DNA Barcoding of Protists
Preamble
Animals, fungi, and plants are recent descendents from just two of some 36 evolutionarily diverse lineages of unicellular and simple multicellular organisms collectively termed “protists”. Protists are ubiquitous in the biosphere, occurring in every drop of water and pinch of soil, and living within animals, plants, and even other protists (Margulis et al.1989). They are critical components of ecosystems and have many direct and indirect economic impacts, being used for food, biomedical research, pharmaceuticals, as proven biomonitoring tools, and as model systems for scientific investigation. Protists are also causative agents in numerous human illnesses, ranging from malaria to paralytic shellfish poisoning. Photosynthetic protists, macroalgae and microalgae, outnumber heterotrophic species by tens of thousands (Corliss 2002) and form the base of aquatic food webs, contribute 50% of global carbon fixation, oxygenate aquatic environments, provide intertidal habitat, and represent an underutilized resource. Unfortunately, algal biodiversity can be negatively impacted by global warming, environmental stress from fisheries and aquaculture, and by invasive species. Conversely, macroalgae foul aquacultural netting, and phytoplankton blooms of nuisance and toxic species (of which there are 30-40 in the Canadian Maritimes alone) can have devastating effects on marine animals. As the value of aquaculture in Atlantic Canada alone is approaching $500 million, the potential for economic damage is great. Understanding genetic diversity in algal species will aid the development of techniques to identify bloom initiation and will facilitate a proactive response. Among heterotrophic protists, ciliates are the most speciose lineage and are major consumers of bacteria, having critical roles in organic carbon utilization and nutrient cycling in both aquatic and terrestrial ecosystems (Corliss 2002). They live symbiotically in the guts of many animals, especially agricultural species where they stabilize bacterial populations, but many parasitize fish with negative impacts on aquaculture, and one causes human illness (Ferguson et al. 1987; Astrofsky et al. 2002; Parama et al. 2004).
Despite considerable diversity and significance, protists, with their pervasive distribution and cryptic habits, are the least understood organisms from a biodiversity perspective. Many display unpredictable and periodic occurrence, are intractable to culturing, and require advanced microscopy for identification. Generally considered to number 110,000 extant species, recent mating and allozyme studies have revealed that many cosmopolitan “species” are actually species complexes, and the true diversity may be three times that currently catalogued (cf. Corliss 2002). Because these sorts of analyses (morphology, breeding studies, and allozymes) are time consuming and not easily automated, there is a critical need for new diagnostic tools. Our research will meet this need by testing the concordance between species currently recognized and those identified through DNA analyses.
Past genetic work on protists has had a predominantly phylogenetic focus with most analyses based on small subunit (SSU) rDNA. This work has provided invaluable insights into the affinities of the major protistan lineages, but SSU rDNA evolves too slowly to resolve recently evolved species. Canada has been a leader in research on whole mitochondrial genomes resulting in data for more than 20 protists (Bullerwell and Gray 2004). This research is powerful for phylogenetics, but too demanding for routine species diagnoses. However, these comprehensive analyses have provided information suggesting that the COI gene is a good candidate for species identification. In fact, COI sequences available for some 150 protists possess much more diversity than exists among the larger number of animal sequences (e.g., Ehara et al. 2000), and indicate that protist COI-5’ is generally free from introns and transposable elements, such that its characterization through standard protocols should be straightforward.
Research plans – years 2 and 3: Development and validation of DNA markers.
Four markers, two mitochondrial and two nuclear, have been selected for evaluation as diagnostic barcodes. The main mitochondrial marker selected is COI-5’, which has the obvious advantage of consistency with the other projects outlined here, thereby facilitating the transfer of developing technology to protists. Work will begin with the design of primers for COI-5’ from culturable target taxa, which will be expedited by the availability of sequences for each group. We also plan work on faster evolving elements of the mitochondrial genome such as the NAD complex (Brunk et al. 2003). Two nuclear markers, ITS1 and LSU(D1/D2) or SSU of the rDNA, will also be analyzed because primer systems are available for most protists, they have been used for species level diagnosis in many lineages (cf. Harper and Saunders 2001), and they are effectively found in single copies. Both nuclear markers will be assessed and the best developed for each lineage will be used to confirm the COI-5’ results, to provide data for those protists that lack mitochondria (which makes them unique among eukaryotes), and to uncover mitochondrial introgression among closely related species.
