Kipling W. Will

The Perils of DNA Barcoding and the Need for Integrative Taxonomy

SYSTEMATIC BIOLOGY that accompany this article, Will and Hebert respond to 10 questions selected by V.S. to reflect the balance of issues raised by the PEET audience (Hebert and Gregory, 2005; Will et al., 2005). Alternatively, you can follow the original debate as all 2 hours of the complete symposium are available to watch as a streaming video from http:// streamer.cen.uiuc.edu/seminars/peet/peet2-3-4.wmv (Windows Media Player required). A CKNOWLEDGEMENTS R EFERENCES Anonymous. 2003. What’s in a name? Economist 366:62. Blaxter, M. 2003. Counting angels with DNA. Nature 421:122– Godfray, H. C. J., and S. Knapp. 2004. Introduction [to a theme issue ’Taxonomy for the twenty-first century’]. Phil. Trans. R. Soc. Lond. B Gotelli, N. J. 2004. A taxonomic wish-list for community ecology. Phil. Trans. R. Soc. Lond. B 359:585–597. Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. deWaard. 2003. Biological identifications through DNA barcodes. Proc. R. Soc. Lond. B 270:313–322. Hebert, P. D. N., and T. R. Gregory. 2005. The promise of DNA barcoding for taxonomy. Syst. Biol. 54:852–859. Hebert, P. D. N., M. Y. Stoeckle, T. S. Zemlak, and C. M. Francis. 2004. Identification of birds through DNA barcodes. PLoS Biol. 2:1657– Holmes, B. 2004. Barcode me. New Scientist 182:32–35. Kurosawa, O., and M. Washizu. 2004. Acquisition and amplification of targeted position of electrostatically stretched DNA. J. Inst. Electro- stat. Jpn. 28:59–64. Lipscomb, D. L., N. Platnick, and Q. D. Wheeler. 2003. The intellectual content of taxonomy: a comment on DNA taxonomy. TREE 18:65–66. May, R. M. 2004. Tomorrow’s taxonomy: Collecting new species in the field will remain the rate-limiting step. Phil. Trans. R. Soc. Lond. B Moritz, C., and C. Cicero. 2004. DNA barcoding: Promise and pitfalls. PLoS Biol. 2:1529–1531. NCBI. 2005. National Center for Biological Information. Gen- Bank statistics available at http://www.ncbi.nlm.nih.gov/About/ tools/restable stat.html. Nicholls, H. 2003. DNA: The barcode of life? Originally published on behalf of Elsevier by BioMedNet. Now available at http://www. uoguelph.ca/∼phebert/media/BioMedNet%20News%20article. pdf. Pennisi, E. 2003. Modernizing the tree of life. Science 300:1692–1697. Rodman, J. E., and J. H. Cody. 2003. The taxonomic impediment over- come: NSF’s Partnerships for Enhancing Expertise in Taxonomy (PEET) as a model. Syst. Biol. 52:428–435. Schindel, D. E., and S. E. Miller. 2005. DNA barcoding a useful tool for taxonomists. Nature 435:17. Tautz, D., P. Arctander, A. Minelli, R. H. Thomas, and A. P. Vogler. 2003. A plea for DNA taxonomy. TREE 18:70–74. Taylor, R. W. 1983. Descriptive taxonomy: Past, present, and future. Pages 93–134 in Australian systematic entomology: A bicentenary perspective (E. Highley, and R. W. Taylor, eds.). CSIRO, Canberra. Vincent, M., Y. Xu, and H. Kong. 2004. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5:795–800. Will, K. W., B. D. Mishler, and Q. D. Wheeler. 2005. The perils of DNA barcoding and the need for integrative taxonomy. Syst. Biol. 54:844– First submitted 23 March 2005; reviews returned 9 June 2005; final acceptance 12 July 2005 Associate Editor: Vincent Savolainen Syst. Biol. 54(5):844–851, 2005 c Society of Systematic Biologists Copyright ISSN: 1063-5157 print / 1076-836X online DOI: 10.1080/10635150500354878 The Perils of DNA Barcoding and the Need for Integrative Taxonomy K IPLING W. W ILL , 1 B RENT D. M ISHLER , 2 AND Q UENTIN D. W HEELER 3 ESPM Department—Insect Biology and 2 Department of Integrative Biology, University of California, Berkeley, California 94720, USA Natural History Museum, Cromwell Road, London, SW7 5BD, UK “Your work, Sir, is both new and good, but what’s new is not good and what’s good is not new.” Samuel Johnson We argue that DNA barcoding has both new and good elements, but unfortunately no elements that are both. We are strongly in favor of the good idea of using DNA for identification, but that is old hat—the use of DNA for identification goes back to the beginning of molecular systematics. The DNA barcoders cannot take any credit for that. Their new idea that DNA barcoding can replace normal taxonomy for naming new species and studying their relationships is worse than bad, it is destructive. Statements by some barcoding proponents suggest an in- evitable replacement of taxonomic research rather than augmentation of technology to taxonomic science, e.g., “a COI-based identification system will undoubtedly Downloaded from http://sysbio.oxfordjournals.org/ at UNIVERSITY OF CALIFORNIA BERKELEY on August 29, 2013 I thank Kipling Will and Paul Hebert for taking part in the de- bate, Mike Irwin and Gail Kampmeier for organizing the fifth biennial PEET conference, and the National Science Foundation for financially supporting this meeting. Kevin Cummings, Martin Hauser, Andrew Miller, Mark Wetzle, and Kazunori Yoshizawa provided specimens and in some cases unpublished DNA sequences that were used dur- ing the species identification demonstration in this session. Martin Hauser, Mathys Meyer, Floyd Shockley, Daniela Takiya, and Jamie Zahniser assisted in the running of the debate. The symposium video was filmed and edited by Ritch Strom on behalf of the office of con- tinuing education at the University of Illinois. Rasplus Jean-Yves, Kevin Johnson, Rod Page, Diana Percy, Vincent Savolainen, Jason Weckstein, and an anonymous reviewer provided comments on an earlier (and considerably different) version of this manuscript. This work was supported by a grant from the National Science Foundation (DEB-0107891). VOL. 54

Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification

So‐called DNA barcodes have recently been proposed to answer the problem of specimen identification and to quantify global biodiversity. We show that this proposition is wanting in terms of rationale, methodology and interpretation of results. In addition to falling short of all its stated goals, the method abandons the benefits of morphological studies in favor of a limited molecular identification system that would ultimately impede our understanding of biodiversity.

Who Will Actually Use DNA Barcoding and What Will It Cost

It is likely that the mere use of the word barcode is responsible for much of the appeal surrounding DNA barcoding, after all DNA-based identification methods (e.g., DeSalle and Birstein, 1996) used prior to Hebert et al.’s (2002) proposal of the term failed to ignite significant attention from the scientific community and none whatsoever from the general public. The term itself is loaded. Product barcodes are scanned using checkout lasers and indeed the image of the “Star Trek tricorder,” a handheld scanner, has been used repeatedly by barcoding proponents in both presentations and papers (Janzen, 2004; Smith, 2005). Savolainen et al. (2005) use just such an allusion to commence their introductory paper of a special issue of Philosophical Transactions of the Royal Society of London devoted to DNA barcoding. Although the need for scanners capable of detecting biological weapons will undoubtedly lead to the development of portable DNA scanners at some point (a stated objective of the U.S. Department of Homeland Security, Directorate for Science and Technology), there is, however, absolutely no indication that they will be affordable or practical for the kinds of mass identification uses that barcoding proponents are selling to the general public as the outcome of this research. Further, a critical difference between a “tricorder” and a portable DNA barcoder would be the DNA component. Whereas Mr. Spock need only wave his tricorder in the general direction of an alien to be told what it is, real-world barcoders will need to actually handle that animal, remove tissue from it, and load it into the barcoder to get an identification. DNA barcoding is intrinsically linked to specimens as samples that must be collected for DNA extraction, be it in a molecular biology laboratory as at present or by a handheld barcoder at some point in the future. This small detail neatly circumscribes what barcoding can, and what it can’t, achieve. Who then would use DNA barcoding? Dan Janzen has written eloquently and spoken passionately about the need to improve biological literacy amongst the general public and that without the ability to “read” nature by identifying its contents, biodiversity is doomed to be underappreciated and so destroyed (see Janzen et al., 2005, for full development of this argument). One wonders what the place for even a portable DNA barcoder is in this vision. The majority of the public observes nature; they don’t sample it by removing the legs from butterflies and throwing them into a barcoder to get identifications. Wildlife protection authorities expend a lot of effort trying to keep people away from wildlife and certainly not handling or vivisecting them. Will the advent of a portable barcoder result in a complete turn around by wildlife authorities encouraging the wholesale handling of wildlife by the public? For smaller or more delicate plants and animals (which would include those most difficult for an interested amateur to identify without a barcode), being “identified” with a barcoder is likely to be fatal. Not simply due to the handling or picking necessarily, but due to the dissection necessary to get a tissue sample. Further, such barcode identifications will not share the benefits of traditionally collected specimens because an inexperienced member of the public is likely to be simply, and passingly curious, and not inclined to retain each “specimen” as a voucher with rigorous locality data for future study or reference. Although proponents suggest to compensate new species vouchers with free identifications (Janzen, 2004), one needs only think of the vast majority of people who toss aside cans they could recycle for a refund. The inconvenience of handling and sending a specimen in decent condition is far greater than recycling. Clearly such barcodes are unlikely to inspire or benefit the vast majority that needs to be reached to protect biodiversity. Amongst the wider public, the largest group identifying species on a regular basis is birdwatchers. DNA barcoding will be of limited use to them unless it is proposed that a 12-gauge shotgun now become standard equipment for such “twitchers.” Even for professional bird identifications, such as those prepared for biological impact studies, trapping is secondary to visual identifications due to the stress that handling inflicts on birds. Mammal identification for the general public is based on direct visual identifications as well as the interpretation of trail signs, scats, or hair. Perhaps some identifications could come through

