Non-photosynthetic predators are sister to red algae

Abstract

Rhodophyta (red algae) is one of three lineages of Archaeplastida1, a supergroup that is united by the primary endosymbiotic origin of plastids in eukaryotes2,3. Red algae are a diverse and species-rich group, members of which are typically photoautotrophic, but are united by a number of highly derived characteristics: they have relatively small intron-poor genomes, reduced metabolism and lack cytoskeletal structures that are associated with motility, flagella and centrioles. This suggests that marked gene loss occurred around their origin4; however, this is difficult to reconstruct because they differ so much from the other archaeplastid lineages, and the relationships between these lineages are unclear. Here we describe the novel eukaryotic phylum Rhodelphidia and, using phylogenomics, demonstrate that it is a closely related sister to red algae. However, the characteristics of the two Rhodelphis species described here are nearly opposite to those that define red algae: they are non-photosynthetic, flagellate predators with gene-rich genomes, along with a relic genome-lacking primary plastid that probably participates in haem synthesis. Overall, these findings alter our views of the origins of Rhodophyta, and Archaeplastida evolution as a whole, as they indicate that mixotrophic feeding—that is, a combination of predation and phototrophy—persisted well into the evolution of the group.

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Fig. 1: Cell morphology of R. limneticus.
Fig. 2: The evolutionary position of Rhodelphis.
Fig. 3: Genomic insights into Rhodelphis cell biology.

Data availability

Raw transcriptome and genome reads from R. limneticus and R. marinus are deposited in GenBank (PRJNA544719), along with full SSU rRNA gene sequences for R. marinus (MK966712) and R. limneticus (MK966713). Assembled transcriptomes and genomes, along with raw light and electron-microscopy images, individual gene alignments, concatenated and trimmed alignments, single-gene trees, and maximum-likelihood and Bayesian tree files for the 151-taxon and 153-taxon datasets have been deposited in Dryad (https://doi.org/10.5061/dryad.tr6d8q2). The family Rhodelphidae (urn:lsid:zoobank.org:act:80B5C004-2954-4A57-A411-482BCD29E85D), genus Rhodelphis (urn:lsid:zoobank.org:act:6D09D4D9-D9FC-4D0C-8FB2-55FD9DDEAD53) and species Rhodelphis limneticus (urn:lsid:zoobank.org:act:695ACD0B-8151-4609-97FC-A044A312BE22) and Rhodelphis marinus (urn:lsid:zoobank.org:act:84233191-4710-43D1-A2DA-914B8E7B7E01) have been registered with the Zoobank database (http://zoobank.org/).

Code availability

All unpublished code is available upon reasonable request from the corresponding authors.

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Acknowledgements

We thank L. Nguyen-Ngoc, H. Doan-Nhu, E. S. Gusev, Y. Dubrovsky and the staff of the Russian-Vietnam Tropical Centre, Coastal Branch for assistance with sample collection and trip management; S. A. Karpov for assistance with interpretation of transmission electron microscopy images; Compute/Calcul Canada for computational resources, especially the Orcinus (Westgrid) and Mammouth Parallèle II (Calcul Québec) clusters. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to P.J.K. (grant 227301). Field work in Vietnam is part of the project ‘Ecolan 3.2’ of the Russian-Vietnam Tropical Centre. R.M.R.G. was supported by a fellowship from the Canadian Institutes of Health Research and a grant from the Tula Foundation to the Centre for Microbial Diversity and Evolution. Cell isolation and culturing, generation of material for sequencing, light and electron microscopy and analysis were supported by the Russian Science Foundation to D.V.T. (grant 18-14-00239). F.H. is supported by an EMBO fellowship (ALTF 1260–2016).

Author information

R.M.R.G., D.V.T. and P.J.K. designed the study. D.V.T. isolated and cultured cells. D.V.T. and F.H. generated material for sequencing and F.H. removed contaminant sequences from the genome assembly. D.V.T. and A.P.M. performed microscopy experiments. R.M.R.G. performed phylogenomic, genomic and transcriptomic analyses. E.H. performed phylogenetic analysis of plastid proteins. R.M.R.G., D.V.T. and P.J.K. wrote the manuscript with input from all authors.

Correspondence to Ryan M. R. Gawryluk or Denis V. Tikhonenkov or Patrick J. Keeling.

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Competing interests

The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Geoffrey McFadden and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Cell structure of R. limneticus.

