The Barcode Blog

If you hear this sound  watch your step–a venomous rattlesnake may be nearby! Prompt administration of the correct antivenom can be life-saving.

The essential first step in toxinological research is reproducible analyses of venom toxins. However, in December 2005 Toxicon 46: 711-715 Pook and McEwing, University of Wales, report that reproducibility of toxin analyses is commonly compromised by “misidentification of species due to insufficient or changing knowledge of current snake systematics.”

Their article describes a breakthrough in toxinological research: DNA barcoding of dried venoms to provide an accurate, permanent “label” that is independent of future taxonomic changes.  This short article is a powerful demonstration of how a standardized approach to identifying species by DNA that uses a public database linked to vouchered specimens, i.e. DNA barcoding, can benefit science and society. While some taxonomists will continue to wring their hands, others may be excited by this work, as it demonstrates how DNA barcoding ADDED TO (and not replacing) standard systematic practice can improve the accessibility and usefulness of ongoing taxonomic work for the larger biological community. I believe this article is enormously important, and I quote here at length.

From the Introduction: “Erroneous or uncertain taxonomy confuses the interpretation of results with respect to intraspecific and interspecific variation. Genuine logistical problems are encountered by non-specialists in taxonomy and snake systematics, in being able to keep up with taxonomic changes. The main hindrances include unresolved taxonomy for some species groups, and new data that change the definition of taxonomic units in others, with the result that the use of systematic information in the toxinological and clinical literature is disorganised. The problem is compounded further by changes in the concept of a particular species, and hence the interpretability of the venom used. Taxonomic confusion, however, has serious implications in snake venom research. The development of effective antivenom treatments and treatment strategies for envenomized patients necessitates a sound taxonomic framework. Accurate clarification of the identity of a given venom is paramount, even after taxonomic revisions involving the species concerned.”   

Pook and McEwing “propose a solution to the long-term problem of species identification in toxinological work, utilising mitochondrial haplotypes isolated directly from snake venoms to provide a means of identification that will remain useful even in the face of radical changes in our understanding of the species concerned.” 

From the conclusion: “The barcode strategy is dependent on the availablity of known mtDNA haplotype standards against which the sequences being investigated can be compared. Mitochondrial haplotype standards should be gene sequences from snakes of confirmed identity, which have been accessioned to a museum collection as voucher specimens.”

Pest and invasive species in agricultural crops and shipped goods pose enormous economic and biosecurity threats. Rapidly and reliably identifying pests and invasives in all life stages is likely to be one of the most economically important applications of DNA barcoding. In March 2006 Ann Entomol Soc Am 99: 204 Scheffer, Lewis, and Joshi from the USDA and the Philippine Department of Agriculture apply DNA barcoding to invasive leafminer flies in the Philippines. They analyzed 258 specimens from the Philippines and compared these to a database of 307 sequences previously collected from worldwide populations. Bootstrap values for all species were 100%. In addition, a total of 7 distinct clusters with 98-100% bootstrap support were found in 3 “morphospecies”, suggesting discovery of new species, similar to findings of unrecognized biodiversity in all DNA barcode surveys to date. They conclude “DNA barcoding of economically and medically important species offers a powerful means of rapid identification”. 

With DNA barcoding, all can identify this Liriomyza sativae pupae

Results so far in animals suggest the major limitation to DNA barcoding is very young species pairs. Young species pairs may not have accumulated enough differences in the 648 bp barcode region of COI to form distinct lineages in neighbor-joining analysis. Because overall rates of mitochondrial DNA evolution differ among animal groups, and the relative rates of specific mitochondrial genes also differ, it may turn out that COI barcoding will be more effective in separating young species pairs in some groups than in others.

20 My of genetic divergence and morphologic stasis 

In April 2006 Systematic Biology Mueller compares complete mitochondrial genomes of 27 salamander species. The genetics of salamanders are particularly important as they are notoriously difficult to distinguish and classify due to morphologic stasis and frequent homoplasy. Many new species of salamanders have been discovered and described on the basis of mitochondrial divergence. Mueller found that COI has the highest rate of evolution among all mitochondrial genes in salamanders, suggesting it will be especially effective at resolving young species in this group. Despite these very encouraging results, the paper ends with a worried discussion about using COI for DNA barcoding of salamanders, because its evolutionary rate varies between lineages.

