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Among the 35,000 known species of noctuid moths, a number are destructive agricultural pests, including for example Corn earworm Helicoverpa zea and Tobacco budworm Heliothis virescens. Accurate identification is the essential first step in pest management, but morphologic identification can be difficult, particularly of eggs and larval forms.

In September 2007 African Entomology, researchers from South Africa, Australia, and France analyze COI mtDNA of Busseola sp. larva in Ethiopian sugarcane. The DNA barcode region of COI was amplified from 7 morphologically-indistinguishable larval specimens using standard invertebrate primers (Folmer et al, Mol Marine Biol Biotech 3:294, 1994). Rearing of the larva was attempted, but none of the collected larva developed to the adult stage. Sequence analysis revealed two distinct clusters that matched sequences derived from adult B. fusca and B. phaia. Assefa and co-authors conclude “DNA-based methods were found to be a quick, easy and reliable method for identification of species…[and] may then be solutions for conditions in Africa where there is an acute shortage of experts and rearing facilities to keep field-collected insects alive until emergence of adults for morphological identification.”

“The first part of knowledge is getting the names right.”   Chinese proverb quoted in Evolution of Insects, Grimaldi and Engel, 2005.

Species names are the primary entrance for accessing biological knowledge about organisms. However, the tangled bank of nomenclature created by 250 years of diverse communities of taxonomic specialists working largely in isolation challenges those seeking knowledge. It can be difficult to know what is already known. Identifying even well-studied organisms in backyards, such as North American ants for example, may require graduate-level training. As taxonomic knowledge moves increasingly onto the web, tools that enable non-specialists and specialists alike to access biological knowledge of organisms are beginning to be developed. In my view, the solution will be a combination of information science tools enabling access to biological literature together with a universal library of standardized genetic sequences, ie DNA barcodes, and simple technologies for barcode sequencing. 

An exciting development in taxonomic information science is uBio (Universal Biological Indexer and Organizer) www.ubio.org, “an initiative within the science library community to join international efforts to create and utilize a comprehensive and collaborative catalog of known names of all living (and once-living) organisms. The Taxonomic Name Server (10,699,999 NameBank records so far) catalogs names and classifications to enable tools that can help users find information on living things using any of the names that may be related to an organism.??” 

The uBio site provides a sophisticated and enjoyable illustrated introduction (excerpt at right) to the variety of challenges in retrieving information using organism names. Another feature is Nomenclator Zoologicus, a searchable list of the names of genera and subgenera in zoology from the tenth edition of Linnaeus 1758 to the end of 2004, developed with Zoological Society of London. uBio is helping organize and index Encyclopedia of Life (“a web page for every species”) and Biodiversity Heritage Library (1.124 million pages digitized and on the web so far). 

I close with an example from birds. Some taxonomic confusion reflects the struggle to integrate older works that use outdated taxon names or species limits with modern knowledge. Other discordances reflect lack of consensus among current experts. Given the intensity of scientific study and public interest in birds, it is surprising there is no single authoritative world checklist, especially since most of the differences at the species level reflect minor disagreements about generic assignment, a few cases of splitting/lumping, or differences in spelling. As one step until there is an expert consensus checklist, for those interested in birds, we have prepared an “ABBI Name Lookup” (Excel, 8 MB) file for harmonizing specimen lists that recognizes 2,462 synonyms, alternate and misspellings, and extinct species.

“Amphibians are globally in decline, yet there is still a tremendous amount of unrecognized diversity” observed Vences et al in 2005 Phil Trans R Soc B 360:1859, the first report applying DNA barcoding to amphibian diversity.  Vences and colleagues highlighted the pressing need for fast and reliable identification tools, including for eggs and larva, which are often unrecognizable morphologically.

Here I focus on one technical aspect of DNA barcoding amphibians, namely designing primers that amplify the target sequence from a broad range of species. Previous research had shown remarkable mitochondrial sequence diversity among closely-related amphibians, and even within what appear to be single species, some of which may represent cryptic species. In the 2005 Proc R Soc B paper, researchers used COI primers designed for invertebrates (Folmer et al 1994); suprisingly these “worked in a large proportion of specimens”. They concluded “We support attempts to build up a global and complete cox1 database of [animal] eukaryotes”.

In 2005 Frontiers Zool 2:5 the same group of researchers quantified their PCR amplification success on specimens from 38 individuals representing 20 amphibian species. Using a well-established primer set for vertebrate 16s (Palumbi et al 1991) 38 of 38 (100%) samples amplified; with 3 COI primer sets (1 for invertebrates, 2 for birds), 36 of 38 (95%) amplified, although there was only 50-70% success for the individual COI primer pairs. The authors did not attempt to design new primers for amphibians. They concluded “we strongly advocate use of 16s rRNA as standard DNA marker for vertebrates to complement COI”. This seems reasonable but the advantages of standardizing on a single gene call for an effort to design primers that amplify COI from amphibians before abandoning the field to 16s or some other marker.  

In 2007 Mol Ecol Notes Smith and colleagues from University of Guelph analyzed 83 amphibian COI sequences in GenBank to design new primers. The 3′ ends of the forward and reverse primers bind at 1st or 2nd codon position G-C residues, which they found to be highly conserved among amphibian species, and each primer contains three 2-fold degenerate sites. Using this set, they amplified full-length PCR products from 267 of 377 specimens (71%) representing 39 amphibian species (including Triturus vulgaris illustrated at right), and recovered an additional 34 sequences (9%) using a “mini-barcode” primer set designed for butterflies. The authors comment “many of the specimens…which failed to amplify had been fixed in formalin or were collected more than 15 years ago”, so further work to test these primers on fresh material and a diversity of species is needed.

Amphibians are an exciting group. A comprehensive amphibian DNA barcode library will likely provide many, many new insights. I believe further work will help establish robust primer sets for amphibian COI sequences. 

Reference databases of DNA sequences used for species identification, ie DNA barcode libraries, are most powerful when the morphologic specimens are vouchered in a museum collection. This way, when there are puzzling results, DNA and morphologic specimens can be re-examined. However to date it has been challenging to recover DNA from small organisms without destroying them in the process. 

In Mol Ecol Notes 9 aug 2007 researchers from US Department Agriculture and Smithsonian Institution, National Museum of Natural History, describe a uniform protocol for “nondestructive extraction of DNA from terrestrial arthropods” including ticks, spiders, beetles, flies, and bees. 1 to 4 h in a guanidium thiocyanate extraction buffer yielded amplifiable COI DNA from most specimens. Inspection of specimens after extraction including with phase contrast and scanning electron microscopy demonstrated preservation of most morphologic characters.

In Mol Ecol Notes 27 june 2007, UK researchers (University College, London, NERC Centre for Ecology and Hydrology, Oxford, and UK Environmental Agency) describe a rapid, non-destructive, chemical-free method for DNA recovery from blackflies, including adult, larval, and pupal forms. Hunter et al report brief (1 minute) sonication in sterile water yielded 66% success with COI barcode amplification and preserved morphologic details.

These reports are exciting in the methods they describe and in how they highlight the general value of extracting DNA and determining DNA barcode sequences as an integral part of preparing traditional morphologic vouchers.