From Plant Press Vol. 9, Issue 2 from April 2006.
By W. John Kress and Lee A. Weigt*
“I am standing in a Costa Rican rain forest,” writes tropical ecologist and conservationist Dan Janzen in his foreword to the new book produced by the Department of Botany, Plant Conservation – A Natural History Approach (Krupnick & Kress, 2005; University of Chicago Press). “There are a thousand species of plants within a long stone's throw. Nearly every one of them is a described species with a proper scientific name, a handle that you can plug into Google and come up with something. Nearly all of these species or their near relatives have been studied, sampled, thought about and are in ‘the literature.’ And I cannot identify a single species. Imagine what it would do to any and all aspects of human interactions with wild plants if you could walk up to any plant anywhere - seedling, sapling, 40 m tree, grass, root, pressed leaf, or fallen log - and know in a few seconds its scientific name. I need not describe how today's technology would then let you use that name to get into the warehouse of collective botanical knowledge. That capacity would transform far more than the science of plant biology, the conservation of plants, and the superficial ways we currently make use of the incredible diversity of form, physiology, genetics and chemistry of plants. It would be to plants what the printing press was to stories, education, science, law, medicine and communication.”
Janzen’s vision is not fantasy and it is almost here. A rapid and accurate method is now being developed for the quick identification of plant species based on extracting DNA from a tiny tissue sample of a leaf, flower, or fruit. Appropriately called “DNA barcoding,” referring to the coded labels one finds on grocery store products, DNA barcodes consist of a short sequence of DNA between 400 and 800 base pairs long that can be easily extracted and characterized for all species on the planet. These genetic barcodes will be accessed through a digital library and used to identify unknown plants in the field, garden, or market.
Once fully developed, DNA barcoding has the potential to completely revolutionize our knowledge of plant diversity and our relationship to nature. By harnessing technological advances in electronics and genetics, DNA barcoding will help many people quickly and cheaply recognize known species and retrieve information about them, and will speed discovery of the thousands of species yet to be named. Barcoding has the potential to provide a vital new tool for appreciating and managing the Earth’s immense and changing biodiversity.
The use of short DNA sequences for biological identifications was first proposed by Paul Hebert and colleagues (2003, Phil. Trans., Ser. B. 270; 2004, Proc. Natl. Acad. Sci. USA 101) at the University of Guelph with the ultimate goal of quick and reliable species-level identifications across all forms of life including animals, plants, and microorganisms. Although the usefulness and practicality of such DNA-based approaches have long been accepted for identifying microorganisms for which morphological data are limiting, this concept has been applied far most successfully in animals. Until this past year, plants have been notably absent in the early stages of barcoding efforts.
The formation of the Consortium for the Barcode of Life (CBOL; see http://www.barcoding.si.edu/) to create a database of documented and vouchered reference sequences to serve as a universal DNA barcode library has been a great stimulus for barcoding efforts around the world and for all life forms. Under the leadership of Executive Secretary David Schindel, CBOL, which is housed here at the National Museum of Natural History (NMNH) and funded by the Sloan Foundation, has succeeded in building a community of museums, botanic gardens, aquaria, and research institutions that now numbers over 100 institutes from 39 countries across six continents. The Consortium also manages various working groups, e.g., Technology, Plants, Databasing, Data Analysis, and DNA, which will help to lay the foundation for the massive collecting, taxonomy, and sequencing efforts to come.
Despite a delayed start, DNA barcodes have now been successfully applied to flowering plants. In a 2005 paper entitled “Use of DNA barcodes to identify flowering plants” published in the Proceedings of the National Academy of Sciences members of the Department of Botany and the Laboratories of Analytical Biology at NMNH (Kress et al., 2005) identified two DNA barcodes that have the potential to be a practical and standardized tool for plant species identification in biodiversity assessments, life history and ecological studies, and forensic analyses. As described in their paper, a successful DNA barcode must be 1) short enough to be quickly sequenced (e.g., approximately 400-800 base pairs in length), 2) easily located and amplified in all plant species, and 3) variable enough to provide a unique sequence for each species. The short sequence in the cytochrome c oxidase (CO1) gene, which has been found to be widely applicable in animal barcoding, is not appropriate in the case of plants because of a much slower rate of COI gene evolution in higher plants than in animals.
The two regions identified so far for plants are the nuclear internal transcribed spacer (ITS) and the plastid trnH-psbA intergenic spacer. ITS is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, though short in length (~450 base pairs), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Together, these two DNA regions may unlock the key to DNA barcoding in plants.
Herbarium collections, such as the millions of specimens maintained in herbaria at natural history museums and botanic gardens, provide a documented source for building the plant barcode library. In the Kress et al. (2005) study, dried herbarium specimens over 100 years old were tested and shown to be usable for DNA barcodes. Despite the overall success of ITS and trnH-psbA as plant barcodes, it is recognized that each of these DNA regions also poses some problems, such as the inability to amplify ITS in a number of groups of vascular plants (such as ferns) and the variable length of the plastid spacer that makes it difficult to align among species. More extensive trials of these and other possible plant DNA barcodes are now underway in laboratories around the world, including those at NMNH, the Royal Botanic Gardens at Kew and Edinburgh, New York Botanical Gardens, and the Natural History Museum in London.
