Our research program currently focuses considerable attention on comparative studies of ultrastructural systems in three diverse and ecologically important groups of eukaryotic single-celled predators, namely euglenids, dinoflagellates and cercozoan flagellates. The first two groups have independently acquired sophisticated feeding apparatuses, complex photoreception apparatuses and photosynthetic organelles. The independent but corresponding patterns of morphological change in these groups represent an ideal comparative system for investigating the constraints and reoccurring innovations in cell evolution.

    Research on the diversity of these eukaryotic cells is, in essence, exploratory and progresses in four principal phases: (1) collection, isolation and cultivation of the organisms from nature (e.g. fieldwork at the Bamfield Marine Sciences Centre - click on image below), (2) identification, specimen preparation and description of the organisms using light and electron microscopy, (3) molecular characterization of the organisms and (4) computational analyses associated with digital image processing and molecular phylogenetics. Outcomes of this research will accelerate the discovery and characterization of new and poorly known species of single-cellular eukaryotes from diverse environments (marine sediment interfaces and hydrothermal vent systems) and identify novel ways in which unicellular predators have solved basic biological problems (cell locomotion, nutrition, and photoreception).

    Transmission and scanning electron microscopy have provided a rich source of novel information about euglenid morphology that has implications for developmental biology, paleontology, and the late secondary origin of chloroplasts. For instance, euglenids possess distinct patterns of strips that are a consequence of strips that terminate before reaching the anterior and posterior poles of the cell. In most phototrophic taxa, the maximum number of strips around the cell periphery (P) is reduced exponentially as discrete whorls of terminating strips. The number of posterior whorls (Wp) is constant within a taxon and different states for this character form a transformational series from Wp = 0 to Wp = 4. Because many euglenids lack whorls of strip reduction and only phototrophic euglenids possess them, more informed inferences can be made about the biology of microfossils. For example, a 450 million-year old group of acritarchs known has Moyeria, possess whorls of strip-like structures, so it is now possible to more confidently infer that these microfossils were not only euglenids but phototrophic.

    In order to test primary homology statements (inferences made independently from a cladogram) and to infer ancestral-derived polarities for specific character state transformations, we are involved in building euglenid phylogenies based on molecular sequence data. Because singe gene phylogenies, on their own, are unreliable reflections of the organismal phylogeny, we are generating new data sets of protein sequences from genes like hsp 90, tubulins, and actin. We are also helping build the euglenid data set of nuclear small subunit ribosomal DNA (SSU rDNA) sequences, particularly for phagotrophic euglenids isolated from nature. Previous studies have demonstrated a high degree of concordance between morphological and SSU rDNA data sets and, thus, the utility of morphological characters for inferring the phylogenetic relationships of euglenids. Tree topologies derived from both total evidence and analyses of partitioned data sets permit the construction of synthetic trees, which form frameworks for tracing character evolution. This approach has shed considerable light onto the evolutionary transitions between bacteriotrophy, eukaryotrophy, osmotrophy, and phototrophy, and helped identify the best candidates for apomorphy-based taxon definitions within an updated classification system for euglenids.

    At first glance, the phylogenetic gaps between these groups appear enormous, and consequently, the ancestral states and intermediate stages that led to these extremely varied lineages have been more a matter of speculation than substantiation. We are interested in a variety of phagotrophic (more specifically, myzocytotic) alveolates that do not fit neatly within the three major groups discussed above, such as Acrocoelus, Colpodella, Colponema, Cryptophagus (a.k.a. Rastrimonas), ebridians, ellobiopsids, Oxyrrhis, Parvilucifera, and Perkinsus. The combination of features in these taxa suggest that some of them may be living microfossils and very important for inferring specific states in ancestral alveolates and for understanding early steps in the evolution of apicomplexans, dinoflagellates, and ciliates. For instance, evidence suggests that perkinsids (Cryptophagus, Parvilucifera and Perkinsus), parasites of mollusks and unicellular eukaryotes, are the earliest diverging sister lineages to dinoflagellates. The zoospores of perkinsids have two dissimilar flagella and possess structures traditionally attributed only to apicomplexans such as a microtubular conoid-like apparatus, rhoptries, and micronemes. Moreover, colpodellids are biflagellated predators with a suite of features that are strikingly similar to perkinsids, but colpodellids diverge as the earliest sister group to apicomplexans in small subunit rRNA phylogenies. These data suggest that perkinsids and colpodellids provide extraordinary insights into the characteristics of the last ancestor of dinoflagellates and apicomplexans and the subsequent evolution in both groups.

    Our best hope for understanding the evolution of alveolate parasites is to apply EM and molecular phylogenetics to enigmatic organisms with putative alveolate affinities. This is necessarily a "look-and-discover" based approach to biodiversity as, for instance, it requires the collection and dissection of a wide variety of marine invertebrate hosts without really knowing beforehand what sorts of unicellular organisms might be found. Other enigmatic eukaryotes that may be important pieces of the phylogenetic puzzle will be specifically searched for. Every attempt is made to prepare cells for EM and DNA extraction following the collection of any of these organisms.

    The main goal of this research has been to re-evaluate marine gregarine diversity with electron microscopy and molecular sequence phylogenies. We have focused on the macroevolution of characters associated with the cortex of the trophozoite stage in the gregarine lifecycle. For instance, we are attempting to identifying the key innovations associated with the parasitic lifestyles of various gregarine lineages. Are different cortex morphologies of gregarines associated with different body cavities in their hosts (e.g., coelom, gut lumen, and seminiferous vesicles)? A phylogenetic hypothesis for the internal topology of gregarines is required before the structural diversity within the group can be understood in an evolutionary context.

    Small subunit (SSU) rRNA genes have been amplified, cloned, and sequenced from several marine gregarines, but the phylogenetic signal is largely overshadowed by long-branch artifacts. Accordingly, sequences from other markers such as a variety of protein coding genes (e.g., Hsp 90 and actin) have been pursued with some success. Eventually, a synthetic tree topology will be generated from the morphological and molecular sequence analyses that will facilitate the mapping of morphological character states, an approach that will provide the basis for documenting novel examples of character evolution within the group.

    Gregarine diversity has been relatively unexplored with EM and molecular phylogenetics because gregarines have never been cultivated; single cells must be isolated from the intestines and coelomic spaces of their natural hosts for both DNA extraction and microscopy. This requires the collection and dissection of diverse groups of marine invertebrates such as polychaetes, sipunculans, echiurans, urochordates (salps), enteroneustans, holothuroideans, phoronids, and nemerteans. Marine hosts have been collected from the Bamfield Marine Station on Vancouver Island (BC, Canada), Friday Harbor Laboratories on San Juan Island (WA, USA), and Stanley Park in Vancouver (BC, Canada). Because only a few gregarine cells are usually available from these hosts, PCR amplification is often weak and non-specific so many clones must be screened with restriction digests prior to sequencing. Moreover, the preparation and examination of single cells for scanning and transmission electron microscopy is very challenging.

Adl, S., Leander, B.S., Simpson, A.G.B., and 17 others. 2007. Diversity, nomenclature, and taxonomy of protists. Syst. Biol. 56:684-689.