DNA Barcoding of Animals
PreambleThe animal kingdom includes approximately 1.3 million described species and an estimated total of nearly 10 million. Although our fauna is incompletely known, it is thought that about 1-2% of global biodiversity occurs within Canada, implying that some 100,000- 200,000 animal species occur within our nation’s boundaries. Of course, the vast majority of these species have distributions that also extend either into Eurasia or into more southerly regions of North America or both.
Although it is feasible to create a DNA-based identification system for the entire Canadian fauna, this enterprise cannot be completed with the resources available through the Research Networks program. However, we will make an important beginning on this task by developing a DNA-based identification system for the 10,000 animal species that are of highest concern to Canada. To optimize our efficiency in building a comprehensive identification system, we will analyze a small number of individuals of each species, examining organisms from varied geographic locales whenever possible. We justify this decision by pointing to the fact that effective identification engines have already been created using this approach for a number of animal groups (Ball et al. 2003, Hebert et al. 2003a, Hogg & Hebert 2003). We emphasize that their success reflects the fact that sequence divergences are ordinarily low among individuals of a species (including our own). In those cases where intraspecific diversity is high relative to interspecific divergence, we will sequence additional individuals. With this analytical strategy, we will be able to maximize the number of species that we examine with the resources available. We recognize that our development of an identification system based on a single mitochondrial gene will be insufficient to guide species diagnosis in all cases. For example, introgressive hybridization will impede species identifications, but such situations are rare in the animal kingdom, and they can be resolved by the supplemental analysis of sequence diversity at a small number of nuclear loci. Finally, we note that rates of COI evolution are so slow in Cnidaria and perhaps Porifera that species diagnoses in these phyla will require supplemental analyses.
In the balance of this section, we describe the research programs that we plan to carry out on eight groups of animals where background work has established that COI-based identification systems will be highly effective. The intensity of our work on each group is not simply a function of its taxonomic diversity. We have, instead, directed our greatest effort to assemblages where economic or policy impacts are high and where prior DNA barcoding studies have resolved impediments to rapid progress. Our work plans will require the analysis of COI sequences in approximately 47,000 individuals, but we expect that our analytical capacity will allow 20,000 more analyses. Of course, some of this ‘excess’ capacity will need to be directed towards the resolution of analytical problems and towards increased sample sizes for certain taxa. However, we expect to devote supplemental effort to key groups that currently lack detailed background information, such as parasites, but this will only be decided once we have acquired enough results to ensure that these efforts will be rewarding.
In describing each project, we have covered the core elements proscribed in the application guidelines, but we have not developed a separate milestone section for each project. Instead, we opt for a brief discussion at this point. We have taken this stance because the approaches employed in our varied projects possess strong congruence. Activity in year 1 will focus on the assembly of species lists, on the acquisition of required specimens and on the consolidation of analytical protocols while later work will focus on sequencing. Projects involving the analysis of 100 species will ordinarily be complete in a year, those examining circa 300 species in two years, while those examining 1000 or more species will require the full 5 years to complete. Our budget calls for 42 PDF years and 52 graduate student years to complete the animal projects. If focused solely on analyses, we expect each PDF could analyze 2000 specimens per year (versus 1000 for GS), a figure that exceeds our analytical needs, but we feel that students must have time to explore novel issues that develop out of their work, a factor that will lessen the volume of samples they process. Top
Agricultural and Forestry PestsCanada has long had a strong involvement in monitoring and identification programs that target the arthropod species that inflict damage in agricultural and forestry settings. For example, nearly 50 years ago the Forest Insect Survey reared more than 700,000 lepidopteran larvae belonging to more than 900 different species (Prentice et al. 1958-1966). Shortly thereafter, Canada led the formation of a tri-national North American consortium that aimed to share information on the optimal control strategies for forestry pests (Davidson & Prentice 1967). Despite this long effort, challenges remain for pest monitoring programs. Because the number of insect species that feed on forest trees and crops is extremely large (Danks 1979), the identification of known species requires the maintenance of a large number of taxonomic specialists. Moreover, some important pest groups include assemblages of species whose discrimination is extremely time consuming. Other groups are easily identified at one life stage (usually adults), but their juveniles are difficult or impossible to separate. The implementation of DNA-based diagnostic systems will enable Canada’s taxonomists to focus their efforts on advancing knowledge of little-known groups rather than on routine identifications.
The dynamic nature of species assemblages presents a secondary challenge. The suite of pest species shifts as species ranges alter both naturally and as a result of invasions. This problem is not a minor one. More than 5% of the lepidopteran species that now occur in Ontario owe their presence to human-mediated transport from Eurasia. These invaders typically do greatest damage during their first wave of population expansion, often while they are still taxonomically anonymous. The solution to this problem lies not in the generation of taxonomists with a global knowledge of life, but rather in the development of DNA-based identification systems. For example, the INRA is planning to develop a COI-5’ barcoding system for the aphids of France. Once their work is complete, it will provide an immediately useful tool for the diagnosis of new aphids that might make their way to North America from this region. Conversely, the creation of a COI-5’ database for Canadian aphids will aid the diagnosis of North American invaders in European settings. Moreover, we emphasize that once a COI profile is in place for any region, it will provide an immediate indication of any novel species that is encountered.
Research Plans (circa 6000 analyses)We have already begun the task of building a diagnostic system for the pest insects of agricultural and forestry settings in Canada. This work has so far involved the collection of COI- 5’ sequences for 500 of the most common moth species from the eastern half of Canada. This study has established several important practical facts. Firstly, it has shown that COI-5’ sequence can readily be generated from DNA extracted from a single leg of even the smallest insect. As a result, pinned specimens in museum collections are prime targets for analysis. Our work has also shown that DNA can be readily recovered from specimens up to 50 years old without major analytical complications. In effect, this means that most of the critically identified specimens held in collections across our nation are suitable for analysis. Moreover, our past work has shown that the sequence characterization of just a single individual can provide an excellent diagnostic basis for the recognition of other individuals of the same species.
