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Two recent articles suggest how and how not to learn about applying mtCOI sequences to identifying species.  In Zoologica Scripta 2006 35:441 researchers from Koenig Zoological Research Museum, Bonn, analyze 113 specimens of 61 morphologically-defined species of pholcid (daddy long-legs) spiders. Important for this analysis and for future study, collection locations are given and voucher numbers are provided for each specimen and DNA extract.

(Some pholcid spiders vibrate in their webs when disturbed, moving so rapidly they become invisible; here is a wonderful video)

16s and COI sequences were successfully amplified using a single primer pair for each gene from 79% and 80% of specimens, respectively. It is striking that strong clustering within species was observed despite using short segments of mtDNA (COI, 312 bp; 16s 287 bp), which are less than half as long as the standard 648 bp COI barcode. In NJ trees with either mtDNA sequence, all morphologic conspecifics grouped together and were reciprocally monophyletic (ie no overlaps between species). Likely splits based on large intraspecific distances and differing geographic distributions were observed in 6 (25%) of 24 multiply-sampled morphospecies.

The authors go on to propose graphic and statistical metrics to calibrate how well simple distances can define species limits. They find that mtDNA distances will often diagnose species: “tree-based taxon clustering and statistical taxon analysis indicate that molecular evidence does coincide with morphological hypotheses” and “we disagree with [Meyer and Paulay’s] point that independently of the group of organisms studied, a “barcoding gap” between interspecific and intraspecific distance values would likely disappear in studies featuring both dense within-species sampling and closely related species”, ie distance-based clustering often corresponds to species limits. 

This study uses vouchered specimens from known locations and accurate modern sequencing technology, focuses on a relatively small clade (959 known species pholcid spiders), and analyzes in a positive way how distance measures might be used to define species, helping us learn about DNA barcoding as a tool for species identification.   

In another recent study Syst Biol 55:715 2006 researchers from National University of Singapore examine COI sequences deposited in GenBank from Diptera (flies, mosquitos, and gnats). They found 449 of the 150,000 known species of dipterans represented, with multiple sequences from 127 species, and analyze these to “test two key claims of molecular taxonomy”. The scientists found that there were often large differences in COI within species and also frequent overlaps between species such that some sequences were more closely related to those of another species than to conspecifics. The litany of failures is quite long, including “even when two COI sequences are identical, there is a 6% chance they belong to different species”.

I do not understand why the authors put so much effort into analyzing such a heterogeneous set of data, except that they are worried about molecular taxonomy in general and DNA barcoding in particular. To my reading this study suggests that many GenBank records contain errors, either because current morphologic taxonomy is incorrect (for example, study cited above suggests probable splits in 25% of pholcid spiders), specimens used for GenBank records are incorrectly identified, or because DNA sequences in GenBank contain errors due to human factors or older sequencing technology. There must be some limitations to COI barcode identification of dipteran species, mostly presumably closely-related young species, but this study has not shown where such problems might lie. 

I hope that future studies will use more of the “best practices” demonstrated in Astrin et al’s study of pholcid spiders and so help us learn more about how to apply COI sequences to species identification.    

Thrips are tiny (.5 to 2 mm) plant feeding insects; approximately 4500 species are known, and some are serious agricultural pests. Kladothrips is an Australian genus of at least 35 species which form galls on Acacia trees. In Biol J Linn Soc 2006 88:555 researchers from Flinders University, Australia, apply mtDNA analysis to show that two gall morpho-types of Kladothrips rugosus represent different species. 

Originally described in 1907, K. rugosus is widely distributed across south and western Australia. Two gall types were noted, but no morphologic differences could be found in the thrips themselves. McLeish, Chapman, and Mound found pairwise uncorrected mtCOI p-distances were 0.0-0.6% within gall morphotypes, and 7.4-7.8% between, similar to distances within and among other gall thrips species. The authors aver the usual taxonomic distaste for distance measures (“Distance values are not intended as a means of identifying different species here, which is a problematic approach for species depiction, but as useful descriptors of genetic variation”). I translate this as distance measures can be used help discover new species, but are verboten in official species descriptions. 

The only morphologic differences are that “abdominal segments I-III are as brown as IV-VII, the metathorax is scarcely paler than the brown mesothorax and prothorax, and the sculptured reticles on the posterior half of of tergites II-III are all small and equiangular.” Phew! Not many persons could decipher such abstruse morphologic terminology, whereas DNA-based identification promises more democratic access to species identification. The main limiting factors are technological and likely solvable: establishing reference libraries and developing inexpensive DNA analytic methods. 

