Chapter 17 DNA barcodes and insect biodiversity Robin M. Floyd, John J. Wilson, and Paul D. N. Hebert Biodiversity Institute of Ontario and Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 I know this little thing A myriad men will save, O Death, where is thy sting? Thy victory, O Grave? — Sir Ronald Ross (1857–1932) Insect Biodiversity: Science and Society, 1st edition. Edited by R. Foottit and P. Adler © 2009 Blackwell Publishing, ISBN 978-1-4051-5142-9 417 bout 3500 species of mosquitoes (Diptera: Insects constitute the most diverse group of animals Culicidae) have been described worldwide. In on the planet, with more than 1 million described A 1897, Ronald Ross, a Scottish physician work- species (1,004,898; introduction to this volume) and ing in India, discovered that only members of one millions more either awaiting description or simply mosquito genus, Anopheles, carry the Plasmodium par- undiscovered (Grimaldi and Engel 2005). They affect asite, the single-celled organism that causes malaria in human society in myriad ways, both harmful (e.g., humans. This revelation reflected painstaking efforts, disease vectors, crop pests) and helpful (e.g., pollina- involving the dissection of stomachs from vast numbers tors, biological control agents). Research of insects has of mosquitoes. It was a key breakthrough that paved added immensely to our understanding of evolution, the way for Ross to demonstrate the life cycle of the ecology, and the genetic control of development. Yet, a parasite in the laboratory, work rewarded by the 1902 fundamental requirement in gaining useful knowledge Nobel prize in Medicine. Unusually for a scientist, Ross about any organismal group is the ability to describe, was also a poet, playwright, and novelist; the preceding classify, and subsequently identify its member taxa. verse was written in response to this breakthrough in Groups, such as insects, present great challenges the understanding of malaria (Carey 1995). to the taxonomic enterprise simply because of their Sadly, Ross’s hope that this knowledge would quickly diversity. The identification of species by traditional allow malarial control proved too optimistic; the dis- morphologicalmethodsiscomplexandusuallyrequires ease still causes more than 1 million deaths per year, specialist knowledge. The recognition, description, and mainly in tropical Africa and Asia, despite numerous naming of new species is more so; yet, the number of eradication efforts (Greenwood et al. 2005). This sit- undescribed insect species far outweighs the number uation continues, in part, because the evolutionary of taxonomic specialists (Grissell 1999), whose work- dynamics of both Plasmodium and its insect vectors force is in decline (Godfray 2002). New approaches are far more complicated than initially realized. Only are needed to overcome this ‘taxonomic impediment’ a limited number of species in the genus Anopheles (Weeks and Gaston 1997, Giangrande 2003). These transmit the agents of the human form of malaria. concerns are not purely academic, but have signifi- Anopheles gambiae (sensu stricto), the most important cant practical implications. Agricultural pests cause vector of the Plasmodium parasite in humans, belongs immense damage. Total annual crop losses due to to a complex of morphologically indistinguishable sib- insect pests in North America have been estimated at ling species that nevertheless differ markedly in their US$7.5 billion and far more in the developing world habitat preferences, behavior, and ability to transmit (Yudelman et al. 1998), making it vital to quickly malarial agents (della Torre et al. 2002, Lehmann et al. identify destructive species before invasions become 2003). Although these species are likely in the midst uncontrollable. There is also a basic scientific need to of speciation (a process expected to result in mor- describe biological diversity before the destruction of phologically cryptic species complexes), they can be natural habitats by human activity causes the loss of readily discriminated on the basis of their ribosomal species on a massive scale. We need a rapid way of DNA sequences (Masendu et al. 2004, della Torre et al. assembling species catalogs, so that conservation pro- 2005, Guelbeogo et al. 2005). Plans are underway to grams can protect those areas of greatest importance control populations of A. gambiae by introducing trans- before they are lost (Myers et al. 2000). genes, a strategy that will depend on knowledge of gene flow and population dynamics within and among these sibling species (Cohuet et al. 2005, Tripet et al. 2005). SPECIES CONCEPTS AND The message from this story is clear: cryptic biolog- RECOGNITION ical diversity matters. Anopheles serves as a pertinent example