We study the biodiversity and evolution of unicellular eukaryotes (~protists), especially free-living protozoa. Free-living protozoa are of profound evolutionary importance; at the level of ‘major lineages’ they more diverse than all other kinds of eukaryotes put together (i.e. animals, plants, fungi, algae and obligate parasites). In fact, all these other kinds of eukaryotes descend directly or indirectly from free-living protozoan ancestors. Free-living protozoa are also of huge ecological significance, representing the major predators of bacteria and other micro-organisms in many ecosystems.

Most of our projects examine poorly-studied protozoa that are especially important for inferring the evolutionary history of eukaryotic life, or that inhabit extraordinary environments and have undergone major adaptive change relative to ‘typical’ eukaryote cells. We also examine the diversity of ecologically important protozoa from sediments, and symbiotic or parasitic associations between protozoa and other marine organisms.

Phylogeny and evolution of eukaryotic life

Scanning Electron Microscopy image of Hemimastix kukwesjijk. Photo by Yana Eglit.

Certain groups of (mostly) free-living protozoa occupy important deep-branching positions on the tree of eukaryotic life. These include the ‘typical excavates’ (see below) as well as various poorly understood flagellates such as ancyromonads, apusomonads, breviates and Mantamonas.

In collaboration with several other research groups (inc. the Roger lab at Dalhousie University, Matt Brown at Mississippi State, and Yuji Inagaki’s group at Tsukuba) we use phylogenomic approaches to infer the tree of eukaryotes including these important overlooked taxa. We have shown that breviates and apusomonads are together closely related to the group containing both animals and fungi (opisthokonts), forming the group now called Obazoa (Brown et al. 2013). Recently, we have identified a new ‘supergroup’ on the eukaryote tree, ‘CRuMs’, that is composed of various free-living protozoa (Brown et al. 2018).

We have also characterised the cytoskeletons of these organisms, primarily using serial transmission electron microscopy (e.g. Heiss et al., 2011, 2013a, 2013b). These studies show that certain distantly related living eukaryotes share several homologous cytoskeletal elements. This implies that all living eukaryotes descend from a common ancestor with a complex flagellar apparatus cytoskeleton, and remarkably, that we will are able to reconstruct much of this cytoskeleton, despite the passage of more than a billion years.

In ongoing research, we are characterising several new or ‘lost’ free-living protozoa that likely represent additional major lineages on the eukaryote tree of life. Recently we completed the first modern study of the Hemimastigophora, employing single-cell transcriptomic methods to allow us to conduct phylogenomic analyses (Lax, Eglit, Eme et al. 2018, Nature 564: 410–4). Hemimastigophora proves to represent (another) new supergroup for which we had no molecular data for at all, and it represents a crucial taxon to consider for understanding the properties of the last eukaryote common ancestor, the location of the root of the eukaryote tree, and many other questions.

Excavates biodiversity phylogeny and evolution (including anaerobes)

Several protist groups have a distinctive type of feeding groove. Some time ago, we characterised these groups (and their immediate relatives) as the ‘excavates’, and proposed that they descended from a common ancestor, forming a ‘supergroup’ called Excavata (see Simpson, 2003 for a review). This is still not resolved: Multigene phylogenetic and phylogenomic analyses identify three robust clades of excavates—Discoba, metamonads and malawimonads (e.g. Simpson et al 2006, 2008; Hampl et al., 2009), however, the monophyly of all excavates is typically not recovered in phylogenomic analyses, most often because malawimonads branch closer to animals and fungi than to metamonads (or Discoba). We have recently conducted a critical examination of the phylogenetic position of malawimonads, combining a detailed study of the flagellar apparatus cytoskeleton with phylogenomic analyses that examined conflicting results from different site- and taxon- samplings (Heiss, Kolisko et al., 2018). We confirm that both provide a strong ‘signal’ that malawimonads are, in fact, most closely related to metamonads.

Metamonads are all anaerobes that have highly modified mitochondria. The group includes the well-known parasites Giardia (a diplomonad) and Trichomonas, as well as the commensalic/symbiotic oxymonads—an oxymonad is the only eukaryote known to have completely lost mitochondria. A long-running and ongoing project of the lab has been to isolate and characterise new free-living metamonads (and other anaerobic excavates), such as Carpediemonas-like organisms and trimastigids (Kolisko et al., 2010, Park et al., 2009, 2010; Zhang et al., 2015). Carpediemonas-like organisms prove to be an extremely diverse paraphyletic group that gave rise to diplomonads (Takishita et al., 2012), while we showed that trimastigids similarly gave rise to oxymonads (Zhang et al., 2015). A current collaboration, primarily with the Roger Lab at Dalhousie University, seeks to trace the evolutionary modifications of the mitochondrion-related organelles of free-living and parasitic metamonads using taxon-rich comparative transcriptomic and genomic datasets (e.g. Leger et al. 2017).

(Extremely) Halophilic Protozoa

Microbiology textbooks present very hypersaline habitats (>25% salt) as being exclusively inhabited by prokaryotes, mostly Haloarchaea, and the alga Dunaliella. In reality, there many heterotrophic protozoa that have been reported from extremely hypersaline habitats—at least 30 morphospecies by a conservative estimate (Park et al., 2009). These include ciliates, amoebae, and several groups of flagellates, some of which have unknown affinities within eukaryotes.

