Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes

Abstract

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes¹. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved^2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

Fig. 1. Phylogenomic analysis of 15 concatenated ribosomal proteins expands Asgard archaea diversity.

ML tree (IQ-TREE, WAG+C60+R4+F+PMSF model) of concatenated protein sequences from at least 5 genes, encoded on a single contig, of a RP15 gene cluster retrieved from publicly available and newly reported Asgard archaeal MAGs. Bootstrap support (100 pseudo-replicates) is indicated by circles at branches, with filled and open circles representing values equal to or larger than 90% and 70% support, respectively. Leaf names indicate the geographical source and isolate name (inner and outer label, respectively) for the MAGs reported in this study. Only the in-group is shown (263 out of 542 total sequences). Scale bar denotes the average number of substitutions per site. AB, Aarhus Bay (Denmark); ABE, ABE vent field, Eastern Lau Spreading Center; ALCG, Asgard Lake Cootharaba Group; Asgard, Asgardarchaeia; Baldr, Baldrarchaeia; GB, Guaymas Basin (Mexico); Gerd, Gerdarchaeales; Hel, Helarchaeales; Heimdall, Heimdallarchaeaceae; Hermod, Hermodarchaeia; Hod, Hodarchaeales; Jord, Jordarchaeia; JZ, Jinze (China); Kari, Kariarchaeaceae; Loki, Lokiarchaeales; Mar, Mariner vent field, Eastern Lau Spreading Center; Njord, Njordarchaeales; Odin, Odinarchaeia; QC, QuCai village (China); QZM, QuZhuoMu village (China); RP, Radiata Pool (New Zealand); SHR, South Hydrate Ridge; Sif, Sifarchaeia; Thor, Thorarchaeia; TNS, Taketomi Island (Japan); WOR: White Oak River (USA); Wukong, Wukongarchaeia.

Fig. 2. Phylogenomic analyses based on 57 concatenated non-ribosomal proteins support the emergence of eukaryotes as a sister to Hodarchaeales.

a, BI based on 278 archaeal taxa, using Euryarchaea and TACK archaea as the outgroup (not shown) (NM57-A175-nDK_sr4 alignment, 15,733 amino acid positions). The concatenation was SR4-recoded and analysed using the CAT+GTR model (4 chains, approximately 25,000 generations). b, Schematic representation of the shift in the position of eukaryotes (grey branches) in ML and BI analyses of this dataset under different treatments. Untreated, unprocessed dataset; Recoding, SR4-recoded dataset; Recoding+FSR, Fast-site removal combined with SR4-recoding (the topology most often recovered after removing 10–50% fastest-evolving sites, in steps of 10%, is shown). The indices 175 and 64 refer to phylogenomic datasets containing 175 and 64 Asgard archaea, respectively. Note that BI was not performed for the 175 untreated dataset owing to computational limitations. For detailed results of phylogenomic analyses, see Supplementary Table 3. Scale bar denotes the average expected number of substitutions per site.

Fig. 3. Eukaryotic signature proteins in Asgard archaea.

Distribution of ESP homologues in Asgard archaea grouped by function. Shaded rectangles above the protein names indicate ESPs newly identified as part of this study. Predicted homologues are depicted by coloured circles: fully filled circles indicate that we detected homologues in at least half of the representative genomes of the clade; half-filled circles indicate that we detected homologues in fewer than half of the representative genomes of the clade. Hodarchaeales ESP homologues are highlighted against a grey background. Accession numbers are available in Supplementary Table 3.

Fig. 4. Genome dynamics, OGT predictions and metabolic reconstruction of Asgard ancestors.

a, Duplication and loss rates inferred for Asgard archaeal ancestors, normalized by proteome size. P values given for each two-sided Wilcoxon-test against the median values of TACK and Euryarchaea (Eury-TACK) ancestors, where *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. No corrections were done for multiple comparisons. b, OGT predictions predicted by genomic features. Right, OGTs within Heimdallarchaeia. Actual values are available in Supplementary Table 5. In a and b, boxplots are represented as a central line denoting the median value, a coloured box containing the first and third quartiles of the dataset, and whiskers representing the lowest and highest values within 1.5 times the interquartile range, and sample sizes are shown within parentheses on the axis labels. c, We predict that the LAsCA transitioned from a hyperthermophilic fermentative lifestyle to a mesophilic mixotroph lifestyle. The LAsCA probably encoded gluconeogenic (Gluconeo.) pathways through the reverse EMP gluconeogenic pathway and through fructose 1,6-bisphosphate aldolase/phosphatase (FBP A/P). The major energy-conserving step in the early Asgard ancestors could have been the ATP synthesis by fermentation of small organic molecules (acetate, formate or formaldehyde). The reverse ribulose monophosphate pathway (rRuMP) was a key pathway in the LAsCA for the generation of reducing power. The WLP appeared only present in the LAsCA. The tricarboxylic acid (TCA) cycle is predicted complete in all three ancestors, the Hodarchaeales common ancestor encoding the most complete ETC, and probably used nitrate as a terminal electron acceptor. Membrane-associated ATP biosynthesis coupled to the oxidation of NADH and succinate and reduction of nitrate could have been present in the LAECA. c, cupredoxin; NR, nitrate reductase; OPPP, oxidative pentose phosphate pathway; PEP, phosphoenolpyruvate; PRK: phosphoribulokinase; Q, quinone; RHP, reductive hexulose-phosphate; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; TMA, trimethylamine.