The fact that analytical protocols are simple, that sample sizes for each species can be small, and that specimens are readily available for analysis means that it will be possible to generate an identification system that provides broad taxonomic coverage. We will focus our work, at least initially, upon the development of a COI-5’ database that provides coverage for all of the important agricultural and forestry pests in four major insect orders (Hymenoptera, Coleoptera, Lepidoptera and Orthoptera). We will initially examine just three individuals per species to enable the analysis of as many species as possible, but will increase these sample sizes if cases are encountered where the diagnosis of closely allied species is problematic.
Because there have been extensive collection efforts over the past 50 years, we will direct little effort to the acquisition of new specimens. We will, instead, focus on the analysis of specimens held in well-identified reference collections that exist in agricultural and forestry laboratories across Canada. For example, Canadian Forest Service regional reference collections, such as that at the Pacific Forestry Centre that includes 145,000 specimens and some 8300 species in our target orders, will provide reliably identified geo-referenced specimens for analysis. Similar collections exist for agricultural pests, such as those of prairie insects overseen by another of our researchers (Dan Johnson, Agriculture & Agri-Food Canada and U. Lethbridge). We emphasize that many research projects in agriculture and forestry depend upon the accurate identification of immature stages that are currently difficult or impossible to identify. Because of the very large number of species available for analysis, we will need to focus our efforts on the taxa of greatest economic concern. Our decisions in this regard will be aided by dialogue with forestry and agricultural entomologists across our nation and by the availability of reports identifying key pest species (e.g. Ives & Wong 1988, Armstrong & Ives 1995). Our analyses will lead to the generation of a COI-based identification system for at least 2000 species of insects in these groups.
Personnel: This research program will involve the collaboration of individuals with three disciplinary backgrounds: insect taxonomy, molecular evolution and pest insect management. We will minimize demands on the taxonomic community by focusing our analytical work on existing well-identified collections of agricultural or forestry pests. For example, Lee Humble (Canadian Forest Service) curates the largest forest insect collection in Western Canada and is keen to see these specimens used for barcoding analysis. Similarly, Dan Johnson (Lethbridge) has extensive research and taxonomic collections of varied prairie pest species (grasshoppers, leafhoppers, beetles) available for analysis. Finally, Georgette Smith, curator of the insect collection in the Atlantic Forestry Centre, is also interested in collaborating in our research programs. As we analyze specimens from collections such as these, we will surely encounter situations where our genetic work will reveal the need for taxonomic reappraisals or verifications. Many insect taxonomists have expressed interest in contributing to this task and we will involve additional experts as the need arises. We have not assembled their curricula vitae because we expect that the time demands on most of these individuals will be modest. However, in some cases, interactions will be more intense because our DNA-based work promises helpful insights into recalcitrant taxonomic problems. For example, Jean-François Landry (Agriculture & Agri-Food Canada) recognizes that this approach will do much to aid the resolution of microlepidopteran taxonomy. The DNA work for this project will be led by Paul Hebert (Guelph) because of his ongoing involvements in the analysis of arthropods from museum collections. the Canadian Barcode of Life Network expects to support this research program with two graduate students and one PDF position.Top
Biological InvadersCanada’s forests, agricultural systems and aquatic ecosystems support more than 1500 alien species (MacIsaac et al. 2002). Invaders, such as these, are now recognized as the second leading threat to global biodiversity and the leading threat to lake communities (Sala et al. 2000). The Great Lakes region has endured an onslaught of invasive species, some of which have reprogrammed food webs, and adversely affected commercial and sport fisheries and raw water users. These problems are not declining - the rate of new species introductions into the Great Lakes tripled during the 1 990s as compared to the previous four decades (Grigorovich et al. 2003). Terrestrial environments are also under siege. The emerald ash-boring beetle, which established itself in southwestern Ontario sometime during the past six years, may eventually have the same destructive impact on ash trees that Dutch elm disease had on the American elm. Likewise, leafy spurge and cheatgrass, among many others, are serious economic and ecological weeds of prairie and western grasslands. The ecological impacts of invaders are not restricted to inland settings. Oyster and blue mussel aquaculture operations are currently threatened in Atlantic Canada by invasive parasites and fouling invertebrates. Total economic damage associated with just 19 pest alien species in Canada may reach $34 billion per year (MacIsaac et al. 2002).
Two complementary strategies are required to combat the flow of invasive species. The first involves gaining knowledge of the vectors and trajectories employed by invaders as they move from one locale to another. Once transit pathways have been identified, management efforts can be directed toward their disruption. Despite such efforts to curb new invaders, they will surely fail on occasion. For example, quarantine inspections at ports of entry do often detect species transported with trade, but their identification is regularly hampered by the lack of systematic tools and personnel (Allen & Humble 2002). DNA barcoding will provide a rapid means of identifying such exotic species and will be of primary importance in providing taxonomic assignments for the immature stages most frequently intercepted. At present, only the most morphologically distinctive invaders can be recognized promptly. A rapid diagnostic system would be particularly valuable for trade in perishable goods, where there is a risk of serious economic loss while identifications are sought. A DNA-based diagnostic system would also reduce the chance of misidentifications during the early phases of the establishment of a new pest species. Such prompt warning might allow the use of control measures that would lead to its eradication. At the very least, rapid taxonomic assignments would enable prompt access to literature concerning optimal control strategies or the likely magnitude of threat that they pose.
Our studies on forestry pests will lead to the development of DNA-based identification systems that will enable the recognition of newly arrived species that are of quarantine significance. This information will be of high value to the Canadian Food Inspection Agency because of its responsibility for quarantine issues. Our studies on aquatic species will provide newly detailed information on the incidence of past invasions and on the dispersal corridors that were employed. This latter research will involve a comparison between the genetic attributes of invasive populations and those of the species at its varied potential source locales. In certain cases, more extensive genetic analyses may be required to reveal dispersal corridors, but several recent studies have employed COI-5’ diversity to gain detailed insights of invasion pathways.
