News

In February 2007 Microbiology Today, scientists report on the Barcoding Protists Workshop held in Portland, Maine in November 2006, which was attended by 40 protist experts from 12 countries (Australia, Canada, Denmark, France, Germany, Japan, Malaysia, Netherlands, Norway, Russia, UK, and USA). The workshop was co-sponsored by the US National Center for Culture of Marine Phytoplankton and the UK NERC Culture Collection of Algae and Protozoa.

According to Williamson et al, “most original descriptions for [over 200,000 named] protist species are based on light microscopy and ink drawings, not only making species identification for some groups an inherently subjective and specialist occupation, but also potentially hiding major genetic diversity.”

 

 

Workshop participants agreed unanimously that “to help resolve many of the contradictions and uncertainties in protist taxonomy, genetic barcoding is the way forward, starting with material, particularly type strains, in internationally recognized culture collections.”

 

In early online 12 Jan 2007 Mol Ecol Notes researchers from Columbia University, American Museum of Natural History, and California State University analyze COI barcode region sequences of 131 individuals representing 19 species of Mopalia chitons. Chitons are molluscs with flattened segmented shells, and most of the 860 known world species are herbivores that graze in tidal zones, although some are found at depths up to 6000 meters.  According to the authors “much of the biology of [Mopalia sp] remains undiscovered” because many “are difficult to distinguish from one another by morphology alone”, making them a good test case for DNA barcoding.

Kelly et al compared three approaches for identifying Mopalia chitons by COI. First, they used a “character based assessment called characteristic attribute organization system (CAOS)”. In this approach, a “guide tree” is generated using maximum likelihood or parsimony, and CAOS identifies sets of characters for each node in the guide tree. CAOS then attempts to assign unknowns based on these characters. If there is insufficient information to assign the query sequence, CAOS stops the analysis. The authors compared CAOS to neighbor-joining distance analysis on Barcode of Life Data Systems (BOLD) site, and to BLAST algorithm. All three approaches had overall accuracy of 100% when provided with the entire data set. CAOS was superior to NJ and BLAST when a skeletonized reference set containing of 50% of the total sequences was used.  

CAOS automatically identifies diagnostic molecular characters, and this will help integrate DNA barcode data into traditional taxonomy. For practical use, diagnostic sequence differences may aid design of solid-state microarrays that detect species in environmental samples, such as the 0.1mm Mopalia mucosa planktonic larva shown here, which might be found floating in seawater, or in the stomach of a krill.    

Krill are shrimp-like crustaceans found throughout the world’s oceans. The Antarctic krill, Euphasia superba, is thought to be the most abundant species on the planet in terms of biomass (500 million metric tonnes corresponding to 5 x 10^14 individuals), is a primary food source for whales, seals, and oceanic birds, and functions as a major planetary carbon sink by excreting waste that sinks to ocean floor. What does this very abundant, central-to-food-web species eat? For many animals, observation of eating behavior is impractical, and analysis of stomach contents or feces may be the only way to determine diet. However, such material may be morphologically unrecognizable. 

In August 2006 Marine Biotech 8:686, researchers from University of Tasmania and Department of Environment, Tasmania, compare DNA sequencing and light microscopy in identifying prey in stomach contents of E. superba. Passmore et al isolated DNA from stomach contents of 6 ethanol preserved krill and, using diatom-specific primers, amplified a 103 bp portion of nuclear small subunit RNA (ssRNA). ssRNA was used because at present it has the best taxonomic representation in GenBank for krill prey species. The researchers sequenced at least 50 clones from each individual krill and found 14 OTUs (operational taxonomic units), with 86% to 100% match to GenBank sequences. These results were compared to microscopic identification of diatom silica skeleton fragments in stomach contents, which involved counting 1000-3000 fragments per individual. Results were similar, although DNA analysis and light microscopy each appeared more sensitive for certain species. This study might be a best case for light microscopy because silica-skeletoned diatoms are not easily digested. As the authors point out, krill also consume a range of protozoa and small zooplankton, and the importance of these sources may be underappreciated.The authors conclude “the application of DNA diet analysis to krill warrants further investigation, particularly for prey that are difficult to study using other methods“.

This work shows the essential need for a comprehensive reference library, so far lacking. A study underway is examining mitochondrial and nuclear genes as barcodes for phytoplankton. Looking ahead, a “massively parallel” pyrosequencing approach could enable rapid and representative analysis of mixed environmental samples, such as stomach contents, without biases resulting from amplification and cloning. 

In early access Dec 2006 Nucl Acid Res, researchers from 9 laboratories in France, Italy, Norway, and Denmark examine a candidate barcode gene for land plants, the group I intron in the chloroplast leucine transfer RNA gene (trnL intron). Prior research has already shown that a simultaneous or tiered multi-gene approach will be needed to distinguish among closely-related land plant species. A project coordinated by Royal Botanic Gardens, Kew aims to identify the best overall approach.

