Brent D. Mishler

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. 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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

The evolution of vegetative desiccation tolerance in land plants

Vegetative desiccation tolerance is a widespread but uncommon occurrence in the plant kingdom generally. The majority of vegetative desiccation-tolerant plants are found in the less complex clades that constitute the algae, lichens and bryophytes. However, within the larger and more complex groups of vascular land plants there are some 60 to 70 species of ferns and fern allies, and approximately 60 species of angiosperms that exhibit some degree of vegetative desiccation tolerance. In this report we analyze the evidence for the differing mechanisms of desiccation tolerance in different plants, including differences in cellular protection and cellular repair, and couple this evidence with a phylogenetic framework to generate a working hypothesis as to the evolution of desiccation tolerance in land plants. We hypothesize that the initial evolution of vegetative desiccation tolerance was a crucial step in the colonization of the land by primitive plants from an origin in fresh water. The primitive mechanism of tolerance probably involved constitutive cellular protection coupled with active cellular repair, similar to that described for modern-day desiccation-tolerant bryophytes. As plant species evolved, vegetative desiccation tolerance was lost as increased growth rates, structural and morphological complexity, and mechanisms that conserve water within the plant and maintain efficient carbon fixation were selected for. Genes that had evolved for cellular protection and repair were, in all likelihood, recruited for different but related processes such as response to water stress and the desiccation tolerance of reproductive propagules. We thus hypothesize that the mechanism of desiccation tolerance exhibited in seeds, a developmentally induced cellular protection system, evolved from the primitive form of vegetative desiccation tolerance. Once established in seeds, this system became available for induction in vegetative tissues by environmental cues related to drying. The more recent, modified vegetative desiccation tolerance mechanism in angiosperms evolved from that programmed into seed development as species spread into very arid environments. Most recently, certain desiccation-tolerant monocots evolved the strategy of poikilochlorophylly to survive and compete in marginal habitats with variability in water availability.

Individuality, Pluralism, and the Phylogenetic Species Concept

The concept of individuality as applied to species, an important advance in the philosophy of evolutionary biology, is nevertheless in need of refinement. Four important subparts of this concept must be recognized: spatial boundaries, temporal boundaries, integration, and cohesion. Not all species necessarily meet all of these. Two very different types of “pluralism” have been advocated with respect to species, only one of which is satisfactory. An often unrecognized distinction between “grouping” and “ranking” components of any species concept is necessary. A phylogenetic species concept is advocated that uses a (monistic) grouping criterion of monophyly in a cladistic sense, and a (pluralistic) ranking criterion based on those causal processes that are most important in producing and maintaining lineages in a particular case. Such causal processes can include actual interbreeding, selective constraints, and developmental canalization. The widespread use of the “biological species concept” is flawed for two reasons: because of a failure to distinguish grouping from ranking criteria and because of an unwarranted emphasis on the importance of interbreeding as a universal causal factor controlling evolutionary diversification. The potential to interbreed is not in itself a process; it is instead a result of a diversity of processes which result in shared selective environments and common developmental programs. These types of processes act in both sexual and asexual organisms, thus the phylogenetic species concept can reflect an underlying unity that the biological species concept can not.

Phylogenetic Relationships of the "Green Algae" and "Bryophytes"

Considerable progress has been made recently, based on classical morphological characters and newly described ultrastructural features, in understanding the phylogenetic relationships of the tracheophytes to the green algae and bryophytes. Recent technological advances in molecular biology, particularly the advent of the polymerase chain reaction (PCR), have allowed nucleotide sequence data relevant to such large-scale phylogenetic questions to accumulate, especially ribosomal RNA gene sequences (both the large and small subunits) from the nucleus and the chloroplast. We present synthetic cladistic analyses of the green plants that combine and compare available morphological and molecular data sets

Desiccation Tolerance in Bryophytes: A Reflection of the Primitive Strategy for Plant Survival in Dehydrating Habitats?

Abstract Bryophytes are a non-monophyletic group of three major lineages (liverworts, hornworts, and mosses) that descend from the earliest branching events in the phylogeny of land plants. We postulate that desiccation tolerance is a primitive trait, thus mechanisms by which the first land plants achieved tolerance may be reflected in how extant desiccation-tolerant bryophytes survive drying. Evidence is consistent with extant bryophytes employing a tolerance strategy of constitutive cellular protection coupled with induction of a recovery/repair mechanism upon rehydration. Cellular structures appear intact in the desiccated state but are disrupted by rapid uptake of water upon rehydration, but cellular integrity is rapidly regained. The photosynthetic machinery appears to be protected such that photosynthetic activity recovers quickly. Gene expression responds following rehydration and not during drying. Gene expression is translationally controlled and results in the synthesis of a number of proteins, collectively called rehydrins. Some prominent rehydrins are similar to Late Embryogenesis Abundant (LEA) proteins, classically ascribed a protection function during desiccation. The role of LEA proteins in a rehydrating system is unknown but data indicates a function in stabilization and reconstitution of membranes. Phylogenetic studies using a Tortula ruralis LEA-like rehydrin led to a re-examination of the evolution of desiccation tolerance. A new phylogenetic analysis suggests that: (i) the basic mechanisms of tolerance seen in modern day bryophytes have changed little from the earliest manifestations of desiccation tolerance in land plants, and (ii) vegetative desiccation tolerance in the early land plants may have evolved from a mechanism present first in spores.

