Predatory colponemids are the sister group to all other alveolates

https://doi.org/10.1016/j.ympev.2020.106839Get rights and content

Highlights

  • Phylogenomics resolves Colponemidia as a sister group to all known alveolates.

  • The ancestor of all alveolates was a biflagellate predator feeding by phagocytosis.

  • Colponemids may illuminate the ancestral states of apicomplexans, dinoflagellates, and ciliates.

  • Colponemids are geographically widespread in freshwater habitats.

Abstract

Alveolates are a major supergroup of eukaryotes encompassing more than ten thousand free-living and parasitic species, including medically, ecologically, and economically important apicomplexans, dinoflagellates, and ciliates. These three groups are among the most widespread eukaryotes on Earth, and their environmental success can be linked to unique innovations that emerged early in each group. Understanding the emergence of these well-studied and diverse groups and their innovations has relied heavily on the discovery and characterization of early-branching relatives, which allow ancestral states to be inferred with much greater confidence. Here we report the phylogenomic analyses of 313 eukaryote protein-coding genes from transcriptomes of three members of one such group, the colponemids (Colponemidia), which support their monophyly and position as the sister lineage to all other known alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but they are diverse and widespread in freshwater habitats around the world. Studied colponemids possess two types of extrusive organelles (trichocysts or toxicysts) for active hunting of other unicellular eukaryotes and potentially play an important role in microbial food webs. Colponemids have generally plesiomorphic morphology and illustrate the ancestral state of Alveolata. We further discuss their importance in understanding the evolution of alveolates and the origin of myzocytosis and plastids.

Introduction

Alveolates are one of the largest major groups of eukaryotes with over ten thousand described species and much larger yet undescribed species diversity based on environmental sequence data (Chambouvet et al., 2008, López-García et al., 2001, de Vargas et al., 2015). These organisms have a special type of cell coverings consisting of a plasmalemma and alveoli — abutting single-membrane flattened sacs, which probably derived from the endomembrane system and always subtend the plasma membrane (Gould et al., 2008). Most alveolates belong to one of three major groups: apicomplexans (e.g., the malaria parasite Plasmodium), dinoflagellates (e.g., the coral endosymbiont Symbiodinium), and ciliates (e.g., the model organisms Tetrahymena and Paramecium). These three groups are among the most ecologically successful eukaryotes on Earth, and are also of evolutionary importance since each lineage also displays a number of distinctive innovations. For example, the apicomplexans infect virtually all known animals and this process is mediated by a complex suite of structures called the apical complex (Katris et al., 2014). Ciliates have evolved a large cell size and structures that make them effective predators, as well as a separation of germ and soma within a single cell. Dinoflagellates have evolved a variety of unique features, including several aspects of genomic organization and function, as well as a wide range of trophic strategies including photosynthetic algae, predators, and parasites. The alveolates have also served as models to understand organelle evolution; mitochondrial genomes and respiratory chains are modified in each of the three groups in different ways (Smith et al., 2007, Nash et al., 2008), and apicomplexans and dinoflagellates (together with their closest relatives collectively known as myzozoans) contain plastids that have adopted diverse functional and genomic states (Janouškovec et al., 2017a).

Reconstructing the deep evolutionary transitions that gave rise to these three well-studied and diverse groups has relied heavily on the discovery and characterization of early-branching lineages, which allow ancestral states to be inferred with much greater confidence. Such deep-branching lineages have been particularly useful in analyses of both main myzozoan groups, apicomplexans and dinoflagellates: sister groups such as perkinsids, chrompodellids, Digyalum, and Platyproteum, have all shed light on the evolution of major characters (Goggin and Barker, 1993, Moore et al., 2008, Cavalier-Smith, 2014, Janouškovec et al., 2015, Janouškovec et al., 2019, Mathur et al., 2019). But no equivalent sister group to ciliates is known, and most importantly, no group has unambiguously been shown to be sister to the alveolates as a whole (the next neighbour group, the stramenopiles, is distantly related and shares little in common with alveolates).