Considering the diversity and ubiquity of protists, it is crucial to constrain both taxonomic and geographic scope of our investigations. All three marine macroalgal lines will be studied: the Chlorophyta (green), Phaeophyceae (brown), and Rhodoplantae (red). From a practical perspective these are the easiest protists to sample comprehensively. Select microalgal lineages will be studied as they comprise a significant phytoplankton component in Canadian waters (notably diatoms, dinoflagellates), and a detailed study of the species-rich ciliate genus Tetrahymena will be implemented. For each, we have identified representative taxa for which barcoding markers will be developed and tested. We will analyze 100-200 isolates per genus, enabling a comparison of intra and interspecific divergences. If results indicate that COI-5’ provides clear diagnosis of species boundaries, further efforts will aim to speed analysis. Because establishing cultures is labour intensive and many cells prove intractable (potentially biasing results), we will develop protocols to enable the generation of DNAsequences from individual field-based colonies and cells to facilitate future research (Edvardsen et al. 2003).
Macroalgae: For the initial screening we have selected the green algae Enteromorpha and Ulva (Hayden et al. 2003), the brown algae Fucus (Serrao et al.1999) and Laminaria (Erting et al. 2004), and red algae from the genera Asteromenia, Callophyllis (Saunders, unpublished), and Mazzaella (Ross et al. 2003), all of which are known to have multiple species, cryptic in many cases, with a range of intrageneric divergence. Preliminary surveys on Fucus, Asteromenia, and Mazzaella have established the utility of COI-5’for species diagnoses in these genera (0-2 nucleotide changes were noted within species, with 5-8 between the very closely related M. linearis and M. splendens, but generally >30 between species). Hence, the outlook is very positive for application of this standard system to brown and red algae.
Microalgae: Diatoms are the most speciose group of protists, are abundant in aquatic and moist habitats, and account for ca. 40% of global aquatic primary production. They are relatively easy to culture and over 1,000 species are available in collections. Initially, primary focus will be given to Pseudo-nitzschia, Thalassiosira, and Chaetoceros, which are known or believed to cause harm to vertebrates (including humans). Species in these genera have been subjected to intensive morphological (e.g., Kaczmarska et al. 2005) and life history (Kaczmarska et al. 2000) studies and contain well-characterized complexes. Some exhibit clonal variation in toxicity and genetic markers would greatly facilitate identification of the most toxic forms. Primers for diatom COI-5’ are already available for several genera (Ehara et al. 2000, Armbrust et al. 2004).
Dinoflagellates are an ubiquitous and diverse group of about 4,000 described living species and the closest relatives of apicomplexan parasites (including malaria, Plasmodium). They will be an interesting test case for DNA barcoding because mRNAs in some species are subjected to extensive RNA editing producing a "double fingerprint" for each mitochondrial gene. Presently, 412 strains are available in culture including representatives of several orders, but with a high concentration of species/strains from economically important genera. Alexandrium spp.are an important cause of “red tides” in Canada. Prior work has shown that different species/strains show varying toxicity to fishes, so discrimination is important. Strain and species-specific markers have been obtained through studies of rDNA diversity (Scholin and Anderson 1996). Our work will compare COI-5’ versus the resolution obtained with rDNA. Cryptophytes and euglenids are less common in the marine environment (diversity increases in freshwater) and will also be subjected to marker development.
Ciliates: Tetrahymena spp. have been subjected to intensive morphological and genetic analyses (Strueder-Kypke et al.2001), motivated by their abundance in microbial ecosystems and their role as parasites in aquaculture (Astrofsky et al. 2002). As a result, the American Type Culture Collection (ATCC) has extensive collections of species and isolates from this genus, facilitating the identification of appropriate markers for species diagnosis.
Research plans – years 3 and 4: comprehensive taxonomic surveys
i. Census of Canadian red algae. We will generate anatomical and barcode data for all recognized species of Rhodoplantae (ca. 500) in Canada. This is an achievable task: COI-5’ barcoding requires only two primers per sample, or 1,000 sequence reactions; even with repetition (10 samples per species covering morphological diversity and biogeography), this can be easily accomplished with today’s technology. The difficulty will be acquiring the necessary collections, notably of microscopic taxa. For the latter, UNB has extensive culturing facilities and we have been culturing microscopic reds (ca. 20 species to date) to facilitate analyses. For larger species, an expansive collecting strategy will be implemented. In the Maritimes we will identify sites in the Bay of Fundy, Atlantic, and the Gulf of St. Lawrence, and for British Columbia, locations will be sampled near the Bamfield Marine Station, Port Hardy, and Victoria. Sites in each region will be selected to include estuarine to exposed habitats, and sampled (intertidal and SCUBA) in the spring, summer, and fall each year. We have collections from Newfoundland & Labrador and the Arctic, and will supplement these with new collections as funding permits. Each collection will consist of a voucher (anatomy) and a silica-dried sample (genetic analyses). Through current research we have acquired 1,100 samples representing 200 known species (ca. 40%) and ca. 25 uncertain taxa (new records & species) from Canadian waters. The result will be the first comprehensive inventory of a marine algal lineage for any nation – establishing a foundation for the conservation of marine biodiversity, helping Canada to meet international obligations under a variety of conventions and treaties, facilitating research from ecologists to molecular biologists, and rendering invasive species easier to document and eradicate.