TRANSITION TO A LAND FLORA: PHYLOGENETIC RELATIONSHIPS OF THE GREEN ALGAE AND BRYOPHYTES

Abstract— Separate cladistic analyses of the green algae, liverworts, and hornworts are presented. Classificatory and evolutionary implications of these analyses, in addition to our previously published cladistic analyses of mosses and the embryophytes as a whole, are discussed. The embryophytes are monophyletic, and are part of a larger monophyletic group that includes some of the green algae (the “charophytes”). Important evolutionary transformations in the early phylogeny of the land plants include: (1) retention of the zygote on the haploid plant (gametophyte), with the sporophyte generation arising de novo by delaying meiosis, (2) independent elaboration of an elongate sporophyte in some liverworts, some hornworts, and in the moss‐tracheophyte clade, (3) independent origin of radial (axial) symmetry in the gametophyte in some liverworts and in the moss‐tracheophyte clade, (4) independent origin of leaves on the gametophyte in some liverworts and in mosses, and (5) the unique development of a branching sporophyte with multiple sporangia in the tracheophytes.

Functional Constraints and rbcL Evidence for Land Plant Phylogeny

Although the proportion of «functional» DNA in eukaryotic genomes is both debatable and subject to definition, most sequences gathered for phylogenetic purposes are indisputably functional. For example, patterns of variation are likely to be strongly constrained in ribosomal RNAs because of their structural and catalytic roles in protein translation, and in protein-coding genes, because of protein function itself. Although seemingly obvious, these concerns are usually ignored by workers producing gene trees. We have examined the extent of functional constraints in land-plant rbcL sequences. Not only do rbcL sequences appear to change with essentially clocklike regularity, but nucleotide-based cladograms imply tbat approximately 97.5% of codon changes on internal branches are functionally neutral (i.e., synonymous or functionally labile)

Character-State Weighting for Restriction Site Data in Phylogenetic Reconstruction, with an Example from Chloroplast DNA

Data derived from cleavage points of various restriction endonucleases in all three genomes present in eukaryotes—nuclear, mitochondrial, and chloroplast— have been used for phylogenetic reconstruction in diverse groups of organisms. Mapped restriction sites, which represent a sampling of a whole genome or of any specific sequence, can be considered estimates of homologous characters and their transformations. In this sense, restriction site data are like any other robustly derived systematic data (e.g., morphological). An important difference is that probabilities for character-state transformations within restriction site characters can now be formulated using hypothetical or empirical estimates of sequence evolution (DeBry and Slade, 1985). Such transformational probabilities can be incorporated into phylogenetic reconstruction using parsimony with the application of maximum likelihood character-state weights (Felsenstein, 1981a).

Evolution of the major moss lineages: phylogenetic analyses based on multiple gene sequences and morphology

Abstract Evolutionary relationships of mosses are still poorly understood, with family, order, and subclass circumscription and relationships remaining especially obscure. Over the past decade, a considerable body of data has accumulated, including information on morphological, developmental, anatomical, and ultrastructural characteristics, as well as nucleotide sequences for a number of nuclear and plastid genes. We have combined data from these different sources to provide an overview of the relationships of the major lineages of mosses. We analyzed a data set that includes 33 moss species and ten outgroup taxa drawn from the liverworts, hornworts, and vascular plants. Molecular data consisted of nucleotide sequences from four DNA regions, (rbcL, trnL-trnF, rps4 and 18S). Morphological data included 41 characters of which many were derived from published anatomical and ultra-structural studies. Combining morphological and molecular data in the analyses showed that mosses, including Sphagnum, Takakia, Andreaea and Andreaeobryum, form a monophyletic group, provided improved resolution of higher level relationships, and further insight into evolutionary patterns in morphology.

Sex expression, skewed sex ratios, and microhabitat distribution in the dioecious desert moss Syntrichia caninervis (Pottiaceae)

The moss Syntrichia caninervis is the dominant soil bryophyte in a blackbrush (Coleogyne ramosissima) community in the southern Nevada Mojave Desert, with a mean cover of 6.3%. A survey of the 10-ha study site revealed an expressed ramet sex ratio of 14♀ : 1♂ (N = 890), with 85% of ramets not expressing sex over their life span, and an expressed population sex ratio of 40♀ : 2♂ : 1♀♂ (female : male : mixed-sex, N = 89), with 52% of populations not expressing sex. A greater incidence of sex expression was associated with shaded microsites, higher soil moisture content, and taller ramets. Shaded microsites had higher surface soil moisture levels than exposed microsites. In the exposed microhabitat, surface soil moisture was positively correlated with ramet height but not with sex expression. Male ramets and populations were restricted to shaded microhabitats, whereas female ramets and populations were found in both shaded and exposed microhabitats, suggesting gender specialization. The rarity of mature sporophytes, found in 0% of the ramets sampled and in only 3% of the populations, is probably due to the rarity of mixed-sex populations. We hypothesize that mixed-sex populations are rare because of factors relating to male rarity and that the differential cost of sex expression reduces the clonal growth capacity of male individuals.