Quantification and Evidence for Mechanically Metered Release of Pygidial Secretions in Formic Acid-Producing Carabid Beetles

Abstract This study is the first to measure the quantity of pygidial gland secretions released defensively by carabid beetles (Coleoptera: Carabidae) and to accurately measure the relative quantity of formic acid contained in their pygidial gland reservoirs and spray emissions. Individuals of three typical formic acid producing species were induced to repeatedly spray, ultimately exhausting their chemical compound reserves. Beetles were subjected to faux attacks using forceps and weighed before and after each ejection of chemicals. Platynus brunneomarginatus (Mannerheim) (Platynini), P. ovipennis (Mannerheim) (Platynini) and Calathus ruficollis Dejean (Sphodrini), sprayed average quantities with standard error of 0.313 ± 0.172 mg, 0.337 ± 0.230 mg, and 0.197 ± 0.117 mg per spray event, respectively. The quantity an individual beetle released when induced to spray tended to decrease with each subsequent spray event. The quantity emitted in a single spray was correlated to the quantity held in the reservoirs at the time of spraying for beetles whose reserves are greater than the average amount emitted in a spray event. For beetles with a quantity less than the average amount sprayed in reserve there was no significant correlation. For beetles comparable in terms of size, physiological condition and gland reservoir fullness, the shape of the gland reservoirs and musculature determined that a similar effort at each spray event would mechanically meter out the release so that a greater amount was emitted when more was available in the reservoir. The average percentage of formic acid was established for these species as 34.2%, 73.5% and 34.1% for for P. brunneomarginatus, P. ovipennis and C. ruficollis, respectively. The average quantities of formic acid released by individuals of these species was less than two-thirds the amount shown to be lethal to ants in previously published experiments. However, the total quantity from multiple spray events from a single individual could aggregate to quantities at or above the lethal level, and lesser quantities are known to act as ant alarm pheromones. Using a model, one directed spray of the formic acid and hydrocarbon mix could spread to an area of 5–8 cm diameter and persisted for 9–22 seconds at a threshold level known to induce alarm behaviors in ants. These results show that carabid defensive secretions may act as a potent and relatively prolonged defense against ants or similar predators even at a sub-lethal dose.