Most recognized faunal invasions involve morphologically distinct species that are readily discriminated from native taxa. However, it seems likely that many other invasions go unrecognized because of the close morphological similarity between the invader and one or more native species. To gain a better sense of the extent of such cryptic faunal invasions, we plan a genetic analysis of key elements of the freshwater fauna. Work will, at least initially, focus on the crustaceans, because they are abundant, diverse and relatively well studied. However, there has not been a comprehensive effort to assess the incidence of crustacean invaders using genetic techniques. We will obtain COI-5’ sequences for 5 individuals from each of the 180 crustacean species known from the Great Lakes. We will subsequently compare the genetic attributes of these populations with those from inland lakes within Ontario. Past work has shown that the diffusion of newly introduced species into isolated inland lakes is slow, so one can assume that populations from these habitats represent relatively uncontaminated gene pools. By contrast, if populations from the Great Lakes consist entirely or partially of invaders, they will possess unique genetic attributes. When such cases are identified, the analysis of reference populations from other continents can establish their source. We expect that these genetic studies will reveal a number of hitherto unrecognized invasions and provide a basis for ascertaining their point of origin. In addition our work will provide baseline data on the genetic composition of the Great Lakes crustacean fauna at the beginning of the 21st century, information that will provide a valuable reference point for future studies.Invasive Species in Forestry (circa 2000 analyses)
We plan work on three model systems to test the efficacy of COI-5’ in the resolution of taxonomic boundaries in forest insect assemblages that include invasive and native taxa. In each case, there are difficulties in both discriminating members of the present fauna and in the recognition of species that currently occur in other regions but present an invasion risk. Our work will involve comprehensive COI-5’ surveys for species in the target groups employing the same analytical strategy as employed in our work on agricultural and forestry pests. Although our initial studies will target the three following groups, we will extend investigations to members of other families (e.g. Cicadellidae, Tenthredinidae, Totricidae) if resources permit.
Scolytid beetles: Bark and ambrosia beetles are one of the most important groups of pest insects in the coniferous forest regions of Canada. The 210+ species involved in causing this damage now include 12 introduced species (Humble 2001).
Coccinellid beetles: Ladybird beetles are important biocontrol agents. The native fauna of 162 species has now been augmented by 9 exotic species. Some of the latter species were intentionally introduced, but are now disrupting the native fauna.
Forest pests of phytosanitary concern: COI-5’ sequences will be generated for forest pests currently regulated by Canada or intercepted in international trade. This work will ensure the availability of DNA diagnostics for their rapid identification. Specimens will be drawn from existing collections in Canada. For example, the Pacific Forestry Centre collection holds numerous northeastern Asian and European invasive species intercepted during quarantine inspections or obtained through international collaborations. Work will focus on taxa that have either become established in other jurisdictions or that pose serious threats to Canada’s forests.
Personnel: This research program will be led by Hugh MacIsaac (Windsor) and Lee Humble (Canadian Forest Service). Hugh holds a DFO Chair in Invasion Biology and is Canada’s leader in the study of invasive species in aquatic ecosystems, while Lee heads the exotics detection program for forest insects in British Columbia. Our studies on forest invaders will be greatly aided by Isabel Leal, a molecular geneticist at the Pacific Forestry Centre. the Canadian Barcode of Life Network expects to provide support for 1 PDF and 2 graduate students to aid work on invasive species.Top
Biting InsectsNo elements of the Canadian insect fauna interact more directly with human populations than those that rely on blood meals to enhance their reproductive output. Although several taxonomic groups are involved, the closest inquilines of humanity (bedbugs, fleas, lice) have been effectively controlled and exert little influence in local settings. However, the situation is different for the other dominant group of biting insects, the Diptera, where Canada has a long standing and deserved reputation as a nation whose wildlands are fly-ridden. Densities of biting Diptera may be particularly high in Canada, but our situation is not unique. These are, by far, the most important insects from a medical and veterinary perspective in a global context. They are, for example, responsible for transmitting major human diseases (e.g. malaria, filariasis, trypanosomiasis). In addition to their role as vectors of disease, biting flies stress hosts through their bloodsucking activities and resultant allergic reactions. The ability to accurately identify biting insects in all their life-history stages is paramount to control programs. Unfortunately, the immature stages of many species are currently difficult or impossible to identify, and even the discrimination of adults requires a high level of taxonomic expertise.
Six families of biting flies occur in Canada: mosquitoes (Culicidae), black flies (Simuliidae), biting midges (Ceratopogonidae), horse and deer flies (Tabanidae), snipe flies (Athericidae) and stable flies (Muscidae). Collectively these taxa contain the most noxious pests of humans and other vertebrates in Canada. Our studies will focus, at least initially, on two of these families, the Culicidae and the Simuliidae, because they are the most important elements of the biting fly fauna, and because their taxonomy is well established.
Mosquitoes are an exceptionally important family of biting flies in Canada because they have historically been involved in the transmission of malaria, St. Louis encephalitis, and eastern/western equine encephalitis. In 2001, the mosquito-borne disease, West Nile virus, also appeared in Ontario and by 2002 it had spread to five provinces. Eight of the 52 mosquito species known from Ontario appear to play a role in its transmission. Their involvement as carriers has elevated the need for detailed knowledge of local mosquito assemblages, but such evaluations are currently hampered by the limited availability of taxonomic experts.
We plan to develop a COI-based identification system for the 80 mosquito species currently known from Canada. We will carry out this work by analyzing 10 individuals of each species, including specimens from varied segments of each species range whenever possible. The identification system created through this work will find immediate practical application because it will make West Nile virus surveillance programs far more accessible. At present, the level of expertise required for mosquito identifications is so high that considerable training is required and the process is time consumptive. As well, many viral surveillance programs involve the bulk analysis of mosquito larvae from a habitat. As a result, it is possible that the number of West Nile ‘carrier’ species has been inflated as a result of cases where a few larvae of a standard carrier species co-occurred with a much larger number of individuals of a second species. DNA-based analysis could easily diagnose such cases in a retrospective fashion. In fact, the future surely lies in a move to DNA diagnostics where single labs will be able to simultaneously process mosquitoes for viral testing and for their species identity at all life history stages.
The taxonomy of Canadian black flies is particularly well established because morphological analyses have been supplemented by cytological analysis of the giant polytene chromosomes found in their larvae. The banding patterns of these chromosomes have revealed numerous groups of sibling species that can currently only be distinguished in this fashion. In fact, cytogenetic examination provides the only means to reliably identify about 25% of the 260 simuliid species found in North America. As only a few specialists have the expertise to prepare and interpret chromosomal banding patterns, the development of a DNA barcoding system will provide a great advance in the accessibility of identifications for this group. We emphasize, as well, that current cytogenetic approaches do not allow the discrimination of simuliid pupae or adults because these life stages lack polytene chromosomes.
