The Barcode Blog

Alien species sometimes damage native landscapes. In Voyage of the Beagle, in entry dated September 19, 1832, Darwin describes the spread of an introduced European thistle Cyanara cardunculus in Banda Oriental, now Uruguay: ”very many (probably several hundred) square miles are covered by one mass of these prickly plants, and are impenetrable by man or beast. Over the undulating plains, where these great beds occur, nothing else can live…I doubt whether any case is on record, of an invasion on so grand a scale of one plant over the aborigines.”

The challenge is to recognize invasive species before they become established. In 11 January 2008 Polar Biology researchers from Stellenbosch University and University of Western Ontario apply DNA barcoding to otherwise unrecognizable moth larvae on sub-Antarctic Marion Island. The indigenous Lepidoptera on Marion Island comprises 2 or 3 flightless moths, and the occassional adult winged moths or butterflies have been assumed to be transients arrived on fresh produce.

In April 2004, 3 noctuid moth larvae were found in an abandoned Wandering Albatross nest, a common habitat for one of the indigenous moth species. The larvae could be tentatively identified only to genus level and so rearing was attempted, with one larva dying after several months of pupating (as an aside, this is one example of how morphologic identifications can be laborious and/or incomplete, even for experts). The final larva was killed and preserved for DNA study; COI DNA barcode region was amplified using standard Folmer primers. The Marion Island moth larva barcode clustered with the 40 or so Black Cutworm Agrotis ipsilon sequences in BOLD, and was distinct from COI sequences of the other 18 Agrotis species in BOLD. Agrotis ipsilon is a common pest that feeds on a wide variety of plants. The authors conclude that Agrotis ipsilon is an established alien species with the potential to disrupt local ecosystems and that ”steps be taken to eradicate the species from Marion Island.”

It is easy to predict that rapid identification of potential invasive alien species will be a major application of DNA barcoding, with direct economic and ecosystem benefits.

Just posted, a freshly minted home page for Barcode of Life activities at Program for the Human Environment, The Rockefeller University, with expanded links to partners and downloadable pdfs and powerpoint files for illustrated flyers ”Top Ten Reasons for Barcoding Life” and “Barcoding Life, Illustrated.” Enjoy!

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Just as DNA analysis regularly overturns seemingly solid eyewitness identifications in crime investigations, routine DNA analysis can also help biologists avoid blunders. In 28 August 2007 Mol Ecol, researchers from University of Colorado, New Mexico State University, Pisces Molecular, and Brigham Young University report that over 20 years of restocking efforts in western US aimed at restoring native populations of endangered greenback cutthroat trout Oncorhynchus clarkii stomias have mostly been restocking a non-native, non-endangered subspecies, Colorado River cutthroat trout O. c. pleuriticus.  They trace the confusion to repeated introductions beginning in the late 1800s of Colorado River cutthroat trout throughout the native range of greenback cutthroat trout. The authors analyzed mitochondrial (COI, ND2) and nuclear (microsatellites, AFLP) DNA from 365 individuals from 15 locations in 3 major river drainage systems in Colorado and surrounding states. Distinct mtDNA lineages corresponding to each subspecies were corroborated by nuclear microsatellite and AFLP data.  For another cautionary tale of repeated misidentification of a widely studied organism, see Siddall and colleagues’ entertaining June 2007 Proc R Soc paper scrutinizing commercially available medicinal leeches sold as Hirudo medicinalis. 

How might the future look with routine application of DNA ID as quality control? Incorporating DNA barcode analysis into Tree of Life studies is one useful approach, exemplified by two recent large-scale evolutionary studies published in January and April 2008 Syst Entomol, one on phylogenetic relationships in Saturnid silkmoths, and one on higher-level relationships among 12 families in ‘bombycoid complex’ of Lepidoptera. Both studies analyze COI barcodes of all specimens, “allowing confirmination of their identification for species present in the BOLD reference library and enabling future identifications of organisms whose identity is still pending.”

On February 27, 2008, Encyclopedia of Life (EOL), ”a web page for every species” officially launched, with over 30,000 species pages, mostly fish so far, and a diversity of links to internet resources including Biodiversity Heritage Library (2.9 million pages digitized). In case you missed it, there is a thrilling, award-winning video.  Following Wikipedia model, EOL users are invited to become “curators” for one or more species pages, and later this year all are invited to submit content (photos, drawing, text, video, for example) for review. For an entertaining brief history of Wikipedia and why it keeps getting better see Nicholson Baker’s review of John Broughton’s Wikipedia: The Missing Manual in March 20, 2008 New York Review of Books.