Extended Data Fig. 1. Cladogram of proposed taxonomic scheme for the ranks of family, order and class for Asgard archaeal lineages employed in this study.

Equivalent names in GTDB are shown in parentheses. Cases with differing or new names have been highlighted in colored bold italics.

Extended Data Fig. 2. Asgard archaea encode homologs of eukaryotic protein complexes involved in N-glycosylation.

The Sec61, the OST and TRAP complexes are depicted according to their eukaryotic composition and localization. On the right-hand side of the panel, dark-colored subunits represent eukaryotic proteins which have prokaryotic homologs in Asgard archaea newly identified as part of this work; Light-colored subunit homologs have been described previously. Figure generated using BioRender (https://www.biorender.com).

Extended Data Fig. 3. Identification of previously undetected vesicular trafficking ESPs in Asgard archaea.

Schematic representation of a eukaryotic cell in which ESPs involved in membrane trafficking and endosomal sorting that have been identified in Asgard archaea are highlighted. Colored subunits have been detected in some Asgard archaea while grey ones seem to be absent from all current representatives. Only major protein complexes are depicted. Additional components can be found in Fig. 2. From left to right, top to bottom: RC, Retromer complex. Retromer is a coat-like complex associated with endosome-to-Golgi retrograde traffic. It is formed by Vacuolar protein sorting-associated protein 35, Vps5, Vps17, Vps26 and Vps29. During cargo recycling, retromer is recruited to the endosomal membrane via the Vps5-Vps17 dimer. Cargo recognition is thought to be mediated primarily through Vps26 and possibly by Vps35. Finally, the BAR domains of Vps5-Vps17 deform the endosomal membrane to form cargo-containing recycling vesicles. Their distribution is sparse, but we have detected Asgard archaeal homologs of all subunits except for Vps17. Interestingly, the Thorarchaeia Vps5-BAR domain is often fused to Vps28, a subunit of the ESCRT machinery complex I, suggesting a functional link between BAR domain proteins and the thorarchaeial ESCRT complex. The best-characterized retromer cargo is Vps10. This transmembrane protein receptor is known in yeast and mammal cells to be involved in the sorting and transport of lipoproteins between the Golgi and the endosome. The Vps10 receptor releases its cargo to the endosome and is recycled back to the Golgi via the retromer complex. CORVET: Class C core vacuole/endosome tethering complex; HOPS: Homotypic fusion and protein sorting complex. Endosomal fusion and autophagy depend on the CORVET and HOPS hexameric complexes; they share the core subunits Vps11, Vps16, Vps18, and Vps33. In addition, HOPS is composed of Vps41 and Vps39. Vps39, found associated to late endosomes and lysosomes, promotes endosomes/lysosomes clustering and their fusion with autophagosomes. AP, Adaptor Proteins. Asgard archaea genomes from diverse phyla encode key functional domains of the AP complexes. The eukaryotic AP tetraheteromeric structure is depicted, each color corresponding to a PFAM functional domain (Medium green: Adaptin, N terminal region; Dark green: Alpha adaptin, C-terminal domain; Light green: Beta2-adaptin appendage, C-terminal sub-domain; Dark pink/clear outline: Clathrin adaptor complex small chain; Light pink/dark outline: C-ter domain of the mu subunit); all five domains were detected in Asgard archaea, although not fused to each other. GARP: Golgi-associated retrograde protein complex. The GARP complex is a multisubunit tethering complex located at the trans-Golgi network where it functions to tether retrograde transport vesicles derived from endosomes^,. GARP comprises four subunits, VPS51, VPS52, VPS53, and VPS54. ESCRT: Endosomal Sorting Complex Required for Transport system. This complex machinery performs a topologically unique membrane bending and scission reaction away from the cytoplasm. While numerous components of the ESCRT-I, II and III systems have been previously detected in Asgard archaea^,,, we here report Asgard homologs for several ESCRT-III regulators Vfa1, Vta1, Ist1, and Bro1. The bottom panel shows where these complexes mainly act in eukaryotic cells. Ub: Ubiquitin; Vps: vacuolar protein sorting. Subunit names in grey indicate that no homologs were detected in Asgard archaea. Domains newly identified as part of this study are indicated with an asterisk. Figure created using BioRender (https://www.biorender.com).