Related to Fig. 1. a, b, Scanning electron microscopy images showing the flagella and mastigonemes on the posterior flagellum. c, Section of an anterior flagellum. d, Section of a posterior flagellum. eg, Arrangement of the transitional zone of a flagellum with transverse plate and cylinder. h, Wide microtubular band 2 accompanies the posterior flagellum. il, Cell sections from anterior to posterior. m, Single microtubules inside the cytoplasm. n, A rhizoplast connects the basal body of the posterior flagellum to the nucleus. o, Area of cell with nucleus, rudiments of glycostyles inside the vesicles and smooth endoplasmic reticulum. p, Contractile vacuole. q, Osmiophilic body. r, s, Phagocytosis of eukaryotic prey and bacteria. t, Cell section showing food vacuole with several engulfed bacterial cells. bc, bacterium; cl, cylinder; cv, contractile vacuole; fv, food vacuole; gl, glycostyles; bb1, basal body of posterior flagellum; bb2, basal body of anterior flagellum; mn, mastigonemes; mrt, microtubule; n, nucleus; nmb, narrow microtubular band; ob, osmiophilic body; pf, posterior flagellum; pr, eukaryotic prey; rgl, rudiments of glycostyles; rp, rhizoplast; ser, sac of smooth endoplasmic reticulum; sm, single microtubule; ss, striated structure; st, satellite of basal body; tp, transverse plate; wmb1, wide microtubular band 1; wmb2, wide microtubular band 2. Scale bars, 5 μm (a, b), 0.5 μm (c, d, io), 0.2 μm (eh, q), 1 μm (p) and 2 μm (rt). These experiments were repeated three (a, b) and seven (ct) times, with similar results.

Extended Data Fig. 2 Cell structure of R. marinus.

a, b, Living cells, obtained by light microscopy. c, Longitudinal section of the cell, d, Region of the cell surface with glycostyles. e, Basal body of posterior flagellum with outgoing fibrils and striated structure. f, Section of the flagellum covered with glycostyles and dark granules. g, Emergence of a posterior flagellum. h, Basal body of the posterior flagellum and mitochondrion with tubular cristae. i, Formation of rudiments of glycostyles in perinuclear space. j, Nucleus, mitochondrion and reserve substance. k, Osmiophilic formation and microtubules. cr, cristae; dg, dark granules; fb, fibril; gl, glycostyles; bb1, basal body of posterior flagellum; bb2, basal body of anterior flagellum; mt, mitochondrion; mrt, microtubules; n, nucleus, of, osmiophilic (dark) formation; pf, posterior flagellum; rgl, rudiments of glycostyles; rs, reserve substance; ser, sac of smooth endoplasmic reticulum; ss, striated structure. Scale bars, 10 μm (a, b), 2 μm (c), 0.2 μm (d, e), 0.5 μm (fi, k) and 1 μm (j). These experiments were repeated ten (a, b) and three (ck) times, with similar results.

Extended Data Fig. 3 Phylogenomic analysis of the concatenated 153/253 dataset.

a, Bayesian tree using the CAT + GTR evolutionary model as implemented in PhyloBayes. b, Maximum-likelihood tree using the LG + C60 + F + G4 model as implemented in IQ-TREE. Black dots denote full statistical support (Bayesian posterior probability = 1.0, maximum-likelihood ultrafast bootstrap and SH-aLRT = 100%); support values <0.7/70% are not shown (indicated by ‘–’). c, Bootstrap support for maximum-likelihood trees (PROT + CAT + LG + F) after progressive removal of the fastest evolving amino acid sites shows that both the Rhodelphis and red algae and the picozoa, Rhodelphis and red algae relationships are relatively robust to data removal. Similar to the 151/253 dataset, support for Archaeplastida paraphyly (Cryptista, green plants and glaucophytes (green and glauc.)) decreases with data removal, whereas Archaeplastida monophyly support increases. Support for Opisthokonta monophyly serves as a control for the presence of sufficient information for phylogenomic inference.

Extended Data Fig. 4 Phylogenomic analysis of the concatenated 151/253 dataset.

a, Bayesian tree using the CAT + GTR evolutionary model as implemented in PhyloBayes. b, Maximum-likelihood tree using the LG + C60 + F + G4 model as implemented in IQ-TREE. Black dots denote full statistical support (Bayesian posterior probability = 1.0, maximum-likelihood ultrafast bootstrap and SH-aLRT = 100%); support values <0.7/70% are not shown.

