M.Y., Gamble, C.C., Kirpekar, R., Young, G., Ahmed, S., and Little, D.P. 2011. Commercial Teas Highlight Plant DNA Barcode Identification Successes and Obstacles. Sci. Rep. 1: 42.

Further information
National Botanic Garden of Wales
Consortium for the Barcode of Life
International Barcode of Life Initiative
Barcode of Life Data System
4th International Barcode of Life Conference

Natasha de Vere
National Botanic Garden of Wales
Llanarthne, Carmarthenshire, SA32 8HG,UK