A better appreciation of the power of a standardized genetic approach for amphibians is Ron et al.’s paper in May 2006 Molecular Phylogenetics and Evolution which uses mitochondrial DNA to look at phylogeny and new species in the Neotropical tungara frog genus Engystomops. The authors observe that “the use of genetic markers in systematics has an enormous potential to facilitate the global inventory of biodiversity”, and conclude, based on their results and those in similar studies, that “the increasing use of molecular techniques will lead to the discovery of a vast number of species of Neotropical amphibians.”

A year ago in Science, Jones et al. described a new species of African monkey, Lophocebus kipunji, documenting their report with field observations, sound recordings, and photographs. This led to legalistic wrangling over whether the species could be said to exist if there was no specimen! Fortunately, the suspense is relieved by Davenport et al. in this month’s Science, in which they provide morphologic and DNA information based on a specimen recovered from a farmer’s trap.  Although the authors relied on mitochondrial DNA evidence to establish the monkey belongs in a new genus, Rungwecebus, the actual sequence data is not listed among diagnostic characters in the species or genus description. Routine inclusion of DNA barcode sequences could improve the usefulness of formal species descriptions, assisting primate conservation efforts that monitor bushmeat trade, for example.

Where is my barcode?

ABI’s smallest sequencer is about the size and weight of a house air conditioning unit [ABI 310: 95 kg (208 lbs); 61 x 56 x 86 cm (24 x 22 x 34 in)]. Researchers who developed the Mars Organic Analyzer for detecting extraterrestrial life recently turned their attention to DNA sequencing. In 9 May 2006 PNAS Blazej, Kumaresan, and Mathies from the University of California, Berkeley, report on a microfabricated DNA sequencer comprised of three 10 cm glass wafers. Using 1 femtomole of DNA template and a 250 nanoliter reaction chamber, the device performs thermal cycling, DNA purification, and capillary electrophoresis, generating reads of up to 556 bases with 99% accuracy. Based on their results “the template/reagent requirements can be reduced an additional 100-fold, and a fully integrated microfluidic genomic sequencing system should also lead to significant infrastructure and labor savings.”

Faster, cheaper, more portable sequencing should facilitate “point-of-use” DNA barcoding devices for identifying specimens in the field, detecting invasive species at the customs station, and sorting through museum drawers for undescribed species, for example.    

In Nature 13 april 2006 “Gardens in full bloom” by Emma Marris highlights the increasing importance of botanical gardens as centers of molecular research. One scientific goal is to compile a working list of known plant species. According to Nature, “plans for the ultimate database inevitably lead to talk of DNA barcoding. If species-specific differences in defined DNA sequences were matched with a species name in some kind of database, an untrained person could use a sequence or a DNA-chip to read the barcode in a botanical sample, send it to the database, and get back a name and all other necessary taxonomic data….Apart from its undoubted geeky appeal, such a technology would in principle save a lot of time and drudgery. Carrying out identifications for colleagues at home and round the world is time consuming and uncompensated. The use of barcoding would free up people to do their own research.”

But Peter Raven, Missouri Botanical garden, is cautious about such a scheme. He worries about how much time and effort it would take and asks “what would one do with barcodes for the 13,000 or so moss species?”

Raven’s question is like a cosmologist asking “why map the distribution of galaxies?” There is likely no way to understand the origins and patterning of biodiversity other than counting species and mapping their distributions. A rapid, simple method for identifying specimens such as DNA barcoding can make this possible. Studying a species-rich group of early terrestrial colonizers such as mosses, which live in some of the coldest and dryest environments as well as in the tropics, and provide habitats for a variety of invertebrates, might be a good place to start.