A handful of predatory protists have been discussed as possible candidates for deep-branching alveolates, based on sequences from one or a small number of genes (Janouškovec et al., 2013, Tikhonenkov et al., 2014, Park and Simpson, 2015). A six gene phylogeny suggested that Acavomonas is the sister taxon to myzozoans and Colponema is even deeper branching, perhaps as a sister lineage to all other alveolates (Janouškovec et al., 2013). The phylogeny of the small subunit ribosomal RNA gene (SSU rDNA) further showed that Palustrimonas and five clades of environmental sequences represent additional deep alveolate lineages (Janouškovec et al., 2013, Park and Simpson, 2015). Their relationships with Acavomonas, Colponema, ciliates, myzozoans are unresolved on SSU rDNA trees, but they all appear evolutionarily distinct (Tikhonenkov et al., 2014, Park and Simpson, 2015). Such diversity of deep-branching taxa could be instrumental in interpreting the alveolate origin and early evolution. In particular, an early-branching position for Colponema could be consistent with it possessing structures and behaviors thought to be plesiomorphic in the group (Mignot and Brugerolle, 1975, O’Kelly, 1993, Tikhonenkov et al., 2014).

Here we report the phylogenomic analyses of transcriptomes of three colponemid strains, which support their monophyly in the phylum Colponemidia, and indicate their position as the sister lineage to all other known alveolates. We further analyse the environmental diversity of colponemids and discuss their role in the ecosystems and importance in understanding the evolution of alveolates.

Section snippets

Collection, culture establishment and microscopy

Clones Colp-22, Colp-26, and Colp-tractor were obtained from samples collected from freshwater bodies in Ukraine (Seret river plankton; Dniester Canyon; 48°40′01.2″N, 25°51′04.2″E), Con Dao Island (Quang Trung lake; bottom detritus within lotuses; 8°41′31.2″N, 106°36′24.1″E), and British Columbia (City of Delta, Burns Bog; water with plant debris and detritus near a sunken tractor; 49°08′41.8″N 122°55′51.3″W), respectively. Colponemid clone Colp-10 was isolated from desert soil in Morocco (same

DNA and RNA extraction, sequencing, and assembling

Cells of strains Colp-7a, Colp-10, Colp-15, Colp-22, Colp-26, and Colp-tractor were grown in clonal laboratory cultures and were harvested following peak abundance after eating most of the prey. Cells were collected by centrifugation (2000g, room temperature) on the 0.8 μm membrane of Vivaclear Mini columns (Sartorium Stedim Biotech Gmng, Germany, Cat. No. VK01P042). Total RNA of strains Colp-7a, Colp-10, Colp-15 was extracted using the RNAqueous®-Micro Kit (ThermoFisher Scientific, cat. #

Phylogenomic analyses

To determine the evolutionary position of colponemids, we generated transcriptomes of Colponema vietnamica strain Colp-7a and two newly-discovered relatives, and incorporated their sequences into a phylogenomic dataset (Materials and Methods). All genes were individually inspected for orthology by computing single-gene phylogenies and removing paralogs and contaminant sequences. The final phylogenomic matrix contained 51 OTUs and 313 protein-coding genes (84,451 amino acid positions). The

Phylogeny and diversity of Colponemidia

Previous phylogenetic analyses of Colponema based on rDNA and a set of six genes failed to conclusively resolve its evolutionary position. SSU and SSU/LSU rDNA phylogenies placed it as a sister to myzozoans, or myzozoans + Acavomonas (Janouškovec et al., 2013, Janouškovec et al., 2017b, Mikhailov et al., 2014, Tikhonenkov et al., 2014, Cavalier-Smith, 2018), or sister to myzozoans + Acavomonas + Palustrimonas (Park and Simpson, 2015). Hsp90 and alpha tubulin phylogenies placed Colponema as a

CRediT roles

Denis V. Tikhonenkov: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Writing - original draft, Writing - review & editing. Jürgen F.H. Strassert: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Jan Janouškovec: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Alexander

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank A.A. Abramov, A.V. Shatilovich, Y.V. Dubrovsky, L. Nguyen-Ngoc, H. Doan-Nhu, E. S. Gusev, A.N. Tsyganov, and the staff of the Russian-Vietnam Tropical Centre, Coastal Branch, Nha Trang, especially Hoan Q. Tran, Tran Duc Dien, and Nguyen Thị Hai Thanh for assistance with sample collection and trip management. Field work in Vietnam is part of the project ‘Ecolan 3.2’ of the Russian-Vietnam Tropical Centre. This work was supported by grants from the Russian Foundation for Basic Research

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  • Cited by (0)

    1

    Shared first authorship, both authors contributed equally.

    2

    Alexander P. Mylnikov is deceased.

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