ii. Census of marine macroalgae in the Bay of Fundy . The unique geography and corresponding tidal range and importance for ecotourism, fisheries, and aquaculture provide overwhelming justification for the monitoring of biodiversity in the Bay of Fundy. The macroalgal flora in this region is not rich (ca. 160 species), which will facilitate a census of all species. At eight sites (seasonality, etc., as noted in previous objective), all of the green, red, and brown algal species will be sampled and analyzed to produce the first comprehensive macroalgal inventory for any region. This is critical to conservation in the Bay, which is experiencing a rapid decline in algal diversity owing to anthropogenic impacts (Bates et al. 2001).
iii. Census of Marine Microalgae in Canada. Using the protocols developed in the early phases of the project, we will perform an inventory of Atlantic and Pacific coastal waters based on seasonal sampling programs. Emphasis will be given to harmful, nuisance, or other economically important species, e.g., the dinoflagellates Alexandrium, Dinophysis, and Prorocentrum, and the diatoms Pseudo-nitzschia and Nitzschia. In addition, several genera from each of the diatom classes with large cells or colonies will be analyzed. Euglenids that are proven bioindicators will be targeted for broader spatial analysis. As well, the widely distributed genus Cryptomonas will be surveyed in the Maritime and Great Lakes regions. We will also develop environmental PCR and RT-PCR to examine the diversity of natural populations of dinoflagellates in various marine environments. Dr. Curtis Suttle at UBC has carried out extensive long-term sampling of diverse environments and has maintained DNA libraries from all sample sites and times. Using this resource, we can estimate the diversity of dinoflagellate COI-5’ to test the coverage provided by the cultures characterized in year 1. Overall, this work will provide evidence for the feasibility of barcoding diverse groups of protists, a foundation for further genetic assessments of microalgae in Canadian waters, a list of toxic species, and a guide to recognizing future invasive species. In the light of the growing number of invasive organisms and the precipitous rate of species extinction, this is an urgent task.
iv. Ciliates of Ontario. Following assessment of the effectiveness of the various markers for species diagnosis within Tetrahymena, we will extend analyses to include three major groups: Class Spirotrichea, conspicuous in aquatic and terrestrial ecosystems (Pierce and Turner, 1992), Class Colpodea, common in terrestrial ecosystems (Foissner, 1993), and Class Oligohymenophorea, important models for genetic and cell research and significant facultative parasites in aquaculture (Parama et al. 2004). Using additional culture collections, and the single cell and environmental strategies as outlined above for the microalgae, we will sample extensively the ciliates in Ontario from terrestrial habitats in coordination with our work on soil fungi, and from freshwater environments varying from temporary ponds to the Great Lakes. This study will set a baseline for ciliate diversity in Ontario, provide an indication of ciliate endemism in divergent habitats, and investigate levels of diversity in stable versus ephemeral habitats from both pristine and impacted sites.
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Personnel
Canada is an acknowledged leader in genetic research on protists and is thus uniquely poised to advance DNA barcoding for these organisms. Gary Saunders (UNB), an EWR Steacie Fellow and CRC in Molecular Systematics and Biodiversity, has an established reputation as a molecular systematist with a broad knowledge of algae and will coordinate the group’s activities and head the macroalgal research. Patrick Keeling (UBC), EWR Steacie Fellow and a CIHR New Investigator with a Distinguished University Scholar Chair, Irena Kaczmarska (Mt. Allison) an internationally recognized expert in diatom ecology, evolution, and classical systematics, and Margaret Beaton (Mt. Allison) with molecular systematic expertise will complete the microalgal research. Denis Lynn (Guelph) will lead out ciliate work. He has published over 100 full papers or chapters in books relating to the systematics and ecology of these organisms, including his collaborative work with Dr. E.B. Small on an illustrated guide to over 600 genera of ciliates, classified by the macrosystem of Lynn and Small (2002), which has recently been updated (Lynn 2004).
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