DNA analysis can also help identify new moss species. In “Cryptic species within the cosmopolitan desiccation-tolerant moss Grimmia laevigata“, Fernandez et al describe 2 cryptic species with overlapping geographic distributions. Their samples were collected only in California, so a world survey might reveal many more hidden species. The authors conclude “the results emphasize the need to make molecular characterization of species a standard part of ecological analyses of populations and communities”.

An exciting paper in Science 28 April 2006 “Population size does not influence mitochondrial genetic diversity in animals” by Eric Bazin, Sylvain Glemin, and Nicolas Galtier from Universite Montpellier, France, calls into question current thinking in population genetics. The authors looked at intraspecific variation in nuclear and mitochondrial DNA using sequence data collected from public databases into Polymorphix database. Contrary to expectations from population genetic theory, there was “no correlation between mtDNA polymorphism and species abundance”. Analysis of non-synonymous (amino acid changing) and synonymous (silent) changes indicated that reduced mitochondrial diversity within species reflects positive selection. They conclude “mtDNA appears to be anything but a neutral marker and probably undergoes frequent adaptive evolution… mtDNA diversity will in many instances, reflect the time since the last event of selective sweep, rather than population history and demography.” Taken together, these findings help explain the general observation of constrained intraspecific mitochondrial variation in animals, even in organisms with enormous population sizes. Recurrent selective sweeps are natural tests of species boundaries and help explain why mtDNA genealogies generally capture the biological discontinuities recognized by taxonomists as species (Avise and Walker PNAS 96:992, 1999), in short, why DNA barcoding works! It is expected that large data sets generated by DNA barcoding surveys will help refine this analysis and identify possible ecological or biological correlates, providing insight into what drives selective sweeps. I close with a question: if a species is morphologically and ecologically stable, does it nonetheless undergo repeated selective sweeps?

150 My of selective sweeps?

“Taxonomy for the twenty-first century” the 29 April 2004 special theme issue of Philosophical Transactions of the Royal Society Biological Sciences is available online. Allen Herre recommends this compilation as “an extremely useful source of information and ideas on what taxonomy is, what its needs are (and our needs of it), and where it is going.”

Introduction by H. C. J. Godfray and S. Knapp
Taxonomic triage and the poverty of phylogeny by Quentin D. Wheeler
A taxonomic wish-list for community ecology by Nicholas J. Gotelli
Protist taxonomy: an ecological perspective by Bland J. Finlay
Stability or stasis in the names of organisms: the evolving codes of nomenclature by Sandra Knapp, Gerardo Lamas, Eimear Nic Lughadha, et al.
Prokaryote diversity and taxonomy: current status and future challenges by Aharon Oren
Taxonomy and fossils: a critical appraisal by Peter L. Forey, Richard A. Fortey, Paul Kenrick, et al.
Automated species identification: why not? by Kevin J. Gaston and Mark A. O’Neill
The promise of a DNA taxonomy by Mark L. Blaxter
Towards a working list of all known plant species by Eimear Nic Lughadha
Biodiversity informatics: managing and applying primary biodiversity data by Jorge Soberón and Townsend Peterson
Unitary or unified taxonomy? by Malcolm J. Scoble
The role of taxonomy in species conservation by Georgina M. Mace
Taxonomy and environmental policy by Cristián Samper
Taxonomy: where are we now? by Peter H. Raven
Now is the time: by Daniel H. Janzen
Tomorrow’s taxonomy: collecting new species in the field will remain the rate-limiting step by Robert M. May
Documenting plant diversity: unfinished business by Peter R. Crane
Taxonomy as a fundamental discipline by Edward O. Wilson

“DNA barcoding of life” the 29 October 2005 issue of Philosophical Transactions of the Royal Society Biological Sciences is devoted to papers presented at the First International Barcode Conference held at The Natural History Museum, London, 7-9 February 2005, a gathering that included 220 participants from 44 countries. By special arrangement with The Royal Society, this issue is available to members and visitors to the Consortium of the Barcode of Life site.