Already a number of significant projects have been initiated to utilize the best plant barcode when it is available. In the Department of Botany, our early work on developing a workable plant barcode centered on the 200 plus species now found on Plummers Island, Maryland, a National Park Service habitat reserve in the Potomac River that has been studied and inventoried by biologists in the Washington, D.C., area for over 100 years, making it an appropriate test site for barcoding trials. We have so far surveyed a broad range of angiosperms in 50 plant families, 72 genera, and 83 species that occur on the island. We will shortly complete the remaining 120 species so that each taxon can be uniquely identified by their ITS and trnH-psbA signature.
In discussions with scientists at the Smithsonian Tropical Research Institute in Panama, we will soon begin a project to produce a DNA barcode library for all the plant species on Barro Colorado Island (BCI), one of the primary research stations for tropical biologists in the world. The experience gained from barcoding the 200 species on Plummers Island will be applied to the over 1,400 species now recorded from BCI. Once completed, the DNA barcode library of plant species will be an invaluable tool for ecologists and evolutionary biologists conducting investigations on BCI. In addition, plans are in place to begin a massive project to barcode the plants of Costa Rica in collaboration with the Asociación Instituto Nacional de Biodiversidad (INBio) and the Área de Conservación Guanacaste (ACG) in Costa Rica as well as the Missouri Botanical Garden.
One of the most exciting barcode projects has recently been launched in collaboration with the United States Botanic Garden here on the Mall in Washington, DC. We are building a DNA barcode library for all plant species that are used by people as medicines. According to World Economic Pants: A Standard Reference (Wiersema & León, 1999; CRC Press), over 1,000 species are currently classified as medicines, which are defined as plants “that serve as sources of specific pharmaceutical agents and those that are widely used, mostly in the crude sense as folklore remedies.” This estimate of the number of medicinal plants is conservative as it does not include numerous species used locally by indigenous peoples. We expect to develop barcodes for nearly 2,000 species that have medicinal value. A universal and simple means of identifying these medicinal species through DNA barcodes will facilitate the use of these species and the easy identification of species in the many forms by which they are applied. David Erickson has joined our team as post-doctoral fellow to spearhead the project.
In addition to the plant barcoding marker work, NMNH has had an active role in numerous barcoding projects from the start with well publicized articles on butterflies and birds. When the idea of DNA barcoding in its present form started circulating, Paul Hebert and Lee Weigt, current head of the Laboratories of Analytical Biology (LAB), met at a genomics conference and upon return Weigt and Noreen Tuross, then Director of LAB, wrote a Smithsonian new initiative proposal to incorporate DNA barcoding into the museum’s methodologies. This idea morphed into a DNA barcoding proposal and included other elements for “enhanced taxonomy” at NMNH. As a result NMNH received an annual increase of $700,000 in the base budget starting in September 2005 that was split between barcoding and basic collections needs. The funding has a three-year ramp up of infrastructure requirements, including two new staff and over a half million dollars of equipment, much of which in now functional at LAB instrumentation rooms at Smithsonian’s Museum Support Center (MSC) in Suitland, Maryland, including new high-throughput automated DNA extractors for both plant and animals tissues. One can extract 96 individuals in less than 30 minutes and the other does high-quality CTAB (plant) or organic (animal) tissue extractions for less than $1 each. Currently, samples can be processed at LAB from tissue through sequencing (both strands of DNA) for $2 per sample, including an archival DNA extract.
As of 2006, all biological departments at NMNH have active DNA barcoding projects. Two global efforts underway in which NMNH is very active are: Fish – Barcode of Life (FISH-BOL) and All Birds Barcoding Initiative (ABBI). Both of these projects aim to be taxonomically comprehensive. FISH-BOL hopes to barcode 22,000 marine and 8,000 freshwater fish species by 2010. ABBI has the same target date for the approximately 10,000 species of birds on the planet. The Feather Identification Lab, directed by Carla Dove in the Bird Division at NMNH, and LAB have teamed up to create a database to utilize DNA to identify remains of birds involved in strikes on aircraft. The desire for this bird strike identification database led to the DNA barcoding of almost all the birds of the United States and Canada, and NMNH is now actively participating in the global effort.
Similar to the Department of Botany’s collaboration with STRI to barcode the plants of BCI, LAB is taking the barcoding project and lab resources to taxonomists in the field in Panama and will participate in two taxonomy workshops at the Bocas Marine Station at STRI. They will focus on sponges in July and tunicates in August of this year and will extract DNA from freshly collected, expertly identified and vouchered specimens, and then bring those extractions back to LAB for subsequent processing and databasing.
Taxonomists, geneticists, biotechnicians, and statisticians are working hard to implement a universal system for DNA barcoding in both plants and animals. Scientists at NMNH are clearly taking a leadership role in international efforts to make barcoding a routine technique for the specialist and layperson as well. By harnessing advances in instrumentation and genetics, barcoding will help many people quickly and inexpensively recognize known species and retrieve information about them, and will speed discovery of the millions of species yet to be named. Barcoding will provide a vital new tool for appreciating and managing the Earth’s immense and changing biodiversity. Within a few years, Janzen’s vision of quick and accurate identification of all plant species in a Costa Rican forest may become a reality. And in the long run, respect for nature and its conservation will proportionally increase as well.
* Lee A. Weigt is the Manager of the Laboratories of Analytical Biology, Smithsonian Institution.
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