Defensive Chemicals of Two Species of Trachypachus Motschulski

Analyses of pygidial gland contents of two species of a previously uninvestigated family of beetles (Trachypachidae) by Gas Chromatography-Mass Spectrometry (GC-MS) revealed that their chemistry is similar to that reported from many members of the family Carabidae. Nevertheless, the composition ofDefensive gland fluids of the two species Trachypachus slevini and T. gibbsii differs sufficiently to distinguish between the two species solely on the basis of theirDefensive chemistry. The major components of T. slevini glandular fluid are methacrylic, tiglic, and octanoic (= caprylic) acids, together with the hydrocarbon (Z)-9-pentacosene. In contrast, the glandular contents of T. gibbsii contain a rather unique mixture of polar and nonpolar compounds, the principal constituents of which are methacrylic and ethacrylic acids (= 2-ethylacrylic acid), together with 2-phenylethanol, 2-phenylethyl methacrylate, 2-phenylethyl ethacrylate, and (Z)-9-pentacosene.

Phylogeny and classification of Hypherpes auctorum (Coleoptera: Carabidae: Pterostichini: Pterostichus)

ABSTRACT Based on an exemplar sample of pterostichine species (Carabidae: Pterostichini), 28S rDNA and COI and COII mtDNA sequence data are used to reconstruct a phylogenetic hypothesis for generic and subgeneric taxa putatively in or related to the subgenus Hypherpes Chaudoir (Coleoptera: Carabidae: Pterostichus Bonelli). The monophyly of Pterostichus is equivocal as the position of the subgenus Bothriopterus Chaudoir varies depending on methods of sequence alignment and gap region treatment. Pterostichus is found to be monophyletic in the combined data analysis if Cyclotrachelus Chaudoir and Tapinopterus Schaum are included in a larger concept of the genus. It is recommended that these be treated as subgenera of Pterostichus. Taxa currently included in Hypherpes are found to form a monophyletic group. No taxon previously suggested as a close relative of Hypherpes was found to be in, or closely related to Hypherpes. The sister-group of Hypherpes remains unclear, but there is some support for a clade of Pseudoferonina Ball + Cryobius Chaudoir as the adelphotaxon. Taxa included in current classifications of Hypherpes compose a group that is in fact a complex of Hypherpes sensu stricto and two other subgenera, Leptoferonia Casey and Anilloferonia Van Dyke, which have been treated as junior synonyms of Hypherpes. Our analyses show that these three taxa are well supported as subgenera and reciprocally monophyletic, with the only change to previous taxonomic concepts of included species being the transfer of Pterostichus rothi (Hatch) from Anilloferonia to Leptoferonia. It is recommended that all three of these subgenera be recognized rather than being subsumed under Hypherpes. In Leptoferonia the DNA data support all species groups that were established by Hacker using morphological characters, with the exception of the inopinus-group. Significant reduction of the compound eyes has occurred independently at least five and possibly seven times in the Hypherpes complex. As many as five separate instances of eye reduction may have occurred in Leptoferonia alone. Maddisons concentrated changes test was used to show that there is a significant correlation between microphthalmy and autapomorphic sequence data as represented by longer than average terminal branch lengths based on Bayesian estimates of change per site. However, taxon pair contrasts show no consistent pattern of absolute difference of evolutionary rate or directionality of differences between small-eyed taxa and their sister species or sister clade. Repeated patterns of allopatric distributions are found for species-pairs of Leptoferonia, which consist of divisions along a north/south axis near the Pacific Coast and in the Sierra Nevada Range, or east/west divisions between coastal species and inland or Sierran species. In addition to allopatric biogeographic patterns, instances of sympatry in closely related species are interpreted to have been the result of two reduced-eye species moving into the deep litter and soil layer, thereby ecologically differentiating from near-surface leaf-litter and log dwelling species. Pterostichus morionides (Chaudoir), which is restricted to the Sierra Nevada Mountains in western North America, is found to be sister to P. adoxus (Say) and P. tristis (Dejean), the only species of Hypherpes in eastern North America. This grouping (mta-clade) was further tested by using a subset of taxa for 18S rDNA, CAD and wg sequence data and was found in some or all most-parsimonious trees for these data. In cases where they did not form a clade, they usually formed a convex group. Although counterintuitive due to the unusual disjunct biogeographic connection of these two areas and the generally dissimilar form of the adults, the mta-clade is very well supported by the DNA sequence data.