We will develop a COI-5’ identification system for the entire simuliid fauna of Canada by analyzing 10 individuals of each of the currently recognized 165 species from varied parts of its range. We will direct additional effort to the discrimination of species in the Simulium venustun and S. vittatum complexes to test the ability of COI-5’ to discriminate members of these recently diverged assemblages. We note that these two complexes also include the most serious human (venustum) and livestock (venustum/vittatum) pests.
Personnel: Our work on this program will involve three researchers. Fiona Hunter (Brock) is well positioned to lead our mosquito project because her laboratory has been very active in identification programs for these organisms linked to the rise of the West Nile virus. Fiona also has the cytogenetic background needed to aid our work on blackflies, a project that will be headed by Douglas Currie (Royal Ontario Museum). The DNA sequencing for these studies will be provided by the Canadian Barcode of Life Network’s Advanced DNA Technology group led by Teresa Crease (Guelph). We additionally plan to provide support for a 2.5 year PDF appointment and I graduate student to aid this work.Top
Freshwater ecosystems include complex assemblages of animal life, but fishes represent the primary target of management programs because of their importance to both recreational and commercial harvests. Indeed, freshwater fishes are heavily exploited in most accessible lakes and rivers throughout Canada. Interest in fishes is further motivated by the fact that some 38% of Canadian species have been listed by COSEWIC (Committee on the Status of Endangered Wildlife in Canada) as being at risk. Therefore, acute management actions are required to ensure their long-term conservation. These conservation threats are exacerbated by the increasing demand to support freshwater fisheries through stocking. While stocking offers the potential of increasing fish biomass in the short-term, this comes at the cost of altering the genetic integrity of locally adapted populations, a factor that can reduce production over the long term (Duchesne & Bernatchez 2002). Past work on a few Canadian fish species has shown that most include two or more lineages showing enough genetic divergence to suggest their isolation for several hundred thousand years, long enough to produce genetic incompatibilities (Bernatchez 1995, Bernatchez et al. 1999). These impacts can be reduced by ensuring that stocking is made using populations that are genetically compatible with the recipient populations (Ryman et al. 1995). Sound management of fish populations also requires accurate quantification of recruitment as well as survival at young life history stages. However, a major constraint faced by managers derives from the difficulties in separating species at the egg, larval and juvenile stages. Clearly, genetic information allowing diagnostic species identification and distinction among regional lineages within species will aid fisheries management (Cabellero & Toro 2002). However, this information is currently lacking for most Canadian fishes (Bernatchez & Wilson 1998).
Research Plans (circa 4000 COl analyses & 4000 Cyt b analyses)
Our research program will involve the development of a DNA-based identification system for the 200 or so freshwater fish species found in Canada. We plan to analyze an average of 20 individuals per species (the number will vary with the species distribution) with the number of samples adjusted and distributed to cover the species’ range. Samples for analysis will largely be acquired through the efforts of annual sampling programs undertaken by provincial and federal resource management agencies. We will collect sequence information for two mitochondrial gene regions, COI-5’ and cytochrome b, to compare the resolution that they provide for both species diagnosis and for the delineation of phylogeographic subunits. This comparison is important because most prior studies on vertebrates, particularly fishes, have focused on the analysis of cytochrome b. Aside from developing a DNA-based identification system for all Canadian freshwater fishes, we will clarify the taxonomic status of certain species. The Atlantic whitefish and the copper redhorse are obvious targets for such analysis because both species are endemic to Canada, are currently regarded as endangered, and their relationship to congeneric species is unclear. This work will address the direct call for further genetic characterization on the former species issued by the Atlantic Whitefish Conservation and Recovery Team (2001). in fact, there is a general need for such work because at least 10 Coregonus species have been listed by COSEWIC.
Personnel: This research program will be led by Louis Bernatchez (Laval) who holds CRC in the conservation genetics of aquatic resources and brings a distinguished history of involvement in studies of Canada’s freshwater fish fauna. Dan Heath (Windsor), who also holds a CRC, will aid in the development of our species-level diagnostic tools, while Arran McPherson (Bedford Institute) will be involved in our work on endangered species. The Canadian Barcode of Life Network expects to support this research program with one PDF and two graduate students.Top
Marine ResourcesWith access to the longest marine coastline of any nation, it should be no surprise that many Canadians continue to derive their livelihood from the harvest of marine resources. Both capture fisheries and aquaculture activities are important sources of income and these primarily involve fishes, crustaceans and molluscs.
There are many situations in which our ability to monitor recruitment and even species presence is currently compromised by difficulties in the diagnosis of larval forms. In other cases, species boundaries remain unclear despite intensive taxonomic efforts. As well, important problems arise in quota management linked to difficulties in the discrimination of closely allied species. Finally, there is a need for forensic capabilities to enable the identification of the species of origin for processed fillets or other products. It is for these reasons that organizations in other nations, such as the Marine Sciences Division of CSIRO (Australia), have devoted substantial effort to the development of genetic diagnostics. Past work of this sort has largely involved allozyme markers or, less often, DNA-based discrimination systems for small groups of closely allied species. Our work will take these endeavors to a new level by developing a DNA-based identification system for all of Canada’s important marine resources based on analysis of the COI-5’ gene. The marker system that we develop will have varied and immediate applications in fisheries management.
Our studies will involve a comprehensive survey of marine fishes, as well as all economically important invertebrates in the Pacific and Atlantic regions of Canada. In the following two sections, we describe the work that will be undertaken.
Although the 538 fish species that occur in the Canadian Atlantic have been intensively studied (Scott & Scott 1988), cases of taxonomic uncertainty remain. For example, two species of sand lance (Ammodytes americanus and A. dubius) are thought to occur, but their separation is uncertain despite their importance as dietary items for many commercial species and their capture for fishmeal. Similarly, two species of Alosa are difficult to discern, and the redfishes (Sebastes) form a species complex whose members are difficult to separate. Taxonomic uncertainties such as these are magnified at the larval stage, where identification to a species level can only be executed by experts with years of training. Moreover, even the most skilled of these individuals encounter problems. For example, the earliest life stages of the Atlantic cod cannot be distinguished from those of several other gadoid fishes.