of the challenges faced by those concerned Although species have long been considered the basic with biodiversity. Life exists in an immense number ‘units of biodiversity’ (Claridge et al. 1997), and the of forms, which are often tiny, difficult to study, and only ‘real’ grouping in the taxonomic hierarchy, the even more difficult to discriminate. Yet, this subtle vari- issue of how best to delimit species remains contro- ation can be crucially important; paraphrasing one versial. Mayden (1997) listed 22 species concepts that article on the subject, what we do not know can hurt have appeared in the literature (though some are essen- us (Besansky et al. 2003). tially synonymous) and that employed varied criteria DNA barcodes and insect biodiversity 419 from ecological niches, mate recognition, genetic Many authors refer to ‘operational taxonomic units’ cohesion, and evolutionary history. These diverse (OTUs) when delimiting taxa by purely phenetic or criteria necessarily lead to ambiguity, which can have heuristic means (Sokal and Sneath 1963). OTUs may important implications for studies of biodiversity and or may not correspond to species in the strict sense, conservation, as differing species concepts can produce but can be used in instances where speed and ease widely varying estimates of taxon richness (Agapow of application are of more practical importance than et al. 2004) Although reproductive isolation is often theoretical considerations (and if there are reasons to considered the most important indicator of species believe that theory and practicality are not directly status, it is seldom directly tested and fails to address in conflict). Taxa diagnosed or delimited by phenetic asexual organisms. In practice, most species continue DNA-sequence divergences can be termed ‘molecu- to be recognized by the presence of one or more appar- lar operational taxonomic units’ (MOTUs – Floyd et al. ently fixed or nonoverlapping diagnostic differences 2002, Blaxter et al. 2005). This approach has become (Davis and Nixon 1992). For most insect groups, the standard for environmental surveys of bacteria and detailed examination of genital morphology has other microorganisms, which could be seen as a capit- represented the gold standard for species definition for ulation to necessity, because these groups are virtually nearly a century, due to the observation of a general impossible to address in any other way (Hagstr¨om et al. phenomenon of rapid and pronounced divergence in 2002, Martinez et al. 2004, Hanage et al. 2005). How- the genitalia between species of animals (Eberhard ever, correspondence between MOTUs and species can 1985). Actual application of this criterion, however, be examined in a number of ways. One approach, is hampered by lack of an appropriate methodology tested with Lepidoptera, is the correlation with previ- to quantify shape variation (Arnqvist 1998) and by ously unassociated morphological or ecological traits, questionable homology assessments. All these factors for example, host plants and caterpillar phenotypes in collectively make species identification an extremely Astraptes fulgerator (Hebert et al. 2004). Where mor- specialized and time-consuming science, and even phological or ecological information is unavailable, a expert taxonomists can have difficulty reaching common situation in many taxonomic studies, congru- consensus. Moreover, this reliance on diagnostic ence with an appropriate nuclear gene is an objective characters that are present only in the adult life stage way to delineate interbreeding groups, and has been creates a serious constraint on identification, as many investigated in tropical beetles (Monaghan et al. 2005) specimens lack these characters (Balakrishnan 2005). and tachinid flies (Smith et al. 2006). Seven of the The life-history stages most commonly intercepted nine methods of delimiting species boundaries recently at ports of entry are larvae and pupae (Scheffer et al. reviewed by Sites and Marshall (2003) require molec- 2006), and damage to specimens collected in the field ular data, which could imply that molecular markers often makes identification difficult or impossible. are becoming increasingly important tools applied by Another option exists – species can be diagnosed by taxonomists, possibly due to objectivity, speed, and the genetic changes that arise between reproductively increased discriminatory capacity. isolated lineages as a result of genetic drift or selection. TheuseofDNAsequencestogaininformationaboutthe taxonomic affinities of an unknown specimen saw its DNA BARCODING earliest adoption in the least morphologically tractable groups such as viruses and bacteria (Theron and Cloete In this chapter, we deal with DNA barcoding, the use 2000,