Rather than cracking the tough nut of an ideal plant barcode, Taberlet and co-authors look at a simple approach “emphasizing the point of view of scientists other than taxonomists“, and test this on food plants in archeological and industrial applications. The chloroplast trnL intron is not the most variable non-coding region in chloroplast DNA and does not differ enough to separate many closely-related plant species. On the plus side, there are robust primers which amplify the intron from diverse species. Like other group I introns, the trnL intron sequence has catalytic activity and a conserved secondary structure with alternating conserved and variable sequence domains. Taking advantage of this feature, the researchers designed primers to amplify one of the variable domains, the P6 loop. Binding sites for both the trnL primers, which amplify the entire intron, and the P6 loop primers are “highly conserved among land plants, from Angiosperms to Bryophytes“. Importantly, the P6 loop is only 10 to 143 bp and can be amplified from degraded DNA.

Using “simulated ePCR” with the large GenBank data set, trnL intron and P6 loop sequences identified to species level 67% and 19% of cases respectively. However, in many practical applications, the number of possible species that need to be distinguished is relatively small and they are taxonomically diverse. Following this reasoning, Taberlet et al tested the intron and its P6 loop on a set of 132 species found in the Arctic and 72 species representing the commonest food plants. With Arctic plants, trnL intron and P6 identified to species level 85% and 47%, respectively. With the food data set, the tiny P6 loop was sufficient to identify 78% to species level. The P6 loop was successfully amplified from a 20,000-year old permafrost sample, from human feces, and from various processed foods including detecting potato, leek, and onion DNA in dried soup mix!

This is an exciting study, and DNA barcoding will likely have multiple applications in food safety. Whether or not these exact gene regions are adopted, a standardized approach will enable widespread and inexpensive use.

Relying on morphologic species descriptions lacking DNA sequences is like diagnosing patients without laboratory tests. An experienced clinician can often make the correct diagnosis from examination alone, but laboratory tests are frequently needed to confirm or point to other causes. Many advances in medicine reflect incorporating laboratory testing into routine evaluation. Two papers on leeches suggest similar benefits to taxonomy from incorporating mtDNA analysis into routine practice.

In May 2005 Conservation Genetics 6:467 researchers at the American Museum of Natural History analyze morphology, mitochondrial COI, and nuclear ND-I sequences of genus Helobdella leeches from Austrialia, New Zealand, South Africa, Hawaii, and South America. The tale starts with a leech discovered in Germany in 1985, H. striata, re-named H. europaea in 1987. 

Authors Siddall and Budinoff found that H. europaea is morphologically and genetically indistinguishable from a leech “discovered” in Australia in 1998, H. papillornata. Including COI sequences in initial species descriptions would have prevented wasted taxonomic effort, and a species native to South America would probably not be given the unfortunate name europaea.

In December 2006 Evol Devel 8:491, scientists from University of Maryland and University of California, Berkeley, apply COI barcoding to another Helobdella leech, H. robusta, a model organism in developmental biology and one of the lucky species selected for genome sequencing. Researchers Bely and Weisblat obtained leech specimens from laboratory cultures and field sites. They found that isolates thought to be H. robusta actually represent four species, 2 of which co-exist at the same locality. The authors observe “the perils of misidentification and taxonomic confusion in the lab are numerous and costly” and conclude with a call for routine application of DNA barcoding and collection of voucher specimens to confirm identity of laboratory organisms. 

For enjoyment or use, a US postage stamp featuring All Birds Barcoding Initiative (ABBI) and the Consortium for the Barcode of Life (CBOL) is available at zazzle.com.

 

 

 

 

 

 

 

 

In early online J Zool Syst Evol Res researchers from Natural History Museum and Imperial College, London, scrutinize “recent advances in DNA taxonomy…that follow the dramatic increase in data generation“. Authors Vogler and Monaghan provide a scientific update to

what has been learned so far: “a key finding from recent studies in animals is that variation in mitochondrial DNA is partitioned as tight clusters of closely related genotypes, which group specimens largely according to traditionally recognized species limits, and which are congruent with nuclear markers”,

the durability of clustering: “it can be expected that denser geographic and taxonomic sampling may result in the discovery of new clusters, and perhaps reduce their divergence from each other, but they are unlikely to erode the clustering altogether”,

the significance of incongruence between DNA-based and morphology-based methods for delimiting species: “the high degree of congruence of mtDNA groups and traditionally defined taxa appears to contradict the reported mismatch of established species boundaries…even well-studied groups may be in need of taxonomic revision before accurate tests of incongruence can be conducted”,

what the future holds: “a standard DNA taxonomic analysis will include broad sampling..followed by large-scale sequencing, and algorithmic procedures for delineating species limits. The taxonomic system will be derived from the data rather than expert opinion“,

and what is needed to harness DNA taxonomy in general and DNA barcoding in particular to speed description of the estimated 80% of earth’s biodiversity that is at yet undescribed: “a feedback loop that [uses] discrepancies between DNA and other data to refine species descriptions..founded in existing theory of evolutionary biology and phylogenetics”

I close with a pictorial analogy. The Coulter counter uses electrical sensing to gain the same information as morphologic diagnosis of blood smears, with dramatic improvements in speed, cost, and necessary expertise. In some situations, DNA sequencing may provide similar improvements over morphologic diagnosis for species-level identification.