Towards writing the encyclopaedia of life: an introduction to DNA barcoding by Vincent Savolainen, Robyn S. Cowan, Alfried P. Vogler, et al.
DNA barcodes for biosecurity: invasive species identification by K.F. Armstrong and S.L. Ball
DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar by M. Alex Smith, Brian L. Fisher, Paul D.N. Hebert
Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding by Daniel H. Janzen, Mehrdad Hajibabaei, John M. Burns, et al.
DNA barcoding Australia’s fish species by Robert D. Ward, Tyler S. Zemlak, Bronwyn H. Innes, et al.
Deciphering amphibian diversity through DNA barcoding: chances and challenges by Miguel Vences, Meike Thomas, Ronald M. Bonett, et al.
The problems and promise of DNA barcodes for species diagnosis of primate biomaterials by Joseph G. Lorenz, Whitney E. Jackson, Jeanne C. Beck, et al.
Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications by Gary W. Saunders
Land plants and DNA barcodes: short-term and long-term goals by Mark W. Chase, Nicolas Salamin, Mike Wilkinson, et al.
Microcoding: the second step in DNA barcoding by R.C. Summerbell, C.A. Lévesque, K.A. Seifert, et al.
The unholy trinity: taxonomy, species delimitation and DNA barcoding by Rob DeSalle, Mary G. Egan, Mark Siddall
Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ribosomal RNA signature sequences by Melanie Markmann and Diethard Tautz
DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers by Michael T. Monaghan, Michael Balke, T. Ryan Gregory, et al.
Defining operational taxonomic units using DNA barcode data by Mark Blaxter, Jenna Mann, Tom Chapman, et al.
An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding by Paul De Ley, Irma Tandingan De Ley, Krystalynne Morris, et al.
Critical factors for assembling a high volume of DNA barcodes by Mehrdad Hajibabaei, Jeremy R. deWaard, Natalia V. Ivanova, et al.
A likelihood ratio test for species membership based on DNA sequence data by Mikhail V. Matz and Rasmus Nielsen
TaxI: a software tool for DNA barcoding using distance methods by Dirk Steinke, Miguel Vences, Walter Salzburger, et al.

Tropical fauna challenge taxonomy because species richness is greater in the tropical than in temperate zones, most tropical species are as yet undescribed, and within-species genetic variation appears to be greater.

Land Animal and Plant Biodiversity World Map

Two recent papers show DNA barcoding aids species identification and discovery in tropical fauna. In January 24, 2006 Proceedings of the National Academy of Sciences USA, Hajibabaei et al examine 4260 specimens representing 521 (71%) of hesperiids (skipper butterflies), sphingids (sphinx moths), and saturniid moths of the of the ACG conservation area in Costa Rica. 510 (98%) of recognized species have distinct barcodes, 11 (2%) have barcodes that overlap with another closely-related species, and 13 recognized species have 2 or more distinct barcode clusters. Associated co-variation in habitat, food plant, and adult and caterpillar morphology indicate these clusters represent cryptic species, a total of 27 new species whose discovery was facilitated by barcoding.

In a similar vein, DNA barcoding revealed cryptic species with unsuspected host-specificity in a genus of presumed generalist tropical parasitoid tachinid flies. Insect parasitoids are a major cause of natural insect mortality and are used as biological control agents. They are thought to represent 8%-25% of all insect species, but understanding species richness and biology is hampered by the very large number of morphologically similar species. A published commentary by Herre emphasizes “…the value of DNA barcoding in uncovering hidden diversity…especially when coupled with traditional taxonomy and a keen appreciation of the fascinating details of basic natural history.”

“Ten Species in One: DNA Barcoding Reveals Cryptic Species in the Neotropical Skipper Butterfly Astraptes fulgerator“ paper by Paul Hebert, Erin Penton, John Burns, Daniel Janzen, and Winnie Hallwachs to be published this fall in Proceeding of the National Academy of Sciences. This exciting study demonstrates the power of DNA barcoding coupled with traditional taxonomic tools in disclosing hidden diversity, a critical issue in global conservation.