Da Cunha et al. determined the quality of the Loki1 genome bin with CheckM [5] and observed 78.21% heterogeneity, which they incorrectly interpreted as a sign of contamination. CheckM measures heterogeneity based on the detection of multiple copies of single-copy marker genes, and in fact, 78.21% of the redundant markers share at least 90% amino acid identity. This confirms our original report, which reported that the Lokiarchaeum genome bin comprises sequences from closely related strains [1].

The authors argue that single-gene trees indicate contamination because Asgard homologs are not always monophyletic. However, given that Loki1–3 representatives belong to the distinct phyla Lokiarchaeota and Heimdallarchaeota, this is not surprising [2]. Furthermore, most deep nodes in these single-gene trees are not significantly supported, which hinders reaching strong conclusions regarding the placement of these sequences.

Da Cunha et al. also state that the single gene–encoding RNA polymerase subunit A (rpoA) represents a contaminant of the Heimdallarchaeote LC_3 bin, because rpoA is encoded by two genes in other Asgard archaea, while some lineages within TACK encode a single gene [2]. BLAST searches for the N- and C-termini of the Heimdallarchaeote LC_3 rpoA gene sequence actually recover best hits to other Asgard archaea, thus indicating that it represents a bona fide Heimdallarchaeote gene. In addition, Heimdallarchaeote LC_3 RpoA forms a strongly supported monophyletic group with the homologs of all other Asgard archaea in phylogenetic analyses [2]. Finally, split/fusion events of archaeal RpoA subunits appear to have occurred more frequently than assumed previously [2], rendering them unsuited for the elucidation of distant evolutionary events.

Moreover, the authors claim that lokiarchaeal EF-2 homologs contain indels indicative of chimerism. However, it is unlikely that chimerism has affected the same gene in all Loki- and Heimdallarchaeota independently while leaving all other genes in these genomes unaffected. The evolution of the EF-2 homologs in archaea is actually a much more complex subject than previously acknowledged. An analysis of the archaeal EF-2 paralogs and their characteristics is already available in [2] and will be discussed in greater detail in a devoted article (Narrowe et al., submitted).

Finally, most of the work by Da Cunha et al. limits itself to the first lokiarchaeal genome bin [1] and disregards the eight additional metagenomic bins from a diversity of Asgard archaea [2]. The currently available Asgard genomes stem from five different samples and metagenomes that were assembled and binned by three independent laboratories using different methodologies. All of them corroborate our original phylogenetic placement of Asgard lineages, their relationship with eukaryotes, and the unique presence of ESPs in all of these genomes.

After running topology tests, Da Cunha et al. argue that only 6 and 11 universal markers statistically rejected 2D and 3D topologies, respectively. They split these two datasets into respective concatenations and reconstructed the corresponding phylogenies (S7 and S8 Figs in [4]). Unsurprisingly, they found a 3D topology for the 6-protein dataset, and a 2D topology, where eukaryotes and Asgard archaea are monophyletic, for the 11-protein concatenation. As the number of individual trees showing a 2D topology is higher when additional longer-branching taxa are included (S1 Fig [4]), the authors incorrectly argue that the topology obtained by the 11-protein dataset is likely the result of a long-branch attraction (LBA) artefact. By definition, LBA artefactually attracts long-branching lineages together, irrespective of their correct phylogenetic placement. Thus, if LBA was affecting the phylogenetic reconstruction, the eukaryotic clade would be attracted outside the archaea, to the distantly related bacterial outgroup, and not well nested within the archaeal clade. Rather, we argue that the 3D topology obtained by Da Cunha et al. for the 6-protein dataset is itself the result of LBA: the branch length connecting the bacterial outgroup to the archaeal and eukaryotic clade is strikingly longer in the 6-protein phylogeny compared to the 11-protein tree (2.1 versus 1.3 average substitution per site). The existence of artefacts in the 3D topology is further supported by the fact that the internal relationships among eukaryotes are less consistent with the accepted phylogeny of organisms than in the 2D tree. For example, the fast-evolving Entamoeba histolytica is attracted toward the base in the 3D tree, whereas it is correctly placed as sister to Dictyostelium discoideum in the 2D tree. The 3D topology reported in Da Cunha et al. is thus most likely incorrect.

Finally, it is important to acknowledge that the 2D topology has been obtained by numerous studies worldwide using different datasets and methods [3,6–14]; several of these studies were published before the publication of the Lokiarchaeum genome.

The claim “removal of the EF-2, is sufficient to break the Eukaryotes-Lokiarchaea affiliation” [4] has been disproven by phylogenetic analyses performed on three different concatenated datasets [2], none of which included elongation factor EF-2 (48 universal single-copy genes, 55 ribosomal proteins, and 16S and 23S rRNA genes, respectively). All analyses clearly retrieved a strongly supported affiliation of eukaryotes with the Asgard superphylum.

The origin of eukaryotes represents a controversial topic, and while we believe it to be refreshing and insightful, our recent work will certainly not be the final word in this debate. However, Da Cunha et al. use inadequate methodology, misinterpret data and ignore previous work [1,2] that already addressed many of their criticisms. In summary, they provide no evidence to falsify the conclusions drawn in Spang et al. and Zaremba-Niedzwiedzka et al.