Extended Data Fig. 5 Internode certainty analyses for the 151/253 and 151/50 datasets.

Internode certainty and internode certainty-all scores were calculated with RAxML v.8.1.6 and mapped onto the 151/253 maximum-likelihood tree topology presented in Extended Data Fig. 4b. a, Internode certainty and internode certainty-all scores for 253 individual bootstrapped maximum-likelihood trees used to generate the concatenated alignment for Extended Data Fig. 4b. b, Internode certainty and internode certainty-all scores for the 50 trees among the 253-tree dataset that have the highest RTC scores, which are expected to improve the robustness of phylogenomic analyses. Internode certainty scores for the sister relationship of Rhodelphis and red algae are higher for the 50 best-supported trees, indicating that they also support this relationship, and with fewer conflicting signals.

Extended Data Fig. 6 Phylogenomic analysis based on concatenation of 50 single-gene datasets with highest RTC scores.

Maximum-likelihood tree using the LG + PMSF + G model as implemented in IQ-TREE (151 taxa, 50 proteins, 21,886 sites). Black dots denote full statistical support (maximum-likelihood ultrafast bootstrap and SH-aLRT = 100%); support values <0.7/70% are not shown. The sister relationship of Rhodelphis and red algae still receives full statistical support with a highly reduced, phylogenetically well-supported dataset.

Extended Data Fig. 7 A coalescence phylogenomic framework recovers Rhodelphis as sister to red algae based on individual gene trees.

Individual bootstrapped gene trees were generated with RAxML v.8.1.6 and used to generate a species tree with ASTRAL-III under default parameters and 100 bootstrap replicates. Support values <0.7/70% are not shown. a, Species trees were made from all 253 single-gene trees from the 151/253 dataset. b, Species trees were made from the 50 trees with the highest relative tree certainty. The sister relationship of Rhodelphis and red algae is recovered with both datasets, and is consistent with concatenated phylogenomic analyses.

Extended Data Fig. 8 Genomic support for Rhodelphis as non-photosynthetic phagotrophs.

a, b, Principal component (PC) plots of gene ontology (GO) category score from free-living phagocytes (a) and photosynthetic organisms (b). a, Rhodelphis associate with phagocytes, but not with photosynthetic eukaryotes. Dashed ellipses represent 95% confidence intervals based on training datasets using a model defined by free-living phagocytes (a; n = 86 GO categories, 474 proteins) and photosynthesis model (b; n = 37 GO categories, 243 proteins). c, Heat map of phagocyte GO terms showing (as in a) that Rhodelphis gene repertoires are similar to phagocytes. Analyses were performed using PredictTrophicMode_Tool.R.

Extended Data Fig. 9 Rhodelphis encode plastid-targeted proteins with N-terminal targeting sequences and homologues of the TIC/TOC plastid import system.

a, An alignment of plastid-type chaperonin 60 (related to Fig. 3b) shows that Rhodelphis nuclear genomes encode plastid-targeted proteins that have clear N-terminal extensions (cTP) relative to plastid-encoded red algal homologues (names in red) and cyanobacterial homologues (names in cyan), but lack a signal peptide (SP) characteristic of proteins targeted to complex secondary or tertiary plastids, as found in Plasmodium (orange). be, Rhodelphis nuclear genomes encode bona fide homologues of plastid protein import subunits TIC20, TIC32, TIC22 and TOC75, which are specific genetic markers for plastid presence.

Extended Data Table 1 Summary of Rhodelphis genome and transcriptome data

Supplementary information

Supplementary Information Formal taxonomic description and detailed morphology. This file provides formal taxonomic descriptions of Rhodelphis limneticus and R. marinus, along with a discussion of evolutionary considerations drawn from the morphological data.

Reporting Summary

Supplementary Table 1 Predicted Rhodelphis plastid-targeted proteins. Rhodelphis proteins with predicted N-terminal plastid transit peptides and/or plastid-type origin are presented. R. marinus and R. limneticus data are presented on separate tabs. Protein sequences are derived from transcriptome datasets, and augmented for R. limneticus by preliminary gene predictions from nuclear genomic data. Note that some protein sequences are incomplete.

Video 1 Rhodelphis actively phagocytose eukaryotic prey. Light microscopic video of a R. limneticus cell actively ingesting a kinetoplastid prey cell (Parabodo caudatus) through its posterior end. The R. marinus culture was lost prior to video documentation, but it feeds in the same fashion.

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