Most near-sun comets are now discovered by amateurs, using images downloaded from the Solar and Heliospheric Observatory (SOHO), a satellite launched December 2, 1995 as part of international collaboration between European Space Agency (ESA) and National Aeronautics and Space Administration (NASA). I expect that EOL and other open-access databases will lead to many more persons contributing to biodiversity science.

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What limits mitochondrial variation within species?  In January 2008 PLoS Biology researchers from Karolinksa Institute, Sweden, and University of Newcastle upon Tyne, United Kingdom, report on an ingenious mouse model that shows strong purifying selection acting within a single generation, or even earlier, during embryogenesis. Stewart and colleagues employed “mtDNA mutator” mice which are homozygous defective for a nuclear gene which encodes a proof-reading subunit of mtDNA polymerase. These mice have increased levels of mtDNA mutations in all tissues, with mutations evenly distributed along all codon positions in mtDNA protein genes, accelerated senescence and “a number of phenotypes associated with mitochondrial diseases.” mtDNA mutator mice were backcrossed to wild-type mice to produce offspring that inherited defective mitochondria but whose nuclear genome is homozygous normal at the mtDNA polymerase locus.  They then sequenced entire mitochondrial genomes from 190 of these progeny individuals in N2 to N6 generations (N2 is the first backcross that is homozygous normal at mtDNA mutator locus). To skip to the conclusion, most of the non-synonomous mitochondrial mutations were eliminated, leaving a pattern of  excess synonymous mutations similar to that seen in human populations (which are the largest dataset so far for mitochondrial variation). The authors conclude that the mitochondrial population bottleneck known to occur at oogenesis, which deposits just one or few mitochondrial genomes per oocyte, means each mitochondrial genome must stand on its own so to speak, with the result that those eggs, embryos, or offspring harboring defective mitochondria will fail to survive. My figure at right tries to illustrate part of this process. 

In the same issue, David Rand, Brown University, provides a lucid commentary on Stewart et al’s research putting it in the context of mitochondrial and evolutionary biology, and suggesting next steps. Among others, he notes “the new mouse study also begs new questions about positive selection on mtDNA. …it is interesting that no signature of a selective sweep leading to fixation of a novel mtDNA variant was evident in the data”.

Purifying selection against deleterious mutations enabled by an embryonic bottleneck may save mtDNA from “mutational meltdown”. Now we need to understand more about the positive selection on mtDNA that presumably occurs when species adapt to new environments or diverge. I believe that growing mtDNA databases in the form of COI barcodes from a diversity of organisms with varying size, lifespan, population size, and reproductive strategy, in a diversity of environments including marine, terrestrial, temperature, and tropical regions will help solve this puzzle.

Plants challenge DNA barcoding. It has been difficult to identify candidate barcode regions that amplify readily and also distinguish among closely-related species. In 7 February 2008 PNAS (open access) researchers from University of Johannesburg; University of Costa Rica; Royal Botanic Gardens, Kew; and Imperial College, London, analyze potential barcode regions on specimens collected in plant biodiversity hotspots in Kruger National Park, South Africa, and Costa Rica. They initially tested eight candidate regions identified in earlier studies (coding regions accD, rpoC1, rpoB, ndhJ, ycf5, and matK, and non-coding trnH-psbA). Amplification was done according to earlier studies except that a different set of matK primers was used which appeared to be more effective. All eight regions were examined in 101 specimens representing 32 species of trees, shrubs, and achlorophyllous parasites from South Africa, and on 71 specimens representing 48 species of Costa Rican orchids (in all, 44 species with 2-7 specimens per species, and 36 species with one sample). Based on their analysis, the coding region matK with the new primer set and the non-coding region trnH-psbA were >90% effective in species identification. For reasons I do not understand, the authors favor unweighted pair group method with arithmetic mean (UPGMA) for analyzing genetic clustering, although they tested neighbor-joining, maximum likelihood, maximum parsimony, and Bayesian methods. Given the presumed advantages of a coding region barcode (ease of alignment, greater higher-level phylogenetic signal), Lahaye et al propose 5′ region of plastid gene matK as a first-pass standard barcode for plants.  

