Our research primarily concerns 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, free-living protozoa are about as diverse as all other kinds of eukaryotes put together (i.e. animals, plants, fungi, algae and obligate parasites). In fact, all other kinds of eukaryotes descend directly or indirectly from free-living protozoan ancestors. Free-living protozoa are also of huge ecological significance, often representing the major predators of bacteria and other micro-organisms within a system. Most of our projects examine poorly-studied protozoa that are of special significance for inferring the evolutionary history of eukaryotic life, or that inhabit extraordinary environments, and have undergone significant adaptive change relative to ‘typical’ eukaryote cells. We also examine the diversity of understudied-but-ecologically-important protozoa from marine sediments, and symbiotic or parasitic associations between protozoa and certain other marine organisms.

Evolutionary examinations of ‘excavates’ (including anaerobes)

A number of 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 are descended from a common ancestor, forming a clade called ‘Excavata’ (see Simpson, 2003 for a review). Excavata is now often treated as one of the 5-6 proposed ‘super-groups’ that would collectively encompass almost all of the known diversity of eukaryotes.

Some multigene phylogenetic and phylogenomic analyses (including Hampl et al., 2009) indeed recover Excavates as monophyletic group. Fascinatingly, however, the monophyly of all excavates has recently come into question again, with the obscure malawimonads branching as a closer relative of animals and fungi in several recent analyses. We are currently conducting a critical examination of the phylogenetic position of malawimonads within eukaryotes using detailed ultrastructural data and phylogenomic analyses (Heiss et al., in prep.).

Several groups of excavates are anaerobes that have highly modified mitochondria, including the well-known parasites Giardia (a diplomonad) and Trichomonas. A long-running project of the lab has been the isolation, characterisation and phylogenetic investigation of free-living anaerobic excavates, especially the Carpediemonas-like organisms, and Trimastigids (Kolisko et al., 2010, Park et al., 2009, 2010; Zhang et al., in prep.). Carpediemonas-like organisms in particular prove to be a diverse paraphyletic group that gave rise to diplomomonads (Takishita et al., 2012). Ongoing collaborations (primarily with the Roger lab at Dalhousie University) aim to trace the modifications of the mitochondrion-related organelles of anaerobic excavates over evolutionary history using taxon-rich comparative transcriptomic and genomic datasets.

The phylogeny and evolution of eukaryotic cells

Certain groups of (mostly) free-living protozoa occupy important ‘deep branching’ positions within the supergroups of eukaryotic life. These include the ‘typical excavates’ as well as various very 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 Mississipi State, and Yuji Inagaki’s group at Tsukuba) we are using phylogenomic approaches to infer the evolutionary tree of eukaryotes including these important overlooked taxa (e.g. Brown et al. 2013). In addition, however, we are also performing very detailed characterisations of the cytoskeletons of these organisms, primarily using serial transmission electron microscopy (e.g. Heiss et al., 2011, 2013a, 2013b).

These data together strongly suggest that some distantly related extant eukaryotes share a large number 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 be able to reconstruct this cytoskeleton in detail today, despite the passage of a billion or more years.

(Extremely) Halophilic Protozoa

Microbiology textbooks give the impression that very hypersaline habitats (>25% salt) are exclusively inhabited by prokayotes, mostly Haloarchaea, and the alga Dunaliella. In reality there many heterotrophic protozoa that have been reported in samples from extremely hypersaline habitats—at least 30 morphologically distinguishable species by our conservative estimate (Park et al., 2009). These include ciliates, amoebae, and several groups of flagellates, some of which have unknown affinities within eukaryotes.

In collaboration with Prof. Byung Cheol Cho of Seoul National University, and/or former Postdoc Jong Soo Park, we have been characterising cultivated isolates of extremely halophilic (or borderline extremely halophilic) protozoa using electron microscopy and molecular phylogenetic techniques, including several new genera (Park et al., 2006, 2009). From this work it is apparent that obligate halophiles (that require salinities above seawater for growth) evolved many times among protozoan eukaryotes (Park and Simpson, in prep.), for example, three times independently in the taxon Heterolobosea alone (see Park et al., 2007, 2009, Park and Simpson, 2011). 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 are likely have secondarily reverted to halotolerance (Harding et al., 2013; Kirby et al., in press). In an ongoing collaboration with Andrew Roger's group at Dalhousie University we are attempting to understand adaptations to halophily in protozoa, primarily using comparative transcriptomic data.

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. Very 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 recently characterised important new cultivations of phagotrophic euglenids with our 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). This approach promises to rapidly capture much more of the taxonomic diversity of phagotrophs than culturing. We have highlighted several current taxonomic, phylogenetic and evolutionary issues, including demonstrating the non-monophyly of several morphologically-defined genera, and identifying which phagotrophic euglenids are most closely related to the osmotrophs.

We are now extending this work to obtain multigene profiles for photodocumented cells, using single-cell whole-genome amplification methods, and/or single-cell transcriptomics. This approach will enable the estimation of multigene phylogenies to get a clearer picture of relationships in euglenids and their biodiversity. A 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.

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

We have recently begun working on protozoa that are (potential) parasites of marine organisms, such as sea urchins and algae.

1) 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). The disease is caused by a facultative pathogen, the amoeba Paramoeba invadens. Paramoeba invadens has not yet been detected in the environment, and thus 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. We have confirmed the distinctiveness of P. invadens using multiple genetic markers (in Feehan et al., 2013), and are now devising and testing molecular methods for detecting P. invadens in tissue and environmental samples. The main goal is to establish where and when P. invadens is present between the disease events.

2) 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 (Lee and Kugrens, unpublished). We have established that these ‘infections’ occur in Codium from Nova Scotian waters, and that they are caused by a single molecular ‘species’. In ongoing work we are further characterising this kinetoplastid morphologically and at the sequence level, and attempting to better understand its geographic spread and its relationship with Codium.