Our work will lead to development of a COI-5’ identification system for all of the fishes of the Atlantic region, as well as all of the economically important bivalves and crustaceans. Most of our analytical work will be directed towards specimens captured through DFO survey trawls or by commercial fishery activities. Whenever possible, we intend to analyze 5 individuals of each species. In cases where this analysis reveals intra-specific sequence divergences that exceed 1.5%, we will analyze an additional 20 individuals to establish if mitochondrial DNA diversity is partitioned into a small number of divergent groups, a result that would suggest the occurrence of sibling species. If such cases are encountered, they will receive much more intensive genetic and morphological analysis. As a result of our work, we will develop a DNA- based identification system that will be able to discriminate all life stages for all of the fishes in the Atlantic Region.
Aside from its diverse fish fauna, the Atlantic region supports an active fishery for 10 crustacean species including lobsters, crabs and shrimps. Although based on a smaller number of species than fish, invertebrate fisheries are important with lobster ($1 billion) and crab ($678 million) exceeding salmonid exports ($578 million). However, with overharvesting already seen in snow crab and northern shrimp, the industry is being encouraged to utilize new species. A recent document (Lemieux et al. 2002) has identified 26 crustacean species of potential commercial interest in Atlantic Canada. These include species such as the jonah crab, rock crab, deep-sea red crab, toad crab, northern stone crab and porcupine crab. Since these crabs are likely to increase in commercial importance, it is important to learn more about their biology. A COI-5’ identification system will enable the identification of juvenile stages of these various species, aiding predictions of commercial stock size and production from larval abundances. In addition to our work on crustaceans, we will develop a DNA barcoding system for Atlantic bivalves. This work is motivated by the growing interest in a fishery for the ocean quahog, Arctica islandica, one of approximately 26 bivalve species on the Scotian shelf. However, because individuals of this species are very long lived (circa 150 years), concerns have been expressed over the establishment of a fishery. A sound scientific judgment on this issue is difficult because there is little known about the recruitment dynamics of this species. Our plans to develop a species- specific marker system will enable the first comprehensive survey of the plankton community for larval Arctica, aiding an assessment of the feasibility of establishing a fishery on this species, an issue of high interest to the commercial sector.
Approximately 415 species of fishes have been reported from Canada’s Pacific waters. Nearly 50 of these species are harvested commercially, while the remainder is an important element of marine food webs. Approximately 90 species of shrimp and 100 species of crabs are also known from the same region. Our work will involve the development of a DNA-based identification system for all these species based on sequence diversity in COI-5’. We emphasize that all of these species have either direct or indirect economic importance with the latter impacts deriving from passage of the Species at Risk legislation. The sample sizes and sample acquisition protocols used in these studies will mirror those used in our study of Atlantic fishes and crustaceans.
Personnel: Work on Atlantic fishes and bivalves will be led by Paul Bentzen (Dalhousie) who holds a DFO Chair in Fisheries Resource Conservation Genetics and by Ellen Kenchington (Bedford Institute) who is Director of the Centre for Marine Biodiversity, while France Dufresne (Rimouski) will work on Atlantic crustaceans. The collections and identification of Pacific fishes and crustaceans will involve a collaboration between Jim Boutillier (DFO) who leads biological survey work on both these groups in the Pacific region and Alex Peden, curator emeritus of fishes at the Royal BC Museum. Paul Hebert (Guelph), who will lead the DNA work on our Pacific collections, brings a long involvement in genetic work on these taxa. The Canadian Barcode of Life Network will deploy one PDF position and one graduate student to advance the research in each region.Top
Parasites represent an intimidating .group for conventional taxonomic approaches. Firstly, they are immensely diverse; more than 50% of all animal species are parasitic in one sense or another. Secondly, many parasites show marked structural simplification that acts as a barrier to their identification. Thirdly, many parasites undergo dramatic reconfigurations in morphology and utilize diverse hosts during their life cycle. The process of connecting these varied stages to gain a sense of whole life cycles is often immensely difficult when pursued with conventional approaches. By contrast, DNA-based identification systems offer an immediate diagnostic tool to connect differing life stages of a species. As well, the prospects for DNA- based discriminations of closely allied species appear high based on past studies.
The development of DNA-based identification systems will make it possible to rapidly gain detailed insights concerning local parasite assemblages. This capability is important because many biodiversity management issues are dependent on knowing what parasites are present in the various hosts within an ecosystem. Knowing the parasite assemblages in natural settings is also key to predicting emerging diseases in both humans and livestock. In addition, parasites are excellent indicators of trophic relationships within ecosystems because each species has a particular mode of transmission (Brooks & Hoberg 2000). In some instances, immatures are simply passed from one host to another, while, in others, larval parasites living in the soil or water actively penetrate suitable hosts. In yet other situations, immature stages parasitize one host, which is subsequently eaten by a second host where the parasite matures.
Our ability to manage parasites within ecosystems depends on knowing the life cycles of each parasite. Until now, painstaking transmission experiments in controlled laboratory settings have offered the sole analytical approach to life cycle documentation. Few people can do this work and the costs are high. As a result, life cycles have been documented for just a tiny fraction of parasite species. For example, life cycles are known for less than 10% of the 5000 described species of trematodes (Brooks 2000, 2003).
The application of microgenomics technology will enhance three critical aspects of our understanding of parasite assemblages. Firstly, it will greatly accelerate the rate at which we are able to document all of the parasitic species in an area. Secondly, it will clarify the true range of hosts used by each parasite species; the current taxonomic literature is replete with species of parasites named as new simply because they occurred in a novel host. Finally, a microgenomic approach will dramatically accelerate the rate at which we can elucidate life cycles.
The high diversity of parasite systems makes it impossible to aim initially for comprehensive taxonomic coverage. We have, instead, opted for a demonstration of the contributions that DNA-based analyses can make to our understanding of parasite systems. We will carry out this work in two ways. Our first study will clarify the extent of genetic divergence between closely allied lineages of parasites on different hosts. Our second study will focus on a detailed analysis of parasite diversity in one model system — fishes in the St. Lawrence River. Through these studies, we expect to demonstrate the powerful insights that can be obtained through microgenomic analysis, creating momentum for similar studies on a broad range of parasitic taxa.