The authors then analyzed the 5′ matK barcode in a much larger sample of orchids: 1,566 specimens representing 1,084 Mesoamerican species. It is exciting that this is the largest test of candidate barcode variation within species for plants to date. They report 212 genetic clusters in UPGMA tree, of which “86 fully matched previously recognized species and a further 25 partially matched taxonomic species…an examination of these clusters reveals cryptic species, which need further taxonomic work”. I am unsure from this short report what “partially matched taxonomic species” are and how many possible cryptic species were identified. I look forward to a more detailed report on the DNA barcodes, morphology, and range distribution of this very large sample of Mesoamerican orchids.  A DNA-based method for identifying non-flowering orchids and other plants could help protect many threatened species. 

Approximately 8,000 - 15,000 species of bivalves (clams, mussels, scallops, oysters, and relatives) are known. According to BOLD Taxonomy Browser www.barcodinglife.org, 620 bivalve species have COI barcode records so far, so this group is relatively unexplored genetically. In September 2007 Zoologica Scripta researchers from University of Bergen, Norway, analyze COI barcode region sequences of 62 deepwater clams dredged in a single offshore region at 69 m to 567 m, morphologically identified as 12 species from 4 genera (Thyasira, Ennucula, Nucula, Yoldiella) representing 3 subclasses of Bivalvia. The COI barcode region was amplified with broad-range primers (Folmer et al 1994). Mean differences within species collected in this single area were small, 0.0 - 0.48%, similar to results in other animal groups, suggesting assignment of specimens to species will be straightforward. This will be helpful in environmental surveys for example, as some species “are infamous for being difficult to determine to species from morphology” and some “remain difficult to identify for the non-expert.” As one example, some Thyasira species are distinguished only by sperm and egg morphology, which is impractical in most circumstances.

mtDNA differences among these bivalves are remarkably large, even among species in the same genus. The differences among congeneric species in this sample (average 22%, range 12-42%) are larger than differences among entire class Aves (according to my analysis with BOLD software, COI differences among birds in different orders, such as penguins and hummingbirds for example, average 20%, with range 14-28%).

Blastn GenBank searches with these divergent mtDNA sequences showed very limited identity to anything, and the closest matches were short stretches (100-150 nucleotides of the 678 full-length barcode sequence) to COI sequences of species outside the phylum Mollusca (I obtained similar results submitting Thyasira sequences for example to the public BOLD Identification Engine at www.barcodinglife.org.)  It will be helpful if Mikkelsen et al deposit their sequences along with associated collecting data (voucher specimen information, images, collection locations) to the BOLD database. I look forward to learning more about these bivalves, and whether their remarkably deep differences in mtDNA are associated with deep physiological, ecological, or other biological differences.

As of 28 january 2008, there are 341,825 barcode records from 35,798 species in the Barcode of Life Database (BOLD) www.barcodinglife.org . What sectors of biodiversity have been analyzed so far? Here I follow the daily updated pages publicly available through “DNA Taxonomy Browser” link on BOLD home page. One can click downward through the taxonomic hierarchy from phyla to species, with a cogent summary at each level showing barcode records so far, contributing institutions and countries, and collection locations. The summary map shows remarkably good coverage of most terrestrial and coastal regions, and representation of nearly all countries. The open oceans are sparsely sampled so far, and remain an exciting terra incognita for biological exploration, including with DNA barcoding.  The global totals of 342K records/36K species work out to about 10 barcodes/species, and the average number of barcodes/species is similar at least down to the class level for most groups I looked at, suggesting a target of roughly 10 specimens per species is being achieved. 

The densest records so far are from Phylum Arthropoda (244,297), particularly insects (230,838), and of these mostly Lepidoptera (moths and butterflies) (169,145); and Phylum Chordata (74,720), particularly mammals (27,186), fish (26,752), and birds (12,770). There is broad sampling of other groups, including records from 376 animal orders in 80 classes representing 25 phyla. In addition, there are a few thousand records from fungi (3 phyla, 8 classes) plants (mostly red algae; 3 phyla, 8 classes), and protists (7 phyla, 11 classes), the latter of which DNA barcoding is likely to reveal as an enormous, deeply diverse group. 

The first paper proposing DNA barcoding was published in February 2003. The results displayed today on BOLD Taxonomy Browser demonstrate amazing progress in a short time, thanks to the inspiration and hard work of many! 