Carabidae from Vietnam (Coleoptera)

Abstract Members of the Vietnamese carabid fauna are arranged and nomenclaturally revised. As the result, 178 species belonging to 74 genera of 12 subfamilies, Brachininae, Omophroninae, Carabinae, Scaritinae, Bembidiinae, Trechinae, Pterostichinae, Harpalinae, Callistinae, Oodinae, Odacanthinae, and Lebiinae, are recorded from Vietnam. Within these, four species, Abacetus leucocetes Bates, 1873, Ago-nom (Metacolpodes) buchanani Hope, 1831, Steno-lopus (Egadromd) difficilis (Hope, 1845), and Phe-ropsophus {Stenaptinus) beckeri Jedlicka, 1930 are newly recorded. A species checklist with taxonomie information, type localities and worldwide distribution are provided.

Molecular phylogeny and Holarctic diversification of the subtribe Calathina (Coleoptera: Carabidae: Sphodrini)

A molecular phylogeny of the subtribe Calathina was inferred from DNA sequence data from the mitochondrial cox1-cox2 region and the nuclear genes 28S and EF-1alpha. All lineages within Calathina from the Holarctic region were represented except for the monotypic subgenus Tachalus. Maximum Parsimony and Bayesian analyses of the combined data set showed that the subtribe is a monophyletic lineage that includes a single genus Calathus, where other taxa currently ranked as independent genera (Lindrothius, Synuchidius, Thermoscelis and Acalathus) are nested within this genus.Neocalathus and Lauricalathus, both subgenera of Calathus, were found to be polyphyletic and in need of taxonomic revision. The subtribe appears to have originated in the Mediterranean Basin and thereafter expanded into most parts of the Palearctic region, the Macaronesian archipelagos (at least five independent colonisation events), the Ethiopian highlands and the Nearctic region (at least two independent events).

Antisymmetric male genitalia in Western Australian populations of Mecyclothorax punctipennis (Coleoptera: Carabidae: Moriomorphini)

Western Australian populations of Mecyclothorax punctipennis (MacLeay) exhibit chiral polymorphism for male genitalic asymmetry. The plesiomorphic genitalic enantiomorph, wherein the male aedeagal median lobe is left side superior when retracted in the abdomen, is rotated 180° to a right side superior position in 23% of males from Western Australia. Conversely, population samples from eastern Australia are monomorphic for the plesiomorphic left side superior condition. Western Australian population samples are significantly heterogeneous for the percentages of chirally reversed males, with right side superior frequencies ranging 0–58%. Conversely, asymmetry of the M. punctipennis female reproductive tract, wherein the apex of the bursa copulatrix is distally expanded toward the right side of the individual, is shown to be monomorphic within the species. Based on the vast disparity in frequencies of left versus right enantiomorphs among populations of Western Australian M. punctipennis, we hypothesize that population demographic factors related to very small population size and differential gene sampling via genetic drift could interact to establish populations fixed for the novel form. When such chiral genitalic substitu tions are coupled with speciation, subsequent diversification stemming from that common ancestor would result in monophyletic lineages characterized by genitalic inversion. This hypothesis is corroborated by the sporadic occurrences of individual males with chirally inverted genitalia throughout the Carabidae, and the known occurrence of eight carabid taxa — individual species to diverse lineages — that are monomorphically characterized by male genitalic inversion.

New and Little Known Species of Loxandrus LeConte 1852 (Coleoptera: Carabidae) from North and South America

AbstractThree new species of beetles in the genus Loxandrus are described: L. straneoi from the southern United States, L. icarus from the Eastern United States, and L. quinarius from central Bolivia. Loxandrus icarus, inhabitant of wet lowland habitat, is unique among eastern North American species in having reduced flight wings. The only other North American species with reduced flight wings, L. omiltemi Allen & Ball, is known from temperate cloud forest habitat in Mexico. Loxandrus quinarius shares a striking phenetic similarity to some members of the African caelostomine genus Strigomerus Chaudoir, and with L. strigomeroides Straneo. Illustrations of the male genitalia of L. strigomeroides and L. mirei Straneo are presented here to assist in identification of these species. The distributional range of L. strigomeroides is newly noted to extend across the Amazon basin. Loxandrus inferus Allen is placed into synonymy under L. velocipes Casey.