The authors found a third genetic cluster in K. rugosus, but were unable to discover any morphologic characters, so did not describe this as a new species. This seems scientifically inconsistent, and the authors seem to agree: “This lack of morphologic divergence has evident problems for traditional taxonomy..we suggest that “morpho-taxonomy” is little more than an historical artifact in the methodology of species recognition, despite commonly providing the most practical methods”

I hope the large data sets emerging from the barcode initiative and other genetic surveys will enable taxonomists to develop consistent methods of species delimitation, whether in thrips or thresher sharks, and the sequences themselves or their diagnostic nucleotide characters will be routinely incorporated into species descriptions.

Insiders can be mistaken, in science and in other fields. At the beginning of the Human Genome Project, “the great majority of scientists dismissed the original proposal with hostility or indifference” (Great 15-year project to decipher genes stirs opposition. New York Times, June 5, 1990). The Times article details some of the initial negative reactions:

“Even if scientists manage to finish the genome project, it will have generated enormous reams of uninterpretable and often useless data”.

“The human genome project is bad science, it’s unthought-out science, it’s hyped science” said Dr. Martin Rechsteiner, a biochemist at the University of Utah. Some critics have begun aggressive letter-writing campaigns, urging colleagues who harbor similar sentiments to write Congress.

“Everybody I talk to thinks this is an incredibly bad idea,” said Dr. Michael Syvanen, a microbiologist at the Medical School of the University of California at Davis and a stout antagonist of the genome project.

Professional societies weighed in as well. A resolution adopted by the Council of the American Society for Biochemistry and Molecular Biology, and endorsed by the Federation of American Societies for Experimental Biology stated: “A large scale, massive effort to ascertain the sequence of the entire genome cannot be adequately justified at the present time… The Council wants to state in the clearest possible terms our opposition to any current proposal that envisions the establishment of one or a few large centers that are designed to map and/or sequence the human genome.” https://www.fasebj.org/cgi/reprint/1/6/502 

This history comes to mind in reading the article by Hickerson, Meyer, and Moritz in October 2006 Syst Biol 55:729. According to their analysis, mathematical modelling predicts that DNA barcoding will often fail to discover young species. Their analysis is based on a classical model of speciation (Bateson-Dobzhansky-Muller) and “well-established population genetic theory”. I should tread lightly here, not being a population biologist! To my reading, these mathematical models are either unsupported or disproved by experimental evidence. The BDM model of biological species formation is “well-characterized, tractable, and its dynamics captures a range of speciation times implicit across many pre- and post-zygotic isolation models”, ie good for modelling, but is not derived from actual genetic data on differences between sister species. Genetic surveys including growing barcode libraries demonstrating limited intraspecific variation in diverse species across enormous differences in population size and generation time indicate that “well established population genetic theory” does not explain intraspecific mitochondrial diversity (Bazin et al 2006 Science 28:570).

Instead of making predictions about why barcoding will fail, I hope the same mathematic rigor will be applied to understanding why barcoding works as well as it does, why the variation within most species is low, why the distances between most species are large, and what determines the exceptions.

In October Proc R Soc B Gomez et al apply DNA barcoding to the cosmopolitan marine bryozoan Celleporella hyalina. Morphologic identification in this genus uses scanning electron microscopy measurements of the 0.2 mm autozooid and its 0.05 mm orifice. To eliminate potential variability associated with colonial development or environmental plasticity, these morphologic measurements are made on cloned F1 progeny grown under controlled laboratory conditions. This example highlights how standard morphologic techniques can be cumbersome and costly, and require highly-trained personnel and expensive equipment. It is unlikely this sort of morphologic identification process can be sped up, while DNA analysis is getting faster, cheaper, and more portable.

The researchers from University of Hull, University of Wales, and Universidad Catolica de la Santisima Concepcion in Chile analyzed mtCOI barcodes in 176 colonies from 33 sites around the globe, revealing at least 10 deeply divergent lineages. Mating compatability in 26 pairwise combinations showed complete reproductive isolation in 23 cases, and 3 were inconclusive due to self-fertilization. Only one of the genetically divergent, reproductively incompatible groups could be reliably separated by morphologic analysis.

It is obviously impractical to do mating studies for routine identification of bryozoans. Instead, standardized genetic analysis, ie DNA barcoding, can first help discover species (as in this case by highlighting lineages that were then subjected to other forms of biological analysis), and then be applied to assign unknown specimens to the newly revealed species. The authors conclude “DNA barcoding clearly identifies biologically meaningful groups in the C. hyalina complex” and speculate that biodiversity is similarly underestimated in other sessile marine invertebrates, including sponges and corals. “Failure to recongize cryptic speciation among sessile benthos therefore may seriously underestimate marine biodiversity as well as impeding attempts to predict the response of marine benthos to environmental change.” I conclude that DNA barcoding is the fastest way forward to help discover and then routinely identify what appear to be the vast numbers of cryptic animal species.