Host Specificity and Molecular Divergences in Parasites (circa 2000 analyses)
This project will examine the levels of genetic divergence between closely allied parasite species isolated from different hosts to provide a newly detailed understanding of the degree of host specialization. Where possible, we will connect these studies with our other animal projects. For example, we will work on mites of the genus Varroa and microsporidians in the genus Nosema, both parasites that cause substantial losses to Canada’s beekeeping industry. The Varroa mites that now occur on Apis mellifera are thought to have arisen just 150 years ago from mites transferred from the Asian honeybee, A. cerana. Since this time, new strains of this mite have evolved varying virulence that justifies an effort to develop a diagnostic tool to distinguish them. Similarly, strains of Nosema vary in their virulence so it would be valuable to be able to discriminate them. Aside from studies on these organisms, we plan a broader investigation of the levels of genetic divergence that characterize other closely allied parasite assemblages. We expect that COI-5’ will provide a very sensitive technique for monitoring diversity both within and between species because it appears that rates of molecular evolution are ordinarily accelerated in parasitic taxa. We will gain many of the specimens required for analysis from members of Canada’s parasitological community and from international collaborators (e.g. University of Edinburgh).
The implications of this work from a practical perspective are substantial. It will provide a newly detailed perspective on the level of parasite interchange between closely allied species, information that will be valuable in managing both present and future zoonoses.
Digenetic trematodes are the dominant parasites of freshwater fishes in Canada. Many species cause pathology, mortality or inferior product quality and, as such, are economically important for commercial and recreational fisheries and aquaculture. A substantial proportion of digenetic parasites of freshwater fish are larval forms (metacercariae) whose taxonomy is poorly understood because they are morphologically indistinct. In fact, many larval forms likely represent multiple species (Gibson 1996). To elucidate the taxonomy of digenetic trematodes infecting fishes, we will focus on the analysis of trematode assemblages in five fish species that are common in the St. Lawrence River. Although the parasite fauna of fishes in this region has been little studied, it is known that diversity is high (Marcogliese, unpublished), with a preponderance of larval digenetic trematodes.
Given that their taxonomy is poorly resolved and that larval stages are often difficult, if not impossible, to identify morphologically, our development of a DNA-based taxonomic system will represent an important advance for biologists, resource managers and conservationists. Using other molecular markers (ITS1, ITS2) in combination with morphotaxonomy of adult forms, we earlier confirmed that one of the most common parasites in the St. Lawrence, the eyefluke Diplostomum spp., an important pathogen in aquaculture internationally that causes blindness in fish, is composed of at least two species that are morphologically inseparable as larvae (Galazzo et al. 2002). Using the results of prior studies such as this, we will ground-truth the ability of COI analyses to diagnose species boundaries in Diplostomum and other genera of digenetic trematodes.
Once COI markers are developed and validated, they will be used to examine parasite diversity at local and regional scales. Using five fish species in the St. Lawrence River, we will examine both the localization of infection sites for larval digenetic trematodes on single host species and the specificity of parasites to particular fish species. The first line of work will involve the analysis of specimens from different organs and tissues from each fish species. The second line of investigation will involve comparisons among individuals of the five fish species at a locality. Finally, we will compare parasite assemblages from a particular host species from different sites to determine geographic variation in parasite abundances.
For host species, we will use spot-tail shiners (Notropis hudsonius), golden shiners (Notemigonus crysoleucas), yellow perch (Perca flavescens) and two stickleback species. Four of these species are important forage fish, while the perch is commercially important. We currently have quantitative data on the parasite fauna of these species in the St. Lawrence River. All are hosts for varied species of Diplostomum, as well as for numerous other larval digenetic trematodes. We will examine fishes from three; major fluvial lakes (St. Francis, St. Louis, St. Pierre) within the St. Lawrence River system plus the Saguenay River. In addition, spottail shiners will be collected from sites throughout the Great Lakes to enable comparisons at a larger geographic scale.
Most digenetic trematodes infect piscivorous birds to complete their life cycles. To verify the species identity of larval forms, we will experimentally infect ring-billed gulls (Larus delawarensis) with metacercariae to obtain the adult parasites needed for morphological comparisons. For those species that do not mature in gulls, adult forms will be obtained from piscivorous birds provided by other working groups in the Canadian Barcode of Life Network, such as the Canadian Wildlife Service, and from avian rehabilitation centres. We also expect that our work will enable the first identifications of the earliest developmental stages of trematodes that are found in mollusks. As these trematodes occur throughout Canada and the northern hemisphere, our results will be relevant at a large geographical scale. As resources permit, we will expand our studies to additional geographic areas and fish species. Samples for this work will be obtained from parasitological colleagues across Canada. As well, we will make use of the vast collections of larval trematodes held in museum collections.
Personnel: Our research program on the parasites of freshwater fishes will be led by Dan Brooks (Toronto), David Marcogliese (Environment Canada) and by Dan McLaughlin (Concordia). Dan Brooks is Canada’s leading parasitologist, an internationally recognized leader in comparative evolutionary studies. David Marcogliese and Dan McLaughlin have been long-term collaborators who have led very important studies on the parasites of fishes. Our work on genetic diversity among parasites on closely allied hosts will be led by Tom Little (University of Edinburgh) and Teresa Crease (Guelph), but it will involve a broader collaboration with members of Canada’s parasitological community. Tom’s research has focused on the identification and consequences of evolutionary arms races between parasites and their hosts, while Teresa brings the analytical capabilities needed to gather the sequence data. We expect that the Canadian Barcode of Life Network will provide funding for 1 PDF to aid the advancement of these projects with a two-year deployment to the host specialization project and a three-year deployment to the fish project. In addition one graduate student position will be funded to aid work on fish parasites.Top
PollinatorsPollination is a crucial ecosystem service that links animal and plant productivity. The value of pollination to Canadian agriculture, through honeybees alone, has been estimated at $782 million. This rises to well over $1 billion when the impacts of other bee pollinators are added. The overall value of pollinators as an ecosystem service to natural and semi-natural ecosystems is priceless. All- terrestrial animal life depends on plant production, that in turn depends on fruits and seeds, most of which (diversity-wise) is insect pollinated. Despite the value of bees to agriculture, forestry and the sustainability of natural communities, Canada lacks basic understanding of its pollinator fauna. There is, for example, no compendium of Canadian bees, such as that in many other countries (e.g. Brazil, Germany, USA). Although it is thought that our fauna includes almost 1000 species, it is certainly the case that intensive study will reveal many currently unrecognized species.
