The phylum Apicomplexa comprises human pathogens such as Plasmodium but is also an under-explored hotspot of evolutionary diversity central to understanding the origins of parasitism and non-photosynthetic plastids. We generated single-cell transcriptomes for all major apicomplexan groups lacking large-scale sequence data. Phylogenetic analysis reveals that apicomplexan-like parasites are polyphyletic and their similar morphologies emerged convergently at least three times. Gregarines and eugregarines are monophyletic, against most expectations, and rhytidocystids and Eleutheroschizon are sister lineages to medically important taxa. Although previously unrecognized, plastids in deep-branching apicomplexans are common, and they contain some of the most divergent and AT-rich genomes ever found. In eugregarines, however, plastids are either abnormally reduced or absent, thus increasing known plastid losses in eukaryotes from two to four. Environmental sequences of ten novel plastid lineages and structural innovations in plastid proteins confirm that plastids in apicomplexans and their relatives are widespread and share a common, photosynthetic origin.

Environmental SSU rDNA surveys have significantly improved our understanding of microeukaryotic diversity. Many of the sequences acquired using this approach are closely related to lineages previously characterized at both morphological and molecular levels, making interpretation of these data relatively straightforward. Some sequences, by contrast, appear to be phylogenetic orphans and are sometimes inferred to represent “novel lineages” of unknown cellular identity. Consequently, interpretation of environmental DNA surveys of cellular diversity rely on an adequately comprehensive database of DNA sequences derived from identified species. Several major taxa of microeukaryotes, however, are still very poorly represented in these databases, and this is especially true for diverse groups of single-celled parasites, such as gregarine apicomplexans. This study attempts to address this paucity of DNA sequence data by characterizing four different gregarine species, isolated from the intestines of crustaceans, at both morphological and molecular levels: Thiriotia pugettiae sp. n. from the graceful kelp crab (Pugettia gracilis), Cephaloidophora cf. communis from two different species of barnacles (Balanus glandula and B. balanus), Heliospora cf. longissima from two different species of freshwater amphipods (Eulimnogammarus verrucosus and E. vittatus), and Heliospora caprellae comb. n. from a skeleton shrimp (Caprella alaskana). SSU rDNA sequences were acquired from isolates of these gregarine species and added to a global apicomplexan alignment containing all major groups of gregarines characterized so far. Molecular phylogenetic analyses of these data demonstrated that all of the gregarines collected from crustacean hosts formed a very strongly supported clade with 48 previously unidentified environmental DNA sequences. This expanded molecular phylogenetic context enabled us to establish a major clade of intestinal gregarine parasites and infer the cellular identities of several previously unidentified environmental SSU rDNA sequences, including several sequences that have formerly been discussed broadly in the literature as a suspected “novel” lineage of eukaryotes.

The evolutionary and ecological importance of predatory flagellates are too often overlooked. This is not only a gap in our understanding of microbial diversity, but also impacts how we interpret their better-studied relatives. A prime example of these problems is found in the alveolates. All well-studied species belong to three large clades (apicomplexans, dinoflagellates, and ciliates), but the predatory colponemid flagellates are also alveolates that are rare in nature and seldom cultured, but potentially important to our understanding of alveolate evolution. Recently we reported the first cultivation and molecular analysis of several colponemid-like organisms representing two novel clades in molecular trees. Here we provide ultrastructural analysis and formal species descriptions for both new species, Colponema vietnamica n. sp. and Acavomonas peruviana n. gen. n. sp. Morphological and feeding characteristics concur with molecular data that both species are distinct members of alveolates, with Acavomonas lacking the longitudinal phagocytotic groove, a defining feature of Colponema. Based on ultrastructure and molecular phylogenies, which both provide concrete rationale for a taxonomic reclassification of Alveolata, we establish the new phyla Colponemidia nom. nov. for the genus Colponema and its close relatives, and Acavomonidia nom. nov. for the genus Acavomonas and its close relatives. The morphological data presented here suggests that colponemids are central to our understanding of early alveolate evolution, and suggest they also retain features of the common ancestor of all eukaryotes.

Metchnikovellidae are a group of unusual microsporidians that lack some of the defining ultrastructural features characteristic of derived Microsporidia and are thought to be one of their earliest-branching lineages. The basal position of metchnikovellids was never confirmed by molecular phylogeny in published research, and thus far no genomic data for this group were available. In this work, we obtain a partial genome of metchnikovellid Amphiamblys sp. using multiple displacement amplification, next-generation sequencing, and metagenomic binning approaches. The partial genome, which we estimate to be close to 90% complete, displays genome compaction on par with gene-dense microsporidian genomes, but contains an unusual repertoire of unique repeat elements. Phylogenetic analyses of multigene datasets place Amphiamblys sp. as the first branch of the microsporidian lineage following the divergence of a mitochondriate microsporidian Mitosporidium. We find evidence for a mitochondrial remnant presumably functionally equivalent to a mitosome in Amphiamblys sp. and the common enzymatic complement for microsporidian anaerobic metabolism. Comparative genomic analyses identify the conservation of components for clathrin vesicle formation as one of the key features distinguishing the metchnikovellid from its highly derived relatives. The presented data confirm the notion of Metchnikovellidae as a less derived microsporidian group, and provide an additional stepping stone for reconstruction of an evolutionary transition from the early diverging parasitic fungi to derived Microsporidia.