By identifying species (leaves) and determining how they are related (branches), taxonomy aims to reconstruct the Tree of Life. To do so taxonomists must distinguish variation within species from that between species, and identify the shared characters that reflect evolutionary ancestry. These tasks require highly-specialized sets of knowledge and skill for each animal and plant group. 

One result is that it has been difficult to compare patterns of diversification between different branches in the Tree. One might ask, how finely and how evenly divided is biodiversity? Are the differences among and within mosquito species (3,400 species), for example, similar to the differences among and within fruit flies (6,200 species) or birds (10,000 species)? Broad application of DNA analysis is beginning to provide some insights. To enable these sorts of comparisons, a standardized locus is needed, as unique genes can solve local branching patterns, but do not allow easy comparisons between branches.

Large-scale surveys of standardized genetic loci, including COI barcoding, commonly reveal distinct groups within what was thought to be a single species. There is also the converse finding that in some cases COI barcode sequences do not distinguish named species, but generally this strikes me as a relatively minor scientific problem that usually involves very closely-related species pairs and can be solved where needed with more DNA sequence, assuming the underlying taxonomy is correct and the named species really are distinct. The greater scientific challenge is finding multiple groups within what appear morphologically to be single species. In many cases so far, organisms with genetically distinct COI barcode clusters show associated biological differences signalling they represent different species.

In December 2007 Mol Ecol 16:4999, researchers from Museum of Comparative Zoology, Harvard, examine genetic differences in Aoraki denticulata, a tiny (2-3 mm) daddy longlegs or harvestman spider found in leaf litter widely through New Zealand.  They determined COI barcode region sequences for 119 individuals from 17 localities in the mountainous northern part of South Island. The two described subspecies A. d. denticulata and A. d. major were genetically distinct. The surprising finding was there were at least 14 distinct clusters within A. d. denticulata, with a different group at almost every site, and 2 clusters at 3 of the sites. The differences between mtDNA clusters were as larger or larger than between other Aoraki species, up to 19.2%, but no morphologic differences were found even with electron microscopic scanning of males. Boyer et al observe “while it is conceivable that some of the geographically widespread populations…represent cryptic species, it is difficult to imagine that morphologically identical individuals from a single sample at a unique geographical point are not conspecific…it is hard to believe that almost every sampled locality would host at least one, if not two, cryptic species.”

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Lumpsuckers are globular, scaleless marine fish with bony tubercles on head and body, and a ventral sucking disc, derived from specialized pelvic fins, which allows them to adhere to environmental substrates. The genus Eumicrotremus comprises 16 species distributed in the Arctic and northern Atlantic and Pacific oceans; the commonest and most widespread in the north Atlantic is the Spiny lumpsucker E. spinosus, which was first described by Fabricius in 1776. A new subspecies E. s. eggvinii was described in 1956, based on a single specimen, and this was later elevated to species level “on the basis of wrinkled skin, numerous dermal warts and a large sucking disk, in addition to the low number of bony tubercles.”

In August 2007 J Fish Biol 71A: 111, researchers from University of Bergen, Norway, analyze DNA and morphologic characters of E. eggvinii (n=16) and E. spinosus (n=67).  All specimens were easily classified by morphologic characters. However, the two species had identical mitochondrial DNA sequences (COI barcode region, COII, cytb) and identical nuclear gene Tmo-4C4. Further genetic testing revealed that E. eggvinii were all males, and E. spinosus were all females. The authors conclude that the two morphologically distinct ”species” represent the sexually dimorphic forms of E. spinosus. 

In this study by Byrkjedal et al, identical mtDNA sequences suggested synonymy, and this in turn suggested that morphologic divergence might represent sexual dimorphism, confirmed by further genetic testing. To my reading, this study suggests DNA testing needs to be as commonplace in taxonomy as recording size, shape, and coloration, and counting rays in fins and placement of tubercles. Every new species should have a representative DNA sequence as part of the species description. For animals, the standard should be a COI barcode. One of the remaining impediments to widespread adoption is that simple protocols for sequencing COI barcode region need to be better disseminated. In this study, the researchers were able to recover COI barcode region using primers designed for invertebrates (Folmer et al 1994), although others have published primer pairs that have greatly increased effectiveness with diverse fish (Ward et al 2005, Ivanova et al 2007). Compiling primer pairs and amplification protocols and displaying this information prominently on the various barcoding web sites will help (see for example SpongeBOL home page www.spongebarcoding.org link to illustrated primer primer!). I close with note this is post #100 since the first DNA barcode blog entry of March 15, 2006!