It is now widely appreciated that pollination is a threatened ecosystem service in various parts of the world and that this problem is expanding (Thomson 2001). Canada is no exception. Honeybees have suffered setbacks from several introduced diseases (acarosis, varroatosis) and they are now further threatened by antibiotic-resistant foul brood, rapidly evolving pesticide- resistant parasites and new pests. Despite diligent efforts by Canadian apiculturists, honeybee pollinator shortages and increasing fees for hive deployment are projected. This fact has generated several practical responses. Researchers have been able to mine the extensive biodiversity of wild bees to develop alternative, special purpose pollinators (Thomson & Goodell 2001, Williams & Thomson 2003). Those now deployed in Canada include alfalfa leaf-cutting bees for forage crops, bumblebees for greenhouse vegetables and blue orchard bees for apples. Hoary squash bees are encouraged for cucurbit pollination and some growers maintain habitat for pollinator assemblages (as in blueberries). Concern for pollinators has helped provoke shifts away from synthetic chemicals towards integrated pest management in both forestry and agricultural settings. Aside from these practical responses in Canada, the threats to pollination systems have led to international policy initiatives. The International Convention on Biological Diversity formally recognized declining pollinators as a global problem. The World Conservation Union subsequently established a Declining Pollinator Task Force that is gaining momentum. However, one of the most important constraints in efforts that seek to protect pollination systems or to develop new pollinators for agriculture lies in the difficulties of discriminating the species that contribute to this activity (Kevan & Imperatriz-Fonesca 2002). Nearly 50% of the bee species in temperate regions are specialists that only collect pollen from a few closely allied plants (Pesenko 1995), and many of these bees are only distinguishable by a few taxonomists. Our research will provide a novel solution to this taxonomic impediment by delivering an accessible identification system for the bee fauna of Canada.
Research Plans (circa 6000 analyses)
Our work will develop a DNA-based identification system for Canada’s bee fauna. The need for this work is great. Allozyme studies have shown that there are numerous cryptic species of bees, even among well-studied groups (Packer & Taylor, 1997). There are two very speciose and problematic taxa of bees that require particular attention - the genus Andrena and the Lasioglossum subgenus Dialictus, each of which is likely to include more than 100 species in Canada. Dialictus, in particular, provides a perfect test case for the utility of DNA barcoding for species diagnosis in ‘nightmare’ taxa. These small bees are currently unidentifiable from most of North America and are ignored in almost all large-scale bee biodiversity surveys, despite representing a very large proportion of all catches both in terms of number of individuals and of species. We are confident that a COI-5’ based analyses of this group will largely resolve this problem. We will also direct special effort toward the genetic characterization of Apis mellifera, because of the ongoing spread of Africanized ‘killer’ bees. These ‘killer’ bees are now well established in North America as far as the southwestern USA. Migratory beekeeping in the USA may bring these bees close to Canada and this could be followed by their unregulated passage. Their identification would be difficult because they can only be recognized by a few experts.
Our work will focus on the most important superfamily of hymenopteran pollinators (Apoidea). This group merits attention because of the strength of taxonomic expertise available, the high level of international interest in this group and the importance of these organisms as pollinators. We will analyze five individuals per species, one from close to its type locality, and the others from as broad a geographic, altitudinal and habitat range as possible. When “conspecific” individuals show more than 1.5% sequence divergence, at least 10 additional individuals will be examined. Analyses will focus largely on pinned specimens held in the Canadian National Collection, and in other private and public collections. We will, as well, access specimens from US museums when this is necessary to obtain individuals from close to the type localities. In all cases, COI-5’ sequences will be generated from PCR-amplified DNA extracted from a single leg. We are confident that it will be possible to recover the target sequence from museum specimens because we have completed a pilot study that recovered COI 5’ from each of 50 trial individuals. This pilot study also reinforced the finding (Danforth et al.
1998) that sequence divergences among allied species were large and that intraspecific divergences were small, indicating that the prospects for a comprehensive diagnostic system are excellent.
Personnel: The pollinator component of the Canadian Barcode of Life Network will be led by Laurence Packer (York), Peter Kevan (Guelph) and James Thomson (Toronto). Laurence has more expertise in bee systematics than anyone else in Canada, while Peter and James have a broad understanding of pollination biology in settings ranging from the high arctic to greenhouses in southern Canada. Our work will also be aided by the involvement of Canada’s leading bee taxonomists (Cory Sheffield - Agriculture Canada, Terence Laverty - Western Ontario, Robin Owen - Calgary). We expect that the Canadian Barcode of Life Network will provide support for 1 graduate student to advance research on the genus Andrena. As well, the Canadian Barcode of Life Network will fund 1 PDF to lead the work on Dialictus and to oversee the assembly of data on other members of the bee fauna.Top
Terrestrial VertebratesNo group of organisms plays a more important role in public perceptions of biodiversity than terrestrial vertebrates. This reflects their importance in recreational pursuits (bird-watching, hunting) and their contributions to the ambience of everyday life. Because these organisms occupy environments that have been intensively modified by human activities, they also include a high proportion of the species that are currently listed as endangered by COSEWIC. Finally, because of their close taxonomic affinity with humans, they occasionally serve as reservoirs for shared diseases (hantavirus, West Nile virus). For all these reasons, terrestrial vertebrates have played a key role in the development of public policy. Their importance justifies the development of a comprehensive COI-5’ diagnostic system for Canada’s terrestrial vertebrates. Although it is certainly the case that most of these species can be diagnosed through morphological inspection, there are many situations in which the availability of diagnostic genetic markers will provide insights that would otherwise be difficult or impossible to gain.