We have characterised numerous cultures of (borderline) extremely halophilic protozoa using electron microscopy and/or molecular phylogenetics, including several new genera (Park et al., 2006, 2009). Some of this work was completed in collaboration with Prof. Byung Cheol Cho of Seoul National University, and/or former postdoc Jong Soo Park. Our research makes clear that obligate halophiles (i.e. organisms requiring salinities above seawater for growth) evolved many times among protozoan eukaryotes, for example, three times independently in the taxon Heterolobosea alone (Park et al., 2007, 2009, Park and Simpson, 2011, 2015; see review by Harding & Simpson, 2018). Almost all the obligate halophiles that we have cultured are distinct at the genus level from marine taxa, although we have also identified two lineages that apparently secondarily reverted to halotolerance (Harding et al., 2013; Kirby et al., 2015).

In collaboration with Andrew Roger's group at Dalhousie University we have used comparative transcriptomic analyses and genomic data to examine adaptations to halophily in representative halophilic protozoa (Harding et al. 2016, 2017). Halocafeteria (a stramenopile) showed differential expression under different salinities in numerous systems (e.g. stress responses, signal transduction systems, sterol metabolism). We also found an overrepresentation of differentially expressed eco-paralogs amongst recent gene duplicates, and two cases of relatively recent lateral gene transfer among the highly differentially expressed genes. Interestingly, Halocafeteria also turns out to possess a full pathway for synthesizing the ‘bacterial’ compatible solutes ectoine and hydroxyectoine.

Biodiversity of phagotrophic euglenids

The major taxon Euglenida contains organisms with a broad range of morphological appearances and various nutritional modes including phagotrophy, osmotrophy, phototrophy and mixotrophy. Phagotrophic euglenids represent the ancestral form that gave rise to all other types (e.g. the phototrophic forms descend from a secondary endosymbiosis involving a phagotrophic euglenid and a green alga), and they are also important micropredators in sediments. Few phagotrophic euglenids have been cultured, however, and the whole assemblage is thus understudied. Current taxonomy is based on a variety of morphological features, but as molecular data are collected, the misleading nature of most of these morphological characters has become obvious.

We have characterised phylogenetically important phagotrophic euglenids with collaborator Won Je Lee, of Kyungnam University, South Korea (Lee and Simpson, 2014a,b). We have also established workflows to isolate single phagotrophic euglenid cells from environmental samples, identify them with high-quality light microscopy and sequence their SSU rRNA genes (Lax & Simpson, 2013; Lax et al. submitted). This approach promises to rapidly capture much more of the taxonomic diversity of phagotrophs than by culturing alone. We have addressed several taxonomic, phylogenetic and evolutionary issues, including identifying which phagotrophic euglenids are most closely related to the osmotrophs, and demonstrating the non-monophyly of several genera, as well as numerous proposed higher taxa. This major expansion of the database of identified euglenids with molecular profiles will also improve our ability to identify euglenids in environmental sequencing surveys, where they are suspected to be badly under-detected.

We are now extending this work to obtain multigene profiles for photodocumented cells using single-cell transcriptomics. This approach will enable the estimation of multigene phylogenies to get a clearer picture of the phylogenetic relationships among euglenids, and their deep-level evolution.

Examples of phagotrophic euglenids. From left to right: Anisonema acinus, Heteronema sp., Notosolenus ostium (without flagella), Ploeotia cf. vitrea (without flagella). Photos by Gordon Lax.

Other projects

Recurrent mass mortalities of the green sea urchin Strongylocentrotus droebachiensis drive transitions between productive kelp beds and ‘barrens’ in shallow water on the Atlantic coast of Nova Scotia (see research by the Scheibling Lab at Dalhousie University). The disease is caused by a facultative pathogen, the amoeba Paramoeba invadens. The source of outbreaks is mysterious, especially as the minimum thermal tolerance of cultured P. invadens is above the local winter minimum water temperature, which argues against local overwintering. In a collaboration with the lab of Bob Scheibling, we confirmed the distinctiveness of P. invadens using multiple gene markers (in Feehan et al., 2013), examined in detail its lower thermal threshold for growth (Buchwald et al. 2015), and have devised and tested PCR and qPCR methods for detecting P. invadens in tissue and environmental samples (Buchwald et al. 2018).

Codium fragile is an invasive alga now present along the Atlantic coast of the USA and Canada. Incubations of Codium utricles frequently produce dense ‘infections’ by kinetoplastids, a type of heterotrophic flagellate. We have established that these ‘infections’ occur in Codium from Nova Scotian waters (from several locations), and that they are caused by a single new species, Allobodo chlorophagus (Goodwin et al. 2018). Allobodo also turns out to represent a new family-level taxon within metakinetoplastids.

We have also recently conducted a project on marine vampyrellids, specifically their biodiversity and feeding on microalgae (More et al. 2018; project run by former postdoc Sebastian Hess). In ongoing work we are examining a collection of unusual alveolate flagellates that consume other eukaryotic cells.