There is already enough information available to make it clear that our efforts to develop a DNA-based identification system for Canada’s vertebrates will succeed. We have carried out the development work needed to ensure success in recovering the COI-5’ fragment by developing a new primer set that amplified all of the 150 taxonomically diverse vertebrates included in a trial study. This pilot work also revealed the regular occurrence of deep sequence divergences among closely allied taxa of birds and mammals, indicating that species diagnosis will ordinarily be straightforward.
Our research will involve the development of a COI-5’-based identification system for all of Canada’s terrestrial vertebrates. In the following sections we discuss the level of effort required to complete this task and the importance of this work. As a general comment, we note that our studies on vertebrates will involve the analysis of larger sample sizes than for most other animal groups. This elevation in sampling effort reflects the fact that many policy concerns in relation to these organisms derive from the endangered status of populations in certain segments of the species range. As such, some understanding of the extent of regional population divergence is critical. We emphasize, as well, that our analyses will not contribute to any further population endangerment; we will analyze specimens held in museum collections or will collect new samples in a non-destructive fashion.
Amphibians/Reptiles (circa 2000 analyses)Forty-six species of amphibians and 45 species of reptiles occur in Canada. COSEWIC has listed half of these species as being at some risk of decline leading to extirpation. Although this partly reflects the fact that many species only penetrate the southern regions of Canada, it mainly reflects the impacts of wetland and forest loss and other habitat degradation.
Numerous Canadian amphibians and reptiles show discontinuous distributions as their ranges are interrupted by geographic barriers. There are also indications that seemingly continuous ranges are in fact composites of distinct entities that dispersed into Canada via different routes after deglaciation (Austin et al. 2002). Together, these results indicate that the populations composing many species represent genetically divergent units with long histories of isolation. This hypothesis needs to be tested to properly focus the efforts of conservation programs. Our studies will provide this information by examining individuals from each geographical region occupied by those species whose ranges feature geographic disjunctions. For other species, we will analyze a minimum of 5 individuals per biotic province. These studies will provide a good sense of the extent and patterns of genetic divergence among populations, information that will be useful in guiding the development of recovery programs or ensuring adequate protection for groups that are not currently threatened. Top
Birds (circa 3000 analyses)Birds are perhaps the best-known group of animals from a taxonomic perspective, with more than 95% of species already named (although ongoing research continues to turn up cryptic species). Phylogenetic relationships and species limits have been explored in many groups using a variety of tools, including pattern, morphology, and a variety of molecular approaches. Because of the rich variety of these supplementary studies, birds are a particularly suitable group with which to test the efficacy of a single gene marker (i.e. COI-5’) to identify species.
The availability of a DNA-based diagnostic system for birds will be a valuable tool for a wide range of research and forensic projects of relevance to human health and bird conservation. One example is the use of DNA to identify the remains in bird strikes on aircraft that cause millions of dollars of damage every year, sometimes with loss of human life. Knowledge of the size and shape of the bird (which is known once it is identified) aids aircraft design (e.g. ballistic resistance). Knowledge of species identity can also help to determine appropriate avoidance activities (changes in flight paths, habitat modifications near the airport, scare tactics). Traditionally, bird remains have been identified by sending material to a museum expert, but often there is simply tissue and blood stuck to a dent. Identifications in the latter case are not possible without molecular techniques, while identifications based on feathers require great expertise and an extensive museum reference collection. By contrast, DNA can be easily sequenced at many different labs, and then identified using web-accessible reference material.
From a conservation/enforcement perspective, a DNA-based identification system will be valuable to confirm the identity of portions of individuals confiscated by enforcement officers, to determine if they are, for example, endangered species, or species for which trade is prohibited. If the animal is intact, this may not be necessary, but if only portions of it are available (e.g. meat from a hunt) genetic testing could quickly and reliably identify its species of origin.
We intend to collect COI-5’ sequences for five individuals of each of the 426 species of birds that breed in Canada. Whenever possible, these samples will examine individuals from different provinces. Because of the high mobility of most bird species, the extent of their regional population differentiation is typically modest so we will employ smaller sample sizes than for other groups of vertebrates. We have already carried out pilot studies on several bird genera that have established that COI-5’ regularly enables diagnosis of even closely allied species. We will obtain the bulk of our samples from tissue banks maintained by organizations such as the Royal Ontario Museum. When additional samples are required, they will be obtained through bird-banding stations (using non-destructive methods, such as the collection of a feather) and through other field activities and collections coordinated by the Canadian Wildlife Service. Top
Mammals (circa 3000 analyses)The Canadian mammal fauna has been intensively studied. This work has revealed 193 species of which 28% are listed as being under threat by COSEWIC. Most of the species in our fauna can be readily distinguished using morphological approaches, but some species groups of bats and shrews are only separable by experts. Moreover, there is a frequent need for diagnostic methods that enable a determination of the species of origin for carcass segments, linked to the enforcement of hunting regulations. As well, because of their limited mobility, many mammal species include several phylogeographic units that can only be distinguished through genetic analysis. The recognition of these subgroups is important for conservation initiatives.
Our work will involve the collection of COI-5’ sequences from 15 individuals of each species. Whenever possible, these samples will include 5 individuals from each of the three provinces/territories that lie at the extreme eastern, western and northern range limits of the species. We will obtain the bulk of the samples needed for our work from tissue banks maintained by organizations such as Parks Canada and The Royal Ontario Museum.
Personnel: Our work on amphibians and reptiles will be led by David Green (McGill) who not only brings a great depth of experience in genetic work on these organisms, but a long involvement with COSEWIC. Charles Francis (Environment Canada), who serves as Chief of Migratory Bird Population Research at the Canadian Wildlife Service, will lead our work on birds. Finally, Brock Fenton (Western Ontario), an internationally recognized expert in mammalian biodiversity, will co-ordinate our work on mammals, but he will be aided in the advancement of this work by Mark Engstrom (Royal Ontario Museum). The Canadian Barcode of Life Network will fund three graduate students to advance work on these groups. As well, a PDF will be supported for the last 2.5 years of the Canadian Barcode of Life Network’S activity to oversee the integration of data and to ensure that our taxon coverage is comprehensive. We expect that the Advanced DNA Technology Group will be responsible for the analysis of the bird and mammal samples, but the amphibians and reptiles will be analyzed at McGill.